Expression of NADPH Oxidase (NOX) 5 in Rabbit Corneal Stromal Cells

To determine whether NOX 5 is expressed in rabbit corneal stromal cells (RCSC). NADPH oxidases (NOXes) are enzymes that preferentially use NADPH as a substrate and generate superoxide. Several isoforms of NOXes function as multi-protein complexes while NOX5 and DUOXs do not require the accessory proteins for their activity and possess calcium binding EF hands.Human NOX5 primers were used to amplify the rabbit NOX5 by RT-PCR. Amplified product was sequenced to confirm its identity. The protein encoded by the NOX5 was identified by western blot analysis. NOX5 siRNA was used to reduce transcript, protein, and calcium stimulated activity. In silico analyses were performed to establish the putative structure, functions, and evolution of rabbit NOX5.NOX activity was measured in RCSC with NADPH rather than NADH as a substrate. RT-PCR with NOX5 primers amplified 288 bp product using RCSC cDNA, which, when sequenced, confirmed its identity to human NOX5 mRNA. This sequence was used to predict the rabbit (Oryctolagus cuniculus) NOX5 gene. NOX5 siRNA reduced amounts of NOX5 mRNA in RCSC and reduced ionomycin stimulated superoxide production. A protein of about 65 to 70 kDa encoded by the NOX5 was detected by western blot analysis. In silico analysis predicted a putative rabbit NOX5 protein containing 801 amino acids. Motif searches predicted the presence of at least 3 putative EF-hands in N-terminus and a NOX domain in C terminal region.The data document that the NOX5 gene was expressed in cells of lagomorphs unlike rodents, making the rabbit an interesting model to study NOX5 functions. The activity of the rabbit NOX5 was calcium stimulated, a trait of NOX5 in general. NOX5 may also prove to be a useful genetic marker for studying the taxonomic position of lagomorphs and the Glires classification

Short interfering RNA against STAT1 attenuates cisplatin-induced ototoxicity in the rat by suppressing inflammation

Cisplatin is widely used for treating various solid tumors. However, this drug produces dose-limiting ototoxicity and nephrotoxicity, which significantly reduce the quality of life of cancer patients. While nephrotoxicity could be alleviated by diuresis, there is currently no approved treatment for hearing loss. Previous studies show that the ROS and inflammation are major contributors to cisplatin-induced hearing loss. In this study, we show that ROS trigger the inflammatory process in the cochlea by activating signal transducer and activator of transcription-1 (STAT1). Activation of STAT1 activation was dependent on ROS generation through NOX3 NADPH oxidase, knockdown of which by siRNA reduced STAT1 activation. Moreover, STAT1 siRNA protected against activation of p53, reduced apoptosis, reduced damage to OHCs and preserved hearing in rats. STAT1 siRNA attenuated the increase in inflammatory mediators, such as TNF-α, inhibition of which protected cells from cisplatin-mediated apoptosis. Finally, we showed that trans-tympanic administration of etanercept, a TNF-α antagonist, protected against OHC damage and cisplatin-induced hearing loss. These studies suggest that controlling inflammation by inhibition of STAT1-dependent pathways in the cochlea could serve as an effective approach to treat cisplatin ototoxicity and improve the overall quality of life for cancer patients

A Claudin-9–Based Ion Permeability Barrier Is Essential for Hearing

Hereditary hearing loss is one of the most common birth defects, yet the majority of genes required for audition is thought to remain unidentified. Ethylnitrosourea (ENU)–mutagenesis has been a valuable approach for generating new animal models of deafness and discovering previously unrecognized gene functions. Here we report on the characterization of a new ENU–induced mouse mutant (nmf329) that exhibits recessively inherited deafness. We found a widespread loss of sensory hair cells in the hearing organs of nmf329 mice after the second week of life. Positional cloning revealed that the nmf329 strain carries a missense mutation in the claudin-9 gene, which encodes a tight junction protein with unknown biological function. In an epithelial cell line, heterologous expression of wild-type claudin-9 reduced the paracellular permeability to Na+ and K+, and the nmf329 mutation eliminated this ion barrier function without affecting the plasma membrane localization of claudin-9. In the nmf329 mouse line, the perilymphatic K+ concentration was found to be elevated, suggesting that the cochlear tight junctions were dysfunctional. Furthermore, the hair-cell loss in the claudin-9–defective cochlea was rescued in vitro when the explanted hearing organs were cultured in a low-K+ milieu and in vivo when the endocochlear K+-driving force was diminished by deletion of the pou3f4 gene. Overall, our data indicate that claudin-9 is required for the preservation of sensory cells in the hearing organ because claudin-9–defective tight junctions fail to shield the basolateral side of hair cells from the K+-rich endolymph. In the tight-junction complexes of hair cells, claudin-9 is localized specifically to a subdomain that is underneath more apical tight-junction strands formed by other claudins. Thus, the analysis of claudin-9 mutant mice suggests that even the deeper (subapical) tight-junction strands have biologically important ion barrier function

Supplementary Material for: The optimization of guideline-directed medical therapy during hospitalization among patients with heart failure with reduced ejection fraction in daily clinical practice

Introduction: Hospitalization due to heart failure (HF) progression is associated with poor prognosis. This highlights the role of the implementation of guideline-directed medical therapy (GDMT) in improving the morbidity and mortality of patients with heart failure with reduced ejection fraction (HFrEF). There is limited data about the intrahospital applicability of GDMT in real-world circumstances. We aimed to assess retrospectively the use of cornerstone GDMT including RASi (ACEI/ARB/ARNI), βB, MRA, and SGLT2i treatment in a consecutive real-world HFrEF patient population admitted with signs and symptoms of heart failure to the HF unit of a Hungarian tertiary cardiac centre between 2019 and 2021. The independent predictors of therapy optimization and the applicability of new HFrEF medication (ARNI, SGLT2i, vericiguat) were also investigated. Methods: Statistical comparison of admission and discharge medication was accomplished with Fisher’s exact test. The independent predictors of the introduction of triple therapy (RASi+βB+MRA) were analyzed using univariate and multivariate logistic regression. The proportion of patients eligible for vericiguat based on the inclusion and exclusion criteria of the VICTORIA trial was also investigated, as well as the number of patients suitable for ARNI and SGLT2i, taking into account the contraindications of application contained in the ESC 2021 HF Guidelines. Results: 238 patients were included. During hospitalization, the use of RASi (69% vs. 89%) (ACEI/ARBs (58% vs. 70%), ARNI (10% vs. 19%)), βBs (69% vs. 85%), and MRAs (61% vs. 95%) increased significantly (p<0.05) compared to at admission, and the use of SGLT2i (3% vs. 11%) also rose (p=0.0005). The application ratio of triple (RASi+βB+MRA; 43% vs. 77%) and quadruple (RASi+βB+MRA+SGLT2i; 2% vs. 11%) therapy increased as well (p<0.0001). The independent predictors of discharge application of triple therapy revealed through multivariate logistic regression analysis were age, duration of hospitalization, eGFR, NTproBNP, and presence of diabetes mellitus. Sixty-eight percent of the cohort would have been suitable for vericiguat, 83% for ARNI, and 84% for SGLT2i. Conclusion: High rates of application of disease-modifying drugs are achievable among hospitalized HFrEF patients in severe clinical condition, thus awareness of the need for the initiation of the former must be raised

Detailed comparison of expressed and native voltage-gated proton channel currents

Two years ago, genes coding for voltage-gated proton channels in humans, mice and Ciona intestinalis were discovered. Transfection of cDNA encoding the human HVCN1 (HV1) or mouse (mVSOP) ortholog of HVCN1 into mammalian cells results in currents that are extremely similar to native proton currents, with a subtle, but functionally important, difference. Expressed proton channels exhibit high H+ selectivity, voltage-dependent gating, strong temperature sensitivity, inhibition by Zn2+, and gating kinetics similar to native proton currents. Like native channels, expressed proton channels are regulated by pH, with the proton conductance–voltage (gH–V) relationship shifting toward more negative voltages when pHo is increased or pHi is decreased. However, in every (unstimulated) cell studied to date, endogenous proton channels open only positive to the Nernst potential for protons, EH. Consequently, only outward H+ currents exist in the steady state. In contrast, when the human or mouse proton channel genes are expressed in HEK-293 or COS-7 cells, sustained inward H+ currents can be elicited, especially with an inward proton gradient (pHo < pHi). Inward current is the result of a negative shift in the absolute voltage dependence of gating. The voltage dependence at any given pHo and pHi is shifted by about −30 mV compared with native H+ channels. Expressed HV1 voltage dependence was insensitive to interventions that promote phosphorylation or dephosphorylation of native phagocyte proton channels, suggesting distinct regulation of expressed channels. Finally, we present additional evidence that speaks against a number of possible mechanisms for the anomalous voltage dependence of expressed H+ channels

A Novel Host Defense System of Airways Is Defective in Cystic Fibrosis

Rationale: The respiratory tract is constantly exposed to airborne microorganisms. Nevertheless, normal airways remain sterile without recruiting phagocytes. This innate immune activity has been attributed to mucociliary clearance and antimicrobial polypeptides of airway surface liquid. Defective airway immunity characterizes cystic fibrosis (CF), a disease caused by mutations in the CF transmembrane conductance regulator, a chloride channel. The pathophysiology of defective immunity in CF remains to be elucidated