FZD7, Regulated by Non-CpG Methylation, Plays an Important Role in Immature Porcine Sertoli Cell Proliferation.

The regulatory role of non-CpG methylation in mammals has been important in whole-genome bisulfite sequencing. It has also been suggested that non-CpG methylation regulates gene expression to affect the development and health of mammals. However, the dynamic regulatory mechanisms of genome-wide, non-CpG methylation during testicular development still require intensive study. In this study, we analyzed the dataset from the whole-genome bisulfite sequencing (WGBS) and the RNA-seq of precocious porcine testicular tissues across two developmental stages (1 and 75 days old) in order to explore the regulatory roles of non-CpG methylation. Our results showed that genes regulated by non-CpG methylation affect the development of testes in multiple pathways. Furthermore, several hub genes that are regulated by non-CpG methylation during testicular development-such as VEGFA, PECAM1, and FZD7-were also identified. We also found that the relative expression of FZD7 was downregulated by the zebularine-induced demethylation of the first exon of FZD7. This regulatory relationship was consistent with the results of the WGBS and RNA-seq analysis. The immature porcine Sertoli cells were transfected with RNAi to mimic the expression patterns of FZD7 during testicular development. The results of the simulation test showed that cell proliferation was significantly impeded and that cell cycle arrest at the G2 phase was caused by the siRNA-induced FZD7 inhibition. We also found that the percentage of early apoptotic Sertoli cells was decreased by transfecting them with the RNAi for FZD7. This indicates that FZD7 is an important factor in linking the proliferation and apoptosis of Sertoli cells. We further demonstrated that Sertoli cells that were treated with the medium collected from apoptotic cells could stimulate proliferation. These findings will contribute to the exploration of the regulatory mechanisms of non-CpG methylation in testicular development and of the relationship between the proliferation and apoptosis of normal somatic cells.

Comprehensive Analysis of the Transcriptome-Wide m6A Methylome in Shaziling Pig Testicular Development.

RNA N6-methyladenosine (m6A) modification is one of the principal post-transcriptional modifications and plays a dynamic role in testicular development and spermatogenesis. However, the role of m6A in porcine testis is understudied. Here, we performed a comprehensive analysis of the m6A transcriptome-wide profile in Shaziling pig testes at birth, puberty, and maturity. We analyzed the total transcriptome m6A profile and found that the m6A patterns were highly distinct in terms of the modification of the transcriptomes during porcine testis development. We found that key m6A methylated genes (AURKC, OVOL, SOX8, ACVR2A, and SPATA46) were highly enriched during spermatogenesis and identified in spermatogenesis-related KEGG pathways, including Wnt, cAMP, mTOR, AMPK, PI3K-Akt, and spliceosome. Our findings indicated that m6A methylations are involved in the complex yet well-organized post-transcriptional regulation of porcine testicular development and spermatogenesis. We found that the m6A eraser ALKBH5 negatively regulated the proliferation of immature porcine Sertoli cells. Furthermore, we proposed a novel mechanism of m6A modification during testicular development: ALKBH5 regulated the RNA methylation level and gene expression of SOX9 mRNA. In addition to serving as a potential target for improving boar reproduction, our findings contributed to the further understanding of the regulation of m6A modifications in male reproduction.

Overexpression of PFKFB3 promotes cell glycolysis and proliferation in renal cell carcinoma.

BACKGROUND: Cancer cells prefer utilizing aerobic glycolysis in order to exacerbate tumor mass and maintain un-regulated proliferative rates. As a key glycolytic activator, phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) has been implicated in multiple tumor type progression. However, the specific function and clinical significance of PFKFB3 in renal cell carcinoma (RCC) are yet not clarified. This investigation assessed PFKFB3 roles in RCC. METHODS: PFKFB3 expression levels were analyzed in clear cell renal cell carcinoma (ccRCC) tissues, together with its relationship with clinical characteristics of ccRCC. Real-time PCR and Western blot assays were employed for determining PFKFB3 expression in different RCC cell lines. Furthermore, we determined the glycolytic activity by glucose uptake, lactate secretion assay and ECAR analysis. CCK-8 assay, clone formation, flow cytometry and EdU assessments were performed for monitoring tumor proliferative capacity and cell-cycle distribution. Furthermore, a murine xenograft model was employed for investigating the effect of PFKFB3 on tumor growth in vivo. RESULTS: PFKFB3 was significantly up-regulated in RCC specimens and cell lines in comparison to normal control. Overexpression of PFKFB3 was directly correlated to later TNM stages, thus becoming a robust prognostic biomarker for ccRCC cases. Furthermore, PFKFB3 knockdown suppressed cell glycolysis, proliferative rate and cell-cycle G1/S conversion in RCC cells. Importantly, in vivo experiments confirmed that PFKFB3 knockdown delayed tumor growth derived from the ACHN cell line. CONCLUSIONS: Such results suggest that PFKFB3 is a key molecular player in RCC progression via mediating glycolysis / proliferation and provides a potential therapeutic target against RCC.

Selenium nanoparticles inhibit tumor metastasis in prostate cancer through upregulated miR-155-5p-related pathway.

Prostate cancer are the most common, malignant and lethal tumors in men, and the complexity of prostate cancer (CaP) is also due to the diverse metastasis profile. Selenium nanoparticles (SeNPs) have been reported to have potent antitumor activity, but whether it impacted the tumor metastasis is not fully clear. Here, we confirmed that SeNPs could inhibit the CaP cell migrations and invasions. Combined with our previous findings, we identified a series of microRNAs that could be upregulated significantly under SeNP treatment, among which miR-155-5p acts as a key component in mediating the SeNP-inhibited migration and invasion of CaP cells, through directly targeting IκB kinase ɛ and Sma- and Mad-related protein 2. The cell-based results were proved in xenograft mice modeling. These results have evidently signified the antitumor potential of SeNPs in the treatment of prostate cancer.

Finite element analysis of female pelvic organ prolapse mechanism: current landscape and future opportunities.

The prevalence of pelvic organ prolapse (POP) has been steadily increasing over the years, rendering it a pressing global health concern that significantly impacts women's physical and mental wellbeing as well as their overall quality of life. With the advancement of three-dimensional reconstruction and computer simulation techniques for pelvic floor structures, research on POP has progressively shifted toward a biomechanical focus. Finite element (FE) analysis is an established tool to analyze the biomechanics of complex systems. With the advancement of computer technology, an increasing number of researchers are now employing FE analysis to investigate the pathogenesis of POP in women. There is a considerable number of research on the female pelvic FE analysis and to date there has been less review of this technique. In this review article, we summarized the current research status of FE analysis in various types of POP diseases and provided a detailed explanation of the issues and future development in pelvic floor disorders. Currently, the application of FE analysis in POP is still in its exploratory stage and has inherent limitations. Through continuous development and optimization of various technologies, this technique can be employed with greater accuracy to depict the true functional state of the pelvic floor, thereby enhancing the supplementation of the POP mechanism from the perspective of computer biomechanics.

Single-Nucleus RNA-Seq Reveals Spermatogonial Stem Cell Developmental Pattern in Shaziling Pigs.

Normal testicular development ensures the process of spermatogenesis, which is a complex biological process. The sustained high productivity of spermatogenesis throughout life is predominantly attributable to the constant proliferation and differentiation of spermatogonial stem cells (SSCs). The self-renewal and differentiation processes of SSCs are strictly regulated by the SSC niche. Therefore, understanding the developmental pattern of SSCs is crucial for spermatogenesis. The Shaziling pig is a medium-sized indigenous pig breed originating from central China. It is renowned for its superior meat quality and early male sexual maturity. The spermatogenic ability of the boars is of great economic importance to the pig industry. To investigate testicular development, particularly the pattern of SSC development in Shaziling pigs, we used single-cell transcriptomics to identify gene expression patterns in 82,027 individual cells from nine Shaziling pig testes at three key postnatal developmental stages. We generated an unbiased cell developmental atlas of Shaziling pig testicular tissues. We elucidated the complex processes involved in the development of SSCs within their niche in the Shaziling pig. Specifically, we identified potential marker genes and cellular signaling pathways that regulate SSC self-renewal and maintenance. Additionally, we proposed potential novel marker genes for SSCs that could be used for SSC isolation and sorting in Shaziling pigs. Furthermore, by immunofluorescence staining of testicular tissues of different developmental ages using marker proteins (UCHL1 and KIT), the developmental pattern of the spermatogonia of Shaziling pigs was intensively studied. Our research enhances the comprehension of the development of SSCs and provides a valuable reference for breeding Shaziling pigs.

Dysregulation and implications of N6-methyladenosine modification in renal cell carcinoma.

Increasing evidence indicates that N6-methyladenosine (m6A) methylation modification serves important functions in biological metabolism. Dysregulation of m6A regulators is related to the progression of different malignancies, including renal cell carcinoma (RCC). Recent studies have reported preliminary findings on the influence of m6A regulator dysregulation on RCC tumorigenesis and development. However, no comprehensive review that integrates and analyzes the roles of m6A modification in RCC has been published to date. In this review, we focus on the dysregulation of m6A regulators as it relates to RCC tumorigenesis and development, as well as possible applications of m6A modification in RCC diagnosis and therapeutics.

Genome-Wide Assessment of Runs of Homozygosity by Whole-Genome Sequencing in Diverse Horse Breeds Worldwide.

In the genomes of diploid organisms, runs of homozygosity (ROH), consecutive segments of homozygosity, are extended. ROH can be applied to evaluate the inbreeding situation of individuals without pedigree data and to detect selective signatures via ROH islands. We sequenced and analyzed data derived from the whole-genome sequencing of 97 horses, investigated the distribution of genome-wide ROH patterns, and calculated ROH-based inbreeding coefficients for 16 representative horse varieties from around the world. Our findings indicated that both ancient and recent inbreeding occurrences had varying degrees of impact on various horse breeds. However, recent inbreeding events were uncommon, particularly among indigenous horse breeds. Consequently, the ROH-based genomic inbreeding coefficient could aid in monitoring the level of inbreeding. Using the Thoroughbred population as a case study, we discovered 24 ROH islands containing 72 candidate genes associated with artificial selection traits. We found that the candidate genes in Thoroughbreds were involved in neurotransmission (CHRNA6, PRKN, and GRM1), muscle development (ADAMTS15 and QKI), positive regulation of heart rate and heart contraction (HEY2 and TRDN), regulation of insulin secretion (CACNA1S, KCNMB2, and KCNMB3), and spermatogenesis (JAM3, PACRG, and SPATA6L). Our findings provide insight into horse breed characteristics and future breeding strategies.

Genetic variation and domestication of horses revealed by 10 chromosome-level genomes and whole-genome resequencing.

Understanding the genetic variations of the horse (Equus caballus) genome will improve breeding conservation and welfare. However, genetic variations in long segments, such as structural variants (SVs), remain understudied. We de novo assembled 10 chromosome-level three-dimensional horse genomes, each representing a distinct breed, and analysed horse SVs using a multi-assembly approach. Our findings suggest that SVs with the accumulation of mammalian-wide interspersed repeats related to long interspersed nuclear elements might be a horse-specific mechanism to modulate genome-wide gene regulatory networks. We found that olfactory receptors were commonly loss and accumulated deleterious mutations, but no purge of deleterious mutations occurred during horse domestication. We examined the potential effects of SVs on the spatial structure of chromatin via topologically associating domains (TADs). Breed-specific TADs were significantly enriched by breed-specific SVs. We identified 4199 unique breakpoint-resolved novel insertions across all chromosomes that account for 2.84 Mb sequences missing from the reference genome. Several novel insertions might have potential functional consequences, as 519 appeared to reside within 449 gene bodies. These genes are primarily involved in pathogen recognition, innate immune responses and drug metabolism. Moreover, 37 diverse horses were resequenced. Combining this with public data, we analysed 97 horses through a comparative population genomics approach to identify the genetic basis underlying breed characteristics using Thoroughbreds as a case study. We provide new scientific evidence for horse domestication, an understanding of the genetic mechanism underlying the phenotypic evolution of horses, and a comprehensive genetic variation resource for further genetic studies of horses.

Integration of RRBS and RNA-seq unravels the regulatory role of DNMT3A in porcine Sertoli cell proliferation.

DNMT3A participates in de novo methylation, yet its impact on the proliferation of testicular Sertoli cells remains unclear. Development-specific methylation has been proven to be associated with cellular development. Therefore, in this study, we simulated DNMT3A expression pattern during testicular development by DNMT3A interference. Then, RRBS and RNA-seq were used to decipher DNMT3A regulatory mechanisms on Sertoli cell proliferation. Immunofluorescence staining revealed the expression of DNMT3A in the Sertoli cells of the prepubertal testis. DNMT3A was demonstrated to inhibit the cell cycle and proliferation of Sertoli cells, while promoting cell apoptosis. After transfected with DNMT3A interference, a total of 560 DEGs and 2,091 DMGs produced by DNMT3A interference were identified between two treated groups, respectively. Integrating the results from RRBS and RNA-seq, the overlapping genes between DMGs and DEGs were found to be enriched in the Gene Ontology (GO) terms related to cellular development and the Apelin signaling pathway. The present study demonstrated the impact of DNMT3A on the proliferation of porcine testicular Sertoli cells, suggesting that DNMT3A primarily acts through the Apelin signaling pathway. These findings provide valuable insights into how DNMT3A influences testicular development and health, offering new perspectives.

Development and validation of a multiparametric MRI-based radiomics signature for distinguishing between indolent and aggressive prostate cancer.

OBJECTIVE: To develop and validate a non-invasive MRI-based radiomics signature for distinguishing between indolent and aggressive prostate cancer (PCa) prior to therapy. METHODS: In all, 139 qualified and pathology-confirmed PCa patients were divided into a training set (n = 93) and a validation set (n = 46). A total of 1576 radiomics features were extracted from the T2WI (n = 788) and diffusion-weighted imaging (n = 788) for each patient. The Select K Best and the least absolute shrinkage and selection operator regression algorithm were used to construct a radiomics signature in the training set. The predictive performance of the radiomics signature was assessed in the training set and then validated in the validation set by receiver operating characteristic curve analysis. We computed the calibration curve and the decision curve to evaluate the calibration and clinical usefulness of the signature. RESULTS: Nine radiomics features were identified to form the radiomics signature. The radiomics score (Rad-score) was significantly different between indolent and aggressive PCa (p < 0.001). The radiomics signature exhibited favorable discrimination between the indolent and aggressive PCa groups in the training set (AUC: 0.853, 95% CI: 0.766 to 0.941) and validation set (AUC: 0.901, 95% CI: 0.793 to 1.000). The decision curve analysis showed that a greater net benefit would be obtained when the threshold probability ranged from 20 to 90%. CONCLUSION: The multiparametric MRI-based radiomics signature can potentially serve as a non-invasive tool for distinguishing between indolent and aggressive PCa prior to therapy. ADVANCES IN KNOWLEDGE: The multiparametric MRI-based radiomics signature has the potential to non-invasively distinguish between the indolent and aggressive PCa, which might aid clinicians in making personalized therapeutic decisions.

Viscera Characteristics of MSTN-Edited Heterozygous Pigs.

Myostatin (MSTN) is a protein that negatively regulates growth of skeletal muscle, and inactivation of MSTN improves the mass of skeletal muscle. Our previous work found that MSTN +/- pigs have higher muscle depth and lower fat depth compared to wild type without any developmental problems. Therefore, MSTN-edited pigs are most likely to appear as heterozygotes in the potential future market, but the characteristics of organs in digestive and reproductive system of pigs with MSTN gene editing remains unclear. Here, we investigated the histological of the organs in the digestive system and reproductive system in MSTN gene heterozygotes at adult stages. The length of intestine was further compared between adult heterozygous and wild type pigs. We found no significant differences in histomorphology of organs, including heart, duodenum, jejunum, ileum, cecum, colon, testis, epididymis, ovaries, oviducts and uterus, between individuals from two genotypes. Moreover, there was no significant difference in the average length of intestine in adult pigs. Our data provide a reference for further clarifying the applications of MSTN gene edited pigs.

Prognostic Impact of MITD1 and Associates With Immune Infiltration in Kidney Renal Clear Cell Carcinoma.

Kidney renal clear cell carcinoma (KIRC) is one of the most malignant diseases with poor survival rate over the world. The tumor microenvironment (TME) is highly related to the oncogenesis, development, and prognosis of KIRC. Thus, making the identification of KIRC biomarkers and immune infiltrates critically important. Microtubule Interacting and Trafficking Domain containing 1(MITD1) was reported to participate in cytokinesis of cell division. In the present study, multiple bioinformatics tools and databases were applied to investigate the expression level and clinical value of MITD1 in KIRC. We found that the expression of MITD1 was significantly increased in KIRC tissues. Further, the KIRC patients with high MITD1 levels showed a worse overall survival (OS) rate and disease free survival (DFS) rate. Otherwise, we found a significant correlation MITD1 expression and the abundance of CD8+ T cells. Functional enrichment analyses revealed that immune response and cytokine-cytokine receptor are very critical signaling pathways which associated with MITD1 in KIRC. In conclusion, our findings indicated that MITD1 may be a potential biomarker and associated with immune infiltration in KIRC.

Effect of Oral Motor Intervention on Oral Feeding in Preterm Infants: A Systematic Review and Meta-Analysis.

Objective This review article aimed to explore the effect of oral motor intervention on oral feeding in preterm infants through a meta-analysis. Method Eligible studies were retrieved from four databases (PubMed, Embase, Cochrane Library, and Web of Science) up to July 2020 and screened based on established selection criteria. Thereafter, relevant data were extracted and heterogeneity tests were conducted to select appropriate effect models according to the chi-square test and I 2 statistics. Assessment of risk of bias was performed among the included studies. Finally, a meta-analysis was carried out to evaluate the effect of oral motor intervention in preterm infants according to four clinical indicators: transition time for oral feeding, length of hospital stay, feeding efficiency, and weight gain. Results Eighteen randomized controlled trials with 848 participants were selected to evaluate the effect of oral motor intervention on preterm infants. The meta-analysis results revealed that oral motor intervention could effectively reduce the transition time to full oral feeds and the length of hospital stay as well as increase feeding efficiency and weight gain. Conclusions Oral motor intervention was an effective way to improve oral feeding in preterm infants. It is worthy to be used widely in hospitals to improve the clinical outcomes of preterm infants and reduce the economic burdens of families and society. Future studies should seek to identify detailed intervention processes and intervention durations for clinical application.

Integrated Microbiome and Metabolome Analysis Reveals a Positive Change in the Intestinal Environment of Myostatin Edited Large White Pigs.

Myostatin (MSTN) functional inactivation can change the proportion of lean meat and fat content in pigs. While both genotype and microbial composition are known to affect the host phenotype, so far there has been no systematic study to detect the changes in the intestinal microbial composition and metabolome of MSTN single copy mutant pigs. Here, we used 16S rDNA sequencing and metabolome analysis to investigate how MSTN gene editing affects changes in the microbial and metabolome composition in the jejunum and the cecum of Large White pigs. Our results showed that Clostridium_sensu_stricto_1, Bifidobacterium, Lachnospiraceae_UCG-007, Clostridium_sensu_stricto_6, Ruminococcaceae_UCG-002, and Ruminococcaceae_UCG-004 were significantly upregulated; while Treponema_2 and T34_unclassified were significantly downregulated in the jejunum of MSTN pigs. Similarly, Phascolarctobacterium, Ruminiclostridium_9, Succinivibrio, Longibaculum, and Candidatus_Stoquefichus were significantly upregulated, while Barnesiella was significantly downregulated in the cecum of MSTN pigs. Moreover, metabolomics analysis showed significant changes in metabolites involved in purine, sphingolipid and tryptophan metabolism in the jejunum, while those associated with glycerophospholipid and pyrimidine metabolism were changed in the cecum. Spearman correlation analysis further demonstrated that there was a significant correlation between microflora composition and metabolites. Our analyses indicated the MSTN editing affects the composition of metabolites and microbial strains in the jejunum and the cecum, which might provide more useable nutrients for the host of MSTN± Large White pigs.

HNF1A inhibition induces the resistance of pancreatic cancer cells to gemcitabine by targeting ABCB1.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with poor prognosis, and gemcitabine-based chemotherapy remains an effective option for the majority of PDAC patients. Hepatocyte nuclear factor 1α (HNF1A) is a tumor-suppressor in PDAC, but its role in gemcitabine chemoresistance of PDAC has not been clarified. METHODS: The function of HNF1A in gemcitabine was detected by overexpression and knockdown of HNF1A in vitro and in vitro. The regulatory network between HNF1A and ABCB1 was further demonstrated by luciferase assays, deletion/mutation reporter construct assays and CHIP assays. FINDINGS: Here, we found that HNF1A expression is significantly associated with gemcitabine sensitivity in PDAC cell lines. Moreover, we identified that HNF1A overexpression enhanced gemcitabine sensitivity of PDAC both in vitro and in vitro, while inhibition of HNF1A had the opposite effect. Furthermore, by inhibiting and overexpressing HNF1A, we revealed that HNF1A regulates the expression of MDR genes (ABCB1 and ABCC1) in PDAC cells. Mechanistically, we demonstrated that HNF1A regulates ABCB1 expression through binding to its specific promoter region and suppressing its transcription levels. Finally, the survival analyses revealed the clinical value of HNF1A in stratification of gemcitabine sensitive pancreatic cancer patients. INTERPRETATION: Our study paved the road for finding novel treatment combinations using conventional cytotoxic agents with functional restoration of the HNF1A protein, individualized treatment through HNF1A staining and improvement of the prognosis of PDAC patients. FUND: National Natural Science Foundations of China and National Natural Science Foundation of Guangdong Province.