@article {6585, title = {Base editing of key residues in the BCL11A-XL-specific zinc finger domains derepresses fetal globin expression.}, journal = {Mol Ther}, volume = {32}, year = {2024}, month = {2024 Mar 06}, pages = {663-677}, abstract = {

BCL11A-XL directly binds and represses the fetal globin (HBG1/2) gene promoters, using 3 zinc-finger domains (ZnF4, ZnF5, and ZnF6), and is a potential target for β-hemoglobinopathy treatments. Disrupting BCL11A-XL results in derepression of fetal globin and high HbF, but also affects hematopoietic stem and progenitor cell (HSPC) engraftment and erythroid maturation. Intriguingly, neurodevelopmental patients with ZnF domain mutations have elevated HbF with normal hematological parameters. Inspired by this natural phenomenon, we used both CRISPR-Cas9 and base editing at specific ZnF domains and assessed the impacts on HbF production and hematopoietic differentiation. Generating indels in the various ZnF domains by CRISPR-Cas9 prevented the binding of BCL11A-XL to its site in the HBG1/2 promoters and elevated the HbF levels but affected normal hematopoiesis. Far fewer side effects were observed with base editing- for instance, erythroid maturation in\ vitro was near normal. However, we observed a modest reduction in HSPC engraftment and a complete loss of B cell development in\ vivo, presumably because current base editing is not capable of precisely recapitulating the mutations found in patients with BCL11A-XL-associated neurodevelopment disorders. Overall, our results reveal that disrupting different ZnF domains has different effects. Disrupting ZnF4 elevated HbF levels significantly while leaving many other erythroid target genes unaffected, and interestingly, disrupting ZnF6 also elevated HbF levels, which was unexpected because this region does not directly interact with the HBG1/2 promoters. This first structure/function analysis of ZnF4-6 provides important insights into the domains of BCL11A-XL that are required to repress fetal globin expression and provide framework for exploring the introduction of natural mutations that may enable the derepression of single gene while leaving other functions unaffected.

}, keywords = {Fetal Hemoglobin, gamma-Globins, Gene Editing, Hematopoietic Stem Cells, Humans, Repressor Proteins, Zinc Fingers}, issn = {1525-0024}, doi = {10.1016/j.ymthe.2024.01.023}, author = {Rajendiran, Vignesh and Devaraju, Nivedhitha and Haddad, Mahdi and Ravi, Nithin Sam and Panigrahi, Lokesh and Paul, Joshua and Gopalakrishnan, Chandrasekar and Wyman, Stacia and Ariudainambi, Keerthiga and Mahalingam, Gokulnath and Periyasami, Yogapriya and Prasad, Kirti and George, Anila and Sukumaran, Dhiyaneshwaran and Gopinathan, Sandhiya and Pai, Aswin Anand and Nakamura, Yukio and Balasubramanian, Poonkuzhali and Ramalingam, Rajasekaran and Thangavel, Saravanabhavan and Velayudhan, Shaji R and Corn, Jacon E and Mackay, Joel P and Marepally, Srujan and Srivastava, Alok and Crossley, Merlin and Mohankumar, Kumarasamypet M} } @article {6592, title = {Cryo-EM research in India.}, journal = {Structure}, volume = {32}, year = {2024}, month = {2024 Feb 01}, pages = {113-119}, abstract = {

To celebrate the 50th anniversary of Cell Press and the Cell special issue focusing on structural biology, we want to highlight the rapid progress of cryo-EM related research in India in this collection of Voices. We have asked structural biologists to introduce their research and the national cryo-EM facilities throughout the country.

}, keywords = {Cryoelectron Microscopy, India}, issn = {1878-4186}, doi = {10.1016/j.str.2024.01.002}, author = {Shukla, Arun K and Banerjee, Manidipa and Singh, Appu K and Penmatsa, Aravind and Dutta, Somnath and Anand, Ruchi and Sirajuddin, Minhajuddin and Srivastava, Shanti Swaroop} } @article {6582, title = {Disruption of DNA methylation-mediated cranial neural crest proliferation and differentiation causes orofacial clefts in mice.}, journal = {Proc Natl Acad Sci U S A}, volume = {121}, year = {2024}, month = {2024 Jan 16}, pages = {e2317668121}, abstract = {

Orofacial clefts of the lip and palate are widely recognized to result from complex gene-environment interactions, but inadequate understanding of environmental risk factors has stymied development of prevention strategies. We interrogated the role of DNA methylation, an environmentally malleable epigenetic mechanism, in orofacial development. Expression of the key DNA methyltransferase enzyme DNMT1 was detected throughout palate morphogenesis in the epithelium and underlying cranial neural crest cell (cNCC) mesenchyme, a highly proliferative multipotent stem cell population that forms orofacial connective tissue. Genetic and pharmacologic manipulations of DNMT activity were then applied to define the tissue- and timing-dependent requirement of DNA methylation in orofacial development. cNCC-specific inactivation targeting initial palate outgrowth resulted in OFCs, while later targeting during palatal shelf elevation and elongation did not. Conditional deletion reduced cNCC proliferation and subsequent differentiation trajectory, resulting in attenuated outgrowth of the palatal shelves and altered development of cNCC-derived skeletal elements. Finally, we found that the cellular mechanisms of cleft pathogenesis observed in vivo can be recapitulated by pharmacologically reducing DNA methylation in multipotent cNCCs cultured in vitro. These findings demonstrate that DNA methylation is a crucial epigenetic regulator of cNCC biology, define a critical period of development in which its disruption directly causes OFCs, and provide opportunities to identify environmental influences that contribute to OFC risk.

}, keywords = {Animals, Cell Proliferation, Cleft Lip, Cleft Palate, DNA Methylation, DNA Modification Methylases, Mice, Neural Crest}, issn = {1091-6490}, doi = {10.1073/pnas.2317668121}, author = {Ulschmid, Caden M and Sun, Miranda R and Jabbarpour, Christopher R and Steward, Austin C and Rivera-Gonz{\'a}lez, Kenneth S and Cao, Jocelyn and Martin, Alexander A and Barnes, Macy and Wicklund, Lorena and Madrid, Andy and Papale, Ligia A and Joseph, Diya B and Vezina, Chad M and Alisch, Reid S and Lipinski, Robert J} } @article {6589, title = {Exploring advances in single particle CryoEM with apoferritin: From blobs to true atomic resolution.}, journal = {Int J Biochem Cell Biol}, volume = {169}, year = {2024}, month = {2024 Apr}, pages = {106536}, abstract = {

Deciphering the three-dimensional structures of macromolecules is of paramount importance for gaining insights into their functions and roles in human health and disease. Single particle cryoEM has emerged as a powerful technique that enables direct visualization of macromolecules and their complexes, and through subsequent averaging, achieve near atomic-level resolution. A major breakthrough was recently achieved with the determination of the apoferritin structure at true atomic resolution. In this review, we discuss the latest technological innovations across the entire single-particle workflow, which have been instrumental in driving the resolution revolution and in transforming cryoEM as a mainstream technique in structural biology. We illustrate these advancements using apoferritin as an example that has served as an excellent benchmark sample for assessing emerging technologies. We further explore whether the existing technology can routinely generate atomic structures of dynamic macromolecules that more accurately represent real-world samples, the limitations in the workflow, and the current approaches employed to overcome them.

}, keywords = {Apoferritins, Cryoelectron Microscopy, Humans, Macromolecular Substances}, issn = {1878-5875}, doi = {10.1016/j.biocel.2024.106536}, author = {Premageetha, Gowtham ThambraRajan and Vinothkumar, Kutti R and Bose, Sucharita} } @article {6583, title = {Photostimulated Extended Nitric Oxide (NO) Release from Water-Soluble Block Copolymer to Enhance Antibacterial Activity.}, journal = {Biomacromolecules}, volume = {25}, year = {2024}, month = {2024 Jan 08}, pages = {77-88}, abstract = {

-Nitrosamines are well established motifs to release nitric oxide (NO) under photoirradiation. Herein, a series of amphiphilic -nitrosamine-based block copolymers () are developed to attain controlled NO release under photoirradiation (365 nm, 3.71 mW/cm). The water-soluble forms micellar architecture in aqueous medium and exhibits a sustained NO release of 92-160 μM within 11.5 h, which is 36.8-64.0\% of the calculated value. To understand the NO release mechanism, a small molecular NO donor () resembling the NO releasing functional motif of is synthesized, which displays a burst NO release in DMSO within 2.5 h. The radical nature of the released NO is confirmed by electron paramagnetic resonance (EPR) spectroscopy. The gradual NO release from micellar enhances antibacterial activity over and exhibits a superior bactericidal effect on Gram-positive . In relation to biomedical applications, this work offers a comprehensive insight into tuning light-triggered NO release to improve antibacterial activity.

}, keywords = {Anti-Bacterial Agents, Micelles, Nitric Oxide, Polymers, Staphylococcus aureus}, issn = {1526-4602}, doi = {10.1021/acs.biomac.3c00822}, author = {Paul, Soumya and Ashrit, Priya and Kumar, Manish and Mete, Sourav and Ghosh, Shilpendu and Vemula, Praveen Kumar and Mukherjee, Arindam and De, Priyadarsi} } @article {6593, title = {SMART-lipid nanoparticles enabled mRNA vaccine elicits cross-reactive humoral responses against the omicron sub-variants.}, journal = {Mol Ther}, year = {2024}, month = {2024 Feb 27}, abstract = {

The continual emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has necessitated the development of broad cross-reactive vaccines. Recent findings suggest that enhanced antigen presentation could lead to cross-reactive humoral responses against the emerging variants. Toward enhancing the antigen presentation to dendritic cells (DCs), we developed a novel shikimoylated mannose receptor targeting lipid nanoparticle (SMART-LNP) system that could effectively deliver mRNAs into DCs. To improve the translation of mRNA, we developed spike domain-based trimeric S1 (TS1) mRNA with optimized codon sequence, base modification, and engineered 5{\textquoteright} and 3{\textquoteright} UTRs. In a mouse model, SMART-LNP-TS1 vaccine could elicit robust broad cross-reactive IgGs against Omicron sub-variants, and induced interferon-γ-producing T\ cells against SARS-CoV-2 virus compared with non-targeted LNP-TS1 vaccine. Further, T\ cells analysis revealed that SMART-LNP-TS1 vaccine induced long-lived memory T\ cell subsets, T helper 1 (Th1)-dominant and cytotoxic T\ cells immune responses against the SARS-CoV-2 virus. Importantly, SMART-LNP-TS1 vaccine produced strong Th1-predominant humoral and cellular immune responses. Overall, SMART-LNPs can be explored for precise antigenic mRNA delivery and robust immune responses. This platform technology can be explored further as a next-generation delivery system for mRNA-based immune therapies.

}, issn = {1525-0024}, doi = {10.1016/j.ymthe.2024.02.028}, author = {Mahalingam, Gokulnath and Rachamalla, Hari Krishnareddy and Arjunan, Porkizhi and Karuppusamy, Karthik V and Periyasami, Yogapriya and Mohan, Aruna and Subramaniyam, Kanimozhi and M, Salma and Rajendran, Vigneshwar and Moorthy, Mahesh and Varghese, George M and Mohankumar, Kumarasamypet M and Thangavel, Saravanabhavan and Srivastava, Alok and Marepally, Srujan} } @article {6584, title = {Target-based drug discovery: Applications of fluorescence techniques in high throughput and fragment-based screening.}, journal = {Heliyon}, volume = {10}, year = {2024}, month = {2024 Jan 15}, pages = {e23864}, abstract = {

Target-based discovery of first-in-class therapeutics demands an in-depth understanding of the molecular mechanisms underlying human diseases. Precise measurements of cellular and biochemical activities are critical to gain mechanistic knowledge of biomolecules and their altered function in disease conditions. Such measurements enable the development of intervention strategies for preventing or treating diseases by modulation of desired molecular processes. Fluorescence-based techniques are routinely employed for accurate and robust measurements of activity of molecular targets and for discovering novel chemical molecules that modulate the activity of molecular targets. In the current review, the authors focus on the applications of fluorescence-based high throughput screening (HTS) and fragment-based ligand discovery (FBLD) techniques such as fluorescence polarization (FP), F{\"o}rster resonance energy transfer (FRET), fluorescence thermal shift assay (FTSA) and microscale thermophoresis (MST) for the discovery of chemical probe to exploring target{\textquoteright}s role in disease biology and ultimately, serve as a foundation for drug discovery. Some recent advancements in these techniques for compound library screening against important classes of drug targets, such as G-protein-coupled receptors (GPCRs) and GTPases, as well as phosphorylation- and acetylation-mediated protein-protein interactions, are discussed. Overall, this review presents a landscape of how these techniques paved the way for the discovery of small-molecule modulators and biologics against these targets for therapeutic benefits.

}, issn = {2405-8440}, doi = {10.1016/j.heliyon.2023.e23864}, author = {Kumar, Vikrant and Chunchagatta Lakshman, Puneeth Kumar and Prasad, Thazhe Kootteri and Manjunath, Kavyashree and Bairy, Sneha and Vasu, Akshaya S and Ganavi, B and Jasti, Subbarao and Kamariah, Neelagandan} } @article {3727, title = {Diosgenin enhances liposome-enabled nucleic acid delivery and CRISPR/Cas9-mediated gene editing by modulating endocytic pathways}, journal = {Frontiers in Bioengineering and Biotechnology}, volume = {10}, year = {2023}, month = {01/2023}, doi = {10.3389/fbioe.2022.1031049}, url = {https://doi.org/10.3389\%2Ffbioe.2022.1031049}, author = {Brijesh Lohchania and Abisha Crystal Christopher and Porkizhi Arjunan and Gokulnath Mahalingam and Durga Kathirvelu and Aishwarya Prasannan and Vigneshwaran Venkatesan and Pankaj Taneja and Mohan Kumar KM and Saravanabhavan Thangavel and Srujan Marepally} } @article {3870, title = {High-affinity binding of celastrol to monomeric α-synuclein mitigates in~vitro aggregation.}, journal = {J Biomol Struct Dyn}, year = {2023}, month = {2023 Feb 06}, pages = {1-11}, abstract = {

α-Synuclein (αSyn) aggregation is associated with Parkinson{\textquoteright}s disease (PD). The region αSyn acts as the nucleation {\textquoteright}master controller{\textquoteright} and αSyn as a {\textquoteright}secondary nucleation site{\textquoteright}. They drive monomeric αSyn to aggregation. Small molecules targeting these motifs are promising for disease-modifying therapy. Using computational techniques, we screened thirty phytochemicals for αSyn binding. The top three compounds were experimentally validated for their binding affinity. Amongst them, celastrol showed high binding affinity. NMR analysis confirmed stable αSyn-celastrol interactions involving several residues in the N-terminus and NAC regions but not in the C-terminal tail. Importantly, celastrol interacted extensively with the key motifs that drive αSyn aggregation. Thioflavin-T assay indicated that celastrol reduced αSyn aggregation. Thus, celastrol holds promise as a potent drug candidate for PD.Communicated by Ramaswamy H. Sarma.

}, issn = {1538-0254}, doi = {10.1080/07391102.2023.2175379}, author = {R, Kavya and Aouti, Snehal and Jos, Sneha and Prasad, Thazhe Kootteri and K N, Kumuda and Unni, Sruthi and Padmanabhan, Balasundaram and Kamariah, Neelagandan and Padavattan, Sivaraman and Mythri, Rajeswara Babu} } @article {3717, title = {Systematic review of articular cartilage derived chondroprogenitors for cartilage repair in animal models.}, journal = {J Orthop}, volume = {35}, year = {2023}, month = {2023 Jan}, pages = {43-53}, abstract = {

PURPOSE OF RESEARCH: The potential for cartilage repair using articular cartilage derived chondroprogenitors has recently gained popularity due to promising results from in-vitro and in-vivo studies. Translation of results from in-vitro to a clinical setting requires a sufficient number of animal studies displaying significant positive outcomes. Thus, this systematic review comprehensively discusses the available literature (January 2000-March 2022) on animal models employing chondroprogenitors for cartilage regeneration, highlighting the results and limitations associated with their use.As per Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, a web-based search of PubMed and SCOPUS databases was performed for the following terminologies: "chondroprogenitors", "cartilage-progenitors", and "chondrogenic-progenitors", which yielded 528 studies. A total of 12 studies met the standardized inclusion criteria, which included chondroprogenitors derived from hyaline cartilage isolated using fibronectin adhesion assay (FAA) or migratory assay from explant cultures, further analyzing the role of chondroprogenitors using in-vivo animal models.

PRINCIPAL RESULTS: Analysis revealed that FAA chondroprogenitors demonstrated the ability to attenuate osteoarthritis, repair chondral defects and form stable cartilage in animal models. They displayed better outcomes than bone marrow-derived mesenchymal stem cells but were comparable to chondrocytes. Migratory chondroprogenitors also demonstrated superiority to BM-MSCs in terms of higher chondrogenesis and lower hypertrophy, although a direct comparison to FAA-CPs and other cell types is warranted.

MAJOR CONCLUSIONS: Chondroprogenitors exhibit superior properties for chondrogenic repair; however, limited data on animal studies necessitates further studies to optimize their use before clinical translation for neo-cartilage formation.

}, issn = {0972-978X}, doi = {10.1016/j.jor.2022.10.012}, author = {Vinod, Elizabeth and Padmaja, Kawin and Ramasamy, Boopalan and Sathishkumar, Solomon} } @article {2885, title = {An Amphiphilic Double-Brush Polymer Hydrogel for Sustained Release of Small Molecules and Biologics: Insulin Delivering-Hydrogel to Control Hyperglycemia}, year = {2022}, doi = { https://doi.org/10.1002/cnma.202200184}, author = {Dhayani, Ashish and Bej, Sujoy and Mudnakudu-Nagaraju, Kiran K and Chakraborty, Saheli and Srinath, Preetham and Kumar, Ashok H and PS, Ann Maria and Kristi, Anand and Ramakrishnan, S and Vemula PK} } @article {2542, title = {DDX24 is required for muscle fiber organization and the suppression of wound-induced Wnt activity necessary for pole re-establishment during planarian regeneration.}, journal = {Dev Biol}, volume = {488}, year = {2022}, month = {2022 May 04}, pages = {11-29}, abstract = {

Planarians have a remarkable ability to undergo whole-body regeneration. Successful regeneration outcome is determined by processes like polarity establishment at the wound site, which is followed by pole (organizer) specification. Interestingly, these determinants are almost exclusively expressed by muscles in these animals. However, the molecular toolkit that enables the functional versatility of planarian muscles remains poorly understood. Here we report that SMED_DDX24, a D-E-A-D Box RNA helicase, is necessary for planarian survival and regeneration. We found that DDX24 is enriched in muscles and its knockdown disrupts muscle fiber organization. This leads to defective pole specification, which in turn results in misregulation of many positional control genes specifically during regeneration. ddx24 RNAi also upregulates wound-induced Wnt signalling. Suppressing this ectopic Wnt activity rescues the knockdown phenotype by enabling better anterior pole regeneration. To summarize, our work highlights the role of an RNA helicase in muscle fiber organization, and modulating amputation-induced wnt levels, both of which seem critical for pole re-organization, thereby regulating whole-body regeneration.

}, issn = {1095-564X}, doi = {10.1016/j.ydbio.2022.04.011}, author = {Sarkar, Souradeep R and Dubey, Vinay Kumar and Jahagirdar, Anusha and Lakshmanan, Vairavan and Haroon, Mohamed Mohamed and Sowndarya, Sai and Sowdhamini, Ramanathan and Palakodeti, Dasaradhi} } @article {2425, title = {Decoding the Kinetic Pathways toward a Lipid/DNA Complex of Alkyl Alcohol Cationic Lipids Formed in a Microfluidic Channel.}, journal = {J Phys Chem B}, volume = {126}, year = {2022}, month = {2022 Jan 27}, pages = {588-600}, abstract = {

Complexes of cationic liposomes with DNA have emerged as promising nonviral vectors for delivering genetic information into cells for gene therapy. Kinetics of the liposome/DNA complex (lipoplex) formation on a millisecond time scale are studied by monitoring time evolution of fluorescence of 8-anilino-1-naphthalene sulfonic acid (ANS) and ethidium bromide (EtBr) in a continuous flow microfluidic channel coupled to a fluorescence microscope. The formation of lipoplexes between calf thymus DNA and liposomes based on two novel cationic lipids (Lip1810 and Lip1814) are found to follow a two-step process with kinetic constants for the Lip1814/DNA complex ( = 1120-1383 s, = 0.227-1.45 s) being significantly different from those ( = 68.53-98.5 s, = 32.3-60.19 s) corresponding to formation of the Lip1810/DNA complex. The kinetic pathway leading to the formation of Lip1814/DNA complex is whereas the formation of Lip1810/DNA complex occurs by a . The observed difference in the kinetics of lipoplex formation likely originates from different structures of the lipid/DNA complexes.

}, issn = {1520-5207}, doi = {10.1021/acs.jpcb.1c07263}, author = {Mukherjee, Dipanjan and Hasan, Md Nur and Ghosh, Ria and Ghosh, Gourab and Bera, Arpan and Prasad, Sujanthi Easwara and Hiwale, Ankita and Vemula, Praveen K and Das, Ranjan and Pal, Samir Kumar} } @article {2503, title = {Dual control of dopamine in Drosophila myeloid-like progenitor cell proliferation and regulation of lymph gland growth.}, journal = {EMBO Rep}, year = {2022}, month = {2022 Apr 27}, pages = {e52951}, abstract = {

In Drosophila, definitive haematopoiesis takes place in a specialized organ termed "lymph gland". It harbours multi-potent stem-like blood progenitor cells whose development controls overall growth of this haematopoietic tissue and formation of mature blood cells. With respect to its development, neurotransmitters have emerged as potent regulators of blood-progenitor cell development and function. In this study, we extend our understanding of neurotransmitters and show that progenitors are self-sufficient with regard to synthesizing dopamine, a well-established neurotransmitter. These cells also have modules for dopamine sensing through the receptor and transporter. We found that modulating expression of these components in progenitor cells affected lymph gland growth, which suggested growth-promoting function of dopamine in blood-progenitor cells. Cell-cycle analysis of developing lymph glands revealed an unexpected requirement for intracellular dopamine in moderating the progression of early progenitor cells from S to G2 phase of the cell cycle, while activation of dopamine receptor signalling later in development regulated their progression from G2 and entry into mitosis. The dual capacity in which dopamine operated, first intracellularly to coordinate S/G2 transition and later extracellularly in G2/M transition, was critical for the growth of the lymph gland. Overall, the data presented highlight a novel non-canonical use of dopamine in the myeloid system that reveals an uncharacterized function of intracellular dopamine in cell-cycle phasing with outcomes on haematopoietic growth and immunity as well.

}, issn = {1469-3178}, doi = {10.15252/embr.202152951}, author = {Kapoor, Ankita and Padmavathi, Achalla and Madhwal, Sukanya and Mukherjee, Tina} } @article {3715, title = {Efficient and error-free correction of sickle mutation in human erythroid cells using prime editor-2.}, journal = {Front Genome Ed}, volume = {4}, year = {2022}, month = {2022}, pages = {1085111}, abstract = {

Sickle cell anaemia (SCA) is one of the common autosomal recessive monogenic disorders, caused by a transverse point mutation (GAG \> GTG) at the sixth codon of the beta-globin gene, which results in haemolytic anaemia due to the fragile RBCs. Recent progress in genome editing has gained attention for the therapeutic cure for SCA. Direct correction of SCA mutation by homology-directed repair relies on a double-strand break (DSB) at the target site and carries the risk of generating beta-thalassaemic mutations if the editing is not error-free. On the other hand, base editors cannot correct the pathogenic SCA mutation resulting from A \> T base transversion. Prime editor (PE), the recently described CRISPR/Cas 9 based gene editing tool that enables precise gene manipulations without DSB and unintended nucleotide changes, is a viable approach for the treatment of SCA. However, the major limitation with the use of prime editing is the lower efficiency especially in human erythroid cell lines and primary cells. To overcome these limitations, we developed a modular lenti-viral based prime editor system and demonstrated its use for the precise modelling of SCA mutation and its subsequent correction in human erythroid cell lines. We achieved highly efficient installation of SCA mutation (up to 72\%) and its subsequent correction in human erythroid cells. For the first time, we demonstrated the functional restoration of adult haemoglobin without any unintended nucleotide changes or indel formations using the PE2 system. We also validated that the off-target effects mediated by the PE2 system is very minimal even with very efficient on-target conversion, making it a safe therapeutic option. Taken together, the modular lenti-viral prime editor system developed in this study not only expands the range of cell lines targetable by prime editor but also improves the efficiency considerably, enabling the use of prime editor for myriad molecular, genetic, and translational studies.

}, issn = {2673-3439}, doi = {10.3389/fgeed.2022.1085111}, author = {George, Anila and Ravi, Nithin Sam and Prasad, Kirti and Panigrahi, Lokesh and Koikkara, Sanya and Rajendiran, Vignesh and Devaraju, Nivedhitha and Paul, Joshua and Pai, Aswin Anand and Nakamura, Yukio and Kurita, Ryo and Balasubramanian, Poonkuzhali and Thangavel, Saravanabhavan and Marepally, Srujan and Velayudhan, Shaji R and Srivastava, Alok and Mohankumar, Kumarasamypet M} } @article {3341, title = {Erythroid lineage-specific lentiviral RNAi vectors suitable for molecular functional studies and therapeutic applications.}, journal = {Sci Rep}, volume = {12}, year = {2022}, month = {2022 08 18}, pages = {14033}, abstract = {

Numerous genes exert multifaceted roles in hematopoiesis. Therefore, we generated novel\ lineage-specific RNA interference\ (RNAi) lentiviral\ vectors, H23B-Ery-Lin-shRNA\ and H234B-Ery-Lin-shRNA, to probe the functions of these genes in erythroid cells\ without affecting other hematopoietic lineages. The lineage specificity of these vectors was confirmed by\ transducing multiple hematopoietic cells to express a fluorescent protein. Unlike the previously reported erythroid lineage RNAi vector, our vectors were designed for cloning the short hairpin RNAs (shRNAs) for\ any gene, and they also provide superior knockdown of the target gene expression with a\ single shRNA integration per cell. High-level lineage-specific downregulation of BCL11A and ZBTB7A,\ two\ well-characterized transcriptional repressors of HBG in adult erythroid cells, was achieved with substantial induction of fetal hemoglobin with a single-copy lentiviral vector integration. Transduction of primary healthy donor CD34 cells with these vectors resulted in\ \>80\% reduction in the target\ protein levels and up to 40\% elevation in the γ-chain levels in the differentiated erythroid cells. Xenotransplantation of the human CD34 cells transduced with H23B-Ery-Lin-shBCL11A\ LV in immunocompromised mice showed ~ 60\% reduction in BCL11A protein expression with ~\ 40\% elevation of γ-chain levels in the erythroid cells derived from the transduced CD34 cells. Overall, the novel erythroid lineage-specific lentiviral RNAi vectors described in this study provide a\ high-level knockdown of target gene expression in the erythroid cells, making them suitable for their use in gene therapy for hemoglobinopathies. Additionally, the design of these vectors also makes them ideal for high-throughput RNAi screening for studying normal and pathological erythropoiesis.

}, keywords = {Animals, Cell Line, Tumor, Cell Lineage, DNA-Binding Proteins, Genetic Vectors, Humans, Lentivirus, Mice, RNA Interference, RNA, Small Interfering, Transcription Factors, Transduction, Genetic}, issn = {2045-2322}, doi = {10.1038/s41598-022-13783-0}, author = {Bagchi, Abhirup and Devaraju, Nivedhitha and Chambayil, Karthik and Rajendiran, Vignesh and Venkatesan, Vigneshwaran and Sayed, Nilofer and Pai, Aswin Anand and Nath, Aneesha and David, Ernest and Nakamura, Yukio and Balasubramanian, Poonkuzhali and Srivastava, Alok and Thangavel, Saravanabhavan and Mohankumar, Kumarasamypet M and Velayudhan, Shaji R} } @article {2424, title = {Flow Cytometry Analysis of Planarian Stem Cells Using DNA and Mitochondrial Dyes.}, journal = {Bio Protoc}, volume = {12}, year = {2022}, month = {2022 Jan 20}, pages = {e4299}, abstract = {

Planarians are free-living flatworms that emerged as a crucial model system to understand regeneration and stem cell biology. The ability to purify neoblasts, the adult stem cell population of planaria, through fluorescence-activated cell sorting (FACS) has tremendously increased our understanding of pluripotency, specialization, and heterogeneity. To date, the FACS-based purification methods for neoblasts relied on nuclear dyes that discriminate proliferating cells (\>2N), as neoblasts are the only dividing somatic cells. However, this method does not distinguish the functional states within the neoblast population. Our work has shown that among the neoblasts, the pluripotent stem cells (PSCs) are associated with low mitochondrial content and this property could be leveraged for purification of the PSC-enriched population. Using the mitochondrial dye MitoTracker Green (MTG) and the nuclear dye SiR-DNA, we have described a method for isolation of PSCs that are viable and compatible with downstream experiments, such as transplantation and cell culture. In this protocol, we provide a detailed description for sample preparation and FACS gating for neoblast isolation in planaria.

}, issn = {2331-8325}, doi = {10.21769/BioProtoc.4299}, author = {Haroon, Mohamed Mohamed and Vemula, Praveen Kumar and Palakodeti, Dasaradhi} } @article {3346, title = {Function of FMRP Domains in Regulating Distinct Roles of Neuronal Protein Synthesis.}, journal = {Mol Neurobiol}, volume = {59}, year = {2022}, month = {2022 Dec}, pages = {7370-7392}, abstract = {

The Fragile-X Mental Retardation Protein (FMRP) is an RNA binding protein that regulates translation of mRNAs essential for synaptic development and plasticity. FMRP interacts with a specific set of mRNAs, aids in their microtubule-dependent transport and regulates their translation through its association with ribosomes. However, the biochemical role of FMRP{\textquoteright}s domains in forming neuronal granules and associating with microtubules and ribosomes is currently undefined. We report that the C-terminus domain of FMRP is sufficient to bind to ribosomes akin to the full-length protein. Furthermore, the C-terminus domain alone is essential and responsible for FMRP-mediated neuronal translation repression. However, dendritic distribution of FMRP and its microtubule association is favored by the synergistic combination of FMRP domains rather than individual domains. Interestingly, we show that the phosphorylation of hFMRP at Serine-500 is important in modulating the dynamics of translation by controlling ribosome association. This is a fundamental mechanism governing the size and number of FMRP puncta that contain actively translating ribosomes. Finally through the use of pathogenic mutations, we emphasize the hierarchical contribution of FMRP{\textquoteright}s domains in translation regulation.

}, keywords = {Fragile X Mental Retardation Protein, Fragile X Syndrome, Humans, Microtubules, Neurons, Protein Biosynthesis, Ribosomes, RNA, Messenger}, issn = {1559-1182}, doi = {10.1007/s12035-022-03049-1}, author = {D{\textquoteright}Souza, Michelle Ninochka and Ramakrishna, Sarayu and Radhakrishna, Bindushree K and Jhaveri, Vishwaja and Ravindran, Sreenath and Yeramala, Lahari and Nair, Deepak and Palakodeti, Dasaradhi and Muddashetty, Ravi S} } @article {2465, title = {Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin.}, journal = {Elife}, volume = {11}, year = {2022}, month = {2022 02 11}, abstract = {

Naturally occurring point mutations in the promoter switch hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin in sickle cell patients with hereditary persistence of fetal hemoglobin (HPFH) and ameliorate the clinical severity. Inspired by this natural phenomenon, we tiled the highly homologous proximal promoters using adenine and cytosine base editors that avoid the generation of large deletions and identified novel regulatory regions including a cluster at the -123 region. Base editing at -123 and -124 bp of promoter induced fetal hemoglobin (HbF) to a higher level than disruption of well-known BCL11A binding site in erythroblasts derived from human CD34+ hematopoietic stem and progenitor cells (HSPC). We further demonstrated in vitro that the introduction of -123T \> C and -124T \> C HPFH-like mutations drives gamma-globin expression by creating a de novo binding site for KLF1. Overall, our findings shed light on so far unknown regulatory elements within the promoter and identified additional targets for therapeutic upregulation of fetal hemoglobin.

}, keywords = {Adenine, Anemia, Sickle Cell, beta-Globins, beta-Thalassemia, Cell Line, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems, Cytosine, Fetal Hemoglobin, gamma-Globins, Gene Editing, Hematopoietic Stem Cells, Humans, Point Mutation, Promoter Regions, Genetic}, issn = {2050-084X}, doi = {10.7554/eLife.65421}, author = {Ravi, Nithin Sam and Wienert, Beeke and Wyman, Stacia K and Bell, Henry William and George, Anila and Mahalingam, Gokulnath and Vu, Jonathan T and Prasad, Kirti and Bandlamudi, Bhanu Prasad and Devaraju, Nivedhitha and Rajendiran, Vignesh and Syedbasha, Nazar and Pai, Aswin Anand and Nakamura, Yukio and Kurita, Ryo and Narayanasamy, Muthuraman and Balasubramanian, Poonkuzhali and Thangavel, Saravanabhavan and Marepally, Srujan and Velayudhan, Shaji R and Srivastava, Alok and DeWitt, Mark A and Crossley, Merlin and Corn, Jacob E and Mohankumar, Kumarasamypet M} } @article {2400, title = {Inflammation-specific targeted carriers for local drug delivery to inflammatory bowel disease.}, journal = {Biomaterials}, volume = {281}, year = {2022}, month = {2022 Jan 05}, pages = {121364}, abstract = {

Delivering drugs directly to the inflamed intestinal sites to treat inflammatory bowel disease (IBD), particularly Crohn{\textquoteright}s and ulcerative colitis, is highly challenging. Recent advances in colitis therapy medications are expanding opportunities for improving local on-site drug availability by minimising the associated systemic side-effects. Drug delivery with targeted carrier systems has shown the potential to increase site-specificity, stability, and therapeutic efficacy. Herein, we report the development of a strong anionic charged inflammation targeted nanocarriers (IT-NCs) loaded with an immunosuppressant model drug. This system showed preferential adhesion on a charge-modified surface in vitro, and in both dextran sulfate sodium (DSS) and TNBS colitis mice in vivo models. IT-NCs showed improved colitis phenotype therapeutic efficacy in both animal models compared to free drug. Furthermore, ex vivo study of colon tissue biopsies from patients with colitis revealed that IT-NCs adhered preferentially to inflamed biopsies compared to normal. Together, our results suggest that IT-NCs have promising therapeutic potential as delivery carriers{\textquoteright} in colitis management.

}, issn = {1878-5905}, doi = {10.1016/j.biomaterials.2022.121364}, author = {Kotla, Niranjan G and Singh, Rajbir and Baby, Becca V and Rasala, Swetha and Rasool, Jawad and Hynes, Sean O and Martin, Darrell and Egan, Laurence J and Vemula, Praveen K and Jala, Venkatakrishna R and Rochev, Yury and Pandit, Abhay} } @article {2502, title = {Influence of Hydrophobicity in the Hydrophilic Region of Cationic Lipids on Enhancing Nucleic Acid Delivery and Gene Editing.}, journal = {ACS Appl Bio Mater}, volume = {5}, year = {2022}, month = {2022 Apr 18}, pages = {1489-1500}, abstract = {

Intracellular delivery of biomolecules using non-viral vectors critically depends on the vectors{\textquoteright} ability to allow the escape and release of the contents from the endosomes. Prior findings demonstrated that aromatic/hydrophobic group-containing amino acids such as phenylalanine (F) and tryptophan (W) destabilize cellular membranes by forming pores in the lipid bilayer. Taking cues from these findings, we have developed four α-tocopherol-based cationic amphiphiles by varying the aromatic/hydrophobic amino acids such as glycine (G), proline (P), phenylalanine (F), and tryptophan (W) as head groups and triazole in the linker region to study their impact on endosomal escape for the enhanced transfection efficacy. The lipids tocopherol-triazole-phenylalanine (TTF) and tocopherol-triazole-tryptophan (TTW) exhibited similar potential to commercial transfecting reagents, Lipofectamine (LF) 3000 and Lipofectamine Messenger Max (LFMM), respectively, in transfecting plasmid DNA and messenger RNA in multiple cultured cell lines. The TTW liposome was also found to be effective in delivering Cas9 mRNA and demonstrated equal efficiency of gene editing AAVS1 locus compared to LFMM in CHO, Neuro-2a, and EA.HY926 cell lines. In this current investigation, it is shown that the synthesized cationic lipids with aromatic hydrophobic R group-containing amino acids are safe, economic, and actually more efficient in nucleic acid delivery and genome-editing applications. These findings can be further explored in the genome-editing approach for treating genetic disorders.

}, keywords = {alpha-Tocopherol, Amino Acids, Cations, Gene Editing, Gene Transfer Techniques, Hydrophobic and Hydrophilic Interactions, Lipids, Nucleic Acids, Phenylalanine, Triazoles, Tryptophan}, issn = {2576-6422}, doi = {10.1021/acsabm.1c01226}, author = {Rapaka, Hithavani and Manturthi, Shireesha and Arjunan, Porkizhi and Venkatesan, Vigneshwaran and Thangavel, Saravanabhavan and Marepally, Srujan and Patri, Srilakshmi V} } @article {3343, title = {Initiation of wound healing is regulated by the convergence of mechanical and epigenetic cues.}, journal = {PLoS Biol}, volume = {20}, year = {2022}, month = {2022 09}, pages = {e3001777}, abstract = {

Wound healing in the skin is a complex physiological process that is a product of a cell state transition from homeostasis to repair. Mechanical cues are increasingly being recognized as important regulators of cellular reprogramming, but the mechanism by which it is translated to changes in gene expression and ultimately cellular behavior remains largely a mystery. To probe the molecular underpinnings of this phenomenon further, we used the down-regulation of caspase-8 as a biomarker of a cell entering the wound healing program. We found that the wound-induced release of tension within the epidermis leads to the alteration of gene expression via the nuclear translocation of the DNA methyltransferase 3A (DNMT3a). This enzyme then methylates promoters of genes that are known to be down-regulated in response to wound stimuli as well as potentially novel players in the repair program. Overall, these findings illuminate the convergence of mechanical and epigenetic signaling modules that are important regulators of the transcriptome landscape required to initiate the tissue repair process in the differentiated layers of the epidermis.

}, keywords = {Biomarkers, Caspase 8, Cues, Epigenesis, Genetic, Wound Healing}, issn = {1545-7885}, doi = {10.1371/journal.pbio.3001777}, author = {Bhatt, Tanay and Dey, Rakesh and Hegde, Akshay and Ketkar, Alhad Ashok and Pulianmackal, Ajai J and Deb, Ashim P and Rampalli, Shravanti and Jamora, Colin} } @article {2544, title = {Integration of synthetic and natural derivatives revives the therapeutic potential of temozolomide against glioma- an in vitro and in vivo perspective.}, journal = {Life Sci}, volume = {301}, year = {2022}, month = {2022 May 06}, pages = {120609}, abstract = {

AIMS: Malignant gliomas constitute one of the deadly brain tumors with high degeneration rate. Though temozolomide (TMZ) is the first-line drug for glioma, its efficacy has decreased due to chemo-resistance. Repurposing synthetic and natural compounds have gained increasing interest in glioma. Hence, we combined chloroquine (CHL) a synthetic drug, naringenin (NAR) and phloroglucinol (PGL) (natural derivatives), to investigate whether the apoptotic effect of these drugs both alone and in combination, enhances the anti-tumor effects of TMZ in an in vitro and in vivo orthotopic xenograft glioma model.

MAIN METHODS: The cytotoxic effect of the drugs was assessed in C6 (murine) glioma cells, U-87 MG and LN229 (human) glioblastoma cells, primary astrocytes (isolated from rat brain tissues) and HEK-293\ T cells. Mitochondrial depolarization and alterations in the cell cycle was determined by confocal imaging and flow cytometry. The expression of angiogenic and apoptotic markers was evaluated using qRT-PCR and ELISA. The efficacy of the combinatorial treatment was assessed in an orthotopic xenograft model using U-87 MG cells.

KEY FINDINGS: The combinatorial treatment inhibited cell proliferation, induced apoptosis and contributed to cell cycle arrest in glioma cells. The quadruple combinatorial cocktail down-regulated BCL-2 with a concomitant decrease in VEGF. As observed in vitro, the quadruple combinatorial treatment enhanced the median survival of glioma-induced rats with lower cellularity rate.

SIGNIFICANCE: The combination of CHL, NAR and PGL synergistically potentiated the efficacy of TMZ on glioma in vitro and in vivo. Hence, this combination may characterize an advanced strategy for glioma treatment, thereby providing a possible translation to clinical trial.

}, issn = {1879-0631}, doi = {10.1016/j.lfs.2022.120609}, author = {Daisy Precilla, S and Kuduvalli, Shreyas S and Angeline Praveena, E and Thangavel, Saravanabhavan and Anitha, T S} } @article {3642, title = {Intermittent scavenging of storage lesion from stored red blood cells by electrospun nanofibrous sheets enhances their quality and shelf-life.}, journal = {Nat Commun}, volume = {13}, year = {2022}, month = {2022 Dec 01}, pages = {7394}, abstract = {

Transfusion of healthy red blood cells (RBCs) is a lifesaving process. However, upon storing RBCs, a wide range of damage-associate molecular patterns (DAMPs), such as cell-free DNA, nucleosomes, free-hemoglobin, and poly-unsaturated-fatty-acids are generated. DAMPs can further damage RBCs; thus, the quality of stored RBCs declines during the storage and limits their shelf-life. Since these DAMPs consist of either positive or negative charged species, we developed taurine and acridine containing electrospun-nanofibrous-sheets (Tau-AcrNFS), featuring anionic, cationic charges and an DNA intercalating group on their surfaces. We show that Tau-AcrNFS are efficient in scavenging DAMPs from stored human and mice RBCs ex vivo. We find that intermittent scavenging of DAMPs by Tau-AcrNFS during the storage reduces the loss of RBC membrane integrity and reduces discocytes-to-spheroechinocytes transformation in stored-old-RBCs. We perform RBC-transfusion studies in mice to reveal that intermittent removal of DAMPs enhances the quality of stored-old-RBCs equivalent to freshly collected RBCs, and increases their shelf-life by ~22\%. Such prophylactic technology may lead to the development of novel blood bags or medical device, and may therefore impact healthcare by reducing transfusion-related adverse effects.

}, keywords = {Acridines, Animals, Drug-Related Side Effects and Adverse Reactions, Erythrocytes, Humans, Mice, Nanofibers, Research Personnel}, issn = {2041-1723}, doi = {10.1038/s41467-022-35269-3}, author = {Pandey, Subhashini and Mahato, Manohar and Srinath, Preethem and Bhutani, Utkarsh and Goap, Tanu Jain and Ravipati, Priusha and Vemula, Praveen Kumar} } @article {2399, title = {Microscale engineering of hollow microneedle tips: design, manufacturing, optimization and validation.}, journal = {Drug Deliv Transl Res}, volume = {12}, year = {2022}, month = {2022 Feb}, pages = {350-367}, abstract = {

Transdermal and intradermal drug delivery utilizing microneedles is an emerging front in painless therapeutics. Drug delivery using hollow microneedles is the most preferred method for delivering generic transdermal drugs in the clinical setup. The needle tip must be extremely short as the drug is administered to sub-millimeter depths. Also, they need to be sharp enough to pierce through the skin with minimal skin flexing. There are multiple challenges in engineering a tip profile that is short and sharp at the same time. Stainless steel (SS) hypodermic needles with the lancet tip profile are ubiquitous in subcutaneous and intramuscular injections. They have long bevel lengths that make them inappropriate as microneedles. Thus, designing a unique tip profile and developing the manufacturing technology for microneedle applications are necessary. This article presents the design and optimization of microneedle tip profiles through analytical models. Further, manufacturing strategies for reliably obtaining designed profiles are discussed. The article concludes with experimental validation of improved piercing performance of the optimized tip profile compared to other tip profiles. The article discusses about tip geometries of stainless steel needles for microneedle applications, where depth of delivery is less than 1 mm. Through series of analyses, the optimum needle tip geometry evolved from single plane bevel (SPB) to hex plane bevel (HPB) progressively improving piercing performance.

}, issn = {2190-3948}, doi = {10.1007/s13346-021-01062-w}, author = {Badnikar, Kedar and Jayadevi, Shreyas Nataraja and Pahal, Suman and Vemula, Praveen Kumar and Nayak, Mangalore Manjunatha and Subramanyam, Dinesh Narasimhaiah} } @article {3644, title = {Nanoarchitectonics for Free-Standing Polyelectrolyte Multilayers Films: Exploring the Flipped Surfaces}, journal = {ChemNanoMat}, year = {2022}, month = {dec}, doi = {10.1002/cnma.202200462}, url = {https://doi.org/10.1002\%2Fcnma.202200462}, author = {Suman Pahal and Rakshith Boranna and Abinash Tripathy and Venkanagouda S. Goudar and Vyshnavi T. Veetil and Rajendra Kurapati and Gurusiddappa R. Prashanth and Praveen K. Vemula} } @article {3716, title = {Omicron infection increases IgG binding to spike protein of predecessor variants.}, journal = {J Med Virol}, year = {2022}, month = {2022 Dec 22}, abstract = {

BACKGROUND: SARS-CoV-2 transmission in India in 2020-2022 was driven predominantly by Wild (Wuhan-Hu-1and D614G), Delta, and Omicron variants. The aim of this study was to examine the effect of infections on the humoral immune response and cross-reactivity to spike proteins of Wuhan-Hu-1, Delta, C.1.2., and Omicron.

OBJECTIVES: Residual archival sera (N=81) received between January 2020 and March 2022 were included. Infection status was inferred by a positive SARS-CoV-2 RT-PCR and/or serology (anti-N and anti-S antibodies) and sequencing of contemporaneous samples (N=18) to infer lineage. We estimated the levels and cross-reactivity of infection-induced sera including Wild, Delta, Omicron as well as vaccine breakthrough infections (Delta and Omicron).

RESULTS: We found ~2-fold increase in spike-specific IgG antibody binding in post-Omicron infection compared to the pre-Omicron period, whilst the change in pre- and post-Delta infections were similar. Further investigation of Omicron-specific humoral responses revealed primary Omicron infection as an inducer of cross-reactive antibodies against predecessor variants, in spite of weaker degree of humoral response compared to Wuhan-Hu-1 and Delta infection. Intriguingly, Omicron vaccine-breakthrough infections when compared with primary infections, exhibited increased humoral responses against RBD (7.7-fold) and Trimeric S (Trimeric form of spike protein) (34.6-fold) in addition to increased binding of IgGs towards previously circulating variants (4.2 - 6.5-fold). Despite Delta breakthrough infections showing a higher level of humoral response against RBD (2.9-fold) and Trimeric S (5.7-fold) compared to primary Delta sera, a demonstrably reduced binding (36-49\%) was observed to Omicron spike protein.

CONCLUSIONS: Omicron vaccine breakthrough infection results in increased intensity of humoral response and wider breadth of IgG binding to spike proteins of antigenically-distinct, predecessor variants. This article is protected by copyright. All rights reserved.

}, issn = {1096-9071}, doi = {10.1002/jmv.28419}, author = {Mahalingam, Gokulnath and Periyasami, Yogapriya and Arjunan, Porkizhi and Subaschandrabose, Rajesh Kumar and Mathivanan, Tamil Venthan and Mathew, Roshlin Susan and Ramya Devi, Kt and Premkumar, Prasanna Samuel and Muliyil, Jayaprakash and Srivastava, Alok and Moorthy, Mahesh and Marepally, Srujan} } @article {3347, title = {S. mediterranea ETS-1 regulates the function of cathepsin-positive cells and the epidermal lineage landscape via basement membrane remodeling.}, journal = {J Cell Sci}, volume = {135}, year = {2022}, month = {2022 10 15}, abstract = {

Extracellular matrix (ECM) is an important component of stem cell niche. Remodeling of ECM mediated by ECM regulators, such as matrix metalloproteinases (MMPs) plays a vital role in stem cell function. However, the mechanisms that modulate the function of ECM regulators in the stem cell niche are understudied. Here, we explored the role of the transcription factor (TF) ETS-1, which is expressed in the cathepsin-positive cell population, in regulating the expression of the ECM regulator, mt-mmpA, thereby modulating basement membrane thickness. In planarians, the basement membrane around the gut/inner parenchyma is thought to act as a niche for pluripotent stem cells. It has been shown that the early epidermal progenitors migrate outwards from this region and progressively differentiate to maintain the terminal epidermis. Our data shows that thickening of the basement membrane in the absence of ets-1 results in defective migration of stem cell progeny. Furthermore, the absence of ets-1 leads to a defective epidermal progenitor landscape, despite its lack of expression in those cell types. Together, our results demonstrate the active role of ECM remodeling in regulating tissue homeostasis and regeneration in the planarian Schmidtea mediterranea. This article has an associated First Person interview with one of the co-first authors of the paper.

}, keywords = {Animals, Basement Membrane, Cathepsins, Cell Differentiation, Epidermis, Humans, Matrix Metalloproteinases, Mediterranea, Planarians, Transcription Factors}, issn = {1477-9137}, doi = {10.1242/jcs.259900}, author = {Dubey, Vinay Kumar and Sarkar, Souradeep R and Lakshmanan, Vairavan and Dalmeida, Rimple and Gulyani, Akash and Palakodeti, Dasaradhi} } @article {3645, title = {Single step fabrication of hollow microneedles and an experimental package for controlled drug delivery.}, journal = {Int J Pharm}, volume = {632}, year = {2022}, month = {2022 Dec 24}, pages = {122546}, abstract = {

Hollow microneedle arrays (HMNs) are an excellent choice for managing chronic diseases requiring the administration of multiple drug doses over a prolonged duration. However, HMNs have gained partial success due to limitations in their manufacturing capabilities, and cumbersome processes. In the present study, polymeric HMNs were fabricated using a novel single-step drop-casting process without needing cleanroom facilities, and sophisticated instrumentation. When drop casted on the pyramidal tip stainless steel needles, the optimized polymer solution allowed the reproducible formation of desired height HMMs on a detachable acrylic base. To enable broader applications, the base with HMNs was integrated into an experimental package built to deliver a dose of\ \~{}\ 5\ {\textmu}L per 30{\textdegree} clockwise rotation of the actuator, allowing multiple metered drug dose administrations. The fabricated HMNs were optically imaged, and tested for mechanical integrity and stability. The working and functional utility of the HMNs package in delivering metered drug doses was demonstrated by delivering vitamin B12 (ex vivo) and insulin (in vivo), respectively. The optimized process can be used for the large-scale manufacturing of HMNs and the experimental package shows the potential to be further developed into a wearable device.

}, issn = {1873-3476}, doi = {10.1016/j.ijpharm.2022.122546}, author = {Ghate, Vivek and Renjith, Anu and Badnikar, Kedar and Pahal, Suman and Jayadevi, Shreyas N and Nayak, Manjunatha M and Vemula, Praveen K and Subramanyam, Dinesh N} } @article {2505, title = {The story of rRNA expansion segments: Finding functionality amidst diversity.}, journal = {Wiley Interdiscip Rev RNA}, year = {2022}, month = {2022 Apr 15}, pages = {e1732}, abstract = {

Expansion segments (ESs) are multinucleotide insertions present across phyla at specific conserved positions in eukaryotic rRNAs. ESs are generally absent in bacterial rRNAs with some exceptions, while the archaeal rRNAs have microexpansions at regions that coincide with those of eukaryotic ESs. Although there is an increasing prominence of ribosomes, especially the ribosomal proteins, in fine-tuning gene expression through translation regulation, the role of rRNA ESs is relatively underexplored. While rRNAs have been established as the major catalytic hub in ribosome function, the presence of ESs widens their scope as a species-specific regulatory hub of protein synthesis. In this comprehensive review, we have elaborately discussed the current understanding of the functional aspects of rRNA ESs of cytoplasmic eukaryotic ribosomes and discuss their past, present, and future. This article is categorized under: RNA Structure and Dynamics \> Influence of RNA Structure in Biological Systems Translation \> Ribosome Structure/Function Translation \> Regulation.

}, issn = {1757-7012}, doi = {10.1002/wrna.1732}, author = {Hariharan, Nivedita and Ghosh, Sumana and Palakodeti, Dasaradhi} } @article {2882, title = {Supplementation of articular cartilage-derived chondroprogenitors with bone morphogenic protein-9 enhances chondrogenesis without affecting hypertrophy.}, journal = {Biotechnol Lett}, year = {2022}, month = {2022 Aug 03}, abstract = {

INTRODUCTION: Chondroprogenitors (CPCs) have emerged as a promising cellular therapy for cartilage-related pathologies due to their inherent primed chondrogenic potential. Studies report that the addition of growth factors such as parathyroid hormone (PTH) and Bone Morphogenic Protein (BMP) enhance the chondroinducive potential in chondrocytes and mesenchymal stem cells. This study evaluated if supplementation of the standard culture medium for cell expansion with 1-34 PTH and BMP-9 would enhance the chondrogenic potential of CPCs and reduce their hypertrophic tendency.

METHODS: Human chondrocytes were isolated from patients undergoing total knee replacement for osteoarthritis (n = 3). Following fibronectin adhesion assay, passage 1 CPCs were divided and further expanded under three culture conditions (a) control, i.e., cells continued under standard culture conditions, (b) 1-34 PTH group, additional intermittent 6\ h exposure with 1-34 PTH and (c) BMP-9 group, additional BMP-9 during culture expansion. All the groups were evaluated for population-doubling, cell cycle analysis, surface marker and gene expression for chondrogenesis, hypertrophy, multilineage differentiation and GAG (glycosaminoglycan)/DNA following chondrogenic differentiation.

RESULTS: Concerning growth kinetics, the BMP-9 group exhibited a significantly lower S-phase and population-doubling when compared to the other two groups. Qualitative analysis for chondrogenic potential (Alcian blue, Safranin O staining and Toluidine blue for GAG) revealed that the BMP-9 group exhibited the highest uptake. The BMP-9 group also showed significantly higher COL2A1 expression than the control group, with no change in the hypertrophy marker expression.

CONCLUSION: BMP-9 can potentially be used as an additive for CPCs expansion, to enhance their chondrogenic potential without affecting their low hypertrophic tendency. The mitigating effects of 1-34PTH on hypertrophy would benefit further investigation when used in combination with BMP-9 to enhance chondrogenesis whilst reducing hypertrophy.

}, issn = {1573-6776}, doi = {10.1007/s10529-022-03280-9}, author = {Padmaja, Kawin and Amirtham, Soosai Manickam and Rebekah, Grace and Sathishkumar, Solomon and Vinod, Elizabeth} } @article {2463, title = {Whole genome sequencing delineates regulatory, copy number, and cryptic splice variants in early onset cardiomyopathy.}, journal = {NPJ Genom Med}, volume = {7}, year = {2022}, month = {2022 Mar 14}, pages = {18}, abstract = {

Cardiomyopathy (CMP) is a heritable disorder. Over 50\% of cases are gene-elusive on clinical gene panel testing. The contribution of variants in non-coding DNA elements that result in cryptic splicing and regulate gene expression has not been explored. We analyzed whole-genome sequencing (WGS) data in a discovery cohort of 209 pediatric CMP patients and 1953 independent replication genomes and exomes. We searched for protein-coding variants, and non-coding variants predicted to affect the function or expression of genes. Thirty-nine percent of cases harbored pathogenic coding variants in known CMP genes, and 5\% harbored high-risk loss-of-function (LoF) variants in additional candidate CMP genes. Fifteen percent harbored high-risk regulatory variants in promoters and enhancers of CMP genes (odds ratio 2.25, p = 6.70 {\texttimes} 10 versus controls). Genes involved in α-dystroglycan glycosylation (FKTN, DTNA) and desmosomal signaling (DSC2, DSG2) were most highly enriched for regulatory variants (odds ratio 6.7-58.1). Functional effects were confirmed in patient myocardium and reporter assays in human cardiomyocytes, and in zebrafish CRISPR knockouts. We provide strong evidence for the genomic contribution of functionally active variants in new genes and in regulatory elements of known CMP genes to early onset CMP.

}, issn = {2056-7944}, doi = {10.1038/s41525-022-00288-y}, author = {Lesurf, Robert and Said, Abdelrahman and Akinrinade, Oyediran and Breckpot, Jeroen and Delfosse, Kathleen and Liu, Ting and Yao, Roderick and Persad, Gabrielle and McKenna, Fintan and Noche, Ramil R and Oliveros, Winona and Mattioli, Kaia and Shah, Shreya and Miron, Anastasia and Yang, Qian and Meng, Guoliang and Yue, Michelle Chan Seng and Sung, Wilson W L and Thiruvahindrapuram, Bhooma and Lougheed, Jane and Oechslin, Erwin and Mondal, Tapas and Bergin, Lynn and Smythe, John and Jayappa, Shashank and Rao, Vinay J and Shenthar, Jayaprakash and Dhandapany, Perundurai S and Semsarian, Christopher and Weintraub, Robert G and Bagnall, Richard D and Ingles, Jodie and Mel{\'e}, Marta and Maass, Philipp G and Ellis, James and Scherer, Stephen W and Mital, Seema} } @article {2364, title = {Analysis of whole exome sequencing in severe mental illness hints at selection of brain development and immune related genes.}, journal = {Sci Rep}, volume = {11}, year = {2021}, month = {2021 Oct 26}, pages = {21088}, abstract = {

Evolutionary trends may underlie some aspects of the risk for common, non-communicable disorders, including psychiatric disease. We analyzed whole exome sequencing data from 80 unique individuals from India coming from families with two or more individuals with severe mental illness. We used Population Branch Statistics (PBS) to identify variants and genes under positive selection and identified 74 genes as candidates for positive selection. Of these, 20 were previously associated with Schizophrenia, Alzheimer{\textquoteright}s disease and cognitive abilities in genome wide association studies. We then checked whether any of these 74 genes were involved in common biological pathways or related to specific cellular or molecular functions. We found that immune related pathways and functions related to innate immunity such as antigen binding were over-represented. We also evaluated for the presence of Neanderthal introgressed segments in these genes and found Neanderthal introgression in a single gene out of the 74 candidate genes. However, the introgression pattern indicates the region is unlikely to be the source for selection. Our findings hint at how selection pressures in individuals from families with a history of severe mental illness may diverge from the general population. Further, it also provides insights into the genetic architecture of severe mental illness, such as schizophrenia and its link to immune factors.

}, issn = {2045-2322}, doi = {10.1038/s41598-021-00123-x}, author = {Mahadevan, Jayant and Pathak, Ajai Kumar and Vemula, Alekhya and Nadella, Ravi Kumar and Viswanath, Biju and Jain, Sanjeev and Purushottam, Meera and Mondal, Mayukh} } @article {2362, title = {Astrocytic reactivity triggered by defective autophagy and metabolic failure causes neurotoxicity in frontotemporal dementia type 3.}, journal = {Stem Cell Reports}, volume = {16}, year = {2021}, month = {2021 Nov 09}, pages = {2736-2751}, abstract = {

Frontotemporal dementia type 3 (FTD3), caused by a point mutation in the charged multivesicular body protein 2B (CHMP2B), affects mitochondrial ultrastructure and the endolysosomal pathway in neurons. To dissect the astrocyte-specific impact of mutant CHMP2B expression, we generated astrocytes from human induced pluripotent stem cells (hiPSCs) and confirmed our findings in CHMP2B mutant mice. Our data provide mechanistic insights into how defective autophagy causes perturbed mitochondrial dynamics with impaired glycolysis, increased reactive oxygen species, and elongated mitochondrial morphology, indicating increased mitochondrial fusion in FTD3 astrocytes. This shift in astrocyte homeostasis triggers a reactive astrocyte phenotype and increased release of toxic cytokines, which accumulate in nuclear factor kappa b (NF-κB) pathway activation with increased production of CHF, LCN2, and C3 causing neurodegeneration.

}, issn = {2213-6711}, doi = {10.1016/j.stemcr.2021.09.013}, author = {Chandrasekaran, Abinaya and Dittlau, Katarina Stoklund and Corsi, Giulia I and Haukedal, Henriette and Doncheva, Nadezhda T and Ramakrishna, Sarayu and Ambardar, Sheetal and Salcedo, Claudia and Schmidt, Sissel I and Zhang, Yu and Cirera, Susanna and Pihl, Maria and Schmid, Benjamin and Nielsen, Troels Tolstrup and Nielsen, J{\o}rgen E and Kolko, Miriam and Kobol{\'a}k, Julianna and Dinny{\'e}s, Andr{\'a}s and Hyttel, Poul and Palakodeti, Dasaradhi and Gorodkin, Jan and Muddashetty, Ravi S and Meyer, Morten and Aldana, Blanca I and Freude, Kristine K} } @article {2207, title = {Comprehensive annotation and characterization of planarian tRNA and tRNA-derived fragments (tRFs).}, journal = {RNA}, year = {2021}, month = {2021 Jan 14}, abstract = {

tRNA-derived fragments (tRFs) have recently gained a lot of scientific interest due to their diverse regulatory roles in several cellular processes. However, their function in dynamic biological process such as development and regeneration remains unexplored. Here, we show that tRFs are dynamically expressed during planarian regeneration suggesting a possible role for these small RNAs in the regulation of regeneration. In order to characterise planarian tRFs, we first annotated 457 tRNAs in S.mediterranea combining two tRNA prediction algorithms. Annotation of tRNAs facilitated the identification of three main species of tRFs in planarians - the shorter tRF-5s and itRFs, and the abundantly expressed 5{\textquoteright}-tsRNAs. Spatial profiling of tRFs in sequential transverse sections of planarians revealed diverse expression patterns of these small RNAs, including those that are enriched in the head and pharyngeal regions. Expression analysis of these tRF species revealed dynamic expression of these small RNAs over the course of regeneration suggesting an important role in planarian anterior and posterior regeneration. Finally, we show that 5{\textquoteright}-tsRNA in planaria interact with all three SMEDWI proteins and an involvement of Ago1 in the processing of itRFs. In summary, our findings implicate a novel role for tRFs in planarian regeneration, highlighting their importance in regulating complex systemic processes. Our study adds to the catalogue of post-transcriptional regulatory systems in planarian, providing valuable insights on the biogenesis and the function of tRFs in neoblasts and planarian regeneration.

}, issn = {1469-9001}, doi = {10.1261/rna.077701.120}, author = {Lakshmanan, Vairavan and T N, Sujith and Bansal, Dhiru and Padubidri, Shivaprasad V and Palakodeti, Dasaradhi and Krishna, Srikar} } @article {2292, title = {Cross-diagnostic evaluation of minor physical anomalies in psychiatric disorders.}, journal = {J Psychiatr Res}, volume = {142}, year = {2021}, month = {2021 Jul 20}, pages = {54-62}, abstract = {

BACKGROUND: Minor physical anomalies (MPA) are markers of impaired neurodevelopment during the prenatal stage. Assessing MPA across psychiatric disorders may help understand their shared nature. In addition, MPA in family members would indicate a shared liability and endophenotype potential. We examined familial aggregation of MPA and their role as transdiagnostic and disorder-specific markers of 5 major psychiatric/neuropsychiatric conditions (schizophrenia, bipolar disorder, substance dependence, obsessive-compulsive disorder, and Alzheimer{\textquoteright}s dementia).

METHODS: Modified Waldrop{\textquoteright}s MPA scale was applied on 1321 individuals from 439 transdiagnostic multiplex families and 125 healthy population controls (HC). Stage of fetal development (morphogenetic/phenogenetic)- and anatomical location (craniofacial/peripheral)-based sub-scores were calculated. Familiality and endophenotypic potential of MPA were analyzed with serial negative binomial mixed-effect regression. Cross-diagnostic differences and the effect of family history density (FHD) of each diagnosis on MPA were assessed. Mixed-effects Cox models estimated the influence of MPA on age-at-onset of illness (AAO).

RESULTS: MPA were found to be heritable in families with psychiatric disorders, with a familiality of 0.52. MPA were higher in psychotic disorders after controlling for effects of sex and intrafamilial correlation. Morphogenetic variant MPA was noted to be lower in dementia in comparison to HC. FHD of schizophrenia and bipolar disorder predicted higher, and that of dementia and substance dependence predicted lower MPA. MPA brought forward the AAO [HR:1.07 (1.03-1.11)], and this was more apparent in psychotic disorders.

CONCLUSION: MPA are transmissible in families, are specifically related to the risk of developing psychoses, and predict an earlier age at onset. Neurodevelopmentally informed classification of MPA has the potential to enhance the etiopathogenic and translational understanding of psychiatric disorders.

}, issn = {1879-1379}, doi = {10.1016/j.jpsychires.2021.07.028}, author = {Sreeraj, Vanteemar S and Puzhakkal, Joan C and Holla, Bharath and Nadella, Ravi Kumar and Sheth, Sweta and Balachander, Srinivas and Ithal, Dhruva and Ali, Furkhan and Viswanath, Biju and Muralidharan, Kesavan and Venkatasubramanian, Ganesan and John, John P and Benegal, Vivek and Murthy, Pratima and Varghese, Mathew and Reddy, Yc Janardhan and Jain, Sanjeev} } @article {2215, title = {Decreased dendritic spine density in posterodorsal medial amygdala neurons of proactive coping rats.}, journal = {Behav Brain Res}, volume = {397}, year = {2021}, month = {2021 Jan 15}, pages = {112940}, abstract = {

There are large individual differences in the way animals, including humans, behaviorally and physiologically cope with environmental challenges and opportunities. Rodents with either a proactive or reactive coping style not only differ in their capacity to adapt successfully to environmental conditions, but also have a differential susceptibility to develop stress-related (psycho)pathologies when coping fails. In this study, we explored if there are structural neuronal differences in spine density in brain regions important for the regulation of stress coping styles. For this, the individual coping styles of wild-type Groningen (WTG) rats were determined using their level of offensive aggressiveness assessed in the resident-intruder paradigm. Subsequently, brains from proactive (high-aggressive) and reactive (low-aggressive) rats were Golgi-cox stained for spine quantification. The results reveal that dendritic spine densities in the dorsal hippocampal CA1 region and basolateral amygdala are similar in rats with proactive and reactive coping styles. Interestingly, however, dendritic spine density in the medial amygdala (MeA) is strikingly reduced in the proactive coping rats. This brain region is reported to be strongly involved in rivalry aggression which is the criterion by which the coping styles in our study are dissociated. The possibility that structural differences in spine density in the MeA are involved in other behavioral traits of distinct coping styles needs further investigation.

}, issn = {1872-7549}, doi = {10.1016/j.bbr.2020.112940}, author = {Anilkumar, Shobha and Patel, Deepika and de Boer, Sietse F and Chattarji, Sumantra and Buwalda, Bauke} } @article {2273, title = {Discovery of a body-wide photosensory array that matures in an adult-like animal and mediates eye-brain-independent movement and arousal.}, journal = {Proc Natl Acad Sci U S A}, volume = {118}, year = {2021}, month = {2021 May 18}, abstract = {

The ability to respond to light has profoundly shaped life. Animals with eyes overwhelmingly rely on their visual circuits for mediating light-induced coordinated movements. Building on previously reported behaviors, we report the discovery of an organized, eye-independent (extraocular), body-wide photosensory framework that allows even a head-removed animal to move like an intact animal. Despite possessing sensitive cerebral eyes and a centralized brain that controls most behaviors, head-removed planarians show acute, coordinated ultraviolet-A (UV-A) aversive phototaxis. We find this eye-brain-independent phototaxis is mediated by two noncanonical rhabdomeric opsins, the first known function for this newly classified opsin-clade. We uncover a unique array of dual-opsin-expressing photoreceptor cells that line the periphery of animal body, are proximal to a body-wide nerve net, and mediate UV-A phototaxis by engaging multiple modes of locomotion. Unlike embryonically developing cerebral eyes that are functional when animals hatch, the body-wide photosensory array matures postembryonically in "adult-like animals." Notably, apart from head-removed phototaxis, the body-wide, extraocular sensory organization also impacts physiology of intact animals. Low-dose UV-A, but not visible light (ocular-stimulus), is able to arouse intact worms that have naturally cycled to an inactive/rest-like state. This wavelength selective, low-light arousal of resting animals is noncanonical-opsin dependent but eye independent. Our discovery of an autonomous, multifunctional, late-maturing, organized body-wide photosensory system establishes a paradigm in sensory biology and evolution of light sensing.

}, issn = {1091-6490}, doi = {10.1073/pnas.2021426118}, author = {Shettigar, Nishan and Chakravarthy, Anirudh and Umashankar, Suchitta and Lakshmanan, Vairavan and Palakodeti, Dasaradhi and Gulyani, Akash} } @article {2327, title = {Engineered RNA biosensors enable ultrasensitive SARS-CoV-2 detection in a simple color and luminescence assay.}, journal = {Life Sci Alliance}, volume = {4}, year = {2021}, month = {2021 12}, abstract = {

The continued resurgence of the COVID-19 pandemic with multiple variants underlines the need for diagnostics that are adaptable to the virus. We have developed toehold RNA-based sensors across the SARS-CoV-2 genome for direct and ultrasensitive detection of the virus and its prominent variants. Here, isothermal amplification of a fragment of SARS-CoV-2 RNA coupled with activation of our biosensors leads to a conformational switch in the sensor. This leads to translation of a reporter protein, for example, LacZ or nano-lantern that is easily detected using color/luminescence. By optimizing RNA amplification and biosensor design, we have generated a highly sensitive diagnostic assay that is capable of detecting as low as 100 copies of viral RNA with development of bright color. This is easily visualized by the human eye and quantifiable using spectrophotometry. Finally, this PHAsed NASBA-Translation Optical Method (PHANTOM) using our engineered RNA biosensors efficiently detects viral RNA in patient samples. This work presents a powerful and universally accessible strategy for detecting COVID-19 and variants. This strategy is adaptable to further viral evolution and brings RNA bioengineering center-stage.

}, keywords = {Biosensing Techniques, COVID-19, Humans, Luminescence, Nucleic Acid Amplification Techniques, RNA, RNA, Viral, SARS-CoV-2}, issn = {2575-1077}, doi = {10.26508/lsa.202101213}, author = {Chakravarthy, Anirudh and Nandakumar, Anirudh and George, Geen and Ranganathan, Shyamsundar and Umashankar, Suchitta and Shettigar, Nishan and Palakodeti, Dasaradhi and Gulyani, Akash and Ramesh, Arati} } @article {2323, title = {Genomic characterization and epidemiology of an emerging SARS-CoV-2 variant in Delhi, India.}, journal = {Science}, year = {2021}, month = {2021 Oct 14}, pages = {eabj9932}, abstract = {

Delhi, the national capital of India, has experienced multiple SARS-CoV-2 outbreaks in 2020 and reached population seropositivity of over 50\% by 2021. During April 2021, the city became overwhelmed by COVID-19 cases and fatalities, as a new variant B.1.617.2 (Delta) replaced B.1.1.7 (Alpha). A Bayesian model explains the growth advantage of Delta through a combination of increased transmissibility and reduced sensitivity to immune responses generated against earlier variants (median estimates; {\texttimes}1.5-fold, 20\% reduction). Seropositivity of an employee and family cohort increased from 42\% to 87.5\% between March and July 2021, with 27\% reinfections, as judged by increased antibody concentration after a previous decline. The likely high transmissibility and partial evasion of immunity by the Delta variant contributed to an overwhelming surge in Delhi.

}, issn = {1095-9203}, doi = {10.1126/science.abj9932}, author = {Dhar, Mahesh S and Marwal, Robin and Vs, Radhakrishnan and Ponnusamy, Kalaiarasan and Jolly, Bani and Bhoyar, Rahul C and Sardana, Viren and Naushin, Salwa and Rophina, Mercy and Mellan, Thomas A and Mishra, Swapnil and Whittaker, Charles and Fatihi, Saman and Datta, Meena and Singh, Priyanka and Sharma, Uma and Ujjainiya, Rajat and Bhatheja, Nitin and Divakar, Mohit Kumar and Singh, Manoj K and Imran, Mohamed and Senthivel, Vigneshwar and Maurya, Ranjeet and Jha, Neha and Mehta, Priyanka and A, Vivekanand and Sharma, Pooja and Vr, Arvinden and Chaudhary, Urmila and Soni, Namita and Thukral, Lipi and Flaxman, Seth and Bhatt, Samir and Pandey, Rajesh and Dash, Debasis and Faruq, Mohammed and Lall, Hemlata and Gogia, Hema and Madan, Preeti and Kulkarni, Sanket and Chauhan, Himanshu and Sengupta, Shantanu and Kabra, Sandhya and Gupta, Ravindra K and Singh, Sujeet K and Agrawal, Anurag and Rakshit, Partha and Nandicoori, Vinay and Tallapaka, Karthik Bharadwaj and Sowpati, Divya Tej and Thangaraj, K and Bashyam, Murali Dharan and Dalal, Ashwin and Sivasubbu, Sridhar and Scaria, Vinod and Parida, Ajay and Raghav, Sunil K and Prasad, Punit and Sarin, Apurva and Mayor, Satyajit and Ramakrishnan, Uma and Palakodeti, Dasaradhi and Seshasayee, Aswin Sai Narain and Bhat, Manoj and Shouche, Yogesh and Pillai, Ajay and Dikid, Tanzin and Das, Saumitra and Maitra, Arindam and Chinnaswamy, Sreedhar and Biswas, Nidhan Kumar and Desai, Anita Sudhir and Pattabiraman, Chitra and Manjunatha, M V and Mani, Reeta S and Arunachal Udupi, Gautam and Abraham, Priya and Atul, Potdar Varsha and Cherian, Sarah S} } @article {2311, title = {Glycomic and glycotranscriptomic profiling of mucin-type O-glycans in planarian Schmidtea mediterranea.}, journal = {Glycobiology}, year = {2021}, month = {2021 Sep 06}, abstract = {

O-Glycans on cell surfaces play important roles in cell-cell, cell-matrix, and receptor-ligand interaction. Therefore, glycan-based interactions are important for tissue regeneration and homeostasis. Free-living flatworm Schmidtea mediterranea, because of its robust regenerative potential, is of great interest in the field of stem cell biology and tissue regeneration. Nevertheless, information on the composition and structure of O-glycans in planaria is unknown. Using mass spectrometry and in silico approaches, we characterized the glycome and the related transcriptome of mucin-type O-glycans of planarian S. mediterranea. Mucin-type O-glycans were composed of multiple isomeric, methylated, and unusually extended mono- and di-substituted O-GalNAc structures. Extensions made of hexoses and 3-O methyl hexoses were the glycoforms observed. From glycotranscriptomic analysis, sixty genes belonging to five distinct enzyme classes were identified to be involved in mucin-type O-glycan biosynthesis. These genes shared homology with those in other invertebrate systems. While a majority of the genes involved in mucin-type O-glycan biosynthesis was highly expressed during organogenesis and in differentiated cells, a few select genes in each enzyme class were specifically enriched during early embryogenesis. Our results indicate a unique temporal and spatial role for mucin-type O-glycans during embryogenesis and organogenesis and in adulthood. In summary, this is the first report on O-glycans in planaria. This study expands the structural and biosynthetic possibilities in cellular glycosylation in the invertebrate glycome and provides a framework towards understanding the biological role of mucin-type O-glycans in tissue regeneration using planarians.

}, issn = {1460-2423}, doi = {10.1093/glycob/cwab097}, author = {Subramanian, Sabarinath Peruvemba and Lakshmanan, Vairavan and Palakodeti, Dasaradhi and Subramanian, Ramaswamy} } @article {2214, title = {iRGD conjugated nimbolide liposomes protect against endotoxin induced acute respiratory distress syndrome.}, journal = {Nanomedicine}, volume = {33}, year = {2021}, month = {2021 Jan 05}, pages = {102351}, abstract = {

Acute respiratory distress syndrome (ARDS) is a deadly respiratory illness associated with refractory hypoxemia and pulmonary edema. The recent pandemic outbreak of COVID-19 is associated with severe pneumonia and inflammatory cytokine storm in the lungs. The anti-inflammatory phytomedicine nimbolide (NIM) may not be feasible for clinical translation due to poor pharmacokinetic properties and lack of suitable delivery systems. To overcome these barriers, we have developed nimbolide liposomes conjugated with iRGD peptide (iRGD-NIMLip) for targeting lung inflammation. It was observed that iRGD-NIMLip treatment significantly inhibited oxidative stress and cytokine storm compared to nimbolide free-drug (f-NIM), nimbolide liposomes (NIMLip), and exhibited superior activity compared to dexamethasone (DEX). iRGD-NIMLip abrogated the LPS induced p65 NF-κB, Akt, MAPK, Integrin β3 and β5, STAT3, and DNMT1 expression. Collectively, our results demonstrate that iRGD-NIMLip could be a promising novel drug delivery system to target severe pathological consequences observed in ARDS and COVID-19 associated cytokine storm.

}, issn = {1549-9642}, doi = {10.1016/j.nano.2020.102351}, author = {Pooladanda, Venkatesh and Thatikonda, Sowjanya and Sunnapu, Omprakash and Tiwary, Shristy and Vemula, Praveen Kumar and Talluri, M V N Kumar and Godugu, Chandraiah} } @article {2242, title = {Mechanical instability of adherens junctions overrides intrinsic quiescence of hair follicle stem cells.}, journal = {Dev Cell}, volume = {56}, year = {2021}, month = {2021 Mar 22}, pages = {761-780.e7}, abstract = {

Vinculin, a mechanotransducer associated with both adherens junctions (AJs) and focal adhesions (FAs), plays a central role in force transmission through cell-cell and cell-substratum contacts. We generated the conditional knockout (cKO) of vinculin in murine skin that results in the loss of bulge stem cell (BuSC) quiescence and promotes continual cycling of the hair follicles. Surprisingly, we find that the AJs in vinculin cKO cells are mechanically weak and impaired in force generation despite increased junctional expression of E-cadherin and α-catenin. Mechanistically, we demonstrate that vinculin functions by keeping α-catenin in a stretched/open conformation, which in turn regulates the retention of YAP1, another potent mechanotransducer and regulator of cell proliferation, at the AJs. Altogether, our data provide mechanistic insights into the hitherto-unexplored regulatory link between the mechanical stability of cell junctions and contact-inhibition-mediated maintenance of BuSC quiescence.

}, issn = {1878-1551}, doi = {10.1016/j.devcel.2021.02.020}, author = {Biswas, Ritusree and Banerjee, Avinanda and Lembo, Sergio and Zhao, Zhihai and Lakshmanan, Vairavan and Lim, Ryan and Le, Shimin and Nakasaki, Manando and Kutyavin, Vassily and Wright, Graham and Palakodeti, Dasaradhi and Ross, Robert S and Jamora, Colin and Vasioukhin, Valeri and Jie, Yan and Raghavan, Srikala} } @article {2212, title = {Microneedles for Extended Transdermal Therapeutics: A Route to Advanced Healthcare.}, journal = {Eur J Pharm Biopharm}, volume = {159}, year = {2021}, month = {2021 Feb}, pages = {151-169}, abstract = {

Sustained release of drugs over a pre-determined period is required to maintain an effective therapeutic dose for variety of drug delivery applications. Transdermal devices such as polymeric microneedle patches and other microneedle-based devices have been utilized for sustained release of their payload. Swift clearing of drugs can be prevented either by designing a slow-degrading polymeric matrix or by providing physiochemical triggers to different microneedle-based devices for on-demand release. These long-acting transdermal devices prevent the burst release of drugs. This review highlights the recent advances of microneedle-based devices for sustained release of vaccines, hormones, and antiretrovirals with their prospective safe clinical translation.

}, issn = {1873-3441}, doi = {10.1016/j.ejpb.2020.12.020}, author = {Pahal, Suman and Badnikar, Kedar and Ghate, Vivek and Bhutani, Utkarsh and Nayak, Mangalore Manjunatha and Subramanyam, Dinesh Narasimhaiah and Vemula, Praveen Kumar} } @article {2274, title = {Mitochondrial state determines functionally divergent stem cell population in planaria.}, journal = {Stem Cell Reports}, volume = {16}, year = {2021}, month = {2021 May 11}, pages = {1302-1316}, abstract = {

Mitochondrial state changes were shown to be critical for stem cell function. However, variation in the mitochondrial content in stem cells and the implication, if any, on differentiation is poorly understood. Here, using cellular and molecular studies, we show that the planarian pluripotent stem cells (PSCs) have low mitochondrial mass compared with their progenitors. Transplantation experiments provided functional validation that neoblasts with low mitochondrial mass are the true PSCs. Further, the mitochondrial mass correlated with OxPhos and inhibiting the transition to OxPhos dependent metabolism in cultured cells resulted in higher PSCs. In summary, we show that low mitochondrial mass is a hallmark of PSCs in planaria and provide a mechanism to isolate live, functionally active, PSCs from different cell cycle stages (G0/G1 and S, G2/M). Our study demonstrates that the change in mitochondrial metabolism, a feature of PSCs is conserved in planaria and highlights its role in organismal regeneration.

}, issn = {2213-6711}, doi = {10.1016/j.stemcr.2021.03.022}, author = {Mohamed Haroon, Mohamed and Lakshmanan, Vairavan and Sarkar, Souradeep R and Lei, Kai and Vemula, Praveen Kumar and Palakodeti, Dasaradhi} } @article {2244, title = {Oxylipin biosynthesis reinforces cellular senescence and allows detection of senolysis.}, journal = {Cell Metab}, year = {2021}, month = {2021 Mar 31}, abstract = {

Cellular senescence is a stress or damage response that causes a permanent proliferative arrest and secretion of numerous factors with potent biological activities. This senescence-associated secretory phenotype (SASP) has been characterized largely for secreted proteins that participate in embryogenesis, wound healing, inflammation, and many age-related pathologies. By contrast, lipid components of the SASP are understudied. We show that senescent cells activate the biosynthesis of several oxylipins that promote segments of the SASP and reinforce the proliferative arrest. Notably, senescent cells synthesize and accumulate an unstudied intracellular prostaglandin, 1a,1b-dihomo-15-deoxy-delta-12,14-prostaglandin J2. Released 15-deoxy-delta-12,14-prostaglandin J2 is a biomarker of senolysis in culture and in\ vivo. This and other prostaglandin D2-related lipids promote the senescence arrest and SASP by activating RAS signaling. These data identify an important aspect of cellular senescence and a method to detect senolysis.

}, issn = {1932-7420}, doi = {10.1016/j.cmet.2021.03.008}, author = {Wiley, Christopher D and Sharma, Rishi and Davis, Sonnet S and Lopez-Dominguez, Jose Alberto and Mitchell, Kylie P and Wiley, Samantha and Alimirah, Fatouma and Kim, Dong Eun and Payne, Therese and Rosko, Andrew and Aimontche, Eliezer and Deshpande, Sharvari M and Neri, Francesco and Kuehnemann, Chisaka and Demaria, Marco and Ramanathan, Arvind and Campisi, Judith} } @article {2373, title = {A regulatory network of microRNAs confers lineage commitment during early developmental trajectories of B and T lymphocytes.}, journal = {Proc Natl Acad Sci U S A}, volume = {118}, year = {2021}, month = {2021 11 16}, abstract = {

The commitment of hematopoietic multipotent progenitors (MPPs) toward a particular lineage involves activation of cell type-specific genes and silencing of genes that promote alternate cell fates. Although the gene expression programs of early-B and early-T lymphocyte development are mutually exclusive, we show that these cell types exhibit significantly correlated microRNA (miRNA) profiles. However, their corresponding miRNA targetomes are distinct and predominated by transcripts associated with natural killer, dendritic cell, and myeloid lineages, suggesting that miRNAs function in a cell-autonomous manner. The combinatorial expression of miRNAs miR-186-5p, miR-128-3p, and miR-330-5p in MPPs significantly attenuates their myeloid differentiation potential due to repression of myeloid-associated transcripts. Depletion of these miRNAs caused a pronounced de-repression of myeloid lineage targets in differentiating early-B and early-T cells, resulting in a mixed-lineage gene expression pattern. De novo motif analysis combined with an assay of promoter activities indicates that B as well as T lineage determinants drive the expression of these miRNAs in lymphoid lineages. Collectively, we present a paradigm that miRNAs are conserved between developing B and T lymphocytes, yet they target distinct sets of promiscuously expressed lineage-inappropriate genes to suppress the alternate cell-fate options. Thus, our studies provide a comprehensive compendium of miRNAs with functional implications for B and T lymphocyte development.

}, keywords = {Animals, B-Lymphocytes, Cell Differentiation, Cell Lineage, Gene Expression, Gene Expression Profiling, Gene Regulatory Networks, Hematopoietic Stem Cells, Mice, MicroRNAs, Myeloid Cells, T-Lymphocytes}, issn = {1091-6490}, doi = {10.1073/pnas.2104297118}, author = {Nikhat, Sameena and Yadavalli, Anurupa D and Prusty, Arpita and Narayan, Priyanka K and Palakodeti, Dasaradhi and Murre, Cornelis and Pongubala, Jagan M R} } @article {2270, title = {Repeated victorious and defeat experiences induce similar apical dendritic spine remodeling in CA1 hippocampus of rats.}, journal = {Behav Brain Res}, volume = {406}, year = {2021}, month = {2021 May 21}, pages = {113243}, abstract = {

In this study, apical dendritic spine density of neurons in hippocampal, amygdalar and prefrontal cortical areas was compared in rats that were repeatedly winning or losing social conflicts. Territorial male wild-type Groningen (WTG) rats were allowed multiple daily attacks (\>20 times) on intruder males in the resident-intruder paradigm. Frequent winning experiences are known to facilitate uncontrolled aggressive behavior reflected in aggressive attacks on anesthetized males which was also observed in the winners in this study. Both winners and losers were socially housed during the experiments; winners with females to stimulate territorial behavior, and losers with two other losing male rats. Twenty-four hours after the last social encounter, brains from experienced residential winners and repeatedly defeated intruder rats were collected and neuronal morphology in selected brain regions was studied via Golgi-Cox staining. Results indicate that spine density in the apical dendrites of the hippocampal CA1 reduced similarly in both winners and losers. In addition, winners showed increased spine densities at the proximal segments (20-30 μm) of the basolateral amygdala neurons and losers tended to show a decreased spine density at the more proximal segments of the infralimbic region of prefrontal cortex neurons. No effect of winning and losing was observed in the medial amygdala. The atrophic effect of repeated defeats in hippocampal and prefrontal regions was anticipated despite the fact that social housing of the repeatedly losing intruder males may have played a protective role. The reduction of hippocampal spine density in the winners seems surprising but supports previous findings in hierarchical dominant males in rat colonies. The dominants showed even greater shrinkage of the apical dendritic arbors of hippocampal CA3 pyramidal neurons compared to the stressed subordinates.

}, issn = {1872-7549}, doi = {10.1016/j.bbr.2021.113243}, author = {Patel, Deepika and Anilkumar, Shobha and Chattarji, Sumantra and de Boer, Sietse F and Buwalda, Bauke} } @article {2372, title = {Ribosomal protein S6 kinase beta-1 gene variants cause hypertrophic cardiomyopathy.}, journal = {J Med Genet}, year = {2021}, month = {2021 Dec 16}, abstract = {

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a genetic heart muscle disease with preserved or increased ejection fraction in the absence of secondary causes. Mutations in the sarcomeric protein-encoding genes predominantly cause HCM. However, relatively little is known about the genetic impact of signalling proteins on HCM.

METHODS AND RESULTS: Here, using exome and targeted sequencing methods, we analysed two independent cohorts comprising 401 Indian patients with HCM and 3521 Indian controls. We identified novel variants in ribosomal protein S6 kinase beta-1 () gene in two unrelated Indian families as a potential candidate gene for HCM. The two unrelated HCM families had the same heterozygous missense S6K1 variant (p.G47W). In a replication association study, we identified two S6K1 heterozygotes variants (p.Q49K and p.Y62H) in the UK Biobank cardiomyopathy cohort (n=190) compared with matched controls (n=16 479). These variants are neither detected in region-specific controls nor in the human population genome data. Additionally, we observed an S6K1 variant (p.P445S) in an Arab patient with HCM. Functional consequences were evaluated using representative S6K1 mutated proteins compared with wild type in cellular models. The mutated proteins activated the S6K1 and hyperphosphorylated the rpS6 and ERK1/2 signalling cascades, suggesting a gain-of-function effect.

CONCLUSIONS: Our study demonstrates for the first time that the variants in the gene are associated with HCM, and early detection of the variant carriers can help to identify family members at risk and subsequent preventive measures. Further screening in patients with HCM with different ethnic populations will establish the specificity and frequency of gene variants.

}, issn = {1468-6244}, doi = {10.1136/jmedgenet-2021-107866}, author = {Jain, Pratul Kumar and Jayappa, Shashank and Sairam, Thiagarajan and Mittal, Anupam and Paul, Sayan and Rao, Vinay J and Chittora, Harshil and Kashyap, Deepak K and Palakodeti, Dasaradhi and Thangaraj, Kumarasamy and Shenthar, Jayaprakash and Koranchery, Rakesh and Rajendran, Ranjith and Alireza, Haghighi and Mohanan, Kurukkanparampil Sreedharan and Rathinavel, Andiappan and Dhandapany, Perundurai S} } @article {2363, title = {SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion.}, journal = {Nature}, volume = {599}, year = {2021}, month = {2021 11}, pages = {114-119}, abstract = {

The B.1.617.2 (Delta) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha). In vitro, B.1.617.2 is sixfold less sensitive to serum neutralizing antibodies from recovered individuals, and eightfold less sensitive to vaccine-elicited antibodies, compared with wild-type Wuhan-1 bearing D614G. Serum neutralizing titres against B.1.617.2 were lower in ChAdOx1 vaccinees than in BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies to the receptor-binding domain and the amino-terminal domain. B.1.617.2 demonstrated higher replication efficiency than B.1.1.7 in both airway organoid and human airway epithelial systems, associated with B.1.617.2 spike being in a predominantly cleaved state compared with B.1.1.7 spike. The B.1.617.2 spike protein was able to mediate highly efficient syncytium formation that was less sensitive to inhibition by neutralizing antibody, compared with that of wild-type spike. We also observed that B.1.617.2 had higher replication and spike-mediated entry than B.1.617.1, potentially explaining the B.1.617.2 dominance. In an analysis of more than 130 SARS-CoV-2-infected health care workers across three centres in India during a period of mixed lineage circulation, we observed reduced ChAdOx1 vaccine effectiveness against B.1.617.2 relative to non-B.1.617.2, with the caveat of possible residual confounding. Compromised vaccine efficacy against the highly fit and immune-evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era.

}, keywords = {Antibodies, Neutralizing, Cell Fusion, Cell Line, COVID-19 Vaccines, Female, Health Personnel, Humans, Immune Evasion, India, Kinetics, Male, SARS-CoV-2, Spike Glycoprotein, Coronavirus, Vaccination, Virus Replication}, issn = {1476-4687}, doi = {10.1038/s41586-021-03944-y}, author = {Mlcochova, Petra and Kemp, Steven A and Dhar, Mahesh Shanker and Papa, Guido and Meng, Bo and Ferreira, Isabella A T M and Datir, Rawlings and Collier, Dami A and Albecka, Anna and Singh, Sujeet and Pandey, Rajesh and Brown, Jonathan and Zhou, Jie and Goonawardane, Niluka and Mishra, Swapnil and Whittaker, Charles and Mellan, Thomas and Marwal, Robin and Datta, Meena and Sengupta, Shantanu and Ponnusamy, Kalaiarasan and Radhakrishnan, Venkatraman Srinivasan and Abdullahi, Adam and Charles, Oscar and Chattopadhyay, Partha and Devi, Priti and Caputo, Daniela and Peacock, Tom and Wattal, Chand and Goel, Neeraj and Satwik, Ambrish and Vaishya, Raju and Agarwal, Meenakshi and Mavousian, Antranik and Lee, Joo Hyeon and Bassi, Jessica and Silacci-Fegni, Chiara and Saliba, Christian and Pinto, Dora and Irie, Takashi and Yoshida, Isao and Hamilton, William L and Sato, Kei and Bhatt, Samir and Flaxman, Seth and James, Leo C and Corti, Davide and Piccoli, Luca and Barclay, Wendy S and Rakshit, Partha and Agrawal, Anurag and Gupta, Ravindra K} } @article {2310, title = {Scanning electron microscopy of murine skin ultrathin sections and cultured keratinocytes.}, journal = {STAR Protoc}, volume = {2}, year = {2021}, month = {2021 Sep 17}, pages = {100729}, abstract = {

Generating high-quality electron microscopy images of the skin and keratinocytes can be challenging. Here we describe a simple protocol for scanning electron microscopy (SEM) of murine skin. The protocol enables characterization of the ultrastructure of the epidermis, dermis, hair follicles, basement membrane, and cell-cell junctions. We detail the specific steps for sample preparation and highlight the critical need for proper orientation of the sample for ultrathin sectioning. We also describe the isolation and preparation of primary keratinocyte monolayers for SEM. For complete details on the use and execution of this protocol, please refer to Biswas et\ al. (2021).

}, issn = {2666-1667}, doi = {10.1016/j.xpro.2021.100729}, author = {Banerjee, Avinanda and Biswas, Ritusree and Lim, Ryan and Pasolli, Hilda Amalia and Raghavan, Srikala} } @article {2290, title = {Strategies to target SARS-CoV-2 entry and infection using dual mechanisms of inhibition by acidification inhibitors.}, journal = {PLoS Pathog}, volume = {17}, year = {2021}, month = {2021 07}, pages = {e1009706}, abstract = {

Many viruses utilize the host endo-lysosomal network for infection. Tracing the endocytic itinerary of SARS-CoV-2 can provide insights into viral trafficking and aid in designing new therapeutic strategies. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV-2 spike protein is internalized via the pH-dependent CLIC/GEEC (CG) endocytic pathway in human gastric-adenocarcinoma (AGS) cells expressing undetectable levels of ACE2. Ectopic expression of ACE2 (AGS-ACE2) results in RBD traffic via both CG and clathrin-mediated endocytosis. Endosomal acidification inhibitors like BafilomycinA1 and NH4Cl, which inhibit the CG pathway, reduce the uptake of RBD and impede Spike-pseudoviral infection in both AGS and AGS-ACE2 cells. The inhibition by BafilomycinA1 was found to be distinct from Chloroquine which neither affects RBD uptake nor alters endosomal pH, yet attenuates Spike-pseudovirus entry. By screening a subset of FDA-approved inhibitors for functionality similar to BafilomycinA1, we identified Niclosamide as a SARS-CoV-2 entry inhibitor. Further validation using a clinical isolate of SARS-CoV-2 in AGS-ACE2 and Vero cells confirmed its antiviral effect. We propose that Niclosamide, and other drugs which neutralize endosomal pH as well as inhibit the endocytic uptake, could provide broader applicability in subverting infection of viruses entering host cells via a pH-dependent endocytic pathway.

}, keywords = {Ammonium Chloride, Angiotensin-Converting Enzyme 2, Animals, Antiviral Agents, Cell Line, Chlorocebus aethiops, Chloroquine, Clathrin, COVID-19, Drug Synergism, Endocytosis, Endosomes, Humans, Hydrogen-Ion Concentration, Hydroxychloroquine, Macrolides, Niclosamide, Protein Binding, Protein Domains, SARS-CoV-2, Spike Glycoprotein, Coronavirus, Vero Cells, Virus Internalization}, issn = {1553-7374}, doi = {10.1371/journal.ppat.1009706}, author = {Prabhakara, Chaitra and Godbole, Rashmi and Sil, Parijat and Jahnavi, Sowmya and Gulzar, Shah-E-Jahan and van Zanten, Thomas S and Sheth, Dhruv and Subhash, Neeraja and Chandra, Anchal and Shivaraj, Akshatha and Panikulam, Patricia and U, Ibrahim and Nuthakki, Vijay Kumar and Puthiyapurayil, Theja Parassini and Ahmed, Riyaz and Najar, Ashaq Hussain and Lingamallu, Sai Manoz and Das, Snigdhadev and Mahajan, Bhagyashri and Vemula, Praveen and Bharate, Sandip B and Singh, Parvinder Pal and Vishwakarma, Ram and Guha, Arjun and Sundaramurthy, Varadharajan and Mayor, Satyajit} } @article {2245, title = {Systematic evaluation of the impact of defacing on quality and volumetric assessments on T1-weighted MR-images.}, journal = {J Neuroradiol}, year = {2021}, month = {2021 Mar 13}, abstract = {

BACKGROUND AND PURPOSE: Facial features can be potentially reconstructed from structural magnetic resonance images, thereby compromising the confidentiality of study participants. Defacing methods can be applied to MRI images to ensure privacy of study participants. These methods remove facial features, thereby rendering the image unidentifiable. It is commonly assumed that defacing would not have any impact on quantitative assessments of the brain. In this study, we have assessed the impact of different defacing methods on quality and volumetric estimates.

MATERIALS AND METHODS: We performed SPM-, Freesurfer-, pydeface, and FSL-based defacing on 30 T1-weighted images. We statistically compared the change in quality measurements (from MRIQC) and volumes (from SPM, CAT, and Freesurfer) between non-defaced and defaced images. We also calculated the Dice coefficient of each tissue class between non-defaced and defaced images.

RESULTS: Almost all quality measurements and tissue volumes changed after defacing, irrespective of the method used. All tissue volumes decreased post-defacing for CAT, but no such consistent trend was seen for SPM and Freesurfer. Dice coefficients indicated that segmentations are relatively robust; however, partial volumes might be affected leading to changed volumetric estimates.

CONCLUSION: In this study, we demonstrated that volumes and quality measurements get affected differently by defacing methods. It is likely that this will have a significant impact on the reproducibility of experiments. We provide suggestions on ways to minimize the impact of defacing on outcome measurements. Our results warrant the need for robust handling of defaced images at different steps of image processing.

}, issn = {0150-9861}, doi = {10.1016/j.neurad.2021.03.001}, author = {Bhalerao, Gaurav Vivek and Parekh, Pravesh and Saini, Jitender and Venkatasubramanian, Ganesan and John, John P} } @article {2289, title = {Target identification for small-molecule discovery in the FOXO3a tumor-suppressor pathway using a biodiverse peptide library.}, journal = {Cell Chem Biol}, year = {2021}, month = {2021 Jun 01}, abstract = {

Genetic screening technologies to identify and validate macromolecular interactions (MMIs) essential for complex pathways remain an important unmet need for systems biology and therapeutics development. Here, we use a library of peptides from diverse prokaryal genomes to screen MMIs promoting the nuclear relocalization of Forkhead Box O3 (FOXO3a), a tumor suppressor more frequently inactivated by post-translational modification than mutation. A hit peptide engages the 14-3-3 family of signal regulators through a phosphorylation-dependent interaction, modulates FOXO3a-mediated transcription, and suppresses cancer cell growth. In a crystal structure, the hit peptide occupies the phosphopeptide-binding groove of 14-3-3ε in a conformation distinct from its natural peptide substrates. A biophysical screen identifies drug-like small molecules that displace the hit peptide from 14-3-3ε, providing starting points for structure-guided development. Our findings exemplify "protein interference," an approach using evolutionarily diverse, natural peptides to rapidly identify, validate, and develop chemical probes against MMIs essential for complex cellular phenotypes.

}, issn = {2451-9448}, doi = {10.1016/j.chembiol.2021.05.009}, author = {Emery, Amy and Hardwick, Bryn S and Crooks, Alex T and Milech, Nadia and Watt, Paul M and Mithra, Chandan and Kumar, Vikrant and Giridharan, Saranya and Sadasivam, Gayathri and Mathivanan, Subashini and Sudhakar, Sneha and Bairy, Sneha and Bharatham, Kavitha and Hurakadli, Manjunath A and Prasad, Thazhe K and Kamariah, Neelagandan and Muellner, Markus and Coelho, Miguel and Torrance, Christopher J and McKenzie, Grahame J and Venkitaraman, Ashok R} } @article {2200, title = {tRNA-derived fragments (tRFs): establishing their turf in post-transcriptional gene regulation.}, journal = {Cell Mol Life Sci}, year = {2021}, month = {2021 Jan 02}, abstract = {

Transfer RNA (tRNA)-derived fragments (tRFs) are an emerging class of conserved small non-coding RNAs that play important roles in post-transcriptional gene regulation. High-throughput sequencing of multiple biological samples have identified heterogeneous species of tRFs with distinct functionalities. These small RNAs have garnered a lot of scientific attention due to their ubiquitous expression and versatility in regulating various biological processes. In this review, we highlight our current understanding of tRF biogenesis and their regulatory functions. We summarize the diverse modes of biogenesis through which tRFs are generated and discuss the mechanism through which different tRF species regulate gene expression and the biological implications. Finally, we conceptualize research areas that require focus to strengthen our understanding of the biogenesis and function of tRFs.

}, issn = {1420-9071}, doi = {10.1007/s00018-020-03720-7}, author = {Krishna, Srikar and Raghavan, Srikala and DasGupta, Ramanuj and Palakodeti, Dasaradhi} } @article {2248, title = {Allosteric inhibition of MTHFR prevents futile SAM cycling and maintains nucleotide pools in one-carbon metabolism.}, journal = {J Biol Chem}, volume = {295}, year = {2020}, month = {2020 11 20}, pages = {16037-16057}, abstract = {

Methylenetetrahydrofolate reductase (MTHFR) links the folate cycle to the methionine cycle in one-carbon metabolism. The enzyme is known to be allosterically inhibited by SAM for decades, but the importance of this regulatory control to one-carbon metabolism has never been adequately understood. To shed light on this issue, we exchanged selected amino acid residues in a highly conserved stretch within the regulatory region of yeast MTHFR to create a series of feedback-insensitive, deregulated mutants. These were exploited to investigate the impact of defective allosteric regulation on one-carbon metabolism. We observed a strong growth defect in the presence of methionine. Biochemical and metabolite analysis revealed that both the folate and methionine cycles were affected in these mutants, as was the transsulfuration pathway, leading also to a disruption in redox homeostasis. The major consequences, however, appeared to be in the depletion of nucleotides. C isotope labeling and metabolic studies revealed that the deregulated MTHFR cells undergo continuous transmethylation of homocysteine by methyltetrahydrofolate (CHTHF) to form methionine. This reaction also drives SAM formation and further depletes ATP reserves. SAM was then cycled back to methionine, leading to futile cycles of SAM synthesis and recycling and explaining the necessity for MTHFR to be regulated by SAM. The study has yielded valuable new insights into the regulation of one-carbon metabolism, and the mutants appear as powerful new tools to further dissect out the intersection of one-carbon metabolism with various pathways both in yeasts and in humans.

}, keywords = {Adenosine Triphosphate, Allosteric Regulation, Humans, Methylation, Methylenetetrahydrofolate Reductase (NADPH2), S-Adenosylmethionine, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins}, issn = {1083-351X}, doi = {10.1074/jbc.RA120.015129}, author = {Bhatia, Muskan and Thakur, Jyotika and Suyal, Shradha and Oniel, Ruchika and Chakraborty, Rahul and Pradhan, Shalini and Sharma, Monika and Sengupta, Shantanu and Laxman, Sunil and Masakapalli, Shyam Kumar and Bachhawat, Anand Kumar} } @article {2110, title = {Cortical neurons derived from human pluripotent stem cells lacking FMRP display altered spontaneous firing patterns.}, journal = {Mol Autism}, volume = {11}, year = {2020}, month = {2020 Jun 19}, pages = {52}, abstract = {

BACKGROUND: Fragile X syndrome (FXS), a neurodevelopmental disorder, is a leading monogenetic cause of intellectual disability and autism spectrum disorder. Notwithstanding the extensive studies using rodent and other pre-clinical models of FXS, which have provided detailed mechanistic insights into the pathophysiology of this disorder, it is only relatively recently that human stem cell-derived neurons have been employed as a model system to further our understanding of the pathophysiological events that may underlie FXS. Our study assesses the physiological properties of human pluripotent stem cell-derived cortical neurons lacking fragile X mental retardation protein (FMRP).

METHODS: Electrophysiological whole-cell voltage- and current-clamp recordings were performed on two control and three FXS patient lines of human cortical neurons derived from induced pluripotent stem cells. In addition, we also describe the properties of an isogenic pair of lines in one of which FMR1 gene expression has been silenced.

RESULTS: Neurons lacking FMRP displayed bursts of spontaneous action potential firing that were more frequent but shorter in duration compared to those recorded from neurons expressing FMRP. Inhibition of large conductance Ca-activated K currents and the persistent Na current in control neurons phenocopies action potential bursting observed in neurons lacking FMRP, while in neurons lacking FMRP pharmacological potentiation of voltage-dependent Na channels phenocopies action potential bursting observed in control neurons. Notwithstanding the changes in spontaneous action potential firing, we did not observe any differences in the intrinsic properties of neurons in any of the lines examined. Moreover, we did not detect any differences in the properties of miniature excitatory postsynaptic currents in any of the lines.

CONCLUSIONS: Pharmacological manipulations can alter the action potential burst profiles in both control and FMRP-null human cortical neurons, making them appear like their genetic counterpart. Our studies indicate that FMRP targets that have been found in rodent models of FXS are also potential targets in a human-based model system, and we suggest potential mechanisms by which activity is altered.

}, issn = {2040-2392}, doi = {10.1186/s13229-020-00351-4}, author = {Das Sharma, Shreya and Pal, Rakhi and Reddy, Bharath Kumar and Selvaraj, Bhuvaneish T and Raj, Nisha and Samaga, Krishna Kumar and Srinivasan, Durga J and Ornelas, Loren and Sareen, Dhruv and Livesey, Matthew R and Bassell, Gary J and Svendsen, Clive N and Kind, Peter C and Chandran, Siddharthan and Chattarji, Sumantra and Wyllie, David J A} } @article {2146, title = {Differential flexibility leading to crucial microelastic properties of asymmetric lipid vesicles for cellular transfection: A combined spectroscopic and atomic force microscopy studies.}, journal = {Colloids Surf B Biointerfaces}, volume = {196}, year = {2020}, month = {2020 Sep 21}, pages = {111363}, abstract = {

The role of microscopic elasticity of nano-carriers in cellular uptake is an important aspect in biomedical research. Herein we have used AFM nano-indentation force spectroscopy and F{\"o}rster resonance energy transfer (FRET) measurements to probe microelastic properties of three novel cationic liposomes based on di-alkyl dihydroxy ethyl ammonium chloride based lipids having asymmetry in their hydrophobic chains (Lip1818, Lip1814 and Lip1810). AFM data reveals that symmetry in hydrophobic chains of a cationic lipid (Lip1818) imparts higher rigidity to the resulting liposomes than those based on asymmetric lipids (Lip1814 and Lip1810). The stiffness of the cationic liposomes is found to decrease with increasing asymmetry in the hydrophobic lipid chains in the order of Lip1818 \> Lip1814 \> lip1810. FRET measurements between Coumarin 500 (Donor) and Merocyanine 540 (Acceptor) have revealed that full width at half-maxima (hw) of the probability distribution (P(r)) of donor-acceptor distance (r), increases in an order Lip1818 \< Lip1814 \< Lip1810 with increasing asymmetry of the hydrophobic lipid chains. This increase in width (hw) of the donor-acceptor distance distributions is reflective of increasing flexibility of the liposomes with increasing asymmetry of their constituent lipids. Thus, the results from AFM and FRET studies are complementary to each other and indicates that an increase in asymmetry of the hydrophobic lipid chains increases elasticity and or flexibility of the corresponding liposomes. Cell biology experiments confirm that liposomal flexibility or rigidity directly influences their cellular transfection efficiency, where Lip1814 is found to be superior than the other two liposomes manifesting that a critical balance between flexibility and rigidity of the cationic liposomes is key to efficient cellular uptake. Taken together, our studies reveal how asymmetry in the molecular architecture of the hydrophobic lipid chains influences the microelastic properties of the liposomes, and hence, their cellular uptake efficiency.

}, issn = {1873-4367}, doi = {10.1016/j.colsurfb.2020.111363}, author = {Mukherjee, Dipanjan and Rakshit, Tatini and Singh, Priya and Mondal, Suman and Paul, Debashish and Ahir, Manisha and Adhikari, Arghya and Puthiyapurayil, Theja P and Vemula, Praveen Kumar and Senapati, Dulal and Das, Ranjan and Pal, Samir Kumar} } @article {2151, title = {Dopamine requires unique residues to signal via the serotonin 2A receptor.}, journal = {Neuroscience}, volume = {439}, year = {2020}, month = {2020 07 15}, pages = {319-331}, abstract = {

Serotonin is an important neurotransmitter and neuromodulator. Disruption of the serotonergic system has been implicated in various psychiatric disorders such as schizophrenia and bipolar disorder. Most of the drugs targeting these neurotransmitter systems are classified primarily as agonists or inverse agonists/antagonists, with their described function being limited to activating the canonical signaling pathway(s), or inhibiting the pathway(s) respectively. Previous work with the human 5-HT has shown the receptor to be activated by dopamine, also an endogenous ligand. Dopamine is the cognate ligand of the dopaminergic system, which significantly overlaps with the serotonergic system in the brain. The two systems innervate many of the same brain areas, and the central serotonergic systems also regulate dopamine functions. Our aim was to investigate the downstream signaling set up by the receptor on being activated by dopamine. We show that dopamine is a functionally selective ligand at 5-HT and have examined dopamine as a ligand with respect to some receptor-dependent phenotypes. Our results show that dopamine acts as an agonist at the human serotonin 2A receptor and brings about its activation and internalization. Using in vitro assays, we have established differences in the signaling pathways set up by dopamine as compared to serotonin. Using site-specific mutagenesis we have identified residues important for this functional selectivity, shown by dopamine at this receptor. Our identification of specific residues important in the functional selectivity of dopamine at 5-HT could have far reaching implications for the field of GPCR signaling and drug-design. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.

}, issn = {1873-7544}, doi = {10.1016/j.neuroscience.2019.03.056}, author = {Soman, Shuchita and Bhattacharya, Aditi and Panicker, Mitradas M} } @article {2251, title = {Generic Molding Platform for Simple, Low-Cost Fabrication of Polymeric Microneedles}, journal = {Macromolecular Materials and Engineering}, volume = {305}, year = {2020}, abstract = {

Micromolding technology is widely used for the fabrication of polymer microneedles for transdermal and intradermal drug delivery applications. Geometric features of microneedles in molding are solely determined by geometry of the master mold template. Fabrication of master mold template usually involves costly and cumbersome technologies due to small feature sizes typical of microneedles. In this research, a novel molding platform is designed that is fabricated using low-cost and simple techniques with flexibility of producing large number of microneedle geometries. The proposed molding platform eliminates need for developing multiple mold templates for fabrication of various geometries of polymer microneedles. Utility of this molding platform is demonstrated in polylactic acid-based solid thermoplastic microneedles and polyacrylic acid-based dissolvable microneedles with various aspect ratio settings. Various microneedles fabricated at heights differing with resolution of as low as 100 {\textmu}m are successfully achieved using specified settings in the molding platform. The suitability of fabricated microneedles for drug delivery applications is evaluated by in vitro and in vivo testing.

}, author = {7. Badnikar, K. and Jayadevi, S. N. and Pahal, S. and Sripada, S. and Nayak, M. M. and Vemula, P. K. and Subrahmanyam, D. N.} } @article {2154, title = {Immune Control of Animal Growth in Homeostasis and Nutritional Stress in .}, journal = {Front Immunol}, volume = {11}, year = {2020}, month = {2020}, pages = {1528}, abstract = {

A large body of research implicates the brain and fat body (liver equivalent) as central players in coordinating growth and nutritional homeostasis in multicellular animals. In this regard, an underlying connection between immune cells and growth is also evident, although mechanistic understanding of this cross-talk is scarce. Here, we explore the importance of innate immune cells in animal growth during homeostasis and in conditions of nutrient stress. We report that larvae lacking blood cells eclose as small adults and show signs of insulin insensitivity. Moreover, when exposed to dietary stress of a high-sucrose diet (HSD), these animals are further growth retarded than normally seen in regular animals raised on HSD. In contrast, larvae carrying increased number of activated macrophage-like plasmatocytes show no defects in adult growth when raised on HSD and grow to sizes almost comparable with that seen with regular diet. These observations imply a central role for immune cell activity in growth control. Mechanistically, our findings reveal a surprising influence of immune cells on balancing fat body inflammation and insulin signaling under conditions of homeostasis and nutrient overload as a means to coordinate systemic metabolism and adult growth. This work integrates both the cellular and humoral arm of the innate immune system in organismal growth homeostasis, the implications of which may be broadly conserved across mammalian systems as well.

}, issn = {1664-3224}, doi = {10.3389/fimmu.2020.01528}, author = {P, Preethi and Tomar, Ajay and Madhwal, Sukanya and Mukherjee, Tina} } @article {2068, title = {Lithium response in bipolar disorder correlates with improved cell viability of patient derived cell lines.}, journal = {Sci Rep}, volume = {10}, year = {2020}, month = {2020 May 04}, pages = {7428}, abstract = {

Lithium is an effective, well-established treatment for bipolar disorder (BD). However, the mechanisms of its action, and reasons for variations in clinical response, are unclear. We used neural precursor cells (NPCs) and lymphoblastoid cell lines (LCLs), from BD patients characterized for clinical response to lithium (using the "Alda scale" and "NIMH Retrospective Life chart method"), to interrogate cellular phenotypes related to both disease and clinical lithium response. NPCs from two biologically related BD patients who differed in their clinical response to lithium were compared with healthy controls. RNA-Seq and analysis, mitochondrial membrane potential (MMP), cell viability, and cell proliferation parameters were assessed, with and without in vitro lithium. These parameters were also examined in LCLs from 25 BD patients (16 lithium responders and 9 non-responders), and 12 controls. MMP was lower in both NPCs and LCLs from BD; but it was reversed with in vitro lithium only in LCLs, and this was unrelated to clinical lithium response. The higher cell proliferation observed in BD was unaffected by in vitro lithium. Cell death was greater in BD. However, LCLs from clinical lithium responders could be rescued by addition of in vitro lithium. In vitro lithium also enhanced BCL2 and GSK3B expression in these cells. Our findings indicate cellular phenotypes related to the disease (MMP, cell proliferation) in both NPCs and LCLs; and those related to clinical lithium response (cell viability, BCL2/GSK3B expression) in LCLs.

}, issn = {2045-2322}, doi = {10.1038/s41598-020-64202-1}, author = {Paul, Pradip and Iyer, Shruti and Nadella, Ravi Kumar and Nayak, Rashmitha and Chellappa, Anirudh S and Ambardar, Sheetal and Sud, Reeteka and Sukumaran, Salil K and Purushottam, Meera and Jain, Sanjeev and Viswanath, Biju} } @article {2109, title = {Modulation of β-catenin levels regulates cranial neural crest patterning and dispersal into first pharyngeal arch.}, journal = {Dev Dyn}, year = {2020}, month = {2020 May 19}, abstract = {

BACKGROUND: Vertebrate cranial neural crest cells (CNCCs) are multipotent, proximal to the source CNCC form the cranial ganglia. Distally, in the pharyngeal arches, they give rise to the craniofacial skeleton and connective tissues. Fate choices are made as CNCC pattern into distinct destination compartments. In spite of this importance, the mechanism patterning CNCC is poorly defined.

RESULTS: Here, we report that a novel β-catenin-dependent regulation of N-Cadherin levels may drive CNCC patterning. In mouse embryos, at the first pharyngeal arch axial level, membrane β-catenin levels correlate with the extent of N-cadherin-mediated adhesion and thus suggest the presence of collective and dispersed states of CNCC. Using in vitro human neural crest model and chemical modulators of β-catenin levels, we show a requirement for down-modulating β-catenin for regulating N-cadherin levels and cell-cell adhesion. Similarly, in β-catenin gain-of-function mutant mouse embryos, CNCC fail to lower N-cadherin levels. This indicates a failure to reduce cell-cell adhesion, which may underlie the failure of mutant CNCC to populate first pharyngeal arch.

CONCLUSION: We suggest that β-catenin-mediated regulation of CNCC adhesion, a previously underappreciated mechanism, underlies the patterning of CNCC into fate-specific compartments.

}, issn = {1097-0177}, doi = {10.1002/dvdy.208}, author = {Javali, Alok and Lakshmanan, Vairavan and Palakodeti, Dasaradhi and Sambasivan, Ramkumar} } @article {2108, title = {A novel polyubiquitin chain linkage formed by viral Ubiquitin is resistant to host deubiquitinating enzymes.}, journal = {Biochem J}, volume = {477}, year = {2020}, month = {2020 Jun 26}, pages = {2193-2219}, abstract = {

The Baculoviridae family of viruses encode a viral Ubiquitin (vUb) gene. Though the vUb is homologous to the host eukaryotic Ubiquitin (Ub), its preservation in the viral genome indicates unique functions that are not compensated by the host Ub. We report the structural, biophysical, and biochemical properties of the vUb from Autographa californica multiple nucleo-polyhedrosis virus (AcMNPV). The packing of central helix α1 to the beta-sheet β1-β5 is different between vUb and Ub. Consequently, its stability is lower compared with Ub. However, the surface properties, ubiquitination activity, and the interaction with Ubiquitin-binding domains are similar between vUb and Ub. Interestingly, vUb forms atypical polyubiquitin chain linked by lysine at the 54th position (K54), and the deubiquitinating enzymes are ineffective against the K54-linked polyubiquitin chains. We propose that the modification of host/viral proteins with the K54-linked chains is an effective way selected by the virus to protect the vUb signal from host DeUbiquitinases.

}, issn = {1470-8728}, doi = {10.1042/BCJ20200289}, author = {Negi, Hitendra and Reddy, Pothula Purushotham and Vengayil, Vineeth and Patole, Chhaya and Laxman, Sunil and Das, Ranabir} } @article {2063, title = {N-terminal variant Asp14Asn of the human p70 S6 Kinase 1 enhances translational signaling causing different effects in developing and mature neuronal cells.}, journal = {Neurobiol Learn Mem}, volume = {171}, year = {2020}, month = {2020 May}, pages = {107203}, abstract = {

The ribosomal p70 S6 Kinase 1 (S6K1) has been implicated in the etiology of complex neurological diseases including autism, depression and dementia. Though no major gene disruption has been reported in humans in RPS6KB1, single nucleotide variants (SNVs) causing missense mutations have been identified, which have not been assessed for their impact on protein function. These S6K1 mutations have the potential to influence disease progression and treatment response. We mined the Simon Simplex Collection (SSC) and SPARK autism database to find inherited SNVs in S6K1 and characterized the effect of two missense SNVs, Asp14Asn (allele frequency\ =\ 0.03282\%) and Glu44Gln (allele frequency\ =\ 0.0008244\%), on S6K1 function in HEK293, human ES cells and primary neurons. Expressing Asp14Asn in HEK293 cells resulted in increased basal phosphorylation of downstream targets of S6K1 and increased de novo translation. This variant also showed blunted response to the specific S6K1 inhibitor, FS-115. In human embryonic cell line Shef4, Asp14Asn enhanced spontaneous neural fate specification in the absence of differentiating growth factors. In addition to enhanced translation, neurons expressing Asp14Asn exhibited impaired dendritic arborization and increased levels of phosphorylated ERK 1/2. Finally, in the SSC families tracked, Asp14Asn segregated with lower IQ scores when found in the autistic individual rather than the unaffected sibling. The Glu44Gln mutation showed a milder, but opposite phenotype in HEK cells as compared to Asp14Asn. Although the Glu44Gln mutation displayed increased neuronal translation, it had no impact on neuronal morphology. Our results provide the first characterization of naturally occurring human S6K1 variants on cognitive phenotype, neuronal morphology and maturation, underscoring again the importance of translation control in neural development and plasticity.

}, issn = {1095-9564}, doi = {10.1016/j.nlm.2020.107203}, author = {Venkatasubramani, Janani Priya and Subramanyam, Prakash and Pal, Rakhi and Reddy, Bharath K and Srinivasan, Durga Jeyalakshmi and Chattarji, Sumantra and Iossifov, Ivan and Klann, Eric and Bhattacharya, Aditi} } @article {2152, title = {Optimization of Protocols for Detection of De Novo Protein Synthesis in Whole Blood Samples via Azide-Alkyne Cycloaddition.}, journal = {J Proteome Res}, volume = {19}, year = {2020}, month = {2020 Sep 04}, pages = {3856-3866}, abstract = {

Aberrant protein synthesis and protein expression are a hallmark of many conditions ranging from cancer to Alzheimer{\textquoteright}s. Blood-based biomarkers indicative of changes in proteomes have long been held to be potentially useful with respect to disease prognosis and treatment. However, most biomarker efforts have focused on unlabeled plasma proteomics that include nonmyeloid origin proteins with no attempt to dynamically tag acute changes in proteomes. Herein we report a method for evaluating de novo protein synthesis in whole blood liquid biopsies. Using a modification of the "bioorthogonal noncanonical amino acid tagging" (BONCAT) protocol, rodent whole blood samples were incubated with l-azidohomoalanine (AHA) to allow incorporation of this selectively reactive non-natural amino acid within nascent polypeptides. Notably, failure to incubate the blood samples with EDTA prior to implementation of azide-alkyne "click" reactions resulted in the inability to detect probe incorporation. This live-labeling assay was sensitive to inhibition with anisomycin and nascent, tagged polypeptides were localized to a variety of blood cells using FUNCAT. Using labeled rodent blood, these tagged peptides could be consistently identified through standard LC/MS-MS detection of known blood proteins across a variety of experimental conditions. Furthermore, this assay could be expanded to measure de novo protein synthesis in human blood samples. Overall, we present a rapid and convenient de novo protein synthesis assay that can be used with whole blood biopsies that can quantify translational change as well as identify differentially expressed proteins that may be useful for clinical applications.

}, issn = {1535-3907}, doi = {10.1021/acs.jproteome.0c00299}, author = {Bowling, Heather L and Kasper, Amanda and Patole, Chhaya and Venkatasubramani, Janani Priya and Leventer, Sarah Parker and Carmody, Erin and Sharp, Kevin and Berry-Kravis, Elizabeth and Kirshenbaum, Kent and Klann, Eric and Bhattacharya, Aditi} } @article {2253, title = {Rethinking Exosomes: From Cell-to-Cell Courier Services to Individualized Medicines}, journal = {AAPS News magazine}, year = {2020}, month = {06/2020}, author = {Ghate, V and Chaudhari, P and Maxwell, A and Lewis, S and Pahal, S and Vemula PK} } @article {2077, title = {A sex difference in the response of the rodent postsynaptic density to synGAP haploinsufficiency.}, journal = {Elife}, volume = {9}, year = {2020}, month = {2020 Jan 15}, abstract = {

SynGAP is a postsynaptic density (PSD) protein that binds to PDZ domains of the scaffold protein PSD-95. We previously reported that heterozygous deletion of in mice is correlated with increased steady-state levels of other key PSD proteins that bind PSD-95, although the level of PSD-95 remains constant (Walkup et al., 2016). For example, the ratio to PSD-95 of Transmembrane AMPA-Receptor-associated Proteins (TARPs), which mediate binding of AMPA-type glutamate receptors to PSD-95, was increased in young mice. Here we show that only females and not males show a highly significant correlation between an increase in TARP and a decrease in synGAP in the PSDs of rodents. The data reveal a sex difference in the adaptation of the PSD scaffold to synGAP haploinsufficiency.

}, issn = {2050-084X}, doi = {10.7554/eLife.52656}, author = {Mastro, Tara L and Preza, Anthony and Basu, Shinjini and Chattarji, Sumantra and Till, Sally M and Kind, Peter C and Kennedy, Mary B} } @article {2111, title = {Stress-induced modulation of endocannabinoid signaling leads to delayed strengthening of synaptic connectivity in the amygdala.}, journal = {Proc Natl Acad Sci U S A}, volume = {117}, year = {2020}, month = {2020 01 07}, pages = {650-655}, abstract = {

Even a brief exposure to severe stress strengthens synaptic connectivity days later in the amygdala, a brain area implicated in the affective symptoms of stress-related psychiatric disorders. However, little is known about the synaptic signaling mechanisms during stress that eventually culminate in its delayed impact on the amygdala. Hence, we investigated early stress-induced changes in amygdalar synaptic signaling in order to prevent its delayed effects. Whole-cell recordings in basolateral amygdala (BLA) slices from rats revealed higher frequency of miniature excitatory postsynaptic currents (mEPSCs) immediately after 2-h immobilization stress. This was replicated by inhibition of cannabinoid receptors (CBR), suggesting a role for endocannabinoid (eCB) signaling. Stress also reduced -arachidonoylethanolamine (AEA), an endogenous ligand of CBR. Since stress-induced activation of fatty acid amide hydrolase (FAAH) reduces AEA, we confirmed that oral administration of an FAAH inhibitor during stress prevents the increase in synaptic excitation in the BLA soon after stress. Although stress also caused an immediate reduction in synaptic inhibition, this was not prevented by FAAH inhibition. Strikingly, FAAH inhibition during the traumatic stressor was also effective 10 d later on the delayed manifestation of synaptic strengthening in BLA neurons, preventing both enhanced mEPSC frequency and increased dendritic spine-density. Thus, oral administration of an FAAH inhibitor during a brief stress prevents the early synaptic changes that eventually build up to hyperexcitability in the amygdala. This framework is of therapeutic relevance because of growing interest in targeting eCB signaling to prevent the gradual development of emotional symptoms and underlying amygdalar dysfunction triggered by traumatic stress.

}, keywords = {Administration, Oral, Amidohydrolases, Animals, Basolateral Nuclear Complex, Cannabinoid Receptor Antagonists, Disease Models, Animal, Emotions, Endocannabinoids, Enzyme Inhibitors, Excitatory Postsynaptic Potentials, Humans, Male, Rats, Receptor, Cannabinoid, CB1, Signal Transduction, Stress, Psychological}, issn = {1091-6490}, doi = {10.1073/pnas.1910322116}, author = {Yasmin, Farhana and Colangeli, Roberto and Morena, Maria and Filipski, Sarah and van der Stelt, Mario and Pittman, Quentin J and Hillard, Cecilia J and Teskey, G Campbell and McEwen, Bruce S and Hill, Matthew N and Chattarji, Sumantra} } @article {2064, title = {Temporal specificity and heterogeneity of Drosophila immune cells.}, journal = {EMBO J}, year = {2020}, month = {2020 Mar 12}, pages = {e104486}, abstract = {

Immune cells provide defense against non-self and have recently been shown to also play key roles in diverse processes such as development, metabolism, and tumor progression. The heterogeneity of Drosophila immune cells (hemocytes) remains an open question. Using bulk RNA sequencing, we find that the hemocytes display distinct features in the embryo, a closed and rapidly developing system, compared to the larva, which is exposed to environmental and metabolic challenges. Through single-cell RNA sequencing, we identify fourteen hemocyte clusters present in unchallenged larvae and associated with distinct processes, e.g., proliferation, phagocytosis, metabolic homeostasis, and humoral response. Finally, we characterize the changes occurring in the hemocyte clusters upon wasp infestation, which triggers the differentiation of a novel hemocyte type, the lamellocyte. This first molecular atlas of hemocytes provides insights and paves the way to study the biology of the Drosophila immune cells in physiological and pathological conditions.

}, issn = {1460-2075}, doi = {10.15252/embj.2020104486}, author = {Cattenoz, Pierre B and Sakr, Rosy and Pavlidaki, Alexia and Delaporte, Claude and Riba, Andrea and Molina, Nacho and Hariharan, Nivedita and Mukherjee, Tina and Giangrande, Angela} } @article {2039, title = {Temporal specificity and heterogeneity of the fly immune cells{\textquoteright} transcriptional landscape}, journal = {The EMBO Journal (in press)}, year = {2020}, abstract = {

Immune cells provide defense against the non-self, however recent data suggest roles well beyond innate immunity, in processes as diverse as development, metabolism and tumor progression. Nevertheless, the heterogeneity of these cells remains an open question. Using bulk RNA sequencing we find that the Drosophila immune cells (hemocytes) display distinct features in the embryo, a closed and rapidly developing system, compared to the larva, which is exposed to environmental and metabolic challenges. Through single cell RNA sequencing we identify fourteen hemocyte clusters present in unchallenged larvae and associated with distinct cellular processes e.g. proliferation, phagocytosis, metabolic homeostasis and humoral response. Finally, we characterize the changes occurring in the hemocyte clusters upon wasp infestation that triggers the differentiation of a novel cell type, the lamellocyte. This first molecular atlas provides precious insights and paves the way to study the biology of the Drosophila immune cells in physiological and pathological conditions.

}, author = {Cattenoz, Pierre B. and Sakr, Rosy and Pavlidaki, Alexia and Delaporte, Claude and Riba, Andrea and Molina, Nacho and Hariharan, Nivedita and Mukherjee, Tina and Giangrande, Angela} } @article {2210, title = {VEGFA Promoter Polymorphisms rs699947 and rs35569394 Are Associated With the Risk of Anterior Cruciate Ligament Ruptures Among Indian Athletes: A Cross-sectional Study.}, journal = {Orthop J Sports Med}, volume = {8}, year = {2020}, month = {2020 Dec}, pages = {2325967120964472}, abstract = {

Background: Associations of genetic variants within certain fibril-forming genes have previously been observed with anterior cruciate ligament (ACL) injuries. Evidence suggests a significant role of angiogenesis-associated cytokines in remodeling the ligament fibril matrix after mechanical loading and maintaining structural and functional integrity of the ligament. Functional polymorphisms within the vascular endothelial growth factor A (VEGFA) gene have emerged as plausible candidates owing to their role in the regulation of angiogenic responses.

Hypothesis: VEGFA promoter polymorphisms rs699947 and rs35569394 are associated with ACL injury risk among athletes.

Study Design: Cross-sectional study; Level of evidence, 3.

Methods: A total of 90 Indian athletes with radiologically confirmed or surgically proven isolated ACL tears and 76 matched-control athletes were selected for the present cross-sectional genetic association study. Oral mouthwash samples were collected from all the case and control athletes and genotyped for VEGFA rs699947 and rs35569394 using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method.

Results: The A allele (rs699947) was significantly overrepresented in the ACL group (C vs A allele: odds ratio [OR], 1.68 [95\% CI, 1.08-2.60]; = .021) (CC vs CA + AA: OR, 2.69 [95\% CI, 1.37-5.26]; = .004). There was a greater frequency of the AA genotype in the ACL group in comparison with the control group (OR, 3.38 [95\% CI, 1.23-9.28]; = .016) when only male athletes were compared. Likewise, there was a greater frequency of the I allele (rs35569394) in the ACL group (D vs I allele: OR, 1.64 [95\% CI, 1.06-2.55]; = .025) (DD vs ID + II: OR, 2.61 [95\% CI, 1.31-5.21]; = .006). The A-I haplotype was overrepresented in the ACL group compared with the control group (OR, 1.68 [95\% CI, 1.08-2.60]; χ = 5.320; = .021), and both the polymorphisms were found to be in complete linkage disequilibrium ( = 0.929; logarithm of the odds score = 63.74; D{\textquoteright} = 1.0). Female athletes did not show any difference in genotype or allele frequency.

Conclusion: This is the first study to investigate the association of VEGFA promoter polymorphisms in ACL tears among Indian athletes. Increased frequencies of the A allele (rs699947) and I allele (rs35569394) were observed in the ACL group. These results suggest that sequence variants in the VEGF gene are associated with ACL injury risk among athletes. Further research with long-term follow-ups measuring VEGF expression levels during recovery is warranted to establish its role in ACL injuries and healing.

}, issn = {2325-9671}, doi = {10.1177/2325967120964472}, author = {Shukla, Manish and Gupta, Rahul and Pandey, Vivek and Rochette, Jacques and Dhandapany, Perundurai S and Tiwari, Pramod Kumar and Amrathlal, Rabbind Singh} } @article {1645, title = {Altered steady state and activity-dependent de novo protein expression in fragile X syndrome.}, journal = {Nat Commun}, volume = {10}, year = {2019}, month = {2019 Apr 12}, pages = {1710}, abstract = {

Whether fragile X mental retardation protein (FMRP) target mRNAs and neuronal activity contributing to elevated basal neuronal protein synthesis in fragile X syndrome (FXS) is unclear. Our proteomic experiments reveal that the de novo translational profile in FXS model mice is altered at steady state and in response to metabotropic glutamate receptor (mGluR) stimulation, but the proteins expressed differ under these conditions. Several altered proteins, including Hexokinase\ 1 and Ras, also are expressed in the blood of\ FXS model mice and pharmacological treatments previously reported to ameliorate phenotypes modify their abundance in blood. In addition, plasma levels of Hexokinase\ 1 and Ras differ between FXS patients and healthy volunteers. Our data suggest that brain-based de novo proteomics in FXS model mice can be used to find altered expression of proteins in blood that could serve as disease-state biomarkers in individuals with FXS.

}, issn = {2041-1723}, doi = {10.1038/s41467-019-09553-8}, author = {Bowling, Heather and Bhattacharya, Aditi and Zhang, Guoan and Alam, Danyal and Lebowitz, Joseph Z and Bohm-Levine, Nathaniel and Lin, Derek and Singha, Priyangvada and Mamcarz, Maggie and Puckett, Rosemary and Zhou, Lili and Aryal, Sameer and Sharp, Kevin and Kirshenbaum, Kent and Berry-Kravis, Elizabeth and Neubert, Thomas A and Klann, Eric} } @article {1604, title = {Derivation of iPSC lines from two patients with familial Alzheimer{\textquoteright}s disease from India.}, journal = {Stem Cell Res}, volume = {34}, year = {2019}, month = {2019 Jan}, pages = {101370}, abstract = {

The current prevalence of diagnosable dementia in India is 1\% of people over 60 years (~3.7 million people), but is estimated to increase significantly, as ~15\% world{\textquoteright}s aged population (\>65 years) would be resident here by 2020 (Shah et al., 2016). While several mutations that pose a familial risk have been identified, the ethnic background may influence disease susceptibility, clinical presentation and treatment response. In this study, we report a detailed characterization of two representative HiPSC lines from a well-characterized dementia cohort from India. Availability of these lines, and associated molecular and clinical information, would be useful in the detailed exploration of the genomic contribution(s) to AD.

}, issn = {1876-7753}, doi = {10.1016/j.scr.2018.101370}, author = {Najar, Ashaq H and Sneha, K M and Ashok, Aparna and Babu, Swathy and Subramaniam, Anand G and Kannan, Ramkrishnan and Viswanath, Biju and Purushottam, Meera and Varghese, Mathew and Parvez, Suhel and Panicker, Mitradas M and Mukherjee, Odity and Jain, Sanjeev} } @article {1982, title = {Differential Regulation of Translation by FMRP Modulates eEF2 Mediated Response on NMDAR Activity.}, journal = {Front Mol Neurosci}, volume = {12}, year = {2019}, month = {2019}, pages = {97}, abstract = {

SYNGAP1, a Synaptic Ras-GTPase activating protein, regulates synapse maturation during a critical developmental window. Heterozygous mutation in () has been shown to cause Intellectual Disability (ID) in children. Recent studies have provided evidence for altered neuronal protein synthesis in a mouse model of . However, the molecular mechanism behind the same is unclear. Here, we report the reduced expression of a known translation regulator, FMRP, during a specific developmental period in mice. Our results demonstrate that FMRP interacts with and regulates the translation of mRNA. We further show reduced translation leads to decreased FMRP level during development in which results in an increase in translation. These developmental changes are reflected in the altered response of eEF2 phosphorylation downstream of NMDA Receptor (NMDAR)-mediated signaling. In this study, we propose a cross-talk between FMRP and SYNGAP1 mediated signaling which can also explain the compensatory effect of impaired signaling observed in mice.

}, issn = {1662-5099}, doi = {10.3389/fnmol.2019.00097}, author = {Paul, Abhik and Nawalpuri, Bharti and Shah, Devanshi and Sateesh, Shruthi and Muddashetty, Ravi S and Clement, James P} } @article {1740, title = {Dynamic expression of tRNA-derived small RNAs define cellular states.}, journal = {EMBO Rep}, volume = {20}, year = {2019}, month = {2019 Jul}, pages = {e47789}, abstract = {

Transfer RNA (tRNA)-derived small RNAs (tsRNAs) have recently emerged as important regulators of protein translation and shown to have diverse biological functions. However, the underlying cellular and molecular mechanisms of tsRNA function in the context of dynamic cell-state transitions remain unclear. Expression analysis of tsRNAs in distinct heterologous cell and tissue models of stem vs. differentiated states revealed a differentiation-dependent enrichment of 5{\textquoteright}-tsRNAs. We report the identification of a set of 5{\textquoteright}-tsRNAs that is upregulated in differentiating mouse embryonic stem cells (mESCs). Notably, interactome studies with differentially enriched 5{\textquoteright}-tsRNAs revealed a switch in their association with "effector" RNPs and "target" mRNAs in different cell states. We demonstrate that specific 5{\textquoteright}-tsRNAs can preferentially interact with the RNA-binding protein, Igf2bp1, in the RA-induced differentiated state. This association influences the transcript stability and thereby translation of the pluripotency-promoting factor, c-Myc, thus providing a mechanistic basis for how 5{\textquoteright}-tsRNAs can modulate stem cell states in mESCs. Together our study highlights the role of 5{\textquoteright}-tsRNAs in defining distinct cell states.

}, issn = {1469-3178}, doi = {10.15252/embr.201947789}, author = {Krishna, Srikar and Yim, Daniel Gr and Lakshmanan, Vairavan and Tirumalai, Varsha and Koh, Judice Ly and Park, Jung Eun and Cheong, Jit Kong and Low, Joo Leng and Lim, Michelle Js and Sze, Siu Kwan and Shivaprasad, Padubidri and Gulyani, Akash and Raghavan, Srikala and Palakodeti, Dasaradhi and DasGupta, Ramanuj} } @article {1603, title = {Enhancement of the gut barrier integrity by a microbial metabolite through the Nrf2 pathway.}, journal = {Nat Commun}, volume = {10}, year = {2019}, month = {2019 01 09}, pages = {89}, abstract = {

The importance of gut microbiota in human health and pathophysiology is undisputable. Despite the abundance of metagenomics data, the functional dynamics of gut microbiota in human health and disease remain elusive. Urolithin A (UroA), a major microbial metabolite derived from polyphenolics of berries and pomegranate fruits displays anti-inflammatory, anti-oxidative, and anti-ageing activities. Here, we show that UroA and its potent synthetic analogue (UAS03) significantly enhance gut barrier function and inhibit unwarranted inflammation. We demonstrate that UroA and UAS03 exert their barrier functions through activation of aryl hydrocarbon receptor (AhR)- nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent pathways to upregulate epithelial tight junction proteins. Importantly, treatment with these compounds attenuated colitis in pre-clinical models by remedying barrier dysfunction in addition to anti-inflammatory activities. Cumulatively, the results highlight how microbial metabolites provide two-pronged beneficial activities at gut epithelium by enhancing barrier functions and reducing inflammation to protect from colonic diseases.

}, keywords = {Animals, Basic Helix-Loop-Helix Transcription Factors, Caco-2 Cells, Coumarins, Epithelial Cells, Gene Expression Regulation, HT29 Cells, Humans, Intestinal Mucosa, Macrophages, Mice, Mice, Inbred C57BL, Mice, Knockout, NF-E2-Related Factor 2, Receptors, Aryl Hydrocarbon, Specific Pathogen-Free Organisms, Tight Junction Proteins}, issn = {2041-1723}, doi = {10.1038/s41467-018-07859-7}, author = {Singh, Rajbir and Chandrashekharappa, Sandeep and Bodduluri, Sobha R and Baby, Becca V and Hegde, Bindu and Kotla, Niranjan G and Hiwale, Ankita A and Saiyed, Taslimarif and Patel, Paresh and Vijay-Kumar, Matam and Langille, Morgan G I and Douglas, Gavin M and Cheng, Xi and Rouchka, Eric C and Waigel, Sabine J and Dryden, Gerald W and Alatassi, Houda and Zhang, Huang-Ge and Haribabu, Bodduluri and Vemula, Praveen K and Jala, Venkatakrishna R} } @article {1607, title = {Exome sequencing in families with severe mental illness identifies novel and rare variants in genes implicated in Mendelian neuropsychiatric syndromes.}, journal = {Psychiatry Clin Neurosci}, volume = {73}, year = {2019}, month = {2019 Jan}, pages = {11-19}, abstract = {

AIM: Severe mental illnesses (SMI), such as bipolar disorder and schizophrenia, are highly heritable, and have a complex pattern of inheritance. Genome-wide association studies detect a part of the heritability, which can be attributed to common genetic variation. Examination of rare variants with next-generation sequencing may add to the understanding of the genetic architecture of SMI.

METHODS: We analyzed 32 ill subjects from eight multiplex families and 33 healthy individuals using whole-exome sequencing. Prioritized variants were selected by a three-step filtering process, which included: deleteriousness by five in silico algorithms; sharing within families by affected individuals; rarity in South Asian sample estimated using the Exome Aggregation Consortium data; and complete absence of these variants in control individuals from the same gene pool.

RESULTS: We identified 42 rare, non-synonymous deleterious variants (~5 per pedigree) in this study. None of the variants were shared across families, indicating a {\textquoteright}private{\textquoteright} mutational profile. Twenty (47.6\%) of the variant harboring genes were previously reported to contribute to the risk of diverse neuropsychiatric syndromes, nine (21.4\%) of which were of Mendelian inheritance. These included genes carrying novel deleterious variants, such as the GRM1 gene implicated in spinocerebellar ataxia 44 and the NIPBL gene implicated in Cornelia de Lange syndrome.

CONCLUSION: Next-generation sequencing approaches in family-based studies are useful to identify novel and rare variants in genes for complex disorders like SMI. The findings of the study suggest a potential phenotypic burden of rare variants in Mendelian disease genes, indicating pleiotropic effects in the etiology of SMI.

}, keywords = {Bipolar Disorder, Exome, Female, Genetic Predisposition to Disease, Genetic Variation, Genome-Wide Association Study, Humans, Male, Pedigree, Phenotype, Schizophrenia}, issn = {1440-1819}, doi = {10.1111/pcn.12788}, author = {Ganesh, Suhas and Ahmed P, Husayn and Nadella, Ravi K and More, Ravi P and Seshadri, Manasa and Viswanath, Biju and Rao, Mahendra and Jain, Sanjeev and Mukherjee, Odity} } @article {1841, title = {Generation of a FMR1 homozygous knockout human embryonic stem cell line (WAe009-A-16) by CRISPR/Cas9 editing.}, journal = {Stem Cell Res}, volume = {39}, year = {2019}, month = {2019 Aug}, pages = {101494}, abstract = {

Mutations in FMR1 gene is the cause of Fragile X Syndrome (FXS) leading inherited cause of intellectual disability and autism spectrum disorders. FMR1 gene encodes Fragile X Mental Retardation Protein (FMRP) which is a RNA binding protein and play important role in synaptic plasticity and translational regulation in neurons. We have generated a homozygous FMR1 knockout (FMR1-KO) hESC line using CRISPR/Cas9 based genome editing. It created a homozygous 280 nucleotide deletion at exon1, removing the start codon. This FMR1-KO cell line maintains stem cell like morphology, pluripotency, normal karyotype and ability to in-vitro differentiation.

}, issn = {1876-7753}, doi = {10.1016/j.scr.2019.101494}, author = {Giri, Subhajit and Purushottam, Meera and Viswanath, Biju and Muddashetty, Ravi S} } @article {1602, title = {Generation of a set of isogenic, gene-edited iPSC lines homozygous for all main APOE variants and an APOE knock-out line.}, journal = {Stem Cell Res}, volume = {34}, year = {2019}, month = {2019 Jan}, pages = {101349}, abstract = {

Alzheimer{\textquoteright}s disease (AD) is the most frequent neurodegenerative disease amongst the elderly. The SNPs rs429358 and rs7412 in the APOE gene are the most common risk factor for sporadic AD, and there are three different alleles commonly referred to as APOE-ε2, APOE-ε3 and APOE-ε4. Induced pluripotent stem cells (iPSCs) hold great promise to model AD as such cells can be differentiated in vitro to the required cell type. Here we report the use of CRISPR/Cas9 technology employed on iPSCs from a healthy individual with an APOE-ε3/ε4 genotype to obtain isogenic APOE-ε2/ε2, APOE-ε3/ε3, APOE-ε4/ε4 lines as well as an APOE-knock-out line.

}, issn = {1876-7753}, doi = {10.1016/j.scr.2018.11.010}, author = {Schmid, Benjamin and Prehn, Kennie R and Nimsanor, Natakarn and Garcia, Blanca Irene Aldana and Poulsen, Ulla and J{\o}rring, Ida and Rasmussen, Mikkel A and Clausen, Christian and Mau-Holzmann, Ulrike A and Ramakrishna, Sarayu and Muddashetty, Ravi and Steeg, Rachel and Bruce, Kevin and Mackintosh, Peter and Ebneth, Andreas and Holst, Bj{\o}rn and Cabrera-Socorro, Alfredo} } @article {1613, title = {INDEX-db: The Indian Exome Reference Database (Phase I).}, journal = {J Comput Biol}, volume = {26}, year = {2019}, month = {2019 Mar}, pages = {225-234}, abstract = {

Deep sequencing-based genetic mapping has greatly enhanced the ability to catalog variants with plausible disease association. Confirming how these identified variants contribute to specific disease conditions, across human populations, poses the next challenge. Differential selection pressure may impact the frequency of genetic variations, and thus detection of association with disease conditions, across populations. To understand genotype to phenotype correlations, it thus becomes important to first understand the spectrum of genetic variation within a population by creating a reference map. In this study, we report the development of phase I of a new database of genetic variations called INDian EXome database (INDEX-db), from the Indian population, with an aim to establish a centralized database of integrated information. This could be useful for researchers involved in studying disease mechanisms at clinical, genetic, and cellular levels.

}, issn = {1557-8666}, doi = {10.1089/cmb.2018.0199}, author = {Ahmed P, Husayn and V, Vidhya and More, Ravi Prabhakar and Viswanath, Biju and Jain, Sanjeev and Rao, Mahendra S and Mukherjee, Odity} } @article {1578, title = {Interactions Between Epidermal Keratinocytes, Dendritic Epidermal T-Cells, and Hair Follicle Stem Cells.}, journal = {Methods Mol Biol}, volume = {1879}, year = {2019}, month = {2019}, pages = {285-297}, abstract = {

The interplay of immune cells and stem cells in maintaining skin homeostasis and repair is an exciting new frontier in cutaneous biology. With the growing appreciation of the importance of this new crosstalk comes the requirement of methods to interrogate the molecular underpinnings of these leukocyte-stem cell interactions. Here we describe how a combination of FACS, cellular coculture assays, and conditioned media treatments can be utilized to advance our understanding of this emerging area of intercellular communication between immune cells and stem cells.

}, issn = {1940-6029}, doi = {10.1007/7651_2018_155}, author = {Badarinath, Krithika and Dutta, Abhik and Hegde, Akshay and Pincha, Neha and Gund, Rupali and Jamora, Colin} } @article {1576, title = {KMT1 family methyltransferases regulate heterochromatin-nuclear periphery tethering via histone and non-histone protein methylation.}, journal = {EMBO Rep}, year = {2019}, month = {2019 Mar 11}, abstract = {

Euchromatic histone methyltransferases (EHMTs), members of the KMT1 family, methylate histone and non-histone proteins. Here, we uncover a novel role for EHMTs in regulating heterochromatin anchorage to the nuclear periphery (NP) via non-histone methylation. We show that EHMTs methylate and stabilize LaminB1 (LMNB1), which associates with the H3K9me2-marked peripheral heterochromatin. Loss of LMNB1 methylation or EHMTs abrogates heterochromatin anchorage at the NP We further demonstrate that the loss of EHMTs induces many hallmarks of aging including global reduction of H3K27methyl marks and altered nuclear morphology. Consistent with this, we observe a gradual depletion of EHMTs, which correlates with loss of methylated LMNB1 and peripheral heterochromatin in aging human fibroblasts. Restoration of EHMT expression reverts peripheral heterochromatin defects in aged cells. Collectively, our work elucidates a new mechanism by which EHMTs regulate heterochromatin domain organization and reveals their impact on fundamental changes associated with the intrinsic aging process.

}, issn = {1469-3178}, doi = {10.15252/embr.201643260}, author = {Rao, Radhika Arasala and Ketkar, Alhad Ashok and Kedia, Neelam and Krishnamoorthy, Vignesh K and Lakshmanan, Vairavan and Kumar, Pankaj and Mohanty, Abhishek and Kumar, Shilpa Dilip and Raja, Sufi O and Gulyani, Akash and Chaturvedi, Chandra Prakash and Brand, Marjorie and Palakodeti, Dasaradhi and Rampalli, Shravanti} } @article {1644, title = {Myocardin ablation in a cardiac-renal rat model.}, journal = {Sci Rep}, volume = {9}, year = {2019}, month = {2019 Apr 10}, pages = {5872}, abstract = {

Cardiorenal syndrome is defined by primary heart failure conditions influencing or leading to renal injury or dysfunction. Dilated cardiomyopathy (DCM) is a major co-existing form of heart failure (HF) with renal diseases. Myocardin (MYOCD), a cardiac-specific co-activator of serum response factor (SRF), is increased in DCM porcine and patient cardiac tissues and plays a crucial role in the pathophysiology of DCM. Inhibiting the increased MYOCD has shown to be partially rescuing the DCM phenotype in porcine model. However, expression levels of MYOCD in the cardiac tissues of the cardiorenal syndromic patients and the effect of inhibiting MYOCD in a cardiorenal syndrome model remains to be explored. Here, we analyzed the expression levels of MYOCD in the DCM patients with and without renal diseases. We also explored, whether cardiac specific silencing of MYOCD expression could ameliorate the cardiac remodeling and improve cardiac function in a renal artery ligated rat model (RAL). We observed an increase in MYOCD levels in the endomyocardial biopsies of DCM patients associated with renal failure compared to DCM alone. Silencing of MYOCD in RAL rats by a cardiac homing peptide conjugated MYOCD siRNA resulted in attenuation of cardiac hypertrophy, fibrosis and restoration of the left ventricular functions. Our data suggest hyper-activation of MYOCD in the pathogenesis of the cardiorenal failure cases. Also, MYOCD silencing showed beneficial effects by rescuing cardiac hypertrophy, fibrosis, size and function in a cardiorenal rat model.

}, issn = {2045-2322}, doi = {10.1038/s41598-019-42009-z}, author = {Mittal, Anupam and Rana, Santanu and Sharma, Rajni and Kumar, Akhilesh and Prasad, Rishikesh and Raut, Satish K and Sarkar, Sagartirtha and Saikia, Uma Nahar and Bahl, Ajay and Dhandapany, Perundurai S and Khullar, Madhu} } @article {1617, title = {Natural monomers: A mine for functional and sustainable materials {\textendash} Occurrence, chemical modification and polymerization}, journal = {Progress in Polymer Science}, volume = {92}, year = {2019}, abstract = {

Owing to the natural abundance, variety of structural features, and highly specific functions, natural monomers render themselves as potential candidates for production of high performance functional polymers. The emerging concept of the biorefinery and development of new biosynthetic routes to synthesize a versatile and broad spectrum of natural monomers and polymers continues to gain momentum. The production of high quality polymers from renewable feedstocks requires innovative chemical modifications and catalytic transformations to achieve higher yields in an efficient manner. A fresh look into monomers available from natural resources such as terpenes, rosin, glycerol, furans, tannins, suberin, their derivatives and miscellaneous monomers may inspire future applications with impactful biobased materials. There are also many areas that require urgent discussion and review pertaining to recent developments in the field; this includes monomer sources that give molecules having special structural features. In particular, cardanol, a naturally occurring low-molecular-weight compound is unique as it contains a phenolic head group and a hydrocarbon chain with different degrees of unsaturation. This molecule possesses functional groups that are amenable for classical chemical modification, which is instrumental in developing a wide range of functional monomers and polymers. A large number of soft and hard materials have been developed from cardanol-based monomers. During the past, a large number of industrial grade materials have been developed from plant-based monomers, including development from microbial and fermentation processes (i.e. lactic acid). This review provides a comprehensive study and survey on recent developments on monomers and polymers derived from urushiol and cardanol based monomers and polymers, vegetable oil-based monomers and polymers, microbially produced monomers and polymers. These all represent emerging fronts giving a vast scope while highlighting important potential material and reagent opportunities.

}, url = {https://www.sciencedirect.com/science/article/pii/S0079670018300273}, author = {John, G., and Nagarajan, S., and Vemula, P. K., and Silverman, J.R., and Pillai, C. K. S.} } @article {1614, title = {Post-transcriptional regulation in planarian stem cells.}, journal = {Semin Cell Dev Biol}, volume = {87}, year = {2019}, month = {2019 Mar}, pages = {69-78}, abstract = {

Planarians are known for their immense regenerative abilities. A pluripotent stem cell population provides the cellular source for this process, as well as for the homeostatic cell turnover of the animals. These stem cells, known as neoblasts, present striking similarities at the morphological and molecular level to germ cells, but however, give rise to somatic tissue. Many RNA binding proteins known to be important for germ cell biology are also required for neoblast function, highlighting the importance of post-transcriptional regulation for stem cell control. Many of its aspects, including alternative splicing, alternative polyadenylation, translational control and mRNA deadenylation, as well as small RNAs such as microRNAs and piRNA are critical for stem cells. Their inhibition often abrogates both regeneration and cell turnover, resulting in lethality. Some of aspects of post-transcriptional regulation are conserved from planarian to mammalian stem cells.

}, issn = {1096-3634}, doi = {10.1016/j.semcdb.2018.05.013}, author = {Krishna, Srikar and Palakodeti, Dasaradhi and Solana, Jordi} } @article {1842, title = {Rodent models of social stress and neuronal plasticity: Relevance to depressive-like disorders.}, journal = {Behav Brain Res}, volume = {369}, year = {2019}, month = {2019 Sep 02}, pages = {111900}, abstract = {

Exposure to severe or persistent social stress may lead to the development of psychiatric disorders such as anxiety and depression. These mood disorders are associated with structural alterations of neural architecture in limbic brain regions that control emotion, mood and cognition. Structural remodeling may either be a sign of successful adaptation, or of failure to do so. In neuropsychiatric disorders like depression structural remodeling involves apoptosis, reduced neurogenesis, and structural remodeling of neuronal dendrites which most likely reflects the latter. Here we review key findings from animal models of psychosocial stress that have been used to gain insights into the relation between stress-related behavioral disorders like depression and structural plasticity. Specifically, we focus on models having a high face validity like social defeat stress in the resident-intruder paradigm and chronic stress of social subordination in social housing conditions. Moderate to severe social stress appears to stimulate plasticity and neuronal growth in regions of the amygdala, whereas the effects in the hippocampus and prefrontal cortex tend to be opposite. A major focus of the current review is to characterize social stress induced structural changes in these brain regions, aiming to provide insight in pathways and factors that underlie behavioral effects of stress and depression.

}, issn = {1872-7549}, doi = {10.1016/j.bbr.2019.111900}, author = {Patel, Deepika and Kas, Martien J and Chattarji, Sumantra and Buwalda, Bauke} } @article {1844, title = {Serotonin is essential for eye regeneration in planaria Schmidtea mediterranea.}, journal = {FEBS Lett}, year = {2019}, month = {2019 Sep 17}, abstract = {

Planaria is an ideal system to study factors involved in regeneration and tissue homeostasis. Little is known about the role of metabolites and small molecules in stem cell maintenance and lineage specification in planarians. Using liquid chromatography and mass spectrometry (LC-MS)-based quantitative metabolomics, we determined the relative levels of metabolites in stem cells, progenitors, and differentiated cells of the planarian Schmidtea mediterranea. Tryptophan and its metabolic product serotonin are significantly enriched in stem cells and progenitor population. Serotonin biosynthesis in these cells is brought about by a non-canonical enzyme, phenylalanine hydroxylase (PAH). Knockdown of Smed-pah leads to complete disappearance of eyes in regenerating planaria, while exogenous supply of serotonin and its precursor rescue the eyeless phenotype. Our results demonstrate a key role for serotonin in eye regeneration.

}, issn = {1873-3468}, doi = {10.1002/1873-3468.13607}, author = {Sarkar, Arunabha and Mukundan, Namita and Sowndarya, Sai and Dubey, Vinay Kumar and Babu, Rosana and Lakshmanan, Vairavan and Rangiah, Kannan and Panicker, Mitradas M and Palakodeti, Dasaradhi and Subramanian, Sabarinath Peruvemba and Ramaswamy, Subramanian} } @article {1981, title = {Stromal cells downregulate miR-23a-5p to activate protective autophagy in acute myeloid leukemia.}, journal = {Cell Death Dis}, volume = {10}, year = {2019}, month = {2019 Sep 30}, pages = {736}, abstract = {

Complex molecular cross talk between stromal cells and the leukemic cells in bone marrow is known to contribute significantly towards drug-resistance. Here, we have identified the molecular events that lead to stromal cells mediated therapy-resistance in acute myeloid leukemia (AML). Our work demonstrates that stromal cells downregulate miR-23a-5p levels in leukemic cells to protect them from the chemotherapy induced apoptosis. Downregulation of miR-23a-5p in leukemic cells leads to upregulation of protective autophagy by targeting TLR2 expression. Further, autophagy inhibitors when used as adjuvants along with conventional drugs can improve drug sensitivity in vitro as well in vivo in a mouse model of leukemia. Our work also demonstrates that this mechanism of bone marrow stromal cell mediated regulation of miR-23a-5p levels and subsequent molecular events are relevant predominantly in myeloid leukemia. Our results illustrate the critical and dynamic role of the bone marrow microenvironment in modulating miRNA expression in leukemic cells which could contribute significantly to drug resistance and subsequent relapse, possibly through persistence of minimal residual disease in this environment.

}, issn = {2041-4889}, doi = {10.1038/s41419-019-1964-8}, author = {Ganesan, Saravanan and Palani, Hamenth Kumar and Lakshmanan, Vairavan and Balasundaram, Nithya and Alex, Ansu Abu and David, Sachin and Venkatraman, Arvind and Korula, Anu and George, Biju and Balasubramanian, Poonkuzhali and Palakodeti, Dasaradhi and Vyas, Neha and Mathews, Vikram} } @article {1983, title = {Structural and functional characterization of CMP-N-acetylneuraminate synthetase from Vibrio cholerae.}, journal = {Acta Crystallogr D Struct Biol}, volume = {75}, year = {2019}, month = {2019 Jun 01}, pages = {564-577}, abstract = {

Several pathogenic bacteria utilize sialic acid, including host-derived N-acetylneuraminic acid (Neu5Ac), in at least two ways: they use it as a nutrient source and as a host-evasion strategy by coating themselves with Neu5Ac. Given the significant role of sialic acid in pathogenesis and host-gut colonization by various pathogenic bacteria, including Neisseria meningitidis, Haemophilus influenzae, Pasteurella multocida and Vibrio cholerae, several enzymes of the sialic acid catabolic, biosynthetic and incorporation pathways are considered to be potential drug targets. In this work, findings on the structural and functional characterization of CMP-N-acetylneuraminate synthetase (CMAS), a key enzyme in the incorporation pathway, from Vibrio cholerae are reported. CMAS catalyzes the synthesis of CMP-sialic acid by utilizing CTP and sialic acid. Crystal structures of the apo and the CDP-bound forms of the enzyme were determined, which allowed the identification of the metal cofactor Mg in the active site interacting with CDP and the invariant Asp215 residue. While open and closed structural forms of the enzyme from eukaryotic and other bacterial species have already been characterized, a partially closed structure of V.\ cholerae CMAS (VcCMAS) observed upon CDP binding, representing an intermediate state, is reported here. The kinetic data suggest that VcCMAS is capable of activating the two most common sialic acid derivatives, Neu5Ac and Neu5Gc. Amino-acid sequence and structural comparison of the active site of VcCMAS with those of eukaryotic and other bacterial counterparts reveal a diverse hydrophobic pocket that interacts with the C5 substituents of sialic acid. Analyses of the thermodynamic signatures obtained from the binding of the nucleotide (CTP) and the product (CMP-sialic acid) to VcCMAS provide fundamental information on the energetics of the binding process.

}, keywords = {Amino Acid Sequence, Bacterial Proteins, Binding Sites, Catalytic Domain, Crystallization, Crystallography, X-Ray, Cytidine Diphosphate, Cytidine Monophosphate N-Acetylneuraminic Acid, Cytidine Triphosphate, N-Acylneuraminate Cytidylyltransferase, Protein Interaction Domains and Motifs, Protein Structure, Quaternary, Sialic Acids, Vibrio cholerae}, issn = {2059-7983}, doi = {10.1107/S2059798319006831}, author = {Bose, Sucharita and Purkait, Debayan and Joseph, Deepthi and Nayak, Vinod and Subramanian, Ramaswamy} } @article {1840, title = {Structure-Guided Synthesis and Evaluation of Small-Molecule Inhibitors Targeting Protein-Protein Interactions of BRCA1 tBRCT Domain.}, journal = {ChemMedChem}, volume = {14}, year = {2019}, month = {2019 Sep 18}, pages = {1620-1632}, abstract = {

The tandem BRCT domains (tBRCT) of BRCA1 engage phosphoserine-containing motifs in target proteins to propagate intracellular signals initiated by DNA damage, thereby controlling cell cycle arrest and DNA repair. Recently, we identified Bractoppin, the first small-molecule inhibitor of the BRCA1 tBRCT domain, which selectively interrupts BRCA1-mediated cellular responses evoked by DNA damage. Here, we combine structure-guided chemical elaboration, protein mutagenesis and cellular assays to define the structural features responsible for Bractoppin{\textquoteright}s activity. Bractoppin fails to bind mutant forms of BRCA1 tBRCT bearing K1702A, a key residue mediating phosphopeptide recognition, or F1662R or L1701K that adjoin the pSer-recognition site. However, the M1775R mutation, which engages the Phe residue in the consensus phosphopeptide motif pSer-X-X-Phe, does not affect Bractoppin binding, confirming a binding mode distinct from the substrate phosphopeptide binding. We explored these structural features through structure-guided chemical elaboration and characterized structure-activity relationships (SARs) in biochemical assays. Two analogues, CCBT2088 and CCBT2103 were effective in abrogating BRCA1 foci formation and inhibiting G2 arrest induced by irradiation of cells. Collectively, our findings reveal structural features underlying the activity of a novel inhibitor of phosphopeptide recognition by the BRCA1 tBRCT domain, providing fresh insights to guide the development of inhibitors that target protein-protein interactions.

}, issn = {1860-7187}, doi = {10.1002/cmdc.201900300}, author = {Kurdekar, Vadiraj and Giridharan, Saranya and Subbarao, Jasti and Nijaguna, Mamatha B and Periasamy, Jayaprakash and Boggaram, Sanjana and Shivange, Amol V and Sadasivam, Gayathri and Padigaru, Muralidhara and Potluri, Vijay and Venkitaraman, Ashok R and Bharatham, Kavitha} } @article {1587, title = {Bioresponsive drug delivery systems in intestinal inflammation: State-of-the-art and future perspectives.}, journal = {Adv Drug Deliv Rev}, year = {2018}, month = {2018 Jun 29}, abstract = {

Oral colon-specific delivery systems emerged as the main therapeutic cargos by making a significant impact in the field of modern medicine for local drug delivery in intestinal inflammation. The site-specific delivery of therapeutics (aminosalicylates, glucocorticoids, biologics) to the ulcerative mucus tissue can provide prominent advantages in mucosal healing (MH). Attaining gut mucosal healing and anti-fibrosis are main treatment outcomes in inflammatory bowel disease (IBD). The pharmaceutical strategies that are commonly used to achieve a colon-specific drug delivery system include time, pH-dependent polymer coating, prodrug, colonic microbiota-activated delivery systems and a combination of these approaches. Amongst the different approaches reported, the use of biodegradable polysaccharide coated systems holds great promise in delivering drugs to the ulcerative regions. The present review focuses on major physiological gastro-intestinal tract challenges involved in altering the pharmacokinetics of delivery systems, pathophysiology of MH and fibrosis, reported drug-polysaccharide cargos and focusing on conventional to advanced disease responsive delivery strategies, highlighting their limitations and future perspectives in intestinal inflammation therapy.

}, issn = {1872-8294}, doi = {10.1016/j.addr.2018.06.021}, author = {Kotla, Niranjan G and Rana, Shubhasmin and Sivaraman, Gandhi and Sunnapu, Omprakash and Vemula, Praveen K and Pandit, Abhay and Rochev, Yury} } @article {1149, title = {Discovery biology of neuropsychiatric syndromes (DBNS): a center for integrating clinical medicine and basic science.}, journal = {BMC Psychiatry}, volume = {18}, year = {2018}, month = {2018 Apr 18}, pages = {106}, abstract = {

BACKGROUND: There is emerging evidence that there are shared genetic, environmental and developmental risk factors in psychiatry, that cut across traditional diagnostic boundaries. With this background, the Discovery biology of neuropsychiatric syndromes (DBNS) proposes to recruit patients from five different syndromes (schizophrenia, bipolar disorder, obsessive compulsive disorder, Alzheimer{\textquoteright}s dementia and substance use disorders), identify those with multiple affected relatives, and invite these families to participate in this study. The families will be assessed: 1) To compare neuro-endophenotype measures between patients, first degree relatives (FDR) and healthy controls., 2) To identify cellular phenotypes which differentiate the groups., 3) To examine the longitudinal course of neuro-endophenotype measures., 4) To identify measures which correlate with outcome, and 5) To create a unified digital database and biorepository.

METHODS: The identification of the index participants will occur at well-established specialty clinics. The selected individuals will have a strong family history (with at least another affected FDR) of mental illness. We will also recruit healthy controls without family history of such illness. All recruited individuals (N = 4500) will undergo brief clinical assessments and a blood sample will be drawn for isolation of DNA and peripheral blood mononuclear cells (PBMCs). From among this set, a subset of 1500 individuals (300 families and 300 controls) will be assessed on several additional assessments [detailed clinical assessments, endophenotype measures (neuroimaging- structural and functional, neuropsychology, psychophysics-electroencephalography, functional near infrared spectroscopy, eye movement tracking)], with the intention of conducting repeated measurements every alternate year. PBMCs from this set will be used to generate lymphoblastoid cell lines, and a subset of these would be converted to induced pluripotent stem cell lines and also undergo whole exome sequencing.

DISCUSSION: We hope to identify unique and overlapping brain endophenotypes for major psychiatric syndromes. In a proportion of subjects, we expect these neuro-endophenotypes to progress over time and to predict treatment outcome. Similarly, cellular assays could differentiate cell lines derived from such groups. The repository of biomaterials as well as digital datasets of clinical parameters, will serve as a valuable resource for the broader scientific community who wish to address research questions in the area.

}, issn = {1471-244X}, doi = {10.1186/s12888-018-1674-2}, author = {Viswanath, Biju and Rao, Naren P and Narayanaswamy, Janardhanan C and Sivakumar, Palanimuthu T and Kandasamy, Arun and Kesavan, Muralidharan and Mehta, Urvakhsh Meherwan and Venkatasubramanian, Ganesan and John, John P and Mukherjee, Odity and Purushottam, Meera and Kannan, Ramakrishnan and Mehta, Bhupesh and Kandavel, Thennarasu and Binukumar, B and Saini, Jitender and Jayarajan, Deepak and Shyamsundar, A and Moirangthem, Sydney and Vijay Kumar, K G and Thirthalli, Jagadisha and Chandra, Prabha S and Gangadhar, Bangalore N and Murthy, Pratima and Panicker, Mitradas M and Bhalla, Upinder S and Chattarji, Sumantra and Benegal, Vivek and Varghese, Mathew and Reddy, Janardhan Y C and Raghu, Padinjat and Rao, Mahendra and Jain, Sanjeev} } @article {1598, title = {FMRP Interacts with C/D Box snoRNA in the Nucleus and Regulates Ribosomal RNA Methylation.}, journal = {iScience}, volume = {9}, year = {2018}, month = {2018 Nov 30}, pages = {399-411}, abstract = {

FMRP is an RNA-binding protein that is known to localize in the cytoplasm and in the nucleus. Here, we have identified an interaction of FMRP with a specific set of C/D box snoRNAs in the nucleus. C/D box snoRNAs guide 2{\textquoteright}O methylations of ribosomal RNA (rRNA) on defined sites, and this modification regulates rRNA folding and assembly of ribosomes. 2{\textquoteright}O methylation of rRNA is partial on several sites in human embryonic stem cells, which results in ribosomes with differential methylation patterns. FMRP-snoRNA interaction affects rRNA methylation on several of these sites, and in the absence of FMRP, differential methylation pattern of rRNA is significantly altered. We found that FMRP recognizes ribosomes carrying specific methylation patterns on rRNA and the recognition of methylation pattern by FMRP may potentially determine the translation status of its target mRNAs. Thus, FMRP integrates its function in the nucleus and in the cytoplasm.

}, issn = {2589-0042}, doi = {10.1016/j.isci.2018.11.007}, author = {D{\textquoteright}Souza, Michelle Ninochka and Gowda, Naveen Kumar Chandappa and Tiwari, Vishal and Babu, Rosana Ottakandathil and Anand, Praveen and Dastidar, Sudhriti Ghosh and Singh, Randhir and James, Owen G and Selvaraj, Bhuvaneish and Pal, Rakhi and Ramesh, Arati and Chattarji, Sumantra and Chandran, Siddharthan and Gulyani, Akash and Palakodeti, Dasaradhi and Muddashetty, Ravi S} } @article {1153, title = {Identification of multiple isomeric core chitobiose-modified high-mannose and paucimannose -glycans in the planarian .}, journal = {J Biol Chem}, volume = {293}, year = {2018}, month = {2018 May 04}, pages = {6707-6720}, abstract = {

Cell surface-associated glycans mediate many cellular processes, including adhesion, migration, signaling, and extracellular matrix organization. The galactosylation of core fucose (GalFuc epitope) in paucimannose and complex-type -glycans is characteristic of protostome organisms, including flatworms (planarians). Although uninvestigated, the structures of these glycans may play a role in planarian regeneration. Whole-organism MALDI-MS analysis of -linked oligosaccharides from the planarian revealed the presence of multiple isomeric high-mannose and paucimannose structures with unusual mono-, di-, and polygalactosylated ( = 3-5) core fucose structures; the latter structures have not been reported in other systems. Di- and trigalactosylated core fucoses were the most dominant glycomers. -Glycans showed extensive, yet selective, methylation patterns, ranging from non-methylated to polymethylated glycoforms. Although the majority of glycoforms were polymethylated, a small fraction also consisted of non-methylated glycans. Remarkably, monogalactosylated core fucose remained unmethylated, whereas its polygalactosylated forms were methylated, indicating structurally selective methylation. Using database searches, we identified two potential homologs of the Galβ1-4Fuc-synthesizing enzyme from nematodes (GALT-1) that were expressed in the prepharyngeal, pharyngeal, and mesenchymal regions in The presence of two GALT-1 homologs suggests different requirements for mono- and polygalactosylation of core fucose for the formation of multiple isomers. Furthermore, we observed variations in core fucose glycosylation patterns in different planarian strains, suggesting evolutionary adaptation in fucose glycosylation. The various core chitobiose modifications and methylations create \>60 different glycoforms in These results contribute greatly to our understanding of -glycan biosynthesis and suggest the presence of a GlcNAc-independent biosynthetic pathway in

}, issn = {1083-351X}, doi = {10.1074/jbc.RA117.000782}, author = {Subramanian, Sabarinath Peruvemba and Babu, Ponnusamy and Palakodeti, Dasaradhi and Subramanian, Ramaswamy} } @article {1145, title = {Induction of quiescence (G0) in bone marrow stromal stem cells enhances their stem cell characteristics.}, journal = {Stem Cell Res}, volume = {30}, year = {2018}, month = {2018 May 17}, pages = {69-80}, abstract = {

Several studies have suggested that bone marrow stromal steam cells (BMSC) exist in a quiescent state (G0) within the in vivo niche; however, an explicit analysis of the biology of G0 state-BMSC has not been reported. We hypothesized that induction of G0 in BMSC might enhance their stem cell properties. Thus, we induced quiescence in BMSC in vitro by (a) suspension culture in a viscous medium or (b) culture on soft polyacrylamide substrate; and examined their molecular and functional phenotype. Induction of G0 was confirmed by bromo-deoxyuridine (BrdU) labelling and analysis of cell cycle gene expression. Upon reactivation and re-entry into cell cycle, G0 state-BMSC exhibited enhanced clonogenic self-renewal, preferential differentiation into osteoblastic rather than adipocytic cells and increased ectopic bone formation when implanted subcutaneously in vivo in immune-deficient mice, compared to asynchronous proliferating (pre-G0) BMSC. Global gene expression profiling revealed reprogramming of the transcriptome during G0 state including significant alterations in relevant pathways and expression of secreted factors, suggesting altered autocrine and paracrine signaling by G0 state-BMSC and a possible mechanism for enhanced bone formation. G0 state-BMSC might provide a clinically relevant model for understanding the in vivo biology of BMSC.

}, issn = {1876-7753}, doi = {10.1016/j.scr.2018.05.010}, author = {Rumman, Mohammad and Majumder, Abhijit and Harkness, Linda and Venugopal, Balu and Vinay, M B and Pillai, Malini S and Kassem, Moustapha and Dhawan, Jyotsna} } @article {1581, title = {Infectivity of adeno-associated virus serotypes in mouse testis.}, journal = {BMC Biotechnol}, volume = {18}, year = {2018}, month = {2018 Nov 01}, pages = {70}, abstract = {

BACKGROUND: Recombinant adeno-associated viruses (AAVs) are emerging as favoured transgene delivery vectors for both research applications and gene therapy. In this context, a thorough investigation of the potential of various AAV serotypes to transduce specific cell types is valuable. Here, we rigorously tested the infectivity of a number of AAV serotypes in murine testis by direct testicular injection.

RESULTS: We report the tropism of serotypes AAV2, 5, 8, 9 and AAVrh10 in mouse testis. We reveal unique infectivity of AAV2 and AAV9, which preferentially target intertubular testosterone-producing Leydig cells. Remarkably, AAV2 TM, a mutant for capsid designed to increase transduction, displayed a dramatic alteration in tropism; it infiltrated seminiferous tubules unlike wildtype AAV2 and transduced Sertoli cells. However, none of the AAVs tested infected spermatogonial cells.

CONCLUSIONS: In spite of direct testicular injection, none of the tested AAVs appeared to infect sperm progenitors as assayed by reporter expression. This lends support to the current view that AAVs are safe gene-therapy vehicles. However, testing the presence of rAAV genomic DNA in germ cells is necessary to assess the risk of individual serotypes.

}, issn = {1472-6750}, doi = {10.1186/s12896-018-0479-1}, author = {Rajasekaran, Santhanasabapathy and Thatte, Jayashree and Periasamy, Jayaprakash and Javali, Alok and Jayaram, Manjunath and Sen, Dwaipayan and Krishnagopal, Akshaya and Jayandharan, Giridhara R and Sambasivan, Ramkumar} } @article {1189, title = {Local injections of tacrolimus-loaded hydrogel reduce systemic immunosuppression-related toxicity in vascularized composite allotransplantation.}, journal = {Transplantation}, year = {2018}, month = {2018 May 23}, abstract = {

BACKGROUND: Routine application of vascularized composite allotransplantation (VCA) is hampered by immunosuppression-related health comorbidities. To mitigate these we developed an inflammation-responsive hydrogel for local immunosuppression. Here we report on its long-term effect on graft survival, immunological and toxicological impact.

METHODS: Brown Norway-to-Lewis rat hind limb transplantations were treated either systemically with daily injections of 1 mg/kg tacrolimus or with subcutaneous intragraft injections of hydrogel containing 7 mg tacrolimus, every 70 days. Animals were monitored for rejection or other pathology for 280 days. Systemic and graft tacrolimus levels, regulatory T cells, and donor cell chimerism were measured periodically. At endpoint, markers for kidney, liver and metabolic state were compared to na{\"\i}ve age-matched rats.

RESULTS: Both daily systemic tacrolimus and subcutaneous intragraft tacrolimus hydrogel at 70 day intervals were able to sustain graft survival for \>280 days in 5 out of 6 recipients. In the hydrogel group, 1 graft progressed to grade 3 rejection at postoperative day (POD) 149. In systemic tacrolimus group, 1 animal was euthanized due to lymphoma on POD 275. Hydrogel treatment provided stable graft- and reduced systemic tacrolimus levels, and a 4 times smaller total tacrolimus dose compared with systemic immunosuppression. Hydrogel-treated animals showed preserved kidney function, absence of malignancies or opportunistic infections and increased hematopoietic chimerism compared to systemic immunosuppression.

CONCLUSIONS: Our findings demonstrate that localized immunosuppression with tacrolimus hydrogel is a long-term safe and reliable treatment. It may reduce the burden of systemic immunosuppression in VCA, potentially boosting the clinical application of this surgical intervention.

}, issn = {1534-6080}, doi = {10.1097/TP.0000000000002283}, author = {Dzhonova, Dzhuliya V and Olariu, Radu and Leckenby, Jonathan and Banz, Yara and Prost, Jean-Christophe and Dhayani, Ashish and Vemula, Praveen K and Voegelin, Esther and Taddeo, Adriano and Rieben, Robert} } @article {1592, title = {Local release of tacrolimus from hydrogel-based drug delivery system is controlled by inflammatory enzymes in vivo and can be monitored non-invasively using in vivo imaging.}, journal = {PLoS One}, volume = {13}, year = {2018}, month = {2018}, pages = {e0203409}, abstract = {

BACKGROUND: Local drug delivery systems that adjust the release of immunosuppressive drug in response to the nature and intensity of inflammation represent a promising approach to reduce systemic immunosuppression and its side effects in allotransplantation. Here we aimed to demonstrate that release of tacrolimus from triglycerol monostearate hydrogel is inflammation-dependent in vivo. We further report that by loading the hydrogel with a near-infrared dye, it is possible to monitor drug release non-invasively in an in vivo model of vascularized composite allotransplantation.

MATERIALS AND METHODS: Inflammation was induced by local challenge with lipopolysaccharides in na{\"\i}ve rats 7 days after injection of tacrolimus-loaded hydrogel in the hind limb. Tacrolimus levels in blood and tissues were measured at selected time points. A near-infrared dye was encapsulated in the hydrogel together with tacrolimus in order to monitor hydrogel deposits and drug release in vitro and in vivo in a model of vascularized composite allotransplantation.

RESULTS: Injection of lipopolysaccharides led to increased blood and skin tacrolimus levels (p = 0.0076, day 7 vs. day 12 in blood, and p = 0.0007 in treated limbs, 48 h after injection compared to controls). Moreover, lipopolysaccharides-injected animals had higher tacrolimus levels in treated limbs compared to contralateral limbs (p = 0.0003 for skin and p = 0.0053 for muscle). Imaging of hydrogel deposits and tacrolimus release was achieved by encapsulating near-infrared dye in the hydrogel for 160 days. The correlation of tacrolimus and near-infrared dye release from hydrogel was R2 = 0.6297 and R2 = 0.5619 in blood and grafts of transplanted animals respectively and R2 = 0.6066 in vitro.

CONCLUSIONS: Here we demonstrate the inflammation-responsiveness of a tacrolimus-loaded hydrogel in vivo. Moreover, we show that encapsulating a near-infrared dye in the hydrogel provides a reliable correlation of tacrolimus and dye release from the hydrogel, and an accessible non-invasive method for monitoring drug release from hydrogel deposits.

}, keywords = {Animals, Drug Delivery Systems, Humans, Hydrogels, Immunosuppressive Agents, Inflammation, Male, Rats, Rats, Inbred BN, Rats, Inbred Lew, Tacrolimus}, issn = {1932-6203}, doi = {10.1371/journal.pone.0203409}, author = {Dzhonova, Dzhuliya and Olariu, Radu and Leckenby, Jonathan and Dhayani, Ashish and Vemula, Praveen Kumar and Prost, Jean-Christophe and Banz, Yara and Taddeo, Adriano and Rieben, Robert} } @article {1583, title = {Mutation burden profile in familial Alzheimer{\textquoteright}s disease cases from India.}, journal = {Neurobiol Aging}, volume = {64}, year = {2018}, month = {2018 04}, pages = {158.e7-158.e13}, abstract = {

This study attempts to identify coding risk variants in genes previously implicated in Alzheimer{\textquoteright}s disease (AD) pathways, through whole-exome sequencing of subjects (N\ = 17) with AD, with a positive family history of dementia (familial AD). We attempted to evaluate the mutation burden in genes encoding amyloid precursor protein metabolism and previously linked to risk of dementias. Novel variants were identified in genes involved in amyloid precursor protein metabolism such as PSEN1 (chr 14:73653575, W161C, tgg \> tgT), PLAT (chr 8:42039530,G272R), and SORL1 (chr11:121414373,G601D). The mutation burden assessment of dementia-related genes for all 17 cases revealed 45 variants, which were either shared across subjects, or were present in just the 1 patient. The study shows that the clinical characteristics, and genetic correlates, obtained in this sample are broadly comparable to the other studies that have investigated familial forms of AD. Our study identifies rare deleterious genetic variations, in the coding region of genes involved in amyloid signaling, and other dementia-associated pathways.

}, keywords = {Aged, Alzheimer Disease, Amyloid beta-Protein Precursor, Genetic Association Studies, Genetic Predisposition to Disease, Genetic Variation, Humans, India, LDL-Receptor Related Proteins, Membrane Transport Proteins, Middle Aged, Mutation, Presenilin-1, Risk, Signal Transduction, Tissue Plasminogen Activator, Whole Exome Sequencing}, issn = {1558-1497}, doi = {10.1016/j.neurobiolaging.2017.12.002}, author = {Syama, Adhikarla and Sen, Somdatta and Kota, Lakshmi Narayanan and Viswanath, Biju and Purushottam, Meera and Varghese, Mathew and Jain, Sanjeev and Panicker, Mitradas M and Mukherjee, Odity} } @article {1152, title = {PAI1 mediates fibroblast-mast cell interactions in skin fibrosis.}, journal = {J Clin Invest}, volume = {128}, year = {2018}, month = {2018 May 01}, pages = {1807-1819}, abstract = {

Fibrosis is a prevalent pathological condition arising from the chronic activation of fibroblasts. This activation results from the extensive intercellular crosstalk mediated by both soluble factors and direct cell-cell connections. Prominent among these are the interactions of fibroblasts with immune cells, in which the fibroblast-mast cell connection, although acknowledged, is relatively unexplored. We have used a Tg mouse model of skin fibrosis, based on expression of the transcription factor Snail in the epidermis, to probe the mechanisms regulating mast cell activity and the contribution of these cells to this pathology. We have discovered that Snail-expressing keratinocytes secrete plasminogen activator inhibitor type 1 (PAI1), which functions as a chemotactic factor to increase mast cell infiltration into the skin. Moreover, we have determined that PAI1 upregulates intercellular adhesion molecule type 1 (ICAM1) expression on dermal fibroblasts, rendering them competent to bind to mast cells. This heterotypic cell-cell adhesion, also observed in the skin fibrotic disorder scleroderma, culminates in the reciprocal activation of both mast cells and fibroblasts, leading to the cascade of events that promote fibrogenesis. Thus, we have identified roles for PAI1 in the multifactorial program of fibrogenesis that expand its functional repertoire beyond its canonical role in plasmin-dependent processes.

}, issn = {1558-8238}, doi = {10.1172/JCI99088}, author = {Pincha, Neha and Hajam, Edries Yousaf and Badarinath, Krithika and Batta, Surya Prakash Rao and Masudi, Tafheem and Dey, Rakesh and Andreasen, Peter and Kawakami, Toshiaki and Samuel, Rekha and George, Renu and Danda, Debashish and Jacob, Paul Mazhuvanchary and Jamora, Colin} } @article {1595, title = {Prevention of pesticide-induced neuronal dysfunction and mortality with nucleophilic poly-Oxime topical gel.}, journal = {Sci Adv}, volume = {4}, year = {2018}, month = {2018 Oct}, pages = {eaau1780}, abstract = {

Organophosphate-based pesticides inhibit acetylcholinesterase (AChE), which plays a pivotal role in neuromuscular function. While spraying in the field, farmworkers get exposed to pesticides through the dermal route. Internalized pesticide inhibits AChE, which leads to neurotoxicity, cardiotoxicity, cognitive dysfunction, loss of endurance, and death in severe cases. Here, we present a nucleophilic pyridine-2-aldoxime-functionalized chitosan-based topical gel (-Oxime gel) that rapidly deactivates organophosphates, methyl parathion (MPT), on the skin of rats, which leads to reduced AChE inhibition in the blood and tissues. Testing the robustness of -Oxime gel, we report reduction in AChE inhibition following repeated dermal administration of MPT in the presence of -Oxime gel. Furthermore, -Oxime gel prevented MPT-induced neuromuscular dysfunction, loss of endurance, and locomotor coordination. We observe a 100\% survival in rats following topical MPT administration in the presence of -Oxime gel. This prophylactic gel may therefore help farmworkers by limiting pesticide-induced toxicity and mortality.

}, issn = {2375-2548}, doi = {10.1126/sciadv.aau1780}, author = {Thorat, Ketan and Pandey, Subhashini and Chandrashekharappa, Sandeep and Vavilthota, Nikitha and Hiwale, Ankita A and Shah, Purna and Sreekumar, Sneha and Upadhyay, Shubhangi and Phuntsok, Tenzin and Mahato, Manohar and Mudnakudu-Nagaraju, Kiran K and Sunnapu, Omprakash and Vemula, Praveen K} } @article {1577, title = {Quantification of Neurotransmitters from Intact and Regenerating Planarians Using UHPLC-MS/SRM Method.}, journal = {Methods Mol Biol}, volume = {1774}, year = {2018}, month = {2018}, pages = {555-570}, abstract = {

Freshwater planarian species S. mediterranea is an emerging stem cell model because of its capability of regenerating large portions of missing body parts. It is one of the best model systems available to address the basic biological mechanisms in the regeneration processes. Absolute quantification of metabolites from planarians is imperative to understand their role in the regeneration processes. Here we describe a stable isotope dilution ultrahigh performance liquid chromatography/mass spectrometry/selected reaction monitoring (UHPLC-MS/SRM) assay for a sensitive and quantitative assessment of neurotransmitters (NTs) in planaria. We used this method for the simultaneous quantification of 16 NTs from both intact and regenerating planarians.

}, keywords = {Animals, Chromatography, High Pressure Liquid, Neurotransmitter Agents, Planarians, Regeneration, Stem Cells, Tandem Mass Spectrometry}, issn = {1940-6029}, doi = {10.1007/978-1-4939-7802-1_25}, author = {Rangiah, Kannan and Palakodeti, Dasaradhi} } @article {1590, title = {Repeated social stress leads to contrasting patterns of structural plasticity in the amygdala and hippocampus.}, journal = {Behav Brain Res}, volume = {347}, year = {2018}, month = {2018 07 16}, pages = {314-324}, abstract = {

Previous studies have demonstrated that repeated immobilization and restraint stress cause contrasting patterns of dendritic reorganization as well as alterations in spine density in amygdalar and hippocampal neurons. Whether social and ethologically relevant stressors can induce similar patterns of morphological plasticity remains largely unexplored. Hence, we assessed the effects of repeated social defeat stress on neuronal morphology in basolateral amygdala (BLA), hippocampal CA1 and infralimbic medial prefrontal cortex (mPFC). Male Wistar rats experienced social defeat stress on 5 consecutive days during confrontation in the resident-intruder paradigm with larger and aggressive Wild-type Groningen rats. This resulted in clear social avoidance behavior one day after the last confrontation. To assess the morphological consequences of repeated social defeat, 2 weeks after the last defeat, animals were sacrificed and brains were stained using a Golgi-Cox procedure. Morphometric analyses revealed that, compared to controls, defeated Wistar rats showed apical dendritic decrease in spine density on CA1 but not BLA. Sholl analysis demonstrated a significant dendritic atrophy of CA1 basal dendrites in defeated animals. In contrast, basal dendrites of BLA pyramidal neurons exhibited enhanced dendritic arborization in defeated animals. Social stress failed to induce lasting structural changes in mPFC neurons. Our findings demonstrate for the first time that social defeat stress elicits divergent patterns of structural plasticity in the hippocampus versus amygdala, similar to what has previously been reported with repeated physical stressors. Therefore, brain region specific variations may be a universal feature of stress-induced plasticity that is shared by both physical and social stressors.

}, keywords = {Amygdala, Animals, Atrophy, Avoidance Learning, CA1 Region, Hippocampal, Dendritic Spines, Dominance-Subordination, Male, Neuronal Plasticity, Prefrontal Cortex, Pyramidal Cells, Rats, Wistar, Stress, Psychological}, issn = {1872-7549}, doi = {10.1016/j.bbr.2018.03.034}, author = {Patel, D and Anilkumar, S and Chattarji, S and Buwalda, B} } @article {1151, title = {Rudhira/BCAS3 is essential for mouse development and cardiovascular patterning.}, journal = {Sci Rep}, volume = {8}, year = {2018}, month = {2018 Apr 04}, pages = {5632}, abstract = {

Rudhira/Breast Carcinoma Amplified Sequence 3 (BCAS3) is a cytoskeletal protein that promotes directional cell migration and angiogenesis in vitro and is implicated in human carcinomas and coronary artery disease. To study the role of Rudhira during development in vivo, we generated the first knockout mouse for rudhira and show that Rudhira is essential for mouse development. Rudhira null embryos die at embryonic day (E) 9.5 accompanied by severe vascular patterning defects in embryonic and extra-embryonic tissues. To identify the molecular processes downstream of rudhira, we analyzed the transcriptome of intact knockout yolk sacs. Genome-wide transcriptome analysis showed that Rudhira functions in angiogenesis and its related processes such as cell adhesion, extracellular matrix organization, peptidase activity and TGFβ signaling. Since Rudhira is also expressed in endothelial cells\ (ECs), we further generated Tie2Cre-mediated endothelial knockout (CKO) of rudhira. CKO embryos survive to E11.5 and similar to the global knockout, display gross vascular patterning defects, showing that endothelial Rudhira is vital for development. Further, Rudhira knockdown ECs in culture fail to sprout in a spheroid-sprouting assay, strongly supporting its role in vascular patterning. Our study identifies an essential role for Rudhira in blood vessel remodeling and provides a mouse model for cardiovascular development.

}, issn = {2045-2322}, doi = {10.1038/s41598-018-24014-w}, author = {Shetty, Ronak and Joshi, Divyesh and Jain, Mamta and Vasudevan, Madavan and Paul, Jasper Chrysolite and Bhat, Ganesh and Banerjee, Poulomi and Abe, Takaya and Kiyonari, Hiroshi and VijayRaghavan, K and Inamdar, Maneesha S} } @article {1588, title = {The Sodium Sialic Acid Symporter From Has Altered Substrate Specificity.}, journal = {Front Chem}, volume = {6}, year = {2018}, month = {2018}, pages = {233}, abstract = {

Mammalian cell surfaces are decorated with complex glycoconjugates that terminate with negatively charged sialic acids. Commensal and pathogenic bacteria can use host-derived sialic acids for a competitive advantage, but require a functional sialic acid transporter to import the sugar into the cell. This work investigates the sodium sialic acid symporter (SiaT) from (SiaT). We demonstrate that SiaT rescues an strain lacking its endogenous sialic acid transporter when grown on the sialic acids -acetylneuraminic acid (Neu5Ac) or -glycolylneuraminic acid (Neu5Gc). We then develop an expression, purification and detergent solubilization system for SiaT and demonstrate that the protein is largely monodisperse in solution with a stable monomeric oligomeric state. Binding studies reveal that SiaT has a higher affinity for Neu5Gc over Neu5Ac, which was unexpected and is not seen in another SiaT homolog. We develop a homology model and use comparative sequence analyses to identify substitutions in the substrate-binding site of SiaT that may explain the altered specificity. SiaT is shown to be electrogenic, and transport is dependent upon more than one Na ion for every sialic acid molecule. A functional sialic acid transporter is essential for the uptake and utilization of sialic acid in a range of pathogenic bacteria, and developing new inhibitors that target these transporters is a valid mechanism for inhibiting bacterial growth. By demonstrating a route to functional recombinant SiaT, and developing the and assay systems, our work underpins the design of inhibitors to this transporter.

}, issn = {2296-2646}, doi = {10.3389/fchem.2018.00233}, author = {North, Rachel A and Wahlgren, Weixiao Y and Remus, Daniela M and Scalise, Mariafrancesca and Kessans, Sarah A and Dunevall, Elin and Claesson, Elin and Soares da Costa, Tatiana P and Perugini, Matthew A and Ramaswamy, S and Allison, Jane R and Indiveri, Cesare and Friemann, Rosmarie and Dobson, Renwick C J} } @article {1147, title = {Substrate-bound outward-open structure of a Na-coupled sialic acid symporter reveals a new Na site.}, journal = {Nat Commun}, volume = {9}, year = {2018}, month = {2018 May 01}, pages = {1753}, abstract = {

Many pathogenic bacteria utilise sialic acids as an energy source or use them as an external coating to evade immune detection. As such, bacteria that colonise sialylated environments deploy specific transporters to mediate import of scavenged sialic acids. Here, we report a substrate-bound 1.95 {\r A} resolution structure and subsequent characterisation of SiaT, a sialic acid transporter from Proteus mirabilis. SiaT is a secondary active transporter of the sodium solute symporter (SSS) family, which use Na gradients to drive the uptake of extracellular substrates. SiaT adopts the LeuT-fold and is in an outward-open conformation in complex with the sialic acid N-acetylneuraminic acid and two Na ions. One Na binds to the conserved Na2 site, while the second Na binds to a new position, termed Na3, which is conserved in many SSS family members. Functional and molecular dynamics studies validate the substrate-binding site and demonstrate that both Na sites regulate N-acetylneuraminic acid transport.

}, issn = {2041-1723}, doi = {10.1038/s41467-018-04045-7}, author = {Wahlgren, Weixiao Y and Dunevall, Elin and North, Rachel A and Paz, Aviv and Scalise, Mariafrancesca and Bisignano, Paola and Bengtsson-Palme, Johan and Goyal, Parveen and Claesson, Elin and Caing-Carlsson, Rhawnie and Andersson, Rebecka and Beis, Konstantinos and Nilsson, Ulf J and Farewell, Anne and Pochini, Lorena and Indiveri, Cesare and Grabe, Michael and Dobson, Renwick C J and Abramson, Jeff and Ramaswamy, S and Friemann, Rosmarie} } @article {1580, title = {Targeting Phosphopeptide Recognition by the Human BRCA1 Tandem BRCT Domain to Interrupt BRCA1-Dependent Signaling.}, journal = {Cell Chem Biol}, volume = {25}, year = {2018}, month = {2018 06 21}, pages = {677-690.e12}, abstract = {

Intracellular signals triggered by DNA breakage flow through proteins containing BRCT (BRCA1 C-terminal) domains. This family, comprising 23 conserved phosphopeptide-binding modules in man, is inaccessible to small-molecule chemical inhibitors. Here, we develop Bractoppin, a drug-like inhibitor of phosphopeptide recognition by the human BRCA1 tandem (t)BRCT domain, which selectively inhibits substrate binding with nanomolar potency in\ vitro. Structure-activity exploration suggests that Bractoppin engages BRCA1 tBRCT residues recognizing pSer in the consensus motif, pSer-Pro-Thr-Phe, plus an abutting hydrophobic pocket that is distinct in structurally related BRCT domains, conferring selectivity. In cells, Bractoppin inhibits substrate recognition detected by F{\"o}rster resonance energy transfer, and diminishes BRCA1 recruitment to DNA breaks, in turn suppressing damage-induced G2 arrest and assembly of the recombinase, RAD51. But damage-induced MDC1 recruitment, single-stranded DNA (ssDNA) generation, and TOPBP1 recruitment remain unaffected. Thus, an inhibitor of phosphopeptide recognition selectively interrupts BRCA1 tBRCT-dependent signals evoked by DNA damage.

}, issn = {2451-9448}, doi = {10.1016/j.chembiol.2018.02.012}, author = {Periasamy, Jayaprakash and Kurdekar, Vadiraj and Jasti, Subbarao and Nijaguna, Mamatha B and Boggaram, Sanjana and Hurakadli, Manjunath A and Raina, Dhruv and Kurup, Lokavya Meenakshi and Chintha, Chetan and Manjunath, Kavyashree and Goyal, Aneesh and Sadasivam, Gayathri and Bharatham, Kavitha and Padigaru, Muralidhara and Potluri, Vijay and Venkitaraman, Ashok R} } @article {1160, title = {Cytoplasmic poly (A)-binding protein critically regulates epidermal maintenance and turnover in the planarian .}, journal = {Development}, volume = {144}, year = {2017}, month = {2017 09 01}, pages = {3066-3079}, abstract = {

Identifying key cellular events that facilitate stem cell function and tissue organization is crucial for understanding the process of regeneration. Planarians are powerful model system to study regeneration and stem cell (neoblast) function. Here, using planaria, we show that the initial events of regeneration, such as epithelialization and epidermal organization are critically regulated by a novel cytoplasmic poly A-binding protein, SMED-PABPC2. Knockdown leads to defects in epidermal lineage specification, disorganization of epidermis and ECM, and deregulated wound healing, resulting in the selective failure of neoblast proliferation near the wound region. Polysome profiling suggests that epidermal lineage transcripts, including , are translationally regulated by SMED-PABPC2 Together, our results uncover a novel role for SMED-PABPC2 in the maintenance of epidermal and ECM integrity, critical for wound healing and subsequent processes for regeneration.

}, keywords = {Animals, Cell Lineage, Cell Proliferation, Cytoplasm, Epidermis, Epithelium, Extracellular Matrix, Gene Knockdown Techniques, Homeostasis, Models, Biological, Planarians, Poly(A)-Binding Protein I, Regeneration, RNA, Messenger, Wound Healing}, issn = {1477-9129}, doi = {10.1242/dev.152942}, author = {Bansal, Dhiru and Kulkarni, Jahnavi and Nadahalli, Kavana and Lakshmanan, Vairavan and Krishna, Srikar and Sasidharan, Vidyanand and Geo, Jini and Dilipkumar, Shilpa and Pasricha, Renu and Gulyani, Akash and Raghavan, Srikala and Palakodeti, Dasaradhi} } @article {1163, title = {Hierarchies in light sensing and dynamic interactions between ocular and extraocular sensory networks in a flatworm.}, journal = {Sci Adv}, volume = {3}, year = {2017}, month = {2017 Jul}, pages = {e1603025}, abstract = {

Light sensing has independently evolved multiple times under diverse selective pressures but has been examined only in a handful among the millions of light-responsive organisms. Unsurprisingly, mechanistic insights into how differential light processing can cause distinct behavioral outputs are limited. We show how an organism can achieve complex light processing with a simple "eye" while also having independent but mutually interacting light sensing networks. Although planarian flatworms lack wavelength-specific eye photoreceptors, a 25 nm change in light wavelength is sufficient to completely switch their phototactic behavior. Quantitative photoassays, eye-brain confocal imaging, and RNA interference/knockdown studies reveal that flatworms are able to compare small differences in the amounts of light absorbed at the eyes through a single eye opsin and convert them into binary behavioral outputs. Because planarians can fully regenerate, eye-brain injury-regeneration studies showed that this acute light intensity sensing and processing are layered on simple light detection. Unlike intact worms, partially regenerated animals with eyes can sense light but cannot sense finer gradients. Planarians also show a "reflex-like," eye-independent (extraocular/whole-body) response to low ultraviolet A light, apart from the "processive" eye-brain-mediated (ocular) response. Competition experiments between ocular and extraocular sensory systems reveal dynamic interchanging hierarchies. In intact worms, cerebral ocular response can override the reflex-like extraocular response. However, injury-regeneration again offers a time window wherein both responses coexist, but the dominance of the ocular response is reversed. Overall, we demonstrate acute light intensity-based behavioral switching and two evolutionarily distinct but interacting light sensing networks in a regenerating organism.

}, issn = {2375-2548}, doi = {10.1126/sciadv.1603025}, author = {Shettigar, Nishan and Joshi, Asawari and Dalmeida, Rimple and Gopalkrishna, Rohini and Chakravarthy, Anirudh and Patnaik, Siddharth and Mathew, Manoj and Palakodeti, Dasaradhi and Gulyani, Akash} } @article {1204, title = {Horse Liver Alcohol Dehydrogenase: Zinc Coordination and Catalysis.}, journal = {Biochemistry}, volume = {56}, year = {2017}, month = {2017 07 18}, pages = {3632-3646}, abstract = {

During catalysis by liver alcohol dehydrogenase (ADH), a water bound to the catalytic zinc is replaced by the oxygen of the substrates. The mechanism might involve a pentacoordinated zinc or a double-displacement reaction with participation by a nearby glutamate residue, as suggested by studies of human ADH3, yeast ADH1, and some other tetrameric ADHs. Zinc coordination and participation of water in the enzyme mechanism were investigated by X-ray crystallography. The apoenzyme and its complex with adenosine 5{\textquoteright}-diphosphoribose have an open protein conformation with the catalytic zinc in one position, tetracoordinated by Cys-46, His-67, Cys-174, and a water molecule. The bidentate chelators 2,2{\textquoteright}-bipyridine and 1,10-phenanthroline displace the water and form a pentacoordinated zinc. The enzyme-NADH complex has a closed conformation similar to that of ternary complexes with coenzyme and substrate analogues; the coordination of the catalytic zinc is similar to that found in the apoenzyme, except that a minor, alternative position for the catalytic zinc is \~{}1.3 {\r A} from the major position and closer to Glu-68, which could form the alternative coordination to the catalytic zinc. Complexes with NADH and N-1-methylhexylformamide or N-benzylformamide (or with NAD and fluoro alcohols) have the classical tetracoordinated zinc, and no water is bound to the zinc or the nicotinamide rings. The major forms of the enzyme in the mechanism have a tetracoordinated zinc, where the carboxylate group of Glu-68 could participate in the exchange of water and substrates on the zinc. Hydride transfer in the Michaelis complexes does not involve a nearby water.

}, keywords = {2,2{\textquoteright}-Dipyridyl, Adenosine Diphosphate Ribose, Alcohol Dehydrogenase, Animals, Catalytic Domain, Crystallography, X-Ray, Formamides, Horses, Kinetics, Liver, Models, Molecular, NAD, Phenanthrolines, Protein Binding, Protein Conformation, Water, Zinc}, issn = {1520-4995}, doi = {10.1021/acs.biochem.7b00446}, author = {Plapp, Bryce V and Savarimuthu, Baskar Raj and Ferraro, Daniel J and Rubach, Jon K and Brown, Eric N and Ramaswamy, S} } @article {1199, title = {The miR-124 family of microRNAs is crucial for regeneration of the brain and visual system in the planarian Schmidtea mediterranea}, journal = {Development}, volume = {144}, year = {2017}, pages = {3211{\textendash}3223}, abstract = {

Brain regeneration in planarians is mediated by precise spatiotemporal control of gene expression and is crucial for multiple aspects of neurogenesis. However, the mechanisms underpinning the gene regulation essential for brain regeneration are largely unknown. Here, we investigated the role of the miR-124 family of microRNAs in planarian brain regeneration. The miR-124 family (miR-124) is highly conserved in animals and regulates neurogenesis by facilitating neural differentiation, yet its role in neural wiring and brain organization is not known. We developed a novel method for delivering anti-miRs using liposomes for the functional knockdown of microRNAs. Smed-miR-124 knockdown revealed a key role for these microRNAs in neuronal organization during planarian brain regeneration. Our results also demonstrated an essential role for miR-124 in the generation of eye progenitors. Additionally, miR-124 regulates Smed-slit-1, which encodes an axon guidance protein, either by targeting slit-1 mRNA or, potentially, by modulating the canonical Notch pathway. Together, our results reveal a role for miR-124 in regulating the regeneration of a functional brain and visual system.

}, issn = {0950-1991}, doi = {10.1242/dev.144758}, url = {http://dev.biologists.org/content/144/18/3211}, author = {Sasidharan, Vidyanand and Marepally, Srujan and Elliott, Sarah A. and Baid, Srishti and Lakshmanan, Vairavan and Nayyar, Nishtha and Bansal, Dhiru and S{\'a}nchez Alvarado, Alejandro and Vemula, Praveen Kumar and Palakodeti, Dasaradhi} } @article {1156, title = {Stimulation of hair follicle stem cell proliferation through an IL-1 dependent activation of γδT-cells.}, journal = {Elife}, volume = {6}, year = {2017}, month = {2017 Dec 04}, abstract = {

The cutaneous wound-healing program is a product of a complex interplay among diverse cell types within the skin. One fundamental process that is mediated by these reciprocal interactions is the mobilization of local stem cell pools to promote tissue regeneration and repair. Using the ablation of epidermal caspase-8 as a model of wound healing in , we analyzed the signaling components responsible for epithelial stem cell proliferation. We found that IL-1α and IL-7 secreted from keratinocytes work in tandem to expand the activated population of resident epidermal γδT-cells. A downstream effect of activated γδT-cells is the preferential proliferation of hair follicle stem cells. By contrast, IL-1α-dependent stimulation of dermal fibroblasts optimally stimulates epidermal stem cell proliferation. These findings provide new mechanistic insights into the regulation and function of epidermal cell-immune cell interactions and into how components that are classically associated with inflammation can differentially influence distinct stem cell niches within a tissue.

}, issn = {2050-084X}, doi = {10.7554/eLife.28875}, author = {Lee, Pedro and Gund, Rupali and Dutta, Abhik and Pincha, Neha and Rana, Isha and Ghosh, Subhasri and Witherden, Deborah and Kandyba, Eve and MacLeod, Amanda and Kobielak, Krzysztof and Havran, Wendy L and Jamora, Colin} } @article {1166, title = {Water-mediated intermolecular interactions in 1,2-O-cyclohexylidene-myo-inositol: a quantitative analysis.}, journal = {Acta Crystallogr C Struct Chem}, volume = {73}, year = {2017}, month = {2017 Jan 01}, pages = {20-27}, abstract = {

The syntheses of new myo-inositol derivatives have received much attention due to their important biological activities. 1,2-O-Cyclohexylidene-myo-inositol is an important intermediate formed during the syntheses of certain myo-inositol derivatives. We report herein the crystal structure of 1,2-O-cyclohexylidene-myo-inositol dihydrate, CHO{\textperiodcentered}2HO, which is an intermediate formed during the syntheses of myo-inositol phosphate derivatives, to demonstrate the participation of water molecules and hydroxy groups in the formation of several intermolecular O-H...O interactions, and to determine a low-energy conformation. The title myo-inositol derivative crystallizes with two water molecules in the asymmetric unit in the space group C2/c, with Z = 8. The water molecules facilitate the formation of an extensive O-H...O hydrogen-bonding network that assists in the formation of a dense crystal packing. Furthermore, geometrical optimization and frequency analysis was carried out using density functional theory (DFT) calculations with B3LYP hybrid functionals and 6-31G(d), 6-31G(d,p) and 6-311G(d,p) basis sets. The theoretical and experimental structures were found to be very similar, with only slight deviations. The intermolecular interactions were quantitatively analysed using Hirshfeld surface analysis and 2D (two-dimensional) fingerplot plots, and the total lattice energy was calculated.

}, issn = {2053-2296}, doi = {10.1107/S2053229616018581}, author = {Purushothaman, Gayathri and Juvale, Kapil and Kirubakaran, Sivapriya and Vemula, Praveen Kumar and Thiruvenkatam, Vijay} } @article {1167, title = {Blue protein with red fluorescence.}, journal = {Proc Natl Acad Sci U S A}, volume = {113}, year = {2016}, month = {2016 10 11}, pages = {11513-11518}, abstract = {

The walleye (Sander vitreus) is a golden yellow fish that inhabits the Northern American lakes. The recent sightings of the blue walleye and the correlation of its sighting to possible increased UV radiation have been proposed earlier. The underlying molecular basis of its adaptation to increased UV radiation is the presence of a protein (Sandercyanin)-ligand complex in the mucus of walleyes. Degradation of heme by UV radiation results in the formation of Biliverdin IXα (BLA), the chromophore bound to Sandercyanin. We show that Sandercyanin is a monomeric protein that forms stable homotetramers on addition of BLA to the protein. A structure of the Sandercyanin-BLA complex, purified from the fish mucus, reveals a glycosylated protein with a lipocalin fold. This protein-ligand complex absorbs light in the UV region (λ of 375 nm) and upon excitation at this wavelength emits in the red region (λ of 675 nm). Unlike all other known biliverdin-bound fluorescent proteins, the chromophore is noncovalently bound to the protein. We provide here a molecular rationale for the observed spectral properties of Sandercyanin.

}, keywords = {Biliverdine, Crystallography, X-Ray, Fluorescence, Models, Molecular, Proteins, Recombinant Proteins}, issn = {1091-6490}, doi = {10.1073/pnas.1525622113}, author = {Ghosh, Swagatha and Yu, Chi-Li and Ferraro, Daniel J and Sudha, Sai and Pal, Samir Kumar and Schaefer, Wayne F and Gibson, David T and Ramaswamy, S} } @article {1170, title = {Genome-Wide Analysis of Polyadenylation Events in Schmidtea mediterranea.}, journal = {G3 (Bethesda)}, volume = {6}, year = {2016}, month = {2016 10 13}, pages = {3035-3048}, abstract = {

In eukaryotes, 3{\textquoteright} untranslated regions (UTRs) play important roles in regulating posttranscriptional gene expression. The 3{\textquoteright}UTR is defined by regulated cleavage/polyadenylation of the pre-mRNA. The advent of next-generation sequencing technology has now enabled us to identify these events on a genome-wide scale. In this study, we used poly(A)-position profiling by sequencing (3P-Seq) to capture all poly(A) sites across the genome of the freshwater planarian, Schmidtea mediterranea, an ideal model system for exploring the process of regeneration and stem cell function. We identified the 3{\textquoteright}UTRs for \~{}14,000 transcripts and thus improved the existing gene annotations. We found 97 transcripts, which are polyadenylated within an internal exon, resulting in the shrinking of the ORF and loss of a predicted protein domain. Around 40\% of the transcripts in planaria were alternatively polyadenylated (ApA), resulting either in an altered 3{\textquoteright}UTR or a change in coding sequence. We identified specific ApA transcript isoforms that were subjected to miRNA mediated gene regulation using degradome sequencing. In this study, we also confirmed a tissue-specific expression pattern for alternate polyadenylated transcripts. The insights from this study highlight the potential role of ApA in regulating the gene expression essential for planarian regeneration.

}, keywords = {3{\textquoteright} Untranslated Regions, Animals, Computational Biology, Genome, Helminth, Genome-Wide Association Study, High-Throughput Nucleotide Sequencing, MicroRNAs, Molecular Sequence Annotation, Platyhelminths, Poly A, Polyadenylation, Reproducibility of Results, RNA Interference, RNA Processing, Post-Transcriptional, RNA, Messenger}, issn = {2160-1836}, doi = {10.1534/g3.116.031120}, author = {Lakshmanan, Vairavan and Bansal, Dhiru and Kulkarni, Jahnavi and Poduval, Deepak and Krishna, Srikar and Sasidharan, Vidyanand and Anand, Praveen and Seshasayee, Aswin and Palakodeti, Dasaradhi} } @article {616, title = {Mechanistic implications from structures of yeast alcohol dehydrogenase complexed with coenzyme and an alcohol.}, journal = {Arch Biochem Biophys}, volume = {591}, year = {2016}, month = {2016 Feb 1}, pages = {35-42}, abstract = {

Yeast alcohol dehydrogenase I is a homotetramer of subunits with 347 amino acid residues, catalyzing the oxidation of alcohols using NAD(+) as coenzyme. A new X-ray structure was determined at 3.0 {\r A} where both subunits of an asymmetric dimer bind coenzyme and trifluoroethanol. The tetramer is a pair of back-to-back dimers. Subunit A has a closed conformation and can represent a Michaelis complex with an appropriate geometry for hydride transfer between coenzyme and alcohol, with the oxygen of 2,2,2-trifluoroethanol ligated at 2.1 {\r A} to the catalytic zinc in the classical tetrahedral coordination with Cys-43, Cys-153, and His-66. Subunit B has an open conformation, and the coenzyme interacts with amino acid residues from the coenzyme binding domain, but not with residues from the catalytic domain. Coenzyme appears to bind to and dissociate from the open conformation. The catalytic zinc in subunit B has an alternative, inverted coordination with Cys-43, Cys-153, His-66 and the carboxylate of Glu-67, while the oxygen of trifluoroethanol is 3.5 {\r A} from the zinc. Subunit B may represent an intermediate in the mechanism after coenzyme and alcohol bind and before the conformation changes to the closed form and the alcohol oxygen binds to the zinc and displaces Glu-67.

}, keywords = {Alcohol Dehydrogenase, Binding Sites, Catalysis, Coenzymes, Computer Simulation, Enzyme Activation, Models, Chemical, Models, Molecular, NAD, Protein Binding, Protein Conformation, Saccharomyces cerevisiae Proteins, Substrate Specificity, Trifluoroethanol}, issn = {1096-0384}, doi = {10.1016/j.abb.2015.12.009}, author = {Plapp, Bryce V and Charlier, Henry A and Ramaswamy, S} } @article {1171, title = {Melatonin and Human Cardiovascular Disease.}, journal = {J Cardiovasc Pharmacol Ther}, year = {2016}, month = {2016 Jul 21}, abstract = {

The possible therapeutic role of melatonin in the pathophysiology of coronary artery disorder (CAD) is increasingly being recognized. In humans, exogenous melatonin has been shown to decrease nocturnal hypertension, improve systolic and diastolic blood pressure, reduce the pulsatility index in the internal carotid artery, decrease platelet aggregation, and reduce serum catecholamine levels. Low circulating levels of melatonin are reported in individuals with CAD, arterial hypertension, and congestive heart failure. This review assesses current literature on the cardiovascular effects of melatonin in humans. It can be concluded that melatonin deserves to be considered in clinical trials evaluating novel therapeutic interventions for cardiovascular disorders.

}, issn = {1940-4034}, doi = {10.1177/1074248416660622}, author = {Pandi-Perumal, Seithikurippu R and BaHammam, Ahmed S and Ojike, Nwakile I and Akinseye, Oluwaseun A and Kendzerska, Tetyana and Buttoo, Kenneth and Dhandapany, Perundurai S and Brown, Gregory M and Cardinali, Daniel P} } @article {2271, title = {A quantitative metabolomics peek into planarian regeneration.}, journal = {Analyst}, volume = {140}, year = {2015}, month = {2015 May 21}, pages = {3445-64}, abstract = {

The fresh water planarian species Schmidtea mediterranea is an emerging stem cell model because of its capability to regenerate a whole animal from a small piece of tissue. It is one of the best model systems to address the basic mechanisms essential for regeneration. Here, we are interested in studying the roles of various amines, thiols and nucleotides in planarian regeneration, stem cell function and growth. We developed mass spectrometry based quantitative methods and validated the differential enrichment of 35 amines, 7 thiol metabolites and 4 nucleotides from both intact and regenerating planarians. Among the amines, alanine in sexual and asparagine in asexual are the highest (\>1000 ng/mg) in the intact planarians. The levels of thiols such as cysteine and GSH are 651 and 1107 ng mg(-1) in planarians. Among the nucleotides, the level of cGMP is the lowest (0.03 ng mg(-1)) and the level of AMP is the highest (187 ng mg(-1)) in both of the planarian strains. We also noticed increasing levels of amines in both anterior and posterior regenerating planarians. The blastema from day 3 regenerating planarians also showed higher amounts of many amines. Interestingly, the thiol (cysteine and GSH) levels are well maintained during planarian regeneration. This suggests an inherent and effective mechanism to control induced oxidative stress because of the robust regeneration and stem cell proliferation. Like in intact planarians, the level of cGMP is also very low in regenerating planarians. Surprisingly, the levels of amines and thiols in head regenerating blastemas are \~{}3 times higher compared to those for tail regenerating blastemas. Thus our results strongly indicate the potential roles of amines, thiols and nucleotides in planarian regeneration.

}, keywords = {Animals, Calibration, Chromatography, High Pressure Liquid, Limit of Detection, Metabolomics, Planarians, Reference Standards, Regeneration, Reproduction, Asexual, Species Specificity, Tandem Mass Spectrometry}, issn = {1364-5528}, doi = {10.1039/c4an02037e}, author = {Natarajan, Nivedita and Ramakrishnan, Padma and Lakshmanan, Vairavan and Palakodeti, Dasaradhi and Rangiah, Kannan} }