Biology subjects

Ali, R.

Publications and source records attributed to Ali, R..

At least 19 recordsLinked to original sources

bioRxiv2026

CRISPR-mediated Correction of Oncogenic AS-NMD in Splicing Factor Mutant Cancer

Alternative splicing coupled to nonsense-mediated mRNA decay (AS-NMD) evolved as a master regulator of gene expression. Dysregulated AS-NMD has been identified as the root of many human maladies, from developmental defects to deadly cancer. Poison exons (PEs) are highly conserved alternative exons that contain a premature termination codon and elicit AS-NMD when included in a transcript. Cancer cells often exploit the inclusion of PEs to downregulate tumor suppressors or the exclusion of PEs to upregulate oncoproteins. Therefore, PEs have drawn significant attention as a novel therapeutic avenue for cancer and other diseases. Here, we examine a therapeutic proof-of-concept for manipulating PE-mediated oncogenic AS-NMD using a CRISPR-based approach. Using paired guide RNA, we successfully deleted a PE of a tumor suppressor (EZH2) from the genome of SRSF2-mutated leukemia. This editing resulted in EZH2 mRNAs without a PE, escaped AS-NMD, and restored the protein expression. This subsequently reinstated H3K27 histone methylation and rescued defective chromatin regulation associated with impaired hematopoietic stem cell differentiation. Finally, we showed the preferential advantages of CRISPR over the antisense technology we recently developed targeting the PE of EZH2. Therefore, the CRISPR strategy shows compelling evidence as a therapeutic approach targeting PE in cancer and other human diseases.

cancer biology↗

bioRxiv2026

Genetic dependency of atrial fibrillation-associated risk genes across tissue types: Discovering novel therapeutic targets

BackgroundAddressing the underlying causes of atrial fibrillation (AFib) is critically important. While potential AFib-related genes have been recognized, the impact of modifying these genes in humans remains poorly understood. ObjectiveWe assessed the cellular dependencies of 309 genes previously associated with AFib through genome-wide association studies using data from the Cancer Dependency Map project, aiming to prioritize potential therapeutic targets with minimal off-target effects. MethodsWe analyzed CRISPR-Cas9 knockout (CHRONOS scores) and RNA interference (RNAi) knockdown (DEMETER2 scores) screening data from 1,927 human cell lines across 24 tissue types, focusing on tissues associated with AFib initiation, presentation, and progression: autonomic ganglia, central nervous system (CNS), and soft tissue. We examined the expression and dependency scores of the AFib-associated genes, identifying significant correlations between gene expression and cellular dependency within specific tissues using Pearson correlation coefficients and controlling the false discovery rate (FDR) at 5%. ResultsOut of the 309 AFib-associated genes, 206 genes (66.7%) had CHRONOS dependency scores and 229 (74.1%) had DEMETER2 dependency scores available. Several genes showed significant negative dependency scores (CHRONOS < -0.5) across multiple tissues, indicating potential off-target effects if inhibited. In contrast, we identified 12 genes with significant expression-driven dependencies within AFib-associated tissues. In CNS cell lines, HAND2 (R = -0.456, FDR = 0.002) and VGLL2 (R = -0.434, FDR = 0.005) showed significant negative correlations between gene expression and cellular dependency. In soft tissue cell lines, BEST3 (R = -0.679, FDR = 0.001) and PITX2 (R = -0.679, FDR = 0.001) also demonstrated strong negative correlations. Additionally, ERBB4 in CNS lines showed a significant negative correlation (R = -0.361, FDR = 0.048). These findings suggest that inhibiting these genes may selectively affect high-expressing cells in AFib-associated tissues while minimizing effects on other tissues. ConclusionOur analysis identified HAND2, VGLL2, BEST3, and ERBB4 as potential therapeutic targets for AFib, demonstrating significant expression-driven dependencies in AFib-associated tissues with no pan-tissue essentiality. These results provide a quantitative basis for developing targeted therapies with reduced off-target effects. CONDENSED ABSTRACTAtrial fibrillation (AFIB) is one of the most common cardiac arrhythmias with numerous known risk factors. Although many AFIB-associated genes have been identified, the impact of screening or the effects of modifying these genes in humans remain poorly understood. We examined CRISPR knockout and RNAi knockdown screen data from nearly 2,000 human cell lines to assess the cellular dependencies of 309 genes associated with AFIB, previously identified through genome-wide association studies. Some genes demonstrate broad cell dependencies across various tissue types, indicating potential off-target effects if inhibited. Conversely, HAND2, VGLL2, BEST3, and ERBB4 were identified as genes of interest because their genetic knockouts specifically impacted high-expressing cells from tissue lineages pertinent to AFIB and/or were not pan-dependent. Overall, analyses of genetic screen data identified AFIB-associated genes whose knockout or knockdown selectively affected cell lines of relevant tissue lineages, prioritizing targets for potential AFIB treatments.

genomics↗

bioRxiv2026

Diet-dependent microbiota and diet-independent immunometabolic responses to probiotic supplementation in broiler chickens

Probiotics are widely used in food animal production to support gut health and immune function, but the indicators of probiotic efficacy and the conditions under which they translate to host benefit remain unclear. Microbiota composition is the most accessible data supporting probiotic effects, yet whether compositional change reliably predicts host outcomes is not well understood. We investigated this question in broiler chickens fed two nutritionally similar basal diets, with or without a commercial probiotic. Microbiota composition was profiled across 6 gastrointestinal regions using 16S rRNA sequencing. To assess systemic functional effects, an in vitro assay building on prior observations of elevated circulating immune cell ATP in probiotic-fed animals was developed. In this assay, serum from each treatment group was applied to a chicken T-lymphocyte cell line before ATP quantitation. Basal diet was the primary driver of microbial community structure, with probiotic-induced compositional shifts observed predominantly in one diet context but minimally in the other. Despite this difference, serum from probiotic-supplemented animals increased T-lymphocyte ATP production across both diets, supporting prior findings and revealing a systemic immunometabolic response independent of broad microbiota restructuring. Functional predictions revealed enrichment of pathways related to mevalonate and carbohydrate metabolism in probiotic-supplemented birds within the more responsive diet context, driven largely by Lactobacillaceae family taxa. These findings demonstrate that basal diet modulates the detectability and nature of probiotic effects on the microbiota, but not the physiological host response. This disconnect has implications for how probiotic efficacy is evaluated and for microbiome targeted interventions across species. ImportanceProbiotics are used widely in food animal production to support gut health and immune function, yet predicting which probiotic preparations will produce meaningful effects remains a challenge. Microbiota composition, profiled by 16S rRNA sequencing, is the most accessible measure of probiotic activity, but it captures only one aspect of the host-microbe dynamic. These data demonstrate that probiotic-induced compositional changes vary substantially between basal diets, while the host immunometabolic response is consistent across diets, demonstrating that compositional readouts alone cannot reliably predict host outcomes. The findings have practical implications for how probiotic efficacy is evaluated and inform the broader effort to design microbiome targeted interventions across both veterinary and human contexts.

microbiology↗

bioRxiv2025

Generalized brain-state modeling with KenazLBM

The large-scale functional state of a human brain remains difficult to characterize, much less predict. Regardless, techniques have been engineered to electrically neuromodulate the brain to treat a subset of neurologic and psychiatric disorders with moderate efficacy. Accurate characterization of a brains instantaneous functional state has stymied the development of more effective neuromodulation paradigms. Advanced computational methods are required to address this gap and enable large-scale neuroscience. Here we define the concept of generalized brain-state modeling across humans as Large Brain-State Modeling (LBM) and present KenazLBM as the worlds first example. KenazLBM can instantaneously characterize the functional state of a persons brain with raw iEEG data, and predict future brain-states. KenazLBM was trained on over 17.9 billion unique multichannel tokens from people undergoing intracranial electroencephalography (iEEG) recordings, and has learned to interrelate brain-states between people into a common interpretable topology. Most importantly, the model generalizes to unseen subject data with significant recording channel heterogeneity from the training set. We offer KenazLBM as a first generalized brain-state model to serve as a new paradigm of basic neuroscience inquiry and potential translation into neuromodulation therapeutics.

neuroscience↗

bioRxiv2025

Functional genomics and tumor microenvironment analysis reveal prognostic biological subtypes in Mantle cell lymphoma

Mantle cell lymphoma (MCL) is a genetically and clinically heterogeneous B-cell malignancy. We studied two MCL cohorts with differing treatment patterns: one enriched for immunochemotherapy, the other for chemotherapy alone. TP53 alterations were consistently associated with poor prognosis, whereas ATM mutations correlated with improved outcomes following rituximab-based chemotherapy. Based on recurrent genetic events, six clusters were identified and refined into three prognostic groups: high-risk (TP53 mutations and deletions at 17p13.3, 13q14.2, and 19p13.3), intermediate-risk (ATM and epigenetic regulator mutations, or gains at 8q/17q/15q), and low-risk (lacking TP53 alterations, rare ATM mutations without 11q deletions, gains at 3q, deletions at 6q). Transcriptomic analysis revealed enrichment of proliferation, metabolism-promoting gene signatures in high-risk; angiogenesis and NOTCH signaling in intermediate-risk; and proinflammatory-related (i.e., IFN, TNF) in low-risk MCLs. Multi-proteomic spatial profiling using imaging mass cytometry (IMC) demonstrated enrichment of CD8 T cells with high expression of exhaustion markers and dominant population of myeloid cells skewed toward an M2-like phenotype. Compared to ATM-perturbed tumors, TP53-perturbed tumors exhibited enriched SOX11 tumor cells and enhanced tumor-immune cell interactions. Functional analysis revealed that p53 represses BCR signaling through PTPN6 activation. Collectively, these findings highlight distinct molecular and immune landscapes and reveal therapeutic vulnerabilities in high-risk TP53-altered MCL.

cancer biology↗

bioRxiv2025

Integrative multi-omic analysis reveals oral microbiome-metabolome signatures of obesity

Obesity is a major global health challenge and a leading risk factor for cardiometabolic disorders. The global surge in obesity, driven by industrialization and the widespread consumption of low-fiber, ultra-processed food, highlights an urgent need for deeper biological insights. While the gut microbiome has been studied in the context of obesity, the contribution of the oral microbiome--the second largest microbial ecosystem in the human body-remains largely underexplored. Here, we report findings from a deeply-phenotyped prospective-cohort of 628 Emirati adults, leveraging amplicon sequencing of mouthwash samples and multi-omics profiling and functional and metabolic activity analysis of 97 obese individuals and 95 matched controls, making this the most comprehensive multi-omics analysis of the oral microbiome. We identified significant differences in oral microbial diversity, composition, functional pathways, and metabolic profiles between obese and non-obese groups. Integrated multi-omics analysis of the 192 matched metagenomes and metabolome samples uncovered significant metabolic reprogramming and altered energy regulation in obesity. Specifically, the oral microbiome of obese participants were enriched for the proinflammatory Streptococcus parasanguinis and Actinomyces oris, and the lactate-producing Oribacterium sinus. Many microbial pathways involved in dietary carbohydrate metabolism, histidine degradation, as well as the production of obesogenic biomolecules were also enriched in obese participants; however, B-vitamin and heme production pathways were depleted. Consequently, metabolites resulting from these pathways such as lactate, histidine derivatives, choline, uridine, and uracil were elevated in obesity. These consistent microbiome-metabolite shifts were strongly associated with prominent obesity-associated cardiometabolic markers, including serum triglycerides and alkaline phosphatases, establishing a robust link between oral microbiome and obesity. These findings provide the most comprehensive insights into how disrupted microbial-metabolic cross-talk in the oral cavity may contribute to obesity and related cardiometabolic disease risk, underscoring the potential of targeting of oral microbiome-host interactions as a novel avenue for obesity prevention and intervention.

microbiology↗

bioRxiv2024

ADAT2/3-mediated tRNA editing promotes cancer cell growth and tumorigenicity

Transfer RNAs (tRNAs) are subject to various chemical modifications that influence their stability or function. Adenosine to Inosine (A-to-I) editing in the tRNA anticodon at position A34 is an important modification that expands anticodon-codon recognition at the wobble position and is required for normal mRNA translation. The relevance of tRNA editing in cancer remains unexplored. Here we show that the genes encoding the ADAT2/3 deaminase complex, responsible for A-to-I tRNA editing in humans, are commonly amplified and/or overexpressed in several tumor types including liposarcoma (LPS). We find that knockdown of the ADAT complex suppresses LPS cell growth and tumorigenicity. Mechanistically, we find that decreased tRNA editing upon ADAT2 depletion leads to defective translation of a subset of mRNAs. Thus, ADAT-mediated tRNA modification promotes oncogenesis by enhancing the translation of growth promoting mRNAs that are enriched in NNC codons that lack cognate tRNAs and therefore depend on A-I tRNA editing for decoding and mRNA translation. Our results uncover an oncogenic role of tRNA editing and identify ADAT2/3 as a potential new cancer therapeutic target.

cancer biology↗

bioRxiv2024

Human macula formation involves two waves of retinoic acid signaling suppression via CYP26A1 regulating cell cycle exit and cone specification

The human macula is a specialized, cone-rich region of the eye, critical for high-acuity vision, yet the pathways regulating its development remain poorly understood. RA-catabolizing enzyme CYP26A1 establishes the chick high-acuity area via upregulation of FGF8. However, detailed analysis of this pathway and its functions has not been performed in early human fetal tissue. Fluorescent in situ hybridization revealed striking biphasic CYP26A1 expression but little FGF8 in the presumptive macula region between post-conception weeks (PCW)6-17. Pharmacological RA signaling inhibition in human retinal organoids mimicking the two waves of CYP26A1 revealed early RA inhibition prompted early cell cycle exit and increased cone genesis, while late inhibition altered cone subtype specification. Conversely, recombinant FGF8 had no effect on photoreceptor fate. This work provides spatiotemporal examination of CYP26A1 across human macular development, as well as experimental evidence for the different roles of RA signaling inhibition in a human model of retinal development.

developmental biology↗

bioRxiv2023

Indigenous probiotics Lactobacillus reuteri and Enterococcus faecium exhibit positive growth performance and disease prevention against extended-spectrum cephalosporin and fluoroquinolones resistant Salmonella enterica in broiler chicks.

ABSTRACTThe rapid increase in antibiotic resistance poses a global threat to public health, necessitating the development of effective antimicrobial alternatives. This study compared an indigenous probiotic mix containing Lactobacillus reuteri and two strains of Enterococcus faecium to a commercial probiotic blend ProtexinR on the growth performance, mortality rate, histomorphology, serum immunoglobulins, and intestinal microflora of broiler chickens challenged with two multi drug resistant Salmonella serovars, Typhimurium and Enteritidis. Two hundred and forty day-old broiler chicks were randomly assigned to six treatment groups for 4 weeks: the treatment groups were; birds continuously supplemented with only indigenous probiotic strains (108 CFU/mL) (IPRO-); birds challenged with Salmonella serovars 106 (CFU/mL) (PC+); birds continuously supplemented with indigenous probiotic strains and challenged with Salmonella serovars (IPRO+); birds supplemented with ProtexinR and challenged with Salmonella serovars (CM+); birds supplemented with only ProtexinR (CM-); and birds with no Salmonella challenge or probiotics (negative control; PC-). The results revealed that IPRO- diets significantly improved feed conversion ratio (FCR) and increased body weight (BW) (P [≤] 0.05). No effect of probiotic treatments was observed on IPRO- and CM- on relative organ weights as compared to the negative control (PC-). The Salmonella- challenged group PC+ had the highest (20%) mortality rate and lowest BW. The IPRO- had significantly lower FCR (1.55) compared to PC- (1.86) and PC+ (1.95). The broilers in the IPRO- group showed significantly increased serum concentrations of IgA and IgG relative to both control groups (P [≤] 0.05). Morphological analysis of the ileum revealed significant increases (P [≤] 0.05) in the villus height and villus height/crypt depth in birds fed IPRO- compared with the PC+. Cecal Lactobacillus and Enterococcus counts were the highest (P [≤] 0.05) and Salmonella counts were the lowest (P [≤] 0.05) in the IPRO- group compared to the Salmonella infected group PC+. These results indicated that indigenous probiotic strains Lactobacillus reuteri and Enterococcus faecium can be an effective and low-cost alternative compared to commercial probiotics in the Pakistan poultry industry.

microbiology↗

bioRxiv2023

The role of structural vs cellular remodeling in arrhythmogenesis: personalized computer models of atrial fibrillation.

Atrial fibrillation (AF) is a progressive disease involving both structural and functional remodeling. To investigate the contribution of cell-scale functional remodeling to AF pathogenesis, we combined personalized 3D anatomical models with pathology-specific ionic models. The latter were developed using recordings in myocytes isolated from patients in sinus rhythm, paroxysmal, postoperative, and persistent AF. To quantify AF dynamics, we developed a novel algorithm for locating RDs by backtracking the conduction velocity field from the wavebreak regions. We demonstrate that our novel algorithm is at least 700 times faster than the traditional phase singularity analysis. The inducibility of simulated AF was not pathology-dependent, but pathological models demonstrate a more extensive arrhythmogenic substrate compared to the sinus rhythm. AF driver locations depend on electrophysiological remodeling; differences between pathology-specific models are explained by differences in wavebreak patterns. Specifically, RDs tend to dwell in the regions with the highest wavebreak probability.

physiology↗

bioRxiv2022

Genomic insights into metabolic flux in ruby-throated hummingbirds

Hummingbirds are very well adapted to sustain efficient and rapid metabolic shifts. They oxidize ingested nectar to directly fuel flight when foraging but have to switch to oxidizing stored lipids derived from ingested sugars during the night or long-distance migratory flights. Understanding how this organism moderates energy turnover is hampered by a lack of information regarding how relevant enzymes differ in sequence, expression, and regulation. To explore these questions, we generated a chromosome level de novo genome assembly of the ruby-throated hummingbird (A. colubris) using a combination of long and short read sequencing and scaffolding using other existing assemblies. We then used hybrid long and short-read RNA-sequencing for a comprehensive transcriptome assembly and annotation. Our genomic and transcriptomic data found positive selection of key metabolic genes in nectivorous avian species and a deletion of critical genes (GLUT4, GCK) involved in glucostasis in other vertebrates. We found expression of fructose-specific GLUT5 putatively in place of insulin-sensitive GLUT4, with predicted protein models suggesting affinity for both fructose and glucose. Alternative isoforms may even act to sequester fructose to preclude limitations from transport in metabolism. Finally, we identified differentially expressed genes from fasted and fed hummingbirds suggesting key pathways for the rapid metabolic switch hummingbirds undergo.

genomics↗

bioRxiv2021

Progressive protein aggregation in PRPF31 patient retinal pigment epithelium cells: the mechanism and its reversal through activation of autophagy

Mutations in pre-mRNA processing factor 31 (PRPF31), a core protein of the spliceosomal tri-snRNP complex, cause autosomal-dominant retinitis pigmentosa (adRP). It has remained an enigma why mutations in ubiquitously expressed tri-snRNP proteins result in retina-specific disorders, and so far, the underlying mechanism of splicing factors-related RP is poorly understood. Here, we used iPSC technology to generate retinal organoids and RPE models from three patients with severe and very severe PRPF31-adRP, normal individuals and a CRISPR/Cas9-corrected isogenic control. To fully assess the impacts of PRPF31 mutations, quantitative proteomics analyses of retinal organoids and RPE cells was carried out showing RNA splicing, autophagy and lysosome, unfolded protein response (UPR) and visual cycle-related pathways to be significantly affected. Strikingly, the patient-derived RPE and retinal cells were characterised by the presence of large amounts of cytoplasmic aggregates containing the mutant PRPF31 and misfolded, ubiquitin-conjugated proteins including key visual cycle proteins, which accumulated progressively with time. Mutant PRPF31 variant was not incorporated into splicing complexes, but reduction of PRPF31 wildtype levels led to tri-snRNP assembly defects in Cajal bodies of PRPF31 patient retinal cells with reduced U4/U6 snRNPs and accumulation of U5, smaller nuclear speckles and reduced formation of active spliceosomes giving rise to global splicing dysregulation. Moreover, the impaired waste disposal mechanisms further exacerbated aggregate formation, and targeting these by activating the autophagy pathway using Rapamycin resulted in reduction of cytoplasmic aggregates and improved cell survival. Our data demonstrate that it is the progressive aggregate accumulation that overburdens the waste disposal machinery rather than direct PRPF31-initiated mis-splicing, and thus relieving the RPE cells from insoluble cytoplasmic aggregates presents a novel therapeutic strategy that can be combined with gene therapy studies to fully restore RPE and retinal cell function in PRPF31-adRP patients. HighlightsO_LIPRPF31 RP mutations lead to formation of insoluble aggregates containing the mutant PRPF31 and misfolded, ubiquitin conjugated proteins including key visual cycle proteins (e.g. RLBP1) in RPE cells, which accumulate progressively with time and affect tight junctions and cell survival. C_LIO_LIMutant PRPF31 is predominantly localised in cytoplasmic aggregates of patient specific RPE and retinal cells and is not able to be incorporated into splicing complexes to cause direct mis-splicing. C_LIO_LIHigh-throughput quantitative proteomics identifies significantly altered RNA splicing, visual perception, retinoid metabolism, waste disposal and unfolded protein response pathways in patient RPE cells, and autophagy and lysosome, unfolded protein response (UPR) and visual cycle-related pathways in photoreceptor cells. C_LIO_LIAccumulation of PRPF31 mutant variant as cytoplasmic aggregates reduces wildtype PRPF31 in the nucleus leading to tri-snRNP assembly defects, characterised by accumulation of U5 and reduction of U4/U6 snRNPs in Cajal bodies, altered morphology of nuclear speckles and consequently downregulation of active spliceosomes (Bact and C complexes) in PRPF31 patient RPE and retinal cells. C_LIO_LIProteomic study of insoluble aggregates identifies other RP-linked splicing factors and multiple key retinal-specific proteins, whose variants are linked to retinitis pigmentosa, within the aggregates of patient RPE cells. C_LIO_LIPRPF31 patient RPE cells have impaired waste disposal and proteasome mediated degradation, which together with the impaired autophagy pathway, further exacerbate aggregate formation. C_LIO_LIPhagocytosis of photoreceptor outer segment fragments (POS) shed daily by RPE cells accelerates aggregation of key proteins indicating enhanced cytoplasmic aggregate formation under physiological conditions in patient RPE cells. C_LIO_LIActivation of autophagy via administration of rapamycin results in reduction of cytoplasmic aggregates in RPE cells, correct localisation of mislocated and misfolded proteins to the nucleus, thereby improving cell survival. C_LI

cell biology↗

bioRxiv2021

Spatiotemporal Control of Actomyosin Contractility by MRCKβ Signaling Drives Phagocytosis

Phagocytosis requires myosin-generated contractile force to regulate actin dynamics. However, little is known about the molecular mechanisms that guide this complex morphodynamic process. Here we show that particle binding to Mer Tyrosine Kinase (MerTK), a widely expressed phagocytic receptor, stimulates phosphorylation of the Cdc42 GEF Dbl3 in the retinal pigment epithelium (RPE), triggering activation of MRCK{beta} and its co-effector N-WASP that cooperate to deform the membrane into cups. Continued MRCK{beta} activity then drives recruitment of a mechanosensing bridge enabling transmission of the cytoskeletal force required for cup closure and particle internalization. MRCK{beta} is also required for Fc receptor-mediated phagocytosis by macrophages. In vivo, MRCK{beta} is essential for RPE phagocytosis of photoreceptor debris and, hence, retinal integrity. MerTK-independent activation of MRCK{beta} signaling by a phosphomimetic Dbl3 mutant rescues phagocytosis in retinitis pigmentosa RPE cells lacking functional MerTK. Thus, conserved MRCK{beta} signaling controls spatiotemporal regulation of actomyosin contractility to guide actomyosin dynamics-driven phagocytosis and represents the principle phagocytic effector pathway downstream of MerTK.

cell biology↗

bioRxiv2021

Large-scale Genetic Characterization of a Model Sulfate Reducing Bacterium

Sulfate-reducing bacteria (SRB) are obligate anaerobes that can couple their growth to the reduction of sulfate. Despite the importance of SRB to global nutrient cycles and their damage to the petroleum industry, our molecular understanding of their physiology remains limited. To systematically provide new insights into SRB biology, we generated a randomly barcoded transposon mutant library in the model SRB Desulfovibrio vulgaris Hildenborough (DvH) and used this genome-wide resource to assay the importance of its genes under a range of metabolic and stress conditions. In addition to defining the essential gene set of DvH, we identified a conditional phenotype for 1,137 non-essential genes. Through examination of these conditional phenotypes, we were able to make a number of novel insights into our molecular understanding of DvH, including how this bacterium synthesizes vitamins. For example, we identified DVU0867 as an atypical L-aspartate decarboxylase required for the synthesis of pantothenic acid, provided the first experimental evidence that biotin synthesis in DvH occurs via a specialized acyl carrier protein and without methyl esters, and demonstrated that the uncharacterized dehydrogenase DVU0826:DVU0827 is necessary for the synthesis of pyridoxal phosphate. In addition, we used the mutant fitness data to identify genes involved in the assimilation of diverse nitrogen sources, and gained insights into the mechanism of inhibition of chlorate and molybdate. Our large-scale fitness dataset and RB-TnSeq mutant library are community-wide resources that can be used to generate further testable hypotheses into the gene functions of this environmentally and industrially important group of bacteria.

microbiology↗

bioRxiv2019

An endophytic bacteria (Pseudomonas aeruginosa) strain translocated and protected tomato (Solanum lycopersicum L.) plants from its parasitic weed Phelipanche aegyptiaca

Phelipanche aegyptiaca is an obligate holo-parasitic weed lacking a functional photosynthetic system, which subsists on roots of a wide range of host crops, causing severe losses in yield quality and quantity. The parasite and its host are connected through their vascular system, forming a unique ecological system that enables the exchange of various substances. In a previous study, it was suggested that endophytic bacteria, which naturally inhabit the internal tissues of plants, can also be transmitted from the parasitic weed to its host and vice versa. In the current study, we investigate the characteristics of a previously isolated Pseudomonas sp. PhelS10 strain, using both biochemical and molecular methods. Our results revealed that production of Pseudomonas aeruginosa quinolone signal (PQS) was 2.1 times higher than that of the standard Pseudomonas aeruginosa strain (PAO1), which contributed to a 22% higher biofilm formation capability. PhelS10 strain was detected in the xylem of tomato plants using FISH analysis. In addition, PhelS10 strain was found in the parasitic weeds inner tissues, confirming the hypothesis that endophytic bacteria traffic between the plant host and its parasitic weed.

microbiology↗

bioRxiv2019

Topoisomerase II inhibitors induce cGAS-STING dependent inflammation resulting in cytokine induction and immune checkpoint activation

Tumours with genomic instability demonstrate enhanced immunogenicity and potential for response to immune checkpoint blockade (ICB). We previously demonstrated activation of the cGAS-STING pathway following loss of DNA repair, resulting in cytokine induction, lymphocytic infiltration and immune checkpoint activation. Here we explore the role of chemotherapies in inducing this innate immune response, identifying topoisomerase II (topo-II) inhibitors, particularly doxorubicin and epirubicin, as potent inducers of a cGAS-STING dependent interferon response. Mechanistically, topo-II inhibition resulted in significant induction of cytoplasmic DNA and subsequent micronuclei formation, a requirement for efficient cGAS-STING activation and consequent cytokine and immune checkpoint gene induction. Importantly, increased cytokine and immune checkpoint gene expression, as well as increased immune cell infiltration, was also observed in patient derived breast tumour biopsies following topo-II inhibitor-based treatment. Taken together, this study indicates topo-II inhibitors such as doxorubicin, may be best placed to induce immunogenic inflammation, and thereby increase responses to ICB therapies.\n\nSignificanceThis work demonstrates how topo-II inhibitors induce STING-pathway activation, cytokine induction and immune checkpoint protein upregulation in cancer cells and provides a rationale for combining topo-II inhibitors with ICB therapy in early breast cancer.

cancer biology↗

bioRxiv2018

Biased signaling downstream of epidermal growth factor receptor regulates proliferative versus apoptotic response to ligand

Inhibition of EGFR signaling by small molecule kinase inhibitors and monocloncal antibodies has proven effective in the treatment of multiple cancers. In contrast, metastatic breast cancers (BC) derived from EGFR-expressing mammary tumors are inherently resistant to EGFR-targeted therapies. Mechanisms that contribute to this inherent resistance remain poorly defined. Here we show that in contrast to primary tumors, ligand-mediated activation of EGFR in metastatic BC is dominated by STAT1 signaling. This change in downstream signaling leads to apoptosis and growth inhibition in response to EGF in metastatic BC cells. Mechanistically, these changes in downstream signaling result from an increase in the internalized pool of EGFR in metastatic cells, increasing physical access to the nuclear pool of STAT1. Along these lines, an EGFR mutant that is defective in endocytosis is unable to elicit STAT1 phosphorylation and apoptosis. Additionally, inhibition of endosomal signaling using an EGFR inhibitor linked to a nuclear localization signal specifically prevents EGF-induced STAT1 phosphorylation and cell death, without affecting EGFR:ERK1/2 signaling. Pharmacologic blockade of ERK1/2 signaling through the use of the allosteric MEK1/2 inhibitor, trametinib, dramatically biases downstream EGFR signaling toward a STAT1 dominated event, resulting in enhanced EGF-induced apoptosis in metastatic BC cells. Importantly, combined administration of trametinib and EGF also facilitated an apoptotic switch in EGFR-transformed primary tumor cells, but not normal mammary epithelial cells. These studies reveal a fundamental distinction for EGFR function in metastatic BC. Furthermore, the data demonstrate that pharmacological biasing of EGFR signaling toward STAT1 activation is capable of revealing the apoptotic function of this critical pathway.

cancer biology↗

bioRxiv2018

HLA-B*39:06 Efficiently Mediates Type 1 Diabetes in a Mouse Model Incorporating Reduced Thymic Insulin Expression

Type 1 diabetes (T1D) is characterized by T cell-mediated destruction of the insulin-producing {beta}cells of the pancreatic islets. Among the loci associated with T1D risk, those most predisposing are found in the MHC region. HLA-B*39:06 is the most predisposing class I MHC allele and is associated with an early age of onset. To establish an NOD mouse model for the study of HLA-B*39:06, we expressed it in the absence of murine class I MHC. HLA-B*39:06 was able to mediate the development of CD8 T cells, support lymphocytic infiltration of the islets, and confer T1D susceptibility. Because reduced thymic insulin expression is associated with increased T1D risk in patients, we incorporated this in our model as well, finding that HLA-B*39:06-transgenic NOD mice with reduced thymic insulin expression have an earlier age of disease onset and a higher overall prevalence as compared to littermates with typical thymic insulin expression. This was despite virtually indistinguishable blood insulin levels, T cell subset percentages, and TCR V{beta} family usage, indicating that reduced thymic insulin expression does not impact T cell development on a global scale. Rather, we propose that it allows the thymic escape of insulin-reactive HLA-B*39:06-restricted T cells which participate in {beta} cell destruction. We also found that in mice expressing either HLA-B*39:06 or HLA-A*02:01 in the absence of murine class I MHC, HLA transgene identity alters TCR V{beta} usage, which may contribute to varying diabetogenic CD8 T cell repertoires in the presence of different HLA class I alleles.

immunology↗