Prolonging photoperiod promotes testosterone synthesis of Leydig cells by directly targeting local melatonin system in rooster testes†.

The study investigated the effects of prolonging photoperiod on the synthesis of testosterone and melatonin in roosters, and the effect of melatonin on testosterone synthesis in rooster Leydig cells as well as its molecular mechanisms. We randomly divided one hundred and twenty 20-week-old roosters into three groups and provided 6, 12.5 and 16 h light, respectively. The results showed that prolonging photoperiod promoted testosterone synthesis, decreased melatonin production, and inhibited the expression of melatonin membrane receptors MEL1A, MEL1B, MEL1C, and aralkylamine N-acetyltransferase (AANAT) in rooster testes. Subsequently, rooster Leydig cells were isolated and treated with 0, 0.1, 1, 10, and 100 ng/mL melatonin for 36 h. The results suggested that melatonin inhibited testosterone synthesis in rooster Leydig cells, and silencing MEL1A and MEL1B relieved the inhibition of melatonin on testosterone synthesis. Additionally, melatonin reduced the intracellular cyclic adenosine monophosphate (cAMP) level and the phosphorylation level of cAMP-response element binding protein (CREB), and CREB overexpression alleviated the inhibition of melatonin on testosterone synthesis. Furthermore, pretreatment with cAMP activator forskolin or protein kinase A (PKA) activator 8-bromo-cAMP blocked the inhibition of melatonin on CREB phosphorylation and testosterone synthesis. These results indicated that prolonging photoperiod promoted testosterone synthesis associated with the decrease in melatonin production and membrane receptors and biosynthetic enzyme of melatonin in rooster testes, and melatonin inhibited testosterone synthesis of rooster Leydig cells by inhibiting the cAMP/PKA/CREB pathway via MEL1A and MEL1B. This may be evidence that prolonging photoperiod could promote testosterone synthesis through the inhibition of the local melatonin pathway in rooster testes.

Study of microRNA Expression Profile in Different Regions of Ram Epididymis.

The regional expression of epididymal genes provides a guarantee for sperm maturation. As a class of endogenous non-coding small RNAs, microRNAs (miRNAs) play an important role in spermatogenesis, maturation and fertilization. Currently, the regulatory role of miRNA in the epididymis is poorly understood. Here, transcriptome sequencing was used to analyse miRNA expression profiles in three regions of the epididymis of rams, including caput, corpus and cauda. The results showed that there were 13 known miRNAs between the caput and corpus controls, 29 between the caput and cauda and 22 differences between the corpus and cauda. Based on the analysis of miRNA target genes by GO and KEGG, a negative regulation network of miRNA-mRNA was constructed in which let-7, miR-541-5p, miR-133b and miR-150 may play an important regulatory role in the maturation regulation of ram epididymal sperm. This research provides a reference for studying the regulation mechanism of sperm maturation in male epididymis and improving semen quality and male reproductive performance.

Adenosine deaminase acting on RNA-1 (ADAR1) inhibits hepatitis B virus (HBV) replication by enhancing microRNA-122 processing.

Adenosine deaminases acting on RNA-1 (ADAR1) involves adenosine to inosine RNA editing and microRNA processing. ADAR1 is known to be involved in the replication of various viruses, including hepatitis C and D. However, the role of ADAR1 in hepatitis B virus (HBV) infection has not yet been elucidated. Here, for the first time, we demonstrated ADAR1 antiviral activity against HBV. ADAR1 has two splicing isoforms in human hepatocytes: constitutive p110 protein and interferon-α (IFN-α)-responsive p150 protein. We found that overexpression of ADAR1 decreased HBV RNA in an HBV culture model. A catalytic-site mutant ADAR1 also decreased HBV RNA levels, whereas another adenosine deaminases that act on the RNA (ADAR) family protein, ADAR2, did not. Moreover, the induction of ADAR1 by stimulation with IFN-α also reduced HBV RNA levels. Decreases in endogenous ADAR1 expression by knock-down or knock-out increased HBV RNA levels. A major hepatocyte-specific microRNA, miRNA-122, was found to be positively correlated with ADAR1 expression, and exogenous miRNA-122 decreased both HBV RNA and DNA, whereas, conversely, transfection with a miRNA-122 inhibitor increased them. The reduction of HBV RNA by ADAR1 expression was abrogated by p53 knock-down, suggesting the involvement of p53 in the ADAR1-mediated reduction of HBV RNA. This study demonstrated, for the first time, that ADAR1 plays an antiviral role against HBV infection by increasing the level of miRNA-122 in hepatocytes.

Self-assembled bifunctional nanoflower-enabled CRISPR/Cas biosensing platform for dual-readout detection of Salmonella enterica.

Sensitive detection and point-of-care test of bacterial pathogens is of great significance in safeguarding the public health worldwide. Inspired by the characteristics of horseradish peroxidase (HRP), we synthesized a hybrid nanoflower with peroxidase-like activity via a three-component self-assembled strategy. Interestingly, the prepared nanozyme not only could act as an alternative to HRP for colorimetric biosensing, but also function as a unique signal probe that could be recognized by a pregnancy test strip. By combining the bifunctional properties of hybrid nanoflower, isothermal amplification of LAMP, and the specific recognition and non-specific cleavage properties of CRISPR/Cas12a system, the dual-readout CRISPR/Cas12a biosensor was developed for sensitive and rapid detection of Salmonella enterica. Moreover, this platform in the detection of Salmonella enterica had limits of detection of 1 cfu/mL (colorimetric assay) in the linear range of 101-108 cfu/mL and 102 cfu/mL (lateral flow assay) in the linear range of 102-108 cfu/mL, respectively. Furthermore, the developed biosensor exhibited good recoveries in the spiked samples (lake water and milk) with varying concentrations of Salmonella enterica. This work provides new insights for the design of multifunctional nanozyme and the development of innovative dual-readout CRISPR/Cas system-based biosensing platform for the detection of pathogens.

A comprehensive analysis on the source-exposure and diffusion rule of VOCs for end-life vehicles dismantling activities.

In recent years, the rapid development of technology has posed significant challenges to the waste management practices of the retired vehicle industry. How to minimize the environmental impact during the recycle process of scrap vehicle has emerged as a prevalent and pressing concern. This study employed statistical analysis and positive matrix factorization (PMF) model to assess the origin of Volatile Organic Compounds (VOCs) at a scrap vehicle dismantling location situated in China. The quantification of potential hazards to human health arising from identified sources was achieved through the integration of source characteristics with exposure risk assessment. In addition, fluent simulation was employed to examine the spatiotemporal dispersion of the pollutant concentration field and velocity profile. The findings of the study revealed that the activities of parts cutting, disassembling air conditioning and refined dismantling were responsible for 89.98 %, 84.36 %, and 78.63 % of the total air pollution accumulation, respectively. Additionally, it should be noted that the aforementioned sources accounted for 59.40 %, 18.44 %, and 4.86 % of the aggregate non-cancer risk. The cumulative cancer risk was determined to be the disassembling air conditioning process, with a contribution of 82.71 %. Meanwhile, the average concentration of VOC in soil around the disassembling air conditioning area is 8.4 times higher than the background value. The simulation revealed that pollutants were primarily dispersed within the factory at a height ranging from 0.75 m to 2 m, which corresponds to the human respiratory zone and the concentration of pollutants in the vehicle cutting area was observed to exceed normal levels by over 10 times. These findings of this study may serve as a foundation for improving of environmental protection measures of industrialization.

Medial patellofemoral ligament reconstruction using a digital tensiometer to determine graft tension: Surgical technique and mid-term follow-up.

PURPOSE: The optimal method for achieving proper graft tension during patellofemoral ligament reconstruction is a topic of debate. In the past, a digital tensiometer was used to simulate the knee structure, and a tension of approximately 2N was identified as suitable for restoring the patellofemoral track. However, it is unclear whether this tension level is sufficient during the actual surgery. The objective of this study was to verify the efficacy of graft tension using a digital tensiometer for medial patellofemoral ligament (MPFL) reconstruction and to conduct a mid-term follow-up. METHODS: The study enrolled 39 patients who had experienced recurrent patellar dislocation. Preoperative computed tomography scans and X-rays confirmed patellar instability, patellar tilt angle patellar congruence angle and the history of dislocation and patellar apprehension test. Knee function was evaluated using preoperative and postoperative Lysholm and Kujala scores. RESULTS: The study included 39 knees, comprising 22 females and 17 males, with an average age of 21.10 ± 7.26. The patients were followed up for at least 24 months through telephone or face-to-face questionnaires. All patients had a preoperative history of ≥2 patellar dislocations, none of which were surgically treated. During surgery, all patients underwent isolated MPFL reconstruction and lateral retinacula release. The mean Kujala and Lysholm scores were 91.28 ± 4.90 and 90.67 ± 5.15, respectively. The mean PTA and PCA were 11.5 ± 2.63 and 2.38 ± 3.58, respectively. The study found that a tension of approximately 27.39 ± 5.57N (14.3-33.5N) was required to restore the patellofemoral track in patients with recurrent patellar dislocation. No patients required reoperation during the follow-up period. Overall, 36 out of 39 patients (92.31%) reported no pain when completing daily activities at the last follow-up. CONCLUSION: In conclusion, a tension level of approximately 27.39 ± 5.57N is necessary to restore normal patellofemoral relationships during clinical practice, which indicates that using a tension of 2N is too low. The use of a tensiometer during patellofemoral ligament reconstruction is a more accurate and reliable surgical procedure for treating recurrent patellar dislocation.

Study on the region-specific expression of epididymis mRNA in the rams.

The epididymis is divided into three regions including the caput, corpus and cauda. Gene expression profiles in different regions indicate the different functions of epididymis which are crucial for sperm maturation. In this study, three one-year-old rams was used as the experimental animal. Transcriptome sequencing technology was used to sequence mRNA in the caput, corpus and cauda of the epididymis. Based on the spatiotemporal-specific expression pattern in the epididymis, the mRNA expression profiles of the three parts of the epididymis were analysed. Region-specifically expressed genes were analysed by GO and KEGG analyses to screen the key genes involved in sheep sperm maturation. We obtained 129, 54 and 99 specifically expressed genes in the caput, corpus and cauda, respectively. And twenty specific expressed genes related to sperm maturation were used to construct functional networks. The heatmap showed that 6 genes of LCN protein family were highly expressed in the head of epididymis of sheep. We infer that sperm maturation is gradual in the epididymis and that there are significant differences in epididymal gene expression patterns between different species. This provides a data resource for analysing the regulatory mechanism of epididymis genes related to sperm maturation in rams.

Genome-Wide Identification of mRNAs, lncRNAs, and Proteins, and Their Relationship With Sheep Fecundity.

The exploration of multiple birth-related genes has always been a significant focus in sheep breeding. This study aimed to find more genes and proteins related to the litter size in sheep. Ovarian specimens of Small Tail Han sheep (multiple births) and Xinji Fine Wool sheep (singleton) were collected during the natural estrus cycle. Transcriptome and proteome of ovarian specimens were analyzed. The transcriptome results showed that "steroid hormone biosynthesis" and "ovarian steroidogenesis" were significantly enriched, in which HSD17B1 played an important role. The proteome data also confirmed that the differentially expressed proteins (DEPs) were enriched in the ovarian steroidogenesis pathway, and the CYP17A1 was the candidate DEP. Furthermore, lncRNA MSTRG.28645 was highly expressed in Small Tailed Han sheep but lowly expressed in Xinji fine wool sheep. In addition, MSTRG.28645, a hub gene in the co-expression network between mRNAs and lncRNAs, was selected as one of the candidate genes for subsequent verification. Expectedly, the overexpression and interference of HSD17B1 and MSTRG.28645 showed a significant effect on hormone secretion in granulosa cells. Therefore, this study confirmed that HSD17B1 and MSTRG.28645 might be potential genes related to the fecundity of sheep. It was concluded that both HSD17B1 and MSTRG.28645 were critical regulators in the secretion of hormones that affect the fecundity of the sheep.

Development of a 3D Collagen Model for the In Vitro Evaluation of Magnetic-assisted Osteogenesis.

Magnetic stimulation has been applied to bone regeneration, however, the cellular and molecular mechanisms of repair still require a better understanding. A three-dimensional (3D) collagen model was developed using plastic compression, which produces dense, cellular, mechanically strong native collagen structures. Osteoblast cells (MG-63) and magnetic iron oxide nanoparticles (IONPs) were incorporated into collagen gels to produce a range of cell-laden models. A magnetic bio-reactor to support cell growth under static magnetic fields (SMFs) was designed and fabricated by 3D printing. The influences of SMFs on cell proliferation, differentiation, extracellular matrix production, mineralisation and gene expression were evaluated. Polymerase chain reaction (PCR) further determined the effects of SMFs on the expression of runt-related transcription factor 2 (Runx2), osteonectin (ON), and bone morphogenic proteins 2 and 4 (BMP-2 and BMP-4). Results demonstrate that SMFs, IONPs and the collagen matrix can stimulate the proliferation, alkaline phosphatase production and mineralisation of MG-63 cells, by influencing matrix/cell interactions and encouraging the expression of Runx2, ON, BMP-2 and BMP-4. Therefore, the collagen model developed here not only offers a novel 3D bone model to better understand the effect of magnetic stimulation on osteogenesis, but also paves the way for further applications in tissue engineering and regenerative medicine.

Effect of kisspeptin on the proliferation and apoptosis of bovine granulosa cells.

Previous studies have shown that kisspeptin (Kp-10) is expressed in mammalian ovaries; however, the expression and role of Kp-10 in bovine ovarian granulosa cells are still unclear. In this study, we assessed the expression of Kp-10 and its effects on the proliferation and apoptosis of bovine granulosa cells. Immunohistochemical analysis showed that Kp-10 was expressed in the cytoplasm of bovine ovarian granulosa cells. Moreover, MTT assays showed that 100nM Kp-10 significantly inhibited the viability of granulosa cells (P<0.05). Flow cytometry analysis showed that Kp-10 could significantly increase accumulation of cells in the G1 phase, decrease accumulation of cells in the S phase, and promote apoptosis in bovine granulosa cells (P<0.05). Additionally, Kp-10 decreased the mRNA levels of Bcl-2, an anti-apoptotic gene; increased the mRNA levels of caspase-3, a pro-apoptotic gene; and increased the mRNA levels of Fas and Fasl (P< 0.05). Thus, our findings demonstrated for the first time that Kp-10 inhibited proliferation and promoted apoptosis in bovine ovarian granulosa cells. These findings provide insights into our understanding of the role of Kp-10 in mediating the proliferation of bovine granulosa cells.