Biology subjects

Khan, S. H.

Publications and source records attributed to Khan, S. H..

7 recordsLinked to original sources

bioRxiv2026

Translesion synthesis protein ImuA from Mycolicibacterium smegmatis is a hexameric helicase-nuclease

Translesion DNA synthesis (TLS) enables DNA replication across damaged DNA and promotes stress-induced mutagenesis that contributes to antibiotic resistance in bacteria. The conserved ImuABC mutasome is essential for TLS in many bacterial species, yet the molecular function of its accessory protein, ImuA, has remained elusive. Here we show that Mycolicibacterium smegmatis ImuA assembles into a hexameric complex, likely arranged as a dimer of trimers, with dual enzymatic activities that reshape current models of its role in DNA damage tolerance. We show that ImuA functions as an ATP-dependent helicase that preferentially unwinds DNA substrates containing single-stranded DNA overhangs and identify amino acids required for both hexamer formation and helicase activity. Unexpectedly, ImuA also possesses ATP-independent 5' exonuclease activity, selectively processing ssDNA substrates with free 5' ends. We show a basic patch on the N-terminus is essential for stabilizing both the nuclease motif and oligomerization. Together, these findings identify ImuA as an active DNA-processing enzyme rather than a passive accessory factor and establish oligomerization as a prerequisite for its function. Our work provides a mechanistic framework for understanding how ImuA may function within the ImuABC mutasome to coordinate DNA processing during translesion synthesis.

biochemistry↗

bioRxiv2026

Enriched-GF: A Reproducible High-Yield Autologous Blood-Derived Growth Factor Method for Regenerative Medicine

BackgroundPlatelet-derived Growth factors play key roles in tissue repair and regeneration, yet conventional platelet-rich plasma (PRP) formulations release these mediators inconsistently in vivo due to variability in platelet yield and activation dynamics. To overcome this limitation, direct administration of concentrated platelet-derived growth factor preparations has gained interest, though current manufacturing approaches for human platelet lysate (hPL), growth factor concentrates (GFC), and conditioned serum remain constrained by batch variability, incomplete platelet degranulation, and reliance on anticoagulants. Here, we examine alternative platelet activation workflows to establish a standardized, efficient, and reproducible method for high-yield growth factor recovery suitable for translational and clinical applications. MethodsNine GFC production protocols were compared, employing different combinations of freeze-thaw (FT) cycling, glass bead (GB) agitation, calcium (Ca2) activation, and a novel Enriched Growth Factor (Enriched-GF) method. The objective was to identify a protocol capable of maximizing growth factor yield within a three-hour workflow. Optimal Ca2 concentrations and GB conditions were determined from prior optimization studies and integrated into the Enriched-GF processing scheme. Platelet concentrates (n = 10 per protocol) were processed under each condition, and growth factor levels were quantified using ELISA. ResultsGrowth factor yields differed significantly across protocols. The greatest and most consistent increases in growth factor release were observed with the Enriched-GF method combining GB activation, FT cycling, and Ca2 stimulation. This approach resulted in markedly elevated concentrations of key regenerative mediators, including enhanced EGF release, a 4.5-fold increase in PDGF, maximal TGF-{beta} liberation, and a four-fold increase in FGF2 relative to conventional platelet lysate or conditioned serum preparations. These results were reproducible across independent donor pools, demonstrating robustness and batch-to-batch consistency. ConclusionWe describe a rapid and reproducible method for producing highly concentrated platelet-derived growth factors using a combined GB-FT-Ca2 activation strategy. The Enriched-GF protocol consistently outperformed existing platelet lysate, conditioned serum, and conventional GFC preparation methods, yielding a standardized product with enhanced growth factor content. This Enriched-GF approach offers a clinically practicable solution for applications in regenerative medicine requiring reliable and high-yield growth factor delivery. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/712883v2_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@ac60c3org.highwire.dtl.DTLVardef@104d8eborg.highwire.dtl.DTLVardef@13d5afcorg.highwire.dtl.DTLVardef@120645c_HPS_FORMAT_FIGEXP M_FIG C_FIG Schematic overview of platelet concentrate preparation from whole blood and the generation of different platelet lysates and growth factor-enriched serum using freeze-thaw, calcium gluconate, and glass bead activation methods.

biochemistry↗

bioRxiv2026

Brief, repeated isoflurane anaesthesia elicits sex-dependent behavioural, cerebrovascular, and microglial alterations in mice

General anaesthetics are indispensable tools in biomedical research, yet growing evidence shows that their effects on brain function extend beyond the transient loss of consciousness for which they are used. Although numerous preclinical studies have examined the impact of a single anaesthetic exposure, far less is known about the cumulative consequences of repeated administration protocols, despite their widespread use in longitudinal imaging, behavioural testing, and disease model studies. Here, we show that brief, repeated isoflurane exposures induce alterations in behaviour, cerebrovascular patterns, and microglial phenotype in adult CD1 mice, with several responses exhibiting pronounced sex specificity. Repeated anaesthesia suppressed locomotor activity selectively in females and impaired ethologically important maintenance behaviours, including nesting and burrowing, in both sexes. These behavioural effects were accompanied by sex-dependent changes in cerebral blood flow dynamics, with females displaying elevated cerebrovascular flow, yet males showing higher reactivity to isoflurane exposure. At the molecular level, repeated isoflurane exposure altered endothelial marker expression without modifying capillary density and shifted microglia toward a hyporeactive state. Together, these data reveal that even short, routine anaesthetic events produce biologically meaningful and sex-dependent effects on neural, vascular, and immune systems. These results indicate that the anaesthetic regimen is an often overlooked but influential factor that deserves careful consideration in pre-clinical neuroscience.

neuroscience↗

bioRxiv2022

Ligand-induced shifts in conformational ensembles that predict transcriptional activation

Nuclear receptors function as ligand-regulated transcription factors whose ability to regulate diverse physiological processes is closely linked with conformational changes induced upon ligand binding. Understanding how conformational populations of nuclear receptors are shifted by various ligands could illuminate strategies for the design of synthetic modulators to regulate specific transcriptional programs. Here, we investigate ligand-induced conformational changes using a reconstructed, ancestral nuclear receptor. By making substitutions at a key position, we engineer receptor variants with altered ligand specificities. We use atomistic molecular dynamics (MD) simulations with enhanced sampling to generate ensembles of wildtype and engineered receptors in combination with multiple ligands, followed by conformational analysis and prediction of ligand activity. We combine cellular and biophysical experiments to allow correlation of MD-based predictions with functional ligand profiles, as well as elucidation of mechanisms underlying altered transcription in receptor variants. We determine that conformational ensembles accurately predict ligand responses based on observed population shifts, even within engineered receptors that were constitutively active or transcriptionally unresponsive in experiments. These studies provide a platform which will allow structural characterization of physiologically-relevant conformational ensembles, as well as provide the ability to design and predict transcriptional responses in novel ligands.

biochemistry↗

bioRxiv2021

Evolutionary tuning of a key helix drove androgen selectivity

The genetic and biophysical mechanisms by which new protein functions evolve are central concerns in evolutionary biology and molecular evolution. Despite much speculation, we know little about how protein function evolves. Here, we use ancestral proteins to trace the evolutionary history of ligand recognition in a sub-class of steroid receptors (SRs), an ancient family of ligand-regulated transcription factors that enable long-range cellular communication central to multicellular life. The most ancestral members of this family display promiscuous ligand binding due to their large ligand binding pockets, while more recently evolved SRs tend to have smaller cavities. Less obvious, however, are the forces driving the selectivity of highly similar ligands. A key example is the divergence between the progesterone and androgen receptors (PR, AR), which display a high degree of sequence similarity and yet display differential ligand preferences. This work uses the ancestral steroid receptor 2 (AncSR2), the common ancestor of all 3-ketosteroids and the ancestral androgen receptor 1 (AncAR1), the seminal androgen receptor, to explore the biophysical mechanisms that drove the evolution of androgen specificity. We determine that ligand specificity in androgen receptors is driven by changes in the conformational dynamics of the receptor as well as altered binding pocket interactions, with helix 10 (H10) playing a critical role in tuning ligand specificity.

evolutionary biology↗

bioRxiv2020

Structural analysis of Sub16 sedolisin of Trichophyton rubrum reveals a flexible nature of its pro domain

Trichophyton rubrum is one of the leading causes of superficial skin infections worldwide. It is a keratinolytic fungus specialized in colonization of keratinized tissue of skin, hair and nails for long periods of time. The fungus encodes a wide repertoire of secreted proteases in its genome that not only aid in nutrient acquisition but also establishment of infection on the host. The proteases are synthesized in prepro form that requires removal of the prosegment for activation. In order to gain insights into the structural association of the pro domain with the catalytic domain, we investigate the structural features of the pro domain of the secreted sedolisin member Sub16 of T. rubrum. Our results show that the pro domain of Sub16 may have inherent flexibility in the absence of the associated catalytic domain which is stabilized in complex with catalytic domain. This is the first report of structural investigation on a stand-alone pro domain of sedolisin family of subtilases that will help in design of further structural studies of this protein.

biophysics↗

bioRxiv2018

Different features of cholera in malnourished and non-malnourised children: analysis of 10-year surveillance data from a large diarrheal disease hospital in urban Bangladesh

Background: Malnourished children are more prone to infectious diseases including severe diarrhea compared to non-malnourished children. Understanding of the differences in the presentation of severe diarrhea such as cholera in children with varying nutritional status may help in the early identification and management these children. However, data are scarce on differences in the presentation in such children. Thus, we aimed to identify the clinical differentials among children with cholera with or without malnutrition.\n\nMethods: Data were extracted from diarrheal disease surveillance system (DDSS) of the Dhaka Hospital of icddr,b for the period, January 2008 to December 2017. Among under-five children, cholera positive (culture confirmed) and malnourished children (weight-for-age, weight-for-length or height-for-age Z score (WAZ, WHZ or HAZ) <-2) were considered as the cases (n=305) and children with cholera but non-malnourished (WAZ, HAZ, and WHZ [≥]-2.00 to [≤]+2.00) were the controls (n=276).\n\nResults: A total of 14,403 under-five children were enrolled in the surveillance system during the study period. After adjusting for potential covariates such as maternal illiteracy and slum dwelling, it was revealed that under-five malnourished children with cholera significantly more often presented to the hospital during evening hours (6 pm to 12 mid-night) (OR=1.64, 95% CI=1.16-2.31, P<0.05), had fathers who were illiterate (OR=1.70, 95% CI=1.11-2.62, P<0.05), presented with history of cough within last 7 days (OR=1.64, 95% CI=1.10-2.43, P<0.05), dehydrating diarrhea (OR=1.70, 95% CI=1.15-2.53, P<0.05), and had longer hospitalization (OR=1.50, 95% CI=1.05-2.14, P<0.05).\n\nConclusions: The study results underscore the importance of understanding of the basic differences in the presentation of severe cholera in malnourished children for prompt identification and the subsequent management of these children. These observations may help policy makers in formulating better case management strategy.\n\nAuthor SummaryMalnourished children are more vulnerable to infectious diseases including cholera in comparison to the non-malnourished children. They often have suboptimal immune function, though there is no precise information on whether there is any difference in associated factor(s) or clinical course of cholera in under-five children with varying nutritional status. Therefore, this study was conducted to elucidate these insights by using the surveillance data of the Dhaka hospital of icddr,b. Among all the under-five children with cholera, 305 malnourished (WAZ or WLZ or HAZ <-2) children constituted as the cases (malnourished), and another 276 non-malnourished (WAZ, HAZ, and WHZ [≥]-2.00GtoG[≤]+2.00) cholera children formed the comparison group.\n\nIn this study we revealed that care seeking at evening time was more common in the malnourished children with cholera compared to those without malnutrition. Dehydrating diarrhea was about two folds higher and prolonged hospitalization was frequent in malnourished children with cholera than their counterparts. These key findings may help policy makers in formulating better case management strategy in the near future.

microbiology↗