Biology subjects

Yadav, S.

Publications and source records attributed to Yadav, S..

At least 73 records · Page 4Linked to original sources

bioRxiv2024

Neural Decoding and Feature Selection Techniques for Closed-Loop Control of Defensive Behavior

ObjectiveMany psychiatric disorders involve excessive avoidant or defensive behavior, such as avoidance in anxiety and trauma disorders or defensive rituals in obsessive-compulsive disorders. Developing algorithms to predict these behaviors from local field potentials (LFPs) could serve as foundational technology for closed-loop control of such disorders. A significant challenge is identifying the LFP features that encode these defensive behaviors. ApproachWe analyzed LFP signals from the infralimbic cortex and basolateral amygdala of rats undergoing tone-shock conditioning and extinction, standard for investigating defensive behaviors. We utilized a comprehensive set of neuro-markers across spectral, temporal, and connectivity domains, employing SHapley Additive exPlanations for feature importance evaluation within Light Gradient-Boosting Machine models. Our goal was to decode three commonly studied avoidance/defensive behaviors: freezing, bar-press suppression, and motion (accelerometry), examining the impact of different features on decoding performance. Main resultsBand power and band power ratio between channels emerged as optimal features across sessions. High-gamma (80-150 Hz) power, power ratios, and inter-regional correlations were more informative than other bands that are more classically linked to defensive behaviors. Focusing on highly informative features enhanced performance. Across 4 recording sessions with 16 subjects, we achieved an average coefficient of determination of 0.5357 and 0.3476, and Pearson correlation coefficients of 0.7579 and 0.6092 for accelerometry jerk and bar press rate, respectively. Utilizing only the most informative features revealed differential encoding between accelerometry and bar press rate, with the former primarily through local spectral power and the latter via inter-regional connectivity. Our methodology demonstrated remarkably low time complexity, requiring <110 ms for training and <1 ms for inference. SignificanceOur results demonstrate the feasibility of accurately decoding defensive behaviors with minimal latency, using LFP features from neural circuits strongly linked to these behaviors. This methodology holds promise for real-time decoding to identify physiological targets in closed-loop psychiatric neuromodulation.

bioengineering↗

bioRxiv2024

Genomic introgression between critically endangered and stable species of Darwin's tree finches on the Galapagos Islands

Natural hybridisation among rare or endangered species and stable congenerics is increasingly topical for the conservation of species-level diversity under anthropogenic impacts. Evidence for beneficial genes being introgressed into or selected for in hybrids raises concurrent questions about its evolutionary significance. In Darwins tree finches on the island of Floreana (Galapagos Islands, Ecuador), the Critically Endangered medium tree finch (Camarhynchus pauper) undergoes introgression with the stable small tree finch (Camarhynchus parvulus), and hybrids regularly backcross with C. parvulus. Earlier studies in 2005-2013 documented an increase in the frequency of Camarhynchus hybridisation on Floreana using field-based and microsatellite data. With single nucleotide polymorphism (SNP) data from the same Floreana tree finches sampled in 2005 and 2013 (n = 95), we examine genome-wide divergence across parental and hybrid birds and evidence for selection in hybrids. In assessing previous estimates of introgression we found that just 18% of previously assigned hybrid birds based on microsatellites were assigned to hybrids using SNPs. Over half of the previously assigned hybrids (63%) were reassigned to C. parvulus, though parental species showed concordance with prior assignments. Of 4869 private alleles found in hybrid birds, 348 were at a high frequency ([≥]0.30) that exceeded their parental species of origin 89-96% of the time. Across the two years, 3436 (70.6%) private alleles underwent a substantial ([≥]0.30) allele frequency increase or decrease. Of these, 28 private alleles were identified as candidate loci under selection via local PCA genome scans and outlier tests. Alleles were annotated to genes associated with inflammation, immunity, brain function and development. We provide evidence that introgression among a critically endangered and stable species of Darwins tree finch is being retained by selection across years and may aid in the retention of genetic diversity in birds threatened with extinction.

evolutionary biology↗

bioRxiv2024

A TRANSLAMINAR SPACETIME CODE SUPPORTS TOUCH-EVOKED TRAVELING WAVES

Linking sensory-evoked traveling waves to underlying circuit patterns is critical to understanding the neural basis of sensory perception. To form this link, we performed simultaneous electrophysiology and two-photon calcium imaging through transparent NeuroGrids and mapped touch-evoked cortical traveling waves and their underlying microcircuit dynamics. In awake mice, both passive and active whisker touch elicited traveling waves within and across barrels, with a fast early component followed by a variable late wave that lasted hundreds of milliseconds post-stimulus. Strikingly, late-wave dynamics were modulated by stimulus value and correlated with task performance. Mechanistically, the late wave component was i) modulated by motor feedback, ii) complemented by a sparse ensemble pattern across layer 2/3, which a balanced-state network model reconciled via inhibitory stabilization, and iii) aligned to regenerative Layer-5 apical dendritic Ca2+ events. Our results reveal a translaminar spacetime pattern organized by cortical feedback in the sensory cortex that supports touch-evoked traveling waves. GRAPHICAL ABSTRACT AND HIGHLIGHTS O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=66 SRC="FIGDIR/small/593381v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@c98840org.highwire.dtl.DTLVardef@1105364org.highwire.dtl.DTLVardef@d2f2c9org.highwire.dtl.DTLVardef@1418744_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIWhisker touch evokes both early- and late-traveling waves in the barrel cortex over 100s of milliseconds C_LIO_LIReward reinforcement modulates wave dynamics C_LIO_LILate wave emergence coincides with network sparsity in L23 and time-locked L5 dendritic Ca2+ spikes C_LIO_LIExperimental and computational results link motor feedback to distinct translaminar spacetime patterns C_LI

neuroscience↗

bioRxiv2024

Altered Expression of microRNAs Implicated in Hematopoietic Dysfunction in the Extracellular Vesicles of Bone Marrow-Mesenchymal Stromal Cells in Aplastic Anemia

Recently, we have reported that extracellular vesicles (EVs) from the bone marrow mesenchymal stromal cells (BM-MSC) of aplastic anemia (AA) patients inhibit hematopoietic stem and progenitor cell (HSPC) proliferative and colony-forming ability and promote apoptosis. One mechanism by which AA BM-MSC EVs might contribute to these altered HSPC functions is through microRNAs (miRNAs) encapsulated in EVs. However, little is known about the role of BM-MSC EVs derived miRNAs in regulating HSPC functions in AA. Therefore, we performed miRNA profiling of EVs from BM-MSC of AA (n=6) and normal controls (NC) (n=6), to identify differentially expressed miRNAs carried in AA BM-MSC EVs. DEseq2 analysis identified 34 significantly altered mature miRNAs in AA BM-MSC EVs. Analysis of transcriptome dataset of AA HSPC genes identified that 235 differentially expressed HSPC genes were targeted by these 34 EV miRNAs. The pathway enrichment analysis of 235 HSPC genes revealed their involvement in pathways associated with cell cycle, proliferation, apoptosis, and hematopoiesis regulation, thus highlighting that AA BM-MSC EV miRNAs could potentially contribute to impaired HSPC functions in AA.

molecular biology↗

bioRxiv2024

In-silico evaluation of Mirror repeats in some selected genes ofCandida albicans

All cellular processes in a living cell are controlled by its genetic material. DNA in majority of the domains acts as a regulatory molecule by controlling various vital functions. The genetic makeup of DNA or RNA (in viruses) is unique in all the domains. Their uniqueness is determined by the presence of various types of repetitive patterns of bases. These includes inverted repeats, tandem repeats, VNTRs, palindromes etc. Among many repetitive pattern types, Mirror repeats (MR) found to be dispersed throughout the genes or genomes. These sequences are associated with various functional features like their involvement in H-DNA formation, in replication & transcription, nervous system related diseases development etc. The major focus of this investigation is to identify MR sequences from some selected genes of Candida albicans using a bioinformatics based pipeline. The approach refers to as FPCB which utilized some manual steps to extract out MR sequence from any targeted gene or genome. The current study find out that the identified Mirror repeats found to be dispersed throughout the selected genes along with variable length. Among them the maximum & minimum MR sequences were reported in the gene FAS2 (108) & HIS1 (15) respectively. The present study will be helpful to provide a new insight for molecular as well as computational based studies of Candida albicans as well as other related fungal groups.

bioinformatics↗

bioRxiv2023

Saturation genome editing-based functional evaluation and clinical classification of BRCA2 single nucleotide variants

Germline BRCA2 loss-of function (LOF) variants identified by clinical genetic testing predispose to breast, ovarian, prostate and pancreatic cancer. However, variants of uncertain significance (VUS) (n>4000) limit the clinical use of testing results. Thus, there is an urgent need for functional characterization and clinical classification of all BRCA2 variants. Here we report on comprehensive saturation genome editing-based functional characterization of 97% of all possible single nucleotide variants (SNVs) in the BRCA2 DNA Binding Domain hotspot for pathogenic missense variants that is encoded by exons 15 to 26. The assay was based on deep sequence analysis of surviving endogenously targeted haploid cells. A total of 7013 SNVs were characterized as functionally abnormal (n=955), intermediate/uncertain, or functionally normal (n=5224) based on 95% agreement with ClinVar known pathogenic and benign standards. Results were validated relative to batches of nonsense and synonymous variants and variants evaluated using a homology directed repair (HDR) functional assay. Breast cancer case-control association studies showed that pooled SNVs encoding functionally abnormal missense variants were associated with increased risk of breast cancer (odds ratio (OR) 3.89, 95%CI: 2.77-5.51). In addition, 86% of tumors associated with abnormal missense SNVs displayed loss of heterozygosity (LOH), whereas 26% of tumors with normal variants had LOH. The functional data were added to other sources of information in a ClinGen/ACMG/AMP-like model and 700 functionally abnormal SNVs, including 220 missense SNVs, were classified as pathogenic or likely pathogenic, while 4862 functionally normal SNVs, including 3084 missense SNVs, were classified as benign or likely benign. These classified variants can now be used for risk assessment and clinical care of variant carriers and the remaining functional scores can be used directly for clinical classification and interpretation of many additional variants. SummaryGermline BRCA2 loss-of function (LOF) variants identified by clinical genetic testing predispose to several types of cancer. However, variants of uncertain significance (VUS) limit the clinical use of testing results. Thus, there is an urgent need for functional characterization and clinical classification of all BRCA2 variants to facilitate current and future clinical management of individuals with these variants. Here we show the results from a saturation genome editing (SGE) and functional analysis of all possible single nucleotide variants (SNVs) from exons 15 to 26 that encode the BRCA2 DNA Binding Domain hotspot for pathogenic missense variants. The assay was based on deep sequence analysis of surviving endogenously targeted human haploid HAP1 cells. The assay was calibrated relative to ClinVar known pathogenic and benign missense standards and 95% prevalence thresholds for functionally abnormal and normal variants were identified. Thresholds were validated based on nonsense and synonymous variants. SNVs encoding functionally abnormal missense variants were associated with increased risks of breast and ovarian cancer. The functional assay results were integrated into a ClinGen/ACMG/AMP-like model for clinical classification of the majority of BRCA2 SNVs as pathogenic/likely pathogenic or benign/likely benign. The classified variants can be used for improved clinical management of variant carriers.

genomics↗

bioRxiv2023

Isogenic iPSC-derived proximal and distal lung-on-chip models: Tissue- and virus-specific immune responses in human lungs

Micro-physiological systems (MPS) are set to play a vital role in preclinical studies, particularly in the context of future viral pandemics. Nonetheless, the development of MPS is often impeded by the scarcity of reliable cell sources, especially when seeking various organs or tissues from a single patient for comparative analysis of the host immune response. Herein, we developed human airway-on-chip and alveolus-on-chip models using induced pluripotent stem cell (iPSC)-derived isogenic lung progenitor cells. Both models demonstrated the replication of two different respiratory viruses, namely SARS-CoV-2 and Influenza, as well as related cellular damage and innate immune responses-on-chip. Our findings reveal distinct immune responses to SARS-CoV-2 in the proximal and distal lung-on-chip models. The airway chips exhibited a robust interferon (IFN)-dependent immune response, whereas the alveolus chips exhibited dysregulated IFN activation but a significantly upregulated chemokine pathway. In contrast, Influenza virus infection induced a more pronounced immune response and cellular damage in both chip models compared to SARS-CoV-2. Thus, iPSC-derived lung-on-chip models may aid in quickly gaining insights into viral pathology and screening potential drugs for future pandemics.

bioengineering↗

bioRxiv2023

Factors affecting macromolecule orientations in thin films formed in cryo-EM

The formation of a vitrified thin film embedded with randomly oriented macromolecules is an essential prerequisite for cryogenic sample electron microscopy. Most commonly, this is achieved using the plunge freeze method described nearly 40 years ago. Although this is a robust method, the behaviour of different macromolecules shows great variation upon freezing and often needs to be optimized to obtain an isotropic, high-resolution reconstruction. For a macromolecule in such a film, the probability of encountering the air-water interface in the time between blotting and freezing and adopting preferred orientations is very high. 3D reconstruction using preferentially oriented particles often leads to anisotropic and uninterpretable maps. Currently, there are no general solutions to this prevalent issue, but several approaches largely focusing on sample preparation with the use of additives and novel grid modifications have been attempted. In this study, the effect of physical and chemical factors on the orientations of macromolecules was investigated through an analysis of select well-studied macromolecules and important parameters that determine the behaviour of proteins on cryo-EM grids were revealed. These insights highlight the nature of interactions that cause preferred orientations and can be utilized to systematically address orientation bias for any given macromolecule and provide a framework to design small-molecule additives to enhance sample stability and behaviour.

biochemistry↗

bioRxiv2023

Myeloid Drp1 Deficiency Limits Revascularization in Ischemic Muscles via Inflammatory Macrophage Polarization and Metabolic Reprograming

In the preclinical model of peripheral arterial disease (PAD), M2-like anti-inflammatory macrophage polarization and angiogenesis are required for revascularization. The regulation of cell metabolism and inflammation in macrophages is tightly linked to mitochondrial dynamics. Drp1, a mitochondrial fission protein, has shown context-dependent macrophage phenotypes with both pro- and anti-inflammatory characteristics. However, the role of macrophage Drp1 in reparative neovascularization remains unexplored. Here we show that Drp1 expression was significantly increased in F4/80+ macrophages within ischemic muscle at day 3 after hindlimb ischemia (HLI), an animal model of PAD. Myeloid-specific Drp1-/- mice exhibited reduced limb perfusion recovery, angiogenesis and muscle regeneration after HLI. These effects were associated with an increase in pro-inflammatory M1-like macrophages, p-NFkB and TNF, and reduced anti-inflammatory M2-like macrophages and p-AMPK in ischemic muscle of myeloid Drp1-/- mice. In vitro, Drp1-/- macrophages under hypoxia serum starvation (HSS), an in vitro PAD model, demonstrated enhanced glycolysis via reducing p-AMPK as well as mitochondrial dysfunction and excessive mitochondrial ROS, resulting in increased M1-gene and reduced M2-gene expression. Conditioned media from HSS-treated Drp1-/- macrophages exhibited increased secretion of pro-inflammatory cytokines and suppressed angiogenic responses in cultured endothelial cells. Thus, Drp1 deficiency in macrophages under ischemia drives inflammatory metabolic reprogramming and macrophage polarization, thereby limiting revascularization in experimental PAD.

cell biology↗

bioRxiv2023

Organic matter degradation in the deep, sulfidic waters of the Black Sea: Insights into the ecophysiology of novel anaerobic bacteria

Our knowledge about the physiology of deep sea (>1,000 m) microorganisms involved in organic matter (OM) degradation is still scare due to the lack of available isolates, especially from sulfidic environments. In this study, we successfully cultivated and characterized the physiology of a wide range of novel piezotolerant anaerobic bacteria affiliated with the phyla Fusobacteriota, Bacillota, Spirochaetota, Bacteroidota, Cloacimonadota, Planctomycetota, Mycoplasmatota and Chloroflexota involved in OM degradation in deep sulfidic waters of the Black Sea. The novel taxa are specialized in degrading specific types of OM and cover a wide range of physiological categories, including primary degraders, fermenters, and terminal oxidizers. This is the first report which demonstrates this for such a diverse physiological group from any sulfidic marine habitat. Collectively, this study provides a step forward in our understanding of the microbes thriving in the extreme conditions of the deep sulfidic waters of the Black Sea.

microbiology↗

bioRxiv2023

Beadex, the Drosophila LIM only protein, is required for the growth of the larval neuromuscular junction and sensorimotor activities

The appropriate growth of the neurons, accurate organization of their synapses, and successful neurotransmission are indispensable for sensorimotor activities. These processes are highly dynamic and tightly regulated. Extensive genetic, molecular, physiological, and behavioural studies have identified many molecular players and investigated their roles in various neuromuscular processes. In this paper, we show that Beadex (Bx), the Drosophila LIM only (LMO) protein, is required for motor activities and neuromuscular growth of Drosophila. Bx7, a null allele, adult flies are flightless, with reduced walking and jumping activities. The larvae of Bx7, and the RNAi-mediated neuronal-specific knockdown of Bx show drastically reduced crawling behaviour, a diminished synaptic span of the neuromuscular junctions and an increased spontaneous neuronal firing with altered motor patterns in the central pattern generators (CPGs). Microarray studies identified multiple targets of Beadex that are involved in different cellular and molecular pathways, including those associated with the cytoskeleton and mitochondria, that could be responsible for the observed neuromuscular defects. With genetic interaction studies, we further show that Highwire (Hiw), a negative regulator of synaptic growth at the NMJs, negatively regulates Bx, as the latters deficiency was able to rescue the phenotype of the Hiw null mutant, HiwDN. Thus, our data indicates that Beadex functions downstream of Hiw to regulate the larval synaptic growth and physiology.

developmental biology↗

bioRxiv2023

Progesterone induces meiosis through two obligate co-receptors with PLA2 activity

The steroid hormone progesterone (P4) regulates multiple aspects of reproductive and metabolic physiology. Classical P4 signaling operates through nuclear receptors that regulate transcription. In addition, P4 signals through membrane P4 receptors (mPRs) in a rapid nongenomic modality. Despite the established physiological importance of P4 nongenomic signaling, the details of its signal transduction cascade remain elusive. Here, using Xenopus oocyte maturation as a well- established physiological readout of nongenomic P4 signaling, we identify the lipid hydrolase ABHD2 (/{beta} hydrolase domain-containing protein 2) as an essential mPR{beta} co-receptor to trigger meiosis. We show using functional assays coupled to unbiased and targeted cell-based lipidomics that ABHD2 possesses a phospholipase A2 (PLA2) activity that requires mPR{beta}. This PLA2 activity bifurcates P4 signaling by inducing clathrin-dependent endocytosis of mPR{beta}, resulting in the production of lipid messengers that are G-protein coupled receptors agonists. Therefore, P4 drives meiosis by inducing an ABHD2 PLA2 activity that requires both mPR{beta} and ABHD2 as obligate co-receptors. Significance StatementNongenomic progesterone signaling is important for many physiological functions yet the details of its signaling remain elusive. Here we define the early signaling steps downstream of membrane progesterone receptor {beta} (mPR{beta}) during Xenopus oocyte meiosis. We show that progesterone requires two cell membrane receptors to work in unison to signal. The co-receptor complex possesses lipase activity that produces lipid messenger and induces receptor endocytosis to trigger meiosis progression. Our findings have broad physiological implications because nongenomic progesterone signaling operates in many tissues and regulates reproduction and metabolism.

cell biology↗

bioRxiv2023

In-Silico analysis of Human Insulin Gene for Mirror repeat sequences

The genetic material contains all the instructions necessary for the development, functioning & regulation of biological processes. This is also considering as a regulatory molecule. The structure constitute by its unique pattern of bases make it unique for every living being consider from bacteria to human. The varied pattern of bases in genomes or genes represents different types of repeats. These repeats types constitute the major fraction of the genomes. Among varied types Mirror repeats (MRs) play crucial roles in the genome especially in formation of H-DNA structure by Non Watson pairing. Some studies suggest that mirror sequences responsible for neurological diseases also. Now days in modern era of technology in silico techniques were develop to extract any type of repeat sequences from any gene or genome. The major focus of current work is on identification of mirror repeats (MRs) from human insulin gene sequence using an in silico approach. The approach is refer to as FPCB (FASTA Parallel Complement Analysis) were utilized to extract MR sequences. From the current study a total no of 210 MR sequenced were identified in Human insulin gene. The identified repeats vary in their length and majority of them are imperfect in nature. The maximum no of MR were reported in the region 3 & minimum is in the region 8 (8). In the remaining regions the no of MR sequences were lies in between maximum & minimum values. These sequences may be helpful in many molecular level studies.

bioinformatics↗

bioRxiv2023

Identification of Mirror Repeats in Viral Genomes using FPCB Analysis

The majority of living domains consist of DNA as genetic material with the minor exception of viruses. The unique nature of every species determines by its unique pattern of genome or gene products. The genomic features become an evident example of evolutionary study also. Different types of repeat patterns are observed in genomes of living domains including human beings whose two third portion of the genome is repetitive. Among the varied type of repeat sequences Mirror Repeats (MR) play crucial roles at the genetic level in every species. The major focus of our research is on identification & to check the distribution of mirror repeat. For this, we employed a bioinformatics-based approach refer as FASTA PARALLEL COMPLEMENT BLAST (FPCB) to identify unique mirror repeat (MR) sequences in some selected viral genomes from three different categories (Animal, Plant & Human). The identified repeats vary in their length as well as found to be distributed throughout the selected viral genomes. The maximum no of MR were reported in the case of Dengue virus (229) & minimum is in the case of TMV (97). In the remaining selected viruses - HCV, HPV, HTLV-1, PVY, Rabies virus 178, 156, 175, 203 & 204 MR sequences were reported. These sequences can be utilized in many ways like in molecular diagnosis, drug delivery target as well as evolutionary study, etc. The present research also helps in the development of novel tools of bioinformatics to study mirror repeats and their functional perspective in the context of their occurrence in all domains.

bioinformatics↗

bioRxiv2023

The bi-directional extracellular electron transfer process aids iron cycling by Geoalkalibacter halelectricus in a highly saline-alkaline condition

Bi-directional extracellular electron transfer (EET) is crucial to upholding microbial metabolism with insoluble electron acceptors or donors in anoxic environments. Investigating bi-directional EET-capable microorganisms is desired to understand the cell-cell and microbe-mineral interactions and their role in mineral cycling besides leveraging their energy generation and conversion, biosensing, and bio-battery applications. Here, we report on iron cycling by haloalkaliphilic Geoalkalibacter halelectricus via bi-directional EET under haloalkaline conditions. It efficiently reduces Fe3+-oxide (Fe2O3) to Fe0 at a 2.29{+/-}0.07 mM/day rate linked to acetate oxidation via outward EET and oxidizes Fe0 to Fe3+ with a 0.038{+/-}0.002 mM/day rate via inward EET to reduce fumarate. Bioelectrochemical cultivation confirmed its outward and inward EET capabilities. It produced 895{+/-}23 A/cm2 current by linking acetate oxidation to anode reduction via outward EET and reduced fumarate by drawing electrons from the cathode (-2.5{+/-}0.3 A/cm2) via inward EET. The cyclic voltammograms of G. halelectricus biofilms revealed redox moieties with different formal potentials, suggesting the involvement of different membrane components in bi-directional EET. The cyclic voltammetry and GC-MS analysis of the cell-free spent medium revealed the lack of soluble redox mediators, suggesting direct electron transfer by G. halelecctricus in achieving bi-directional EET. By reporting on the first haloalkaliphilic bacterium capable of oxidizing and reducing insoluble Fe0 and Fe3+-oxide, respectively, this study advances the limited understanding of the metabolic capabilities of extremophiles to respire on insoluble electron acceptors or donors via bi-directional EET and invokes the possible role of G. halelectricus in iron cycling in barely studied haloalkaline environments. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/536630v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1c8080aorg.highwire.dtl.DTLVardef@187d9d9org.highwire.dtl.DTLVardef@1ef6066org.highwire.dtl.DTLVardef@a2081b_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Delineating the functional role of the PPE50 (Rv3135) - PPE51 (Rv3136) gene cluster in the pathophysiology of Mycobacterium tuberculosis

The extraordinary success of Mycobacterium tuberculosis (M. tb) has been attributed to its ability to modulate host immune responses. The genome of M. tb encodes multiple immunomodulatory factors, including several proteins of the multigenic PE_PPE family, which comprise about 10% of its coding potential. The presence of these proteins in pathogenic mycobacteria strongly suggests that they play a role in disease pathogenesis. To understand its role in M. tb physiology we have begun to characterise the PPE50 (Rv3135)-PPE51 (Rv3136) gene cluster, one of nine PPE-PPE clusters in the M. tb genome. We demonstrate that this cluster encodes a co-transcriptional unit and that PPE50 and PPE51 interact both in vitro and in vivo, the first demonstration of PPE-PPE interaction. THP-1 macrophages infected with recombinant M. smegmatis strains expressing PPE50 and PPE51 showed less intracellular viability than the control strain containing the vector alone, the decline in viable counts correlating with an increase in transcript levels of inducible nitric oxide synthase (iNOS2). Macrophages infected with the recombinant strains exhibited an upregulation in levels of the anti-inflammatory cytokine IL-10, indicating an immunomodulatory role for these proteins. Using pull-down assays, we discovered TLR1 to be the cognate receptor for PPE50, with signalling through the receptor being indicated by an increase in IRAK1 phosphorylation. All the phenotypes observed on infection of THP-1 macrophages including the decrease in CFUs, the increase in iNOS2 and IL-10 levels, as well as signalling through the receptor, were reversed on treatment of macrophages with an anti-TLR1 antibody prior to infection, validating the functional outcome of PPE50-TLR1 interaction. Our data points to a TLR1 dependent role for the PPE50-PPE51 cluster in promoting bacillary persistence, via CFU reduction and a concomitant upregulation of the anti-inflammatory response - a two-pronged strategy to circumvent host immune surveillance.

microbiology↗

bioRxiv2023

Antibodies generated in vitro and in vivo elucidate design of a thermostable ADDomer COVID-19 nasal nanoparticle vaccine

COVID-19 continues to damage populations, communities and economies worldwide. Vaccines have reduced COVID-19-related hospitalisations and deaths, primarily in developed countries. Persisting infection rates, and highly transmissible SARS-CoV-2 Variants of Concern (VOCs) causing repeat and breakthrough infections, underscore the ongoing need for new treatments to achieve a global solution. Based on ADDomer, a self-assembling protein nanoparticle scaffold, we created ADDoCoV, a thermostable COVID-19 candidate vaccine displaying multiple copies of a SARS-CoV-2 receptor binding motif (RBM)-derived epitope. In vitro generated neutralising nanobodies combined with molecular dynamics (MD) simulations and electron cryo-microscopy (cryo-EM) established authenticity and accessibility of the epitopes displayed. A Gigabody comprising multimerized nanobodies prevented SARS-CoV-2 virion attachment with picomolar EC50. Antibodies generated by immunising mice cross-reacted with VOCs including Delta and Omicron. Our study elucidates nasal administration of ADDomer-based nanoparticles for active and passive immunisation against SARS-CoV-2 and provides a blueprint for designing nanoparticle reagents to combat respiratory viral infections.

biochemistry↗

bioRxiv2023

Structure of the Commander endosomal trafficking complex linked to X-linked intellectual disability/Ritscher-Schinzel syndrome

The Commander complex is required for endosomal recycling of diverse transmembrane cargos and is mutated in Ritscher-Schinzel syndrome. It comprises two subassemblies; Retriever composed of VPS35L, VPS26C and VPS29, and the CCC complex which contains ten subunits COMMD1-COMMD10 and two coiled-coil domain-containing (CCDC) proteins CCDC22 and CCDC93. Combining X-ray crystallography, electron cryomicroscopy and in silico predictions we have assembled a complete structural model of Commander. Retriever is distantly related to the endosomal Retromer complex but has unique features preventing the shared VPS29 subunit from interacting with Retromer-associated factors. The COMMD proteins form a distinctive hetero-decameric ring stabilised by extensive interactions with CCDC22 and CCDC93. These adopt a coiled-coil structure that connects the CCC and Retriever assemblies and recruits a sixteenth subunit, DENND10, to form the complete Commander complex. The structure allows mapping of disease-causing mutations and reveals the molecular features required for the function of this evolutionarily conserved trafficking machinery.

biochemistry↗