Biology subjects

Pandey, M.

Publications and source records attributed to Pandey, M..

36 records · Page 2Linked to original sources

bioRxiv2024

Phosphoglucomutase A mediated regulation of carbon flux is essential for antibiotic and disease persistence in Mycobacterium tuberculosis

The long-term survival of Mtb mandates judicious utilization of the available resources inside the host. Uninterrupted access to host-derived nutrients holds the key to the success of Mtb. Phosphoglucomutase enzyme besides synthesizing glycogen, which serves as a nutrient reservoir, also helps modulate the carbon flux in different pathogens. Studies on the role of glycogen metabolism in disease progression, reactivation, and drug susceptibility in tuberculosis are severely lacking. To investigate this, we generated an Mtb strain ({Delta}pgmA) devoid of the gene that encodes for the enzyme phosphoglucomutase A (pgmA). The absence of pgmA impedes the ability of the pathogen to survive under nutrient-limiting and reactivation conditions. In the current study, we demonstrate that the absence of cell membrane-associated glycolipids in {Delta}pgmA compromised the cell wall integrity and increased the susceptibility of {Delta}pgmA to various stresses. Interestingly, in comparison to the wild type, low cAMP levels in {Delta}pgmA imparted an enhanced growth phenotype on cholesterol. Differential gene expression and carbon flux analysis suggest that stored carbon in the form of glycogen is essential for the survival of Mtb under nutrient-limiting conditions. Finally, we demonstrate that the pgmA gene of Mtb is essential for the growth of Mtb inside the host. Overall, this study unveils the significance of pgmA-mediated regulation of membrane glycolipids and its implication on antibiotic and disease persistence in tuberculosis. Additionally, information derived from this study will help design anti-TB strategies that are novel, short, and more efficient.

microbiology↗

bioRxiv2024

Streptococcus pyogenes pharyngitis elicits diverse antibody responses to key vaccine antigens influenced by the imprint of past infections.

Knowledge gaps regarding human immunity to Streptococcus pyogenes have impeded vaccine development. To address these gaps and evaluate vaccine candidates, we established a human challenge model of S. pyogenes pharyngitis. Here, we analysed antibody responses in serum and saliva against 19 antigens to identify characteristics distinguishing 19 participants who developed pharyngitis and 6 who did not. Pharyngitis elicited serum IgG responses to key vaccine antigens and a muted mucosal IgA response, whereas the 6 participants without pharyngitis had more pronounced IgA responses and minimal IgG responses. Serum IgG responses to pharyngitis in adult participants resembled those observed in children and were inversely correlated with the magnitude of pre-existing responses. While a straightforward correlate of protection was not evident, baseline antibody signatures distinguished clinical and immunological outcomes following experimental challenge. This highlights the influence of a complex humoral imprint from previous exposure, relevant for interpreting immunogenicity in forthcoming vaccine trials.

immunology↗

bioRxiv2024

Current limitations in predicting mRNA translation with deep learning models

BackgroundThe design of nucleotide sequences with defined properties is long-standing problem in bioengineering. An important application is protein expression, be it in the context of research or the production of mRNA vaccines. The rate of protein synthesis depends on the 5 untranslated region (5UTR) of the mRNAs, and recently, deep learning models were proposed to predict the translation output of mRNAs from the 5UTR sequence. At the same time, large data sets of endogenous and reporter mRNA translation have become available. ResultsIn this study we use complementary data obtained in two different cell types to assess the accuracy and generality of currently available models of translation. We find that while performing well on the data sets on which they were trained, deep learning models do not generalize well to other data sets, in particular of endogenous mRNAs, which differ in many properties from reporter constructs. ConclusionsThese differences limit the ability of deep learning models to uncover mechanisms of translation control and to predict the impact of genetic variation. We suggest directions that combine high-throughput measurements and machine learning to unravel mechanisms of translation control and improve construct design.

bioinformatics↗

bioRxiv2023

PROTACable is an Integrative Computational Pipeline of 3-D Modeling and Deep Learning to Automate the De Novo Design of PROTACs

Proteolysis-targeting chimeras (PROTACs) that engages two biological targets at once is a promising technology in degrading clinically relevant protein targets. Since factors that influence the biological activities of PROTACs are more complex than those of a small molecule drug, we explored a combination of computational chemistry and deep learning strategies to forecast PROTAC activity and enable automated design. A new method named PROTACable was developed for de novo design of PROTACs, which includes a robust 3-D modeling workflow to model PROTAC ternary complexes using a library of E3 ligase and linker and an SE(3)-equivariant graph transformer network to predict the activity of newly designed PROTACs. PROTACable is available at https://github.com/giaguaro/PROTACable/.

bioinformatics↗

bioRxiv2023

Disruption of IL-17 signaling in the respiratory mucosa results in invasive streptococcal infection

Streptococcus pyogenes infection of the upper respiratory tract and skin can lead to severe invasive streptococcal disease (ISD). Previous studies have demonstrated that the deficiency of IL-17 in mice (IL-17-/-) reduces mucosal immunity against S. pyogenes. However, the impact of IL-17 deficiency on the development of ISD is unknown. Here, we model single or repeated non-lethal, intranasal (IN) S. pyogenes M1 strain infections in immunocompetent and IL-17-/-mice to assess bacterial dissemination following a final IN or skin challenge. Immunocompetent mice that received a single S. pyogenes IN infection displayed long-lasting mucosal immunity and no systemic infection. However, in the absence of IL-17, a single IN infection resulted in the dissemination of S. pyogenes to the spleens, which was further exacerbated by repeated IN infections. Interestingly, immunity following skin challenge did not show a correlation with IL-17 and was instead associated with the activation of germinal center responses and the accumulation of neutrophils in the spleen. Our results highlight the critical role of IL-17 in preventing ISD following S. pyogenes infection of the respiratory mucosa.

immunology↗

bioRxiv2023

Hypothetical gene Rv0495c regulates redox homeostasis in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) has evolved sophisticated surveillance mechanisms to regulate and neutralize redox imbalances and associated lethal consequences. Failing this, the accumulated ROS induces toxicity by oxidizing a variety of biological molecules including proteins, nucleic acids and lipids. In the present study we identified Mtbs Rv0495c gene as an important regulator of oxidized cytosolic environment. Compared to wild type Mtb strain lacking the Rv0495c gene, {Delta}Rv0495c, had increased ROS and NAD+/NADH ratio creating a highly oxidized intracellular environment. {Delta}Rv0495c strain demonstrated slow growth phenotype under in vitro and ex-vivo growth conditions and demonstrated enhanced susceptibility to drugs, oxidative, nitrosative and hypoxic growth conditions. In addition, the increase in the superoxide radicals triggered a Fenton-like reaction rendering the {Delta}Rv0495c susceptible to free iron. The increase in the intracellular ROS levels of the {Delta}Rv0495c was further corroborated by an increase in the expression of proteins involved in antioxidant defense and enhanced ROS-mediated oxidation and degradation of mycobacterial lipids. This superoxide-induced lipid degradation resulted in altered colony morphology and loss of membrane integrity in the {Delta}Rv0495c. Surprisingly, despite showing a growth defect phenotype in an ex-vivo macrophage infection model, the absence of the Rv0495c gene in Mtb enhanced the pathogenicity and augmented the ability of the Mtb to grow inside the host. Gene expression analysis revealed a Rv0495c mediated immunomodulation of the host controls inflammation and helps creates a favorable niche for long-term survival of Mtb inside the host. In summary, the current study underscores the fact that the truce in the war between the host and the pathogen favors long-term disease persistence in tuberculosis. We believe targeting Rv0495c could potentially be explored as a strategy to potentiate the current anti-TB regimen.

microbiology↗

bioRxiv2023

VapC12 ribonuclease toxin modulates host immune response during Mycobacterium tuberculosis infection

Mechanistic understanding of antibiotic persistence is a prerequisite in controlling the emergence of MDR cases in Tuberculosis (TB). We have reported that the cholesterol-induced activation of VapC12 ribonuclease is critical for disease persistence in TB. In this study, we observed that relative to the wild type, mice infected with {Delta}vapC12 induced a proinflammatory response, had a higher pathogen load, and responded better to the anti-TB treatment. In a high-dose infection model, all the mice infected with {Delta}vapC12 succumbed early to the disease. Finally, we reported that the above phenotype of {Delta}vapC12 was dependent on the presence of the TLR4 receptor. Overall, the data suggest that the inability of {Delta}vapC12 to resolve neutrophil-mediated inflammation reduced bacterial killing by altering the T-cell response. In conclusion, our findings suggest the role of the VapC12 toxin in modulating the hosts innate immune response in ways that favor the long-term survival of the pathogen inside the host.

microbiology↗

bioRxiv2022

MAP Kinase and mammalian target of rapamycin are main pathways of gallbladder carcinogenesis: Results from bioinformatic analysis of Next Generation Sequencing data from a hospital-based cohort.

BackgroundGallbladder Cancer (GBC) is one of the most common cancers of the biliary tract and the third commonest gastrointestinal (GI) malignancy worldwide. The disease is characterized by the late presentation and poor outcome despite treatment, and hence, newer therapies and targets need to be identified. MethodsThe current study investigated various functionally enriched pathways in GBC pathogenesis involving the genes identified through Next Generation Sequencing (NGS). The Pathway enrichment analysis and Gene Ontology (GO) were carried out after NGS, followed by the construction of the protein-protein interaction (PPI) network to discover associations among the genes. ResultsOf the thirty-three patients with GBC who were screened through next-generation sequencing (NGS), 27somatic mutations were identified. These mutations involved a total of 14 genes. The p53 and KRAS were commonly found to be mutated, while mutations in other genes were seen in one case each, the mean number of mutations were 1.2, and maximum mutation in a single case (eight) was seen in one case. The bioinformatics analysis identified MAP kinase, PI3K-AKT, EGF/EGFR, and Focal Adhesion PI3K-AKT-mTOR signaling pathways and cross-talk between these. ConclusionThe results suggest that the complex crosstalk between the mTOR, MAPK, and multiple interacting cell signaling cascades can promote GBC progression, and hence, mTOR - MAPK targeted treatment will be an attractive option.

cancer biology↗

bioRxiv2022

Ligand Binding Prediction using Protein Structure Graphs and Residual Graph Attention Networks

MotivationComputational prediction of ligand-target interactions is a crucial part of modern drug discovery as it helps to bypass high costs and labor demands of in vitro and in vivo screening. As the wealth of bioactivity data accumulates, it provides opportunities for the development of deep learning (DL) models with increasing predictive powers. Conventionally, such models were either limited to the use of very simplified representations of proteins or ineffective voxelization of their 3D structures. Herein, we present the development of the PSG-BAR (Protein Structure Graph -Binding Affinity Regression) approach that utilizes 3D structural information of the proteins along with 2D graph representations of ligands. The method also introduces attention scores to selectively weight protein regions that are most important for ligand binding. ResultsThe developed approach demonstrates the state-of-the-art performance on several binding affinity benchmarking datasets. The attention-based pooling of protein graphs enables identification of surface residues as critical residues for protein-ligand binding. Finally, we validate our model predictions against an experimental assay on a viral main protease (Mpro)- the hallmark target of SARS-CoV-2 coronavirus. AvailabilityThe code for PSG-BAR is made available at https://github.com/diamondspark/PSG-BAR Contactacherkasov@prostatecentre.com

bioinformatics↗

bioRxiv2022

A phasevarion controls multiple virulence traits, including expression of vaccine candidates, in Streptococcus pneumoniae

Streptococcus pneumoniae is the most common cause of bacterial illness worldwide. Current vaccines based on the polysaccharide capsule (PCV-13 and PPSV-23) are only effective against a limited number of the >100 capsular serotypes. A universal vaccine based on conserved protein antigens requires a thorough understanding of gene expression in S. pneumoniae. Restriction-Modification (R-M) systems, classically described as a defence against bacteriophage, are almost ubiquitous in the bacterial domain, and roles other than phage defence. All S. pneumoniae strains encode the SpnIII R-M system. This system contains a phase-variable methyltransferase that randomly switches specificity, and controls expression of multiple genes; a phasevarion. We aimed to determine the role of the SpnIII phasevarion during pneumococcal pathobiology and determine if phase-variation resulted in differences in expression of protein antigens that are being investigated as vaccine candidates. Using locked S. pneumoniae strains that express a single SpnIII methyltransferase specificity, we found significant differences in clinically relevant traits, including survival in blood, and adherence to and invasion of human cells. Crucially, we also observed differences in expression of numerous proteinaceous vaccine candidates, which complicates selection of protein antigens for inclusion in a universal protein-based pneumococcal vaccine. This study will inform future vaccine design against S. pneumoniae by ensuring only stably expressed candidates are included in a rationally designed vaccine. Significance StatementS. pneumoniae is the worlds foremost bacterial pathogen. S. pneumoniae encodes a randomly expressed epigenetic regulator, a phasevarion (phase-variable regulon), that results in random expression of multiple genes. Previous work demonstrated that the pneumococcal SpnIII phasevarion switches between six different expression states, generating six unique phenotypic variants in a pneumococcal population. Here, we show that this phasevarion generates multiple phenotypic differences relevant to pathobiology. Importantly, expression of conserved protein antigens varies with phasevarion switching. As capsule expression, a major pneumococcal virulence factor, is also controlled by the phasevarion, our work will inform the selection of the best candidates to include in a rationally designed, universal pneumococcal vaccine.

microbiology↗

bioRxiv2020

miR-125-chinmo pathway regulates dietary restriction dependent enhancement of lifespan in Drosophila

Dietary restriction (DR) extends healthy lifespan in diverse species. Age and nutrient-related changes in the abundance of microRNAs (miRNAs) and their processing factors have been linked to organismal longevity. However, the mechanisms by which they modulate lifespan and the tissue-specific role of miRNA mediated networks in DR dependent enhancement of lifespan remains largely unexplored. We show that two neuronally enriched and highly conserved microRNAs, miR-125 and let-7 mediate the DR response in Drosophila melanogaster. Functional characterization of miR-125 demonstrates its role in neurons while its target chinmo acts both in neurons and the fat body to modulate fat metabolism and longevity. Proteomic analysis revealed that Chinmo exerts its DR effects by regulating expression of FATP, CG2017, CG9577, CG17554, CG5009, CG8778, CG9527 and FASN1. Our findings identify miR-125 as a conserved effector of DR pathway and open up the avenue for this small RNA molecule and its downstream effectors to be considered as potential drug candidates for treatment of late-onset diseases and biomarkers for healthy aging in humans.

molecular biology↗

bioRxiv2020

Flagellar targeting of an arginine kinase requires the conserved Lipidated Intraflagellar Transport (LIFT) pathway in Trypanosoma brucei

Both intraflagellar transport (IFT) and lipidated intraflagellar transport (LIFT) pathways are essential for cilia/flagella biogenesis, motility and sensory functions. In the LIFT pathway, lipidated cargoes are transported into the cilia through the coordinated actions of cargo carrier proteins such as Unc119 or PDE6{delta}, as well as small GTPases Arl13b and Arl3 in the cilium. Our previous studies revealed a single Arl13b ortholog in the evolutionarily divergent Trypanosoma brucei. TbArl13 catalyses two TbArl3 homologs, TbArl3A and TbArl3C, suggesting the presence of a conserved LIFT pathway in these protozoan parasites. Only a single homolog to the cargo carrier protein Unc119 was identified in T. brucei genome, but its function in lipidated protein transport has not been characterized. In this study, we exploited the proximity-based biotinylation approach to identify binding partners of TbUnc119. We showed that TbUnc119 binds to a flagellar arginine kinase TbAK3 in a myristoylation-dependent manner and is responsible for its targeting and enrichment in the flagellum. Interestingly, only TbArl3A, but not TbArl3C interacts with TbUnc119 in a GTP-dependant manner, suggesting functional specialization of Arl3-GTPases in T. brucei. This study establishes the function of TbUnc119 as a myristoylated cargo carrier and supports the presence of a conserved LIFT pathway in T. brucei.

cell biology↗

bioRxiv2020

Effect of dioxins in milk on 3D-cultured primary buffalo granulosa cells, a pilot study for a prospective RT-LAMP colour reaction for dioxin toxicity

Dioxins are highly toxic environmental persistent organic pollutants. In several countries, their presence was also reported in cow and human milk samples in the range of 0.023-26.46 and 0.88-19.0 pg/gm of fat, respectively. The detection of dioxns in food samples has been relied on several expensive technologies, which do not represent their toxic effects on consumers. However, mammalian cell based bioassays have such potential to detect the toxins, while representing their toxic effects. Therefore, we tried a three-dimensionally (3D) cultured buffalo granulosa cell based RT-LAMP colour reaction for detecting the presence of added model dioxin, 2,3,7,8-tetrachlorodibenzodioxin (TCDD), in commercial milk. The 3D spheroids on the fifth day of culture were treated with different concentrations of TCDD (i.e. 0.02- 20 pg/ml) directly as well as indirectly through milk fat. After 24hrs of treatment, gene expression studies were performed on certain granulosa cell-specific (CYP19A1, ER-beta, FSHR and LHR) and selective TCDD-responsive (CYP1A1, CYP1B1 and AHR) genes to identify the potential dioxin responsive gene for further RT-LAMP reaction. As the AHR expression in 3D cultured buffalo granulosa cells appears to be a potential gene marker for sensing the added TCDD in the milk, a colour based RT-LAMP reaction was successfully attempted for its expression. However, future studies are needed to develop a dose-responsive colour reaction by considering the treatment time less than 24 hrs.

pharmacology and toxicology↗

bioRxiv2019

Host cholesterol dependent activation of VapC12 toxin enriches persister population during Mycobacterium tuberculosis infection

A worldwide increase in the frequency of multidrug-resistant and extensively drug-resistant cases of tuberculosis is mainly due to therapeutic noncompliance associated with a lengthy treatment regimen. Depending on the drug susceptibility profile, the treatment duration can extend from 6 months to 2 years. This protracted regimen is attributed to a supposedly non-replicating and metabolically inert subset of the Mycobacterium tuberculosis (Mtb) population, called persisters. The mechanism underlying stochastic generation and enrichment of persisters is not fully known. We have previously reported that the utilization of host cholesterol is essential for mycobacterial persistence. In this study, we have demonstrated that cholesterol-induced activation of a ribonuclease toxin (VapC12) inhibits translation by targeting proT tRNA in Mtb. This results in cholesterol-specific growth modulation that increases the frequency of the generation of persisters in a heterogeneous Mtb population. Also, a null mutant strain of this toxin ({Delta}vapC12) failed to persist and demonstrated an enhanced growth phenotype in a guinea pig model of Mtb infection. Thus, we have identified a novel strategy through which cholesterol-specific activation of a toxin-antitoxin (TA) module in Mtb enhances persister formation during infection. In addition to identifying the mechanism, the study provides opportunity for targeting persisters, a new paradigm facilitating tuberculosis drug development.

microbiology↗

bioRxiv2019

Deamidation in Moxetumomab Pasudotox Leading to Conformational Change and Immunotoxin Activity Loss

Asparagine deamidation is a common posttranslational modification in which asparagine is converted to aspartic acid or isoaspartic acid. By introducing a negative charge, deamidation could potentially impact the binding interface and biological activities of protein therapeutics. We identified a deamidation variant in moxetumomab pasudotox, an immunotoxin Fv fusion protein drug derived from a 38-kilodalton (kDa) truncated Pseudomonas exotoxin A (PE38) for the treatment of hairy-cell leukemia. Although the deamidation site, Asn-358, was outside of the binding interface, the modification had a significant impact on the biological activity of moxetumomab pasudotox. Surprisingly, the variant eluted earlier than its unmodified form on anion exchange chromatography, which suggests a higher positive charge. Here we describe the characterization of the deamidation variant with differential scanning calorimetry and hydrogen-deuterium exchange mass spectrometry, which revealed that the Asn-358 deamidation caused the conformational changes in the catalytic domain of the PE38 region. These results provide a possible explanation for why the deamidation affected the biological activity of moxetumomab pasudotox and suggest an approach that can be used for process control to ensure product quality and process consistency.\n\nStatement of SignificanceAsparagine deamidation can have a potentially significant impact on protein therapeutics. Previous studies have revealed that deamidation at a single site significantly reduces the biological activity of moxetumomab pasudotox, a recombinant anti-CD22 immunotoxin developed for the treatment of B-cell malignancies. Surprisingly, despite the fact that deamidation introduced a negative charge, the deamidated variant eluted earlier than its unmodified form on anion exchange chromatography. In order to understand these observations, we isolated the deamidated variant using an anion exchange column and characterized it by differential scanning calorimetry and hydrogen-deuterium exchange mass spectrometry. The results revealed the conformational change caused by the deamidation, which explains the diminished biological activity of the variant and its early elution time on anion exchange chromatography.

biophysics↗

bioRxiv2019

Epidemiological consequences of enduring strain-specific immunity requiring repeated episodes of infection

Group A Streptococcus (GAS) skin infections are caused by a diverse array of strain types and are highly prevalent in Indigenous and other disadvantaged populations. The role of strain-specific immunity in preventing GAS infections is poorly understood, representing a critical knowledge gap in vaccine development. A recent GAS murine challenge study showed evidence that sterilising strain-specific and enduring immunity required two skin infections by the same GAS strain within three weeks. This mechanism of developing enduring immunity may be a significant impediment to the accumulation of immunity in populations.\n\nWe used a mathematical model of GAS transmission to investigate the epidemiological consequences of enduring strain-specific immunity developing only after two infections with the same strain within a specified interval. Accounting for uncertainty when correlating murine timeframes to humans, we varied this maximum inter-infection interval from 3 to 420 weeks to assess its impact on prevalence and strain diversity. Model outputs were compared with longitudinal GAS surveillance observations from northern Australia, a region with endemic infection. We also assessed the likely impact of a targeted strain-specific multivalent vaccine in this context.\n\nOur model produced patterns of transmission consistent with observations when the maximum inter-infection interval for developing enduring immunity was 19 weeks. Our vaccine analysis suggests that the leading multivalent GAS vaccine may have limited impact on the prevalence of GAS in populations in northern Australia if strain-specific immunity requires repeated episodes of infection.\n\nOur results suggest that observed GAS epidemiology from disease endemic settings is consistent with enduring strain-specific immunity being dependent on repeated infections with the same strain, and provide additional motivation for relevant human studies to confirm the human immune response to GAS skin infection.\n\nAuthor summaryGroup A Streptococcus (GAS) is a ubiquitous bacterial pathogen that exists in many distinct strains, and is a major cause of death and disability globally. Vaccines against GAS are under development, but their effective use will require better understanding of how immunity develops following infection. Evidence from an animal model of skin infection suggests that the generation of enduring strain-specific immunity requires two infections by the same strain within a short time frame. It is not clear if this mechanism of immune development operates in humans, nor how it would contribute to the persistence of GAS in populations and affect vaccine impact. We used a mathematical model of GAS transmission, calibrated to data collected in an Indigenous Australian community, to assess whether this mechanism of immune development is consistent with epidemiological observations, and to explore its implications for the impact of a vaccine. We found that it is plausible that repeat infections are required for the development of immunity in humans, and illustrate the difficulties associated with achieving sustained reductions in disease prevalence with a vaccine.

epidemiology↗

bioRxiv2019

Carbohydrate, glutathione, and polyamine metabolism are central to Aspergillus flavus oxidative stress responses over time

The primary and secondary metabolites of fungi are critical for adaptation to environmental stresses, host pathogenicity, competition with other microbes, and reproductive fitness. Drought-derived reactive oxygen species (ROS) have been shown to stimulate aflatoxin production and regulate development in Aspergillus flavus, and may function in signaling with host plants. Here, we have performed global, untargeted metabolomics to better understand the role of aflatoxin production in oxidative stress responses, and also explore isolate-specific oxidative stress responses over time. Two field isolates of A. flavus, AF13 and NRRL3357, possessing high and moderate aflatoxin production, respectively, were cultured in medium with and without supplementation with 15mM H2O2, and mycelia were collected following 4 and 7 days in culture for global metabolomics. Overall, 389 compounds were described in the analysis which were examined for differential accumulation. Significant differences were observed in both isolates in response to oxidative stress and when comparing sampling time points. The moderate aflatoxin-producing isolate, NRRL3357, showed extensive stimulation of antioxidant mechanisms and pathways including polyamines metabolism, glutathione metabolism, TCA cycle, and lipid metabolism while the highly aflatoxigenic isolate, AF13, showed a less vigorous response to stress. Carbohydrate pathway levels also imply that carbohydrate repression and starvation may influence metabolite accumulation at the later timepoint. Higher conidial oxidative stress tolerance and antioxidant capacity in AF13 compared to NRRL3357, inferred from their metabolomic profiles and growth curves over time, may be connected to aflatoxin production capability and aflatoxin-related antioxidant accumulation. The coincidence of several of the detected metabolites in H2O2-stressed A. flavus and drought-stressed hosts suggests their potential role in the interaction between these organisms and their use as markers/targets to enhance host resistance through biomarker selection or genetic engineering.\n\nAuthor SummaryAspergillus flavus is a fungal pathogen of several important crops including maize and peanut. This pathogen produces carcinogenic mycotoxins known as aflatoxins during infection of plant materials, and is particularly severe under drought stress conditions. This results in significant losses in crop value and poses a threat to food safety and security globally. To combat this, understanding how this fungus responds to environmental stresses related to drought can allow us to identify novel methods of mitigating aflatoxin contamination. Here, we analyzed the accumulation of a broad series of metabolites over time in two isolates of A. flavus with differing stress tolerance and aflatoxin production capabilities in response to drought-related oxidative stress. We identified several metabolites and mechanisms in A. flavus which allow it to cope with environmental oxidative stress and may influence aflatoxin production and fungal growth. These may serve as potential targets for selection in breeding programs for the development of new cultivars, or for alteration using genetic engineering approaches to mitigate excessive aflatoxin contamination under drought stress.

microbiology↗

bioRxiv2018

Experimental analysis of exome-scale mutational signature of glycidamide, the reactive metabolite of acrylamide

Acrylamide, a probable human carcinogen, is ubiquitously present in the human environment, with sources including heated starchy foods, coffee and cigarette smoke. Humans are also exposed to acrylamide occupationally. Acrylamide is genotoxic, inducing gene mutations and chromosomal aberrations in various experimental settings. Covalent haemoglobin adducts were reported in acrylamide-exposed humans and DNA adducts in experimental systems. The carcinogenicity of acrylamide has been attributed to the effects of glycidamide, its reactive and mutagenic metabolite capable of inducing rodent tumors at various anatomical sites. In order to characterize the pre-mutagenic DNA lesions and global mutation spectra induced by acrylamide and glycidamide, we combined DNA-adduct and whole-exome sequencing analyses in an established exposure-clonal immortalization system based on mouse embryonic fibroblasts. Sequencing and computational analysis revealed a unique mutational signature of glycidamide, characterized by predominant T:A>A:T transversions, followed by T:A>C:G and C:G>A:T mutations exhibiting specific trinucleotide contexts and significant transcription strand bias. Computational interrogation of human cancer genome sequencing data indicated that a combination of the glycidamide signature and an experimental benzo[a]pyrene signature are nearly equivalent to the COSMIC tobacco-smoking related signature 4 in lung adenocarcinomas and squamous cell carcinomas. We found a more variable relationship between the glycidamide- and benzo[a]pyrene-signatures and COSMIC signature 4 in liver cancer, indicating more complex exposures in the liver. Our study demonstrates that the controlled experimental characterization of specific genetic damage associated with glycidamide exposure facilitates identifying corresponding patterns in cancer genome data, thereby underscoring how mutation signature laboratory experimentation contributes to the elucidation of cancer causation.\n\nA 40-word summaryInnovative experimental approaches identify a novel mutational signature of glycidamide, a metabolite of the probable human carcinogen acrylamide. The results may elucidate the cancer risks associated with exposure to acrylamide, commonly found in tobacco smoke, thermally processed foods and beverages.

genomics↗