Biology subjects

Khosravi, A.

Publications and source records attributed to Khosravi, A..

5 recordsLinked to original sources

bioRxiv2026

Purification of filamentous phage ssDNA genomes using Chaotropic salt/silica methods

Filamentous phages play important roles in molecular biology and bacterial pathogenesis. There is therefore great interest in the genomes of the phages, including M13 and Pf, which infect Escherichia coli and Pseudomonas aeruginosa, respectively. However, efficient methods for purifying single-stranded DNA (ssDNA) from filamentous phages are scarce and rely mainly on repurposed commercial kits developed for dsDNA. These kits yield variable amounts of ssDNA, varying considerably from one phage to another, their solutions are proprietary and troubleshooting, and customizing the protocol to improve purification is not possible. Here, we developed and optimized two protocols based on ssDNA binding to silica columns in the presence of guanidine hydrochloride and potassium iodide chaotropic salts, for filamentous phages M13KO7 and Pf4. The protocols achieved 60 to 100% recovery, equal to or higher than the Monarch Spin gDNA Extraction and DNeasy Blood and Tissue kits, with similar A260/280 and A260/230 ratios, intact DNA in agarose gels, and no detectable host genomic DNA by PCR. Recovery depended critically on the chaotropic salts and on ethanol, and did not increase at lower pH, consistent with a water-exclusion model in which hydrophobic interactions and hydrogen bonding dominate over electrostatic repulsion. Phage-specific differences in recovery indicate that optimization may be needed for some phages, which the fully customizable nature of these protocols facilitates. Comparing the virion ssDNA with the double-stranded replicative form (RF) revealed genome variants in both phages, including a shorter M13KO7 form lacking the kanamycin resistance-p15A insert, and illustrates the value of analyzing both RF and ssDNA forms to study filamentous phage biology. Implementing these protocols in routine filamentous phage workflows should facilitate their characterization and engineering.

molecular biology↗

bioRxiv2026

Phage-Mediated Iron Acquisition by Pseudomonas aeruginosa

Iron acquisition is critical to bacterial growth and pathogenesis. Here, we describe a previously unrecognized mechanism by which the major human pathogen Pseudomonas aeruginosa acquires iron through a unique partnership with bacteriophages (phages). Pf is a filamentous phage that infects P. aeruginosa and is associated with chronic infections. We reveal that Pf contributes to P. aeruginosa pathogenesis by promoting iron uptake. We demonstrate that Pf phage is highly induced under iron-deplete growth conditions and that once induced, Pf virions directly bind and locally concentrate free iron. Pf-bound iron is more efficiently utilized by P. aeruginosa than unbound iron, enhancing bacterial growth. We further demonstrate that Pf-mediated iron acquisition depends on type IV pili, which facilitate Pf attachment and confers strain selective uptake of phage-bound iron, providing a competitive fitness advantage in polymicrobial settings. Together, these findings identify an unrecognized role for filamentous phages in mediating iron acquisition and reveal a novel phage-bacterium partnership that operates through selective kin cooperation.

microbiology↗

bioRxiv2025

Pseudomonas superinfection drives Pf phage transmission within airway infections in patients with cystic fibrosis

Pf bacteriophages, lysogenic viruses that infect Pseudomonas aeruginosa (Pa), are implicated in the pathogenesis of chronic Pa infections; phage-infected (Pf+) strains are known to predominate in people with cystic fibrosis (pwCF) who are older and have more severe disease. However, the transmission patterns of Pf underlying the progressive dominance of Pf+ strains are unclear. In particular, it is unknown whether phage transmission commonly occurs horizontally between bacteria within the airway via viral particles or if Pf+ bacteria are mostly acquired via new Pseudomonas infections. Here, we have studied Pa genomic sequences from 3 patient cohorts totaling 663 clinical isolates from 105 pwCF. We identify Pf+ isolates and analyze transmission patterns of Pf within patients between genetically similar groups of bacteria called "clone types". We find that Pf is predominantly passed down vertically within Pa lineages and rarely via horizontal transfer between clone types within the airway. Conversely, we find extensive evidence of Pa superinfection by a new, genetically distinct Pa that is Pf+. Finally, we find that clinical isolates show reduced activity of the type IV pilus and reduced susceptibility to Pf in vitro. These results cast new light on the transmission of virulence-associated phages in the clinical setting.

microbiology↗

bioRxiv2024

Assessment of Stiffness-Dependent Autophagosome Formation and Apoptosis in Embryonal Rhabdomyosarcoma Tumor Cells

Summary/AbstractRemodeling of the extracellular matrix (ECM) eventually causes the stiffening of tumors and changes to the microenvironment. The stiffening alters the biological processes in cancer cells due to altered signaling through cell surface receptors. Autophagy, a key catabolic process in normal and cancer cells, is thought to be involved in mechano-transduction and the level of autophagy is probably stiffness-dependent. Here, we provide a methodology to study the effect of matrix stiffness on autophagy in embryonal rhabdomyosarcoma cells. To mimic stiffness, we seeded cells on GelMA hydrogel matrices with defined stiffness and evaluated autophagy-related endpoints. We also evaluated autophagy dependent pathways, apoptosis, and cell viability. Specifically, we utilized immunocytochemistry and confocal microscopy to track autophagosome formation through LC3 lipidation. This approach suggests that the use GelMA hydrogels with defined stiffness represent a novel method to evaluate the role of autophagy in embryonal rhabdomyosarcoma and other cancer cells. Workflow O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/583012v1_ufig1.gif" ALT="Figure 1"> View larger version (92K): org.highwire.dtl.DTLVardef@127453borg.highwire.dtl.DTLVardef@1325f8dorg.highwire.dtl.DTLVardef@1f2de0org.highwire.dtl.DTLVardef@fd0494_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2019

Cell Generation Dynamics Underlying Naïve T Cell Homeostasis in Adult Humans

Thymic involution and proliferation of naive T cells both contribute to shaping the naive T cell repertoire as humans age, but a clear understanding of the roles of each throughout a human lifespan has been difficult to determine. By measuring nuclear bomb test-derived 14C in genomic DNA we determined the turnover rates of CD4+ and CD8+ naive T cell populations and defined their dynamics in healthy individuals ranging from 20-65 years of age. We demonstrate that naive T cell generation decreases with age, and that this could be explained by a combination of declining cell loss, peripheral division and thymic production during adulthood. We investigated putative mechanisms underlying age-related changes in homeostatic regulation of naive T cell turnover using mass cytometry to profile candidate signaling pathways involved in T cell activation and proliferation in CD4+ naive T cells relative to CD31 expression, a marker of thymic proximity. We show that basal NF-{kappa}B phosphorylation inversely correlated with CD31 expression and thus is decreased in peripherally expanded naive T cell clones. Functionally we found that NF-{kappa}B signaling was essential for naive T cell proliferation to the homeostatic growth factor IL-7, and reduced NF-{kappa}B phosphorylation in CD4+CD31- naive T cells is linked to reduced homeostatic proliferation potential. Our results reveal an age-related decline in naive T cell turnover as a putative regulator of naive T cell diversity and identify a molecular pathway that restricts proliferation of peripherally expanded naive T cell clones that accumulate with age.

immunology↗