Biology subjects

Ashraf, M.

Publications and source records attributed to Ashraf, M..

12 recordsLinked to original sources

bioRxiv2026

Expanding the therapeutic window of bacteria for cancer therapy

Engineered bacteria enable self-amplifying therapeutic delivery and immune activation, yet these same features drive systemic toxicity and off-target growth. Here, we create a "tumorobe" that overcomes these limitations by integrating reduced immunogenicity, tumour-restricted growth, and enhanced payload release. Specifically, we identify lipopolysaccharide modifications that reduce immunogenicity while preserving fitness, restrict growth to tumours using a hypoxia-lactate "AND" gate, and enhance extracellular payload release via lipoprotein deletion. This strain expands the maximum tolerated dose in a route-dependent manner by up to 1,000-fold. Across multiple murine models, including colorectal cancer and glioblastoma, the strain drives robust antitumour efficacy as both a standalone therapeutic and an immunotherapeutic delivery platform, achieving durable complete responses in up to 75% of colorectal tumours when armed with a cytokine payload. Together, these results establish a broadly applicable platform for safe and effective bacterial cancer therapy.

synthetic biology↗

bioRxiv2026

NanoPro - a Framework for Probiotically-Guided Nanovector Therapy

The 'antigen problem', i.e. the disconnect between the broad spectrum of therapeutic modes enabled by the range of viral and synthetic nanoparticles for which workable receptor-targeting architectures exist and the relative dearth of addressable target receptors on most tumors, is a long-standing challenge in targeted tumor nanotherapy. Here we provide in vitro-proof-of-principle for NanoPro, a platform for guiding nanoscale therapeutics to otherwise antigenically indistinct tumors via probiotically-released tumor cell-binding tags, ProTags. These consist of an orthogonal antigen (GFP or ALFA) fused to the heparin-binding domain of placenta growth factor-2 and serve as attachment receptors for ProTag-targeted nanovectors. ProTag release from Escherichia coli Nissle 1917 was facilitated by deletion of the membrane-tethering lpp gene, which increased ProTag supernatant concentrations 16.5-fold compared to the parental strain without impacting viability. HBD-dependent decoration with ProTag in bacterial supernatant followed by treatment with receptor-targeted aGFP- or aALFA-lentiviral vectors resulted in majority transduction of MC-38 and MDA-MB-468 tumor cell lines with antigen specificities of 96.5% and 98.5%, respectively, depending on the choice of nanobody. This ProTag-mediated interaction was notably robust, with selective transduction still observed at 625-fold supernatant dilution, warranting further exploration of the utility of the NanoPro system for tumor-restricted delivery of nanotherapeutics.

synthetic biology↗

bioRxiv2025

Evolved microbial diversity enables combinatoric biosensing in complex environments

Whole-cell biosensors (WCBs) offer rapid, cost-effective monitoring of environmental contamination and human disease. Current WCB efforts to optimize detection of single target analytes under laboratory conditions have achieved vastly improved performance, setting the stage for WCB deployment in complex environments. We propose a framework that leverages the cross-specificity of single-target WCBs to quantify multiple targets using supervised machine learning. Specifically, we engineer six sensors for heavy metal contaminants in laboratory E. coli. We then evolve the strain to generate five chassis with improved growth in seawater conditions. We transform the chassis with the sensors, creating a set of 30 variants. The variant dynamic responses are characterized with microfluidics, revealing significant diversity. Leveraging this diversity, we construct a consortium to combinatorically quantify multiple analytes with machine learning, outperforming single-target biosensors in over 90% of our test samples. These results form a generalizable framework that facilitates WCB translation toward settings beyond the laboratory.

bioengineering↗

bioRxiv2024

Single-cell transcriptome analysis reveals CD34 as a novel marker of human sinoatrial node pacemaker cardiomyocytes

The sinoatrial node (SAN) regulates the heart rate throughout life. Failure of this primary pacemaker results in life-threatening, slow heart rhythm. Despite its important function, the cellular and molecular composition of the human SAN is not completely resolved. Particularly, no cell surface marker to identify and isolate SAN pacemaker cells has been reported to date. Here we used single-nuclei/cell RNA sequencing of fetal and human pluripotent stem cell (hPSC)- derived SAN cells and show that the SAN consists of three subtypes of pacemaker cells, including Core SAN, SAN, and Transitional Cells. Our study identified a host of novel Core SAN markers including MYH11, BMP4, and the cell surface antigen CD34. We demonstrate that sorting for CD34+ cells from cardiac hPSC differentiations enriches for SAN cells with a functional pacemaker phenotype. This novel SAN pacemaker cell surface marker is highly valuable for future hPSC- based disease modelling, drug discovery, cell replacement therapies, as well as the delivery of therapeutics to SAN cells in vivo using antibody-drug conjugates.

cell biology↗

bioRxiv2023

Phenotypic Switching of Vascular Smooth Muscle Cells in Duchenne Muscular Dystrophy

BackgroundExtensive studies have been conducted in skeletal muscle and myocardium affected by Duchenne Muscular Dystrophy (DMD) disease but there is a significant gap of research in the role of vascular smooth muscle cells (VSMCs) in DMD. Here, we investigated the role of dystrophin deficiency in the maintenance of VSMCs contractile phenotype. Methods12-14 months old mdx mice and DMD induced pluripotent stem cells (iPSC) derived VSMCs were used as disease models. Morphological and immunohistochemistry analyses were performed to determine histological changes and the expression of contractile markers. Transmission Electron Microscopy (TEM) was used to assess ultrastructural changes in the VSMCs. Mito-tracker staining and TUNEL staining were performed to determine mitochondria fission-fusion and apoptosis respectively. mRNA Sequencing for normal iPSC derived VSMCs (WT-VSMCs) and DMD iPSC derived VSMCs (DMD-VSMCs) with or without oxidative stress was performed. KEGG signaling pathway enrichment, Go function enrichment and Gene set enrichment analysis (GESA) were conducted to explore the potential mechanism responsible for these changes. In addition, transcription factor enrichment analysis was performed to unravel mechanistic pathways of regulatory networks. ResultsSpontaneous abnormal VSMCs proliferation, loss of vascular structure and degenerative changes occurred in VSMCs in aorta from 12-14 months old mdx mice. The DMD-VSMCs showed maturation defect, loss of mitochondrial hemostasis, and increased vulnerability to oxidative stress compared with WT-VSMCs. Transcriptome analysis revealed dysregulation of smooth muscle proliferation, differentiation, and vascular development in DMD-VSMCs. Transcriptional factor, target, and motif discovery analysis of the dysregulated gene set suggested potential contributions of transcriptional factors GADD45A, SOX9, TIA1, RBBP9 and FOXM to the phenotypes of DMD-VSMCs. Under oxidative stress, initiation of apoptotic process was significantly enhanced in DMD-VSMCs while their response to hypoxia and oxidative stress was downregulated. ConclusionsDystrophin deficiency induced VSMCs phenotype switching and disrupted mitochondrial metabolism. The findings in this study underscore the importance of vascular dysfunction in DMD disease and therapeutic interventions to restore VSMC phenotype may ameliorate the propensity of disease progression. It is suggested that the transcriptome analysis may allow the discovery of potential signaling pathways involved in the dysregulation of transcription factors.

cell biology↗

bioRxiv2023

Patient and cell-type specific hiPSC-modeling of a truncating titin variant associated with atrial fibrillation

BackgroundProtein truncating mutations in the titin gene are associated with increased risk of atrial fibrillation (AF). However, little is known regarding the underlying pathophysiology. MethodsWe identified a heterozygous titin truncating variant in a patient with unexplained early-onset AF using whole exome sequencing. We used atrial and ventricular patient induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), CRISPR/Cas9 genetic correction, and engineered heart tissue (EHT) constructs to evaluate the impact of the titin truncating variant on electrophysiology, sarcomere structure, contractility, and gene expression. ResultsWe generated atrial and ventricular iPSC-CMs from the AF patient with the titin truncating variant and a CRISPR/Cas9 genome corrected isogenic control. We demonstrate that the titin truncating variant increases susceptibility to pacing-induced arrhythmia (prevalence of arrhythmogenic phenotypes, 85.7% versus 14.2%; P = 0.03), promotes sarcomere disorganization (mean {+/-} SEM, 66.3 {+/-} 6.8% versus 88.0 {+/-} 2.9%; P = 0.04) in atrial iPSC-CMs, and reduces contractile force (0.013 {+/-} 0.003 mN versus 0.027 {+/-} 0.004 mN; P < 0.01) in atrial EHTs compared to isogenic controls. In ventricular iPSC-CMs, this variant led to altered electrophysiology (90.0% versus 33.3%; P = 0.02) and sarcomere organization (62.0 {+/-} 3.9% versus 82.9 {+/-} 2.9%; P < 0.01) with no change in EHT contractility compared to isogenic controls. RNA-sequencing revealed an upregulation of cell adhesion and extracellular matrix genes in the presence of the titin truncating variant for both atrial and ventricular EHTs. ConclusionsIn a patient with early-onset unexplained AF and normal ventricular function, iPSC-CMs with a titin truncating variant showed structural and electrophysiological abnormalities in both atrial and ventricular preparations, while only atrial EHTs demonstrated reduced contractility. Whole transcriptome sequencing showed upregulation of genes involved in cell-cell and cell-matrix interactions in both atrial and ventricular EHTs. Together, these findings suggest titin truncating variants promote the development of AF through remodeling of atrial cardiac tissue and provide insight into the chamber-specific effects of titin truncating variants.

cell biology↗

bioRxiv2022

Dynamic Phosphorylation Regulates Eukaryotic Translation Initiation Factor 4A Activity During the Cell Cycle

The eukaryotic translation initiation factor 4A (eIF4A) resolves mRNA structures to support protein synthesis, yet little is known about its regulation. Here we analyzed eIF4A phosphorylation during alternate stages of the cell cycle, and found three residues near the DEAD box motif (T73, T146, and S177) underwent substantial phosphorylation changes. Phosphomimetic mutations T73D and T146D led to G2/M phase arrest, and abolished eIF4A interaction with RNA, suggesting eIF4A activity is needed for completion of cell division. In addition to these repressive events, we found that S177, a site immediately adjacent to the DEAD-box, showed diametrically opposed phosphorylation, with only phosphorylated S177 present during G1/S arrest and dephosphorylated S177 peptides during G2/M arrest. Phosphomimetic S177D eIF4A increased polysome levels and enhanced normally reduced eIF4A-eIF4G-interaction during G2/M, while phosphodeficient S177A decreased polysome levels and reduced growth, suggesting phosphorylation of S177 enhances eIF4A-mediated translation during G1/S. Together these results suggest that dynamic phosphorylation of eIF4A S177 serves to stimulate translation during G1/S, while inhibitory phosphorylation of additional sites holds the potential to rapidly transition eIF4A to an inactive state and turn off translation. These results also suggest an important role for eIF4A in coupling translation to cell cycle stages.

molecular biology↗

bioRxiv2020

An in vitro assessment of anti-SARS-CoV-2 activity of oral preparations of iodine complexes (RENESSANS)

Since the emergence of CoVID-19 pandemic in China in late 2019, scientists are striving hard to explore non-toxic, viable anti-SARS-CoV-2 compounds or medicines. We determined In Vitro anti-SARS-CoV-2 activity of oral formulations (syrup and capsule) of an Iodine-complex (Renessans). A monolayer of vero cells were exposed to SARS-CoV-2 in the presence and absence of different concentrations (equivalent to 50, 05 and 0.5 g/ml of I2) of Renessans. Anti-SARS-CoV-2 activity of each of the formulation was assessed in the form of cell survival, SARS-CoV-2-specific cytopathic effect (CPE) and genome quantization. With varying concentrations of syrup and capsule, a varying rate of inhibition of CPE, cells survival and virus replication was observed. Compared to 0.5 g/ml concentration of Renessans syrup, 5 and 50 g/ml showed comparable results where there was a 100% cell survival, no CPEs and a negligible viral replication ({Delta}CT= 0.11 and 0.13, respectively). This study indicates that Renessans, containing iodine, may have potential activity against SARS-CoV-2 which needs to be further investigated in human clinical trials.

microbiology↗

bioRxiv2020

Evaluation of cytotoxic, antiviral effect and mutagenic potential of a micronutrient combination in vitro cell culture

Background Micronutrients are essential for the body to produce enzymes, hormones and other substances essential for proper growth and development. Iodine, potassium iodide and ascorbic acid are ones of the most important in global public health terms. However, their lack represents a major threat to the human and animal health.Methodology Antiviral, cytotoxic and mutagenic activity of commercially available micronutrient combination consisting of iodine, ascorbic acid, potassium iodide and excipients was evaluated. Commercial preparation of micronutrient combination was compared with self-prepared micronutrient preparation. MTT assay was used to evaluate the cytotoxicity of all the preparation. Confluent monolayer of Chicken Embryo Fibroblasts grown in 96-well cell culture plates were treated with ten concentrations of each preparation in triplicate manner and was used to determine the viability of the cells and cell survival percentage. Antiviral efficacy was determined against influenza virus H9N1 strain by virus infection and subsequent cell viability assay. Furthermore, mutagenicity was measured by bacterial reverse mutation analysis by Ames test using two strains of Salmonella typhimurium TA100 and TA 98 with and without S9. After appropriate incubation, number of revertant colonies per plate were counted in triplicate manner and MF was calculated.Results Our combination showed cytotoxicity at doses higher than 11.71ug/ml while showed significant antiviral efficacy at concentrations of 11.71ug/ml, 5.86ug/ml, 2.93ug/ml and 1.4ug/ml which faded away at lower dilutions. Commercially available preparation was found to be non-mutagenic in our experiment.Conclusions Combination consisting of iodine, ascorbic acid, potassium iodide is effective in the treatment of viral infections.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pharmacology and toxicology↗

bioRxiv2020

Human iPS Cells Derived Skeletal Muscle Progenitor Cells Promote Myoangiogenesis and Restore Dystrophin in Duchenne Muscular Dystrophic Mice

Background and ObjectiveDuchenne muscular dystrophy (DMD) is caused by mutations of the gene that encodes the protein dystrophin. Loss of dystrophin leads to severe and progressive muscle-wasting in both skeletal and heart muscles. Human induced pluripotent stem cells (hiPSCs) and their derivatives offer important opportunities to treat a number of diseases. Here, we investigated whether givinostat, a histone deacetylase inhibitor (HDACi), could reprogram hiPSCs into muscle progenitor cells (MPC) for DMD treatment. Methods and ResultsMPC generated by CHIR99021 and givinostat (Givi) small molecules from multiple hiPSCs expressed myogenic makers (Pax7, desmin) and were differentiated into myotubes expressing MF20 upon culture in specific differentiation medium. These MPC exhibited superior proliferation and migration capacity determined by CCK-8, colony and migration assays compared to control-MPC generated by CHIR99021 and fibroblast growth factor (FGF). Upon transplantation in hind limb of Mdx/SCID mice with cardiotoxin (CTX) induced injury, these MPC showed higher engraftment and restoration of dystrophin than treatment with control-MPC and human myoblasts. In addition, treated muscle with these MPC showed significantly limited infiltration of inflammatory cells and reduced muscle necrosis and fibrosis. A number of these cells were engrafted under basal lamina expressing Pax7, which were capable of generating new muscle fibers after additional injury. Extracellular vesicles released from these cells promoted angiogenesis after reinjury. ConclusionWe successfully generated integration free MPC from multiple hiPS cell lines using CHIR99021 and Givi. Givinostat induced MPC showed marked and impressive regenerative capabilities and restored dystrophin in injured tibialis muscle compared to control MPC. Additionally, MPC generated by Givi also seeded the stem cell pool in the treated muscle. It is concluded that hiPSCs pharmacologically reprogrammed into MPC with a small molecule, Givi with anti-oxidative, anti-inflammatory and muscle gene promoting properties might be an effective cellular source for treatment of muscle injury and restoration of dystrophin in DMD.

cell biology↗

bioRxiv2019

Agronomic, physiological and molecular characterization of rice mutants revealed key role of ROS and catalase in high temperature stress tolerance

Plants adapt to harsh environments particularly high temperature stress by regulating their physiological and biochemical processes, which are key tolerance mechanisms. Thus, identification of heat-tolerant rice genotypes and reliable selection indices are crucial for rice improvement programs. Here, we evaluated the response of a rice mutant population for high-temperature stress at the seedling and reproductive stages based on agronomic, physiological and molecular traits. The estimate of variance components revealed significant differences (P<0.001) among genotypes, treatments and their interaction for almost all traits. Principal component analysis showed significant diversity among the genotypes and traits under high-temperature stress. The mutant HTT-121 was identified as the most heat tolerant mutant with higher grain yield, panicle fertility, cell membrane thermo-stability (CMTS) and antioxidant enzyme levels under heat stress conditions. Various seedling-based morpho-physiological traits (leaf fresh weight, relative water contents, malondialdehyde, CMTS) and biochemical traits (superoxide dismutase, catalase and hydrogen peroxide) explained variations in grain yield that could be used as selection indices for heat tolerance in rice at early growth stages. Notably, heat sensitive mutants showed a significant accumulation of ROS level, reduced activities of catalase and upregulation of OsSRFP1 expression under heat stress, suggesting their key role in regulating heat tolerance in rice. The heat-tolerant mutants identified in this study could be used in breeding programs and the development of mapping populations to unravel the underlying genetic architecture for heat-stress adaptability.\n\nSummary text for table of contentsHeat stress probably due to changing climate scenario has become a serious threat for global rice production. On the other side, efforts to develop high yielding cultivars have led to the reduced genetic variability to withstand harsh environmental conditions. This study aimed to identify novel heat tolerant mutants developed through gamma irradiation which will provide a unique genetic resource for breeding programs. Further, we have identified reliable selection indices for screening heat-tolerant rice germplasm at early growth stages.

physiology↗

bioRxiv2019

Solid State High Throughput Screening Microscopy

We introduce a solid state high throughput screening (ssHTS) imaging modality that uses a novel Newtonian telescope design to image multiple spatially separated samples without moving parts or robotics. Conventional high-throughput imaging modalities either require movement of the sample to the focal plane of the imaging system1-3 or movement of the imaging system itself4,5, or use a wide-field approach to capture several samples in one frame. Schemes which move the sample or the imaging system can be mechanically complex and are inherently slow, while wide-field imaging systems have poor light collection efficiency and resolution compared to systems that image a single sample at a given time point. Our proposed ssHTS system uses a large parabolic reflector and an imaging lenses positioned at their focal distances above each sample. A fast LED array sequentially illuminate samples to generate images that are captured with a single camera placed at the focal point of the reflector. This optical configuration allows each sample to completely fill a sensors field of view. Since each LED illuminates a single sample and LED switch times are very fast, images from spatially separated samples can be captured at rates limited only by the cameras frame rate. The system is demonstrated by imaging cardiac monolayer and Caenorhabditis elegans (C. elegans) preparations.

bioengineering↗