Biology subjects

Mukhopadhyay, S.

Publications and source records attributed to Mukhopadhyay, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Kidney cystogenesis in embryonic- and adult-onset ADPKD is suppressed from lack of adenylyl cyclase targeting to cilia

Multiple cellular pathways are dysregulated in autosomal dominant polycystic kidney disease (ADPKD), but mechanisms initiating cyst formation are unknown. ADPKD is caused by mutations in Pkd1/Pkd2 genes that encode for polycystins that localize to primary cilia. The primary cilium is a miniscule subcellular compartment for generating signaling outputs that profoundly affect cellular function. Severe cystogenesis from polycystin loss is mostly cilia dependent. However, the polycystin-repressed ciliary signals that promote cyst growth are unknown and have been challenging to uncouple from downstream cystogenic pathways. Here we aimed at differentiating ciliary adenylyl cyclase signaling from total cellular changes in second messenger cAMP implicated in cystogenesis. We studied an Ankyrin repeat and MYND domain protein, ANKMY2 that we previously implicated in maturation and ciliary localization of adenylyl cyclases in fibroblasts. We studied kidney-specific conditional knockout mouse models of Ankmy2/Pkd1 and ciliary localization of adenylyl cyclases in kidney epithelial cells. We found suppression of early postnatal renal cystogenesis and prolonged survival in an embryonic onset Pkd1 deletion model from ANKMY2 loss. Phosphorylated CREB formation, from elevated cellular cAMP levels, remained unaffected. Cyst load in male mice in an adult inducible conditional Pkd1 deletion model was suppressed from ANKMY2 loss. Mechanistically, ANKMY2 determined ciliary trafficking of adenylyl cyclases in kidney epithelial cells without disrupting cilia. Further, ANKMY2 loss prevented ciliary length increase in ADPKD mouse models irrespective of cyst load or sex. Cilia length increase was seen preceding cystogenesis. Our results suggest that targeting of adenylyl cyclases to renal epithelial cilia promotes PC1/2-inhibited cilia-dependent cyst activation distinct from cyst progression involving cellular cAMP.

cell biology↗

bioRxiv2025

Mitragynine and 7-Hydroxymitragynine: Bidirectional Effects on Breathing in Rats

The use of kratom as an alternative to conventional opioids has surged, driven largely by anecdotal reports of its efficacy for pain relief and opioid withdrawal management. The growing prevalence of kratom products enriched with 7-hydroxymitragynine (7-HMG), an active metabolite of mitragynine (MG), necessitates evaluating the respiratory effects of these alkaloids and determining if naloxone reverses their potential respiratory depressant effects. Respiratory parameters were measured in awake, freely moving female and male Sprague-Dawley rats using whole body plethysmography. To minimize handling-induced artifacts and ensure precise respiratory recordings, drugs were administered intravenously. Morphine and 7-HMG induced significant respiratory depression, evidenced by reductions in breathing frequency, tidal volume, and minute volume. In contrast, MG administration unexpectedly increased respiratory frequency. Naloxone fully reversed the respiratory depression induced by both morphine and 7-HMG but did not alter the respiratory stimulant effects produced by MG. These findings demonstrate that 7-HMG exhibits significant respiratory depressant properties similar to classical opioids, and importantly, such depressant effects are effectively antagonized by naloxone. Conversely, MG exerts respiratory stimulant effects through mechanisms independent of opioid receptor pathways. Collectively, these data highlight crucial pharmacological distinctions between kratom alkaloids, underscoring the risk associated with high 7-HMG-containing kratom products and suggesting that the predominant alkaloid MG may offer a safer respiratory profile. Significance StatementThe prevalence of kratom products containing 7-hydroxymitragynine (7-HMG), a {micro}-opioid receptor agonist, underscores the need to evaluate respiratory effects of kratom-related alkaloids and their reversal by naloxone. 7-HMG induced significant respiratory depression comparable to morphine, which was reversed by naloxone. Conversely, mitragynine, kratoms most abundant alkaloid, unexpectedly increased respiratory frequency unaffected by naloxone. These findings highlight critical pharmacological differences between kratom-related alkaloids, emphasizing potential risks associated with products containing high concentrations of 7-HMG.

pharmacology and toxicology↗

bioRxiv2025

Collectin-11 regulates osteoclastogenesis and bone maintenance via a complement-dependent mechanism.

The human developmental disorder 3MC syndrome is characterized by skeletal deformities associated with a deficiency of the pattern recognition molecule collectin-11 (CL-11), yet the underlying molecular and cellular mechanisms remain unclear. Here, we demonstrate that CL-11 deletion alone does not cause bone abnormalities in mice; however, combined deficiencies involving CL-11 and complement components MASP-2 (lectin pathway), CFB, or C3 (alternative amplification pathway) lead to significant vertebral bone loss and spinal curvature by 12 weeks of age. Ex vivo osteoclast (OCL) differentiation from bone marrow-derived cells of these double-knockout (DKO) mice was markedly impaired, but differentiation capacity was substantially restored by supplementation with CL-11. Furthermore, CL-11 and membrane attack complex (C5b-9) deposition were co-localized to OCLs and their precursors in normal bone tissues from embryonic stages to adulthood. These findings identify CL-11 as a critical osteoclastogenesis and bone maintenance regulator in conjunction with complement system-mediated signalling pathways and highlight CL-11 as a potential therapeutic target in diseases involving dysregulated osteoclast function and bone remodelling. Significance statementOur research in mice provides new insights into how mutations in the immune surveillance molecule collectin-11 contribute to skeletal abnormalities in humans. Evidence from our study suggests that normal osteoclasts interact with collectin-11 and complement, and that the disruption of this cooperation results in impaired bone maintenance in adulthood. These findings not only advance our understanding of osteoclast function but also highlight the therapeutic potential of targeting collectin-11 in conditions associated with osteoclast dysfunction.

immunology↗

bioRxiv2025

Arabidopsis ecotype screening reveals novel sources of clubroot resistance and insights into resistance inheritance

Clubroot, caused by Plasmodiophora brassicae, poses a persistent threat to Brassicaceae crops, particularly in regions where resistant cultivars are under strong selection pressure. To identify new sources of resistance and better understand the underlying genetic mechanisms, we evaluated 60 Arabidopsis thaliana ecotypes against the highly virulent Canadian pathotype 3A. Using stringent phenotyping criteria, pathogen DNA quantification, and survival analysis, we identified eight resistant ecotypes, including two novel sources, Marce-1 and DraII-6. DraII-6 exhibited exceptionally low disease symptoms and a high survival rate. While the resistance gene RPB1/WeiTsing was present in most ecotypes, its expression in DraII-6 was significantly elevated at early infection stages, suggesting a potential role in pathogen suppression. However, genetic analysis of F1 and F2 progeny from a DraII-6 x Col-0 cross revealed a recessive resistance pattern, supporting the hypothesis that RPB1 alone may not be sufficient to confer resistance to clubroot in DraII-6. Our findings highlight the complexity of clubroot resistance and the need for further research into gene regulation and resistance networks beyond RPB1, particularly in the context of translating Arabidopsis-based insights to Brassica crops. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/649024v1_ufig1.gif" ALT="Figure 1"> View larger version (98K): org.highwire.dtl.DTLVardef@1e30ef3org.highwire.dtl.DTLVardef@1ea7aa4org.highwire.dtl.DTLVardef@119c0a3org.highwire.dtl.DTLVardef@1b62891_HPS_FORMAT_FIGEXP M_FIG C_FIG Natural variability of Arabidopsis thaliana ecotypes in response to the Canadian clubroot pathotype 3A. The abstract also celebrates the first author, Melaine Gonzalez-Garcia, who is submitting this manuscript just one week before welcoming her first child.

plant biology↗

bioRxiv2025

Dissection of Gαs and Hedgehog signaling crosstalk reveals therapeutic opportunities to target adenosine receptor 2b in Hedgehog-dependent tumors

Basal cell carcinoma (BCC), the most common human cancer, is driven by hyperactivation of Hedgehog Smoothened (SMO) and GLI transcription. Gs and protein kinase A (PKA) negatively regulate Hedgehog signaling, offering an alternative BCC development and treatment pathway. Here, using histology alongside bulk and single-cell RNA sequencing, we find that mouse BCC-like tumors that originate from Gs pathway inactivation are strikingly similar to those driven by canonical Hedgehog SMO. Interestingly, mutations that reduce Gs and PKA activity are present in human BCC. Tumors from Gs pathway inactivation are independent of the canonical Hedgehog regulators SMO and GPR161, establishing them as an SMO-independent oncogenic Hedgehog signaling model. Finally, we demonstrate that activation of the Gs-coupled adenosine 2B receptor counteracts oncogenic SMO, reducing Hedgehog signaling and tumor formation and offering a potential therapeutic strategy for BCC.

cancer biology↗

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

Spatiotemporal dynamics of primary and motile cilia throughout lung development

Cilia are specialized structures found on a variety of mammalian cells, with variable roles in the transduction of mechanical and biological signals (by primary cilia, PC), as well as the generation of fluid flow (by motile cilia). Their critical role in the establishment of a left-right axis in early development is well described, as is the innate immune function of multiciliated upper airway epithelium. By contrast, the dynamics of ciliary status during organogenesis and postnatal development is largely unknown. In this study, we define the progression of ciliary status within the endothelium, epithelium, and mesenchyme of the lung. Remarkably, we find that endothelial cells (ECs) lack PC at all stages of development, except in low numbers in the most proximal portions of the pulmonary arteries. In the lung epithelium, a proximodistal ciliary gradient is established over time, as the uniformly mono-ciliated epithelium transitions into proximal, multiciliated cells, and the distal alveolar epithelium loses its cilia. Mesenchymal cells, interestingly, are uniformly ciliated in early development, but with restriction to PDGFR+ fibroblasts in the adult alveoli. This dynamic process in multiple cellular populations both challenges prior assertions that PC are found on all cells, and highlights a need to understand their spatiotemporal functions. HighlightsO_LIPrimary cilia are found broadly throughout early embryonic tissues. C_LIO_LIPrimary cilia are observed in both epithelial and mesenchymal cells in the early lung. C_LIO_LIPulmonary endothelial cells largely do not possess primary cilia during embryonic development. C_LIO_LIDifferential multiciliation and loss of epithelial cilia in a proximal-distal axis. C_LIO_LIMaintenance of cilia in adult pulmonary PDGFR+ fibroblasts. C_LI

developmental biology↗

bioRxiv2024

De novo assembly and annotation of the Empoasca fabae mitochondrial genome

This study presents the assembly and annotation of the full-length mitochondrial genome for the leafhopper species Empoasca fabae Harris, 1841. The mitogenome was obtained from a contig-level assembly with the identified mitochondrial genome being 14,873 bp in length. The base composition was A (38.8%), T (39.1%), C (11.7%), and G (10.4%). The mitogenome comprised 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and showed a unique, non-AT-rich D-loop region. Nearly all PCGs being with an ATN start codon, while two begin with TCG and GTG. Phylogenetic analysis confirmed the placement of E. fabae within the subfamily Typhlocybinae, clustering with other species in the Empoasca genus.

genomics↗

bioRxiv2024

Chaperone-mediated heterotypic phase separation prevents the amyloid formation of the pathological Y145Stop variant of the prion protein

Biomolecular condensates formed via phase separation of proteins and nucleic acids are crucial for the spatiotemporal regulation of a diverse array of essential cellular functions and the maintenance of cellular homeostasis. However, aberrant liquid-to-solid phase transitions of such condensates are associated with several fatal human diseases. Such dynamic membraneless compartments can contain a range of molecular chaperones that can regulate the phase behavior of proteins involved in the formation of these biological condensates. Here, we show that a heat shock protein 40 (Hsp40), Ydj1, exhibits a holdase activity by potentiating the phase separation of a disease-associated stop codon mutant of the prion protein (Y145Stop) either by recruitment into Y145Stop condensates or via Y145Stop-Ydj1 two-component heterotypic phase separation that prevents the conformational conversion of Y145Stop into amyloid fibrils. Utilizing site-directed mutagenesis, multicolor fluorescence imaging, single-droplet steady-state and picosecond time-resolved fluorescence anisotropy, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy, we delineate the complex network of interactions that govern the heterotypic phase separation of Y145Stop and Ydj1. We also show that the properties of such heterotypic condensates can further be tuned by RNA that promotes the formation of multicomponent multiphasic protein-RNA condensates. Our vibrational Raman spectroscopy results in conjunction with atomic force microscopy imaging reveal that Ydj1 effectively redirects the self-assembly of Y145Stop towards a dynamically-arrested non-amyloidogenic pathway, preventing the formation of typical amyloid fibrils. Our findings underscore the importance of chaperone-mediated heterotypic phase separation in regulating aberrant phase transitions and amyloid formation associated with a wide range of deadly neurodegenerative diseases.

biophysics↗

bioRxiv2024

Structure-guided secretome analysis of gall-forming microbes offers insights into effector diversity and evolution

Phytopathogens secrete effector molecules to manipulate host immunity and metabolism. Recent advances in structural genomics have identified fungal effector families whose members adopt similar folds despite sequence divergence, highlighting their importance in virulence and immune evasion. To extend the scope of comparative structure-guided analysis to more evolutionarily distant phytopathogens with similar lifestyles, we used AlphaFold2 to predict the 3D structures of the secretome from selected plasmodiophorid, oomycete, and fungal gall-forming pathogens. Clustering protein folds based on structural homology revealed species-specific expansions and a low abundance of known orphan effector families. We identified novel sequence-unrelated but structurally similar (SUSS) effector clusters, rich in conserved motifs such as CCG and RAYH. We demonstrate that these motifs likely play a central role in maintaining the overall fold. We also identified a SUSS cluster adopting a nucleoside hydrolase-like fold conserved among various gall-forming microbes. Notably, ankyrin proteins (ANK) were significantly expanded in gall-forming plasmodiophorids, with most being highly expressed during clubroot disease, suggesting a role in pathogenicity. Subsequently, we screened ANK proteins against Arabidopsis immunity hubs using AlphaFold-Multimer and verified one of the positive results by Y2H and BiFC assays to show that the ankyrin effector PBTT_00818 targets host MPK3 and a zinc-binding dehydrogenase. These findings suggest a potential new mechanism in which ANK effectors target multiple host proteins involved in stress sensing, opening a novel avenue to study the role of ANK in host-pathogen interactions. Altogether, this study advances our understanding of secretome landscapes in gall-forming microbes and provides a valuable resource for broadening structural phylogenomic studies across diverse phytopathogens.

microbiology↗

bioRxiv2024

Refinement of the Brassica napus NLRome using RenSeq

Canola (Brassica napus L.), a valuable oilseed crop, faces significant challenges from diseases such as clubroot and blackleg, threatening global yields. To improve disease resistance, its crucial to accurately annotate nucleotide-binding leucine-rich repeat receptors (NLRs), which are key components of plant immune systems. This study focuses on refining the NLR repertoire (NLRome) of the Westar cultivar using Resistance gene enrichment sequencing (RenSeq). Initially, only 345 NLR genes were identified in the Westar genome annotation, a figure significantly lower than other canola cultivars. By employing RenSeq, we expanded the annotated NLR genes to 715, including 287 full NLRs and 428 partial NLRs, thus providing a more comprehensive understanding of the NLR diversity in this cultivar. Our findings reveal crucial genomic regions with previously misannotated NLR genes and identify key integrated domains that may contribute to enhanced disease resistance. This refined NLRome not only advances the fundamental understanding of B. napus genetics but also offers valuable insights for breeding programs and biotechnological applications aimed at improving crop resilience. The data generated in this study is available in public repositories for further research and application.

plant biology↗

bioRxiv2024

Majority-LCL: Towards Malaria Cell Detection using Label Contrastive Learning and Majority voting Ensembling

Malaria is a contagious disease caused by Plasmodium, a group of single-celled parasites, and is most commonly transmitted by an infected female Anopheles mosquito. More than 40 percent of the global population is at risk, with approximately 219 million reported cases and around 435000 deaths recorded in 2017 alone. Despite the availability of several advanced diagnostic tools, accurate malaria diagnosis remains challenging in resource-constrained settings, where microscopists often struggle to improve diagnostic accuracy. Deep learning-based cell image classification helps reduce incorrect diagnostic conclusions by enabling automated analysis. This research aims to improve diagnostic accuracy by classifying malaria-infected cells using a majority voting ensemble framework combined with triplet loss aided label contrastive learning. Experimental results demonstrate the effectiveness of the proposed method on microscopic cell images in terms of accuracy, precision, recall, and other evaluation metrics.

bioinformatics↗

bioRxiv2024

RAF2Net: Automated grading of Renal cell Carcinoma utilizing Attention-enhanced deep learning models through Feature Fusion

It is anticipated that the number of instances of kidney cancer will continue to rise globally, which motivates changes to the current diagnostic framework in order to address emerging issues. Renal cell carcinoma (RCC) accounts for 80-85% of all renal tumors and is the most common kind of kidney cancer. Based on kidney histopathology images, this study presented a completely automated, robust, and computationally efficient Renal Cell Carcinoma Grading Network (RAF 2Net). Our suggested model incorporates 3 different Mobilenet backbones with intelligent feature fusion. Moreover, the attention blocks help us give more importance to the important pixels, which are majorly responsible for classification. For comparison purposes, Similar tests were conducted using transfer learning methods with pre-trained ImageNet weights and deep learning models created from scratch. To show the efficacy of the suggested method, we have computed evaluation parameters like Accuracy, Precision, F score, Recall, Confusion Matrix, and TSNE. Based on the provided KMC dataset, the experimental result demonstrates that the proposed RAF 2Net outperforms the nine most recent classification methods regarding prediction Accuracy, Recall, Precision, and F score with a value greater than 92%.

bioinformatics↗

bioRxiv2024

Structure-function relationship of PE11 esterase of Mycobacterium tuberculosis with respect to its role in virulence

The lipolytic enzymes of Mycobacterium tuberculosis play a critical role in immunomodulation and virulence. Among these proteins, PE11 which also belongs to the PE/PPE family, is the smallest ([~]10.8 kDa) and play a significant role in cell wall remodelling and virulence. PE11 is established to be an esterase, but its enzymatic and structural properties are not yet characterized. In this study, using homology modelling we deduced the putative structure which shows the presence of both -helix and {beta}-sheet structures which is in close agreement with that observed by CD spectra of the purified protein. PE11 was found to contain a GX3SX4G motif homologous to canonical GxSxG motif present in many serin hydrolases. The catalytic triad appears to be located within this motif as substitution of Serine26 and Glycine31 residues abrogated its enzymatic activity. Gel-filtration chromatography data indicate that PE11 possibly exists as dimer and tetramer showing positive cooperativity for binding its substrates. In addition, PE11 esterase activity was found to be critical for cell wall remodelling, antibiotic resistance and conferring survival advantages to M. tuberculosis. Our data suggest that PE11 can be targeted for designing potential therapeutic strategies.

microbiology↗

bioRxiv2024

Chaperone-mediated heterotypic phase separation regulates liquid-to-solid phase transitions into amyloid fibrils

Biomolecular condensates formed via the phase separation of proteins and nucleic acids are thought to regulate a myriad of cellular processes with exquisite spatiotemporal precision. However, such highly dynamic, viscoelastic, mesoscopic, intracellular membraneless bodies can undergo aberrant liquid-to-solid transitions into a range of amyloid-like species. The formation of such pathological assemblies necessitates their clearance by the cellular protein quality control machinery comprising molecular chaperones. Nonetheless, the mechanism underlying the chaperone-mediated regulation of protein homeostasis within biomolecular condensates remains elusive. Here, we present a unique case demonstrating that a heat shock protein 40 (Hsp40), Ydj1, promotes the heterotypic phase separation of intrinsically disordered tau via intermolecular electrostatic and hydrophobic interactions. Through a diverse array of tools involving high-resolution fluorescence imaging, single-droplet steady-state and picosecond time-resolved fluorescence anisotropy, and single-molecule FRET (Forster resonance energy transfer), we elucidate the diverse structural conformations of tau present within phase-separated heterotypic condensates that are otherwise predisposed to aggregation. Our vibrational Raman spectroscopy and electron microscopy data show that the presence of Ydj1 in tau-Ydj1 condensates abolishes the formation of amyloid fibrils, unlike tau-only droplets. By sequentially deleting segments, we identify amyloidogenic hexapeptide motifs located in the hydrophobic microtubule-binding region of tau that foster contacts with the peptide-binding regions of Ydj1, promoting the formation of tau-Ydj1 binary condensates. Additionally, we show that the underlying network of interactions governing these condensates can be further tuned by RNA. Our results underscore an intriguing interplay of molecular drivers that govern chaperone-associated phase separation, with broader implications for the chaperoning of a wide range of intrinsically disordered proteins involved in physiology and disease.

biophysics↗

bioRxiv2024

A Dose-Response Model for Accurate Detection and Quantification of Transcriptome-Wide Gene Knockdown for Oligonucleotide-Based Medicines

Synthetic antisense oligonucleotides and siRNAs are a class of Oligonucleotide-Based Medicines (OBMs) that can hybridize with pre-mRNA and mRNA, recruit a mechanism-of-action specific enzymatic complex, and knockdown target gene expression. This class of molecules provides an excellent substrate for designing precision gene-modulatory therapeutics; however, quantifying on- and off-target dose response as measured by next-generation sequencing for this class of therapeutics has remained under-powered and ambiguous. Often in silico predictions of off-targets (ranked by edit tolerance) are used as putative off-target analysis in ASO and siRNA drug design. We construct a simple, effective theory of transcriptional dynamics and enzymatic activity in order to describe the transcriptome-wide response to these oligonucleotides. We establish rigorous quantification methods of off-target analysis in oligonucleotide drug design. We also extend the DESeq work [1, 2] of Negative Binomial noise in gene expression measurements to describe noise, including outliers, in OBM-dose response NGS experiments. We demonstrate the performance of our model on both synthetic and experimental Digital Gene Expression (DGE) data of dose response in ASO-treated cells. We present our analysis package, DoReSeq, as a freely available resource for the community. We hope this will elevate the standards of off-target analysis for such an important class of precision therapeutics.

pharmacology and toxicology↗

bioRxiv2024

Telomere-to-telomere genome assembly of the clubroot pathogen Plasmodiophora brassicae

Plasmodiophora brassicae (Woronin, 1877), a biotrophic, obligate parasite, is the causal agent of clubroot disease in brassicas. The clubroot pathogen has been reported in more than 80 countries worldwide, causing economic losses of hundreds of millions every year. Despite its widespread impact, very little is known about the molecular strategies it employs to induce the characteristic clubs in the roots of susceptible hosts during infection, nor about the mechanisms it uses to overcome genetic resistance. Here, we provide the first telomere-to-telomere complete genome of Plasmodiophora brassicae. We generated [~] 27 Gb of Illumina, Oxford Nanopore, and PacBio HiFi data from resting spores of strain Pb3A and produced a 25.3 Mb assembly comprising 20 chromosomes, with an N50 of 1.37 Mb. The BUSCO score, the highest reported for any member of the group Rhizaria (Eukaryota: 88.2%), highlights the limitations within the Eukaryota database for members of this lineage. Using available transcriptomic data and protein evidence, we annotated the Pb3A genome, identifying 10,521 protein-coding gene models. This high-quality, complete genome of Plasmodiophora brassicae will serve as a crucial resource for the plant pathology community to advance the much-needed understanding of the evolution of the clubroot pathogen. SIGNIFICANCEPlasmodiophora brassicae (Woronin, 1877) is a devastating plant pathogen, member of the Rhizaria group, and is putting at risk the oilseed rape and cruciferous vegetable industry worldwide. Here, we present the first telomere-to-telomere genome of P. brassicae, and the first complete genome for a member of the Rhizaria taxon. We also provide a high-quality genome annotation featuring an enhanced gene model relative to previous studies. This complete genome will be of great interest to researchers focused on the ecology and evolution of the clubroot pathogen, as well as those studying other members of the Rhizaria group.

microbiology↗

bioRxiv2024

Dynamic Adaptation in Extant Porins Facilitates Antibiotic Tolerance in Energetic Escherichia coli

Bacteria can tolerate antibiotics despite lacking the genetic components for resistance. The prevailing notion is that tolerance results from depleted cellular energy or cell dormancy. In contrast to this view, many cells in the tolerant population of Escherichia coli can exhibit motility - a phenomenon that requires cellular energy, specifically, the proton-motive force (PMF). As these motile-tolerant cells are challenging to isolate from the heterogeneous tolerant population, their survival mechanism is unknown. Here, we discovered that motile bacteria segregate themselves from the tolerant population under micro-confinement, owing to their unique ability to penetrate micron-sized channels. Single-cell measurements on the motile-tolerant population showed that the cells retained a high PMF, but they did not survive through active efflux alone. By utilizing growth assays, single-cell fluorescence studies, and chemotaxis assays, we showed that the cells survived by dynamically inhibiting the function of existing porins in the outer membrane. A drug transport model for porin-mediated intake and efflux pump-mediated expulsion suggested that energetic tolerant cells withstand antibiotics by constricting their porins. The novel porin adaptation we have uncovered is independent of gene expression changes and may involve electrostatic modifications within individual porins to prevent extracellular ligand entry.

microbiology↗