Transduction of Adeno-Associated Virus Vectors Targeting Hair Cells and Supporting Cells in the Neonatal Mouse Cochlea

Adeno-associated virus (AAV) is the preferred vector for gene therapy of hereditary deafness, and different viral serotypes, promoters and transduction pathways can influence the targeting of AAV to different types of cells and the expression levels of numerous exogenous genes. To determine the transduction and expression patterns of AAV with different serotypes or promoters in hair cells and supporting cells in the neonatal mouse cochlea, we examined the expression of enhanced green fluorescent protein (eGFP) for five different types of AAV vectors [serotypes 2, 9, and Anc80L65 with promoter cytomegalovirus (CMV)-beta-Globin and serotypes 2 and 9 with promoter chicken beta-actin (CBA)] in in vitro cochlear explant cultures and we tested the transduction of AAV2/2-CBA, AAV2/9-CBA, and AAV2/Anc80L65-CMV by in vivo microinjection into the scala media of the cochlea. We found that each AAV vector had its own transduction and expression characteristics in hair cells and supporting cells in different regions of the cochlea. There was a tonotopic gradient for the in vitro transduction of AAV2/2-CBA, AAV2/9-CBA, AAV2/2-CMV, and AAV2/9-CMV in outer hair cells (OHCs), with more OHCs expressing eGFP at the base of the cochlea than at the apex. AAV2/2-CBA in vitro and AAV2/Anc80L65-CMV in vivo induced more supporting cells expressing eGFP at the apex than in the base. We found that AAV vectors with different promoters had different expression efficacies in hair cells and supporting cells of the auditory epithelium. The CMV-beta-Globin promoter could drive the expression of the delivered construct more efficiently in hair cells, while the CBA promoter was more efficient in supporting cells. The in vitro and in vivo experiments both demonstrated that AAV2/Anc80L65-CMV was a very promising vector for gene therapy of deafness because of its high transduction rates in hair cells. These results might be useful for selecting the appropriate vectors for gene delivery into different types of inner ear cells and thus improving the effectiveness of gene therapy

Temperature Management With Paracetamol in Acute Stroke Patients: Evidence From Randomized Controlled Trials

Whether or not paracetamol can improve functional outcomes in patients with acute stroke has been examined in several clinical trials. The inconsistent results of these trials have caused great controversy regarding the need for further studies. In the present meta-analysis, we have aimed to address this controversy. The main databases (Medline, Embase, and Cochrane Library) were searched for randomized controlled trials involving the use of paracetamol in acute stroke patients. Pooled relative risks (RRs) or mean differences (MDs) and 95% confidence intervals (CIs) were calculated using a random-effects model. A total of 1,836 patients were pooled from four phase II and two phase III trials. The use of paracetamol resulted in a significant reduction in body temperature after 24 h (MD, −0.21; 95% CI, −0.28 to −0.13; P < 0.001) and mortality rate after 7–14 days (RR, 0.62; 95% CI, 0.41–0.93; P = 0.02) when compared with the placebo group; however, no effect of paracetamol was observed in the modified Rankin Scale score (RR, 1.07; 95% CI, 0.91–1.27; P = 0.40) or Barthel Index score (RR, 0.98; 95% CI, 0.91–1.06; P = 0.63) at 30 or 90 days. No significant differences were observed with respect to serious adverse events between the paracetamol and the placebo groups (P > 0.05). Subgroup analyses were performed to detect the source of the heterogeneity, which showed that ischemic stroke, serious condition at baseline, and late time-to-treatment had adverse impacts on the effect of paracetamol post stroke. In conclusion, temperature management with paracetamol in acute stroke patients is safe. Although paracetamol reduced the mortality rate in the early stage of stroke, it did not appear to affect long-term mortality and functional recovery. It should be noted that this conclusion is based on the results from studies of poor quality. A large clinical trial with a focus on early treatment of patients with acute stroke is warranted

Permeability Prediction Model Modified on Kozeny-Carman for Building Foundation of Clay Soil

Clay soil is a common building foundation material, and its permeability is very important for the safety of foundation pits and the later settlement of buildings. However, the traditional Kozeny-Carman (K-C) equation shows serious discrepancies when predicting the permeability of clay in building foundation treatment. Therefore, solving the application of K-C equation in clay is a problem faced by the engineers and scholars. In this paper, the influence of clay mineralogy on pore structure and permeability is analyzed, and then the effective e (eeff) and effective SSA (Seff) are proposed. Based on the eeff and Seff, the permeability prediction model modified on Kozeny-Carman is built. Then, seepage experiments are conducted on two types of clay samples to test this prediction model; at the same time, the MIP combining freeze-drying methods are used to obtain the Seff and eeff. Through the discussion of the test results, three main conclusions are obtained: (1) there are invalid pores in clay due to the influence of clay mineral, this is the reason for which K-C equation is unsuitable for clay; (2) the eeff and Seff can reflect the structural state of clay during seepage; (3) the results of the permeability prediction model in this paper agree well with the test results, which indicates that this prediction model is applicable to clay. The research results of this paper are significant to solve the academic problem that K-C equation is not applicable to clay and significant to ensure the safety of building foundation pits in clay areas

Massively Parallel Sequencing of a Chinese Family with DFNA9 Identified a Novel Missense Mutation in the LCCL Domain of COCH

DFNA9 is a late-onset, progressive, autosomal dominantly inherited sensorineural hearing loss with vestibular dysfunction, which is caused by mutations in the COCH (coagulation factor C homology) gene. In this study, we investigated a Chinese family segregating autosomal dominant nonsyndromic sensorineural hearing loss. We identified a missense mutation c.T275A p.V92D in the LCCL domain of COCH cosegregating with the disease and absent in 100 normal hearing controls. This mutation leads to substitution of the hydrophobic valine to an acidic amino acid aspartic acid. Our data enriched the mutation spectrum of DFNA9 and implied the importance for mutation screening of COCH in age related hearing loss with vestibular dysfunctions

Correction: Chen, S., et al. Identification of an Essential Region for Translocation of Clostridium difficile Toxin B. Toxins 2016, 8, 241

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Evaluate the value of prolonging the duration of tiopronin for injection administration in preventing hepatotoxicity

Abstract As part of supportive therapy, prophylaxis with tiopronin for injection (TI) against common hepatotoxicity complications has often been used. However, methods to prevent hepatotoxicity have not been established. Therefore, our study was aimed to find out the relationship between the periods of TI prophylaxis and post-treatment hepatotoxicity, and evaluated the value of prolonging the duration of TI administration in preventing hepatotoxicity. Hepatotoxicity was detected through liver transaminases, bilirubin, alkaline phosphatase, and clinical features of liver insufficiency. Multivariable logistic regressions were conducted to examine the association of the periods of TI prophylaxis and post-treatment hepatotoxicity. Between January 2022 and March 2023, a total of 452 patients with gynecological cancer were enrolled in the study, of which 93 (20.58%) participants were post-treatment hepatotoxicity positive. TI with different prevention days were no significant difference among participants with or without post-treatment hepatotoxicity in crude model (P > 0.05). The P-value, the odds ratios (OR) and 95% confidence intervals (CI) of participants with TI prophylaxis for 1 day for post-treatment hepatotoxicity were 0.040, 3.534 (1.061–11.765) in fully adjusted model. Past history of hepatotoxicity is a confounding variable, and there was no significant difference for post-treatment hepatotoxicity when stratified by past history of hepatotoxicity (P > 0.05). The study indicate that the periods of TI prophylaxis is not associated with post-treatment hepatotoxicity, suggesting that prolonged the periods of TI prophylaxis might be an invalid method for the prevention of post-treatment hepatotoxicity

Identification of an Essential Region for Translocation of Clostridium difficile Toxin B

Clostridium difficile toxin A (TcdA) and toxin B (TcdB) are the major virulence factors involved in C. difficile-associated diarrhea and pseudomembranous colitis. TcdA and TcdB both contain at least four distinct domains: the glucosyltransferase domain, cysteine protease domain, receptor binding domain, and translocation domain. Few studies have investigated the translocation domain and its mechanism of action. Recently, it was demonstrated that a segment of 97 amino acids (AA 1756–1852, designated D97) within the translocation domain of TcdB is essential for the in vitro and in vivo toxicity of TcdB. However, the mechanism by which D97 regulates the action of TcdB in host cells and the important amino acids within this region are unknown. In this study, we discovered that a smaller fragment, amino acids 1756–1780, located in the N-terminus of the D97 fragment, is essential for translocation of the effector glucosyltransferase domain into the host cytosol. A sequence of 25AA within D97 is predicted to form an alpha helical structure and is the critical part of D97. The deletion mutant TcdB∆1756–1780 showed similar glucosyltransferase and cysteine protease activity, cellular binding, and pore formation to wild type TcdB, but it failed to induce the glucosylation of Rho GTPase Rac1 of host cells. Moreover, we found that TcdB∆1756–1780 was rapidly degraded in the endosome of target cells, and therefore its intact glucosyltransferase domain was unable to translocate efficiently into host cytosol. Our finding provides an insight into the molecular mechanisms of action of TcdB in the intoxication of host cells

<i>Monascus</i> Yellow Pigment Production by Coupled Immobilized-Cell Fermentation and Extractive Fermentation in Nonionic Surfactant Micelle Aqueous Solution

Microbial fermentation with immobilized cells possesses many advantages. However, this fermentation mode is restricted to the production of extracellular products. Our previous study demonstrated that the extractive fermentation of Monascus spp. in nonionic surfactant micelle aqueous solution can export Monascus pigments that are supposed to be mainly intracellular products to extracellular culture broth and, in the meantime, extracellularly enhance the production of yellow pigments at a low pH condition; consequently, this makes the continuous production of yellow pigments with immobilized Monascus cells feasible. In this study, immobilized-cell fermentation and extractive fermentation in Triton X-100 micelle aqueous solution were successfully combined to continuously produce Monascus yellow pigments extracellularly. We examined the effects of cell immobilization and Triton X-100 on cell growth, pigment production, and pigment composition. In the repeated-batch extractive fermentation with immobilized cells, the biomass in Ca-alginate gel beads continued to grow and reached 21.2 g/L after seven batches, and dominant yellow pigments were produced extracellularly and stable for each batch. The mean productivity of the extracellular yellow pigments reached up to 22.31 AU410 nm/day within the first four batches (13 days) and 19.7 AU410 nm/day within the first seven batches (25 days). The results also provide a new strategy for producing such intracellular products continuously and extracellularly

Novel Cysteine Desulfidase CdsB Involved in Releasing Cysteine Repression of Toxin Synthesis in Clostridium difficile

Clostridium difficile, a major cause of nosocomial diarrhea and pseudomembranous colitis, still poses serious health-care challenges. The expression of its two main virulence factors, TcdA and TcdB, is reportedly repressed by cysteine, but molecular mechanism remains unclear. The cysteine desulfidase CdsB affects the virulence and infection progresses of some bacteria. The C. difficile strain 630 genome encodes a homolog of CdsB, and in the present study, we analyzed its role in C. difficile 630Δerm by constructing an isogenic ClosTron-based cdsB mutant. When C. difficile was cultured in TY broth supplemented with cysteine, the cdsB gene was rapidly induced during the exponential growth phase. The inactivation of cdsB not only affected the resistance of C. difficile to cysteine, but also altered the expression levels of intracellular cysteine-degrading enzymes and the production of hydrogen sulfide. This suggests that C. difficile CdsB is a major inducible cysteine-degrading enzyme. The inactivation of the cdsB gene in C. difficile also removed the cysteine-dependent repression of toxin production, but failed to remove the Na2S-dependent repression, which supports that the cysteine-dependent repression of toxin production is probably attributable to the accumulation of cysteine by-products. We also mapped a δ54 (SigL)-dependent promoter upstream from the cdsB gene, and cdsB expression was not induced in response to cysteine in the cdsR::ermB or sigL::ermB strain. Using a reporter gene fusion analysis, we identified the necessary promoter sequence for cysteine-dependent cdsB expression. Taken together, these results indicate that CdsB is a key inducible cysteine desulfidase in C. difficile which is regulated by δ54 and CdsR in response to cysteine and that cysteine-dependent regulation of toxin production is closely associated with cysteine degradation