10 research outputs found
Exploring the secrets of virus entry: the first respiratory syncytial virus carrying beta lactamase
BackgroundRespiratory Syncytial Virus (RSV) presents a significant health threat, especially to young children. In-depth understanding of RSV entry mechanisms is essential for effective antiviral development. This study introduces an innovative RSV variant, featuring the fusion of the beta-lactamase (BlaM) enzyme with the RSV-P phosphoprotein, providing a versatile tool for dissecting viral entry dynamics.MethodsUsing the AlphaFold2 algorithm, we modeled the tertiary structure of the P-BlaM chimera, revealing structural similarities with both RSV-P and BlaM. Functional assessments, utilizing flow cytometry, quantified beta-lactamase activity and GFP expression in infected bronchial epithelial cells. Western blot analysis confirmed the integrity of P-BlaM within virions.ResultsThe modeled P-BlaM chimera exhibited structural parallels with RSV-P and BlaM. Functional assays demonstrated robust beta-lactamase activity in recombinant virions, confirming successful P-BlaM incorporation as a structural protein. Quercetin, known for its antiviral properties, impeded viral entry by affecting virion fusion. Additionally, Ulixertinib, an ERK-1/2 inhibitor, significantly curtailed viral entry, implicating ERK-1/2 pathway signaling.ConclusionsOur engineered RSV-P-BlaM chimera emerges as a valuable tool, illuminating RSV entry mechanisms. Structural and functional analyses unveil potential therapeutic targets. Quercetin and Ulixertinib, identified as distinct stage inhibitors, show promise for targeted antiviral strategies. Time-of-addition assays pinpoint quercetin’s specific interference stage, advancing our comprehension of RSV entry and guiding future antiviral developments
Adipose tissue-derived WNT5A regulates vascular redox signaling in obesity via USP17//RAC1-mediated activation of NADPH oxidases
Obesity is associated with changes in the secretome of adipose tissue (AT), which affects the vasculature through endocrine and paracrine mechanisms. Wingless-related integration site 5A (WNT5A) and secreted frizzled-related protein 5 (SFRP5), adipokines that regulate noncanonical Wnt signaling, are dysregulated in obesity. We hypothesized that WNT5A released from AT exerts endocrine and paracrine effects on the arterial wall through noncanonical RAC1-mediated Wnt signaling. In a cohort of 1004 humans with atherosclerosis, obesity was associated with increased WNT5A bioavailability in the circulation and the AT, higher expression of WNT5A receptors Frizzled 2 and Frizzled 5 in the human arterial wall, and increased vascular oxidative stress due to activation of NADPH oxidases. Plasma concentration of WNT5A was elevated in patients with coronary artery disease compared to matched controls and was independently associated with calcified coronary plaque progression. We further demonstrated that WNT5A induces arterial oxidative stress and redox-sensitive migration of vascular smooth muscle cells via Frizzled 2–mediated activation of a previously uncharacterized pathway involving the deubiquitinating enzyme ubiquitin-specific protease 17 (USP17) and the GTPase RAC1. Our study identifies WNT5A and its downstream vascular signaling as a link between obesity and vascular disease pathogenesis, with translational implications in humans
Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Gene Editing Technique in Xenotransplantation
Genetically modified pigs have been considered favorable resources in xenotransplantation. Microinjection of randomly integrating transgenes into zygotes, somatic cell nuclear transfer, homologous recombination, zinc finger nucleases, transcription activator-like effector nucleases, and most recently, clustered regularly interspaced short palindromic repeats-cas9 (CRISPR/Cas9) are the techniques that have been used to generate these animals. Here, we provide an overview of the CRISPR approaches that have been used to modify genes which are vital in improving xenograft survival rate, including cytidine monophosphate-N-acetylneuraminic acid hydroxylase, B1,4N-acetylgalactosaminyltransferase, isoglobotrihexosylceramide synthase, class I MHC, von Willebrand factor, C3, and porcine endogenous retroviruses. In addition, we will mention the importance of potential candidate genes which could be targeted using CRISPR/Cas9
Adipose tissue-derived WNT5A regulates vascular redox signaling in obesity via USP17/RAC1-mediated activation of NADPH oxidases
Obesity is associated with changes in the secretome of adipose tissue
(AT), which affects the vasculature through endocrine and paracrine
mechanisms. Wingless-related integration site 5A (WNT5A) and secreted
frizzled-related protein 5 (SFRP5), adipokines that regulate
noncanonical Wnt signaling, are dysregulated in obesity. We hypothesized
that WNT5A released from AT exerts endocrine and paracrine effects on
the arterial wall through noncanonical RAC1-mediated Wnt signaling. In a
cohort of 1004 humans with atherosclerosis, obesity was associated with
increased WNT5A bioavailability in the circulation and the AT, higher
expression of WNT5A receptors Frizzled 2 and Frizzled 5 in the human
arterial wall, and increased vascular oxidative stress due to activation
of NADPH oxidases. Plasma concentration of WNT5A was elevated in
patients with coronary artery disease compared to matched controls and
was independently associated with calcified coronary plaque progression.
We further demonstrated that WNT5A induces arterial oxidative stress and
redox-sensitive migration of vascular smooth muscle cells via Frizzled
2-mediated activation of a previously uncharacterized pathway involving
the deubiquitinating enzyme ubiquitin-specific protease 17 (USP17) and
the GTPase RAC1. Our study identifies WNT5A and its downstream vascular
signaling as a link between obesity and vascular disease pathogenesis,
with translational implications in humans