Biology subjects

Mohammad, N.

Publications and source records attributed to Mohammad, N..

7 recordsLinked to original sources

bioRxiv2025

Attomolar Detection of HIV-1 with Label-Free RCA-rCRISPR on Smartphone

Human Immunodeficiency Virus-1 (HIV) remains a major global public health challenge, having led to over 42.3 million deaths since its discovery in the early 1980s. Despite progress in prevention and treatment, around 60% of people with HIV (PWH) remain undiagnosed in resource-limited regions, disproportionately affecting vulnerable populations and underserved communities across the world. This illustrates the critical need for accessible, accurate, and equipment-free diagnostic tools to enhance detection and thus provide opportunities to curb its spread. Here, we developed a low-cost, robust, and label-free rolling circle amplification (RCA)-rCRISPR diagnostic platform for detecting HIV viral load with minimal instrumentation. Our strategy, combining the integration of RNA-detecting RCA reaction with plasmid reporter-based ratiometric CRISPR (rCRISPR), enables sensitive detection of unprocessed RNA targets without the need for intensive sample pre-treatment. This label-free RCA-rCRISPR diagnostic platform detected HIV RNA down to single-digit aM sensitivity (~3000 copies/mL) from PWH-derived HIV samples ex vivo. Unlike typical RCA, which requires sample fragmentations to break long RNA target sequences, our design harnesses the triple functions of the phi29 DNA polymerase (namely exonuclease activity, polymerization, and strand displacement), enabling the detection of the entire HIV genome without pre-fragmentation. For point-of-care (POC) applications, we constructed an all-in-one smartphone-based minigel electrophoresis device to facilitate equipment-free HIV viral load testing, making it accessible to resource-limited communities. Additionally, the assay has demonstrated the ability for point mutation detection (BRAF mutation in canine urothelial carcinoma), showcasing the robustness of our strategy for broad disease diagnostic applications.

bioengineering↗

bioRxiv2025

Carboxyl Terminal Domain Missense Mutations Alter Distinct Gating Properties of the Cardiac Sodium Channel

Voltage-gated sodium channels undergo reversible voltage/time-dependent transitions from closed to open and inactivated states. The voltage setpoints and efficiency of cardiac sodium channel Nav1.5 state transitions are crucial for tuning the initiation and conduction of myocardial action potentials. The channels cytoplasmic carboxyl-terminal domain (CTD) regulates gating by intramolecular interactions and by serving as a hub for the binding of accessory proteins. We have investigated the roles of the CTD in intrinsic and FGF homologous factor (FHF)-modulated Nav1.5 gating through structure-guided CTD subdomain mutagenesis. The EF-hand module within the CTD was found to exert the most profound effects on channel gating, strongly influencing voltage-dependence of inactivation and activation, accelerating inactivation from the closed state, decelerating inactivation from the open state, minimizing persistent sodium current, and serving as the binding domain for FHF proteins. Nav1.5D1788K bearing a missense mutation in the EF-hand motif displayed a depolarizing shift in voltage dependence of activation and generated greatly enhanced persistent sodium current without altering the voltage dependence of channel inactivation. Reciprocally, Nav1.5L1861A bearing a different missense mutation in the EF-hand underwent closed-state inactivation at more negative membrane potential and at an accelerated rate, but did not display other phenotypes associated with CTD deletion. Nav1.5V1776A/T1778A bearing mutations in the juxtamembrane region between the EF-hand and the channel pore helices displayed wild-type intrinsic gating properties, while FHF modulation of inactivation gating was impaired. Our channel physiology studies together with prior structural data suggest that the voltage and rate of channel inactivation from the closed state are governed by an intramolecular hydrophobic interaction of the CTD EF-hand with the cytoplasmic inactivation loop helix and the extension of this binding interface upon FHF-induced restructuring of the juxtamembrane region, while a distinct CTD intramolecular electrostatic interaction modulates voltage-dependent activation and minimizes persistent sodium current.

physiology↗

bioRxiv2024

A Dual-Functional Needle-Based VOC Sensing Platform for Rapid Vegetable Quality Examination

Volatile organic compounds (VOCs) are common constituents of fruits, vegetables, and crops, and are closely associated with their quality attributes, such as firmness, sugar level, ripeness, translucency, and pungency levels. While VOCs are vital for assessing vegetable quality, traditional detection methods, such as Gas Chromatography-Mass Spectrometry (GC-MS) and Proton Transfer Reaction Mass Spectrometry (PTR-MS) are limited by expensive equipment, complex sample preparation, and slow turnaround time. Additionally, the transient nature of VOCs complicates their detection using these methods. Here, we developed a paper-based colorimetric sensor array combined with needles that could induce vegetable VOC release in a minimally invasive fashion and analyze VOCs in situ with a smartphone reader device. The colorimetric sensor array was optimized using sulfur compounds as main targets and classified fourteen different vegetable VOCs, including sulfoxides, sulfides, mercaptans, thiophenes, and aldehydes. By combining principal components analysis (PCA) analysis, the integrated sensor platform proficiently discriminated between four vegetable subtypes originating from two major categories within 2 min of testing time. This rapid and minimally invasive sensing technology holds great promise for conducting field-based vegetable quality monitoring. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/628229v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@12bf541org.highwire.dtl.DTLVardef@f2a809org.highwire.dtl.DTLVardef@f5f5b7org.highwire.dtl.DTLVardef@1d7027f_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2024

Novel Transgenic Humanized Alpha-1 Antitrypsin Deficiency Mouse Model on Murine SERPINA1 Null Background

Alpha-1 antitrypsin deficiency (AATD) is a rare genetic disorder caused by accumulation of misfolded -1 antitrypsin within hepatocytes. AATD patients are prone to develop liver disease that remains undiagnosed until the late stages of the disease. Due to challenges in manipulating the -1 antitrypsin genes in mice, determining a true loss of function of -1 antitrypsin in previous AATD mouse models has been challenging. Here, we report generation and liver characterization of a new humanized transgenic mouse model for AATD with a background of a CRISPR-Cas9 generated SERPINA1-null mouse. Male and female transgenic mice for normal (Pi*M) and mutant (Pi*Z) variants of human -1 antitrypsin at 4-6 months of age were subjected to this study. The accumulation of human -1 antitrypsin in the hepatocytes and fibrotic features of the liver were monitored by performing an in vivo study. We demonstrate a strong liver phenotype satisfying clinically relevant manifestations of liver pathology associated with AATD, including hepatic accumulation of human -1 antitrypsin globules, liver deposition of extracellular matrix proteins, hepatic ER stress, and liver fibrosis in Pi*Z mice, in addition to mild systemic inflammation. In addition to major phenotypic criteria of AATD-associated liver fibrosis, accompanying single-nucleus RNA-seq data demonstrate activation of pathways associated with liver metabolic changes, inflammation, and regeneration. Data from this study suggest our humanized transgenic AATD mouse model could provide a suitable model to study -1 antitrypsin loss of function, replicate the pathophysiology of AATD associated liver disease, and evaluate therapeutic reagents against this disease. NEW & NOTEWORTHYWe have characterized a new humanized transgenic mouse model of -1 antitrypsin deficiency with a SERPINA1-null background that shows strong manifestations of liver disease. Our data explores the altered phenotype of -1 antitrypsin deficient hepatocytes and suggests a relationship between liver cell types during disease progression. This model may become a useful tool for investigating -1 antitrypsin loss of function, pathogenic mechanisms, and for drug discovery aimed at both prevention and treatment of the disease.

molecular biology↗

bioRxiv2023

A Ratiometric Nonfluorescent CRISPR Assay Utilizing Cas12a-Induced Plasmid Supercoil Relaxation

AbstractMost CRISPR-based biosensors rely on labeled reporter molecules and expensive equipment for signal readout. A recent alternative approach quantifies analyte concentration by sizing native {lambda} DNA reporters, providing a simple and label-free solution for ultrasensitive detection. However, this method faces challenges in accurately quantifying size reduction of long DNA reporters via gel electrophoresis due to the sensitivity of DNA band shift to other interferences such as gel distortion. To overcome these limitations, here we developed a simple and robust ratiometric signaling strategy using CRISPR-Cas12a-induced supercoil relaxation of dsDNA plasmid reporters. In the presence of target, we observed that the fraction of supercoiled plasmid DNA decreased, and the amount of relaxed conformation (circular) increased over time. The relative percentage of supercoiled DNA to the relaxed circular DNA was analyzed by gel electrophoresis to generate an intensity-based ratiometric signal for more accurate target concentration quantification. This simple and inexpensive method is [~]100 times more sensitive when compared with the typical fluorescent reporter system. This self-referenced strategy solves the potential application limitations of previously demonstrated DNA sizing-based CRISPR-Dx without compromising the sensitivity. Finally, we demonstrated the applicability of ratiometric sensing strategy using model DNA targets such as AAV and HPV 16, highlighting its feasibility for point-of-use CRISPR-Dx applications. Table of Contents (TOC) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/570032v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@2c81b3org.highwire.dtl.DTLVardef@1f7453aorg.highwire.dtl.DTLVardef@e1eb06org.highwire.dtl.DTLVardef@cf103_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Rapid Adeno-Associated Virus Genome Quantification with Amplification-Free CRISPR-Cas12a

Efficient manufacturing of recombinant Adeno-Associated Viral (rAAV) vectors to meet rising clinical demand remains a major hurdle. One of the most significant challenges is the generation of large amounts of empty capsids without the therapeutic genome. There is no standardized analytical method to accurately quantify the viral genes, and subsequently the empty-to-full ratio, making the manufacturing challenges even more complex. We propose the use of CRISPR diagnostics (CRISPR-Dx) as a robust and rapid approach to determine AAV genome titers. We designed and developed the CRISPR-AAV Evaluation (CRAAVE) assay to maximize sensitivity, minimize time-to-result, and provide a potentially universal design for quantifying multiple transgene constructs encapsidated within different AAV serotypes. We also demonstrate an on-chip CRAAVE assay with lyophilized reagents to minimize end user assay input. The CRAAVE assay was able to detect AAV titers as low as 7e7 vg/mL with high precision (<3% error) in quantifying unknown AAV titers when compared with conventional quantitative PCR (qPCR) method. The assay only requires 30 minutes of assay time, shortening the analytical workflow drastically. Our results suggest CRISPR-Dx could be a promising tool for efficient rAAV genome titer quantification and has the potential to revolutionize biomanufacturing process analytical technology (PAT). Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=183 HEIGHT=200 SRC="FIGDIR/small/567134v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1bfce18org.highwire.dtl.DTLVardef@17393c9org.highwire.dtl.DTLVardef@eb7c1forg.highwire.dtl.DTLVardef@2203cd_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2018

Comparison between QST and ΦST indices in an endangered Boswellia serrata Roxb: Implications for conservation

Boswellia serrata, an economically important indigenous tree of dry deciduous forests, provides oleoresin gum of pharmaceutical significance and excellent pulp for paper industries, but faces threat to extinction due to poor natural regeneration and commercial exploitation. 240 individuals of the species representing 12 locations of its natural distribution in central India were investigated to compare the genetic differentiation indices, QST for GBH and wood fiber length and {phi}ST for neutral (RAPD+ISSR) markers. The comparison for paired locations was more informative than for metapopulation. The most paired locations were either under the stabilizing selection (QST (L) < {Phi}ST (L)) or in the genetic drift (QST(L) = {Phi}ST (L)) whereas a relatively small number of paired locations was under the divergent selection (QS T(L) > {Phi}ST (L)). The comparison for the metapopulation generating only a single trend of QST (P) > {Phi}ST (P) is, therefore, misleading. For conservation, the genetically deficit locations (QST (L) < {Phi}ST (L) and QST (L) = {Phi}ST (L)) of B. serrata warrant for reinforcement of their genetic diversity by introduction of genotypes from other genetically divergent locations (QST (L) > {Phi}ST (L)), which would check the fragmentation and genetic drift, resulting in reproductive vigour, natural regeneration and reverse the endangered status of the species.

genetics↗