Biology subjects

Sharma, N.

Publications and source records attributed to Sharma, N..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

MSH3 is a genetic modifier of somatic repeat instability in X-linked dystonia parkinsonism

X-linked dystonia parkinsonism (XDP) is a progressive adult-onset neurogenerative disorder caused by the insertion of a SINE-VNTR-Alu (SVA) retrotransposon in TAF1 gene. One element of the SVA is a tandem polymorphic CCCTCT repeat tract whose length inversely correlates with the age of disease onset. Previous observations that the repeat exhibits length-dependent somatic expansion and that XDP onset is modified by variation in DNA repair gene MSH3 indicated that somatic repeat expansion is an important disease driver. Here, we sought to uncover genetic modifiers of CCCTCT instability in XDP patients and to provide a mechanistic link between somatic instability and disease. We determined quantitative metrics of both repeat expansion and repeat contraction in blood. Using genetic association analyses of exome sequencing data, as well as directed sequencing of a variant MSH3 repeat, we found that MSH3 modifies repeat expansion and contraction in blood as well as age at onset. MSH3 alleles associated with earlier disease onset were associated with more expansion and less contraction. Conversely, alleles associated with later disease onset were associated with less expansion and more contraction. Notably, MSH3 repeat alleles were also similarly associated with expansion and contraction in brain tissues. Our findings provide key evidence that MSH3s role(s) in CCCTCT repeat dynamics underlies its impact on clinical disease and indicate that therapeutic strategies to lower or inhibit MSH3 are predicted to both slow CCCTCT expansion and promote CCCTCT contraction, impacting the disease course prior to clinical onset.

genetics↗

bioRxiv2025

ATLS2021-PA (A Sphingolipid mimetic): Upcoming Host Directed Therapy for Acute and MDR Tuberculosis

Several reports have shown that reduced Sphingolipids content in the lungs largely contributes to the outcome of Tuberculosis diseases. In this context, our pioneer study has advocated that Sphingosine -1 phosphate (a central Sphingolipid metabolite) can help host in limiting Mycobacterium tuberculosis burden in lungs by their virtue of tweaking M1 retuning of infected macrophages. This indicated that S1P may serve as anti-tubercular regimen, however allergic and autoimmune manifestation of S-1P refrain its further use as anti-tubercular Drugs. In view of this limitation, and high demand of developing newer host directed anti tubercular regimen, we explored whether enhancing / boosting Sphingolipids levels via de novo pathways by our patented drugs ATLS2021 and PA would also help host in controlling mycobacterial infection. Our results prudently demonstrated that ATLS2021 was effective in controlling survival of both wild type and MDR strain of TB in various models we tested. Our results further demonstrated the influence of ATLS2021 on NO mediated killing of mycobacteria which indicated one of most possible anti-mycobacterial mechanism of ATLS2021. Additionally, ATLS2021 lowered the IC50 value of Rifampicin for both wild-type and MDR TB by sensitizing the MDR clinical isolate of Mycobacterium tuberculosis to Rifampicin-mediated killing. ATLS2021 induced immunogenic responses in blood derived CD14+ macrophages from both healthy donors as well as MDR-TB patient demonstrating immune adjuvant potential of ATLS2021. Real time PCR data demonstrated that ATLS2021 enhanced the expression of almost all key enzymes involved in the Sphingolipid biosynthesis pathways in the CD14 positive monocytes from healthy donors and MDR TB patients. Taken together these results potentially advocated that ATLS2021 based approach is potentially immunogenic interventions against TB.

pharmacology and toxicology↗

bioRxiv2025

Mapping the dimerization specificity of bZIP transcription factors in bread wheat

Dimerization plays a pivotal role in target-binding interactions among various transcription factors (TFs), including bZIPs, bHLHs, MYBs, and zinc finger TFs. The bZIP family is defined by an -helix class of TFs containing a basic region that binds to the major groove of double-stranded DNA (dsDNA), followed by a leucine zipper motif that mediates dimerization through coiled-coil structures, forming homo- or heterodimers. In polyploid bread wheat (Triticum aestivum cv.), bZIPs and their homeologs interact through the dimerization motifs to form specific bZIP pairs with distinct regulatory functions. The dimerization pattern of bZIPs is critical for gene regulation, however, the mechanisms underlying their specificity remain poorly understood. Here, we focus on the dimerization specificity of coiled-coil - helical bZIP proteins in bread wheat, highlighting the leucine zipper region as a key dimerization motif. We identified 265 bZIPs containing the bZIP motif, following dimerization criteria established in humans, Drosophila, and Arabidopsis based on the presence of specific amino acids at the a, d, e, and g positions in heptads. Eight key bZIP TFs were cloned, expressed, and purified for the structural analysis. This study not only provides the dimerization trend exhibited by bZIPs during thermal stability analysis but also provides the reversibility pattern followed by the thermal denaturation exposure at 85{degrees}C. Our study also points towards that how amino acid substitutions and the length of the leucine zipper influence the formation of coiled-coil structures among bZIP homeologs of polyploid wheat. Overall, the study advances our understanding of the role of leucine zippers, in determining the specificity and stability of bZIP dimers, which in turn governs DNA binding and gene regulation in plants.

biochemistry↗

bioRxiv2025

Discovering Symbolic Cognitive Models from Human and Animal Behavior

Symbolic models play a key role in cognitive science, expressing computationally precise hypotheses about how the brain implements a cognitive process. Identifying an appropriate model typically requires a great deal of effort and ingenuity on the part of a human scientist. Here, we adapt FunSearch Romera-Paredes et al. (2024), a recently developed tool that uses Large Language Models (LLMs) in an evolutionary algorithm, to automatically discover symbolic cognitive models that accurately capture human and animal behavior. We consider datasets from three species performing a classic reward-learning task that has been the focus of substantial modeling effort, and find that the discovered programs outperform state-of-the-art cognitive models for each. The discovered programs can readily be interpreted as hypotheses about human and animal cognition, instantiating interpretable symbolic learning and decision-making algorithms. Broadly, these results demonstrate the viability of using LLM-powered program synthesis to propose novel scientific hypotheses regarding mechanisms of human and animal cognition.

animal behavior and cognition↗

bioRxiv2025

Distinct region-specific neutralization profiles of contemporary HIV-1 clade C against best-in-class broadly neutralizing antibodies

Broadly neutralizing antibodies (bnAb) have been clinically proven to be an excellent choice for HIV-1 prevention. However, the relative effectiveness of best-in-class bnAbs against regionally relevant circulating HIV-1 forms is not clear. In the present study, we compared the degree of neutralization sensitivity of contemporary HIV-1 Indian clade C with that of South African origin. Phylogenetic analysis revealed that these clade C viruses continue to evolve distinctly from one another. Env-pseudotyped viruses prepared using contemporary HIV-1 clade C env genes (N=115) obtained from nine geographically distinct sites in India (between 2020-2023) were found to be most sensitive to V3-directed bnAbs 10-1074 and BG18, and second generation CD4 binding site (CD4bs) directed bnAbs (VRC07, N6 and 1-18), however they were found to be significantly resistant to V1/V2 apex directed bnAbs. Moreover, we observed that the degree of sensitivity varied between contemporary Indian and South African clade C viruses. Differences in degree of neutralization susceptibility were associated with differences observed in key residues that form bnAb contact sites, gp120 loop lengths and the number of N-linked glycans in the V4 hypervariable region. Interestingly, the second generation CD4bs bnAbs (VRC07, N6, 1-18) showed neutralization of VRC01 and 3BNC117 resistant viruses but with 2-7-fold reduced potency compared to the VRC01 sensitive counterparts, likely due to the enrichment of resistance associated residues observed in loop D. Predictive analysis indicated that combination of BG18, N6 and PGDM1400 can provide over 95% neutralization coverage at 1g/mL of contemporary India clade C, an observation found to be distinct to that reported for the Africa clade C viruses. Taken together, we found distinct neutralization patterns and env signatures associated with resistance to key bnAbs. Our study highlights that towards achieving clinical effectiveness, both the complementarity of bnAb classes and the regionally relevant HIV forms need to be considered. Author summaryWhile the development of vaccines to prevent HIV infection remains a global priority, their potential effectiveness is limited by the extraordinarily diversified circulating forms of HIV-1. The prospect of best-in-class bnAbs as potential prevention option has been demonstrated in several studies including the Phase II Antibody Mediated Prevention (AMP) trial; however, to be broadly applicable, bnAbs will need to overcome the substantial variability of HIV env. The present study highlights that the contemporary HIV-1 clade C viruses are evolving to be less sensitive to the best-in-class bnAbs and HIV-1 clade C that predominates in India and South Africa vary in their degree of susceptibility to best-in-class clinically relevant bnAbs. This indicates differences in the antigenic properties between globally circulating HIV-1 clade C at a population level. Overall, the outcome of this study highlights the need for periodic assessment of sequence and neutralization profiles of the circulating regionally relevant HIV-1 forms towards prioritizing the bnAb combination suitable for effective intervention.

immunology↗

bioRxiv2024

Distinct roles of the two BRCA2 DNA binding domains in DNA damage repair and replication fork preservation

Homologous recombination (HR) is a highly conserved tool for the removal of DNA double-strand breaks (DSBs) and the preservation of stalled and damaged DNA replication forks. Successful completion of HR requires the tumor suppressor BRCA2. Germline mutations in BRCA2 lead to familial breast, ovarian, and other cancers, underscoring the importance of this protein for maintaining genome stability. BRCA2 harbors two distinct DNA binding domains, one that possesses three oligonucleotide/oligosaccharide binding (OB) folds (known as the OB-DBD), and with the other residing in the C-terminal recombinase binding domain (termed the CTRB-DBD) encoded by the last gene exon. Here, we employ a combination of genetic, biochemical, and cellular approaches to delineate contributions of these two DNA binding domains toward HR and the maintenance of stressed DNA replication forks. We show that OB-DBD and CTRB-DBD confer ssDNA and dsDNA binding capabilities to BRCA2, respectively, and that BRCA2 variants mutated in either DNA binding domain are impaired in the ability to load the recombinase RAD51 onto ssDNA pre-occupied by RPA. While the CTRB-DBD mutant is modestly affected for HR, it exhibits a strong defect in the protection of stressed replication forks. In contrast, the OB-DBD is indispensable for both BRCA2 functions. Our study thus defines the unique contributions of the two BRCA2 DNA binding domains in genome maintenance.

biochemistry↗

bioRxiv2024

MHC class I / II restricted T cell epitopes from clinical isolate of Mycobacterium tuberculosis: A potential candidate for vaccine development for Tuberculosis

Tuberculosis is major challenge to the health care system with TB associated death rates increasing annually. Optimum management of TB (particularly latent or MDR cases) warrants use of immunological approaches like subunit or peptide-based vaccination for tailoring effector immunity in patients. Since MHC class I is a potent enhancer element of host immunity and effective in clearing large variety of intracellular pathogens or tumors. In this context, we explore whether MHC-I restricted peptides from clinical isolates of M. tuberculosis can be used as an adjuvant for augmenting host immune responses. In the present study, we have synthesized various peptides from clinical isolates of M. tuberculosis which were having high affinity for Class I MHC molecules as potential immune enhancer for T cell or iNKT cell populations. We have evaluated the immunogenic potential of various MHC class I restricted epitopes (Rv2588c, Rv1357, Rv0148, Rv2973, Rv2557 and Rv2445) which were derived from clinical isolates of M. tuberculosis on increased proliferation of T or iNKT cells, release of IFN gamma secreted by T cells as well as NO as indicative parameters of immuno-stimulation. As expected, FACS and ELISA data clearly revealed that these peptides were potentially immunogenic for PBMCs from both healthy as well as 10 HC PTB patients. Our data clearly demonstrated a significant immune response in the PBMC from w PTB patients over healthy individuals which mimicked booster response. Our cytokine and nitric oxide data further revealed the influence of these peptides on sensitizing innate immune response as well. SignificanceOur study demonstrates the significance of MHC class I restricted peptides from M. tuberculosis for inducing potential immunogenic responses in host that may qualify them as potent vaccine candidate. To the best of our knowledge this is the first immune monitoring protocol describing the impact of synthetic novel MHC class I restricted T-cell epitope (Rv2588c, Rv0148) on cell mediated and innate immune response in PBMC populations and suggests their potential as vaccine candidate

immunology↗

bioRxiv2024

rpcFold: residual parallel convolutional neural network to decipher RNA folding from RNA sequence

Precise secondary structure information offers deeper insights into the functionality of many RNA molecules. Earlier approaches, which primarily relied on the free-energy minimization, proved inadequate, as RNA often adopts complex folds, especially with increasing length. We present rpcFold, a residual parallel convolutional neural network-based model for RNA secondary structure prediction. From the nucleotide sequences, we compute a base-pairing possibility score using a locally weighted Gaussian function. It captures the intricate canonical and noncanonical pairing patterns, which enhances the modelling of short- and long-range dependencies. The features are mapped into an image that is the input to rpcFold. Our sliding window mechanism accommodates sequences of arbitrary length, and hence explicitly addresses the prediction of pseudoknots, which are often overlooked in prior works. The performance of rpcFold is demonstrated on nested and on non-nested (pseudoknot) base-pairs. While tested on within-family and cross-family benchmark datasets, rpcFold shows improved performance over existing state-of-the-art methods. Additionally, for long nucleotide sequences with complex pseudoknots, rpcFold achieves 71.1% F1-score in jointly predicting nested and non-nested pairs. Performance on unseen RNA families further confirms the robustness and adaptability of our approach. The prediction accuracy of rpcFold, particularly in long-range RNAs and pseudoknots, improves our understanding of RNA functions.

bioinformatics↗

bioRxiv2024

Prevalence and diversity of Helicobacter species in captive wild carnivores, and their implications for conservation management of endangered species

Helicobacter species colonize the gastrointestinal tract of various hosts, and have always been of interest due to their potential zoonotic transmission and impact on health of humans and animals. Comprehensive studies involving wild animals from different locations are lacking, hindering our understanding of their natural host range, prevalence, and genetic diversity. Here we investigated the prevalence and genetic diversity of Helicobacter species in thirteen wild carnivore species across twenty-two zoos and rescue centers in India by targeting partial 16S rRNA gene. We used sequences obtained from positive samples for phylogenetic analysis, and further evaluated factors influencing Helicobacter prevalence with generalised linear mixed models. We analysed 985 faecal samples of which 286 (29%) tested positive for Helicobacter, spanning all the host species included in this study. Helicobacter prevalence is strongly related to host species and location, and varied from 7.3% in common leopard to 80% in Indian fox. Phylogenetic analysis identified 59 unique genotypes clustered into 11 groups of three major Helicobacter types: enterohepatic, gastric, and unsheathed. Diverse Helicobacter species associated with diseases in humans and domestic animals were observed, such as H. canis, H. bilis, along with several novel strains/ species which require formal classification. Overall, this study highlights the wide occurrence and high genetic diversity of Helicobacter species in captive wild carnivores in India. Our findings underscore the need for regular health assessments in captive facilities to monitor Helicobacter infections, which could impact the health and management of endangered species. Future research should explore Helicobacter presence in biotic and abiotic factors in zoo and free-ranging populations following One Health approaches.

microbiology↗

bioRxiv2024

Graph-structured populations elucidate the role of deleterious mutations in long-term evolution

Birth-death models have long been employed to understand the interplay of genetic drift and natural selection. While well-mixed populations remain unaffected by the choice of replacement rules, the evolutionary outcomes in spatially structured populations are strongly impacted by this choice. Moving parent individuals to vacant sites gives rise to new update rules, leading to new fixation categories for spatial graphs. We discover a new category of graphs, amplifiers of fixation, where a structure has a higher probability of fixation for mutants than the well-mixed population, regardless of their fitness value. Under death-Birth updating with parents moving to vacant sites, the star graph is an amplifier of fixation. For very large population sizes, the probability to fix deleterious mutants on the star graph converges to a non-zero value, in contrast to the result from well-mixed populations where the probability goes to zero. Additionally, most random graphs are amplifiers of fixation for death-Birth updating, with parent individuals replacing dead individuals. Conversely, most random graphs are suppressors of fixation- graphs with lower fixation probability for mutants regardless of their fitnesses- for Birth-death updating with offspring replacing dead individuals. When subjected to long-term evolution, amplifiers of fixation, despite being more efficient at fixing beneficial mutants, attain lower fitness than the well-mixed population, whereas suppressors attain higher fitness despite their inferior ability to fix beneficial mutants. These surprising findings can be explained by their deleterious mutant regime. Therefore, the deleterious mutant regime can be as crucial as the beneficial mutant regime for adaptive evolution.

evolutionary biology↗

bioRxiv2024

Mitochondrial Dynamics and Bioenergetics in iPSC-Derived Neurons with Familial Alzheimer's Disease Mutations

Mitochondrial (MT) dysfunction is a hallmark of Alzheimers Disease (AD), but the specific defects across forms of AD are unknown. We measured multiple parameters of MT dynamics and function, and neurite degeneration, in iPSC-derived human neurons possessing natural and engineered mutations in PS1, PS2, and APP genes. Mutations in all three genes altered MT function measured by basal, ATP-linked, and maximal oxygen consumption rate; and spare respiratory capacity, with PS1/PS2 alleles being more severe than APP mutations. Electron flow through Complexes I-IV was decreased in PS1/PS2 mutations but; in contrast, APP alleles had only modest impairments of CI and CII. We measured aspects of MT dynamics including fragmentation, and neurite degeneration, both of which were dramatic in PS1/PS2 alleles, but essentially absent in APP alleles. The marked differences in MT pathology may occur from the distinct ways APP is processed into A{beta} and may correlate with the disease severity.

neuroscience↗

bioRxiv2024

A general approach to reduce off-target radioactivity in vivo via Tetrazine-Knock-Out (TKO)

Monoclonal antibodies have had a remarkable impact on cancer therapy due to their high target specificity. However, their large molecular weight results in slow blood clearance, which can take weeks to clear from circulation. As companion nuclear imaging and diagnostic tools, these characteristics force delayed imaging and the use of isotopes with long half-lives such as 89Zr. For optimal clinical application, it is desirable that radioimmunoconjugates remain in the blood for just enough time to accumulate adequately in target tissues, while non-targeted or circulating radioactivity is ideally rapidly excreted from the body to maximize imaging contrast and minimize radiation dose to healthy tissues. We addressed the current challenges of antibody-based imaging by developing rituximab radioimmunoconjugates that accumulate sufficient activity for tumor imaging within 24 h of administration, while clearing circulating radioactivity via administration of a small molecule clearing agent. Rituximab, an anti-CD20 monoclonal antibody, is used as standard first-line therapy for diffuse large B-cell lymphoma. CD20 is expressed by 95% of B-lymphocytes and their malignant counterparts, making it a therapeutic target for B-cell malignancies. We attached 125I, 68Ga, and 89Zr to rituximab using a "clickable" linker containing trans-cyclooctene and tested the ability of tetrazines to induce the inverse electron demand Diels-Alder reaction (iEDDA) after antibody administration. This "tetrazine-knock-out" (TKO) approach liberates the radioactivity from rituximab in the bloodstream, resulting in its rapid renal excretion which enhances target-to-background ratios, and minimizes off-target radiation exposure. Due to the internalization of the radioimmunoconjugate in CD20+ tumor cells, no substantial clearance was observed from Raji xenografts. We characterized different leaving groups, several cellular models and antibodies with distinct internalizaing properties. The TKO approach opens opportunities to use radiolabeled antibodies for low-abundance or heterogeneously expressed biologic targets and may allow radioimmunotherapy (RIT) for targets traditionally untenable due to dose-limiting toxicities. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/596510v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@11bc2f0org.highwire.dtl.DTLVardef@19b4f72org.highwire.dtl.DTLVardef@17cee32org.highwire.dtl.DTLVardef@fa4491_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Differential control of intestine function by genetically defined enteric neurons

The function of the intestine is regulated by direct innervation from a combination of enteric, sensory, and autonomic neurons. A central question in neurobiology is how these distinct peripheral neuron populations collectively control intestinal function. However, disambiguating the functions of intestine-innervating neuronal populations has been a challenge. Using intersectional genetic approaches in mice, we enable precise manipulations of defined neuronal populations within the intestinal tract. We examined enteric neurons, which represent the majority of intestine-innervating neurons, by genetically isolating neuronal subclasses, identifying their morphological specializations, and defining subclass-specific influences on intestinal functions. We further found that food consumption can be modulated by select enteric neuron populations via the spinal sensory afferent pathway. Taken together, the presented molecular genetic characterization of intestine-innervating neurons establishes a foundation for detailed studies of the enteric nervous system and its interactions with the broader neural networks of the body.

neuroscience↗

bioRxiv2024

Cytosolic Class I J-domain proteins aid mitochondrial protein import and influence homeostasis in Arabidopsis thaliana.

Mitochondrial homeostasis heavily relies on import of numerous nuclear encoded proteins. Multiple chaperone machineries operate at the cytosolic and mitochondrial side of the bilayer to ensure the efficacy of this process. While the mechanisms and components involved in translocation of polypeptides into the mitochondria are known in great detail, the cytosolic events of mitochondrial protein import are poorly understood in plants. This study explores the role of two cytosolic Class I J-domain proteins (JDPs), atDjA1 and atDjA2, in mitochondrial homeostasis in Arabidopsis thaliana. We show that atDjA1 and atDjA2 uniquely interacted with outer mitochondrial surface receptors, Tom9, Metaxin, and OM64, which guide the import of numerous mitochondrial precursors. Knockout of atDjA1 or atDjA2 impeded seed germination under multiple abiotic stress conditions, with atDjA1 mutant being more sensitive. Both the mutants demonstrated decreased respiratory rates and enhanced accumulation of reactive oxygen species (ROS), indicating compromised mitochondrial function. Further, knockout of atDjA1 resulted in a significant decline in the number of active mitochondria as well as import of MTS-GFP. While atDjA2 mutants did not show a reduction in mitochondrial number, both the mutants had an activated mitochondrial retrograde signaling (MRS) further underscoring compromised mitochondrial homeostasis in these mutants. Our findings suggest that evolutionarily conserved cytosolic Class I JDPs preserve mitochondrial homeostasis by targeting specific receptors on the outer mitochondrial surface, facilitating the import of cytosolically synthesized mitochondrial precursors in Arabidopsis thaliana.

plant biology↗

bioRxiv2024

Evolutionary origin of synovial joints

Synovial joints, characterized by reciprocally congruent and lubricated articular surfaces separated by a cavity, are hypothesized to have evolved from continuous cartilaginous joints for increased mobility and improved load bearing. To test the evolutionary origins of synovial joints, we examine the morphology, genetic, and molecular mechanisms required for the development and function of the joints in elasmobranchs and cyclostomes. We find the presence of cavitated and articulated joints in elasmobranchs, such as the little skate (Leucoraja erinacea) and bamboo shark (Chiloscyllium plagiosum), and the expression of lubrication-related proteoglycans such as aggrecan and glycoproteins such as hyaluronic acid receptor (CD44) at the articular surfaces in little skates. Sea lampreys (Petromyozon marinus), a representative of cyclostomes, are devoid of articular cavities but express proteoglycan-linking proteins throughout their cartilaginous skeleton, suggesting that the expression of proteoglycans is primitively not limited to the articular cartilage. Analysis of the development of joints in the little skate reveals the expression of growth differentiation factor-5 (Gdf5) and {beta}-catenin at the joint interzone before the process of cavitation, indicating the involvement of BMP and Wnt-signaling pathway, and reliance on muscle contraction for the process of joint cavitation, similar to tetrapods. In conclusion, our results show that synovial joints are present in elasmobranchs but not cyclostomes, and therefore, synovial joints originated in the common ancestor of extant gnathostomes. A review of fossils from the extinct clades along the gnathostome stem further shows that synovial joints likely arose in the common ancestor of gnathostomes. Our results have implications for understanding how the evolution of synovial joints around 400 mya in our vertebrate ancestors unlocked motor behaviors such as feeding and locomotion. Author summaryWe owe our mobility and agility to synovial joints, characterized by a lubricated joint cavity between the bony elements. Due to the cavity, synovial joints function by bones sliding relative to each other, allowing an extensive range of motion and heightened stability compared to fused or cartilaginous joints that function by bending. Using histological and protein expression analysis, we show that reciprocally articulated, cavitated, and lubricated joints are present in elasmobranchs such as skates and sharks but not in cyclostomes such as the sea lamprey. Furthermore, the development of the little skate joints relies on genetic regulatory mechanisms such as BMP and Wnt-signalling, similar to tetrapods. Thus, our results show that synovial joints are present in elasmobranchs but not in cyclostomes. In conclusion, synovial joints originated in the common ancestor of jawed vertebrates. Furthermore, a review of fossil taxa along the gnathostome stem shows that cavitated joints that function by relative sliding of articulating surfaces originated at the common ancestor of all gnathostomes. Our results have consequences for understanding how the evolution of cavitated and lubricated joints in ancient vertebrates impacted behaviors like feeding and locomotion 400 million years ago.

evolutionary biology↗

bioRxiv2024

Efficacy of auxin-inducible protein degradation in C. elegans tissues using different auxins and TIR1-expressing strains

The auxin-inducible degradation system has emerged as a powerful tool to deplete proteins of interest in cells and tissues of various model organisms, including C. elegans 2-5. Here, we present a detailed protocol to perform AID-driven spatiotemporal depletion of specific proteins in C. elegans tissues. First, we introduced the AID degron and a fluorescent reporter at two conserved proteins: (a) the transcription factor CFI-1 (human ARID3), which is expressed in the nucleus of multiple C. elegans neurons and head muscle cells 6,7, and (b) the broadly expressed translation initiation factor Y47D3A.21 (human DENR) that localizes in the cytoplasm. Second, we provide a step-by-step guide on how to generate C. elegans strains suitable for AID-mediated protein (CFI-1 and DENR) depletion. Third, we find that the degree of CFI-1 and DENR depletion in C. elegans tissues is comparable upon treatment with either natural auxin (indole-3-acetic acid (IAA) or a water-soluble synthetic auxin analog (K-NAA). Last, we compare the degree of AID-mediated CFI-1 depletion in C. elegans neurons when the transport inhibitor response 1 (TIR1), component of the SCF ubiquitin ligase complex, is provided in neurons or all somatic cells. Altogether, this protocol provides side-by-side comparisons of different auxins and TIR1-expressing lines. Such comparisons may benefit future studies of AID-mediated protein depletion in C. elegans. Graphical abstractImage provided as pdf (together with Figures). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/575916v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@adf68eorg.highwire.dtl.DTLVardef@1160cfcorg.highwire.dtl.DTLVardef@aeb593org.highwire.dtl.DTLVardef@12c63e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIEfficient protein depletion in C. elegans tissues upon treatment with either natural or synthetic auxins. C_LIO_LIPansomatic TIR1 expression leads to efficient depletion of CFI-1 and DENR. C_LIO_LIPanneuronal TIR1 expression leads to neuron-specific, yet variable CFI-1 depletion. C_LIO_LIThe AID system is compatible with fluorescence microscopy, Western blotting and behavioral assays. C_LI

developmental biology↗

bioRxiv2024

Inflammatory Cytokines Can Induce Synthesis Of Type-I Interferon

Type I interferon (IFN) is induced in virus infected cells, secreted and it inhibits viral replication in neighboring cells. IFN is also an important player in many non-viral diseases and in the development of normal immune cells. Although the signaling pathways for IFN induction by viral RNA or DNA have been extensively studied, its mode of induction in uninfected cells remains obscure. Here, we report that inflammatory cytokines, such as TNF- and IL-1{beta}, can induce IFN-{beta} through activation of the cytoplasmic RIG-I signaling pathway. However, RIG-I is activated not by RNA, but by PACT, the protein activator of PKR. In cell lines or primary cells expressing RIG-I and PACT, activation of the MAPK, p38, by cytokine signaling, leads to phosphorylation of PACT, which binds to primed RIG-I and activates its signaling pathway. Thus, a new mode of type I IFN induction by ubiquitous inflammatory cytokines has been revealed. Key pointsO_LICytochalasin D followed by TNF- / IL-1{beta} treatment activates IFN-{beta} expression. C_LIO_LIIFN-{beta} expression happens due to activation of RIG-I signaling. C_LIO_LIInteraction between RIG-I and PACT activates IFN-{beta} expression. C_LI

cell biology↗

bioRxiv2023

Clustered Regularly Interspaced Short Palindromic Repeats-Cas system regulates Salmonella virulence

ObjectivesInvestigating the type 1-E CRISPR-Cas-mediated regulation of Salmonella pathogenesis. MethodsWe assessed the pathogenicity of the wild-type and CRISPR-Cas knockout strains using infection models. The mechanisms were elucidated using antimicrobial assays and expression analysis. ResultsCRISPR-Cas knockout strains were defective in invasion and proliferation in intestinal epithelial cells and macrophages. However, proliferation defects were not observed in the Gp91phox-/- macrophages, suggesting the systems role in antioxidant defence. The knockout strains show hampered colonization in in-vivo infection models, possibly due to increased sensitivity against innate immune barriers like antimicrobial peptides, complement proteins and oxidative stress. The expression studies of various virulence regulators: pmr genes, anti-oxidant genes, SPI-1 and SPI-2 encoded master regulators, and effectors showed repressed expression in the knockout strains. Some of these genes could be directly regulated by the CRISPR-spacers owing to partial complementarity between the sequences. ConclusionOverall, our study shows that the CRISPR-Cas system positively regulates Salmonella pathogenesis by regulating the expression of different virulence factors.

microbiology↗