%0 Journal Article %J J Med Virol %D 2022 %T Age-stratified adeno-associated virus serotype 3 neutralizing and total antibody prevalence in hemophilia A patients from India. %A Daniel, Hubert D-J %A Kumar, Sanjay %A Kannangai, Rajesh %A J, Farzana %A Joel, Joseph N %A Abraham, Aby %A Lakshmi, Kavitha M %A Agbandje-McKenna, Mavis %A Coleman, Kirsten E %A Srivastava, Arun %A Srivastava, Alok %A Abraham, Asha M %K Adult %K Animals %K Antibodies, Neutralizing %K Antibodies, Viral %K Child %K Dependovirus %K Genetic Vectors %K Hemophilia A %K Humans %K Prevalence %K Serogroup %X

Gene therapy using an adeno-associated virus (AAV) vector offers a new treatment option for individuals with monogenetic disorders. The major bottleneck is the presence of pre-existing anti-AAV antibodies, which impacts its use. Even very low titers of neutralizing antibodies (NAb) to capsids from natural AAV infections have been reported to inhibit the transduction of intravenously administered AAV in animal models and are associated with limited efficacy in human trials. Assessing the level of pre-existing NAb is important for determining the primary eligibility of patients for AAV vector-based gene therapy clinical trials. Techniques used to screen AAV-antibodies include AAV capsid enzyme-linked immunosorbent assay (ELISA) and transduction inhibition assay (TIA) for detecting total capsid-binding (TAb) and Nab, respectively. In this study, we screened 521 individuals with hemophilia A from India for TAb and NAb using ELISA and TIA, respectively. The prevalence of TAb and NAb in hemophilia A patients from India were 96% and 77.5%, respectively. There was a significant increase in anti-AAV3 NAb prevalence with age in the hemophilia A patient group from India. There was a trend in anti-AAV3 TAb positivity between the pediatric age group (94.4%) and the adult age group (97.4%).

%B J Med Virol %V 94 %P 4542-4547 %8 2022 Sep %G eng %N 9 %R 10.1002/jmv.27859 %0 Generic %D 2022 %T An Amphiphilic Double-Brush Polymer Hydrogel for Sustained Release of Small Molecules and Biologics: Insulin Delivering-Hydrogel to Control Hyperglycemia %A Dhayani, Ashish %A Bej, Sujoy %A Mudnakudu-Nagaraju, Kiran K %A Chakraborty, Saheli %A Srinath, Preetham %A Kumar, Ashok H %A PS, Ann Maria %A Kristi, Anand %A Ramakrishnan, S %A Vemula PK %G eng %R https://doi.org/10.1002/cnma.202200184 %0 Journal Article %J J Orthop %D 2022 %T Assessment of the inherent chondrogenic potential of human articular cartilage-derived chondroprogenitors in pellet culture using a novel whole pellet processing approach. %A Johnson, Noel Naveen %A Amirtham, Soosai Manickam %A Sandya Rani, B %A Sathishkumar, Solomon %A Rebekah, Grace %A Vinod, Elizabeth %X

Purpose: Cartilage-derived chondroprogenitors have been reported to possess the biological potential for cartilage repair. However, its inherent chondrogenic potential in pellet culture needs evaluation. In-vitro cartilage regeneration models based on pellet cultures have been employed to evaluate the chondrogenic potential of stem cells. Evaluation of the degree of differentiation routinely involves paraffin embedding, sectioning, and immunohistochemical staining of the pellet. However, since chondrogenic differentiation is commonly non-uniform, processing random sections could lead to inaccurate conclusions. The study aimed at assessing the inherent lineage bias of chondroprogenitors with and without chondrogenic induction, using a novel whole pellet processing technique.

Methods: Human chondroprogenitors (n=3) were evaluated for MSC markers and processed in pellet cultures either with stromal medium (uninduced) or chondrogenic differentiation medium (induced) for 28 days. The whole pellets and the conventional paraffin-embedded sectioned pellets were subjected to Collagen type II immunostaining and assessed using confocal laser microscopy. The staining intensities of the whole pellet were compared to the paraffin sections and revalidated using qRT-PCR for COL2A1 expression.

Results: Uninduced and induced pellets displayed Collagen type II in all the layers with comparable fluorescence intensities. COL2A1 expression in both pellets was comparable to confocal results. The study demonstrated that uninduced chondroprogenitors in pellet culture possess promising inherent chondrogenic potential. Confocal imaging of whole pellets displayed different degrees of chondrogenic differentiation in the entire pellet, thus its probable in-vivo behavior.

Conclusion: The novel approach presented in this study could serve as an efficient in-vitro alternative for understanding translational application for cartilage repair.

%B J Orthop %V 31 %P 45-51 %8 2022 May-Jun %G eng %R 10.1016/j.jor.2022.03.007 %0 Journal Article %J Sci Adv %D 2021 %T Adiponectin receptor 1 variants contribute to hypertrophic cardiomyopathy that can be reversed by rapamycin. %A Dhandapany, Perundurai S %A Kang, Soojeong %A Kashyap, Deepak K %A Rajagopal, Raksha %A Sundaresan, Nagalingam R %A Singh, Rajvir %A Thangaraj, Kumarasamy %A Jayaprakash, Shilpa %A Manjunath, Cholenahally N %A Shenthar, Jayaprakash %A Lebeche, Djamel %X

Hypertrophic cardiomyopathy (HCM) is a heterogeneous genetic heart muscle disease characterized by hypertrophy with preserved or increased ejection fraction in the absence of secondary causes. However, recent studies have demonstrated that a substantial proportion of individuals with HCM also have comorbid diabetes mellitus (~10%). Whether genetic variants may contribute a combined phenotype of HCM and diabetes mellitus is not known. Here, using next-generation sequencing methods, we identified novel and ultrarare variants in adiponectin receptor 1 () as risk factors for HCM. Biochemical studies showed that variants dysregulate glucose and lipid metabolism and cause cardiac hypertrophy through the p38/mammalian target of rapamycin and/or extracellular signal-regulated kinase pathways. A transgenic mouse model expressing an variant displayed cardiomyopathy that recapitulated the cellular findings, and these features were rescued by rapamycin. Our results provide the first evidence that variants can cause HCM and provide new insights into regulation.

%B Sci Adv %V 7 %8 2021 Jan %G eng %N 2 %R 10.1126/sciadv.abb3991 %0 Journal Article %J Sci Rep %D 2021 %T Analysis of whole exome sequencing in severe mental illness hints at selection of brain development and immune related genes. %A Mahadevan, Jayant %A Pathak, Ajai Kumar %A Vemula, Alekhya %A Nadella, Ravi Kumar %A Viswanath, Biju %A Jain, Sanjeev %A Purushottam, Meera %A Mondal, Mayukh %X

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.

%B Sci Rep %V 11 %P 21088 %8 2021 Oct 26 %G eng %N 1 %R 10.1038/s41598-021-00123-x %0 Journal Article %J J Neurosci %D 2021 %T APOE4 Affects Basal and NMDAR-Mediated Protein Synthesis in Neurons by Perturbing Calcium Homeostasis. %A Ramakrishna, Sarayu %A Jhaveri, Vishwaja %A Konings, Sabine C %A Nawalpuri, Bharti %A Chakraborty, Sumita %A Holst, Bjørn %A Schmid, Benjamin %A Gouras, Gunnar K %A Freude, Kristine K %A Muddashetty, Ravi S %X

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.

%B J Neurosci %V 41 %P 8686-8709 %8 2021 Oct 20 %G eng %N 42 %R 10.1523/JNEUROSCI.0435-21.2021 %0 Journal Article %J Stem Cell Reports %D 2021 %T Astrocytic reactivity triggered by defective autophagy and metabolic failure causes neurotoxicity in frontotemporal dementia type 3. %A Chandrasekaran, Abinaya %A Dittlau, Katarina Stoklund %A Corsi, Giulia I %A Haukedal, Henriette %A Doncheva, Nadezhda T %A Ramakrishna, Sarayu %A Ambardar, Sheetal %A Salcedo, Claudia %A Schmidt, Sissel I %A Zhang, Yu %A Cirera, Susanna %A Pihl, Maria %A Schmid, Benjamin %A Nielsen, Troels Tolstrup %A Nielsen, Jørgen E %A Kolko, Miriam %A Kobolák, Julianna %A Dinnyés, András %A Hyttel, Poul %A Palakodeti, Dasaradhi %A Gorodkin, Jan %A Muddashetty, Ravi S %A Meyer, Morten %A Aldana, Blanca I %A Freude, Kristine K %X

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.

%B Stem Cell Reports %V 16 %P 2736-2751 %8 2021 Nov 09 %G eng %N 11 %R 10.1016/j.stemcr.2021.09.013 %0 Journal Article %J Aust N Z J Psychiatry %D 2020 %T Adverse childhood experiences in families with multiple members diagnosed to have psychiatric illnesses. %A Someshwar, Amala %A Holla, Bharath %A Pansari Agarwal, Preeti %A Thomas, Anza %A Jose, Anand %A Joseph, Boban %A Raju, Birudu %A Karle, Hariprasad %A Muthukumaran, M %A Kodancha, Prabhath G %A Kumar, Pramod %A Reddy, Preethi V %A Kumar Nadella, Ravi %A Naik, Sanjay T %A Mitra, Sayantanava %A Mallappagiri, Sreenivasulu %A Sreeraj, Vanteemar S %A Balachander, Srinivas %A Ganesh, Suhas %A Murthy, Pratima %A Benegal, Vivek %A Reddy, Janardhan Yc %A Jain, Sanjeev %A Mahadevan, Jayant %A Viswanath, Biju %X

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.

%B Aust N Z J Psychiatry %V 54 %P 1086-1094 %8 2020 Nov %G eng %N 11 %R 10.1177/0004867420931157 %0 Journal Article %J Proteomics %D 2020 %T The Aging Metabolome-Biomarkers to Hub Metabolites. %A Sharma, Rishi %A Ramanathan, Arvind %X

Aging biology is intimately associated with dysregulated metabolism, which is one of the hallmarks of aging. Aging-related pathways such as mTOR and AMPK, which are major targets of anti-aging interventions including rapamcyin, metformin, and exercise, either directly regulate or intersect with metabolic pathways. In this review, numerous candidate bio-markers of aging that have emerged using metabolomics are outlined. Metabolomics studies also reveal that not all metabolites are created equally. A set of core "hub" metabolites are emerging as central mediators of aging. The hub metabolites reviewed here are nicotinamide adenine dinucleotide, reduced nicotinamide dinucleotide phosphate, α-ketoglutarate, and β-hydroxybutyrate. These "hub" metabolites have signaling and epigenetic roles along with their canonical roles as co-factors or intermediates of carbon metabolism. Together these hub metabolites suggest a central role of the TCA cycle in signaling and metabolic dysregulation associated with aging.

%B Proteomics %V 20 %P e1800407 %8 2020 03 %G eng %N 5-6 %R 10.1002/pmic.201800407 %0 Journal Article %J J Biol Chem %D 2020 %T Allosteric inhibition of MTHFR prevents futile SAM cycling and maintains nucleotide pools in one-carbon metabolism. %A Bhatia, Muskan %A Thakur, Jyotika %A Suyal, Shradha %A Oniel, Ruchika %A Chakraborty, Rahul %A Pradhan, Shalini %A Sharma, Monika %A Sengupta, Shantanu %A Laxman, Sunil %A Masakapalli, Shyam Kumar %A Bachhawat, Anand Kumar %K Adenosine Triphosphate %K Allosteric Regulation %K Humans %K Methylation %K Methylenetetrahydrofolate Reductase (NADPH2) %K S-Adenosylmethionine %K Saccharomyces cerevisiae %K Saccharomyces cerevisiae Proteins %X

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

%B J Biol Chem %V 295 %P 16037-16057 %8 2020 11 20 %G eng %N 47 %R 10.1074/jbc.RA120.015129 %0 Journal Article %J Mol Cell Biol %D 2020 %T Anabolic SIRT4 Exerts Retrograde Control over TORC1 Signaling by Glutamine Sparing in the Mitochondria. %A Shaw, Eisha %A Talwadekar, Manasi %A Rashida, Zeenat %A Mohan, Nitya %A Acharya, Aishwarya %A Khatri, Subhash %A Laxman, Sunil %A Kolthur-Seetharam, Ullas %X

Anabolic and catabolic signaling mediated via mTOR and AMPK (AMP-activated kinase) have to be intrinsically coupled to mitochondrial functions for maintaining homeostasis and mitigate cellular/organismal stress. Although glutamine is known to activate mTOR, whether and how differential mitochondrial utilization of glutamine impinges on mTOR signaling has been less explored. Mitochondrial SIRT4, which unlike other sirtuins is induced in a fed state, is known to inhibit catabolic signaling/pathways through the AMPK-PGC1α/SIRT1-peroxisome proliferator-activated receptor α (PPARα) axis and negatively regulate glutamine metabolism via the tricarboxylic acid cycle. However, physiological significance of SIRT4 functions during a fed state is still unknown. Here, we establish SIRT4 as key anabolic factor that activates TORC1 signaling and regulates lipogenesis, autophagy, and cell proliferation. Mechanistically, we demonstrate that the ability of SIRT4 to inhibit anaplerotic conversion of glutamine to α-ketoglutarate potentiates TORC1. Interestingly, we also show that mitochondrial glutamine sparing or utilization is critical for differentially regulating TORC1 under fed and fasted conditions. Moreover, we conclusively show that differential expression of SIRT4 during fed and fasted states is vital for coupling mitochondrial energetics and glutamine utilization with anabolic pathways. These significant findings also illustrate that SIRT4 integrates nutrient inputs with mitochondrial retrograde signals to maintain a balance between anabolic and catabolic pathways.

%B Mol Cell Biol %V 40 %8 2020 Jan 03 %G eng %N 2 %R 10.1128/MCB.00212-19 %0 Journal Article %J Nat Commun %D 2019 %T Altered steady state and activity-dependent de novo protein expression in fragile X syndrome. %A Bowling, Heather %A Bhattacharya, Aditi %A Zhang, Guoan %A Alam, Danyal %A Lebowitz, Joseph Z %A Bohm-Levine, Nathaniel %A Lin, Derek %A Singha, Priyangvada %A Mamcarz, Maggie %A Puckett, Rosemary %A Zhou, Lili %A Aryal, Sameer %A Sharp, Kevin %A Kirshenbaum, Kent %A Berry-Kravis, Elizabeth %A Neubert, Thomas A %A Klann, Eric %X

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.

%B Nat Commun %V 10 %P 1710 %8 2019 Apr 12 %G eng %N 1 %R 10.1038/s41467-019-09553-8 %0 Journal Article %J Microb Biotechnol %D 2018 %T Automation aided optimization of cloning, expression and purification of enzymes of the bacterial sialic acid catabolic and sialylation pathways enzymes for structural studies. %A Bairy, Sneha %A Gopalan, Lakshmi Narayanan %A Setty, Thanuja Gangi %A Srinivasachari, Sathya %A Manjunath, Lavanyaa %A Kumar, Jay Prakash %A Guntupalli, Sai R %A Bose, Sucharita %A Nayak, Vinod %A Ghosh, Swagatha %A Sathyanarayanan, Nitish %A Caing-Carlsson, Rhawnie %A Wahlgren, Weixiao Yuan %A Friemann, Rosmarie %A Ramaswamy, S %A Neerathilingam, Muniasamy %X

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.

%B Microb Biotechnol %V 11 %P 420-428 %8 2018 Mar %G eng %N 2 %R 10.1111/1751-7915.13041