RNA m6A modification regulates L1 retrotransposons in human spermatogonial stem cell differentiation in vitro and in vivo.

The maintenance of genome integrity in the germline is crucial for mammalian development. Long interspersed element type 1 (LINE-1, L1) is a mobile genetic element that makes up about 17% of the human genome and poses a threat to genome integrity. N6-methyl-adenosine (m6A) plays an essential role in regulating various biological processes. However, the function of m6A modification in L1 retrotransposons and human germline development remains largely unknown. Here we knocked out the m6A methyltransferase METTL3 or the m6A reader YTHDF2 in human embryonic stem cells (hESCs) and discovered that METTL3 and YTHDF2 are crucial for inducing human spermatogonial stem cells (hSSCs) from hESCs in vitro. The removal of METTL3 or YTHDF2 resulted in increased L1 retrotransposition and reduced the efficiency of SSC differentiation in vitro. Further analysis showed that YTHDF2 recognizes the METTL3-catalyzed m6A modification of L1 retrotransposons and degrades L1 mRNA through autophagy, thereby blocking L1 retrotransposition. Moreover, the study confirmed that m6A modification in human fetal germ cells promotes the degradation of L1 retrotransposon RNA, preventing the insertion of new L1 retrotransposons into the genome. Interestingly, L1 retrotransposon RNA was highly expressed while METTL3 was significantly downregulated in the seminal plasma of azoospermic patients with meiotic arrest compared to males with normal fertility. Additionally, we identified some potentially pathogenic variants in m6A-related genes in azoospermic men with meiotic arrest. In summary, our study suggests that m6A modification serves as a guardian of genome stability during human germline development and provides novel insights into the function and regulatory mechanisms of m6A modification in restricting L1 retrotransposition.

Application of multi-layer porcine small intestinal submucosa for the reconstruction of deep corneal defects in cats.

OBJECTIVE: This study documented the application of porcine small intestinal submucosa (SIS) as a stand-alone scaffold for treating deep corneal defects in cats. METHODS: Medical records of 20 cats with deep stromal ulcers, perforations, or corneal sequestra that underwent surgical treatment with SIS grafts between 2021 and 2022 were retrospectively reviewed. Data on re-epithelialization time, corneal transparency score, and complications were collected to analyze the reconstruction of deep corneal defects after SIS biomaterial implantation. RESULTS: All cats were unilaterally affected. The corneal defects varied in size, with a median diameter of 8.3 mm (range: 3-15 mm). Re-epithelialization of the SIS graft was completed 16-32 days after surgery (median, 22.3 days). No, mild, or moderate corneal transparency was detected in 90% of the cases. Complications were observed in eight cases (40%), including aqueous leakage (10%), partial SIS malacia (25%), and persistent bullous keratopathy (5%). The follow-up period ranged 90-725 days, with a median duration of 255 days. The SIS graft was successfully applied as a single scaffold in 17 of 20 cases (85%). CONCLUSION: The results of this study suggest that the application of commercial SIS is an effective surgical technique for managing deep corneal defects in cats.

Prophylatic use of IV nalmefene to prevent epidural opioid-induced pruritus: A multicenter, randomized clinical trial.

STUDY OBJECTIVE: The incidence of pruritus from neuraxial opioids is about 60%. Pruritus causes discomfort and decreases the quality of recovery. This randomized double-blinded clinical trial was aimed to evaluate the prophylactic effects of a single dose IV nalmefene on the incidence and severity of epidural opioid-induced pruritus within 24 h after surgeries. DESIGN: A two-center, randomized, double blinded, controlled clinical trial. SETTING: The study was conducted from March 2022 to February 2023 at two tertiary care hospitals in China. PATIENTS: Patients aged between 18 and 80 years-old who underwent elective surgeries and received epidural analgesia intra- and post-operatively were screened for study enrollment. A total of 306 patients were enrolled, 302 patients underwent randomization and 296 patients were included in the final analysis. INTERVENTIONS: The nalmefene group was prophylactically given 0.5 µg/kg nalmefene intravenously while the control group was given the same volume of saline. MEASUREMENTS: The primary endpoint was the incidence of pruritus within 24 h after surgeries. The secondary endpoints included time of the first patient-reported pruritus, severity of pruritus after surgeries, severity of acute pain scores after surgeries and other anesthesia/analgesia related side effects. MAIN RESULTS: Pruritus occurred in 51 of the 147 (34.69%) patients in the control group and 35 of the 149 (23.49%) patients in the nalmefene group (odds ratio, 0.58; 95% CI, 0.35 to 0.96; P = 0.034) within 24 h postoperatively. Nalmefene group demonstrated delayed onset of pruritus, reduced severity of pruritus and decreased vomiting within 24 h after surgery. There were no significant differences in postoperative analgesia and the incidence of other anesthesia/analgesia associated side effects. CONCLUSIONS: A single dose of 0.5 µg/kg nalmefene intravenously significantly reduced the incidence and severity of epidural-opioid induced pruritus within 24 h after surgery without affecting the efficacy of epidural analgesia. TRIAL REGISTRATION: Chinese Clinical Trial Registry (www.chictr.org.cn) and the registration number is ChiCTR2100050463. Registered on August 27th, 2021.

Engineered mitochondria exert potent antitumor immunity as a cancer vaccine platform.

The preferable antigen delivery profile accompanied by sufficient adjuvants favors vaccine efficiency. Mitochondria, which feature prokaryotic characteristics and contain various damage-associated molecular patterns (DAMPs), are easily taken up by phagocytes and simultaneously activate innate immunity. In the current study, we established a mitochondria engineering platform for generating antigen-enriched mitochondria as cancer vaccine. Ovalbumin (OVA) and tyrosinase-related protein 2 (TRP2) were used as model antigens to synthesize fusion proteins with mitochondria-localized signal peptides. The lentiviral infection system was then employed to produce mitochondrial vaccines containing either OVA or TRP2. Engineered OVA- and TRP2-containing mitochondria (OVA-MITO and TRP2-MITO) were extracted and evaluated as potential cancer vaccines. Impressively, the engineered mitochondria vaccine demonstrated efficient antitumor effects when used as both prophylactic and therapeutic vaccines in murine tumor models. Mechanistically, OVA-MITO and TRP2-MITO potently recruited and activated dendritic cells (DCs) and induced a tumor-specific cell-mediated immunity. Moreover, DC activation by mitochondria vaccine critically involves TLR2 pathway and its lipid agonist, namely, cardiolipin derived from the mitochondrial membrane. The results demonstrated that engineered mitochondria are natively well-orchestrated carriers full of immune stimulants for antigen delivery, which could preferably target local dendritic cells and exert strong adaptive cellular immunity. This proof-of-concept study established a universal platform for vaccine construction with engineered mitochondria bearing alterable antigens for cancers as well as other diseases.