Biology subjects

Chauhan, D.

Publications and source records attributed to Chauhan, D..

10 recordsLinked to original sources

bioRxiv2026

The atypical RHO-GTPase RND3/RHOE interacts with FLRT3 to regulate cortical migration and folding

Cortical gyrification, characterized by the formation of sulcal fissures and gyral peaks, is a distinctive feature of large mammalian brains and is associated with the emergence of higher cognitive functions. This contrasts with small mammals, like mice, which generally display smooth (lissencephalic) cortices. Gyrification results from the integration of several complex processes such as neuron progenitor amplification and migration. However, the underlying molecular mechanisms remain poorly understood. Here, we show that genetic ablation of Rnd3, encoding the atypical RHO-GTPase RND3/RHOE, induces spontaneous cortical sulci in a subset of mouse embryos without increasing neural progenitor amplification. Instead, RND3 regulates cortical neuron migration, as RND3 overexpression delays neuronal migration, whereas its loss accelerates this process. Similar phenotypes reported in the double Flrt1/3 mutants, suggested a functional interaction between the transmembrane FLRTs and RND3 during cortex development. We demonstrate that Rnd3 and Flrt3 are co-expressed in migrating cortical neurons and interact through a {beta}-strand-mediated binding mechanism, involving a conserved motif in RND3. Disruption of this motif abolishes FLRT3 binding, impairs RND3-mediated regulation of neuronal migration and promotes sulcus formation, indicating that FLRT3-RND3 suppresses cortical folding by regulating neuronal migration in mice. Consistently, Rnd3 expression is reduced in the outer subventricular zone of the prospective sulcal regions in the gyrencephalic ferret brain, supporting the notion that RND3 downregulation contributes to sulci development. Together, these findings identify FLRT3-RND3 signalling as a conserved negative regulator of cortical neuron migration and cortical folding.

neuroscience↗

bioRxiv2026

Targeted mutagenesis in Ehrlichia canis deleting the phage head-to-tail connector protein gene and its assessment as a vaccine candidate preventing canine ehrlichiosis

Ehrlichia canis is primarily a Rhipicephalus sanguineus tick-borne rickettsial pathogen initially identified as causing canine monocytic ehrlichiosis, and infections in people have also been reported in Venezuela, Mexico, and parts of Europe. It is of high importance to have a vaccine suitable in protecting the canine host, which will aid in lessening E. canis infections also in people. Gene inactivation mutations in the phage head-to-tail connector protein genes (phtcp) from E. chaffeensis and A. marginale caused attenuated growth, and prior infection with the mutated bacteria induced protective immunity against wild-type bacterial infections in natural hosts, independent of blood-borne infection or tick-transmission infection. In the current study, we describe the development of targeted mutagenesis for the first time in E. canis genome and with a novel modification to avoid introducing antibiotic resistance cassettes to delete the phtcp ortholog from E. canis. The mutated E. canis was then assessed for its in vivo growth and the induction of host immunity exerted following the mutant infection aiding to protect against wild-type infection challenge in the canine host. We assessed systemic pathogen loads, hematological parameters, IgG immune responses, and plasma cytokines following the mutant infection relative to uninfected dogs. Similarly, the assessments were carried out following wild-type pathogen infections in dogs with or without prior mutant infection challenges. The study demonstrates that prior infection of dogs with the mutant induces immunity to prevent infection establishment by wild-type E. canis. Similarly, the mutant infection resulted in clear biological differences compared to the wild-type infection. This study establishes that the molecular genetic methods are broadly applicable to pathogens belonging to the family Anaplasmataceae and that the modified live vaccines with phtcp gene orthologs are valuable in reducing the diseases caused by the tick-borne rickettsial pathogens belong to Anaplasmataceae, including E. canis.

microbiology↗

bioRxiv2025

The control of prickle formation in Rubus

Prickles on blackberry and raspberry canes make pruning, harvesting, and handling more difficult and can increase labor costs for growers. The trait has been challenging to improve in these clonal crops because it is recessive and linked to undesirable agronomic traits. In blackberry and red raspberry, breeding programs have used recessive mutants at the S locus to generate prickleless cultivars for the last century. In this study, we identified independent loss-of-function mutations in a WUSCHEL-LIKE HOMEOBOX transcription factor, WOX1, as the genetic basis of the prickleless S locus in both blackberry and red raspberry. We mapped the S locus using integrated genome-wide association, bulked segregant analysis, and identity-by-descent analyses informed by breeding pedigrees. Additionally, we generated a genome sequence from Luther Burbanks prickleless blackberry variety Burbank Thornless that contained an additional allele of WOX1. To verify the genes role, we used gene editing to knock out WOX1 in an elite prickled commercial blackberry line. All edited plants were prickleless and lacked glandular trichomes, confirming that WOX1 controls a joint developmental pathway. Other plant traits were unchanged, indicating WOX1 is a specific and safe target for improvement. Gene editing can enable breeders to remove prickles directly from elite varieties, reducing the need for extensive breeding cycles and delivering safer, easier-to-harvest cultivars to growers.

plant biology↗

bioRxiv2024

Cytoplasmic expression of the cell cycle regulator cyclin D1 in radial glial progenitor cells modulates brain cortex development

During nervous system development, the interplay between cell cycle regulation and neurogenesis is fundamental to achieve the correct timing for neuronal differentiation. However, the molecular players regulating this transition are poorly understood. Among these, the cell-cycle regulatory cyclins and their cyclin-dependent kinases (Cdks) play a pivotal role. In the present work we uncover an unknown function of cyclin D1 (Ccnd1) during cortex development which is independent of cell cycle regulation and that relies on its cytoplasmic localization and membrane association. We show that Ccnd1 is localized in the cytoplasm of the radial glial process (RGP) of neuron progenitors in different regions of the developing brain, including the cortex. Cytoplasmic Ccnd1 is enriched at the distal tip of the RGP, adjacent to the meningeal basement membrane, and overlaps with {beta}1-integrin at the plasma membrane. CCND1 knock-out animals show an abnormal cortical layering in which the distribution of Tbr2+ and Ctip2+ cells are affected without displaying proliferation defects. This is consistent with a cytoplasmic function of Ccnd1 as overexpression by in utero electroporation of a dominant negative Ccnd1, unable to activate Cdks, and targeted to the cytoplasmic membranes, reproduces some of these Tbr2 and Ctip2 defects. Finally, we provide evidence that cytoplasmic Ccnd1 affects neuron morphology and that it is required for the proper detachment of the RGP from the meningeal basement membrane by a mechanism involving the phosphorylation of the integrin effector protein paxillin. Hence, we propose that Ccnd1 has an important cytoplasmic function for cortical development independently of cell cycle regulation. Significant StatementA key developmental step during nervous system formation is the transition from proliferating progenitors to postmitotic neurons. However, the molecular mechanisms regulating this process are not fully understood. Cyclin D1 (Ccnd1) is a canonical regulator of cell cycle in the cell nucleus. Surprisingly, we show that Ccnd1 is also located in the radial glial process of neuron progenitors and associated to the plasma membrane in different regions of the developing mouse brain. We uncover a novel function for this cytoplasmic Ccnd1 and show that it is required for proper cortical layering, independent of cell cycle regulation. Mechanistically, we provide evidence that this function is mediated by the integrin effector paxillin. We propose therefore that cytoplasmic Ccnd1 is important for cortex development independent of cell cycle regulation.

neuroscience↗

bioRxiv2024

Advancing Tolerogenic Immunotherapy: A Multi-Epitope Vaccine Design Targeting the CYP2D6 Autoantigen in Autoimmune Hepatitis Through Immuno-Informatics

Juvenile autoimmune hepatitis (JAIH) is a rare autoimmune disorder affecting children, characterized by the immune systems misguided attack on liver cells, primarily targeting the CYP2D6 autoantigen. This repeated attack leads to hepatic inflammation, fibrosis, and eventual liver failure. Current therapeutic strategies predominantly rely on immunosuppressive agents or whole B cell depletion antibodies, which render patients susceptible to infections and cancers. Hence, there is an urgent need for antigen-specific therapies to mitigate the severity of autoimmune hepatitis. Tolerogenic antigens represent a promising avenue in immunotherapy, capable of dampening autoimmunity. Here, we present a novel computationally designed multi-epitope tolerogenic vaccine tailored to target CYP2D6, aimed at inducing tolerogenic dendritic cells (DCs) and halting autoimmune progression in JAIH patients. To validate our approach, we have developed a similar vaccine for testing in mouse models of JAIH. The selected tolerogenic epitopes exhibit antigenicity without allergenicity or toxicity, and specifically induce IL-10 production (restricted to CD4+ T cell epitopes). In our vaccine design, tolerogenic poly-epitopes are linked with Toll-like receptor (TLR)-4-agonist, the 50S ribosomal unit, and IL-10, effectively programming DCs towards a tolerogenic state. Molecular docking and dynamic simulations have confirmed strong binding affinities and stable complexes between the vaccine structures, TLR4 and IL-10 receptor alpha (IL-10RA), indicating their potential for in vivo DC interaction and programming. Consequently, this innovative vaccine approach demands further exploration through wet lab experiments to assess its tolerogenicity, safety, and efficacy, thereby laying the groundwork for potential application in clinical settings.

immunology↗

bioRxiv2023

Postnatal maternal care moderates the effects of prenatal bisphenol exposure on offspring neurodevelopmental, behavioral, and transcriptomic outcomes

Bisphenols (BPs), including BPA and "BPA-free" structural analogs, are commonly used plasticizers that are present in many plastics and are known endocrine disrupting chemicals. Prenatal exposure to BPA has been associated with negative neurodevelopmental and behavioral outcomes in children and rodent models. Prenatal BPA exposure has also been shown to impair postnatal maternal care provisioning, which can also affect offspring neurodevelopment and behavior. However, there is limited knowledge regarding the biological effects of prenatal exposure to bisphenols other than BPA and the interplay between prenatal BP exposure and postnatal maternal care on adult behavior. The purpose of the current study was to determine the interactive impact of prenatal BP exposure and postnatal maternal care on neurodevelopment and behavior. Our findings suggest that the effects of prenatal BP exposure on eye-opening, adult attentional set shifting and anxiety-like behavior in the open field are dependent on maternal care in the first five days of life. Interestingly, maternal care might also attenuate the effects of prenatal BP exposure on eye opening and adult attentional set shifting. Finally, transcriptomic profiles in male and female medial prefrontal cortex and amygdala suggest that the interactive effects of prenatal BP exposure and postnatal maternal care converge on estrogen receptor signaling and are involved in biological processes related to gene expression and protein translation and synthesis. Overall, these findings indicate that postnatal maternal care plays a critical role in the expression of the effects of prenatal BP exposure on neurodevelopment and adult behavior. Understanding the underlying biological mechanisms involved might allow us to identify potential avenues to mitigate the adverse effects of prenatal BP exposure and improve health and well-being in human populations.

neuroscience↗

bioRxiv2022

Intrabacterial regulation of a cytotoxic effector by its cognate metaeffector promotes Legionella pneumophila virulence

Legionella pneumophila is a natural pathogen of unicellular protozoa that can opportunistically infect macrophages and cause Legionnaires Disease. Intracellular replication is driven by hundreds of bacterial effector proteins that are translocated into infected host cells by a Dot/Icm type IV secretion system. L. pneumophila effectors are temporally regulated in part by a unique family of translocated regulatory effectors, termed metaeffectors, which bind and modulate the function of a cognate effector in host cells. We discovered that regulation of the cytotoxic effector SidI by its metaeffector, MesI, is critical for L. pneumophila virulence in natural and opportunistic hosts. MesI binds and negatively regulates SidI activity in vitro but how dysregulation of SidI impairs L. pneumophila intracellular replication is unclear. Using a chromosomally-encoded inducible expression system, we discovered that dysregulation of SidI, via loss of MesI, was toxic to L. pneumophila. SidI enzymatic activity was required for toxicity since L. pneumophila growth was unaffected by induced expression of a catalytically inactive sidI allele. We found MesI translocation into host cells was dispensable for intracellular replication and that MesI-deficient bacteria were rapidly degraded within host cells. Together, our data suggest a unique role for intrabacterial effector regulation by a translocated metaeffector in L. pneumophila virulence. ImportanceLegionella pneumophila replicates within phagocytic host cells using hundreds of effector protein virulence factors, which canonically subvert the function of host proteins and pathways. L. pneumophila encodes a unique family of translocated effectors called metaeffectors, which bind and regulate the function of a cognate effector in host cells. The metaeffector MesI promotes L. pneumophila virulence by regulating the cytotoxic effector SidI; however, the MesI regulatory mechanism is poorly understood. We discovered a unique intrabacterial role for MesI in L. pneumophila virulence. When uncoupled from MesI, SidI was toxic to L. pneumophila in vitro and triggered robust bacterial degradation in host cells. Importantly, translocation of MesI was dispensable for intracellular replication, demonstrating that intrabacterial regulation of SidI contributes to L. pneumophila virulence. These data show a unique and important role for MesI regulation of SidI within the bacterium, which challenges the dogma that effectors function exclusively within host cells.

microbiology↗

bioRxiv2022

Effector-mediated subversion of proteasome activator (PA)28αβ enhances lysosomal targeting of Legionella pneumophila within cytokine-activated macrophages

Legionella pneumophila is a natural pathogen of amoebae that causes Legionnaires Disease in immunocompromised individuals via replication within macrophages. L. pneumophila virulence and intracellular replication hinges on hundreds of Dot/Icm-translocated effector proteins, which are essential for biogenesis of the replication permissive Legionella-containing vacuole (LCV). However, effector activity can also enhance mammalian host defense via effector-triggered immunity. The L. pneumophila effector LegC4 is important for virulence in amoebae but enhances host defense against L. pneumophila in the mouse lung and, uniquely, within macrophages activated with either tumor necrosis factor (TNF) or interferon (IFN)-{gamma}. The mechanism by which LegC4 potentiates cytokine-mediated host defense in macrophages is unknown. Here, we found that LegC4 enhances cytokine-mediated phagolysosomal fusion with Legionella-containing vacuole (LCV) and binds host proteasome activator (PA)28, which forms a heterooligomer with PA28{beta} to facilitate ubiquitin-independent proteasomal degradation of oxidant-damaged (carbonylated) proteins. We found that oxidative stress was sustained in the presence of LegC4 and that the LegC4 restriction phenotype was relieved in PA28{beta}-deficient macrophages and in the lungs of mice in vivo. Our data also show that oxidative stress is sufficient for LegC4-mediated restriction in macrophages producing PA28{beta}. PA28{beta} has been traditionally associated with antigen presentation; however, our data support a novel mechanism whereby effector-mediated subversion of PA28{beta} enhances cell-autonomous host defense against L. pneumophila under inflammatory and oxidative stress conditions. This work provides a solid foundation to evaluate induced proteasome regulators as mediators of innate immunity. Author SummaryPro-inflammatory cytokines induce antimicrobial host defense pathways within macrophages to control intracellular pathogens. We discovered that the Legionella pneumophila effector protein LegC4 potentiates pathogen clearance within cytokine-activated macrophages. Here, we show a central role for host proteasome activator (PA)28{beta} in LegC4 restriction. PA28{beta} is upregulated by cytokine signaling and under oxidative stress conditions to facilitate proteasomal degradation of oxidant-damaged proteins. We found that LegC4 binds PA28{beta} and that LegC4 restriction was lost in PA28{beta}-deficient (Psme1/2-/-) macrophages and a mouse model of Legionnaires Disease in vivo. Furthermore, oxidative stress was sustained in the presence of LegC4 and was sufficient for LegC4 restriction in PA28{beta}-producing cells macrophages. Finally, we found that L. pneumophila replication was attenuated within PA28{beta}-deficient macrophages irrespective of LegC4. These findings support a model whereby subversion of host proteostasis machinery triggers cell-autonomous host defense within macrophages under inflammatory and oxidative stress conditions.

microbiology↗

bioRxiv2021

Long Noncoding RNA RROL Provides Chromatin Scaffold for MYC-WDR82 Interaction to Impact Lipid Metabolism and Tumor Cell Growth in Multiple Myeloma

Long noncoding RNAs (lncRNA) can drive the tumorigenesis and be susceptible to therapeutic intervention. To define the landscape of therapeutically actionable lncRNA dependencies in multiple myeloma (MM), we coupled our extensive lncRNA transcriptomic profile with lncRNA targeted CRISPR interference viability screen and identified RNA Regulator of Lipogenesis (RROL) as a leading lncRNA dependency in MM. RROL shares its origin with the microRNA locus MIR17HG, however supports the proliferation and survival of MM cells in a microRNA- and DROSHA- independent manner. We found that RROL provides a chromatin scaffold for the functional interaction between c-MYC and WDR82 to promote the regulation of the lipogenic pathways via the transcriptional control of the rate-limiting enzyme ACC1 in MM cells. Inhibition of RROL with clinically applicable antisense molecules disrupts its transcriptional and functional activities causing potent anti-tumor effects both in vitro and in vivo in two pre-clinical animal models. This study establishes lncRNA RROL as a therapeutically actionable dependency with a unique mechanism of action in support of myeloma cell growth.

cancer biology↗

bioRxiv2019

Priming enables a NEK7-independent route of NLRP3 activation

The NLRP3 inflammasome plays a central role in antimicrobial defense, as well as in sterile inflammatory conditions. NLRP3 activity is governed by two independent signals. The first signal primes NLRP3, allowing it to respond to its activation signal. In the murine system, the mitotic spindle kinase NEK7 has been identified as a crucial factor in relaying the activation signal to NLRP3. Here we show that the requirement for NEK7 can be bypassed by TAK1-dependent post-translational priming. Under pro-inflammatory conditions that activate TAK1, NEK7 was dispensable for NLRP3 inflammasome formation in human and murine cells. Intriguingly, dissecting the NEK7 requirement in iPSC-derived primary human macrophages revealed that this NEK7-independent mechanism constitutes the predominant NLRP3 priming pathway in these cells. In summary, our results suggest that NEK7 functions as an NLRP3 priming - rather than activation - factor that can work in synergy or redundancy with other priming pathways to accelerate inflammasome activation.

immunology↗