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Nagarajan, A.

Publications and source records attributed to Nagarajan, A..

12 recordsLinked to original sources

Surface-induced tau condensation generates a selective microenvironment around microtubules

Tau is a neuron-specific microtubule-associated protein that can self-associate into pathological insoluble aggregates or phase separate into condensates whose (patho)physiological role is debated. Recent studies suggest that intracellular surfaces can locally promote biomolecular condensation, even at low molecular concentrations. While microtubules in neurons provide an abundant tau-interaction surface, their role in tau phase separation remains unclear. Through a dialogue between experiments and theory, we demonstrate that tau forms multilayered condensates on microtubules at physiological concentrations via a prewetting-like transition. Concomitant tau-microtubule and tau-tau interactions explain the experimentally observed cooperative binding of the innermost tau layer directly adsorbed to the microtubule. The formation of this layer is dictated by the spacing of tubulin dimers within the microtubule lattice. Additional tau layers, driven by tau-tau interactions and independent of lattice spacing, are finite in thickness and unstable away from the microtubule surface. While the microtubule-adsorbed tau can selectively restrict proteins from the microtubule surface, the multilayered tau condensates can recruit tau interactors, such as RNA or soluble tubulin, highlighting the distinct roles of the condensate layers. Our results suggest that a prewetting-like transition constitutes a general physical mechanism for organizing liquid-like biomolecular layers of defined composition on charged intracellular surfaces.

biophysics↗

Synergetic hallmark knockouts immortalize bovine muscle stem cells for cellular agriculture

Achieving scalable and sustainable production of cultivated meat hinges on developing robust livestock muscle and fat cell lines that can proliferate and differentiate effectively, while meeting regulatory standards and consumer expectations. In this study, bovine satellite cells (BSCs) were immortalized using CRISPR/Cas9 to knockdown PTEN (phosphatase and tensin homolog), TP53 (cellular tumor antigen) and SMAD4 (SMAD [Sma and Mad proteins] family member 4). The resulting cell line, named "CriBSC2," exhibited consistent growth, maintained muscle cell characteristics, and successfully differentiated into multinucleated myotubes after more than 150 cell divisions. In contrast, another cell line, "CriBSC1," achieved immortalization with TP53 knockout alone but lacked differentiation capacity. CriBSC2 were further cultured on gelatin scaffolds to evaluate their anchorage-dependent responses, laying the groundwork for their potential application in tissue engineering for cellular agriculture. Ultimately, CriBSC2 cells demonstrate suitable proliferation and differentiation capabilities crucial for advancing cellular agriculture and future food technologies.

cell biology↗

Comparative Transcriptomics of Adherent and Suspension Chicken Fibroblast Cell Lines for the Optimization of Cultivated Meat Processes

For the cultivated meat industry, food-relevant cell lines with optimal attributes for industrial bioprocesses are needed. One key trait is suspension-proficiency, or the ability of cells to proliferate in non-adherent culture conditions, to achieve high viable cell densities and streamline cell harvests. The limited success with suspension adaptations of animal cells for cultivated meat restricts the number of non-adherent cell lines suitable for cost-efficient biomass production. Here, we investigated transcriptional profiles of chicken embryonic fibroblasts (DF-1s) as they progressed from adherent to suspension culture using time-series RNA sequencing. From this analysis, we identified that DF-1s adapt to their non-adherent conditions by suppressing non-essential metabolism and tightly regulating cell junctions in response to oxidative stress and cell detachment. Based on these findings, we propose a novel mechanism enabling suspension proficiency in DF-1s, where mitogen-activated protein kinase (MAPK) p38{delta} sequesters TEA domain (TEAD) proteins to the cytoplasm and promotes Yes-associated protein (YAP) binding to Forkhead box protein 1 (FoxO1), simultaneously causing cell-matrix dissociation and antioxidant production. The outcome of this work is a deeper understanding of mechanisms that drive the generation of suspension cell lines, with a goal to translate these findings to other food-relevant cell lines and develop expanded cell options for the field.

cell biology↗

Membrane Interfacial Potential Governs Surface Condensation andFibrillation of α-Synuclein in Neurons

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) are essential for cellular organization. -Synuclein, an amyloidogenic protein linked to Parkinsons Disease (PD), undergoes phase separation at high concentrations, but the influence of lipid membranes on this process remains unclear. Here, combining in vitro reconstitution, cell biology, and simulations, we show that membranous interfaces promote -Synuclein condensation at physiologically relevant sub-critical concentrations ([~]10 nM) without crowding agents. Notably, condensation occurs only on membranes with a specific stoichiometry of lipids, underscoring the role of interfacial potential. These condensates serve as nucleation sites for fibril formation, leading to membrane deformation and rupture. A lattice gas model reveals this behavior as a prewetting-like transition, where an attractive membrane induces local phase separation below the bulk saturation concentration. Indeed altering interfacial potential by lipid composition and membrane depolarization not only drastically changes -Synuclein puncta size and number but also triggers their release from neurons. These findings reveal the crucial role of lipid membrane interfaces in regulating -Synuclein condensation, aggregation and release, shedding light on a potential mechanism of their cell-to-cell propagation during neurodegeneration.

biophysics↗

Extracellular Vesicles from hiPSC-derived NSCs Protect Human Neurons against Abeta-42 Oligomers Induced Neurodegeneration, Mitochondrial Dysfunction and Tau Phosphorylation

BackgroundAlzheimers disease (AD) is characterized by the accumulation of amyloid beta-42 (A{beta}-42) in the brain, causing various adverse effects. Thus, therapies that reduce A{beta}-42 toxicity in AD are of great interest. One promising approach is to use extracellular vesicles from human induced pluripotent stem cell-derived neural stem cells (hiPSC-NSC-EVs) because they carry multiple therapeutic miRNAs and proteins capable of protecting neurons against A{beta}-42-induced toxicity. Therefore, this in vitro study investigated the proficiency of hiPSC-NSC-EVs to protect human neurons from A{beta}-42 oligomers (A{beta}-42o) induced neurodegeneration. MethodsWe isolated hiPSC-NSC-EVs using chromatographic methods and characterized their size, ultrastructure, expression of EV-specific markers and proficiency in getting incorporated into mature human neurons. Next, mature human neurons differentiated from two different hiPSC lines were exposed to 1 {micro}M A{beta}-42o alone or with varying concentrations of hiPSC-NSC-EVs. The protective effects of hiPSC-NSC-EVs against A{beta}-42o-induced neurodegeneration, oxidative stress, mitochondrial dysfunction, impaired autophagy, and tau phosphorylation were ascertained using multiple measures and one-way ANOVA with Newman-Keuls multiple comparisons post hoc tests. ResultsA significant neurodegeneration was observed when human neurons were exposed to A{beta}-42o alone. Neurodegeneration was associated with 1) elevated levels of reactive oxygen species (ROS), mitochondrial superoxide, malondialdehyde (MDA) and protein carbonyls (PCs), 2) increased expression of proapoptotic Bax and Bad genes and proteins, and genes encoding mitochondrial complex proteins, 3) diminished mitochondrial membrane potential and mitochondria, 4) reduced expression of the antiapoptotic gene and protein Bcl-2, and autophagy-related proteins, and 5) increased phosphorylation of tau. However, the addition of an optimal dose of hiPSC-NSC-EVs (6 x 109 EVs) to human neuronal cultures exposed to A{beta}-42o significantly reduced the extent of neurodegeneration, along with diminished levels of ROS, superoxide, MDA and PCs, normalized expressions of Bax, Bad, and Bcl-2, and autophagy-related proteins, higher mitochondrial membrane potential and mitochondria, enhanced expression of genes linked to mitochondrial complex proteins, and reduced tau phosphorylation. ConclusionsAn optimal dose of hiPSC-NSC-EVs could significantly decrease the degeneration of human neurons induced by A{beta}-42o. The results support further research into the effectiveness of hiPSC- NSC-EVs in AD, particularly their proficiency in preserving neurons and slowing disease progression.

neuroscience↗

Late-life dietary folate restriction reduces biosynthetic processes without compromising healthspan in mice

Folate is a vitamin required for cell growth and is present in fortified foods in the form of folic acid to prevent congenital abnormalities. The impact of low folate status on life-long health is poorly understood. We found that limiting folate levels with the folate antagonist methotrexate increased the lifespan of yeast and worms. We then restricted folate intake in aged mice and measured various health metrics, metabolites, and gene expression signatures. Limiting folate intake decreased anabolic biosynthetic processes in mice and enhanced metabolic plasticity. Despite reduced serum folate levels in mice with limited folic acid intake, these animals maintained their weight and adiposity late in life, and we did not observe adverse health outcomes. These results argue that the effectiveness of folate dietary interventions may vary depending on an individuals age and sex. A higher folate intake is advantageous during the early stages of life to support cell divisions needed for proper development. However, a lower folate intake later in life may result in healthier aging.

molecular biology↗

Transcriptomic network analysis of brain and bone reveals shared molecular mechanisms underlyingAlzheimer's Disease and related dementias and Osteoporosis

Alzheimers disease and related dementias (ADRD) and Osteoporosis (OP) are two prevalent diseases of aging with numerous epidemiological associations, but the underlying molecular mechanisms contributing to this association are unknown. We used WGCNA (weighted gene co-expression network analysis) to develop transcriptomic networks in bone and brain tissue using two different studies to discover common molecular mechanisms. We used RNA-sequencing data from the dorsolateral prefrontal cortex tissue of autopsied brains in 629 participants from ROSMAP (Religious Orders Study and the Memory and Aging Project), including a subset of 298 meeting criteria for inclusion in five ADRD categories and the full set in a secondary analysis, and RNA array data from transiliac bone in 84 participants from the Oslo study of postmenopausal women. After developing each network, we analyzed associations between modules (groups of co-expressed genes) with multiple bone and neurological traits, examined overlap in modules between networks, and performed pathway enrichment analysis to discover conserved mechanisms. We discovered three modules in ROSMAP that showed significant associations with ADRD and bone related traits and four modules in Oslo that showed significant associations with multiple bone outcomes. We found significant module overlap between the two networks, most notably among those modules linked to canonical Wnt signaling and skeletal tissue homeostasis and development. These results were preserved with a network from the full ROSMAP cohort (n=629), which included a broader spectrum of participants. Our results require validation in experimental studies but show support for Wnt signaling as an important driver of pathology in OP and ADRD. We additionally show a strong link between Dementia with Lewy bodies and bone outcomes. These results have translational significance in the development of novel treatments and biomarkers for both ADRD and OP.

neuroscience↗

Identification of a genetic region in linked to tolerance to MRSA infection using Collaborative Cross mice.

Staphylococcus aureus (S. aureus) colonizes humans asymptomatically but can also cause opportunistic infections, ranging from mild skin infections to severe life-threatening conditions. Resistance and tolerance are two ways a host can survive an infection. Resistance is limiting the pathogen burden, while tolerance is limiting the health impact of a given pathogen burden. In previous work, we established that collaborative cross (CC) mouse line CC061 is highly susceptible to Methicillin-resistant S. aureus infection (MRSA, USA300), while CC024 is tolerant. To identify host genes involved in tolerance after S. aureus infection, we crossed CC061 mice and CC024 mice to generate F1 and F2 populations. Survival after MRSA infection in the F1 and F2 generations was 65% and 45% and followed a complex dominant-recessive inheritance pattern. Colonization in F2 animals was more extreme than in their parents, suggesting successful segregation of genetic factors. We identified a QTL peak on chromosome 7 for survival and weight change after infection. In this QTL, the WSB allele was present in CC024 mice and contributed to their MRSA tolerant phenotype. Two genes, C5ar1 and C5ar2, have high-impact variants in this region. The complement factor, C5a, is an anaphylatoxin that can trigger a massive immune response by binding to its receptors, C5ar1 and C5ar2. We hypothesize that C5a may have altered binding to variant receptors in CC024 mice, reducing damage caused by the cytokine storm and resulting in the ability to tolerate a higher pathogen burden and longer survival. ImportanceStaphylococcus aureus causes a wide range of diseases in humans. Resistance and tolerance are two ways a host can survive an infection. Resistance is limiting the pathogen burden, while tolerance is limiting the health impact of a given pathogen burden. Tolerance mechanisms are poorly understood in context of host-pathogen interaction. To identify host genes involved in tolerance after S. aureus infection, we crossed CC061 mice and CC024 mice. The genetic factors controlling tolerance were well segregated in the F2 population. Using QTL mapping, we identified a significant peak on chromosome 7 for survival and weight change after infection. Two genes, C5ar1 and C5ar2, have high-impact variants in this region. We hypothesize that C5a may have altered binding to variant receptors in CC024 mice, reducing damage caused by the cytokine storm and resulting in the ability to tolerate a higher pathogen burden and longer survival.

genetics↗

Collaborative Cross mice have diverse phenotypic responses to infection with Methicillin-resistant Staphylococcus aureus USA300

Staphylococcus aureus (S. aureus) is an opportunistic pathogen causing diseases ranging from mild skin infections to life threatening conditions, including endocarditis, pneumonia, and sepsis. To identify host genes modulating this host-pathogen interaction, we infected 25 Collaborative Cross (CC) mouse strains with methicillin-resistant S. aureus (MRSA) and monitored disease progression for seven days using a surgically implanted telemetry system. CC strains varied widely in their response to intravenous MRSA infection. We identified eight susceptible CC strains with high bacterial load, tissue damage, and reduced survival. Among the surviving strains, six with minimal colonization were classified as resistant, while the remaining six tolerated higher organ colonization ( tolerant). The kidney was the most heavily colonized organ, but liver, spleen and lung colonization were better correlated with reduced survival. Resistant strains had higher pre-infection circulating neutrophils and lower post-infection tissue damage compared to susceptible and tolerant strains. We identified four CC strains with sexual dimorphism: all females survived the study period while all males met our euthanasia criteria earlier. In these CC strains, males had more baseline circulating monocytes and red blood cells. We identified several CC strains that may be useful as new models for endocarditis, myocarditis, pneumonia, and resistance to MRSA infection. Quantitative Trait Locus (QTL) analysis identified two significant loci, on Chromosomes 18 and 3, involved in early susceptibility and late survival after infection. We prioritized Npc1 and Ifi44l genes as the strongest candidates influencing survival using variant analysis and mRNA expression data from kidneys within these intervals. Author SummaryMethicillin-resistant Staphylococcus aureus is a human opportunistic pathogen that can cause life-threatening diseases. To study the influence of host genetics on the outcome of MRSA infection, we infected a collection of genetically diverse mice. We identified different phenotypes for survival, organ colonization, and tissue damage, and classified CC strains into MRSA susceptible, tolerant, and resistant categories. We identified several parameters that correlated with these phenotypes. Four CC strains exhibited strong sexual dimorphism in infection outcome: females lived longer, and males had higher baseline circulating monocytes and red blood cells. Several of the CC strains we characterize may represent better animal models for diseases caused by MRSA. QTL analysis identified two genes, Npc1 and Ifi44l, as strong candidates for involvement in early susceptibility and late survival after MRSA infection. Our data suggests a strong involvement of host genetics in MRSA infection outcome.

microbiology↗

Taxonomic and metagenomic analyses define the development of the microbiota in the chick

Chicks are ideal to follow the development of the intestinal microbiota and to understand how a pathogen perturbs this developing population. Taxonomic/metagenomic analyses captured the development of the chick microbiota in unperturbed chicks and in chicks infected with Salmonella enterica serotype Typhimurium (STm) during development. Taxonomic analysis suggests that colonization by the chicken microbiota takes place in several waves. The cecal microbiota stabilizes at day 12 post-hatch with prominent Gammaproteobacteria and Clostridiales. Introduction of S. Typhimurium at day 4 post-hatch disrupted the expected waves of intestinal colonization. Taxonomic and metagenomic shotgun sequencing analyses allowed us to identify species present in uninfected chicks. Untargeted metabolomics suggested different metabolic activities in infected chick microbiota. This analysis, and GS-MS on ingesta confirmed that lactic acid in cecal content coincides with the stable presence of Enterococci in STm infected chicks. Unique metabolites including 2-isopropylmalic acid, an intermediate in the biosynthesis of leucine, was present only in the cecal content of STm infected chicks. Metagenomic data suggested that the microbiota in STm infected chicks contained a higher abundance of genes, from STm itself, involved in branched chain amino acid synthesis. We generated a deletion mutant in ilvC (STM3909) encoding ketol-acid-reductoisomerase, a gene required for the production of L-isoleucine and L-valine. {Delta}ilvC mutants are disadvantaged for growth during competitive infection with the wild type. Providing the ilvC gene in trans restored growth of the {Delta}ilvC mutant. Our integrative approach identified biochemical pathways used by STm to establish a colonization niche in the chick intestine during development. IMPORTANCEChicks are an ideal model to follow the development of the intestinal microbiota and to understand how a pathogen perturbs this developing population. Using taxonomic and metagenomic analyses we captured the development of the chick microbiota to 19 days post-hatch in unperturbed chicks and in chicks infected with Salmonella enterica serotype Typhimurium (STm). We show that normal development of the microbiota takes place in waves, and is altered in the presence of a pathogen. Metagenomics and metabolomics suggested that branched chain amino acid biosynthesis is especially important for Salmonella growth in the infected chick intestine. Salmonella mutants unable to make L-isoleucine and L-valine colonize the chick intestine poorly. Restoration of the pathway for biosynthesis of these amino acids restored the colonizing ability of Salmonella. Integration of multiple analyses allowed us to correctly identify biochemical pathways used by Salmonella to establish a niche for colonization in the chick intestine during development.

microbiology↗

Elucidating Mechanisms of Tolerance to Salmonella Typhimurium Across Long-Term Infections Using the Collaborative Cross

Understanding the molecular mechanisms underlying resistance and tolerance to pathogen infection may present the opportunity to develop novel interventions. Resistance is the absence of clinical disease with low pathogen burden, while tolerance is minimal clinical disease in the face of high pathogen burden. Salmonella is a worldwide health concern. We studied 18 strains of Collaborative Cross mice that survive acute Salmonella Typhimurium (STm) infections. We infected these strains orally and monitored them for three weeks post-infection. Five strains cleared STm by the end of the experiment (resistant), while 6 strains maintained a bacterial load and survived to the end of the experiment (tolerant). The remaining 7 strains survived longer than 7 days but succumbed to infection before the end of the study period and were called "delayed susceptible" to differentiate them from strains that do not survive to day 7 (susceptible). Tolerant strains were colonized in Peyers patches, mesenteric lymph node, spleen and liver, while resistant strains had significantly reduced bacterial colonization. Tolerant strains had lower pre-infection core body temperatures than both delayed susceptible and resistant strains and had disrupted circadian patterns of body temperature post-infection sooner than resistant strains. Tolerant strains had higher circulating total white blood cells than resistant strains, driven by increased numbers of neutrophils. Tolerant strains had more severe tissue damage and higher circulating levels of MCP-1 and IFN-{gamma}, but lower levels of ENA-78 than resistant strains. QTL analysis revealed 1 significant association and 6 suggestive associations. RNA-seq analysis identified 22 genes that are differentially regulated in tolerant versus resistant animals that overlapped with the QTLs we identified and allowed us to identify the top 5 canonical pathways. Fibrinogen genes (Fga, Fgb, and Fgg) were found across the QTL, RNA, and top canonical pathways making them the best candidate genes for differentiating tolerance and resistance. Author SummaryAn infected host can respond in multiple ways to bacterial infection including resistance and tolerance. Resistance is a decrease in pathogen load, while in tolerance mild clinical signs are present despite high pathogen load. We infected a collection of 18 strains of genetically diverse mice with Salmonella Typhimurium for up to three weeks. Five strains were resistant, 6 strains were tolerant, and the remaining 7 strains survived an intermediate amount of time ("delayed susceptible"). Tolerant strains maintained bacterial load across several organs, while resistant strains reduced bacterial load. Tolerant strains had the lowest pre-infection core body temperatures and the most rapid disruption in circadian patterns of body temperature post-infection. Tolerant strains had higher circulating neutrophils, higher circulating levels of MCP-1 and IFN-{gamma}, but lower levels of ENA-78 than resistant strains, in addition to more severe tissue damage than resistant strains. QTL analysis revealed multiple associated regions, and gene expression analysis identified 22 genes that are differentially regulated in tolerant versus resistant animals in these regions. Fibrinogen genes (Fga, Fgb, and Fgg) were found across the QTL, RNA, and top canonical pathways making them the best candidate genes for differentiating tolerance and resistance.

genetics↗

Genetic background influences survival of infections with Salmonella enterica serovar Typhimurium in the Collaborative Cross

Salmonella infections typically cause self-limiting gastroenteritis, but in some individuals these bacteria can spread systemically and cause disseminated disease. Salmonella Typhimurium (STm), which causes severe systemic disease in most inbred mice, has been used as a model for disseminated disease. To screen for new infection phenotypes across a range of host genetics, we orally infected 32 Collaborative Cross (CC) mouse strains with STm and monitored their disease progression for seven days by telemetry. Our data revealed a broad range of phenotypes across CC strains in many parameters including survival, bacterial colonization, tissue damage, complete blood counts (CBC), and serum cytokines. Eighteen CC strains survived to day 7, while fourteen susceptible strains succumbed to infection before day 7. Several CC strains had sex differences in survival and colonization. Surviving strains had lower pre-infection baseline temperatures and were less active during their daily active period. Core body temperature disruptions were detected earlier after STm infection than activity disruptions, making temperature a better detector of illness. All CC strains had STm in spleen and liver, but susceptible strains were more highly colonized. Tissue damage was weakly negatively correlated to survival. We identified loci associated with survival on Chromosomes (Chr) 1, 2, 4, 7. Polymorphisms in Ncf2 and Slc11a1, known to reduce survival in mice after STm infections, are located in the Chr 1 interval, and the Chr 7 association overlaps with a previously identified QTL peak called Ses2. We identified two new genetic regions on Chr 2 and 4 associated with susceptibility to STm infection. Our data reveal the diversity of responses to STm infection across a range of host genetics and identified new candidate regions for survival of STm infection. Author SummarySalmonella Typhimurium (STm) infections typically cause self-limiting diarrheal symptoms, but in some individuals, the bacteria can spread throughout the body and cause life-threatening infection. We used a population of genetically different mice (Collaborative Cross) to identify their range of responses to STm infection. We identified a broad range of outcomes across these different mice, including a group of mice susceptible to lethal infection and a group that survived our 7 day study. We found that mice that survived STm infection had a cooler core body temperature before infection than susceptible mice, while remaining active. Thus, body temperature, rather than activity, appears to be a better predictor of poor outcomes after STm infection. We identified several regions of the mouse genome that are associated with outcome after STm infection. One of these regions, mouse Chromosome (Chr) 1 has genes that are already known to influence susceptibility to STm infection. Two other regions that we identified to influence survival after STm infection, located on mouse Chr 2 and 4, are novel and contain numerous genes of interest that may be linked to susceptibility. Our work defines the utility of exploring how host genetic diversity influences infection outcomes with bacterial pathogens.

genetics↗