Biology subjects

Ozturk, T.

Publications and source records attributed to Ozturk, T..

6 recordsLinked to original sources

bioRxiv2025

CRISPR/Cas13-mediated Dynamin 2 reduction therapy in a canine model of DNM2-related centronuclear myopathy

We determined the potential of CRISPR/Cas13 technology as a therapeutic approach for centronuclear myopathies (CNMs) by reducing the expression of a single protein, DNM2. CNMs are severe congenital rare muscle disorders that result in muscle hypotrophy and weakness, with no cure. CNMs frequently result from mutations in either BIN1, MTM1, or DNM2 genes, with DNM2 being a key GTPase that plays a pivotal role in muscle membrane interactions with MTM1 and BIN1. Previous studies indicate that reducing DNM2 transcript expression by half could correct CNM phenotypes regardless the genetic forms, paving the way for a broad-spectrum CNM-therapy. We evaluated CRISPR/Cas13X.1-mediated DNM2 transcript knockdown, as a therapeutic application in a unique naturally-occurring canine CNM model harboring the DNM2R465W/+ mutation, the most frequent pathogenic variant in patients. We show that in vivo intramuscular AAV-mediated CRISPR/Cas13X.1 injections, led to a reduction in DNM2 transcript and protein levels at one and two months post-treatment. Our results demonstrate the feasibility of CRISPR/Cas13-based therapy for CNM in a large animal model, paving the way for advancing this approach towards clinical trials.

molecular biology↗

bioRxiv2025

Metabolic heterogeneity coupled with a resistance gene generates antibiotic heteroresistance

Heteroresistance is a form of antibiotic resistance in which a phenotypically unstable subpopulation of resistant cells exists within a majority population of susceptible cells. The impact of the extracellular environment on mechanisms of heteroresistance is unclear. Studying fosfomycin heteroresistance in an Enterobacter cloacae complex isolate, we observed that glucose availability greatly increased the frequency of the resistant subpopulation. Glucose downregulated the glycerol and fosfomycin importer GlpT, whose expression was heterogenous at the single cell level. This heterogeneous expression of GlpT, in combination with the expression of the fosfomycin resistance gene fosA, which acted as a resistance enhancer, led to the generation of heteroresistance. Correspondingly, the frequency of the fosfomycin resistant subpopulation was increased in murine models of hyperglycemia/diabetes. These data demonstrate how metabolic heterogeneity and carbon source availability can impact antibiotic resistance phenotypes in the infection environment.

microbiology↗

bioRxiv2025

Sulbactam-durlobactam susceptibility among cefiderocol heteroresistant Acinetobacter

The ATTACK clinical trial for treatment of carbapenem-resistant Acinetobacter baumannii-calcoaceticus complex (CRAB) isolates determined treatment with sulbactam-durlobactam to be efficacious and safe. However, other newly introduced {beta}-lactam antibiotics, including the novel cephalosporin cefiderocol, have been compromised upon clinical introduction by a type of antibiotic resistance called heteroresistance, in which only a small subpopulation of total cells exhibit phenotypic resistance. Therefore, we sought to test for sulbactam-durlobactam heteroresistance, as well as whether sulbactam-durlobactam was effective against cefiderocol heteroresistant CRAB isolates. We did not observe heteroresistance (or conventional resistance) to sulbactam-durlobactam among the 107 carbapenem-resistant Acinetobacter isolates tested, consistent with the efficacy of this new antibiotic in the ATTACK trial. Further, sulbactam-durlobactam was active against cefiderocol heteroresistant CRAB, highlighting that this antibiotic may be prioritized in relation to cefiderocol in treating CRAB infections.

microbiology↗

bioRxiv2024

Targeted-SMN insufficiency in Skeletal Muscle Stem Cells mediates non-cell autonomous loss of motor neurons at long term

Spinal Muscular Atrophy (SMA) is due to a deficit in SMN protein encoded by the SMN1 gene. SMN-targeted disease modifying treatments have greatly improved the clinical outcomes of this neuromuscular disease. However, uncertainties remain regarding their long-term efficacy and non-neuronal tissue involvement in disease progression. We found that SMA type II patient muscles display a reduced number of quiescent PAX7+ Muscle Stem Cells (MuSC). In SMA mice, we showed that SMN is an important regulator of myogenic progenitor fate during early postnatal growth. In Pax7 Cre-driven conditional knockout mouse models, we demonstrated that high levels of SMN are required to ensure the maintenance of the quiescent MuSC pool in adult muscle. We further established that depletion of SMN-deficient MuSC yielded neuromuscular junctions remodeling followed by a non-cell autonomous loss of motor neurons in the long term. Overall, our findings demonstrate that MuSC are a crucial therapeutic target for SMA treatment. HIGHLIGHTSO_LISMN regulates myogenic lineage progression and quiescent MuSC pool establishment during postnatal growth C_LIO_LIBoth Smn alleles are necessary for the survival of quiescent MuSC in adult muscle C_LIO_LIDepletion of SMN-deficient MuSC leads to NMJ remodeling and non-cell autonomous loss of MN C_LI

pathology↗

bioRxiv2022

A single-molecule method for measuring fluorophore labeling yields for the study of membrane protein oligomerization in membranes

Membrane proteins are often structured as higher-order oligomers. Yet, the role of these specific assemblies is not always apparent, raising the question of whether differential oligomerization states can be linked to modulation of function. To better understand this hypothetical regulatory mechanism, there is an ongoing effort to quantify equilibrium reactions of membrane proteins in membranes. Single-molecule photobleaching analysis is particularly useful for this as it provides a binary readout of fluorophores attached to protein subunits at dilute conditions. The subunit capture method adds consideration of the Poisson probability of protein partitioning into liposomes from large equilibrium membranes. If the liposome size distribution is known, then the capture statistics can be modeled with accuracy to quantify oligomerization as a function of membrane density to obtain binding isotherms, as was demonstrated for the dimeric chloride/proton antiporter CLC-ec1. However, any quantification of stoichiometry also critically requires knowing the probability that a subunit is fluorescently labeled. Since labeling uncertainty is often unavoidable, we tested an alternate approach to estimate labeling yields using the photobleaching probability of an intrinsic dimeric control, the disulfide cross-linked R230C/L249C CLC-ec1. By iterative fitting of the experimental dimeric photobleaching probability distribution to a dimer model while varying labeling parameters, we predict the labeling yields measured by direct absorbance measurements of the purified protein before reconstitution. Finally, the average predicted labeling yield over multiple samples is used to estimate the dissociation constant of CLC-ec1 dimerization reactions, eliminating the need to quantify fluorophore labeling a priori. This approach can be generalized to study dimerization reactions where an irreversible dimeric control can be prepared. Thus, our study maps out a new method for quantifying fluorophore occupancy in samples that cannot be purified directly and improves quantification of membrane protein stoichiometry in membranes.

biophysics↗

bioRxiv2022

Heteroresistance via beta-lactamase gene amplification threatens the beta-lactam pipeline

It is unclear how bacteria rapidly adapt to resist novel drugs upon their introduction. Heteroresistance (HR) is a form of antibiotic resistance where a phenotypically unstable minority resistant subpopulation co-exists with a susceptible population. We observed HR to cefiderocol, a novel beta-lactam developed to resist beta-lactamases including extended-spectrum-beta-lactamases (ESBLs), among clinical isolates collected before its use. The resistant subpopulation in Enterobacter was a continuum; increasing gene copy number of an otherwise ineffective ESBL mediated increased resistance in decreasing numbers of cells. The factors that control the magnitude of amplification are unclear. We observed that ESBL activity controlled the level of amplification, thus increased copy number can compensate for poor enzymatic activity. A Klebsiella isolate from a clinical treatment failure also demonstrated amplification, highlighting the relevance of this phenomenon. These data provide insights into factors controlling dynamics of HR and how bacteria use phenotypic resistance to flexibly confront new antibiotic threats.

microbiology↗