Biology subjects

Ahmed, K.

Publications and source records attributed to Ahmed, K..

15 recordsLinked to original sources

bioRxiv2026

Unbiased Long-Read Whole-Genome Sequencing Enables High-Resolution Mapping of Transgene Concatenation and Off-target Genomic Disruption in a Mouse Model

Transgenic mouse models are indispensable for dissecting disease mechanisms; yet, their interpretability is frequently compromised by cryptic genomic alterations introduced during transgenesis. Thus, robust quality control strategies are needed to elucidate integration architecture and evaluate model performance when such unintended events occur. Here, we applied unbiased whole-genome long-read sequencing using the PacBio Revio to investigate a mouse model exhibiting unexpected transgene silencing, originally designed to recapitulate autosomal-dominant hereditary macular dystrophy driven by upregulation of a ZZEF1-ALOX15 fusion gene. Long-read sequencing analysis revealed a [≥]29-kb head-to-tail concatemer containing more than three copies of the transgene vector. Reconstruction of transgene-genome junctions revealed off-target integration of the concatemer into the calcium-sensing receptor gene (Casr), along with exogenous E. coli DNA, that together defined final transgene architecture. 5-methylcytosine profiling identified hypermethylation of the transgene promoter and additional phenotyping indicated disruption of endogenous Casr function resulting from the rearrangement. Our workflow enabled direct detection of transgene concatenation and off-target mapping. These findings establish long-read sequencing as a powerful and scalable quality control standard for genetically engineered animal models, uniquely capable of uncovering hidden genomic complexity, resolving aberrant phenotypes, and enhancing the reliability of in vivo disease modelling.

genomics↗

bioRxiv2026

Omics-analyses of Fermented Onion pickle in Shaping Gut Microbiota and Immune Response in Women: A Community-Based Trial in Pakistan

A fermented-food intervention trial conducted in Pakistan suggested beneficial changes in the composition of the gut microbiota in healthy women. Using a subset (n=17) of the same participants, this study further investigates the impact of fermented food (onion pickle) on gene expression levels using RNA transcriptomics, with a focus on host-microbiome interactions. After consuming pickles (50g/day) for eight weeks, blood and stool samples of participants were collected at baseline and post-intervention to assess inflammatory markers, 16S rRNA gene sequencing, clinical parameters, and RNA sequencing. Among inflammatory biomarkers, lipocalin (LCN-2) levels significantly decreased (pre=86.5{+/-}80.1ng/mL, post=61.0{+/-}59.0 ng/mL, p=0.04, paired T-test). Additionally, the intervention downregulated pathways (p<0.05) involved host responses to microbial stimuli, including response to bacterial origin, chemotaxis, and response to lipopolysaccharide. In gut microbiota, observed -diversity significantly increased post-intervention (p=0.02). Linear discriminant analysis effect size (LEfSe) revealed differential expressions (LDA [≥] 2.0) of Olsenella and Coriobacteriales at week-8, where Olsenella sp. showed a significant negative correlation with LCN-2 (R=-0.36, p<0.05, Spearmans correlation). These findings suggest that fermented onion pickle consumption for eight weeks modestly alters gut microbial diversity and composition and is associated with reduced inflammatory markers and altered host immune-related gene expression, potentially improving intestinal health. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/711246v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@18679e0org.highwire.dtl.DTLVardef@1ab62eeorg.highwire.dtl.DTLVardef@ec318forg.highwire.dtl.DTLVardef@a9b94c_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

A cross model spatial and single-cell atlas reveals the conserved involvement of osteopontin in polycystic kidney disease

Polycystic kidney disease (PKD) arises from mutations in cilia-associated genes, such as PKD1 and PKD2, expressed in renal epithelial cells, leading to progressive kidney dysfunction and end- stage kidney disease (ESKD). PKD patients exhibit significant heterogeneity in disease progression, largely due to genetic and environmental modifiers. Like patients, mouse models of PKD also exhibit significant heterogeneity with regards to the gene mutated, age of disease onset, and rate of disease progression. To elucidate the cellular and molecular consequences of these variables, we constructed an integrated single-cell and spatial transcriptomics atlas across mouse models of PKD, mapping changes in cell type composition, gene expression, and intercellular signaling networks across the whole atlas and within individual models. Consistently across models, single cell RNA sequencing (scRNAseq) data revealed increased Spp1 (osteopontin) expression and signaling from PKD-enriched clusters to Ly6clo monocytes. Global deletion of Spp1 in Pkd1RC/RC mice resulted in reduced cyst severity, improved kidney function, and reduced Ly6clo monocyte numbers, suggesting that SPP1 signaling to Ly6clo monocytes promotes PKD progression. We also created a freely available, searchable website (https://bmblx.bmi.osumc.edu/scPKD/) that can be used to identify cross- and intra-model specific changes in gene expression, guiding researchers to new therapeutic targets for treating PKD.

bioinformatics↗

bioRxiv2025

The Role of SARS-CoV-2 Nucleocapsid Protein Persistence in Inducing Chronic Type I Interferon and Mitochondrial Dysfunction.

The potential mechanisms that link SARS-CoV-2 infection to post-acute sequelae of SARS-CoV-2 symptoms and ultimately transition to new onset of autoimmune disease remain poorly understood. Here, we report the consequences of SARS-CoV-2 nucleocapsid (N) protein persistence in the absence of detectable SARS-CoV-2 replication in otherwise healthy individuals, a set of systemic lupus erythematosus (SLE) patients, and a case of a 60-year-old man who developed a new onset of lupus nephritis seven months following mild COVID-19 disease. We have identified that N protein persistence in peripheral blood mononuclear cells (PBMC) did not correlate with detectable SARS-CoV-2 RNA in blood but is associated with significantly increased secretion of type I interferons (IFN) and presence of tubuloreticular structures in peripheral leukocytes. We further demonstrate that the N protein colocalizes in the mitochondrial fraction of PBMCs from individuals positive for N protein alongside mitochondrial antiviral signaling protein (MAVS). In vitro expression of the N protein in a monocytic cell line showed that the N protein itself was directly capable of interacting with MAVS in the absence of viral RNA, and this interaction was enhanced if the cell was exposed to oxidative stress. We show that the type I IFN signature in the presence of N protein expression was MAVS, but not STING signaling pathway-dependent. Our findings suggest a mechanism for the onset and promotion of type I IFN signature after COVID-19; in our model, the SARS-CoV-2 N protein can independently trigger sustained type I IFN production via direct activation of MAVS and its spontaneous oligomerization. This long-lasting type I IFN generation may create a chronic inflammatory milieu, favoring autoimmunity with SLE-like symptoms in susceptible individuals. One Sentence SummaryThe persistence of SARS-CoV-2 nucleocapsid in the absence of viral replication is associated with an induction of mitochondria-mediated inflammatory milieu, which can favor autoimmunity in susceptible individuals.

immunology↗

bioRxiv2025

Structural and Conformational Impact of Deleterious Spike Protein Mutations in SARS-CoV-2 Omicron Lineages

BackgroundA large number of mutations in the Spike (S) protein of the SARS-CoV-2 omicron variant have been noted to alter the receptor binding domain (RBD) and increase the binding surface and enhance the opening of the binding pocket. The cumulative effect of S1 and S2 subunit mutations can influence the conformational dynamics of the binding surface, facilitating the release of viral genome into host cells. AimThis study investigates the deleterious mutations across all Omicron lineages identified in our analysis and their effect on the conformational stability of RBD opening. MethodsWhole Genome Sequencing of 231 SARS-CoV-2 positive patients in Karachi, Pakistan, were performed using Illumina Miseq instrument and raw reads were analyzed using viralrecon pipeline. The mutational effects of omicron variant on the stability of S protein, including wild-type (7FG7), close (6VXX) and open (6VYB) states, were assessed through MD simulations. ResultsFour deleterious missense mutations (Tyr505His, Asn764Lys, Asp950Asn, Asn969Lys) were identified in the S1 and S2 subunit of the S protein of omicron variant. In the wildtype and open state mutant models, Tyr505His, Asp950Asn and Asn969Lys caused destabilizing effects, higher RMSDs vs. wild-type, and fluctuations in the RBD (438-510) region and S2 subunit (946-1010), compared to the native structure. These mutations increased the binding pocket propensity to open in mutant model compared to the native open conformation (6VYB). This structural change promoted trimer opening in the open state through -helix movement in the S2 subunit away from the RBD region. In the closed state, only S2 subunit mutations (Asp950Asn and Asn969Lys) lead to predicted destabilization through the movement of protomer C towards protomer B (RBD region). These S2 subunit mutations are predicted to stabilize the RBD "down" conformation potentially enhancing spike antigenic heterogeneity. ConclusionThis study highlighted the cumulative effect of S1 (Tyr505His) and S2 (Asp950Asn and Asn969Lys) subunits mutations on different S protein states, potentially controlling its conformational dynamics and presentation to host receptors. Future experimental studies are needed to elucidate the biological significance of these alterations, particularly by establishing a link between the identified mutations and their impact on viral biology.

bioinformatics↗

bioRxiv2024

Analysis of differential expression of matrix metalloproteins and defensins in the nasopharyngeal milieu of mild and severe COVID-19 cases

IntroductionA subset of COVID-19 disease patients suffers a severe form of the illness, however, underlying early pathophysiological mechanisms associated with the severe form of COVID-19 disease remain to be fully understood. Several studies showed the association of COVID-19 disease severity with the changes in the expression profile of various matrix metalloproteinases (MMPs) and defensins. However, the link between the changes in the expression of matrix metalloproteinase (MMPs) and defensins (DA) in the nasopharyngeal milieu, during early phases of infection, and disease severity remains poorly understood. Therefore, we performed differential gene expression analysis of matrix metalloproteinases (MMPs) and defensins in the nasopharyngeal swab samples collected from mild and severe COVID-19 cases within three days of infection and examined the association between MMP and DA expression and disease severity. Material and MethodA total of 118 previously collected nasopharyngeal samples from mild and severe COVID-19 patients (as per the WHO criteria) were used in this study. To determine the viral loads and assess the mRNA expression of matrix metalloproteinase (MMPs) and defensins, a real-time qPCR assay was used. To assess statistically significant differences in the mean expression of viral loads and the cytokines in between the severe and mild groups, an unpaired T-test was applied. The Pearson correlation test was used to assess the correlation between cytokine expressions. In addition, a multivariable logistic regression analysis was carried out with all the variables from the data set using severity as the outcome variable. ResultsOur results showed that the expression of DA3 and MMP2 to be considerably lower in the severe group than in the mild group. Furthermore, there was a significant association between MMP1 and DA4 and DA6 (r=0.5, p=0.0001); as well as between MMP7 and DA1 and DA6 (r=0.5, p=0.00). Additionally, the regression analysis shows a significant correlation (p 0.05) between MMP2 and the severity of COVID-19 disease. ConclusionThe early detection of changes in the expression of MMPs and defensins may act as a useful biomarker/predictor for possible severe COVID-19 disease, which may be useful in the clinical management of patients to reduce COVID-19-associated morbidity and mortality.

microbiology↗

bioRxiv2024

BCG activation of trained immunity is associated with induction of cross reactive COVID-19 antibodies in a BCG vaccinated population.

BackgroundDuring the current COVID-19 pandemic, the rate of morbidity and mortality was considerably lower in BCG vaccinated countries like Pakistan. BCG has been shown to provide cross protection to both disseminated TB as well as non related viral infections in BCG vaccinated children which is consistent with COVID-19 morbidity in the younger age group. Recently, this cross protection was attributed to trained immunity (TI) associated with BCG recall responses in the innate arm of the immune system. Little is known about the longevity of BCG Trained Immunity (TI) beyond early childhood. ObjectiveTo assess the BCG-induced recall responses in healthy individuals by cytokines secreted from the TI network and its potential role in providing cross-protection against COVID-19 and other viral infections. Study DesignIn this cross-sectional study, healthy young adults and adolescents (n=20) were recruited from 16-40 years of age, with no prior history of TB treatment, autoimmune, or chronic inflammatory condition. MethodsBCG-induced cytokine responses were assessed using prototypic markers for cells of the TI network {macrophages [M1 (TNF, IFN{gamma}), M2 (IL10)], NK (IL2), Gamma delta ({gamma}{delta}) T (IL17, IL4)} and SARS CoV2 IgG antibodies against RBD using short-term (12 hrs.) cultures assay. ResultsSignificant differences were observed in the magnitude of recall responses to BCG with macrophage cytokines showing the highest mean levels of TNF (9148 pg/ml) followed by IL10 (488 pg/ml) and IFN{gamma} (355 pg/ml). The ratio of unstimulated vs.BCG-stimulated cytokines was 132 fold higher for TNF, 40 fold for IL10, and 27 fold for IFN{gamma}. Furthermore, SARS-CoV-2 antibodies were also detected in unstimulated plasma which showed cross reactivity with BCG. ConclusionThe presence of cross reactive antibodies to SARS-CoV-2 and the relative ratio of pro-and anti-inflammatory cytokines secreted by activated TI cellular network may play a pivotal role in protection in the early stages of infection as observed during the COVID-19 pandemic in the younger age groups resulting in lower morbidity and mortality.

immunology↗

bioRxiv2023

Mesothelial cells exhibit characteristics of perivascular cells in an in vitro angiogenesis assay

Mesothelial cells have been shown to have remarkable plasticity towards mesenchymal cell types during development and in disease situations. Here, we have characterized the potential of mesothelial cells to undergo changes towards perivascular cells using an in vitro angiogenesis assay. We demonstrate that GFP-labelled mesothelial cells (GFP-MCs) aligned closely and specifically with endothelial networks formed when human dermal microvascular endothelial cells (HDMECs) were cultured in the presence of VEGF-A165 on normal human dermal fibroblasts (NHDFs) for a 7-day period. The co-culture with GFP-MCs had a positive effect on branch point formation indicating that the cells supported endothelial tube formation. We interrogated the molecular response of the GFP- MCs to the angiogenic co-culture by qRT-PCR and found that the pericyte marker Ng2 was upregulated when the cells were co-cultured with HDMECs on NHDFs, indicating a change towards a perivascular phenotype. When GFP-MCs were cultured on the NHDF feeder layer, they upregulated the epithelial-mesenchymal transition marker Zeb1 and lost their circularity while increasing their size, indicating a change to a more migratory cell type. We analysed the pericyte- like behavior of the GFP-MCs in a 3D cardiac microtissue (spheroid) with cardiomyocytes, cardiac fibroblasts and cardiac endothelial cells where the mesothelial cells showed alignment with the endothelial cells. These results indicate that mesothelial cells have the potential to adopt a perivascular phenotype and associate with endothelial cells to potentially support angiogenesis.

cell biology↗

bioRxiv2022

Omicron BA.1 and BA.2 neutralizing activity elicited by a comprehensive panel of human vaccines

The SARS-CoV-2 Omicron variant of concern comprises three sublineages designated BA.1, BA.2, and BA.3, with BA.2 steadily replacing the globally dominant BA.1. We show that the large number of BA.1 and BA.2 spike mutations severely dampen plasma neutralizing activity elicited by infection or seven clinical vaccines, with cross-neutralization of BA.2 being consistently more potent than that of BA.1, independent of the vaccine platform and number of doses. Although mRNA vaccines induced the greatest magnitude of Omicron BA.1 and BA.2 plasma neutralizing activity, administration of a booster based on the Wuhan-Hu-1 spike sequence markedly increased neutralizing antibody titers and breadth against BA.1 and BA.2 across all vaccines evaluated. Our data suggest that although BA.1 and BA.2 evade polyclonal neutralizing antibody responses, current vaccine boosting regimens may provide sufficient protection against Omicron-induced disease.

immunology↗

bioRxiv2021

SARS-CoV-2 spike conformation determines plasma neutralizing activity

Numerous safe and effective COVID-19 vaccines have been developed that utilize various delivery technologies and engineering strategies. The influence of the SARS-CoV-2 spike (S) glycoprotein conformation on antibody responses induced by vaccination or infection in humans remains unknown. To address this question, we compared plasma antibodies elicited by six globally-distributed vaccines or infection and observed markedly higher binding titers for vaccines encoding a prefusion-stabilized S relative to other groups. Prefusion S binding titers positively correlated with plasma neutralizing activity, indicating that physical stabilization of the prefusion conformation enhances protection against SARS-CoV-2. We show that almost all plasma neutralizing activity is directed to prefusion S, in particular the S1 subunit, and that variant cross-neutralization is mediated solely by RBD-specific antibodies. Our data provide a quantitative framework for guiding future S engineering efforts to develop vaccines with higher resilience to the emergence of variants and longer durability than current technologies.

immunology↗

bioRxiv2020

Harnessing tumor immune ecosystem dynamics to personalize radiotherapy

Radiotherapy is a pillar of cancer care and augments the response to immunotherapies. However, little is known regarding the relationships between the tumor immune ecosystem (TIES) and intrinsic radiosensitivity, and a pressing question in oncology is how to optimize radiotherapy to improve patient responses to immune therapies. To address this challenge, we profiled over 10,000 primary tumors for their metrics of radiosensitivity and immune cell infiltrate (ICI), and applied a new integrated in silico model that mimics the dynamic relationships between tumor growth, ICI flux and the response to radiation. We then validated this model with a separate cohort of 59 lung cancer patients treated with radiotherapy. These analyses explain radiation response based on its effect on the TIES and quantifies the likelihood that radiation can promote a shift to anti-tumor immunity. Dynamic modeling of the relationship between tumor radiosensitivity and the TIES may provide opportunity to personalize combined radiation and immunotherapy approaches.

cancer biology↗

bioRxiv2019

Convolutional neural networks for mild diabetic retinopathy detection: An experimental study

Currently, Diabetes and the associated Diabetic Retinopathy (DR) instances are increasing at an alarming rate. Numerous previous research has focused on automated DR detection from fundus photography. The classification of severe cases of pathological indications in the eye has achieved over 90% accuracy. Still, the mild cases are challenging to detect due to CNN inability to identify the subtle features, discrimnative of disease. The data used (i.e. annotated fundus photographies) was obtained from 2 publicly available sources - Messidor and Kaggle. The experiments were conducted with 13 Convolutional Neural Networks architectures, pre-trained on large-scale ImageNet database using the concept of Transfer Learning. Several performance improvement techniques were applied, such as: (i) fine-tuning, (ii) data augmentation, and (iii) volume increase. The results were measured against the standard Accuracy metric on the testing dataset. After the extensive experimentation, the maximum Accuracy of 86% on No DR/Mild DR classification task was obtained for ResNet50 model with fine-tuning (un-freeze and re-train the layers from 100 onwards), and RMSProp Optimiser trained on the combined Messidor + Kaggle (aug) datasets. Despite promising results, Deep learning continues to be an empirical approach that requires extensive experimentation in order to arrive at the most optimal solution. The comprehensive evaluation of numerous CNN architectures was conducted in order to facilitate an early DR detection. Furthermore, several performance improvement techniques were assessed to address the CNN limitation in subtle eye lesions identification. The model also included various levels of image quality (low/high resolution, under/over-exposure, out-of-focus etc.), in order to prove its robustness and ability to adapt to real-world conditions.

bioinformatics↗

bioRxiv2019

Hyperactive TORC1 sensitizes yeast cells to endoplasmic reticulum stress by compromising cell wall integrity

The disruption of protein folding homeostasis in the endoplasmic reticulum (ER) results in an accumulation of toxic misfolded proteins and activates a network of signaling events collectively known as the unfolded protein response (UPR). While UPR activation upon ER stress is well characterized, how other signaling pathways integrate into the ER proteostasis network is unclear. Here, we sought to investigate how the target of rapamycin complex 1 (TORC1) signaling cascade acts in parallel with the UPR to regulate ER stress sensitivity. Using S. cerevisiae, we found that TORC1 signaling is attenuated during ER stress and constitutive activation of TORC1 increases sensitivity to ER stressors such as tunicamycin and inositol deprivation. This phenotype is independent of the UPR. Transcriptome analysis revealed that TORC1 hyperactivation results in cell wall remodelling. Conversely, hyperactive TORC1 sensitizes cells to cell wall stressors, including the antifungal caspofungin. Elucidating the crosstalk between the UPR, cell wall integrity, and TORC1 signaling may uncover new paradigms through which the response to protein misfolding is regulated, and thus have crucial implications for the development of novel therapeutics against pathogenic fungal infections.\n\nIMPORTANCEThe prevalence of pathogenic fungal infections, coupled with the emergence of new fungal pathogens, has brought these diseases to the forefront of global health problems. While antifungal treatments have advanced over the last decade, patient outcomes have not substantially improved. These shortcomings are largely attributed to the evolutionary similarity between fungi and humans, which limits the scope of drug development. As such, there is a pressing need to understand the unique cellular mechanisms that govern fungal viability. Given that Saccharomyces cerevisiae is evolutionarily related to a number of pathogenic fungi, and in particular to the Candida species, most genes from S. cerevisiae are highly conserved in pathogenic fungal strains. Here we show that hyperactivation of TORC1 signaling sensitizes S. cerevisiae cells to both endoplasmic reticulum stress and cell wall stressors by compromising cell wall integrity. Therefore, targeting TORC1 signaling and endoplasmic reticulum stress pathways may be useful in developing novel targets for antifungal drugs.

cell biology↗

bioRxiv2018

Sfp1 regulates the SAGA component Tra1 in response to proteotoxic stress in Saccharomyces cerevisiae

Proteotoxic stress triggers transcriptional responses that allow cells to compensate for the accumulation of toxic misfolded proteins. Chromatin remodeling regulates gene expression in response to the accumulation of misfolded polyQ proteins associated with Huntingtons disease (HD). Tra1 is an essential component of both the SAGA/SLIK and NuA4 transcription co-activator complexes and is linked to multiple cellular processes associated with misfolded protein stress, including the heat shock response. Cells with compromised Tra1 activity display phenotypes distinct from deletions encoding components of the SAGA and NuA4 complexes, indicating a potentially unique regulatory role of Tra1 in the cellular response to protein misfolding. Here, we employed a yeast model of HD to define how the expression of toxic polyQ expansion proteins affects Tra1 expression and function. Expression of expanded polyQ proteins, mimics deletion of SAGA/NuA4 components and results in growth defects under stress conditions. Moreover, deleting genes encoding SAGA and, to a lesser extent, NuA4 components exacerbates polyQ toxicity. Also, cells carrying a mutant Tra1 allele displayed increased sensitivity to polyQ toxicity. Interestingly, expression of polyQ proteins also upregulated the expression of TRA1 and other genes encoding SAGA components, revealing a feedback mechanism aimed at maintaining Tra1and SAGA functional integrity. Moreover, deleting the TORC1 (Target of Rapamycin) effector SFP1 specifically abolished upregulation of TRA1 upon expression of polyQ proteins. While Sfp1 is known to adjust ribosome biogenesis and cell size in response to stress, we identified a new role for Sfp1 in the control of Tra1, linking TORC1 and cell growth regulation to functions of the SAGA acetyltransferase complex during misfolded protein stress.

genetics↗

bioRxiv2018

Tsc1-mTOR signaling controls the structure and function of midbrain dopamine neurons

mTOR complex 1 (mTORC1) is a central coordinator of cell growth and metabolism. Mutations in regulators of mTORC1 cause syndromic disorders with a high prevalence of cognitive and psychiatric conditions. To elucidate the cellular origins of these manifestations, we conditionally deleted the gene encoding the mTORC1 negative regulator Tsc1 from mouse midbrain dopamine neurons, which modulate motor, affective, and cognitive behaviors that are frequently affected in psychiatric disorders. Loss of Tsc1 and constitutive activation of mTORC1 strongly impacted the properties of dopamine neurons, causing somatodendritic hypertrophy, reduced intrinsic excitability, altered axon terminal ultrastructure, and severely impaired dopamine release. These perturbations were associated with selective deficits in cognitive flexibility, which could be prevented by genetic reduction of the obligatory mTORC1 protein Raptor. Our results establish a critical role for mTORC1 in setting the functional properties of midbrain dopamine neurons, and indicate that dopaminergic dysfunction may underlie cognitive inflexibility in mTOR-related syndromes.

neuroscience↗