Biology subjects

Tiwari, P.

Publications and source records attributed to Tiwari, P..

At least 19 recordsLinked to original sources

bioRxiv2026

Species Distribution, Antifungal Susceptibility, and Machine Learning-Based Prediction of Optimal Therapies Against Candida Species

Invasive candidiasis has become more common in recent decades, particularly within ICUs. Identifying specific Candida spp., and testing their sensitivity to antifungal drugs is crucial for effective treatment that helps healthcare providers to detect potential treatment challenges early. This study aimed to identify Candida spp. at their spp. level present in blood, assess their susceptibility to antifungal agents, and apply a random forest classifier to predict the best antifungal drug for a given species. The isolates were classified at the species complex level through MALDI-TOF MS. The strains' susceptibility to antifungal agents such as AMB, FLZ, VOZ, ITZ, POZ, CAS, MFN, and ANF was assessed using CLSI guidelines. Out of the 354 Candida spp. isolated from blood, C. utilis was found to be the predominant species, followed by C. tropicalis, and C. albicans. This study demonstrated that MFN and ANF exhibited high potency, with an impressive MIC90 of 0.12 {micro}g/mL against all isolates, surpassing CAS, which had a slightly higher overall MIC90 of 0.25 {micro}g/mL. FLZ exhibited the highest MIC90 value among the azoles at 16 {micro}g/mL, while VOZ, ITZ, and POZ all had MIC90 values of 0.5 {micro}g/mL. AMB MIC90 values were 2 {micro}g/mL against all isolated Candida species. The Random Forest AI/ML model showed perfect overall prediction, with the actual and predicted best antifungal drugs matching for all fungal species. This study is particularly essential because rare Candida spp. becoming more common and exhibiting higher MICs to AMB and FLZ and required suitable remedies for controlling the infection.

microbiology↗

bioRxiv2026

CryoEM reveals the dynamic conformational landscape and C -terminal gating mechanism of Mycobacterial Class Ib ribonucleotide reductase

Ribonucleotide reductases (RNRs) are essential enzymes that catalyze the reduction of ribonucleoside diphosphates to deoxyribonucleoside diphosphates and function as multi-subunit, asymmetric heterotetrameric (2{beta}2) complexes. Despite their central role in DNA synthesis, the mechanism of subunit association and regulation within this asymmetric assembly has remained incompletely understood. Here, we employed cryo-electron microscopy (cryo-EM) to investigate the apo state assembly and dynamics of a class Ib RNR complex from Mycobacterium thermoresistibile. The 3.8 [A] structure provides a detailed architecture of the complex, including order-disorder transition in the C-terminal tail of the {beta} subunit, which is critical for long-range radical transfer. The alternative {beta}-hairpin conformation in the subunit suggests its role in stabilizing the 2{beta}2 interaction. Structural analysis of the 2{beta}2 complex identified seven distinct conformational states, highlighting substantial heterogeneity and variability in -{beta} subunit association. The observed structural heterogeneity supports a model for apo-state dynamics and suggests {beta}-subunit movements toward the subunit that may facilitate productive {beta} and potential '{beta}' interactions. Complementary thermodynamic analyses further support a model in which conformational flexibility is central to -{beta} subunit recognition and stabilization. Together, these findings illuminate the dynamic conformational landscape that governs subunit association and radical transfer in class Ib RNRs. Given the importance of mycobacteria as pathogens of significant importance in humans and animals, this first structural characterization of a mycobacterial class Ib RNR provides a foundation for future structure-based inhibitor design. Significance StatementRNRs are essential for DNA synthesis in all life forms. Here, we present cryo-EM structures of the ligand-free mycobacterial 2{beta}2 complex, capturing multiple modes of asymmetric subunit association. These structures show that the enzyme samples multiple conformations that likely correspond to different activity states, potentially associated with processes such as DNA repair or replication. Notably, this flexibility arises intrinsically from subunit interactions, even in the absence of substrates or regulatory nucleotides. A 3.8 [A] structure reveals ordering in the {beta}-subunit C-terminal tail critical for long-range radical transfer, while an alternative {beta}-hairpin in the subunit may stabilize the complex. These results provide a structural framework for RNR function and inform future drug design.

biochemistry↗

bioRxiv2026

Chronic postnatal chemogenetic activation of forebrain excitatory neurons modulates adult glial function and metabolism in male mice

Early adversity increases vulnerability for adult psychopathology. Across multiple pre-clinical models of early adversity, there are reports of glial dysfunction and disrupted amino acid neurotransmission, along with maladaptive behavioral responses in adulthood. Disrupted G-protein coupled receptor signaling is known to phenocopy specific consequences of early life adversity. Enhanced Gq signaling in the forebrain excitatory neurons in early postnatal life programs anxio-depressive behaviors in adulthood, accompanied by altered neuronal glutamate and GABA metabolism in mouse models. We hypothesized that enhancing Gq signaling in forebrain excitatory neurons in early postnatal life may also impact glial function in adulthood. Our results show that postnatal hM3Dq-mediated chemogenetic activation of CaMKII-positive forebrain excitatory neurons not only increases anxiety-like behavior, but also evokes bidirectional transcriptional regulation of multiple glia-associated genes in the neocortex and hippocampi. While Gfap, Aldh1l1, S100{beta}, Eaat1, Eaat2 and Eaat3, mRNA levels were reduced in the neocortex, they were enhanced in the hippocampus, and a similar pattern was noted for GFAP protein levels. Transient, postnatal chemogenetic activation of CaMKII-positive neurons did not alter astrocyte cell density in both the neocortex and the hippocampus. Using (1H-(13C)) NMR spectroscopy, we observed a significant decline in astrocyte-specific glutamate and GABA neurotransmitter turnover, and a reduction in astrocyte metabolic flux within the neocortex and the hippocampus in adulthood in animals with a history of postnatal chemogenetic activation of forebrain excitatory neurons. Our findings indicate that chemogenetically driving Gq signaling transiently during the postnatal window in forebrain excitatory neurons results in persistent changes well into adulthood, with enhanced anxiety-like behaviors and disrupted glial function and metabolism, phenocopying specific changes in glial function noted following early adversity.

neuroscience↗

bioRxiv2026

TRACKING EVOLUTIONARY COSTS OF IMMUNE ADAPTATION AGAINST SINGLE VERSUS COINFECTING PATHOGENS

Immunity against pathogens is costly and can adversely affect fitness through resource allocation or physiological trade-offs. Infection by multiple pathogens may further worsen the effects if hosts require the activation of multiple immune responses, which in turn can elevate the overall immunological costs. Poor body condition and stressful environments can also exacerbate these trade-offs. In this study, we experimentally tested these possibilities using Tribolium castaneum populations that were evolving against either a single or a set of coinfecting bacterial pathogens. Contrary to our expectations, none of these evolved beetles showed any measurable trade-offs between pathogen resistance vs major fitness traits, such as reproduction or lifespan. Instead, they appeared to increase reproductive success and resistance to starvation, suggesting improved body condition that could mask underlying fitness costs. However, evolved beetles showed reduced quinone production, an externally secreted antimicrobial defence, indicating trade-offs between internal vs external immunity. Finally, we identified reproductive costs only under limited resource availability, but not under suboptimal resource quality, suggesting that trade-offs can be highly condition-dependent. Overall, this study provides a unique comparison across pathogens and infection types, highlighting the importance of analysing variation in life-history traits within relevant ecological contexts to understand fitness costs of evolving immunity.

evolutionary biology↗

bioRxiv2025

Identification of optimal fluorophores for use in the Drosophila embryo

The use of fluorescent proteins has become ubiquitous throughout the life sciences as a key method for understanding molecular, cellular and tissue scale processes. Genetically encodable fluorophores have enabled stable genetic lines to be generated in a large array of organisms. There are now suites of fluorophores available, particularly in the green and red spectra. Yet, which fluorophore works best in vivo can depend on a range of factors, both extrinsic (e.g. pH, temperature) and intrinsic (e.g. photobleaching, brightness). While fluorophores have been well characterised in cell culture, such measures within in vivo systems are more limited. Here, we present a quantitative screen of nine green and eight red fluorophore lines in Drosophila, with the fluorescent protein expressed from the same genomic location and imaged under identical conditions. We analyse the expression of the fluorophores in both early and late Drosophila embryos. We provide a quantitative analysis of the bleaching and folding rates. We find amongst the green fluorophores that the suitable choice - e.g., mEGFP, mNeonGreen, mStayGold - depends on timing and imaging requirements. For the red fluorophores, mScarlet-I performed consistently well, though no particular fluorophore stood out as ideal under all conditions. These results provide a powerful database for selecting optimal fluorophores for imaging in the Drosophila embryo in green and red channels.

developmental biology↗

bioRxiv2025

Identification of Potential Oral Cancer-Specific Biomarker in Oral Potentially Malignant Disorders for Early Detection of Malignancy: A Systematic Review

Oral malignancies are increasing and significantly contribute to cancer-related mortality. Oral Potentially Malignant Diseases (OPMDs) share several characteristics with Oral Cancer (OC) and have the potential to undergo malignant transformation. Beyond clinical and visual similarities, OC and OPMDs also exhibit common molecular alterations at the genomic and transcriptional levels. Identifying commonly mutated genes between OC and OPMDs may enhance early detection strategies. Therefore, we conducted a systematic review analyzing mutated genes from 25 studies (selected from 331 studies using the PRISMA flowchart) that investigated gene mutations in OC and OPMDs through whole-exome sequencing analysis. Across these studies, we identified a total of 7,564 mutated genes: 6,885 from OC studies and 579 from OPMD studies. Of these, 311 genes were found to be common between OCs and OPMDs. To further assess their relevance, these genes were categorized into High (N=5), Medium (N=20), and Low-frequency (N=386) groups, and their distribution was compared between OC and OPMD studies. Based on these comparisons, four genes: TP53, PIK3CA, FAT1, and TTN were identified as showing comparable mutation patterns between OC and OPMDs, suggesting their potential role as early biomarkers for malignant transformation. Furthermore, FAT1 and TTN may serve as promising candidates for assessing the likelihood of Oral Cancer development in OPMDs.

cancer biology↗

bioRxiv2025

CREB5 promotes immunotherapy resistance via tumor-intrinsic collagenmatrix deposition

Treatment with immune checkpoint inhibitors induces remarkable clinical responses in several cancer types. However, most cancer patients fail to respond to immunotherapy, and patients who initially respond often exhibit acquired resistance. Understanding the universe of immune evasion strategies will enable design of more effective immunotherapies. Here, we identify genes that drive immune evasion using genome-scale in vivo CRISPR gain-of-function screens in tumors treated with anti-PD-1 antibodies and found that the transcription factor CREB5 drives immune checkpoint blockade resistance. Using transcriptional profiling and functional studies, we show that CREB5 promotes a mesenchymal-like phenotype in melanoma characterized by upregulation of extracellular matrix genes including collagen and collagen-stabilizing factors. Using engineered tumor models and knockout mice, we found that immunotherapy resistance is functionally mediated by tumor-intrinsic collagen deposition. Collagen is the major ligand for the inhibitory receptor LAIR1, broadly expressed on T cells, B cells, NK cells, and myeloid cells. Deletion of LAIR1 in mice or overexpression of the decoy receptor LAIR2 in tumors abrogated the resistance induced by CREB5 overexpression, demonstrating that collagen-LAIR1 inhibitory signaling drives resistance to immune checkpoint inhibitors. These observations define a transcriptional program that remodels the tumor microenvironment to promote immunotherapy resistance via extracellular matrix deposition and indicates that targeting this pathway may enhance immunotherapy efficacy. One-Sentence Summary: In vivo gain-of-function screening in immunotherapy-treated mice reveals a transcription factor, Creb5, that drives the mesenchymal state in melanoma and facilitates immune escape by promoting tumor-intrinsic collagen matrix deposition.

cancer biology↗

bioRxiv2025

Systematic evaluation of single-cell multimodal data integration for comprehensive human reference atlas.

The integration of multimodal single-cell data enables comprehensive organ reference atlases, yet its impact remains largely unexplored, particularly in complex tissues. We generated a benchmarking dataset for the renal cortex by integrating 3 and 5 scRNA-seq with joint snRNA-seq and snATAC-seq, profiling 119,744 high-quality nuclei/cells from 19 donors. To align cell identities and enable consistent comparisons, we developed the interpretable machine learning tool scOMM (single-cell Omics Multimodal Mapping) and systematically assessed integration strategies. "Horizontal" integration of scRNA and snRNA-seq improved cell-type identification, while "vertical" integration of snRNA-seq and snATAC-seq had an additive effect, enhancing resolution in homogeneous populations and difficult-to-identify states. Global integration was especially effective in identifying adaptive states and rare cell types, including WFDC2-expressing Thick Ascending Limb and Norn cells, previously undetected in kidney atlases. Our work establishes a robust framework for multimodal reference atlas generation, advancing single-cell analysis and extending its applicability to diverse tissues.

genomics↗

bioRxiv2024

EEG data quality in large scale field studies in India and Tanzania

There is a growing imperative to understand the neurophysiological impact of our rapidly changing and diverse technological, social, chemical, and physical environments. To untangle the multidimensional and interacting effects requires data at scale across diverse populations, taking measurement out of a controlled lab environment and into the field. Electroencephalography (EEG), which has correlates with various environmental factors as well as cognitive and mental health outcomes, has the advantage of both portability and cost-effectiveness for this purpose. However, with numerous field researchers spread across diverse locations, data quality issues and researcher idle time due to insufficient participants can quickly become unmanageable and expensive problems. In programs we have established in India and Tanzania, we demonstrate that with appropriate training, structured teams, and daily automated analysis and feedback on data quality, non-specialists can reliably collect EEG data alongside various survey and assessments with consistently high throughput and quality. Over a 30-week period, research teams were able to maintain an average of 25.6 subjects per week, collecting data from a diverse sample of 7,933 participants ranging from Hadzabe hunter-gatherers to office workers. Furthermore, data quality, computed on the first 2,400 records using two common methods, PREP and FASTER, was comparable to benchmark datasets from controlled lab conditions. Altogether this resulted in a cost per subject of under $50, a fraction of the cost typical of such data collection, opening up the possibility for large-scale programs particularly in low- and middle-income countries. Significance StatementWith wide human diversity, a rapidly changing environment and growing rates of neurological and mental health disorders, there is an imperative for large scale neuroimaging studies across diverse populations that can deliver high quality data and be affordably sustained. Here we demonstrate, across two large-scale field data acquisition programs operating in India and Tanzania, that with appropriate systems it is possible to generate high throughput EEG data of quality comparable to controlled lab settings. With effective costs of under $50 per subject, this opens new possibilities for low- and middle-income countries to implement large-scale programs, and to do so at scales that previously could not be considered.

neuroscience↗

bioRxiv2024

Disrupting CD38-driven T cell dysfunction restores sensitivity to cancer immunotherapy

A central problem in cancer immunotherapy with immune checkpoint blockade (ICB) is the development of resistance, which affects 50% of patients with metastatic melanoma1,2. T cell exhaustion, resulting from chronic antigen exposure in the tumour microenvironment, is a major driver of ICB resistance3. Here, we show that CD38, an ecto-enzyme involved in nicotinamide adenine dinucleotide (NAD+) catabolism, is highly expressed in exhausted CD8+ T cells in melanoma and is associated with ICB resistance. Tumour-derived CD38hiCD8+ T cells are dysfunctional, characterised by impaired proliferative capacity, effector function, and dysregulated mitochondrial bioenergetics. Genetic and pharmacological blockade of CD38 in murine and patient-derived organotypic tumour models (MDOTS/PDOTS) enhanced tumour immunity and overcame ICB resistance. Mechanistically, disrupting CD38 activity in T cells restored cellular NAD+ pools, improved mitochondrial function, increased proliferation, augmented effector function, and restored ICB sensitivity. Taken together, these data demonstrate a role for the CD38-NAD+ axis in promoting T cell exhaustion and ICB resistance, and establish the efficacy of CD38 directed therapeutic strategies to overcome ICB resistance using clinically relevant, patient-derived 3D tumour models.

cancer biology↗

bioRxiv2023

A glucanotransferase that uses two sub-sites and four catalytic aspartates/glutamates to disproportionate oligosaccharides ranging in length from maltotriose to starch

PF0272 (PfuAmyGT) from Pyrococcus furiosus is a 656 residues-long, homodimeric, three-domain GH57 glycoside hydrolase [homologous to TLGT from Thermococcus litoralis (PDB ID: 1K1X)]. It is proposed to be either an -amylase (EC 3.2.1.1), or a 4--glucanotransferase (EC 2.4.1.25). We demonstrate that PfuAmyGT is an exo-amylase-cum-glucanotransferase capable of transferring glucose, and dis-proportionating oligosaccharides, by excising glucose from malto-oligosaccharides (ranging in length from maltotriose to amylose/starch), and transferring it to malto-oligosaccharides (ranging in length from glucose to maltoheptaose and, possibly, even longer lengths). Convention holds that glucanotransferases transfer sugars through the serial and alternating binding of donors and acceptors to the same site, with covalent retention of excised sugars between such bindings. We present evidence of multiple behaviors in PfuAmyGT that challenge this view: (i) Production of free glucose, indicating scope for release of excised glucose; (ii) Higher activity with longer donors, indicating processivity; (iii) Accelerated activity with shorter acceptors, indicating a dependence upon rapid acceptor turnover; and (iv) Evidence of four catalytic glutamates/aspartates (E131, D222, E224, D362), indicating possible separation of excision and ligation functions. These behaviours collectively indicate the binding of donors and acceptors to separate sub-sites that have different substrate length preferences, supporting our own previous proposal regarding separate tunnel (internal) and groove (surface) binding sites. Although PfuAmyGTs mechanism of function remains to be fully elucidated, this paper definitively demonstrate coupling of exo-amylase and glucanotransferase functions involving separate sub-sites for donor and acceptor binding.

biochemistry↗

bioRxiv2023

Acetyl transferase EP300 deficiency leads to chronic replication stress mediated by defective fork protection at stalled replication forks

Mutations in the epigenetic regulator and global transcriptional activator, E1A binding protein (EP300), is being increasingly reported in aggressive hematological malignancies including adult T-cell leukemia/lymphoma (ATLL). However, the mechanistic contribution of EP300 dysregulation to cancer initiation and progression are currently unknown. Independent inhibition of EP300 in human cells results in the differential expression of genes involved in regulating the cell cycle, DNA replication and DNA damage response. Nevertheless, specific function played by EP300 in DNA replication initiation, progression and replication fork integrity has not been studied. Here, using ATLL cells as a model to study EP300 deficiency and an p300-selective PROTAC degrader, degrader as a pharmacologic tool, we reveal that EP300-mutated cells display prolonged cell cycle kinetics, due to pronounced dysregulations in DNA replication dynamics leading to persistent genomic instability. Aberrant DNA replication in EP300-mutated cells is characterized by elevated replication origin firing due to increased replisome pausing genome-wide. We demonstrate that EP300 deficiency results in nucleolytic degradation of nascently synthesized DNA at stalled forks due to a prominent defect in fork stabilization and protection. This in turn results in the accumulation of single stranded DNA gaps at collapsed replication forks, in EP300-deficient cells. Inhibition of Mre11 nuclease rescues the ssDNA accumulation indicating a dysregulation in downstream mechanisms that restrain nuclease activity at stalled forks. Importantly, we find that the absence of EP300 results in decreased expression of BRCA2 protein expression and a dependency on POLD3-mediated error-prone replication restart mechanisms. The overall S-phase abnormalities observed lead to under-replicated DNA in G2/M that instigates mitotic DNA synthesis. This in turn is associated with mitotic segregation defects characterized by elevated micronuclei formation, accumulation of cytosolic DNA and transmission of unrepaired inherited DNA lesions in the subsequent G1-phase in EP300-deficient cells. We demonstrate that the DNA replication dynamics of EP300-mutated cells ATLL cells recapitulate features of BRCA-deficient cancers. Altogether these results suggest that mutations in EP300 cause chronic DNA replication stress and defective replication fork restart results in persistent genomic instability that underlie aggressive chemo-resistant tumorigenesis in humans.

cancer biology↗

bioRxiv2022

A hybrid approach for predicting transcription factors

Transcription factors (TFs) are essential DNA-binding proteins that regulate the rate of transcription of several genes and controls the expression of genes inside a cell. The prediction of TFs with high precision is important for understanding number of biological processes such as cell-differentiation, intracellular signaling, cell-cycle control. In this study, we developed a hybrid method that combine alignment-based and alignment-free methods for predicting transcription factors with higher accuracy. All models have been trained, tested and evaluated on a large dataset that contain 19406 TFs and 523560 non-TFs protein sequences. In order to avoid biasness in evaluation, dataset is divided in training and validation/independent dataset, where 80% data was used for training and remaining 20% for external validation. In case of alignment-free methods, models are developed based on machine learning techniques using compositional features of a protein. Our best alignment-free model obtained AUC 0.97 on independent dataset. In case of alignment-based method, we used BLAST at different cut-off to predict transcription factors. Though alignment-based method shows excellent performance but unable to cover all transcription factor due to no-hits. In order to combine power of both, we developed a hybrid method that combine alignment-free and alignment-based method; achieved maximum AUC of 0.99 on independent dataset. The method proposed in this study perform better than existing methods. We incorporated the best models in the webserver/standalone package "TransFacPred" (https://webs.iiitd.edu.in/raghava/transfacpred). Key PointsO_LITranscription factors (TFs) are vital DNA-binding proteins. C_LIO_LIA hybrid method for the prediction of TFs using sequence information. C_LIO_LIComputer-aided model were developed using machine-learning algorithm to predict TFs. C_LIO_LIAlignment-based and alignment-free approaches were used for the prediction. C_LIO_LIA user-friendly webserver, python- and Perl-based standalone package available. C_LI

bioinformatics↗

bioRxiv2022

Chronic hM3Dq-DREADD mediated chemogenetic activation of parvalbumin-positive inhibitory interneurons in postnatal life alters anxiety and despair-like behavior in adulthood in a task and sex-dependent manner

Animal models of early adversity or neurodevelopmental disorders are associated with altered parvalbumin (PV)-positive inhibitory interneuron number and function, correlated with a dysregulated excitation-inhibition (E/I) balance that is implicated in the pathophysiology of neuropsychiatric disorders. We sought to address whether altering neuronal activity of PV-positive interneurons during the postnatal developmental window influences the emergence of anxio-depressive behaviors in adulthood, which are known to be perturbed in models of early adversity and neurodevelopmental disorders. We used a PV-Cre::hM3Dq-DREADD bigenic mouse line that selectively expresses the hM3Dq-DREADD receptor in PV-positive interneurons, and chemogenetically enhanced Gq signaling in PV-positive interneurons during the postnatal window via administration of the DREADD agonist, clozapine-N-oxide. Immunofluorescence studies indicated the selective expression of hM3Dq-DREADD in PV-positive interneurons in limbic circuits, and revealed a reduction in expression of the neuronal activity marker, c-Fos, in these circuits, following chemogenetic hM3Dq-DREADD mediated activation of PV-positive inhibitory interneurons. We noted no change in either growth or sensorimotor reflex milestones following chronic hM3Dq-DREADD mediated chemogenetic activation of PV-positive inhibitory interneurons in postnatal life. Adult male and female PV-Cre::hM3Dq-DREADD bigenic mice with a history of postnatal chemogenetic activation of PV-positive interneurons exhibited a reduction in anxiety and despair-like behavior in adulthood, which was noted in both a behavioral task and sex-dependent manner. These results indicate that altering neuronal activity within PV-positive interneurons during the critical postnatal developmental window can shape the emergence of anxio-depressive behaviors in adulthood, and suggest that interactions with sex as a variable play a key role in determining behavioral outcomes.

neuroscience↗

bioRxiv2022

Dilated cardiomyopathy mutation E525K in human beta-cardiac myosin stabilizes the interacting heads motif and super-relaxed state of myosin

The auto-inhibited, super-relaxed (SRX) state of cardiac myosin is thought to be crucial for regulating contraction, relaxation, and energy conservation in the heart. We used single ATP turnover experiments to demonstrate that a dilated cardiomyopathy (DCM) mutation (E525K) in human beta-cardiac myosin increases the fraction of myosin heads in the SRX state (with slow ATP turnover), especially in physiological ionic strength conditions. We also utilized FRET between a C-terminal GFP tag on the myosin tail and Cy3ATP bound to the active site of the motor domain to estimate the fraction of heads in the closed, interacting-heads motif (IHM); we found a strong correlation between the IHM and SRX state. Negative stain EM and 2D class averaging of the construct demonstrated that the E525K mutation increased the fraction of molecules adopting the IHM. Overall, our results demonstrate that the E525K DCM mutation may reduce muscle force and power by stabilizing the auto-inhibited SRX state. Our studies also provide direct evidence for a correlation between the SRX biochemical state and the IHM structural state in cardiac muscle myosin. Furthermore, the E525 residue may be implicated in crucial electrostatic interactions that modulate this conserved, auto-inhibited conformation of myosin. Significance StatementDilated cardiomyopathy can be caused by single point mutations in cardiac muscle myosin, the motor protein that powers contraction of the myocardium. We found that the E525K DCM mutation in the cardiac myosin heavy chain stabilizes the auto-inhibited, super-relaxed state, suggesting a mechanism by which this mutation reduces muscle force and power. The E525K mutation also highlights critical electrostatic interactions important for forming the conserved, auto-inhibited conformational state of striated muscle myosins.

biophysics↗

bioRxiv2021

Chronic hM4Di-DREADD mediated chemogenetic inhibition of forebrain excitatory neurons in postnatal or juvenile life does not alter adult mood-related behavior

G-protein coupled receptors (GPCRs) coupled to Gi-signaling, in particular downstream of monoaminergic neurotransmission, are posited to play a key role during developmental epochs (postnatal and juvenile), in shaping the emergence of adult anxio-depressive behaviors and sensorimotor gating. To address the role of Gi-signaling in these developmental windows, we used a CamKII-tTA::TRE hM4Di bigenic mouse line to express the hM4Di-DREADD in forebrain excitatory neurons and enhanced Gi-signaling via chronic administration of the DREADD agonist, CNO in the postnatal (PNCNO: postnatal day 2-14) or juvenile (JCNO: postnatal day 28-40) window. We confirmed that the expression of the HA-tagged hM4Di-DREADD was restricted to CamKII-positive neurons in the forebrain, and administration of CNO in postnatal or juvenile windows evoked inhibition in forebrain circuits of the hippocampus and cortex, as indicated by a decline in expression of the neuronal activity marker, c-fos. hM4Di-DREADD mediated inhibition of CamKII-positive forebrain excitatory neurons in postnatal or juvenile life did not impact the weight profile of mouse pups, and also did not influence the normal ontogeny of sensory reflexes. Further, postnatal or juvenile hM4Di-DREADD mediated inhibition of CamKII-positive forebrain excitatory neurons did not alter anxiety or despair-like behaviors in adulthood, and did not impact sensorimotor gating. Collectively, these results indicate that chemogenetic induction of Gi-signaling in CamKII-positive forebrain excitatory neurons in postnatal and juvenile temporal windows does not appear to impinge on the programming of anxio-depressive behaviors in adulthood.

neuroscience↗

bioRxiv2021

Condensation of the Drosophila Nerve Cord is Oscillatory and depends on Coordinated Mechanical Interactions

During development, organs must form with precise shapes and sizes. Organ morphology is not always obtained through growth; a classic counterexample is condensation of the nervous system during Drosophila embryogenesis. The mechanics underlying such condensation remain poorly understood. Here, we combine in toto live-imaging, biophysical and genetic perturbations, and atomic force microscopy to characterize the condensation of the Drosophila ventral nerve cord (VNC) during embryonic development at both subcellular and tissue scales. This analysis reveals that condensation is not a unidirectional continuous process, but instead occurs through oscillatory contractions alternating from anterior and posterior ends. The VNC mechanical properties spatially and temporally vary during its condensation, and forces along its longitudinal axis are spatially heterogeneous, with larger ones exerted between neuromeres. We demonstrate that the process of VNC condensation is dependent on the coordinated mechanical activities of neurons and glia. Finally, we show that these outcomes are consistent with a viscoelastic model of condensation, which incorporates time delays due to the different time scales on which the mechanical processes act, and effective frictional interactions. In summary, we have defined the complex and progressive mechanics driving VNC condensation, providing insights into how a highly viscous tissue can autonomously change shape and size.

developmental biology↗

bioRxiv2020

Scaling of Internal Organs during Drosophila Embryonic Development

Many species show a diverse range of sizes; for example, domestic dogs have large variation in body mass. Yet, the internal structure of the organism remains similar, i.e. the system scales to organism size. Drosophila melanogaster has been a powerful model system for exploring scaling mechanisms. In the early embryo, gene expression boundaries scale very precisely to embryo length. Later in development, the adult wings grow with remarkable symmetry and scale well with animal size. Yet, our knowledge of whether internal organs initially scale to embryo size remains largely unknown. Here, we utilise artificially small Drosophila embryos to explore how three critical internal organs - the heart, hindgut and ventral nerve cord (VNC) - adapt to changes in embryo morphology. We find that the heart scales precisely with embryo length. Intriguingly, reduction in cardiac cell length, rather than number, appears to be important in controlling heart length. The hindgut - which is the first chiral organ to form - displays scaling with embryo size under large-scale changes in the artificially smaller embryos but shows few hallmarks of scaling within wild-type size variation. Finally, the VNC only displays weak scaling behaviour; even large changes in embryo geometry result in only small shifts in VNC length. This suggests that the VNC may have an intrinsic minimal length, which is largely independent of embryo length. Overall, our work shows that internal organs can adapt to embryo size changes in Drosophila. but the extent to which they scale varies significantly between organs.

developmental biology↗