Biology subjects

Javed, M.

Publications and source records attributed to Javed, M..

5 recordsLinked to original sources

bioRxiv2026

Prime Editing Corrects the HBB Codon 8/9 (+G) Mutation in Patient-Derived Induced Pluripotent Stem Cells and Restores β-Globin Expression in iPSC-Derived Erythroid Cells

Homozygosity for the HBB codon 8/9 (+G) frameshift (c.27dup (p.Ser10ValfsTer14)) causes transfusion-dependent {beta}-thalassaemia and is common in South Asia. Prime editing can reverse this insertion without double-strand breaks or donor DNA, but its efficiency depends on pegRNA design. We derived Sendai-reprogrammed iPSCs from a homozygous patient, optimised PEmax editing (spacer, pegRNA extension, secondary nick, MLH1dn) and compared patient, PEmax-treated and control iPSC-derived erythroid cells by flow cytometry, colony assays, RT-qPCR, western blotting and cation-exchange HPLC. Patient iPSCs had a normal 46,XY karyotype, expressed pluripotency markers, formed all three germ layers and were Sendai-free by passage 15. A PAM-disrupting spacer with a +68 nicking sgRNA gave the highest intended-edit frequency 8.4%; MLH1dn added little. PEmax-treated cultures matured and formed colonies more like control than patient cultures, restored HBB transcript (about 4-fold control iPSC-derived cells) and detectable {beta}-globin protein, and contained an HPLC fraction consistent with HbA ( undetectable in patient cells). Fetal haemoglobin (about 80%) and embryonic globin remained predominant. Prime editing corrects HBB c.27dupG in patient iPSCs and restores {beta}-globin expression, with partial restoration of adult haemoglobin in a fetal/embryonic-type erythroid background. Validation in additional donors, haematopoietic stem cells and in vivo is required.

cell biology↗

bioRxiv2025

Food-activated Microneedle Sensor for Real-time, Colorimetric Spoilage Monitoring of Pre-packaged Food

At a time of growing food insecurity, developing technologies to reduce food waste is critical. We report an inexpensive, colorimetric spoilage sensor for real-time food product assessment. The sensor is composed of dehydrated gelatin microneedles that exhibit high mechanical integrity in their base state. However, once exposed to fluid-rich food environments, they rapidly transition to a hydrogel sensing state. Food-derived anthocyanins embedded within these microneedles enable pH-based spoilage monitoring. When applied to sealed fish products, these microneedles non-destructively penetrate through packaging and are rehydrated by the underlying fish matrix. As the product ages, a defined colour shift occurs, demonstrating strong correlation with quantitative spoilage markers. When applied to unsealed fish products for rapid testing, the large microneedle sensing interface enables accelerated colorimetric sensing. Finally, successful fresh versus spoiled categorization of smartphone-acquired images of the sensor using machine learning removes readout ambiguity, empowering consumers with independent real-time product monitoring.

molecular biology↗

bioRxiv2024

A Rapid, Cost-Effective Method for High-Yield DNA Purification from PCR Products and Agarose Gels.

Nucleic acid purification is essential for molecular biology workflows, enabling successful downstream applications like cloning, sequencing, and PCR amplification. While commercial kits are widely used for DNA extraction from PCR products and agarose gels, their high costs can burden resource-limited laboratories. To provide a viable alternative, we developed an optimized, cost-effective in-house protocol for high-yield DNA purification. This study evaluates the performance of our protocol for plasmid DNA and PCR products, comparing it with commercial kits from Qiagen, Thermo Fisher, and WizBio in terms of cost, time, DNA concentration, and purity. Results from gel electrophoresis demonstrated that the in-house protocol significantly enhances PCR product clarity and reduces background smearing, yielding high-purity DNA compatible with sensitive applications. Restriction-digested plasmid samples showed successful ligation and transformation in E. coli (DH5), with Sanger sequencing chromatogram further confirming the sequence integrity of the purified DNA. Our findings highlight the in-house protocol as a cost-effective, efficient, and reliable alternative to commercial kits, delivering high-quality DNA suitable for various molecular applications. This method offers an accessible and practical solution for laboratories seeking to optimize DNA purification under budget constraints.

molecular biology↗

bioRxiv2023

Metabolic alterations unravel the materno fetal immune responses with disease severity in pregnant women infected with SARS-CoV-2

BackgroundPregnancy being immune compromised state, COVID-19 disease poses high risk of premature delivery and threat to fetus. Plasma metabolome regulates immune cellular responses and we aimed to analyze the plasma secretome, metabolome and immune cells in COVID-19 positive pregnant mother and cord blood. MethodsCOVID-19 RT-PCR positive pregnant females (n=112) asymptomatic (n=82), or with mild (n=21) or moderate (n=9) disease and control healthy pregnant (n=10) females were included. Mothers blood and cord blood (n=80) was analysed for untargeted metabolome profiling and plasma cytokines by high-resolution mass spectrometry (MS) and multiplex cytokine bead array. Immune scan in mothers was done using flow cytometry. ResultsIn asymptomatic SARS-CoV-2 infection, --the amino acid metabolic pathways such as glycine, serine, L-lactate and threonine metabolism was upregulated, riboflavin and tyrosine metabolism, downregulated. In mild to moderate disease, the pyruvate and NAD+ metabolism (energy metabolic pathways) were mostly altered. In addition to raised TNF-, IFN-, IFN-{gamma}, IL-6 cytokine storm, IL-9 was increased in both mothers and neonates. Pyruvate and NAD+ metabolic pathways along with IL-9 and IFN-{gamma} had impact on non-classical monocytes, increased CD4 T cells and B cells but depleted CD8+ T cells. Cord blood mimicked the mothers metabolomic profiles by showing altered valine, leucine, isoleucine, glycine, serine, threonine in asymptomatic and NAD+ and riboflavin metabolism in mild and moderate disease subjects. ConclusionsOur results demonstrate a graduated immune-metabolomic interplay in mother and fetus in pregnant females with different degrees of severity of COVID-19 disease. IL-9 and IFN- {gamma} regulated pyruvate, lactate TCA metabolism and riboflavin metabolism with context to disease severity are hall marks of this materno-fetal metabolome. HighlightsO_LISARS-CoV-2 infection alters energy consumption metabolic pathways during pregnancy. C_LIO_LIPregnant women with mild to moderate COVID-19 show increased energy demands, and consume stored glucose by upregulating pyruvate and NAD+ metabolism. C_LIO_LIIncreased TNF- and IL-9 in mild COVID-19 disease involve TCA cycle to produce lactate and consume stored glucose by up regulating pyruvate and nicotinamide and nicotinate metabolism. C_LIO_LIWith mild to moderate disease, raised IL-9 and TNF-, decreased riboflavin pathway, exhaustion of T and B cells cause pathogenesis. C_LIO_LICord blood mimics the metabolic profile of mothers peripheral blood, SARS- CoV-2 infection reshapes immune-metabolic profiles of mother-infant dyad. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=141 HEIGHT=200 SRC="FIGDIR/small/540101v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@657cc3org.highwire.dtl.DTLVardef@e1e76forg.highwire.dtl.DTLVardef@12153f2org.highwire.dtl.DTLVardef@10e5332_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2018

Multi-omics approach identifies novel pathogen-derived prognostic biomarkers in patients with Pseudomonas aeruginosa bloodstream infection

Pseudomonas aeruginosa is a human pathogen that causes health-care associated blood stream infections (BSI). Although P. aeruginosa BSI are associated with high mortality rates, the clinical relevance of pathogen-derived prognostic biomarker to identify patients at risk for unfavorable outcome remains largely unexplored. We found novel pathogen-derived prognostic biomarker candidates by applying a multi-omics approach on a multicenter sepsis patient cohort. Multi-level Cox regression was used to investigate the relation between patient characteristics and pathogen features (2298 accessory genes, 1078 core protein levels, 107 parsimony-informative variations in reported virulence factors) with 30-day mortality. Our analysis revealed that presence of the helP gene encoding a putative DEAD-box helicase was independently associated with a fatal outcome (hazard ratio 2.01, p = 0.05). helP is located within a region related to the pathogenicity island PAPI-1 in close proximity to a pil gene cluster, which has been associated with horizontal gene transfer. Besides helP, elevated protein levels of the bacterial flagellum protein FliL (hazard ratio 3.44, p < 0.001) and of a bacterioferritin-like protein (hazard ratio 1.74, p = 0.003) increased the risk of death, while high protein levels of a putative aminotransferase were associated with an improved outcome (hazard ratio 0.12, p < 0.001). The prognostic potential of biomarker candidates and clinical factors was confirmed with different machine learning approaches using training and hold-out datasets. The helP genotype appeared the most attractive biomarker for clinical risk stratification due to its relevant predictive power and ease of detection.

microbiology↗