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Tripathi, S.

Publications and source records attributed to Tripathi, S..

At least 37 records · Page 2Linked to original sources
bioRxiv2025

Granular component sub-phases direct ribosome biogenesis in the nucleolus

The hierarchical, multiphase organization of the nucleolus underlies ribosome biogenesis. Ribonucleoprotein particles that regulate ribosomal subunit assembly are heterogeneously disposed in the granular component (GC) of the nucleolus. However, the molecular origins of the GCs spatial heterogeneity and its association with ribosomal subunit assembly remain poorly understood. Here, using super-resolution microscopy, we uncover that key GC biomolecules, including nucleophosmin (NPM1), surfeit locus protein 6 (SURF6), and ribosomal RNA (rRNA), are heterogeneously localized within sub-phases in the GC. In vitro reconstitution showed that these GC biomolecules form multiphase condensates with SURF6/rRNA-rich core and NPM1-rich shell, providing a mechanistic basis for GCs spatial heterogeneity. SURF6s association with rRNA is weakened upon ribosome subunit assembly, enabling NPM1 to extract assembled subunits from condensates--suggesting an assembly-line-like mechanism of subunit efflux from the GC. Our results establish a framework for understanding the heterogeneous structure of the GC and reveal how its distinct sub-phases facilitate ribosome subunit assembly.

biochemistry↗
bioRxiv2025

High Dimensional Proteomic Multiplex Imaging of the Central Nervous System Using the COMET(TM) System

Sequential multiplex methodologies such as Akoya CODEX, Miltenyi MACSima, Rarecyte Orion, and others require modification of the antibodies by conjugation to an oligo or a specific fluorophore which means the use of off-the-shelf reagents is not possible. Modifications of these antibodies are typically performed via reduction chemistry and thus require verification and validation post-modification. Fixed panels are therefore developed due to various limitations including spectral overlap that creates spectral unmixing issues, steric hindrance, harsh antibody removal, and tissue degradation throughout the labeling. As such, a complex interrogation evaluating multiple study hypotheses and/or endpoints requires the development of sequential panels, reconstruction, and realignment of the tissue that necessitate a z-stack strategy. Standardized antibody panels are typically fixed and require substantial validation efforts to modify a single target and thus do not evolve with the pace of research interests. To increase the throughput of profiling cells within the human central nervous system (CNS), we developed and validated a CNS-specific library with an associated analysis platform using the newly developed Lunaphore COMETTM platform. The COMETTM is an automated staining/imaging instrument integrating a reagent deck for staining buffers and off-the-shelf label-free primary antibodies and fluorophore-labeled secondary antibodies, which feed into a circular plate holding up to 4 slides that are automatically imaged in microscope-operated control software. For this study, standard formalin fixed paraffin embedded histology slides are used. However, the COMET is capable of imaging fresh-frozen samples using specialized settings. Our methodologies address an unmet need in the neuroscience field while leveraging prior developmental efforts in the domain of immunology spatial profiling. Cataloging and validating a large series of antibodies on the COMET along with developing CNS autofluorescence management strategies while optimizing standard operating procedures have allowed for the visualization at the subcellular level. Forty analytes can be used to analyze one specimen which has clinical utility in cases in which the CNS can only be sampled by biopsy. CNS biopsies, depending on the anatomical location, can have limited available volume to a degree that requires prioritization and restriction to select analysis. In-depth bioinformatic imaging analysis can be done using standard bioinformatic tools and software such as Visiopharm(R). These results establish a general framework for imaging and quantifying cell populations and networks within the CNS while providing the scientific community with standard operating procedures.

bioinformatics↗
bioRxiv2024

A multi-level formalism to capture the characteristics of hybrid phenotypes in Epithelial-Mesenchymal Plasticity

Epithelial-mesenchymal plasticity (EMP) is a cell-fate switching program that enables cells to adopt a spectrum of phenotypes ranging from epithelial (E) to mesenchymal (M), including intermediate hybrid E/M states. Hybrid E/M phenotypes are conducive to cancer metastasis, as they are associated with metastatic initiation, cancer stemness, drug resistance, and collective migration. Boolean models of the gene regulatory networks (GRNs) underlying EMP have yielded valuable insights into the dynamics of E and M phenotypes. However, these models are limited in their ability to capture hybrid phenotypes effectively, as they restrict gene expression to binary states. In contrast, hybrid E/M cells often exhibit partial expression of epithelial and mesenchymal markers. To overcome this limitation, we modified a threshold-based Boolean formalism to incorporate intermediate gene expression levels. The resulting multilevel model reveals novel hybrid steady-states characterized by partial expression of both E and M genes, thereby expanding the phenotypic landscape beyond that represented by traditional Boolean approaches. Notably, these hybrid states exhibit lower frustration compared to their counterparts in classical Boolean models. Furthermore, by resolving dynamical degeneracy, we demonstrate that the hybrid states identified by the multilevel model are more stable. These findings suggest that introducing minimal additional complexity into Boolean models can uncover previously hidden qualitative features of phenotypic landscapes governed by GRNs. Significance StatementHybrid epithelial-mesenchymal phenotypes drive metastasis through collective migration, stemness, and drug resistance. However, traditional Boolean models of epithelial-mesenchymal plasticity (EMP) networks cannot capture the partial gene expression characteristic of these biologically crucial hybrid states. We developed a multilevel Boolean formalism that extends classical binary models to include intermediate expression levels. This approach successfully captures hybrid states with partial expression of both epithelial and mesenchymal genes, exhibiting lower frustration and greater stability than traditional Boolean hybrid phenotypes. While conventional Boolean hybrid states depend on modeling artifacts, multilevel hybrid states emerge independently of such uncertainties. Critically, plasticity experiments revealed that multilevel hybrid states show significantly higher transition probabilities to other hybrid states compared to traditional models, creating a stable "hybrid cloud" that maintains metastatic properties while allowing adaptive responses to environmental stress. This multilevel framework provides a biologically meaningful characterization of hybrid phenotypes essential for understanding metastasis.

systems biology↗
bioRxiv2024

Pelvic Ring Fractures: Does Spinopelvic Alignment Affect Sacral and Lumbopelvic Fixation Stability?

Background ContextPelvic ring is a complex boney and ligamentous complex and its structural integrity is paramount to be able to withstand the high forces. Pelvic ring fractures, which can result from low-energy impacts or high-velocity injuries and falls from great heights, can severely impair the structural integrity of the pelvic ring. These injuries can be fatal, especially in geriatric patients, with mortality rates ranging between 10% and 16%. Pelvic ring injuries require immediate stabilization, typically performed through internal fixation, to maintain circulatory balance and achieve anatomical restoration of the pelvis. MethodsA previously validated FE model of the spine, pelvis, and femur model was used to evaluate how different fixation techniques for pelvic ring injury affect spinopelvic biomechanics. The SS, PI, PT and LL of the normal, intact model was modified to generate two more SS models for this study. One modified model with an SS of 20{o} was considered the low SS model (SSD), while the second modified model with an SS of 32{o} was termed the high SS model (SSI). A unilateral pelvic ring fracture was simulated by resecting the left side of the sacrum and pelvis. The fractures were stabilized using two different posterior stabilization techniques 1) The fractures were stabilized using two different posterior stabilization techniques, and 2) L5-Ilium posterior screw fixation without cross connector (L5_PF_WO_CC). ResultsSSI demonstrated the higher range of motion, followed by Normal model and least by SSD model at right Sacroiliac Joint. No differences were seen between with and without cross connector model for range of motion at right SIJ. For L5-S1 motion models treated with cross connectors demonstrated the least motion compared to those treated without cross connectors for all configurations and loading conditions. For the sacrum fracture, models with high sacral slopes recorded the least horizontal displacement, followed by decreased sacral slope models for both configurations and loading conditions.

bioengineering↗
bioRxiv2024

Structural and functional analysis of cancer-associated missense variants in the retinoblastoma protein (Rb) pocket domain

The retinoblastoma tumor suppressor (Rb) is a multifunctional protein that primarily regulates the cell cycle but also has roles in cellular differentiation, DNA damage response and apoptosis. The loss of Rb is a key event in the development or progression of many cancers. Essential functions of Rb occur through its pocket domain, which is necessary for regulating binding interactions with E2F transcription factors and transcription repressors that bind via an LxCxE motif. The pocket domain is the most highly-conserved region of the multidomain protein, as well as the most frequent site of mutations. To understand what effects cancer missense mutations have on Rbs pocket domain, we used fluorescence polarization and differential scanning fluorimetry to quantify changes, caused by 75 cancer-associated missense variants, to E2F transactivation domain (E2FTD) binding, LxCxE binding, and changes to the thermostability of the protein. We find that 43% of the missense variants we tested reduce Rb-E2FTD binding. Many of these variants are not located at the E2F binding site, yet they destabilize the fold of the protein and show temperature-sensitive binding effects. We also find that 21% of tested mutations reduce LxCxE binding, and several mutations selectively disrupt either E2FTD or LxCxE binding. Protein X-ray crystallography of four missense variants reveals how different mutations destabilize the protein fold and inhibit E2FTD or LxCxE binding. Taken together, this work provides the first understanding of the multiple ways through which stability, structure and function of Rbs pocket domain is altered by a large number of missense mutations seen in cancer.

biochemistry↗
bioRxiv2024

Statins attenuate Wnt/β-catenin signaling by targeting SATB family proteins in colorectal cancer

Colorectal cancer is the second leading cause of cancer-related deaths worldwide, highlighting the need for improved treatments and advanced molecular research. A recent therapeutic approach focuses on repurposing drugs to target dysregulated pathways involved in tumorigenesis. Among these, statins, commonly known for lowering cholesterol, have attracted attention for their potential anti-cancer properties. Here, we provide direct evidence for the same by assessing the impact of statin treatment on lipid, transcript, and protein levels. Our findings reveal that statins specifically target key components of the Wnt/{beta}-catenin pathway, a major factor in adenoma formation, including the SATB (Special AT-rich Binding protein) family proteins. While SATB1 is recognized as a regulator of tumorigenesis, particularly under Wnt signaling, SATB2 appears to exert an opposing role. We demonstrate that statin treatment reciprocally alters the expression pattern of these proteins. Furthermore, a human clinical trial evaluating statins as an anti-cancer therapy supports the hypothesis that differential expression of SATB proteins is crucial in tumorigenic outcomes. In conclusion, this modulation by statin treatment suggests promising new therapeutic avenues through drug repurposing.

cancer biology↗
bioRxiv2024

Selective promotion of oligonucleotides in the course of evolution

Evolution shapes the structure and content of genomes, yet the contribution of local sequence composition to variant selection remains poorly understood. While traditional models emphasize protein function or cross-species conservation, we propose that intra-genomic patterns of oligonucleotide (k-mer) frequencies also reflect selective forces. To explore this, we developed kGain score, a metric that quantifies the frequency shift of a k-mer upon single-nucleotide substitution, using the surrounding genomic context as a baseline. We hypothesize that variants arising in high-kGain contexts are more likely to persist due to evolutionary favorability. We validated this hypothesis across multiple systems. In E. coli and S. cerevisiae long-term evolution experiments, we found that fixed, essential, and parallel mutations consistently show elevated kGain scores. This trend held in SARS-CoV-2 variants of concern and in an in-house antibiotic adaptation experiment, where a high-kGain fusA Y515N mutation conferred resistance and maintained fitness when overexpressed, demonstrating a causal link between kGain and adaptive potential. To enable cross-species generalization, we trained a transformer-based neural network regressor on LTEE-derived mutations to predict kGain from sequence alone. The model achieved high correlation in held-out in-domain data (Pearson r = 0.81) and accurately predicted kGain trends in out-of-domain data (Pearson r = 0.82), demonstrating that k-mer-based sequence constraints learned from one genome can be effectively transferred to others. Together, our results establish kGain as a biologically meaningful, scalable metric for probing within-genome sequence selection, offering a complementary lens to existing conservation-based frameworks for understanding evolutionary fitness and variant persistence.

evolutionary biology↗
bioRxiv2024

Evaluating Paraspinal Muscle Response and Compensation via Musculoskeletal Modeling in Spinal Stenosis Surgeries

IntroductionLumbar spinal stenosis is a common cause of lower back pain and weakness in elderly patients. The gold standard treatment for this is lumbar laminectomy which involves widespread muscle damage to the multifidus, a complete loss of the posterior tension band which contains the supraspinous and interspinous ligaments. However, in recent years minimally invasive techniques such as bilateral and unilateral laminotomy have become more popular and are showing efficacy in the decompression of spinal stenosis. Due to its minimally invasive approach, the muscle retraction required for laminotomy is less intensive than that required for laminectomy. The overall body of literature on the surgical treatment of spinal stenosis is sparse in its interrogation of the biomechanical outcomes of these techniques and to our knowledge, there are no current publications that incorporate muscle forces. MethodsA previously validated thoracolumbar ribcage finite element (FE) model was used for this study. Three different surgeries, traditional laminectomy, unilateral and bilateral midline sparing approaches at L4-L5 segment were simulated by removing the spinous process, supraspinous, and interspinous ligaments. The segmental range of motion (ROM) for all models were acquired and input into a musculoskeletal modelling software to calculate muscle forces. ResultsUnilateral and bilateral laminotomy showed similar muscle forces for every muscle group in both flexion and extension motion. While comparing the muscle forces in laminotomy to the laminectomy in extension motion displayed an increase in Iliocostalis lumborum (IL) by 12 % and multifidus (MF) by 16% and decrease in transverse abdominus (TA) by 138% and erector spine (ES) by 12%. For flexion, there was an increase in IL by 35%, and MF by 12%. ConclusionOur results highlight that laminectomy, which involves the removal of paraspinal muscles and posterior ligamentous structures to relieve stenosis, can lead to increased instability and necessitate muscle compensation, particularly in adjacent and thoracic spine segments. Conversely, midline sparing approaches such as laminotomies, are associated with decreased muscle compensation across spinal segments and enhanced stability.

bioengineering↗
bioRxiv2024

Soft Tissue Mechanics in Hip Distraction after Total Knee Arthroplasty: A Finite Element Analysis

INTRODUCTIONImprovement in diagnostic and surgical techniques in hip arthroscopy have led to a surge in hip distraction procedures over the recent years with the predicted annual frequency being four out of every 10,000 orthopedic procedures in 2017. Due to the large traction force required to achieve the appropriate joint spacing intra-operatively, an emergence of traction-related neurological and soft tissue injuries have surfaced. Pre-existing hip joint pathologies and surgical procedures disrupt the biomechanical stability of the joint and significantly increase the risk of iatrogenic damage. Furthermore, patients with total knee arthroplasties are often subject to intra-articular ligament releases, leading to reduced stability; however, it is not well understood how this may impact their outcomes of hip arthroscopic procedures. The current study aims to investigate the biomechanical behavior of various instrumented knee joints subjected to traction forces to aid clinical understanding and advancements of hip arthroscopy techniques. METHODSA validated finite element (FE) model of the pelvis and lower extremity was developed from computed tomography (CT) scans of a healthy 45-year-old female. Three different models were assembled according to different TKA techniques performed: Bi-Cruciate Retaining (BCR) model, Posterior-Cruciate Retaining (PCR) model, and Posterior Stabilized (PS) model. The BCR model is noted by retaining all native ligaments of the knee joint (ACL, PCL, MCL, and LCL), whereas the PCR model was subject to ACL removal and the PS model required ACL and PCL removal (Figure 1). The pelvis was encastered to prevent translation under the traction forces as motion of the patients trunk is restrained, intraoperatively. To simulate the loading condition of hip distraction, an axial force was coupled to the distal fibula and tibia and incrementally increased from 100N to 500N. Joint spacing and ligament strain in the hip and knee joint were analyzed to assess the effects of traction forces. O_FIG O_LINKSMALLFIG WIDTH=174 HEIGHT=200 SRC="FIGDIR/small/580129v2_fig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@1494014org.highwire.dtl.DTLVardef@e0fdeorg.highwire.dtl.DTLVardef@133cb56org.highwire.dtl.DTLVardef@16aaf02_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Meshed parts of the A.) Posterior Cruciate retaining total knee replacement system and the B.) Bi-cruciate retaining total knee replacement system. C_FIG RESULTSThe medial and lateral compartment stiffness of the knee joint was analyzed under hip distraction for the three different TKA scenarios. The BCR model displayed the greatest average knee complex stiffness. Release of the ACL resulted in a larger decrease of stiffness compared to release of the PCL. There was no change in forces required for hip distraction as result of changes in the knee joint stiffness (Figure 3). The PCR and PS models were subject to excess knee joint distraction that exceeded 12 mm and ligament strain greater than 20% before adequate hip joint distraction of 10 mm was achieved. The BCR model remained below 10 mm of knee distraction and 15% ligament strain at 10 mm of hip joint distraction. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=182 SRC="FIGDIR/small/580129v2_fig3.gif" ALT="Figure 3"> View larger version (90K): org.highwire.dtl.DTLVardef@107c292org.highwire.dtl.DTLVardef@77351corg.highwire.dtl.DTLVardef@40011aorg.highwire.dtl.DTLVardef@88e9c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 3.C_FLOATNO FE models of instrumented TKA. A) PCR TKA with simulated ACL release. B) PS TKA with simulated ACL and PCL releases. C) BCR TKA with intact ligaments. C_FIG DISCUSSIONOur study reveals patients undergoing hip distraction with a prior TKA may experience increased soft tissue damage or iatrogenic dislocation due to reduced knee joint stability. The PCR and PS models outline a trend suggesting patients who have undergone ligament sacrificing TKAs experience large reductions in knee joint stability, causing strain levels that are indicative of soft tissue injury. The BCR TKA was indicated to be the safest under the distraction conditions as joint spacing and strain levels were largely reduced comparatively; however, when surpassing 10 mm of knee joint distraction at forces greater than 350 N, the strain levels in the ACL suggest minor injury may occur.

bioengineering↗
bioRxiv2024

Biomechanical Implications of Spinopelvic Alignment on Femoral Head Cartilage and the Proximal Femoral Physis in Slipped Capital Femoral Epiphysis: A Theoretical Finite Element Analysis

BackgroundSlipped capital femoral epiphysis (SCFE) is a prevalent pediatric hip disorder. Recent studies suggest the spines sagittal profile may influence the proximal femoral growth plates slippage, an aspect not extensively explored. This study utilizes finite element analysis to investigate how different spinopelvic alignments affect shear stress and potential slippage at the growth plate. MethodsA finite element model was developed from CT scans of a healthy adult male lumbar spine, pelvis, and femurs. The model was subjected to various sagittal alignments through rotational boundary conditions. Simulations of two-leg stance, one-leg stance, walking heel strike, ascending stairs heel strike, and descending stairs heel strike were conducted. Parameters measured included hip joint contact area, stress, and maximum Tresca (shear) stress on the growth plate. FindingsPosterior pelvic tilt cases indicated larger shear stresses compared to the anterior pelvic tilt variants except in two leg stance. Two leg stance resulted in decreases in the posterior tilted pelvi variants compared to anterior tilted pelvi, however a combination of posterior pelvic tilt and high pelvic incidence indicated larger shear stresses on the growth plate. One leg stance and heal strike resulted in higher shear stress on the growth plate in posterior pelvic tilt variants compared to anterior pelvic tilt, with a combination of posterior pelvic tilt and high pelvic incidence resulting in the largest shear stress. InterpretationOur findings suggest that posterior pelvic tilt and high pelvic incidence can lead to increased shear stress at the growth plate. Activities performed in patients with these alignments may predispose to biomechanical loading that shears the growth plate, potentially causing slippage.

bioengineering↗
bioRxiv2023

Assessing Protein Surface-Based Scoring for Interpreting Genomic Variants

Clinical genomics sequencing is rapidly expanding the number of variants that need to be functionally elucidated. Interpreting genetic variants (i.e., mutations) usually begins by identifying how they affect protein-coding sequences. Still, the 3D protein molecule is rarely considered for large-scale variant analysis, nor for their effects on how proteins interact with each other and their environment. We propose a standardized approach to scoring protein surface property changes as a new dimension for functionally and mechanistically interpreting genomic variants. Further, it directs hypothesis generation for functional genomics research to learn more about the encoded proteins function. We developed a novel method leveraging 3D structures and time-dependent simulations to score and statistically evaluate protein surface property changes. We evaluated positive controls composed of 8 thermophilic versus mesophilic orthologs and variants that experimentally change protein solubility, which all showed large and statistically significant differences in charge distribution (p<0.01). We scored static 3D structures and dynamic ensembles for 43 independent variants (23 pathogenic and 20 uninterpreted) across four proteins. Focusing on the potassium ion channel, KCNK9, average local surface potential shifts were 0.41 kBT/ec with an average p-value of 1x10-2. In contrast, dynamic ensemble shifts averaged 1.15 kBT/ec and an average p-value of 1x10-5, enabling the identification of changes far from mutated sites. This study demonstrates that an objective assessment of how mutations affect electrostatic distributions of protein surfaces can aid in interpreting genomic variants discovered through clinical genomics sequencing.

bioinformatics↗
bioRxiv2023

Regulation of chromatin transcription dynamics by DNA supercoiling

Transcription has a mechanical component, as the translocation of the transcription machinery or RNA polymerase (RNAP) on DNA or chromatin is dynamically coupled to the chromatin torsion. This posits chromatin mechanics as a possible regulator of eukaryotic transcription, however, the modes and mechanisms of this regulation are elusive. Here, we first take a statistical mechanics approach to model the torsional response of topology-constrained chromatin. Our model recapitulates the experimentally observed weaker torsional rigidity of chromatin compared to bare DNA, and proposes structural transitions of nucleosomes into chirally distinct states as the driver of the contrasting torsional mechanics. Coupling chromatin mechanics with RNAP translocation in stochastic simulations, we reveal a complex interplay of DNA supercoiling and nucleosome dynamics in governing RNAP velocity. Nucleosomes play a dual role in controlling the transcription dynamics. The steric barrier aspect of nucleosomes in the gene body counteracts transcription via hindering RNAP motion, whereas the chiral transitions facilitate RNAP motion via driving a low restoring torque upon twisting the DNA. While nucleosomes with low dissociation rates are typically transcriptionally repressive, highly dynamic nucleosomes offer less of a steric barrier and enhance the transcription elongation dynamics of weakly transcribed genes via buffering DNA twist. We use the model to predict transcription-dependent levels of DNA supercoiling in segments of the budding yeast genome that are in accord with available experimental data. The model unveils a paradigm of DNA supercoiling-mediated interaction between genes and makes testable predictions that will guide experimental design.

biophysics↗
bioRxiv2023

Reconstruction of single cell lineage trajectories and identification of diversity in fates during the epithelial-to-mesenchymal transition

Exploring the complexity of the epithelial-to-mesenchymal transition (EMT) unveils a diversity of potential cell fates; however, the exact timing and intricate mechanisms by which early cell states diverge into distinct EMT trajectories remain unclear. Studying these EMT trajectories through single cell RNA sequencing is challenging due to the necessity of sacrificing cells for each measurement. In this study, we employed optimal-transport (OT) analysis to reconstruct the past trajectories of different cell fates during TGF-beta-induced EMT in the MCF10A cell line. Our analysis revealed three distinct trajectories leading to low EMT, partial EMT, and high EMT states. Cells along partial EMT trajectory showed substantial variations in the EMT signature and exhibited pronounced stemness. Throughout this EMT trajectory, we observed a consistent downregulation of the EED and EZH2 genes. This finding was validated by recent inhibitor screens of EMT regulators and CRISPR screen studies. Moreover, we applied our analysis of early-phase differential gene expression to gene sets associated with stemness and proliferation, pinpointing ITGB4, LAMA3, and LAMB3 as genes differentially expressed in the initial stages of the partial versus high EMT trajectories. We also found that CENPF, CKS1B, and MKI67 showed significant upregulation in the high EMT trajectory. While the first group of genes aligns with findings from previous studies, our work uniquely pinpoints the precise timing of these upregulations. Finally, the latter group of genes represents newly identified regulators, shedding light on potential targets for modulating EMT trajectories. Significance StatementIn our study, we investigated cellular trajectories during EMT using a time-series scRNAseq dataset. OT analysis was used to infer cell-to-cell connections from scRNAseq data, allowing us to predict cell linkages and overcome limitations of sequencing such as the need to sacrifice cells for each measurement. This approach allowed us to identify diverse EMT responses under uniform treatment, a significant advancement over previous studies limited by the static nature of scRNAseq data. Our analysis identified a broad set of genes involved in the EMT process, uncovering novel insights such as the upregulation of cell cycle genes in cells predisposed to a high EMT state and the enhancement of cell adhesion marker genes in cells veering towards a partial EMT state. This work enriches our understanding of the dynamic processes of EMT, showcasing the varied cellular fates within the same experimental setup.

systems biology↗
bioRxiv2023

SARS-CoV-2 Nsp13 is a viral RHIM protein promoting cell death linked to Z-RNA sensing and ZBP1-RIPK3 signaling

RHIM is a protein motif in cell death proteins that assembles higher-order signaling complexes and triggers regulated cell death, which in itself limits virus spread and additionally triggers inflammation for mounting immune responses. A few DNA viruses employ viral RHIMs mimicking host RHIMs. However, these viral RHIMs counteract host cell death by interacting with host RHIM proteins and blocking complex formation to alleviate antiviral defenses. Whether RNA viruses operate such viral RHIMs remains unknown. RHIM-protein signaling promotes lung damage and cytokine storm in respiratory RNA virus infections, arguing the presence of viral RHIMs. Here, we report the novel viral RHIMs in Nsp13 and Nsp14 of SARS-CoV-2 and other bat RNA viruses, providing the basis for bats as the hosts for their evolution. Nsp13 promoted cell death in bat and human cells, however, viral RHIM of Nsp13 is more critical for human cell death than bat cells, suggesting species-specific regulation. The conformation of RNA-binding channel in Nsp13 is critical for cell death in bat and human cells. Nsp13 showed RHIM-dependent interactions with ZBP1 and RIPK3 and promoted the formation of large insoluble complexes of ZBP1 and RIPK3. Also, Nsp13 promoted ZBP1-RIPK3 signaling-mediated cell death dependent on intracellular RNA ligands. Intriguingly, the SARS-CoV-2 genome consists of bona fide Z-RNA-forming segments. These SARS-CoV-2 Z-RNA segments promoted Nsp13-dependent cell death, further revealing Nsp13s association with Z-RNA sensing and ZBP1-RIPK3 signaling. Our findings reveal the functional viral RHIMs of bat-originated RNA viruses regulating host cell death associated with Z-RNA sensing and ZBP1-RIPK3 signaling activation. These observations allow the understanding of mechanisms of cellular damage and cytokine storm in SARS-CoV-2 and other bat-originated RNA virus infections. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/554434v2_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@f71c5aorg.highwire.dtl.DTLVardef@1666aa5org.highwire.dtl.DTLVardef@e37d82org.highwire.dtl.DTLVardef@145020f_HPS_FORMAT_FIGEXP M_FIG C_FIG One-sentence summaryBat-associated RNA viruses employ viral RHIMs and regulate host cell death.

immunology↗
bioRxiv2023

SARS-CoV-2 variants of concern exhibit differential gastro-intestinal tropism and pathogenesis in the Syrian golden hamster model.

Severe Acute Respiratory Syndrome Virus-2 (SARS-CoV-2) is a respiratory virus that uses angiotensin-converting enzyme 2 (ACE2) protein as an entry receptor. Thus, ACE2 expression levels in different tissues should dictate viral tropism. Notably, human ACE2 mRNA and protein levels are most abundant in the gastrointestinal tract, a pattern mirrored in Syrian golden hamsters. This prompted us to investigate the gastrointestinal tropism of SARS-CoV-2 variants of concern, including the Wuhan-like Hong Kong strain, highly pathogenic Delta, and highly transmissible but mildly pathogenic Omicron variants in hamsters. Delta was the most pathogenic in the respiratory and gastrointestinal tracts, followed by ancestral Wuhan-like and Omicron strains. In the gastrointestinal tract, viral RNA load was significant in the proximal organs such as the oesophagus and stomach, highest in the SI, and minimal to undetectable in the colon. Additionally, all three variants reduced fecal microbial diversity, with the Delta causing the highest decrease in observed features and phylogenetic diversity. Our findings highlight Deltas stronger preference for the gastrointestinal tract, suggesting a link between high virulence and gastrointestinal tropism of SARS-CoV-2 variants of concern.

pathology↗
bioRxiv2023

Regulation of the circadian clock in C. elegans by clock gene homologs kin-20 and lin-42

Circadian rhythms are endogenous oscillations present in nearly all organisms from prokaryotes to humans, allowing them to adapt to cyclical environments close to 24 hours. Circadian rhythms are regulated by a central clock, which is based on a transcription-translation feedback loop. One important protein in the central loop in metazoan clocks is PERIOD, which is regulated in part by Casein kinase 1{varepsilon}/{delta} (CK1{varepsilon}/{delta}) phosphorylation. In the nematode Caenorhabditis elegans, period and casein kinase 1{varepsilon}/{delta} are conserved as lin-42 and kin-20, respectively. Here we studied the involvement of lin-42 and kin-20 in circadian rhythms of the adult nematode using a bioluminescence-based circadian transcriptional reporter. We show that mutations of lin-42 and kin-20 generate a significantly longer endogenous period, suggesting a role for both genes in the nematode circadian clock, as in other organisms. These phenotypes can be partially rescued by overexpression of either gene under their native promoter. Both proteins are expressed in neurons and seam cells, a population of epidermal stem cells in C. elegans that undergo multiple divisions during development. Depletion of LIN-42 and KIN-20 specifically in neuronal cells after development was sufficient to lengthen the period of oscillating sur-5 expression. Therefore, we conclude that LIN-42 and KIN-20 are critical regulators of the adult nematode circadian clock through neuronal cells.

neuroscience↗
bioRxiv2023

A broadly protective CHO cell expressed recombinant spike protein subunit based vaccine (IMT-CVAX) against SARS-CoV-2

Protective immunity induced by COVID-19 vaccines is mediated mainly by spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we report the development of a recombinant prefusion stabilized SARS-CoV-2 spike protein-subunit-based COVID-19 vaccine produced in the mammalian cell line. The gene encoding ectodomain (ECD) of the spike protein was engineered and cloned into Freedom pCHO 1.0, a mammalian expression vector, and subsequently expressed in the Chinese Hamster Ovary suspension cell line (CHO-S). The recombinant S protein ectodomain (hereafter referred to as IMT-CVAX) was purified using a combination of tangential flow filtration and liquid chromatography. Biochemical and biophysical characterization of IMT-CVAX was done to ensure its vital quality attributes. Intramuscular immunization of mice with two doses of adjuvanted IMT-CVAX elicited a strong anti-Spike IgG response. In pseudovirus-based assays, IMT-CVAX- immune mice sera exhibited a broad-spectrum neutralization of several SARS-CoV-2 variants of concern (VoCs). Golden Syrian Hamster immunized with IMT-CVAX provided excellent protection against SARS-CoV-2 infection, and, hamster immune sera neutralized the live SARS-CoV-2 virus. The adjuvanted IMT-CVAX induced robust Tfh-cells response and germinal center (GC) reaction in human ACE2 receptor-expressing transgenic mice. The findings of this study may pave the way for developing next-generation protein subunit-based vaccines to combat the existing SARS-CoV-2 and its emerging VoCs. The IMT-CVAX is produced using a scalable process and can be used for large-scale vaccine production in an industrial setup.

microbiology↗