Biology subjects

Rath, A.

Publications and source records attributed to Rath, A..

8 recordsLinked to original sources

bioRxiv2026

Using Arabian Sea Coastal Water as a Model to Study Plasmid-mediated AMR Dissemination driven by Anthropogenic Impacts

Background Antimicrobial resistance (AMR) is a growing global public health threat. The water bodies act as both reservoirs and medium for dissemination of AMR. Coastal water driven by anthropogenic effects accumulate antibiotics, heavy metals, microplastics, and disinfectants which act as stressors, increasing the overall AMR. Therefore, it is necessary to understand the AMR entities and its transmission in coastal water bodies. Methods and Results Mumbai, mirroring most cosmopolitan cities of the world, was used as a model to understand how rapid urbanization disseminate AMR in coastal waters of the Arabian Sea and can pose substantial risk to human health. Bacteria isolated from coastal waters were screened and found to be multi-drug resistant. Several isolates carried multiple plasmids of high molecular weight (>10 kb). Plasmid sequencing and analysis using various bioinformatic tools identified plasmid replicons like IncFIA, IncFII, rep7a and repUS43. Analysis showed a high burden of antibiotic resistance genes (ARGs) conferring resistance to antibiotic classes such as monobactams (blaTEM-116), carbapenems (blaNDM-5), vancomycin (vanA), disinfectants and antiseptics (qacG). Additionally, clinically relevant mecA and mecR1 associated with methicillin-resistant Staphylococcus sp., rpsL associated with streptomycin resistant Mycobacterium tuberculosis and the colistin-resistance genes mcr-10.1 and mcr-10.4 were also detected. We also report for the first-time presence of a plasmid-borne mupB gene in coastal waters, that confers high resistance to mupirocin. Conclusions Despite the moderate sample size, the presence of diverse plasmid replicons and clinically important ARGs suggests urban coastal environment as a potent reservoir of AMR, with potential for global transmission through active waterway trade and transport.

bioinformatics↗

bioRxiv2026

Host response and cell fates of human U937 monocytes upon infection with vancomycin-resistant E. faecium

In recent years, Enterococcus faecium has become a high-priority human opportunistic pathogen, especially due to its antibiotic and environmental resistance, promoting hospital-acquired infections. However, its interaction with host cells remains poorly understood. In this study, we investigated the interaction of four recently collected, clinical vancomycin-resistant Enterococcus faecium (VREfm) isolates with the human U937 cell line. Early after infection, we observed close bacterial association with U937 monocytes and features suggestive of internalization. We thus investigated host cell fates after washing at 2 hours post infection (hpi) to concentrate on effects induced by internalized bacteria or those with direct host cell contact. Whereas viability was not impaired at 6 hpi, moderate cell death was induced to an isolate-specific extent at 24 hpi, which was abrogated when direct host-pathogen contact as prevented via boyden chamber cell culture inserts, but was enhanced by TNF-R1 deficiency. Host mRNA sequencing revealed a consistent pattern of differentially expressed genes across all VREfm isolates, suggesting a shared innate immune and stress-related response at 6 hpi, with functionally enriched biological processes related to leukocyte activation, cytokine signaling, programmed cell death, and RNA stability. Together, these data show that VREfm triggers an early pro-inflammatory transcriptional response in U937 monocytes without immediate cytotoxicity in U937 monocytes, while late-stage isolate-specific cell death induction depends on direct host cell contact or internalization and involves TNF-R1 mediated regulation. Our findings may assist future efforts to understand how VREfm triggers host cell damage that contributes to severe inflammation and systemic infection such as persistent bacteremia.

microbiology↗

bioRxiv2025

Comparing AI and Human Mentoring in Supporting Dental Student Well-being: A Mixed-Methods Study

BackgroundDental education is associated with high psychological distress among students, exacerbated by intensive clinical training and variable institutional support. Traditional mentoring programs mitigate such challenges, yet faculty shortages and resource constraints necessitate exploration of alternative models. Artificial intelligence (AI) platforms such as ChatGPT offer potential supplemental mentoring support; however, their comparative effectiveness in health professions education remains underexplored. MethodsThis comparative, convergent mixed-methods study was conducted at SEGi University, Malaysia, involving 31 Year 4 dental students receiving human mentoring and 36 Year 5 students utilizing ChatGPT as an AI mentor. Quantitative outcomes were assessed using the Depression, Anxiety, and Stress Scale (DASS-21), the Perceived Mentor Support Scale (PMSS), and the Mentoring Effectiveness Scale (MES). Qualitative data were collected through focus groups and analysis of ChatGPT interaction logs. Statistical analyses included paired t-tests and ANOVA, while thematic analysis was used for qualitative data integration. ResultsYear 4 students demonstrated significant reductions in stress, anxiety, and depression scores (p < 0.001) and greater improvements in perceived mentor support (p < 0.001) and mentoring effectiveness (p < 0.001) compared to Year 5 students. Year 5 students reported modest gains following ChatGPT mentoring, highlighting strengths in accessibility and immediacy but limitations in emotional resonance. Qualitative themes underscored human mentors superior capacity for emotional attunement, while ChatGPT was valued for availability and practical support. ConclusionsWhile AI-based mentoring via ChatGPT shows promise in enhancing mentoring accessibility, human mentorship remains critical for addressing the emotional and relational needs of dental students. Careful integration of AI tools into hybrid mentorship models could optimize support systems, particularly in resource-constrained educational environments. Future research should explore ethical frameworks and training strategies to enhance empathetic AI mentorship in health professions education.

scientific communication and education↗

bioRxiv2024

Interplay of ferroptotic and apoptotic cell death and its modulation by BH3-mimetics

Ferroptosis and apoptosis are widely considered to be independent cell death modalities. Ferroptotic cell death is a consequence of insufficient radical detoxification and progressive lipid peroxidation, which is counteracted by glutathione peroxidase 4 (GPX4). Apoptotic cell death can be triggered by a wide variety of stresses, including oxygen radicals, and can be suppressed by anti-apoptotic members of the BCL-2 protein family. Mitochondria are the main interaction site of BCL-2 family members and likewise a major source of oxygen radical stress. We therefore studied if ferroptosis and apoptosis might intersect and possibly interfere with one another. Indeed, cells dying from impaired GPX4 activity displayed hallmarks of both ferroptotic and apoptotic cell death, with the latter including (transient) membrane blebbing, submaximal cytochrome-c release and caspase activation. Targeting BCL-2, MCL-1 or BCL-XL with BH3-mimetics under conditions of moderate ferroptotic stress in many cases synergistically enhanced overall cell death and frequently skewed primarily ferroptotic into apoptotic outcomes. Surprisingly though, in other cases BH3-mimetics, most notably the BCL-XL inhibitor WEHI-539, counter-intuitively suppressed cell death and promoted cell survival following GPX4 inhibition. Further studies revealed that most BH3-mimetics possess previously undescribed antioxidant activities that counteract ferroptotic cell death at commonly employed concentration ranges. Our results therefore show that ferroptosis and apoptosis can intersect. We also show that combining ferroptotic stress with BH3-mimetics, context-dependently can either enhance and convert cell death outcomes between ferroptosis and apoptosis or can also suppress cell death by intrinsic antioxidant activities.

cancer biology↗

bioRxiv2021

A calibrated cell-based functional assay to aide classification of MLH1 DNA mismatch repair gene variants

PURPOSEFunctional assays provide important evidence for classifying the disease significance of germline variants in the DNA mismatch repair genes. We sought to develop a cell-based approach for testing the function of variants of uncertain significance (VUS) in the MLH1 gene. METHODSUsing CRISPR gene editing, we knocked-in MLH1 VUS into the endogenous MLH1 loci in human embryonic stem cells. We examined their impact at the RNA and protein level, including their ability to maintain stability of microsatellite sequences and instigate a DNA damage response. We calibrated these assays by testing well-established pathogenic and benign control variants. RESULTSFive VUS resulted in functionally abnormal protein, 15 VUS resulted in functionally normal protein, and one VUS showed mixed results. Furthermore, we converted the functional outputs into a single odds in favor of pathogenicity score for each VUS. CONCLUSIONOur CRISPR-based functional assay successfully models phenotypes observed in patients in a cellular context. Using this approach, we generated evidence for or against pathogenicity for utilization by variant classification expert panels. Ultimately, this information will assist in proper diagnosis and disease management for suspected Lynch syndrome patients.

cancer biology↗

bioRxiv2020

Characterization of novel pollen-expressed transcripts reveals their potential roles in pollen heat stress response in Arabidopsis thaliana.

The male gametophyte is the most heat-sensitive of all plant tissues. In recent years, long noncoding RNAs (lncRNAs) have emerged as important components of cellular regulatory networks involved in most biological processes, including response to stress. While examining RNAseq datasets of developing and germinating Arabidopsis thaliana pollen exposed to heat stress (HS), we identified 66 novel and 246 recently-annotated intergenic expressed loci (XLOCs) of unknown function, with the majority encoding lncRNAs. Comparison to HS in cauline leaves and other RNAseq experiments, indicated 74% of the 312 XLOCs are pollen-specific, and at least 42% are HS-responsive. Phylogenetic analysis revealed 96% of the genes evolved recently in Brassicaceae. We found that 50 genes are putative targets of microRNAs, and that 30% of the XLOCs contain small open reading frames (ORFs) with homology to protein sequences. Finally, RNAseq of ribosome-protected RNA fragments together with predictions of periodic footprint of the ribosome P-sites indicated that 23 of these ORFs are likely to be translated. Our findings indicate that many of the 312 unknown genes might be functional, and play significant role in pollen biology, including the HS response.

plant biology↗

bioRxiv2019

Casilio-ME: Enhanced CRISPR-based DNA demethylation by RNA-guided coupling methylcytosine oxidation and DNA repair pathways

We have developed a methylation editing toolbox, Casilio-ME, that enables not only RNA-guided methylcytosine editing by targeting TET1 to genomic sites, but also by co-delivering TET1 and protein factors that couple methylcytosine oxidation to DNA repair activities, and/or promote TET1 to achieve enhanced activation of methylation-silenced genes. Delivery of TET1 activity by Casilio-ME1 robustly altered the CpG methylation landscape of promoter regions and activated methylation-silenced genes. We augmented Casilio-ME1 to simultaneously deliver the TET1-catalytic domain and GADD45A (Casilio-ME2) or NEIL2 (Casilio-ME3) to streamline removal of oxidized cytosine intermediates to enhance activation of targeted genes. Using two-in-one effectors or modular effectors, Casilio-ME2 and Casilio-ME3 remarkably boosted gene activation and methylcytosine demethylation of targeted loci. We expanded the toolbox to enable a stable and expression-inducible system for broader application of the Casilio-ME platforms. This work establishes an advanced platform for editing DNA methylation to enable transformative research investigations interrogating DNA methylomes.

genomics↗

bioRxiv2018

Functional Interrogation of Lynch Syndrome Associated MSH2 Missense Variants Using CRISPR-Cas9 Gene Editing in Human Embryonic Stem Cells

Lynch syndrome (LS) is a hereditary cancer predisposition condition caused by inactivating germline mutations in one of the DNA mismatch repair (MMR) genes. Identifying a deleterious germline mutation by DNA sequencing is important for confirming an LS diagnosis. Frameshift and nonsense mutations significantly alter the protein product and likely impair MMR function. However, the implication of a missense mutation is often difficult to interpret. Referred to as variants of uncertain significance (VUS), their discovery hampers the definitive LS diagnosis. To determine the pathogenic significance of a VUS it is helpful to know its impact on protein function. Functional studies in the test tube and in cellular models have been performed for some VUS, however, these studies have been limited by the artificial nature of the assays. We report here an improved functional assay in which we engineered site-specific MSH2 VUS using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 gene editing in human embryonic stem cells. This approach introduces the variant into the endogenous MSH2 loci, while simultaneously eliminating the wild-type gene. We then characterized the impact of the variants on cellular MMR functions including DNA damage response signaling upon challenge with a DNA alkylating agent and the repair of DNA microsatellites. We classified the MMR functional capability of 8 of 10 VUS under study providing valuable information for determining their likelihood of being bona fide LS mutations. This improved human cell-based assay system for functionally testing MMR gene VUS will facilitate the identification of high risk LS patients.\n\nSignificance StatementUnderstanding how cancer-associated missense variants in MMR genes affect function helps determine whether they truly contribute to disease. Laboratory assays previously utilized are limited by their artificial nature. To improve this, we introduced variants directly into the endogenous MMR loci in hESCs using CRISPR-Cas9 gene editing. This approach allows us to assess each variant while being expressed by its normal regulatory elements in a cellular environment. Our results will help guide the management of patients world-wide who carry these variants. At the same time, this study provides a technical road map for assessing the functional effects of all LS-associated variants, as well as variants linked to other genetic diseases where a cell-based functional assay is available.

genomics↗