Decreased Nrf2 protein level and low sperm quality in intractable spermatocystitis.

ABSTRACT: To investigate the molecular etiology of low sperm quality in patients with intractable spermatocystitis, spermatozoa samples from patients with persistent hematospermia undergoing transurethral seminal vesiculoscopy and healthy volunteers were utilized. Spermatozoa samples were collected from the seminal vesicles through transurethral seminal vesiculoscopy or by masturbation ejaculation. Sperm quality was analyzed by a WLJY-9000 color semen analysis system. Measurement of tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) in the seminal plasma was performed using enzyme-linked immunosorbent assay (ELISA). Measurement of H2O2 in the seminal plasma was performed with a hydrogen peroxide kit. The protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and phosphorylated-Nrf2 (p-Nrf2) were measured by western blot analysis and immunofluorescence assays. Low sperm quality parameters and increased levels of inflammatory cytokines (TNFα, IL-6, and H2O2) in the seminal plasma were detected among the semen samples from the patients with persistent hematospermia. Nrf2 and p-Nrf2 were strongly expressed in the nucleus and periphery of human sperm cells, according to the results of the immunofluorescence assays. The protein levels of Nrf2 and p-Nrf2 were significantly lower in the spermatozoa samples from patients with persistent hematospermia than in those from healthy volunteers with normal sperm motility. The results suggested that Nrf2 signaling might play a role in the low sperm quality of patients with intractable spermatocystitis.

Enzymatic Extraction of Bioactive and Self-Assembling Wool Keratin for Biomedical Applications.

Keratin is widely recognized as a high-quality renewable protein resource for biomedical applications. Despite their extensive existence, keratin resources such as feathers, wool, and hair exhibit high stability and mechanical properties because of their high disulfide bond content. Consequently, keratin extraction is challenging and its application is greatly hindered. In this work, a biological extraction strategy is proposed for the preparation of bioactive keratin and the fabrication of self-assembled keratin hydrogels (KHs). Based on moderate and controlled hydrolysis by keratinase, keratin with a high molecular weight of approximately 45 and 28 kDa that retain its intrinsic bioactivities is obtained. The keratin products show excellent ability to promote cell growth and migration and are conferred with significant antioxidant ability because of their intrinsically high cysteine content. In addition, without the presence of any cross-linking agent, the extracted keratin can self-assemble into injectable hydrogels. The KHs exhibit a porous network structure and 3D culture ability, showing potential in promoting wound healing. This enzyme-driven keratin extraction strategy opens up a new approach for the preparation of keratin that can self-assemble into injectable hydrogels for biomedical engineering.

Fabrication and characterization of high molecular keratin based nanofibrous membranes for wound healing.

Keratin is widely used in the biomaterial application, but the keratin prepared by the physical or chemical approach has relatively low molecular weight and mechanical properties. Here we report the preparation of high molecular keratin (HMK) with molecular weight of 120 kDa via multi-enzyme cascade pathway and its application in wound healing. Briefly, we prepared the soluble keratin from wool by keratinase and improved the molecular weight of keratin by transglutaminase (TGase). The HMK was coelectrospun with poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) and the prepared nanofibrous mats demonstrated improved mechanical properties. Ag nanoparticles (AgNPs) were synthesized on the nanofibers via in situ bioreduction, using the above-mentioned keratinase as the reducing agent. It is demonstrated that the PHBV/HMK/AgNPs nanofibrous mats possess favorable antibacterial properties and good biocompatibility. Moreover, in vivo wound healing assessment, the PHBV/HMK/AgNPs membrane displayed better wound healing ability than the control group. These results indicate that PHBV/HMK/AgNPs mats exhibit significant potential in tissue engineering.

Efficient keratinase expression via promoter engineering strategies for degradation of feather wastes.

Keratinases are promising alternatives over ordinary proteases in several industrial applications due to their unique properties compared with their counterparts in the protease categories. However, their large-scale industrial application is limited by the low expression and poor fermentation efficiency of keratinase. Here, we demonstrate that the expression level of keratinase can be improved by constructing a more efficient enzyme expression system hereby enables the highest production titer as regarding recombinant keratinase production to date. Specially, ten promoters were evaluated and the aprE promoter exhibits a significant promotion of keratinase (kerBv) titer from 165 U/mL to 2605 U/mL in Bacillus subtilis. The batch fermentation mode resulted in a maximum keratinase activity of 7176 U/mL at 36 h in a 5-L fermenter. Furthermore, the extracellular keratinase activity attained up to 16,860 U/mL via fed-batch fermentation within 30 h. The combination of keratinase with l-cysteine brings about 66.4 % degree of degradation of feather. Our work provides a new insight into the development of efficient keratinase fermentation processes with B. subtilis cell factory.