Biology subjects

Joshi, D.

Publications and source records attributed to Joshi, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Distinct Longitudinal Antibody Responses Following Primary Influenza Infection or Vaccination in Infants: A Prospective Birth Cohort Study

The longitudinal effects of initial influenza exposure on the magnitude, persistence, and antigen specificity of antibody responses during infancy remain incompletely understood. We investigated humoral antibody responses following primary influenza infection or seasonal vaccination in a prospective US birth cohort with weekly respiratory surveillance for influenza infection. Influenza-specific serum IgG binding to H1 and H3 haemagglutinin (HA) antigens, HA inhibition (HAI), and live-virus neutralizing antibody responses were assessed longitudinally. To minimize influence of maternally derived antibodies, analyses of primary exposure responses were restricted to infants for whom influenza-specific maternal antibodies had declined below the assay detection limit before exposure. Antibody responses were compared following primary infection and vaccination and examined after subsequent annual vaccination. We found that maternal influenza-specific IgG declined rapidly during early infancy. Among infants without detectable pre-exposure influenza-specific antibodies, primary influenza infection was associated with greater post-exposure antibody responses and less measurable decline over follow-up than primary vaccination. These groups differed in age, calendar season, and exposure characteristics, limiting direct attribution of these differences to route of exposure. In infants with previous influenza infection followed by vaccination, responses after subsequent vaccination initially showed greater binding to the previously infecting subtype among the tested HA antigens. With repeated vaccination, responses became more distributed across the tested antigens. Our findings reveal differences in the magnitude, persistence, and antigen specificity of antibody responses according to the nature and sequence of influenza exposure. By prospectively capturing early-life exposures, this study provides longitudinal evidence linking exposure history to subsequent humoral antibody responses.

immunology↗

bioRxiv2026

Task-Dependent Modulation of Upper-Extremity Muscle Synergies during Object Slippage

Preventing object slippage requires the central nervous system to control and coordinate the muscle groups while adapting to the variations in the slip characteristics. Our study investigates how slip characteristics, such as slip direction, slip distance, and slip speed, affect the modulation of muscle synergies as a manifestation of object slippage. Our results reveal that there are shared synergies existing in the upward and downward slip emulation, and that these shared synergies are dependent on slip characteristics. However, there were no specific synergies found to exist in the upward and downward slip conditions. Our results also indicate the existence of combined synergies, as the motor modules in the upward slip direction were found to have shared synergies with multiple motor modules in the downward slip direction. We also observed that the number of shared synergies varied depending on the slip distance and slip speed. Together, these findings suggest that the CNS flexibly reconfigures a limited set of muscle synergies to accommodate varying slip dynamics, balancing control stability with adaptability.

neuroscience↗

bioRxiv2026

Safe Redosable Low-Immunogenic In Vivo CAR-T Therapy for B Cell Malignancies and Solid Tumors

In vivo CAR-T cell therapy eliminates manufacturing complexities associated with ex vivo autologous approaches, but safety concerns have limited adoption. We developed viroVbot, a next-generation in vivo CAR-T platform, by combining computational immunogenicity prediction (CIMMEXTM) with envelope engineering. Screening 22,562 glycoprotein sequences, we identified 641 vesiculovirus homologs, from which we selected Piry virus glycoprotein (PIRYV) as the optimal candidate. PIRYV exhibited lower MHC-epitope density, reduced human seroprevalence, with decreased T cell activation compared to VSV-G. To enhance targeting specificity, we engineered receptor-binding-deficient PIRYV (ePIRYVRBD) displaying CD3/CD7 nanobodies for T cell-selective transduction. To maximize safety, we engineered CAR-TRAP producer cells to eliminate unwanted B cell transduction and incorporated machine learning-optimized T cell-specific promoters that restrict CAR activation exclusively to lymphocytes. Additional modifications suppressed hepatocyte expression and prevented phagocytic uptake. In humanized xenograft models, viroVbot3 generated potent BCMA/CD19 specific CAR-T responses against multiple myeloma and Claudin18.2-targeting gastric cancer, demonstrating sequential redosing with alternative envelopes. Critically, viroVbot3 exhibited minimal off-target organ biodistribution with CAR expression restricted to T lymphocytes. These findings establish viroVbot as a low-immunogenic platform for scalable in vivo CAR-T manufacturing with capability for sequential redosing across hematologic and solid tumors.

bioengineering↗

bioRxiv2026

Establishment of a healthy control iPSC line from an Eastern Indian donor as a population specific resource for disease modelling

BackgroundiPSCs generated from healthy individuals constitute an important control resource for disease modelling applications but existing biobanks are highly skewed towards populations of European ancestry while well characterized control lines from Indian populations remain limited. Given the extensive genetic diversity of the Indian subcontinent, the availability of ethnically relevant healthy control lines is important for developing accurate disease models and reducing population specific confounding effects. MethodologyWe used peripheral blood mononuclear cells (PBMNCs) of a healthy female donor of Eastern Indian origin for the generation a wild type iPSC line using non-integrating episomal reprogramming vectors. Established colonies were expanded and characterized through morphological assessment, expression of pluripotency and trilineage markers, episomal vector clearance analysis, and chromosomal stability evaluation and mycoplasma contamination analysis. ResultsThe line generated exhibited characteristic pluripotent stem cell morphology and also showed strong expression of pluripotency markers, was free from any contamination and free from the reprogramming vectors confirming an integration free system. The cells maintained a normal diploidy number during characterization. Expression of lineage specific markers associated with ectoderm, mesoderm and endoderm confirmed the developed iPSCs functional capacity to undergo trilineage differentiation. ConclusionWe have developed and validated an iPSC line from an underrepresented Indian population. This well characterized, ethnicity specific iPSC line provides a valuable cell line for establishing a high quality, well characterized control baseline, which is a major missing element in South Asian stem cell repositories and thus will provide a solid foundation for future disease specific modelling and screening.

cell biology↗

bioRxiv2026

Dirty mice better recapitulate key features of mRNA vaccine immunogenicity observed in humans

Although specific pathogen free (SPF) mice have traditionally been used to test candidate vaccines, recent work has demonstrated that "dirty" mice with broad microbial exposure more appropriately recapitulate human immune responses. Using a model where lab mice are co-housed with pet store mice, we modeled SARS-CoV-2 mRNA vaccine responses in dirty and traditional SPF models. We found that dirty mice show reduced serum spike-binding antibody titers after prime and require a second booster dose to reach SPF-level spike antibody titers. Additionally, spike antibodies showed faster waning in dirty mice through 5 months post-vaccination, and neutralizing activity of these antibodies were reduced against Omicron variants, directly comparable with observations in humans. We further investigated the seasonality and consistency of pathogens in cohoused mice, and the impact of serial microbial exposure on our animal model system. We found that pathogen exposure and T cell activation remained consistent over time, and that a single co-housing event was sufficient to provide broad microbial exposure. This work demonstrates that the dirty mouse co-housing system is a promising, translationally representative approach to screen candidate mRNA vaccines for efficacy and durability prior to human clinical trials. Significance statementThe development of mRNA vaccines during the COVID-19 pandemic dramatically reduced hospitalization and death rates for infected individuals. However, booster vaccinations were required to achieve full efficacy, and protection waned over time. Our research leveraged a "dirty" mouse model to test whether SARS-CoV-2 mRNA vaccinations in animals with previous microbial exposure better modelled human immune responses. We found that dirty mice require a booster vaccination for full efficacy and experienced waning serum antibody titer over time. We propose this approach as a future model for robust preclinical mRNA vaccine testing.

immunology↗

bioRxiv2026

Ex vivo maturation of the malaria parasite egress protease SERA6 aids pathway dissection and inhibitor development.

Release (egress) of malaria parasites from host red blood cells (RBC) is a protease-dependent process involving breakdown of the RBC cytoskeleton by a parasite cysteine protease-like protein called SERA6. In the penultimate step of the egress cascade, SERA6 undergoes autoproteolytic maturation triggered upon cleavage by a serine protease called SUB1 and requiring interactions between SERA6 and fragments of another parasite protein called MSA180. Egress can be blocked by treatment of intraerythrocytic parasites with small molecules that prevent the autocatalytic SERA6 maturation step, suggesting that SERA6 is a druggable target. Here we describe the development of a cell-free in vitro system that recapitulates SERA6 maturation. We use the assay to confirm the strict requirement for MSA180 in SERA6 maturation by SUB1 and to show that these 3 components are sufficient for SERA6 maturation. Using a synthetic peptide substrate based on a predicted autocatalytic cleavage site we demonstrate that the fully mature SERA6 is an active proteolytic enzyme and we validate improved small molecule inhibitors of SERA6. Our lead inhibitory compound efficiently blocks egress of asexual blood stage parasites, confirming SERA6 as a new potential antimalarial drug target.

biochemistry↗

bioRxiv2025

Orthogonal and Robust Analytics Enable Reproducible and Scalable Manufacturing of High Purity Extracellular Vesicles Derived from Mesenchymal Stromal Cells

The development of extracellular vesicle (EV) based therapeutics requires robust analytical assays and scalable downstream processing (DSP) strategies to ensure product quality and reproducibility. In this study, we established an analytical toolbox comprising scattering and fluorescence mode nanoparticle tracking analysis (NTA), fluorescence-based flow cytometry (Fl-FC), and multi-detector analytical size exclusion chromatography. Liposomes, selected for their physicochemical similarity to EVs, were used as reference materials to optimize assay parameters, fluorescence labeling conditions, and dynamic range, minimizing artifacts such as photobleaching and masking effects. Using these optimized analytical tools, we designed a scalable DSP workflow for human bone marrow mesenchymal stromal cell (hBM-MSC) derived EVs, incorporating clarification, tangential flow filtration (TFF), ion exchange chromatography (IEX), buffer exchange, and sterile filtration. IEX chromatography resulted in the elution of two cell-secreted populations, with similar scattering signals while eluate 1 showed approximately 30 times higher absorbance signal compared to eluate 2. Transmission electron microscopy revealed that eluate 1 contained non-vesicular extracellular particles (NVEPs), and eluate 2 was enriched in EVs and showed higher expression of positive markers such as CD81 and CD73 using Simple Western. Additionally, the two IEX eluates showed different proteomic and lipidomic profiles. Then the analytical toolbox was utilized to monitor the DSP process in terms of particle recovery and impurity removal throughout the process determining the high purity level of the final EV preparation. Together, these results demonstrate that orthogonal analytics coupled to a scalable DSP yield reproducible MSC-EV preparations while depleting commonly co-isolated NVEPs. This practical framework advances process analytics of MSC-EV manufacturing and supports reporting of identity, purity, and function aligned with existing guidelines in the field. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/672476v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@142ff8aorg.highwire.dtl.DTLVardef@196c0bforg.highwire.dtl.DTLVardef@485c36org.highwire.dtl.DTLVardef@9dff39_HPS_FORMAT_FIGEXP M_FIG C_FIG Liposomes were used as reference materials to optimize single particle analytics as well as multi detector analytical chromatography to assess particle recovery and impurity removal. This optimized analytical toolbox enabled accurate monitoring of downstream processing steps, establishing a robust workflow for reproducible and scalable mesenchymal stromal derived extracellular vesicles bioprocessing.

bioengineering↗

bioRxiv2025

Development of a Stable Human iPSC Line from Peripheral Blood: A Control Resource for Disease Modeling

IntroductionTraditional disease modeling approaches have primarily utilized animal models and immortalized cell lines to investigate disease mechanisms and develop therapeutic strategies. However, previous research indicates that only about 5% of therapeutic interventions tested in animal models eventually receive regulatory approval for human use, highlighting limitations. The discovery of human-induced pluripotent stem cells (iPSCs) by Yamanakas team in 2007 has revolutionized the field with remarkable possibilities for modeling human diseases, drug testing, and regenerative medicine. Differentiated cells derived from iPSCs in two-dimensional (2D) monolayers offer a relatively straightforward system to study disease pathogenesis and underlying molecular mechanisms. ObjectiveThe present study aimed to generate and characterize an induced pluripotent stem cell (iPSC) line from peripheral blood mono-nuclear cells (PBMCs) of a healthy individual intending to serve as an age and gender-matched control for future disease modeling and regenerative medicine research. Materials and MethodsPBMCs were isolated from a healthy 31-year-old male volunteer. Somatic reprogramming was performed using episomal vectors expressing OCT3/4, SOX2, KLF4, and L-MYC. The resulting colonies were cultured and characterized for pluripotency markers by immunocytochemistry demonstrating the ability to differentiate into three germ layers. Karyotype analysis was performed to check the chromosomal abnormalities. InferencesThe resulting iPSC line exhibited pluripotency markers with the differentiation ability into three germ layers. Karyotyping analysis confirmed a normal chromosomal profile in both the donor and the reprogrammed iPSC line. This iPSC line could be a valuable resource as a healthy control for disease modeling and will contribute to advancing stem cell research with potential for regenerative medicine applications.

molecular biology↗

bioRxiv2024

Polycomb Repressive Complex 2 promotes atherosclerotic plaque vulnerability

Key findings1. PRC2 regulates EC shear stress responses. 2. PRC2 governs Klf2/4 suppression downstream of Pcdhg. 3. High PRC2 in ASCVD-prone arterial regions suppresses Klf2/4 to promote ASCVD. 4. Athero-protective Klf2/4 induction upon PRC2 inhibition requires Notch signaling. 5. Tazemetostat, an FDA approved PRC2 inhibitor, slows ASCVD progression and improves markers of plaque stability. Atherosclerotic cardiovascular disease (ASCVD), the leading cause of mortality worldwide, is driven by endothelial cell inflammatory activation and counter-balanced by anti-inflammatory transcription factors Klf2 and Klf4 (Klf2/4). Understanding vascular endothelial inflammation to develop effective treatments is thus essential. Here, we identify, Polycomb Repressive Complex (PRC) 2, which blocks gene transcription by trimethylating histone3 Lysine27 in gene promoter/enhancers, as a potent, therapeutically targetable determinant of vascular inflammation and ASCVD progression. Bioinformatics identified PRC2 as a direct suppressor of Klf2/4 transcription. Klf2/4 transcription requires Notch signaling, which reverses PRC2 modification of Klf2/4 promoter/enhancers. PRC2 activity is elevated in human ASCVD endothelium. Treating mice with established ASCVD with tazemetostat, an FDA approved pharmacological inhibitor of PRC2, slowed plaque progression by 50% and drastically improved markers of plaque stability. This study elucidates a fundamental mechanism of vascular inflammation, thus identifying a potential method for treating ASCVD and possibly other vascular inflammatory diseases.

cell biology↗

bioRxiv2024

Restoring the Multiple Sclerosis Associated Imbalance of Gut Indole Metabolites Promotes Remyelination and Suppresses Neuroinflammation

In multiple sclerosis (MS) the circulating metabolome is dysregulated, with indole lactate (ILA) being one of the most significantly reduced metabolites. We demonstrate that oral supplementation of ILA impacts key MS disease processes in two preclinical models. ILA reduces neuroinflammation by dampening immune cell activation as well as infiltration; and promotes remyelination and in vitro oligodendrocyte differentiation through the aryl hydrocarbon receptor (AhR). Supplementation of ILA, a reductive indole metabolite, restores the gut microbiomes oxidative/reductive metabolic balance by lowering circulating indole acetate (IAA), an oxidative indole metabolite, that blocks remyelination and oligodendrocyte maturation. The ILA-induced reduction in circulating IAA is linked to changes in IAA-producing gut microbiota taxa and pathways that are also dysregulated in MS. Notably, a lower ILA:IAA ratio correlates with worse MS outcomes. Overall, these findings identify ILA as a potential anti-inflammatory remyelinating agent and provide insights into the role of gut dysbiosis-related metabolic alterations in MS progression. One Sentence SummaryIndole lactate, a postbiotic metabolite reduced in MS, corrects gut microbiome metabolic imbalances associated with remyelination and neuroinflammation.

neuroscience↗

bioRxiv2024

PDZ-directed substrate recruitment is the primary determinant of specific 4E-BP1dephosphorylation by PP1-Neurabin

Protein Phosphatase 1 (PP1) relies on association with PP1-interacting proteins (PIPs) to generate substrate-specific PIP/PP1 holoenzymes, but the lack of well-defined substrates has hindered elucidation of the mechanisms involved. We previously demonstrated that the Phactr1 PIP confers sequence specificity on the Phactr1/PP1 holoenzyme by remodelling the PP1 hydrophobic substrate groove. Phactr1 defines a group of "RVxF-{Phi}{Phi}-R-W" PIPs that all interact with PP1 in a similar fashion. Here we use a PP1-PIP fusion approach to address sequence specificity and identify substrates of the RVxF-{Phi}{Phi}-R-W family PIPs. We show that the four Phactr proteins confer identical sequence specificities on their holoenzymes. We identify the 4E-BP and p70 S6K translational regulators as substrates for the Neurabin/Spinophilin PIPs, implicated in neuronal plasticity, pointing to a role for their holoenzymes in mTORC1-dependent translational control. Biochemical and structural experiments show that in contrast to the Phactrs, substrate recruitment and catalytic efficiency of the PP1-Neurabin and PP1-Spinophilin fusions is primarily determined by substrate interaction with the PDZ domain adjoining their RVxF-{Phi}{Phi}-R-W motifs, rather than by recognition of the remodelled PP1 hydrophobic groove. Thus, even PIPs that interact with PP1 in a similar manner use different mechanisms to ensure substrate selectivity.

molecular biology↗

bioRxiv2024

Latrophilin-2 mediates fluid shear stress mechanotransduction at endothelial junctions

Endothelial cell responses to fluid shear stress from blood flow are crucial for vascular development, function and disease. A complex of PECAM-1, VE-cadherin, VEGF receptors (VEGFRs) and PlexinD1 located at cell-cell junctions mediates many of these events. But available evidence suggests that another mechanosensor upstream of PECAM-1 initiates signaling. Hypothesizing that GPCR and G proteins may serve this role, we performed siRNA screening of G subunits and found that Gi2 and Gq/11 are required for activation of the junctional complex. We then developed a new activation assay, which showed that these G proteins are activated by flow. We next mapped the G residues required for activation and developed an affinity purification method that used this information to identify latrophilin-2 (Lphn-2/ADGRL2) as the upstream GPCR. Latrophilin-2 is required for all PECAM-1 downstream events tested. In both mice and zebrafish, latrophilin-2 is required for flow-dependent angiogenesis and artery remodeling. Furthermore, endothelial specific knockout demonstrates that latrophilin plays a role in flow-dependent artery remodeling. Human genetic data reveal a correlation between the latrophilin-2-encoding Adgrl2 gene and cardiovascular disease. Together, these results define a pathway that connects latrophilin-dependent G protein activation to subsequent endothelial signaling, vascular physiology and disease.

molecular biology↗

bioRxiv2024

Evaluation of enzyme activity predictions for variants of unknown significance in Arylsulfatase A

Continued advances in variant effect prediction are necessary to demonstrate the ability of machine learning methods to accurately determine the clinical impact of variants of unknown significance (VUS). Towards this goal, the ARSA Critical Assessment of Genome Interpretation (CAGI) challenge was designed to characterize progress by utilizing 219 experimentally assayed missense VUS in the Arylsulfa-tase A (ARSA) gene to assess the performance of community-submitted predictions of variant functional effects. The challenge involved 15 teams, and evaluated additional predictions from established and recently released models. Notably, a model developed by participants of a genetics and coding bootcamp, trained with standard machine-learning tools in Python, demonstrated superior performance among sub-missions. Furthermore, the study observed that state-of-the-art deep learning methods provided small but statistically significant improvement in predictive performance compared to less elaborate techniques. These findings underscore the utility of variant effect prediction, and the potential for models trained with modest resources to accurately classify VUS in genetic and clinical research.

genetics↗

bioRxiv2024

Rudhira-mediated microtubule stability controls TGFβ signaling during mouse vascular development

The Transforming Growth Factor {beta} (TGF{beta}) signaling pathway is critical for survival, proliferation, and cell migration, and is tightly regulated during cardiovascular development. Smads, key effectors of TGF{beta} signaling, are sequestered by microtubules (MTs) and need to be released for pathway function. Independently, TGF{beta} signaling also stabilizes MTs. Molecular details and the in vivo relevance of this cross-regulation remain unclear, understanding which is important in complex biological processes such as cardiovascular development. Here, we use rudhira/Breast Carcinoma Amplified Sequence 3 (BCAS3), a MT-associated, endothelium-restricted and developmentally essential proto-oncogene, as a pivot to decipher cellular mechanisms in bridging TGF{beta} signaling and MT stability. We show that Rudhira regulates TGF{beta} signaling in vivo, during mouse cardiovascular development, and in endothelial cells in culture. Rudhira associates with MTs and is essential for the activation and release of Smad2/3 from MTs. Consequently, Rudhira depletion attenuates Smad2/3- dependent TGF{beta} signaling thereby impairing cell migration. Interestingly, Rudhira is also a transcriptional target of Smad2/3-dependent TGF{beta} signaling essential for TGF{beta}-induced MT stability. Our study identifies an immediate early physical role and a slower, transcription-dependent role for Rudhira in cytoskeleton-TGF{beta} signaling crosstalk. These two phases of control could facilitate temporally-and spatially restricted targeting of the cytoskeleton and/or TGF{beta} signaling in vascular development and disease. Significance statementThe developmental TGF{beta} pathway is essential for cell migration, cell-cell communication, adhesion, apoptosis, and matrix remodeling. Dysregulation of TGF{beta} signaling leads to aberrant vascular patterning and angiogenesis during mouse embryogenesis. Pathway activation involves phosphorylation and nuclear transport of Smads. Microtubules sequester Smads in the cytoplasm, thereby inhibiting TGF{beta} signaling. Conversely, TGF{beta} signaling stabilizes microtubules. However, the molecular components involved, and biological relevance of this cross-regulation remain unclear. We show that the oncoprotein Rudhira/BCAS3 facilitates Smad-MT dissociation upon ligand-mediated TGF{beta} receptor activation. Interestingly, Smad-dependent TGF{beta} signaling activation enhances rudhira transcription, essential for microtubule stabilization during cardiovascular development. This dual regulation of TGF{beta} signaling and microtubule stability by Rudhira allows sustained temporal control essential for development, and its dysregulation has pathological outcomes.

cell biology↗

bioRxiv2024

Evaluation Of Suitability Analysis of Gangetic Water from Upper, Middle, And Lower Ganga Rivers

This study assesses the suitability of water from distinct segments of the Ganga River in India for drinking, irrigation, and industrial purposes, utilizing various indices. Conducted in MAR-MAY 2023, water samples were collected from multiple sites along the river, and categorized into upper, middle, and lower stretches. The findings revealed differing levels of suitability for various purposes among the sampled sites within each stretch. Parameters for drinking water quality generally adhered to specified limits across all stretches, except for E. coli, emphasizing the importance of disinfection measures. Moreover, the study evaluated irrigation suitability, with most sites falling within the good to excellent range, albeit some exhibiting elevated sodium levels, particularly in the lower stretch. Analysis of major ion abundance revealed an alkaline nature dominated by sodium and potassium ions over calcium and magnesium ions across all stretches. Heavy metal concentrations were either within specified limits or absent, indicating minimal industrial pollution. However, caution is warranted regarding industrial applications due to water aggressiveness, as suggested by indices such as the Langelier saturation index and Ryznar stability index, which hint at potential corrosion issues. Water quality in the Ganga River in India exhibited variability across the upper, middle, and lower stretches, with each segment presenting unique characteristics and suitability levels. Continuous monitoring and control measures, especially concerning the most probable number (MPN) of contaminants, are imperative to ensure the sustainability of water quality and meet the diverse usage needs along the river. The evaluation of Gangetic water from the Upper, Middle, and Lower Ganga Rivers is crucial for understanding water quality in one of Indias vital river basins. This study assesses physical, chemical, and biological parameters to determine suitability for drinking, irrigation, and aquatic life. Factors like pH, dissolved oxygen, turbidity, nutrients, heavy metals, and microbial load are analyzed to reveal water quality and potential risks. Temporal and spatial variations, influenced by natural processes and human activities, are explored. The findings aid in sustainable water management and conservation in the Gangetic basin, addressing the urgent need to safeguard this critical resource.

microbiology↗

bioRxiv2024

Evolution and inhibition of the FIKK effector kinase family in P. falciparum

Among the [~]200 Plasmodium species that infect vertebrates, six infect humans. Of these, P. falciparum causes >95% of all [~]500,000 annual fatalities. Phylogenetically, P. falciparum belongs to the Laverania subgenus, a group of Plasmodium species that infect great apes. Common to Laverania species is the family of FIKK kinases. One million years ago, a single FIKK kinase conserved in all Plasmodium species gained an export element in the Laverania subgenus and expanded into the family of [~]20 atypical FIKK kinases, most of which are exported into the host cell. The fikk genes are conserved in syntenic loci across the Laverania, arguing for a rapid expansion controlling important functions in host cell remodelling and pathogenesis. We provide evidence that the FIKK paralogues evolved specific and mutually exclusive phosphorylation motif preferences, conserved across their Laverania orthologues, in a short evolutionary timeframe. Surprisingly, we find that FIKK13 has evolved exclusive tyrosine-phosphorylation preference, which was thought to be absent in Plasmodium species. Combining a crystal structure with AlphaFold2 predictions, we identify residues that determine kinase-specificity within the FIKK family in a fast-evolving flexible loop. Finally, we show that all expressed members of the FIKK kinase family can be chemically inhibited in vitro using a single compound. Such a pan-specific inhibitor of this kinase family important for virulence could reduce the ability of the parasite to gain escape-mutations and resistance.

microbiology↗

bioRxiv2024

VEGF counteracts shear stress-determined arterial fate specification during capillary remodeling.

Postnatal vascular morphogenesis is a dynamic process that initially involves angiogenesis to form the capillary bed, followed by its partial remodeling into arteries. Vascular endothelial growth factor A (VEGF hereafter), the primary inducer of angiogenesis, is also implicated in arterial specification in developmental contexts. However, it is unclear why arterial patterning is spatiotemporally segregated from angiogenesis, while postnatal arterial specification in animal models with blocked VEGF signaling remains unstudied. Here, we report that VEGF does not induce arterial fate in the capillaries, instead serving as a physiological brake to attenuate fluid shear stress (FSS)-driven capillary-to-arterial cell fate transition. Mouse models with disrupted VEGF signaling reveal impaired angiogenesis but intact and ectopic arterialization. Mechanistically, mechanosensitive transcription factor Sox17 determines the FSS-arterial program, while VEGF suppresses Sox17 transcription activity at arterial promoters and enhancers sites. Angiogenic signaling, while essential for the initial capillary morphogenesis, inhibits arterial specification, thereby protecting the capillary bed from premature arterialization driven by flow. These findings establish a new paradigm in which precise spatiotemporal coordination of environmental stimuli orchestrates angiogenesis, arterial patterning and capillary maintenance during vascular morphogenesis.

developmental biology↗

bioRxiv2024

Mechanism of chaperone coordination during cotranslational protein folding in bacteria

Protein folding is assisted by molecular chaperones that bind nascent polypeptides during mRNA translation. Several structurally-distinct classes of chaperone promote de novo folding, suggesting that their activities are coordinated at the ribosome. We used biochemical reconstitution and structural proteomics to explore the molecular basis for cotranslational chaperone action in bacteria. We found that chaperone binding is disfavoured close to the ribosome, allowing folding to precede chaperone recruitment. Trigger factor subsequently recognises compact folding intermediates exposing extensive non-native surface and dictates DnaJ access to nascent chains. DnaJ uses a large surface to bind structurally diverse intermediates, and recruits DnaK to sequence-diverse solvent-accessible sites. Neither Trigger factor, DnaJ nor DnaK destabilize cotranslational folding intermediates. Instead, the chaperones collaborate to create a protected space for protein maturation that extends well beyond the ribosome exit tunnel. Our findings show how the chaperone network selects and modulates cotranslational folding intermediates.

biochemistry↗