TY - JOUR T1 - High-affinity binding of celastrol to monomeric α-synuclein mitigates in vitro aggregation. JF - J Biomol Struct Dyn Y1 - 2023 A1 - R, Kavya A1 - Aouti, Snehal A1 - Jos, Sneha A1 - Prasad, Thazhe Kootteri A1 - K N, Kumuda A1 - Unni, Sruthi A1 - Padmanabhan, Balasundaram A1 - Kamariah, Neelagandan A1 - Padavattan, Sivaraman A1 - Mythri, Rajeswara Babu AB -

α-Synuclein (αSyn) aggregation is associated with Parkinson's disease (PD). The region αSyn acts as the nucleation 'master controller' and αSyn as a 'secondary nucleation site'. 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.

ER - TY - JOUR T1 - Efficient and error-free correction of sickle mutation in human erythroid cells using prime editor-2. JF - Front Genome Ed Y1 - 2022 A1 - George, Anila A1 - Ravi, Nithin Sam A1 - Prasad, Kirti A1 - Panigrahi, Lokesh A1 - Koikkara, Sanya A1 - Rajendiran, Vignesh A1 - Devaraju, Nivedhitha A1 - Paul, Joshua A1 - Pai, Aswin Anand A1 - Nakamura, Yukio A1 - Kurita, Ryo A1 - Balasubramanian, Poonkuzhali A1 - Thangavel, Saravanabhavan A1 - Marepally, Srujan A1 - Velayudhan, Shaji R A1 - Srivastava, Alok A1 - Mohankumar, Kumarasamypet M AB -

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.

VL - 4 ER - TY - JOUR T1 - Erythroid lineage-specific lentiviral RNAi vectors suitable for molecular functional studies and therapeutic applications. JF - Sci Rep Y1 - 2022 A1 - Bagchi, Abhirup A1 - Devaraju, Nivedhitha A1 - Chambayil, Karthik A1 - Rajendiran, Vignesh A1 - Venkatesan, Vigneshwaran A1 - Sayed, Nilofer A1 - Pai, Aswin Anand A1 - Nath, Aneesha A1 - David, Ernest A1 - Nakamura, Yukio A1 - Balasubramanian, Poonkuzhali A1 - Srivastava, Alok A1 - Thangavel, Saravanabhavan A1 - Mohankumar, Kumarasamypet M A1 - Velayudhan, Shaji R KW - Animals KW - Cell Line, Tumor KW - Cell Lineage KW - DNA-Binding Proteins KW - Genetic Vectors KW - Humans KW - Lentivirus KW - Mice KW - RNA Interference KW - RNA, Small Interfering KW - Transcription Factors KW - Transduction, Genetic AB -

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.

VL - 12 IS - 1 ER - TY - JOUR T1 - Function of FMRP Domains in Regulating Distinct Roles of Neuronal Protein Synthesis. JF - Mol Neurobiol Y1 - 2022 A1 - D'Souza, Michelle Ninochka A1 - Ramakrishna, Sarayu A1 - Radhakrishna, Bindushree K A1 - Jhaveri, Vishwaja A1 - Ravindran, Sreenath A1 - Yeramala, Lahari A1 - Nair, Deepak A1 - Palakodeti, Dasaradhi A1 - Muddashetty, Ravi S KW - Fragile X Mental Retardation Protein KW - Fragile X Syndrome KW - Humans KW - Microtubules KW - Neurons KW - Protein Biosynthesis KW - Ribosomes KW - RNA, Messenger AB -

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'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's domains in translation regulation.

VL - 59 IS - 12 ER - TY - JOUR T1 - Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin. JF - Elife Y1 - 2022 A1 - Ravi, Nithin Sam A1 - Wienert, Beeke A1 - Wyman, Stacia K A1 - Bell, Henry William A1 - George, Anila A1 - Mahalingam, Gokulnath A1 - Vu, Jonathan T A1 - Prasad, Kirti A1 - Bandlamudi, Bhanu Prasad A1 - Devaraju, Nivedhitha A1 - Rajendiran, Vignesh A1 - Syedbasha, Nazar A1 - Pai, Aswin Anand A1 - Nakamura, Yukio A1 - Kurita, Ryo A1 - Narayanasamy, Muthuraman A1 - Balasubramanian, Poonkuzhali A1 - Thangavel, Saravanabhavan A1 - Marepally, Srujan A1 - Velayudhan, Shaji R A1 - Srivastava, Alok A1 - DeWitt, Mark A A1 - Crossley, Merlin A1 - Corn, Jacob E A1 - Mohankumar, Kumarasamypet M KW - Adenine KW - Anemia, Sickle Cell KW - beta-Globins KW - beta-Thalassemia KW - Cell Line KW - Clustered Regularly Interspaced Short Palindromic Repeats KW - CRISPR-Cas Systems KW - Cytosine KW - Fetal Hemoglobin KW - gamma-Globins KW - Gene Editing KW - Hematopoietic Stem Cells KW - Humans KW - Point Mutation KW - Promoter Regions, Genetic AB -

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.

VL - 11 ER - TY - JOUR T1 - Methionine uptake via the SLC43A2 transporter is essential for regulatory T-cell survival. JF - Life Sci Alliance Y1 - 2022 A1 - Saini, Neetu A1 - Naaz, Afsana A1 - Metur, Shree Padma A1 - Gahlot, Pinki A1 - Walvekar, Adhish A1 - Dutta, Anupam A1 - Davathamizhan, Umamaheswari A1 - Sarin, Apurva A1 - Laxman, Sunil KW - Interleukin-2 KW - Methionine KW - Racemethionine KW - Solute Carrier Proteins KW - T-Lymphocytes, Regulatory AB -

Cell death, survival, or growth decisions in T-cell subsets depend on interplay between cytokine-dependent and metabolic processes. The metabolic requirements of T-regulatory cells (Tregs) for their survival and how these are satisfied remain unclear. Herein, we identified a necessary requirement of methionine uptake and usage for Tregs survival upon IL-2 deprivation. Activated Tregs have high methionine uptake and usage to S-adenosyl methionine, and this uptake is essential for Tregs survival in conditions of IL-2 deprivation. We identify a solute carrier protein SLC43A2 transporter, regulated in a Notch1-dependent manner that is necessary for this methionine uptake and Tregs viability. Collectively, we uncover a specifically regulated mechanism of methionine import in Tregs that is required for cells to adapt to cytokine withdrawal. We highlight the need for methionine availability and metabolism in contextually regulating cell death in this immunosuppressive population of T cells.

VL - 5 IS - 12 ER - TY - JOUR T1 - Microscale engineering of hollow microneedle tips: design, manufacturing, optimization and validation. JF - Drug Deliv Transl Res Y1 - 2022 A1 - Badnikar, Kedar A1 - Jayadevi, Shreyas Nataraja A1 - Pahal, Suman A1 - Vemula, Praveen Kumar A1 - Nayak, Mangalore Manjunatha A1 - Subramanyam, Dinesh Narasimhaiah AB -

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.

VL - 12 IS - 2 ER - TY - JOUR T1 - Modulation of biliverdin dynamics and spectral properties by Sandercyanin. JF - RSC Adv Y1 - 2022 A1 - Ghosh, Swagatha A1 - Mondal, Sayan A1 - Yadav, Keerti A1 - Aggarwal, Shantanu A1 - Schaefer, Wayne F A1 - Narayana, Chandrabhas A1 - Subramanian, Ramaswamy AB -

Biliverdin IX-alpha (BV), a tetrapyrrole, is found ubiquitously in most living organisms. It functions as a metabolite, pigment, and signaling compound. While BV is known to bind to diverse protein families such as heme-metabolizing enzymes and phytochromes, not many BV-bound lipocalins (ubiquitous, small lipid-binding proteins) have been studied. The molecular basis of binding and conformational selectivity of BV in lipocalins remains unexplained. Sandercyanin (SFP)-BV complex is a blue lipocalin protein present in the mucus of the Canadian walleye (). In this study, we present the structures and binding modes of BV to SFP. Using a combination of designed site-directed mutations, X-ray crystallography, UV/VIS, and resonance Raman spectroscopy, we have identified multiple conformations of BV that are stabilized in the binding pocket of SFP. In complex with the protein, these conformers generate varied spectroscopic signatures both in their absorption and fluorescence spectra. We show that despite no covalent anchor, structural heterogeneity of the chromophore is primarily driven by the D-ring pyrrole of BV. Our work shows how conformational promiscuity of BV is correlated to the rearrangement of amino acids in the protein matrix leading to modulation of spectral properties.

VL - 12 IS - 31 ER - TY - JOUR T1 - NMDAR mediated dynamic changes in mA inversely correlates with neuronal translation. JF - Sci Rep Y1 - 2022 A1 - Gowda, Naveen Kumar Chandappa A1 - Nawalpuri, Bharti A1 - Ramakrishna, Sarayu A1 - Jhaveri, Vishwaja A1 - Muddashetty, Ravi S KW - Adenosine KW - Neurons KW - Phosphorylation KW - Receptors, N-Methyl-D-Aspartate KW - RNA AB -

Epitranscriptome modifications are crucial in translation regulation and essential for maintaining cellular homeostasis. N6 methyladenosine (mA) is one of the most abundant and well-conserved epitranscriptome modifications, which is known to play a pivotal role in diverse aspects of neuronal functions. However, the role of mA modifications with respect to activity-mediated translation regulation and synaptic plasticity has not been studied. Here, we investigated the role of mA modification in response to NMDAR stimulation. We have consistently observed that 5 min NMDAR stimulation causes an increase in eEF2 phosphorylation. Correspondingly, NMDAR stimulation caused a significant increase in the mA signal at 5 min time point, correlating with the global translation inhibition. The NMDAR induced increase in the mA signal is accompanied by the redistribution of the mA marked RNAs from translating to the non-translating pool of ribosomes. The increased mA levels are well correlated with the reduced FTO levels observed on NMDAR stimulation. Additionally, we show that inhibition of FTO prevents NMDAR mediated changes in mA levels. Overall, our results establish RNA-based molecular readout which corelates with the NMDAR-dependent translation regulation which helps in understanding changes in protein synthesis.

VL - 12 IS - 1 ER - TY - JOUR T1 - Novel Mutations in β- Gene in Indian Patients With Dilated Cardiomyopathy. JF - CJC Open Y1 - 2022 A1 - Rani, Deepa Selvi A1 - Vijaya Kumar, Archana A1 - Nallari, Pratibha A1 - Sampathkumar, Katakam A1 - Dhandapany, Perundurai S A1 - Narasimhan, Calambur A1 - Rathinavel, Andiappan A1 - Thangaraj, Kumarasamy AB -

Background: Heart failure is a hallmark of severe hypertrophic cardiomyopathy and dilated cardiomyopathy (DCM). Several mutations in the gene lead to hypertrophic cardiomyopathy. Recently, causative mutations in the gene have also been detected in DCM from different populations.

Methods: Here, we sequenced the gene in 137 Indian DCM patients and 167 ethnically matched healthy controls to detect the frequency of mutations and their association.

Results: Our study revealed 27 variations, of which 7 mutations (8.0%) were detected exclusively in Indian DCM patients for the first time. These included 4 missense mutations-Arg723His, Phe510Leu, His358Leu, and Ser384Tyr (2.9%); a frameshift mutation-Asn676_T-del (1.5%); and 2 splice-site mutations (IVS17+2T) T>G and (IVS19-1G) G>A (3.6%). Remarkably, all 4 missense mutations altered evolutionarily conserved amino acids. All 4 missense mutations were predicted to be pathogenic by 2 bioinformatics tools-polymorphism phenotyping v2 (PolyPhen-2) and sorting intolerant from tolerant (SIFT). In addition, the 4 homology models of β-MYH7-p.Leu358, p.Tyr384, p.Leu510, and p.His723-displayed root-mean-square deviations of ∼2.55 Å, ∼1.24 Å, ∼3.36 Å, and ∼3.86 Å, respectively.

Conclusions: In the present study, we detected numerous novel, unique, and rare mutations in the gene exclusively in Indian DCM patients (8.0%). Here, we demonstrated how each mutant (missense) uniquely disrupts a critical network of non-bonding interactions at the mutation site (molecular level) and may contribute to development of dilated cardiomyopathy (DCM). Therefore, our findings may provide insight into the understanding of the molecular bases of disease and into diagnosis along with promoting novel therapeutic strategies (through personalized medicine).

VL - 4 IS - 1 ER - TY - JOUR T1 - Single step fabrication of hollow microneedles and an experimental package for controlled drug delivery. JF - Int J Pharm Y1 - 2022 A1 - Ghate, Vivek A1 - Renjith, Anu A1 - Badnikar, Kedar A1 - Pahal, Suman A1 - Jayadevi, Shreyas N A1 - Nayak, Manjunatha M A1 - Vemula, Praveen K A1 - Subramanyam, Dinesh N AB -

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 µL per 30° 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.

VL - 632 ER - TY - JOUR T1 - Whole genome sequencing delineates regulatory, copy number, and cryptic splice variants in early onset cardiomyopathy. JF - NPJ Genom Med Y1 - 2022 A1 - Lesurf, Robert A1 - Said, Abdelrahman A1 - Akinrinade, Oyediran A1 - Breckpot, Jeroen A1 - Delfosse, Kathleen A1 - Liu, Ting A1 - Yao, Roderick A1 - Persad, Gabrielle A1 - McKenna, Fintan A1 - Noche, Ramil R A1 - Oliveros, Winona A1 - Mattioli, Kaia A1 - Shah, Shreya A1 - Miron, Anastasia A1 - Yang, Qian A1 - Meng, Guoliang A1 - Yue, Michelle Chan Seng A1 - Sung, Wilson W L A1 - Thiruvahindrapuram, Bhooma A1 - Lougheed, Jane A1 - Oechslin, Erwin A1 - Mondal, Tapas A1 - Bergin, Lynn A1 - Smythe, John A1 - Jayappa, Shashank A1 - Rao, Vinay J A1 - Shenthar, Jayaprakash A1 - Dhandapany, Perundurai S A1 - Semsarian, Christopher A1 - Weintraub, Robert G A1 - Bagnall, Richard D A1 - Ingles, Jodie A1 - Melé, Marta A1 - Maass, Philipp G A1 - Ellis, James A1 - Scherer, Stephen W A1 - Mital, Seema AB -

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 × 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.

VL - 7 IS - 1 ER - TY - JOUR T1 - Analysis of whole exome sequencing in severe mental illness hints at selection of brain development and immune related genes. JF - Sci Rep Y1 - 2021 A1 - Mahadevan, Jayant A1 - Pathak, Ajai Kumar A1 - Vemula, Alekhya A1 - Nadella, Ravi Kumar A1 - Viswanath, Biju A1 - Jain, Sanjeev A1 - Purushottam, Meera A1 - Mondal, Mayukh AB -

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'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.

VL - 11 IS - 1 ER - TY - JOUR T1 - APOE4 Affects Basal and NMDAR-Mediated Protein Synthesis in Neurons by Perturbing Calcium Homeostasis. JF - J Neurosci Y1 - 2021 A1 - Ramakrishna, Sarayu A1 - Jhaveri, Vishwaja A1 - Konings, Sabine C A1 - Nawalpuri, Bharti A1 - Chakraborty, Sumita A1 - Holst, Bjørn A1 - Schmid, Benjamin A1 - Gouras, Gunnar K A1 - Freude, Kristine K A1 - Muddashetty, Ravi S AB -

Apolipoprotein E (APOE), one of the primary lipoproteins in the brain has three isoforms in humans, APOE2, APOE3, and APOE4. APOE4 is the most well-established risk factor increasing the predisposition for Alzheimer's disease (AD). The presence of the APOE4 allele alone is shown to cause synaptic defects in neurons and recent studies have identified multiple pathways directly influenced by APOE4. However, the mechanisms underlying APOE4-induced synaptic dysfunction remain elusive. Here, we report that the acute exposure of primary cortical neurons or synaptoneurosomes to APOE4 leads to a significant decrease in global protein synthesis. Primary cortical neurons were derived from male and female embryos of Sprague Dawley (SD) rats or C57BL/6J mice. Synaptoneurosomes were prepared from P30 male SD rats. APOE4 treatment also abrogates the NMDA-mediated translation response indicating an alteration of synaptic signaling. Importantly, we demonstrate that both APOE3 and APOE4 generate a distinct translation response which is closely linked to their respective calcium signature. Acute exposure of neurons to APOE3 causes a short burst of calcium through NMDA receptors (NMDARs) leading to an initial decrease in protein synthesis which quickly recovers. Contrarily, APOE4 leads to a sustained increase in calcium levels by activating both NMDARs and L-type voltage-gated calcium channels (L-VGCCs), thereby causing sustained translation inhibition through eukaryotic translation elongation factor 2 (eEF2) phosphorylation, which in turn disrupts the NMDAR response. Thus, we show that APOE4 affects basal and activity-mediated protein synthesis responses in neurons by affecting calcium homeostasis. Defective protein synthesis has been shown as an early defect in familial Alzheimer's disease (AD). However, this has not been studied in the context of sporadic AD, which constitutes the majority of cases. In our study, we show that Apolipoprotein E4 (APOE4), the predominant risk factor for AD, inhibits global protein synthesis in neurons. APOE4 also affects NMDA activity-mediated protein synthesis response, thus inhibiting synaptic translation. We also show that the defective protein synthesis mediated by APOE4 is closely linked to the perturbation of calcium homeostasis caused by APOE4 in neurons. Thus, we propose the dysregulation of protein synthesis as one of the possible molecular mechanisms to explain APOE4-mediated synaptic and cognitive defects. Hence, the study not only suggests an explanation for the APOE4-mediated predisposition to AD, it also bridges the gap in understanding APOE4-mediated pathology.

VL - 41 IS - 42 ER - TY - JOUR T1 - Astrocytic reactivity triggered by defective autophagy and metabolic failure causes neurotoxicity in frontotemporal dementia type 3. JF - Stem Cell Reports Y1 - 2021 A1 - Chandrasekaran, Abinaya A1 - Dittlau, Katarina Stoklund A1 - Corsi, Giulia I A1 - Haukedal, Henriette A1 - Doncheva, Nadezhda T A1 - Ramakrishna, Sarayu A1 - Ambardar, Sheetal A1 - Salcedo, Claudia A1 - Schmidt, Sissel I A1 - Zhang, Yu A1 - Cirera, Susanna A1 - Pihl, Maria A1 - Schmid, Benjamin A1 - Nielsen, Troels Tolstrup A1 - Nielsen, Jørgen E A1 - Kolko, Miriam A1 - Kobolák, Julianna A1 - Dinnyés, András A1 - Hyttel, Poul A1 - Palakodeti, Dasaradhi A1 - Gorodkin, Jan A1 - Muddashetty, Ravi S A1 - Meyer, Morten A1 - Aldana, Blanca I A1 - Freude, Kristine K AB -

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.

VL - 16 IS - 11 ER - TY - JOUR T1 - Comprehensive annotation and characterization of planarian tRNA and tRNA-derived fragments (tRFs). JF - RNA Y1 - 2021 A1 - Lakshmanan, Vairavan A1 - T N, Sujith A1 - Bansal, Dhiru A1 - Padubidri, Shivaprasad V A1 - Palakodeti, Dasaradhi A1 - Krishna, Srikar AB -

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'-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'-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.

ER - TY - JOUR T1 - Correction of amygdalar dysfunction in a rat model of fragile X syndrome. JF - Cell Rep Y1 - 2021 A1 - Fernandes, Giselle A1 - Mishra, Pradeep K A1 - Nawaz, Mohammad Sarfaraz A1 - Donlin-Asp, Paul G A1 - Rahman, Mohammed Mostafizur A1 - Hazra, Anupam A1 - Kedia, Sonal A1 - Kayenaat, Aiman A1 - Songara, Dheeraj A1 - Wyllie, David J A A1 - Schuman, Erin M A1 - Kind, Peter C A1 - Chattarji, Sumantra AB -

Fragile X syndrome (FXS), a commonly inherited form of autism and intellectual disability, is associated with emotional symptoms that implicate dysfunction of the amygdala. However, current understanding of the pathogenesis of the disease is based primarily on studies in the hippocampus and neocortex, where FXS defects have been corrected by inhibiting group I metabotropic glutamate receptors (mGluRs). Here, we observe that activation, rather than inhibition, of mGluRs in the basolateral amygdala reverses impairments in a rat model of FXS. FXS rats exhibit deficient recall of auditory conditioned fear, which is accompanied by a range of in vitro and in vivo deficits in synaptic transmission and plasticity. We find presynaptic mGluR5 in the amygdala, activation of which reverses deficient synaptic transmission and plasticity, thereby restoring normal fear learning in FXS rats. This highlights the importance of modifying the prevailing mGluR-based framework for therapeutic strategies to include circuit-specific differences in FXS pathophysiology.

VL - 37 IS - 2 ER - TY - JOUR T1 - Cross-diagnostic evaluation of minor physical anomalies in psychiatric disorders. JF - J Psychiatr Res Y1 - 2021 A1 - Sreeraj, Vanteemar S A1 - Puzhakkal, Joan C A1 - Holla, Bharath A1 - Nadella, Ravi Kumar A1 - Sheth, Sweta A1 - Balachander, Srinivas A1 - Ithal, Dhruva A1 - Ali, Furkhan A1 - Viswanath, Biju A1 - Muralidharan, Kesavan A1 - Venkatasubramanian, Ganesan A1 - John, John P A1 - Benegal, Vivek A1 - Murthy, Pratima A1 - Varghese, Mathew A1 - Reddy, Yc Janardhan A1 - Jain, Sanjeev AB -

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's dementia).

METHODS: Modified Waldrop'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.

VL - 142 ER - TY - JOUR T1 - Distinct temporal expression of the GW182 paralog TNRC6A in neurons regulates dendritic arborization. JF - J Cell Sci Y1 - 2021 A1 - Nawalpuri, Bharti A1 - Sharma, Arpita A1 - Chattarji, Sumantra A1 - Muddashetty, Ravi S AB -

Precise development of the dendritic architecture is a critical determinant of mature neuronal circuitry. MicroRNA (miRNA)-mediated regulation of protein synthesis plays a crucial role in dendritic morphogenesis, but the role of miRNA-induced silencing complex (miRISC) protein components in this process is less studied. Here, we show an important role of a key miRISC protein, the GW182 paralog TNRC6A, in the regulation of dendritic growth. We identified a distinct brain region-specific spatiotemporal expression pattern of GW182 during rat postnatal development. We found that the window of peak GW182 expression coincides with the period of extensive dendritic growth, both in the hippocampus and cerebellum. Perturbation of GW182 function during a specific temporal window resulted in reduced dendritic growth of cultured hippocampal neurons. Mechanistically, we show that GW182 modulates dendritic growth by regulating global somatodendritic translation and actin cytoskeletal dynamics of developing neurons. Furthermore, we found that GW182 affects dendritic architecture by regulating the expression of actin modulator LIMK1. Taken together, our data reveal a previously undescribed neurodevelopmental expression pattern of GW182 and its role in dendritic morphogenesis, which involves both translational control and actin cytoskeletal rearrangement. This article has an associated First Person interview with the first author of the paper.

VL - 134 IS - 16 ER - TY - JOUR T1 - Engineered RNA biosensors enable ultrasensitive SARS-CoV-2 detection in a simple color and luminescence assay. JF - Life Sci Alliance Y1 - 2021 A1 - Chakravarthy, Anirudh A1 - Nandakumar, Anirudh A1 - George, Geen A1 - Ranganathan, Shyamsundar A1 - Umashankar, Suchitta A1 - Shettigar, Nishan A1 - Palakodeti, Dasaradhi A1 - Gulyani, Akash A1 - Ramesh, Arati KW - Biosensing Techniques KW - COVID-19 KW - Humans KW - Luminescence KW - Nucleic Acid Amplification Techniques KW - RNA KW - RNA, Viral KW - SARS-CoV-2 AB -

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.

VL - 4 IS - 12 ER - TY - JOUR T1 - Genomic characterization and epidemiology of an emerging SARS-CoV-2 variant in Delhi, India. JF - Science Y1 - 2021 A1 - Dhar, Mahesh S A1 - Marwal, Robin A1 - Vs, Radhakrishnan A1 - Ponnusamy, Kalaiarasan A1 - Jolly, Bani A1 - Bhoyar, Rahul C A1 - Sardana, Viren A1 - Naushin, Salwa A1 - Rophina, Mercy A1 - Mellan, Thomas A A1 - Mishra, Swapnil A1 - Whittaker, Charles A1 - Fatihi, Saman A1 - Datta, Meena A1 - Singh, Priyanka A1 - Sharma, Uma A1 - Ujjainiya, Rajat A1 - Bhatheja, Nitin A1 - Divakar, Mohit Kumar A1 - Singh, Manoj K A1 - Imran, Mohamed A1 - Senthivel, Vigneshwar A1 - Maurya, Ranjeet A1 - Jha, Neha A1 - Mehta, Priyanka A1 - A, Vivekanand A1 - Sharma, Pooja A1 - Vr, Arvinden A1 - Chaudhary, Urmila A1 - Soni, Namita A1 - Thukral, Lipi A1 - Flaxman, Seth A1 - Bhatt, Samir A1 - Pandey, Rajesh A1 - Dash, Debasis A1 - Faruq, Mohammed A1 - Lall, Hemlata A1 - Gogia, Hema A1 - Madan, Preeti A1 - Kulkarni, Sanket A1 - Chauhan, Himanshu A1 - Sengupta, Shantanu A1 - Kabra, Sandhya A1 - Gupta, Ravindra K A1 - Singh, Sujeet K A1 - Agrawal, Anurag A1 - Rakshit, Partha A1 - Nandicoori, Vinay A1 - Tallapaka, Karthik Bharadwaj A1 - Sowpati, Divya Tej A1 - Thangaraj, K A1 - Bashyam, Murali Dharan A1 - Dalal, Ashwin A1 - Sivasubbu, Sridhar A1 - Scaria, Vinod A1 - Parida, Ajay A1 - Raghav, Sunil K A1 - Prasad, Punit A1 - Sarin, Apurva A1 - Mayor, Satyajit A1 - Ramakrishnan, Uma A1 - Palakodeti, Dasaradhi A1 - Seshasayee, Aswin Sai Narain A1 - Bhat, Manoj A1 - Shouche, Yogesh A1 - Pillai, Ajay A1 - Dikid, Tanzin A1 - Das, Saumitra A1 - Maitra, Arindam A1 - Chinnaswamy, Sreedhar A1 - Biswas, Nidhan Kumar A1 - Desai, Anita Sudhir A1 - Pattabiraman, Chitra A1 - Manjunatha, M V A1 - Mani, Reeta S A1 - Arunachal Udupi, Gautam A1 - Abraham, Priya A1 - Atul, Potdar Varsha A1 - Cherian, Sarah S AB -

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; ×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.

ER - TY - JOUR T1 - Mechanical instability of adherens junctions overrides intrinsic quiescence of hair follicle stem cells. JF - Dev Cell Y1 - 2021 A1 - Biswas, Ritusree A1 - Banerjee, Avinanda A1 - Lembo, Sergio A1 - Zhao, Zhihai A1 - Lakshmanan, Vairavan A1 - Lim, Ryan A1 - Le, Shimin A1 - Nakasaki, Manando A1 - Kutyavin, Vassily A1 - Wright, Graham A1 - Palakodeti, Dasaradhi A1 - Ross, Robert S A1 - Jamora, Colin A1 - Vasioukhin, Valeri A1 - Jie, Yan A1 - Raghavan, Srikala AB -

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.

VL - 56 IS - 6 ER - TY - JOUR T1 - Microneedles for Extended Transdermal Therapeutics: A Route to Advanced Healthcare. JF - Eur J Pharm Biopharm Y1 - 2021 A1 - Pahal, Suman A1 - Badnikar, Kedar A1 - Ghate, Vivek A1 - Bhutani, Utkarsh A1 - Nayak, Mangalore Manjunatha A1 - Subramanyam, Dinesh Narasimhaiah A1 - Vemula, Praveen Kumar AB -

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.

VL - 159 ER - TY - JOUR T1 - Oxylipin biosynthesis reinforces cellular senescence and allows detection of senolysis. JF - Cell Metab Y1 - 2021 A1 - Wiley, Christopher D A1 - Sharma, Rishi A1 - Davis, Sonnet S A1 - Lopez-Dominguez, Jose Alberto A1 - Mitchell, Kylie P A1 - Wiley, Samantha A1 - Alimirah, Fatouma A1 - Kim, Dong Eun A1 - Payne, Therese A1 - Rosko, Andrew A1 - Aimontche, Eliezer A1 - Deshpande, Sharvari M A1 - Neri, Francesco A1 - Kuehnemann, Chisaka A1 - Demaria, Marco A1 - Ramanathan, Arvind A1 - Campisi, Judith AB -

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.

ER - TY - JOUR T1 - Psychiatric symptoms and syndromes transcending diagnostic boundaries in Indian multiplex families: The cohort of ADBS study. JF - Psychiatry Res Y1 - 2021 A1 - Sreeraj, Vanteemar S A1 - Holla, Bharath A1 - Ithal, Dhruva A1 - Nadella, Ravi Kumar A1 - Mahadevan, Jayant A1 - Balachander, Srinivas A1 - Ali, Furkhan A1 - Sheth, Sweta A1 - Narayanaswamy, Janardhanan C A1 - Venkatasubramanian, Ganesan A1 - John, John P A1 - Varghese, Mathew A1 - Benegal, Vivek A1 - Jain, Sanjeev A1 - Reddy, Yc Janardhan A1 - Viswanath, Biju AB -

Syndromes of schizophrenia, bipolar disorder, obsessive-compulsive disorder, substance use disorders and Alzheimer's dementia are highly heritable. About 10-20% of subjects have another affected first degree relative (FDR), and thus represent a 'greater' genetic susceptibility. We screened 3583 families to identify 481 families with multiple affected members, assessed 1406 individuals in person, and collected information systematically about other relatives. Within the selected families, a third of all FDRs were affected with serious mental illness. Although similar diagnoses aggregated within families, 62% of the families also had members with other syndromes. Moreover, 15% of affected individuals met criteria for co-occurrence of two or more syndromes, across their lifetime. Using dimensional assessments, we detected a range of symptom clusters in both affected and unaffected individuals, and across diagnostic categories. Our findings suggest that in multiplex families, there is considerable heterogeneity of clinical syndromes, as well as sub-threshold symptoms. These families would help provide an opportunity for further research using both genetic analyses and biomarkers.

VL - 296 ER - TY - JOUR T1 - A regulatory network of microRNAs confers lineage commitment during early developmental trajectories of B and T lymphocytes. JF - Proc Natl Acad Sci U S A Y1 - 2021 A1 - Nikhat, Sameena A1 - Yadavalli, Anurupa D A1 - Prusty, Arpita A1 - Narayan, Priyanka K A1 - Palakodeti, Dasaradhi A1 - Murre, Cornelis A1 - Pongubala, Jagan M R KW - Animals KW - B-Lymphocytes KW - Cell Differentiation KW - Cell Lineage KW - Gene Expression KW - Gene Expression Profiling KW - Gene Regulatory Networks KW - Hematopoietic Stem Cells KW - Mice KW - MicroRNAs KW - Myeloid Cells KW - T-Lymphocytes AB -

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.

VL - 118 IS - 46 ER - TY - JOUR T1 - Role of Hypoxia-Mediated Autophagy in Tumor Cell Death and Survival. JF - Cancers (Basel) Y1 - 2021 A1 - Zaarour, Rania F A1 - Azakir, Bilal A1 - Hajam, Edries Y A1 - Nawafleh, Husam A1 - Zeinelabdin, Nagwa A A1 - Engelsen, Agnete S T A1 - Thiery, Jérome A1 - Jamora, Colin A1 - Chouaib, Salem AB -

Programmed cell death or type I apoptosis has been extensively studied and its contribution to the pathogenesis of disease is well established. However, autophagy functions together with apoptosis to determine the overall fate of the cell. The cross talk between this active self-destruction process and apoptosis is quite complex and contradictory as well, but it is unquestionably decisive for cell survival or cell death. Autophagy can promote tumor suppression but also tumor growth by inducing cancer-cell development and proliferation. In this review, we will discuss how autophagy reprograms tumor cells in the context of tumor hypoxic stress. We will illustrate how autophagy acts as both a suppressor and a driver of tumorigenesis through tuning survival in a context dependent manner. We also shed light on the relationship between autophagy and immune response in this complex regulation. A better understanding of the autophagy mechanisms and pathways will undoubtedly ameliorate the design of therapeutics aimed at targeting autophagy for future cancer immunotherapies.

VL - 13 IS - 3 ER - TY - JOUR T1 - Strategies to target SARS-CoV-2 entry and infection using dual mechanisms of inhibition by acidification inhibitors. JF - PLoS Pathog Y1 - 2021 A1 - Prabhakara, Chaitra A1 - Godbole, Rashmi A1 - Sil, Parijat A1 - Jahnavi, Sowmya A1 - Gulzar, Shah-E-Jahan A1 - van Zanten, Thomas S A1 - Sheth, Dhruv A1 - Subhash, Neeraja A1 - Chandra, Anchal A1 - Shivaraj, Akshatha A1 - Panikulam, Patricia A1 - U, Ibrahim A1 - Nuthakki, Vijay Kumar A1 - Puthiyapurayil, Theja Parassini A1 - Ahmed, Riyaz A1 - Najar, Ashaq Hussain A1 - Lingamallu, Sai Manoz A1 - Das, Snigdhadev A1 - Mahajan, Bhagyashri A1 - Vemula, Praveen A1 - Bharate, Sandip B A1 - Singh, Parvinder Pal A1 - Vishwakarma, Ram A1 - Guha, Arjun A1 - Sundaramurthy, Varadharajan A1 - Mayor, Satyajit KW - Ammonium Chloride KW - Angiotensin-Converting Enzyme 2 KW - Animals KW - Antiviral Agents KW - Cell Line KW - Chlorocebus aethiops KW - Chloroquine KW - Clathrin KW - COVID-19 KW - Drug Synergism KW - Endocytosis KW - Endosomes KW - Humans KW - Hydrogen-Ion Concentration KW - Hydroxychloroquine KW - Macrolides KW - Niclosamide KW - Protein Binding KW - Protein Domains KW - SARS-CoV-2 KW - Spike Glycoprotein, Coronavirus KW - Vero Cells KW - Virus Internalization AB -

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.

VL - 17 IS - 7 ER - TY - JOUR T1 - Adverse childhood experiences in families with multiple members diagnosed to have psychiatric illnesses. JF - Aust N Z J Psychiatry Y1 - 2020 A1 - Someshwar, Amala A1 - Holla, Bharath A1 - Pansari Agarwal, Preeti A1 - Thomas, Anza A1 - Jose, Anand A1 - Joseph, Boban A1 - Raju, Birudu A1 - Karle, Hariprasad A1 - Muthukumaran, M A1 - Kodancha, Prabhath G A1 - Kumar, Pramod A1 - Reddy, Preethi V A1 - Kumar Nadella, Ravi A1 - Naik, Sanjay T A1 - Mitra, Sayantanava A1 - Mallappagiri, Sreenivasulu A1 - Sreeraj, Vanteemar S A1 - Balachander, Srinivas A1 - Ganesh, Suhas A1 - Murthy, Pratima A1 - Benegal, Vivek A1 - Reddy, Janardhan Yc A1 - Jain, Sanjeev A1 - Mahadevan, Jayant A1 - Viswanath, Biju AB -

OBJECTIVE: Adverse childhood experiences are linked to the development of a number of psychiatric illnesses in adulthood. Our study examined the pattern of adverse childhood experiences and their relation to the age of onset of major psychiatric conditions in individuals from families that had ⩾2 first-degree relatives with major psychiatric conditions (multiplex families), identified as part of an ongoing longitudinal study.

METHODS: Our sample consisted of 509 individuals from 215 families. Of these, 268 were affected, i.e., diagnosed with bipolar disorder ( = 61), obsessive-compulsive disorder ( = 58), schizophrenia ( = 52), substance dependence ( = 59) or co-occurring diagnoses ( = 38), while 241 were at-risk first-degree relatives who were either unaffected ( = 210) or had other depressive or anxiety disorders ( = 31). All individuals were evaluated using the Adverse Childhood Experiences - International Questionnaire and total adverse childhood experiences exposure and severity scores were calculated.

RESULTS: It was seen that affected males, as a group, had the greatest adverse childhood experiences exposure and severity scores in our sample. A Cox mixed effects model fit by gender revealed that a higher total adverse childhood experiences severity score was associated with significantly increased risk for an earlier age of onset of psychiatric diagnoses in males. A similar model that evaluated the interaction of diagnosis revealed an earlier age of onset in obsessive-compulsive disorder and substance dependence, but not in schizophrenia and bipolar disorder.

CONCLUSION: Our study indicates that adverse childhood experiences were associated with an earlier onset of major psychiatric conditions in men and individuals diagnosed with obsessive-compulsive disorder and substance dependence. Ongoing longitudinal assessments in first-degree relatives from these families are expected to identify mechanisms underlying this relationship.

VL - 54 IS - 11 ER - TY - JOUR T1 - Generic Molding Platform for Simple, Low‐Cost Fabrication of Polymeric Microneedles JF - Macromolecular Materials and Engineering Y1 - 2020 A1 - 7. Badnikar, K. A1 - Jayadevi, S. N. A1 - Pahal, S. A1 - Sripada, S. A1 - Nayak, M. M. A1 - Vemula, P. K. A1 - Subrahmanyam, D. N. AB -

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 µm 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.

VL - 305 IS - 5 ER - TY - JOUR T1 - Genetic, clinical, molecular, and pathogenic aspects of the South Asian-specific polymorphic MYBPC3 variant. JF - Biophys Rev Y1 - 2020 A1 - Arif, Mohammed A1 - Nabavizadeh, Pooneh A1 - Song, Taejeong A1 - Desai, Darshini A1 - Singh, Rohit A1 - Bazrafshan, Sholeh A1 - Kumar, Mohit A1 - Wang, Yigang A1 - Gilbert, Richard J A1 - Dhandapany, Perundurai S A1 - Becker, Richard C A1 - Kranias, Evangelia G A1 - Sadayappan, Sakthivel AB -

Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease characterized by ventricular enlargement, diastolic dysfunction, and increased risk for sudden cardiac death. Sarcomeric genetic defects are the predominant known cause of HCM. In particular, mutations in the myosin-binding protein C gene (MYBPC3) are associated with ~ 40% of all HCM cases in which a genetic basis has been established. A decade ago, our group reported a 25-base pair deletion in intron 32 of MYBPC3 (MYBPC3) that is uniquely prevalent in South Asians and is associated with autosomal dominant cardiomyopathy. Although our studies suggest that this deletion results in left ventricular dysfunction, cardiomyopathies, and heart failure, the precise mechanism by which this variant predisposes to heart disease remains unclear. Increasingly appreciated, however, is the contribution of secondary risk factors, additional mutations, and lifestyle choices in augmenting or modifying the HCM phenotype in MYBPC3 carriers. Therefore, the goal of this review article is to summarize the current research dedicated to understanding the molecular pathophysiology of HCM in South Asians with the MYBPC3 variant. An emphasis is to review the latest techniques currently applied to explore the MYBPC3 pathogenesis and to provide a foundation for developing new diagnostic strategies and advances in therapeutics.

VL - 12 IS - 4 ER - TY - JOUR T1 - Genetic drift and bottleneck do not influence diversity in Toll-like receptor genes at a small spatial scale in a Himalayan passerine. JF - Ecol Evol Y1 - 2020 A1 - Nandakumar, Mridula A1 - Ishtiaq, Farah AB -

Genetic diversity is important for long-term viability of a population. Low genetic diversity reduces persistence and survival of populations and increases susceptibility to diseases. Comparisons of the neutral markers with functional loci such as immune genes [Toll-like receptors; TLR] can provide useful insights into evolutionary potential of a species and how the diversity of pathogens and selection pressures on their hosts are directly linked to their environment. In this study, we compare genetic diversity in neutral (eleven microsatellite loci) and adaptive (seven TLR loci) loci to determine genetic variation in a nonmigratory western Himalayan passerine, the black-throated tit (), distributed across an elevation gradient with varying degree of pathogen-mediated selection pressure. We further compare the diversity in TLR loci with a high-elevation sister species, the white-throated tit (). Our results indicate a lack of population genetic structure in the black-throated tit and signatures of a past bottleneck. In contrast, we found high diversity in TLR loci and locus-specific (TLR7) signatures of pathogen-mediated selection, which was comparable to diversity in the white-throated tit. Levels of diversity at TLR5 locus corresponded very closely with neutral microsatellite variation. We found evidence of positive selection in TLR1LA, TLR5, and TLR7 loci highlighting the importance in pathogen recognition. Our finding demonstrates that reduction in neutral variation does not necessarily lead to reduction in functional genetic diversity and probably helps in revival of population in a widespread species.

VL - 10 IS - 21 ER - TY - JOUR T1 - Lithium response in bipolar disorder correlates with improved cell viability of patient derived cell lines. JF - Sci Rep Y1 - 2020 A1 - Paul, Pradip A1 - Iyer, Shruti A1 - Nadella, Ravi Kumar A1 - Nayak, Rashmitha A1 - Chellappa, Anirudh S A1 - Ambardar, Sheetal A1 - Sud, Reeteka A1 - Sukumaran, Salil K A1 - Purushottam, Meera A1 - Jain, Sanjeev A1 - Viswanath, Biju AB -

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.

VL - 10 IS - 1 ER - TY - JOUR T1 - A novel polyubiquitin chain linkage formed by viral Ubiquitin is resistant to host deubiquitinating enzymes. JF - Biochem J Y1 - 2020 A1 - Negi, Hitendra A1 - Reddy, Pothula Purushotham A1 - Vengayil, Vineeth A1 - Patole, Chhaya A1 - Laxman, Sunil A1 - Das, Ranabir AB -

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.

VL - 477 IS - 12 ER - TY - JOUR T1 - The Role of Dynamic miRISC During Neuronal Development. JF - Front Mol Biosci Y1 - 2020 A1 - Nawalpuri, Bharti A1 - Ravindran, Sreenath A1 - Muddashetty, Ravi S AB -

Activity-dependent protein synthesis plays an important role during neuronal development by fine-tuning the formation and function of neuronal circuits. Recent studies have shown that miRNAs are integral to this regulation because of their ability to control protein synthesis in a rapid, specific and potentially reversible manner. miRNA mediated regulation is a multistep process that involves inhibition of translation before degradation of targeted mRNA, which provides the possibility to store and reverse the inhibition at multiple stages. This flexibility is primarily thought to be derived from the composition of miRNA induced silencing complex (miRISC). AGO2 is likely the only obligatory component of miRISC, while multiple RBPs are shown to be associated with this core miRISC to form diverse miRISC complexes. The formation of these heterogeneous miRISC complexes is intricately regulated by various extracellular signals and cell-specific contexts. In this review, we discuss the composition of miRISC and its functions during neuronal development. Neurodevelopment is guided by both internal programs and external cues. Neuronal activity and external signals play an important role in the formation and refining of the neuronal network. miRISC composition and diversity have a critical role at distinct stages of neurodevelopment. Even though there is a good amount of literature available on the role of miRNAs mediated regulation of neuronal development, surprisingly the role of miRISC composition and its functional dynamics in neuronal development is not much discussed. In this article, we review the available literature on the heterogeneity of the neuronal miRISC composition and how this may influence translation regulation in the context of neuronal development.

VL - 7 ER - TY - JOUR T1 - Altered steady state and activity-dependent de novo protein expression in fragile X syndrome. JF - Nat Commun Y1 - 2019 A1 - Bowling, Heather A1 - Bhattacharya, Aditi A1 - Zhang, Guoan A1 - Alam, Danyal A1 - Lebowitz, Joseph Z A1 - Bohm-Levine, Nathaniel A1 - Lin, Derek A1 - Singha, Priyangvada A1 - Mamcarz, Maggie A1 - Puckett, Rosemary A1 - Zhou, Lili A1 - Aryal, Sameer A1 - Sharp, Kevin A1 - Kirshenbaum, Kent A1 - Berry-Kravis, Elizabeth A1 - Neubert, Thomas A A1 - Klann, Eric AB -

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.

VL - 10 IS - 1 ER - TY - JOUR T1 - BDNF Induced Translation of Limk1 in Developing Neurons Regulates Dendrite Growth by Fine-Tuning Cofilin1 Activity. JF - Front Mol Neurosci Y1 - 2019 A1 - Ravindran, Sreenath A1 - Nalavadi, Vijayalaxmi C A1 - Muddashetty, Ravi S AB -

Dendritic growth and branching are highly regulated processes and are essential for establishing proper neuronal connectivity. There is a critical phase of early dendrite development when these are heavily regulated by external cues such as trophic factors. Brain-derived neurotrophic factor (BDNF) is a major trophic factor known to enhance dendrite growth in cortical neurons, but the molecular underpinnings of this response are not completely understood. We have identified that BDNF induced translational regulation is an important mechanism governing dendrite development in cultured rat cortical neurons. We show that BDNF treatment for 1 h in young neurons leads to translational up-regulation of an important actin regulatory protein LIM domain kinase 1 (Limk1), increasing its level locally in the dendrites. Limk1 is a member of serine/threonine (Ser/Thr) family kinases downstream of the Rho-GTPase pathway. BDNF induced increase in Limk1 levels leads to increased phosphorylation of its target protein cofilin1. We observed that these changes are maintained for long durations of up to 48 h and are mediating increase in number of primary dendrites and total dendrite length. Thus, we show that BDNF induced protein synthesis leads to fine-tuning of the actin cytoskeletal reassembly and thereby mediate dendrite development.

VL - 12 ER - TY - JOUR T1 - Derivation of iPSC lines from two patients with familial Alzheimer's disease from India. JF - Stem Cell Res Y1 - 2019 A1 - Najar, Ashaq H A1 - Sneha, K M A1 - Ashok, Aparna A1 - Babu, Swathy A1 - Subramaniam, Anand G A1 - Kannan, Ramkrishnan A1 - Viswanath, Biju A1 - Purushottam, Meera A1 - Varghese, Mathew A1 - Parvez, Suhel A1 - Panicker, Mitradas M A1 - Mukherjee, Odity A1 - Jain, Sanjeev AB -

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'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.

VL - 34 ER - TY - JOUR T1 - Differential effects of unipolar versus bipolar depression on episodic memory updating. JF - Neurobiol Learn Mem Y1 - 2019 A1 - Dongaonkar, Bhaktee A1 - Hupbach, Almut A1 - Nadel, Lynn A1 - Chattarji, Sumantra AB -

Episodic memories, when reactivated, can be modified or updated by new learning. Since such dynamic memory processes remain largely unexplored in psychiatric disorders, we examined the impact of depression on episodic memory updating. Unipolar and bipolar depression patients, and age/education matched controls, first learned a set of objects (List-1). Two days later, participants in all three groups were either reminded of the first learning session or not followed by the learning of a new set of objects (List-2). Forty-eight hours later, List-1 recall was impaired in unipolar and bipolar patients compared to control participants. Further, as expected, control participants who received a reminder spontaneously recalled items from List-2 during recall of List-1, indicative of an updated List-1 memory. Such spontaneous intrusions were also seen in the unipolar and bipolar patients that received the reminder, suggesting that memory updating was unaffected in these two patient groups despite impaired recall of List 1. Unexpectedly, we observed a trend towards higher intrusions, albeit statistically insignificant, not only in the reminder but also in the no-reminder subgroups of bipolar patients. We probed this further in a second cohort by testing recall of List-2, which was also impaired in both depression groups. Again bipolar patients showed intrusions, but this time in the reverse order from List-1 into List-2, independent of a reminder. Taken together, despite impaired recall, updating of episodic memories was intact and unidirectional in unipolar depression. In contrast, indiscriminate updating, as evidenced by bidirectional interference between episodic memories, was seen in bipolar depression. These findings reveal a novel distinction between unipolar versus bipolar depression using a reactivation-dependent memory updating paradigm.

VL - 161 ER - TY - JOUR T1 - Differential Regulation of Translation by FMRP Modulates eEF2 Mediated Response on NMDAR Activity. JF - Front Mol Neurosci Y1 - 2019 A1 - Paul, Abhik A1 - Nawalpuri, Bharti A1 - Shah, Devanshi A1 - Sateesh, Shruthi A1 - Muddashetty, Ravi S A1 - Clement, James P AB -

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.

VL - 12 ER - TY - JOUR T1 - Exome sequencing in families with severe mental illness identifies novel and rare variants in genes implicated in Mendelian neuropsychiatric syndromes. JF - Psychiatry Clin Neurosci Y1 - 2019 A1 - Ganesh, Suhas A1 - Ahmed P, Husayn A1 - Nadella, Ravi K A1 - More, Ravi P A1 - Seshadri, Manasa A1 - Viswanath, Biju A1 - Rao, Mahendra A1 - Jain, Sanjeev A1 - Mukherjee, Odity KW - Bipolar Disorder KW - Exome KW - Female KW - Genetic Predisposition to Disease KW - Genetic Variation KW - Genome-Wide Association Study KW - Humans KW - Male KW - Pedigree KW - Phenotype KW - Schizophrenia AB -

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 'private' 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.

VL - 73 IS - 1 ER - TY - JOUR T1 - Generation of a set of isogenic, gene-edited iPSC lines homozygous for all main APOE variants and an APOE knock-out line. JF - Stem Cell Res Y1 - 2019 A1 - Schmid, Benjamin A1 - Prehn, Kennie R A1 - Nimsanor, Natakarn A1 - Garcia, Blanca Irene Aldana A1 - Poulsen, Ulla A1 - Jørring, Ida A1 - Rasmussen, Mikkel A A1 - Clausen, Christian A1 - Mau-Holzmann, Ulrike A A1 - Ramakrishna, Sarayu A1 - Muddashetty, Ravi A1 - Steeg, Rachel A1 - Bruce, Kevin A1 - Mackintosh, Peter A1 - Ebneth, Andreas A1 - Holst, Bjørn A1 - Cabrera-Socorro, Alfredo AB -

Alzheimer'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.

VL - 34 ER - TY - JOUR T1 - Natural monomers: A mine for functional and sustainable materials – Occurrence, chemical modification and polymerization JF - Progress in Polymer Science Y1 - 2019 A1 - John, G., A1 - Nagarajan, S., A1 - Vemula, P. K., A1 - Silverman, J.R., A1 - Pillai, C. K. S. AB -

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.

VL - 92 UR - https://www.sciencedirect.com/science/article/pii/S0079670018300273 ER - TY - JOUR T1 - NMDAR mediated translation at the synapse is regulated by MOV10 and FMRP. JF - Mol Brain Y1 - 2019 A1 - Kute, Preeti Madhav A1 - Ramakrishna, Sarayu A1 - Neelagandan, Nagammal A1 - Chattarji, Sumantra A1 - Muddashetty, Ravi S AB -

Protein synthesis is crucial for maintaining synaptic plasticity and synaptic signalling. Here we have attempted to understand the role of RNA binding proteins, Fragile X Mental Retardation Protein (FMRP) and Moloney Leukemia Virus 10 (MOV10) protein in N-Methyl-D-Aspartate Receptor (NMDAR) mediated translation regulation. We show that FMRP is required for translation downstream of NMDAR stimulation and MOV10 is the key specificity factor in this process. In rat cortical synaptoneurosomes, MOV10 in association with FMRP and Argonaute 2 (AGO2) forms the inhibitory complex on a subset of NMDAR responsive mRNAs. On NMDAR stimulation, MOV10 dissociates from AGO2 and promotes the translation of its target mRNAs. FMRP is required to form MOV10-AGO2 inhibitory complex and to promote translation of MOV10 associated mRNAs. Phosphorylation of FMRP appears to be the potential switch for NMDAR mediated translation and in the absence of FMRP, the distinct translation response to NMDAR stimulation is lost. Thus, FMRP and MOV10 have an important regulatory role in NMDAR mediated translation at the synapse.

VL - 12 IS - 1 ER - TY - JOUR T1 - Novel Series of Methyl 3-(Substituted Benzoyl)-7-Substituted-2-Phenylindolizine-1-Carboxylates as Promising Anti-Inflammatory Agents: Molecular Modeling Studies. JF - Biomolecules Y1 - 2019 A1 - Venugopala, Katharigatta N A1 - Al-Attraqchi, Omar H A A1 - Tratrat, Christophe A1 - Nayak, Susanta K A1 - Morsy, Mohamed A A1 - Aldhubiab, Bandar E A1 - Attimarad, Mahesh A1 - Nair, Anroop B A1 - Sreeharsha, Nagaraja A1 - Venugopala, Rashmi A1 - Haroun, Michelyne A1 - Girish, Meravanige B A1 - Chandrashekharappa, Sandeep A1 - Alwassil, Osama I A1 - Odhav, Bharti AB -

The cyclooxygenase-2 (COX-2) enzyme is considered to be an important target for developing novel anti-inflammatory agents. Selective COX-2 inhibitors offer the advantage of lower adverse effects that are commonly associated with non-selective COX inhibitors. In this work, a novel series of methyl 3-(substituted benzoyl)-7-substituted-2-phenylindolizine-1-carboxylates was synthesized and evaluated for COX-2 inhibitory activity. Compound was identified as the most active compound of the series with an IC of 6.71 M, which is comparable to the IC of indomethacin, a marketed non-steroidal anti-inflammatory drug (NSAID). Molecular modeling and crystallographic studies were conducted to further characterize the compounds and gain better understanding of the binding interactions between the compounds and the residues at the active site of the COX-2 enzyme. The pharmacokinetic properties and potential toxic effects were predicted for all the synthesized compounds, which indicated good drug-like properties. Thus, these synthesized compounds can be considered as potential lead compounds for developing effective anti-inflammatory therapeutic agents.

VL - 9 IS - 11 ER - TY - JOUR T1 - Stress Elicits Contrasting Effects on the Structure and Number of Astrocytes in the Amygdala versus Hippocampus. JF - eNeuro Y1 - 2019 A1 - Naskar, Saptarnab A1 - Chattarji, Sumantra AB -

Stress causes divergent patterns of structural and physiological plasticity in the hippocampus versus amygdala. However, a majority of earlier studies focused primarily on neurons. Despite growing evidence for the importance of glia in health and disease, relatively little is known about how stress affects astrocytes. Further, previous work focused on hippocampal astrocytes. Hence, we examined the impact of chronic immobilization stress (2 h/d, 10 d), on the number and structure of astrocytes in the rat hippocampus and amygdala. We observed a reduction in the number of glial fibrillary acidic protein (GFAP)-positive astrocytes in the basal amygdala (BA), 1 d after the end of 10 d of chronic stress. Detailed morphometric analysis of individual dye-filled astrocytes also revealed a decrease in the neuropil volume occupied by these astrocytes in the BA, alongside a reduction in the volume fraction of fine astrocytic protrusions rather than larger dendrite-like processes. By contrast, the same chronic stress had no effect on the number or morphology of astrocytes in hippocampal area CA3. We also confirmed previous reports that chronic stress triggers dendritic hypertrophy in dye-filled BA principal neurons that were located adjacent to astrocytes that had undergone atrophy. Thus, building on earlier evidence for contrasting patterns of stress-induced plasticity in neurons across brain areas, our findings offer new evidence that the same stress can also elicit divergent morphological effects in astrocytes in the hippocampus versus the amygdala.

VL - 6 IS - 1 ER - TY - JOUR T1 - Structural and functional characterization of CMP-N-acetylneuraminate synthetase from Vibrio cholerae. JF - Acta Crystallogr D Struct Biol Y1 - 2019 A1 - Bose, Sucharita A1 - Purkait, Debayan A1 - Joseph, Deepthi A1 - Nayak, Vinod A1 - Subramanian, Ramaswamy KW - Amino Acid Sequence KW - Bacterial Proteins KW - Binding Sites KW - Catalytic Domain KW - Crystallization KW - Crystallography, X-Ray KW - Cytidine Diphosphate KW - Cytidine Monophosphate N-Acetylneuraminic Acid KW - Cytidine Triphosphate KW - N-Acylneuraminate Cytidylyltransferase KW - Protein Interaction Domains and Motifs KW - Protein Structure, Quaternary KW - Sialic Acids KW - Vibrio cholerae AB -

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.

VL - 75 IS - Pt 6 ER - TY - JOUR T1 - Structure-Guided Synthesis and Evaluation of Small-Molecule Inhibitors Targeting Protein-Protein Interactions of BRCA1 tBRCT Domain. JF - ChemMedChem Y1 - 2019 A1 - Kurdekar, Vadiraj A1 - Giridharan, Saranya A1 - Subbarao, Jasti A1 - Nijaguna, Mamatha B A1 - Periasamy, Jayaprakash A1 - Boggaram, Sanjana A1 - Shivange, Amol V A1 - Sadasivam, Gayathri A1 - Padigaru, Muralidhara A1 - Potluri, Vijay A1 - Venkitaraman, Ashok R A1 - Bharatham, Kavitha AB -

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'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.

VL - 14 IS - 18 ER - TY - JOUR T1 - Urolithin A, a Novel Natural Compound to Target PI3K/AKT/mTOR Pathway in Pancreatic Cancer. JF - Mol Cancer Ther Y1 - 2019 A1 - Totiger, Tulasigeri M A1 - Srinivasan, Supriya A1 - Jala, Venkatakrishna R A1 - Lamichhane, Purushottam A1 - Dosch, Austin R A1 - Gaidarski, Alexander A A1 - Joshi, Chandrashekhar A1 - Rangappa, Shobith A1 - Castellanos, Jason A1 - Vemula, Praveen Kumar A1 - Chen, Xi A1 - Kwon, Deukwoo A1 - Kashikar, Nilesh A1 - VanSaun, Michael A1 - Merchant, Nipun B A1 - Nagathihalli, Nagaraj S AB -

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy and is highly resistant to standard treatment regimens. Targeted therapies against , a mutation present in an overwhelming majority of PDAC cases, have been largely ineffective. However, inhibition of downstream components in the KRAS signaling cascade provides promising therapeutic targets in the management of PDAC and warrants further exploration. Here, we investigated Urolithin A (Uro A), a novel natural compound derived from pomegranates, which targets numerous kinases downstream of KRAS, in particular the PI3K/AKT/mTOR signaling pathways. We showed that treatment of PDAC cells with Uro A blocked the phosphorylation of AKT and p70S6K successfully inhibited the growth of tumor xenografts, and increased overall survival of Ptf1a;LSL-Kras;Tgfbr2 (PKT) mice compared with vehicle or gemcitabine therapy alone. Histologic evaluation of these Uro A-treated tumor samples confirmed mechanistic actions of Uro A via decreased phosphorylation of AKT and p70S6K, reduced proliferation, and increased cellular apoptosis in both xenograft and PKT mouse models. In addition, Uro A treatment reprogrammed the tumor microenvironment, as evidenced by reduced levels of infiltrating immunosuppressive cell populations such as myeloid-derived suppressor cells, tumor-associated macrophages, and regulatory T cells. Overall, this work provides convincing preclinical evidence for the utility of Uro A as a therapeutic agent in PDAC through suppression of the PI3K/AKT/mTOR pathway.

VL - 18 IS - 2 ER - TY - JOUR T1 - Automation aided optimization of cloning, expression and purification of enzymes of the bacterial sialic acid catabolic and sialylation pathways enzymes for structural studies. JF - Microb Biotechnol Y1 - 2018 A1 - Bairy, Sneha A1 - Gopalan, Lakshmi Narayanan A1 - Setty, Thanuja Gangi A1 - Srinivasachari, Sathya A1 - Manjunath, Lavanyaa A1 - Kumar, Jay Prakash A1 - Guntupalli, Sai R A1 - Bose, Sucharita A1 - Nayak, Vinod A1 - Ghosh, Swagatha A1 - Sathyanarayanan, Nitish A1 - Caing-Carlsson, Rhawnie A1 - Wahlgren, Weixiao Yuan A1 - Friemann, Rosmarie A1 - Ramaswamy, S A1 - Neerathilingam, Muniasamy AB -

The process of obtaining a well-expressing, soluble and correctly folded constructs can be made easier and quicker by automating the optimization of cloning, expression and purification. While there are many semiautomated pipelines available for cloning, expression and purification, there is hardly any pipeline that involves complete automation. Here, we achieve complete automation of all the steps involved in cloning and in vivo expression screening. This is demonstrated using 18 genes involved in sialic acid catabolism and the surface sialylation pathway. Our main objective was to clone these genes into a His-tagged Gateway vector, followed by their small-scale expression optimization in vivo. The constructs that showed best soluble expression were then selected for purification studies and scaled up for crystallization studies. Our technique allowed us to quickly find conditions for producing significant quantities of soluble proteins in Escherichia coli, their large-scale purification and successful crystallization of a number of these proteins. The method can be implemented in other cases where one needs to screen a large number of constructs, clones and expression vectors for successful recombinant production of functional proteins.

VL - 11 IS - 2 ER - TY - JOUR T1 - Crystal structures and kinetics of N-acetylneuraminate lyase from Fusobacterium nucleatum. JF - Acta Crystallogr F Struct Biol Commun Y1 - 2018 A1 - Kumar, Jay Prakash A1 - Rao, Harshvardhan A1 - Nayak, Vinod A1 - Ramaswamy, S KW - Bacterial Proteins KW - Crystallography, X-Ray KW - Fusobacterium nucleatum KW - Hydrogen Bonding KW - Models, Molecular KW - N-Acetylneuraminic Acid KW - Oxo-Acid-Lyases KW - Protein Conformation KW - Protein Folding KW - Pyruvic Acid KW - Schiff Bases KW - Sequence Alignment KW - Tyrosine AB -

N-Acetyl-D-neuraminic acid lyase (NanA) catalyzes the breakdown of sialic acid (Neu5Ac) to N-acetyl-D-mannosamine (ManNAc) and pyruvate. NanA plays a key role in Neu5Ac catabolism in many pathogenic and bacterial commensals where sialic acid is available as a carbon and nitrogen source. Several pathogens or commensals decorate their surfaces with sialic acids as a strategy to escape host innate immunity. Catabolism of sialic acid is key to a range of host-pathogen interactions. In this study, atomic resolution structures of NanA from Fusobacterium nucleatum (FnNanA) in ligand-free and ligand-bound forms are reported at 2.32 and 1.76 Å resolution, respectively. F. nucleatum is a Gram-negative pathogen that causes gingival and periodontal diseases in human hosts. Like other bacterial N-acetylneuraminate lyases, FnNanA also shares the triosephosphate isomerase (TIM)-barrel fold. As observed in other homologous enzymes, FnNanA forms a tetramer. In order to characterize the structure-function relationship, the steady-state kinetic parameters of the enzyme are also reported.

VL - 74 IS - Pt 11 ER - TY - JOUR T1 - Discovery biology of neuropsychiatric syndromes (DBNS): a center for integrating clinical medicine and basic science. JF - BMC Psychiatry Y1 - 2018 A1 - Viswanath, Biju A1 - Rao, Naren P A1 - Narayanaswamy, Janardhanan C A1 - Sivakumar, Palanimuthu T A1 - Kandasamy, Arun A1 - Kesavan, Muralidharan A1 - Mehta, Urvakhsh Meherwan A1 - Venkatasubramanian, Ganesan A1 - John, John P A1 - Mukherjee, Odity A1 - Purushottam, Meera A1 - Kannan, Ramakrishnan A1 - Mehta, Bhupesh A1 - Kandavel, Thennarasu A1 - Binukumar, B A1 - Saini, Jitender A1 - Jayarajan, Deepak A1 - Shyamsundar, A A1 - Moirangthem, Sydney A1 - Vijay Kumar, K G A1 - Thirthalli, Jagadisha A1 - Chandra, Prabha S A1 - Gangadhar, Bangalore N A1 - Murthy, Pratima A1 - Panicker, Mitradas M A1 - Bhalla, Upinder S A1 - Chattarji, Sumantra A1 - Benegal, Vivek A1 - Varghese, Mathew A1 - Reddy, Janardhan Y C A1 - Raghu, Padinjat A1 - Rao, Mahendra A1 - Jain, Sanjeev AB -

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'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.

VL - 18 IS - 1 ER - TY - JOUR T1 - "Just a spoonful of sugar...": import of sialic acid across bacterial cell membranes. JF - Biophys Rev Y1 - 2018 A1 - North, Rachel A A1 - Horne, Christopher R A1 - Davies, James S A1 - Remus, Daniela M A1 - Muscroft-Taylor, Andrew C A1 - Goyal, Parveen A1 - Wahlgren, Weixiao Yuan A1 - Ramaswamy, S A1 - Friemann, Rosmarie A1 - Dobson, Renwick C J AB -

Eukaryotic cell surfaces are decorated with a complex array of glycoconjugates that are usually capped with sialic acids, a large family of over 50 structurally distinct nine-carbon amino sugars, the most common member of which is N-acetylneuraminic acid. Once made available through the action of neuraminidases, bacterial pathogens and commensals utilise host-derived sialic acid by degrading it for energy or repurposing the sialic acid onto their own cell surface to camouflage the bacterium from the immune system. A functional sialic acid transporter has been shown to be essential for the uptake of sialic acid in a range of human bacterial pathogens and important for host colonisation and persistence. Here, we review the state-of-play in the field with respect to the molecular mechanisms by which these bio-nanomachines transport sialic acids across bacterial cell membranes.

VL - 10 IS - 2 ER - TY - JOUR T1 - The Sodium Sialic Acid Symporter From Has Altered Substrate Specificity. JF - Front Chem Y1 - 2018 A1 - North, Rachel A A1 - Wahlgren, Weixiao Y A1 - Remus, Daniela M A1 - Scalise, Mariafrancesca A1 - Kessans, Sarah A A1 - Dunevall, Elin A1 - Claesson, Elin A1 - Soares da Costa, Tatiana P A1 - Perugini, Matthew A A1 - Ramaswamy, S A1 - Allison, Jane R A1 - Indiveri, Cesare A1 - Friemann, Rosmarie A1 - Dobson, Renwick C J AB -

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.

VL - 6 ER - TY - JOUR T1 - Substrate-bound outward-open structure of a Na-coupled sialic acid symporter reveals a new Na site. JF - Nat Commun Y1 - 2018 A1 - Wahlgren, Weixiao Y A1 - Dunevall, Elin A1 - North, Rachel A A1 - Paz, Aviv A1 - Scalise, Mariafrancesca A1 - Bisignano, Paola A1 - Bengtsson-Palme, Johan A1 - Goyal, Parveen A1 - Claesson, Elin A1 - Caing-Carlsson, Rhawnie A1 - Andersson, Rebecka A1 - Beis, Konstantinos A1 - Nilsson, Ulf J A1 - Farewell, Anne A1 - Pochini, Lorena A1 - Indiveri, Cesare A1 - Grabe, Michael A1 - Dobson, Renwick C J A1 - Abramson, Jeff A1 - Ramaswamy, S A1 - Friemann, Rosmarie AB -

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 Å 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.

VL - 9 IS - 1 ER - TY - JOUR T1 - Targeting Phosphopeptide Recognition by the Human BRCA1 Tandem BRCT Domain to Interrupt BRCA1-Dependent Signaling. JF - Cell Chem Biol Y1 - 2018 A1 - Periasamy, Jayaprakash A1 - Kurdekar, Vadiraj A1 - Jasti, Subbarao A1 - Nijaguna, Mamatha B A1 - Boggaram, Sanjana A1 - Hurakadli, Manjunath A A1 - Raina, Dhruv A1 - Kurup, Lokavya Meenakshi A1 - Chintha, Chetan A1 - Manjunath, Kavyashree A1 - Goyal, Aneesh A1 - Sadasivam, Gayathri A1 - Bharatham, Kavitha A1 - Padigaru, Muralidhara A1 - Potluri, Vijay A1 - Venkitaraman, Ashok R AB -

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 Förster 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.

VL - 25 IS - 6 ER - TY - JOUR T1 - Tunable Emission from Fluorescent Organic Nanoparticles in Water: Insight into the Nature of Self‐Assembly and Photoswitching JF - Chemistry – A European Journal Y1 - 2018 A1 - Gulyani Akash A1 - Dey Nilanjan A1 - Bhattacharya Santanu KW - aggregation KW - Fluorescence KW - nanoparticles KW - self-assembly AB -

Abstract Excitation‐dependent tuning of the emission behavior of fluorescent organic nanoparticles (FONs) with two simple luminescent pyrenyl–pyridyl conjugates as model systems is demonstrated. In the case of the compound with a flexible bis‐picolyl moiety, the simultaneous presence of multiple ground‐state species with distinct absorption and emission characteristics can be observed. The relative ratios of these species can easily be modulated, and it is possible to selectively stimulate any one of them individually by choosing an appropriate excitation channel. Moreover, at high concentration, a drastic change in the nature of the self‐assembly is observed, which shifts from donor–acceptor‐type self‐assembly to exciplex‐type self‐agglomeration. On the contrary, the compound containing a rigid terpyridine unit has only a single ground state and shows no such tunable emission. However, it can exhibit multiple emission bands in water, whereby the positions of their emission maxima depend on the extent of aggregation‐induced planarization of the probe molecules. Overall, this work demonstrates multimodal modulation of FON emission and a gives insight into how molecular order can translate into complete switching of nanoparticle self‐assembly and photophysics.

VL - 24 UR - https://onlinelibrary.wiley.com/doi/abs/10.1002/chem.201704607 ER - TY - JOUR T1 - Crystal structure of N-acetylmannosamine kinase from Fusobacterium nucleatum. JF - Acta Crystallogr F Struct Biol Commun Y1 - 2017 A1 - Caing-Carlsson, Rhawnie A1 - Goyal, Parveen A1 - Sharma, Amit A1 - Ghosh, Swagatha A1 - Setty, Thanuja Gangi A1 - North, Rachel A A1 - Friemann, Rosmarie A1 - Ramaswamy, S KW - Adenosine Triphosphate KW - Amino Acid Sequence KW - Bacterial Proteins KW - Binding Sites KW - Cloning, Molecular KW - Crystallography, X-Ray KW - Escherichia coli KW - Fusobacterium nucleatum KW - Gene Expression KW - Genetic Vectors KW - Hexosamines KW - Models, Molecular KW - Phosphotransferases (Alcohol Group Acceptor) KW - Protein Binding KW - Protein Conformation, alpha-Helical KW - Protein Conformation, beta-Strand KW - Protein Interaction Domains and Motifs KW - Protein Multimerization KW - Recombinant Proteins KW - Sequence Alignment KW - Sequence Homology, Amino Acid KW - Substrate Specificity AB -

Sialic acids comprise a varied group of nine-carbon amino sugars that are widely distributed among mammals and higher metazoans. Some human commensals and bacterial pathogens can scavenge sialic acids from their environment and degrade them for use as a carbon and nitrogen source. The enzyme N-acetylmannosamine kinase (NanK; EC 2.7.1.60) belongs to the transcriptional repressors, uncharacterized open reading frames and sugar kinases (ROK) superfamily. NanK catalyzes the second step of the sialic acid catabolic pathway, transferring a phosphate group from adenosine 5'-triphosphate to the C6 position of N-acetylmannosamine to generate N-acetylmannosamine 6-phosphate. The structure of NanK from Fusobacterium nucleatum was determined to 2.23 Å resolution by X-ray crystallography. Unlike other NanK enzymes and ROK family members, F. nucleatum NanK does not have a conserved zinc-binding site. In spite of the absence of the zinc-binding site, all of the major structural features of enzymatic activity are conserved.

VL - 73 IS - Pt 6 ER - TY - JOUR T1 - Cytoplasmic poly (A)-binding protein critically regulates epidermal maintenance and turnover in the planarian . JF - Development Y1 - 2017 A1 - Bansal, Dhiru A1 - Kulkarni, Jahnavi A1 - Nadahalli, Kavana A1 - Lakshmanan, Vairavan A1 - Krishna, Srikar A1 - Sasidharan, Vidyanand A1 - Geo, Jini A1 - Dilipkumar, Shilpa A1 - Pasricha, Renu A1 - Gulyani, Akash A1 - Raghavan, Srikala A1 - Palakodeti, Dasaradhi KW - Animals KW - Cell Lineage KW - Cell Proliferation KW - Cytoplasm KW - Epidermis KW - Epithelium KW - Extracellular Matrix KW - Gene Knockdown Techniques KW - Homeostasis KW - Models, Biological KW - Planarians KW - Poly(A)-Binding Protein I KW - Regeneration KW - RNA, Messenger KW - Wound Healing AB -

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.

VL - 144 IS - 17 ER - TY - JOUR T1 - The miR-124 family of microRNAs is crucial for regeneration of the brain and visual system in the planarian Schmidtea mediterranea JF - Development Y1 - 2017 A1 - Sasidharan, Vidyanand A1 - Marepally, Srujan A1 - Elliott, Sarah A. A1 - Baid, Srishti A1 - Lakshmanan, Vairavan A1 - Nayyar, Nishtha A1 - Bansal, Dhiru A1 - Sánchez Alvarado, Alejandro A1 - Vemula, Praveen Kumar A1 - Palakodeti, Dasaradhi AB -

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.

VL - 144 UR - http://dev.biologists.org/content/144/18/3211 ER - TY - JOUR T1 - Morphology transition in helical tubules of a supramolecular gel driven by metal ions. JF - Chem Commun (Camb) Y1 - 2017 A1 - Lalitha, Krishnamoorthy A1 - Sridharan, Vellaisamy A1 - Maheswari, C Uma A1 - Vemula, Praveen Kumar A1 - Nagarajan, Subbiah AB -

Our aim to access a particular chemical functionality on helical tubules has been achieved by the rational molecular design and synthesis of glucono-appended cardanol derivatives. For the first time, we report a chiral molecular packing with α-helical tubules, and chiral symmetry-breaking upon exposure to Cu that generated the final ordered structure via an in situ morphological transition without undergoing any phase change.

VL - 53 IS - 9 ER - TY - JOUR T1 - Scaling the effect of hydrophobic chain length on gene transfer properties of di-alkyl{,} di-hydroxy ethylammonium chloride based cationic amphiphiles JF - RSC Adv. Y1 - 2017 A1 - Hiwale, Ankita A. A1 - Voshavar, Chandrashekhar A1 - Dharmalingam, Priya A1 - Dhayani, Ashish A1 - Mukthavaram, Rajesh A1 - Nadella, Rasajna A1 - Sunnapu, Omprakash A1 - Gandhi, Sivaraman A1 - Naidu, V. G. M. A1 - Chaudhuri, Arabinda A1 - Marepally, Srujan A1 - Vemula, Praveen Kumar AB -

The success of gene therapy critically depends on the availability of efficient transfection vectors. Cationic lipids are the most widely studied non-viral vectors. The molecular architecture of the cationic lipid determines its transfection efficiency. Variations in alkyl chain lengths of lipids influence self-assembly and liposomal fusion with the cell membrane. These factors determine the transfection ability of the lipid. Thus{,} to probe the effect of asymmetry in hydrophobic chains on transfection efficiency{,} we designed and synthesized a series of cationic lipids by systematically varying one of the two alkyl chains linked to the quaternary nitrogen centre from C18 to C10 and keeping the other alkyl C18 chain constant (Lip1818-Lip1810). Transfection studies in multiple cultured mammalian cells (CHO{,} B16F10 and HeLa) revealed that the lipids with C18:C14 and C18:C12 alkyl chains (Lip1814 & Lip1812) showed 20-30% higher transfection efficacies than their counterparts at 2 : 1 and 4 : 1 lipid to pDNA charge ratios. Cryo-transmission electron images showed unilamellar vesicle structures for the liposomes of lipids. Mechanistic studies involving Small Angle X-ray Scattering (SAXS) revealed that asymmetry in the hydrophobic region has a significant impact on liposomal fusion with the plasma membrane model. Collectively{,} these findings demonstrate that chain length asymmetry in the hydrophobic region of cationic lipids has an important role in their liposome-DNA interactions at optimal 2 : 1 and 4 : 1 lipid to pDNA charge ratios{,} which in turn modulates their gene transfer properties.

VL - 7 UR - http://dx.doi.org/10.1039/C7RA02271A ER - TY - JOUR T1 - Low Oxygen Tension Enhances Expression of Myogenic Genes When Human Myoblasts Are Activated from G0 Arrest. JF - PLoS One Y1 - 2016 A1 - Sellathurai, Jeeva A1 - Nielsen, Joachim A1 - Hejbøl, Eva Kildall A1 - Jørgensen, Louise Helskov A1 - Dhawan, Jyotsna A1 - Nielsen, Michael Friberg Bruun A1 - Schrøder, Henrik Daa KW - Adolescent KW - Cell Cycle Checkpoints KW - Cell Hypoxia KW - Cell Proliferation KW - Cell Separation KW - Cells, Cultured KW - Down-Regulation KW - Female KW - Gene Expression Regulation KW - Humans KW - Ki-67 Antigen KW - Male KW - Muscle Development KW - Muscle Proteins KW - Myoblasts KW - Oxygen KW - Receptors, Notch KW - Resting Phase, Cell Cycle KW - Transforming Growth Factor beta KW - Wnt Signaling Pathway KW - Young Adult AB -

OBJECTIVES: Most cell culture studies have been performed at atmospheric oxygen tension of 21%, however the physiological oxygen tension is much lower and is a factor that may affect skeletal muscle myoblasts. In this study we have compared activation of G0 arrested myoblasts in 21% O2 and in 1% O2 in order to see how oxygen tension affects activation and proliferation of human myoblasts.

MATERIALS AND METHODS: Human myoblasts were isolated from skeletal muscle tissue and G0 arrested in vitro followed by reactivation at 21% O2 and 1% O2. The effect was assesses by Real-time RT-PCR, immunocytochemistry and western blot.

RESULTS AND CONCLUSIONS: We found an increase in proliferation rate of myoblasts when activated at a low oxygen tension (1% O2) compared to 21% O2. In addition, the gene expression studies showed up regulation of the myogenesis related genes PAX3, PAX7, MYOD, MYOG (myogenin), MET, NCAM, DES (desmin), MEF2A, MEF2C and CDH15 (M-cadherin), however, the fraction of DES and MYOD positive cells was not increased by low oxygen tension, indicating that 1% O2 may not have a functional effect on the myogenic response. Furthermore, the expression of genes involved in the TGFβ, Notch and Wnt signaling pathways were also up regulated in low oxygen tension. The differences in gene expression were most pronounced at day one after activation from G0-arrest, thus the initial activation of myoblasts seemed most sensitive to changes in oxygen tension. Protein expression of HES1 and β-catenin indicated that notch signaling may be induced in 21% O2, while the canonical Wnt signaling may be induced in 1% O2 during activation and proliferation of myoblasts.

VL - 11 IS - 7 ER - TY - JOUR T1 - Mechanistic heterogeneity in contractile properties of α-tropomyosin (TPM1) mutants associated with inherited cardiomyopathies. JF - J Biol Chem Y1 - 2015 A1 - Gupte, Tejas M A1 - Haque, Farah A1 - Gangadharan, Binnu A1 - Sunitha, Margaret S A1 - Mukherjee, Souhrid A1 - Anandhan, Swetha A1 - Rani, Deepa Selvi A1 - Mukundan, Namita A1 - Jambekar, Amruta A1 - Thangaraj, Kumarasamy A1 - Sowdhamini, Ramanathan A1 - Sommese, Ruth F A1 - Nag, Suman A1 - Spudich, James A A1 - Mercer, John A KW - Actins KW - Adenosine Triphosphatases KW - Calcium KW - Cardiomyopathies KW - Humans KW - Models, Molecular KW - Myosins KW - Point Mutation KW - Protein Stability KW - Tropomyosin AB -

The most frequent known causes of primary cardiomyopathies are mutations in the genes encoding sarcomeric proteins. Among those are 30 single-residue mutations in TPM1, the gene encoding α-tropomyosin. We examined seven mutant tropomyosins, E62Q, D84N, I172T, L185R, S215L, D230N, and M281T, that were chosen based on their clinical severity and locations along the molecule. The goal of our study was to determine how the biochemical characteristics of each of these mutant proteins are altered, which in turn could provide a structural rationale for treatment of the cardiomyopathies they produce. Measurements of Ca(2+) sensitivity of human β-cardiac myosin ATPase activity are consistent with the hypothesis that hypertrophic cardiomyopathies are hypersensitive to Ca(2+) activation, and dilated cardiomyopathies are hyposensitive. We also report correlations between ATPase activity at maximum Ca(2+) concentrations and conformational changes in TnC measured using a fluorescent probe, which provide evidence that different substitutions perturb the structure of the regulatory complex in different ways. Moreover, we observed changes in protein stability and protein-protein interactions in these mutants. Our results suggest multiple mechanistic pathways to hypertrophic and dilated cardiomyopathies. Finally, we examined a computationally designed mutant, E181K, that is hypersensitive, confirming predictions derived from in silico structural analysis.

VL - 290 IS - 11 ER - TY - JOUR T1 - A quantitative metabolomics peek into planarian regeneration. JF - Analyst Y1 - 2015 A1 - Natarajan, Nivedita A1 - Ramakrishnan, Padma A1 - Lakshmanan, Vairavan A1 - Palakodeti, Dasaradhi A1 - Rangiah, Kannan KW - Animals KW - Calibration KW - Chromatography, High Pressure Liquid KW - Limit of Detection KW - Metabolomics KW - Planarians KW - Reference Standards KW - Regeneration KW - Reproduction, Asexual KW - Species Specificity KW - Tandem Mass Spectrometry AB -

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.

VL - 140 IS - 10 ER -