PPP2R2A promotes testosterone secretion in Hu sheep Leydig cells via activation of the AKT/mTOR signaling pathway.

The serine-threonine protein phosphatase 2A (PP2A) is a heterotrimeric enzyme complex that plays a vital role in regulating male reproductive activities. However, as an essential member of the PP2A family, the physiological functions of PP2A regulatory subunit B55α (PPP2R2A) in testis remain inconclusive. Hu sheep are noted for their reproductive precocity and fertility, and are ideal models for the study of male reproductive physiology. Here, we analyzed the expression patterns of PPP2R2A in the male Hu sheep reproductive tract at different developmental stages and further investigated its role in testosterone secretion and its underlying mechanisms. In this study, we found that there were temporal and spatial differences in PPP2R2A protein expression in the testis and epididymis, especially the expression abundance in the testis at 8 months old (8M) was higher than that at 3 months old (3M). Interestingly, we observed that PPP2R2A interference reduced the testosterone levels in the cell culture medium, which is accompanied by a reduction in Leydig cell proliferation and an elevation in Leydig cell apoptosis. The level of reactive oxygen species in cells increased significantly, while the mitochondrial membrane potential (ΔΨm) decreased significantly after PPP2R2A deletion. Meanwhile, the mitochondrial mitotic protein DNM1L was significantly upregulated, while the mitochondrial fusion proteins MFN1/2 and OPA1 were significantly downregulated after PPP2R2A interference. Furthermore, PPP2R2A interference suppressed the AKT/mTOR signaling pathway. Taken together, our data indicated that PPP2R2A enhanced testosterone secretion, promoted cell proliferation, and inhibited cell apoptosis in vitro, all of which were associated with the AKT/mTOR signaling pathway.

Dietary spirulina supplementation modifies rumen development, fermentation and bacteria composition in Hu sheep when consuming high-fat dietary.

Introduction: This study aims to investigate the long-term effects of spirulina supplementation in a high-fat diet (HFD) on rumen morphology, rumen fermentation, and the composition of rumen microbiota in lambs. Spirulina is a blue-green microalgae that has been shown to have high nutritional value for livestock. Methods: Fifty-four lambs were randomly divided into three groups: a normal chow diet (NCD) group, a high-fat diet (HFD) group, and a high-fat diet supplemented with 3% spirulina (HFD+S) group. Rumen morphology, rumen fermentation, and rumen microbiota were analyzed at the end of the study. Results: Spirulina supplementation improved the concentration of volatile fatty acids and rumen papilla length. Additionally, there was a tendency for an increase in rumen weight and an upregulation of the genes Claudin-1, Claudin-4, and Occludin in the HFD+S group. Pyrosequencing of the 16S ribosomal RNA gene also showed that spirulina supplementation significantly changed the rumen microbiota composition in the HFD group, with a decrease in richness and diversity. Specifically, the relative abundance of Prevotella 9 and Megasphaera was significantly increased in the HFD group compared to the NCD group, while spirulina supplementation reversed these changes. Discussion: This study suggests that 3% spirulina supplementation can improve rumen development and fermentation, and effectively relieve rumen microbe disorders in lambs caused by a high-fat diet. However, further research is needed to confirm the findings and to examine the long-term effects of spirulina supplementation in different types of livestock and under different dietary conditions.

Exploration the role of INHBA in Hu sheep granulosa cells using RNA-Seq.

Activin/inhibin is an important factor for the fecundity of Hu sheep, and it is involved in follicular development in ovaries. Inhibin subunit beta A (INHBA) participates in the synthesis of activin A and inhibin A. In this study, we also noted a positive correlation between INHBA level and the secretion of both activin A and inhibin A in culture medium. Nevertheless, both knockdown and overexpression of INHBA downregulated the expression of Inhibin Subunit Alpha (INHA). Based on RNA-Sequencing, we further examined the effect and molecular mechanism of INHBA knockdown in GCs on mRNA expression. A total of 1,687 differentially expressed genes (DEGs) were identified (Fold change ≥ 2; False-discovory-rates (FDR) ≤ 0.01), of which 602 genes were upregulated and 1,087 genes were downregulated in the INHBA interference group compared with the control groups. Gene Ontology (GO) enrichment indicated that these DEGs were mainly involved in the regulation of cell cycle, protein serine/threonine kinase activity, and actin cytoskeleton reorganization. Moreover, DEGs were significantly enriched in 40 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including P53, progesterone-mediated oocyte maturation, and PI3K-AKT signaling pathways. We also noted a positive correlation between INHBA level and many PI3K/Akt/mTOR pathway-related genes at the gene or/and protein expression. Overall, this study may contribute to a better understanding of the roles of INHBA on GCs of prolific sheep, as well as the molecular effect of low INHBA expression on GCs, clarifying some reproductive failures.

Inflammatory Myofibroblastic Tumor of the Hilar Bile Duct: A Case Report and Literature Review.

Background: Inflammatory myofibroblastic tumor (IMT) is a very rare tumor and occurs seldom in the biliary tract. IMT can occur in any part of the body and in people of any age; however, it most commonly occurs in children or adolescents. Its etiology and pathogenesis are currently unknown. The clinical manifestations of a hilar inflammatory myofibroblastic tumor are atypical, and the imaging examination is nonspecific. The diagnosis is mainly based on histopathology and immunohistochemistry findings, and surgical resection is the preferred treatment method. Case Description: Herein, we report a rare case of hilar bile duct IMT and review the related literature. Our patient was a 54-year-old woman presenting with a 1-day history of upper abdominal pain as the main clinical symptom. She was misdiagnosed as having cholangiocarcinoma before the surgery. She underwent surgery and was ultimately diagnosed with IMT based on histopathology and immunohistochemistry findings. On 1-year follow-up, no tumor recurrence or related complications were noted. Conclusions: We hope this case report helps clinicians gain a deeper understanding of biliary IMT of the hilum.

LncRNA-412.25 activates the LIF/STAT3 signaling pathway in ovarian granulosa cells of Hu sheep by sponging miR-346.

Although long non-coding RNAs (lncRNAs) are reported to regulate follicular development and reproductive disease pathogenesis, the underlying mechanisms have not been elucidated. In this study, lncRNA expression profiling of different-sized healthy follicles from Hu sheep with different prolificacy revealed 50 613 lncRNAs. Numerous lncRNAs were differentially expressed among different comparison groups. This study characterized one novel transcript, lncRNA-412.25 (from healthy follicles with a diameter of >5 mm), which was predominantly expressed in the high prolificacy group and localized to the cytoplasm of granulosa cells (GCs). LncRNA-412.25 knockdown promoted and inhibited Hu sheep GC apoptosis and proliferation, respectively. Interestingly, lncRNA-412.25 could directly bind to miR-346, which can target the gene of leukemia inhibitory factor (LIF). Knockdown of lncRNA-412.25 promoted GC apoptosis by downregulating LIF expression, where this effect was attenuated by miR-346. Moreover, the miR-346 inhibitor mitigated the lncRNA-412.25 knockdown-induced downregulation of phosphorylated protein of signal transducer and activator of transcription 3 (STAT3), which was validated using immunofluorescence analysis. Our results demonstrated that lncRNA-412.25 regulates GC proliferation and apoptosis in Hu sheep by binding to miR-346 and then activating the LIF/STAT3 pathway. These findings provide novel insights into the mechanisms underlying prolificacy in sheep.

PPP2R2A affects embryonic implantation by regulating the proliferation and apoptosis of Hu sheep endometrial stromal cells.

Embryonic implantation is a complex reproductive physiological process in mammals. Although several endometrial proteins affecting embryonic implantation have been reported in the past, there are still potential endometrial proteins that have been neglected, and their specific regulatory mechanisms are unclear. This study demonstrated that protein phosphatase 2A regulatory subunit B55α (PPP2R2A) served as a novel regulator in medication of sheep embryonic implantation in vitro. Our results showed that sheep PPP2R2A encoded 447 amino acids and shared 91.74%-92.36% amino acid sequences with its orthologs compared with other species. Meanwhile, PPP2R2A was widely expressed in sheep uterine tissues, and it could regulate the expression levels of key regulators of embryonic implantation in endometrial stromal cells (ESCs). Knockdown of PPP2R2A significantly inhibited cell proliferation by blocking cell cycle transfer G0/G1 into S phase accompanied by downregulation of CDK2, CDK4, CCND1, CCNE1 and upregulation of P21. In contrast to PPP2R2A overexpression, PPP2R2A interference greatly promoted cell apoptosis and the expression of BAX, CASP3, CASP9 and BAX/BCL-2. Taken together, these results suggest that PPP2R2A, as a novel regulatory factor, affects embryonic implantation via regulating the proliferation and apoptosis of Hu sheep ESCs in vitro.

INHBA transfection regulates proliferation, apoptosis and hormone synthesis in sheep granulosa cells.

Inhibin subunit beta A (INHBA) participates in the synthesis of inhibin A, activin A and activin AB. Here we investigated the effect and molecular mechanism of INHBA on proliferation, apoptosis and hormone synthesis in sheep granulosa cells (GCs) using in vitro transfection. We first noticed that INHBA expression increased with follicle diameter and was widely distributed in ovarian tissue. The proliferation rate of GCs was significantly increased and decreased with overexpression and silence of INHBA, respectively, compared with the negative controls. INHBA transfection affected GC proliferation and apoptosis, regulating the expression of many cell cycle-related and apoptosis-related genes. INHBA overexpression significantly decreased activin and estradiol secretion while increasing inhibin and progesterone secretion. The expression of follicle-stimulating hormone beta subunit was significantly decreased and increased with INHBA overexpression and knockdown, respectively. Notably, silence of INHBA inhibited the expression of many transforming growth factor beta-related genes. Overall, the functional molecule of INHBA gene may be associated with follicular development via regulating proliferation, apoptosis and folliculogenesis-related hormone secretion of sheep GCs. In addition, our findings may contribute to a better understanding of the law of follicular development and thus improve the reproductive performance of female animals.

Effects of SPATA6 on proliferation, apoptosis and steroidogenesis of Hu sheep Leydig cells in vitro.

This study aimed to investigate the expression pattern of spermatogenesis associated protein 6 (SPATA6) in Hu sheep testis and to ascertain the effects of SPATA6 on sheep Leydig cells (LCs) function linked to spermatogenesis. In the present study, we detected a 1970 bp cDNA fragment of SPATA6 included a 1467 bp coding sequence which encoded 487 amino acids. Meanwhile, sheep SPATA6 shared 51.70%-97.41% amino acid sequences with its orthologs compared with other species. In addition, SPATA6 was highly expressed in testis and localized in cytoplasm and nucleus of LCs as well as spermatogenic cells at different stages. Compared to the negative control (NC), SPATA6 interference promoted apoptosis of LCs with the increase of BAX/BCL-2 mRNA and protein levels, while the results of SPATA6 overexpression were on the contrary. Meanwhile, cell cycle was blocked at G2/M phase and CDK1 and CCNB1 were down-regulated after SPATA6 interference. SPATA6 overexpression induced cell cycle transfer G0/G1 into S and G2/M phase with upregulation of CDK1, CDK4, CCND1 and CCND2. Moreover, the secretion of testosterone hormone and the expression of StAR in LCs with SPATA6 overexpression were significantly promoted. Overall, our data suggest that SPATA6 is an important functional molecule of spermatogenesis, via regulating the proliferation, apoptosis and testosterone biosynthesis of Hu sheep LCs. These findings will enhance the understanding of the roles of SPATA6 in sheep spermatogenesis.

lncRNA FDNCR promotes apoptosis of granulosa cells by targeting the miR-543-3p/DCN/TGF-ß signaling pathway in Hu sheep.

Long non-coding RNAs (lncRNAs) regulate the development of follicles and reproductive diseases, but the mechanisms by which lncRNAs regulate ovarian functions and fertility remain elusive. We profiled the expression of lncRNAs in ovarian tissues of Hu sheep with different prolificacy and identified 21,327 lncRNAs. Many of the lncRNAs were differentially expressed in different groups. We further characterized an lncRNA that was predominantly expressed in the ovaries of the low prolificacy FecB+ (LPB+) group and mainly present in granulosa cells (GCs), and the expression of this lncRNA decreased during follicular development, which we named follicular development-associated lncRNA (FDNCR). Next, we found that FDNCR directly binds miR-543-3p, and decorin (DCN) was identified as a target of miR-543-3p. FDNCR overexpression promoted GC apoptosis through increased expression of DCN, which could be attenuated by miR-543-3p. Furthermore, miR-543-3p increased and FDNCR reduced the expression of transforming growth factor-ß (TGF-ß) pathway-related genes, including TGF-ß1 and inhibin beta A (INHBA), which were upregulated upon DCN silencing. Our results demonstrated that FDNCR sponges miR-543-3p in GCs and prevents miR-543-3p from binding to the DCN 3' UTR, resulting in DCN transactivation and TGF-ß pathway inhibition and promotion of GC apoptosis in Hu sheep. These findings provide insights into the mechanisms underlying prolificacy in sheep.

CITED4 mediates proliferation, apoptosis and steroidogenesis of Hu sheep granulosa cells in vitro.

Being a novel target of luteinizing hormone (LH), the effect of CREB-binding protein/P300-interacting trans-activator with ED-rich tail member 4 (CITED4) gene on the proliferation, apoptosis, and steroidogenesis of ovarian granulosa cells (GCs) in Hu sheep was investigated. The presence of CITED4, CREB-binding protein (CBP), CCAAT/enhancer-binding protein alpha (C/EBPα) and -beta (C/EBPß) proteins was demonstrated in GCs and luteal cells. CITED4 protein in GCs was induced by LH, and CITED4 overexpression moderately increased GC responses to LH. In contrast, CITED4 knockdown in GCs decreased prostaglandin (PGE2)-induced LH target gene levels. Moreover, PGE2-stimulated CITED4 mRNA expression was blocked by ERK1/2 inhibition (U0126), suggesting that CITED4 is a downstream target of the ERK1/2 pathway in sheep GCs. In contrast to CITED4 knockdown, CITED4 overexpression promoted GC proliferation, inhibited apoptosis, upregulated cell cycle-related genes, and downregulated apoptosis-related genes. Additionally, CITED4 overexpression induced cell cycle transition from S to G2/M phase. No effect was observed with CITED4 knockdown. CITED4 overexpression increased progesterone (P4) production levels and STAR mRNA expression, whereas CITED4 knockdown decreased P4 production and STAR and 3ß-HSD mRNA expression levels. Thus, our results suggest that CITED4 is involved in regulating the expression of LH-induced genes and the ERK1/2 pathway and the proliferation, apoptosis, and steroidogenesis in Hu sheep GCs by modulating the expression of related genes. These findings will help understand the role of CITED4 in follicular development and ovulation of pre-ovulatory follicles.

The C-reactive protein to albumin ratio is an excellent prognostic predictor for gallbladder cancer.

A number of inflammation indicators based on C-reactive protein (CRP) and albumin have been widely used to predict the prognosis in several types of tumors, but their functions in gallbladder cancer (GBC) have rarely been explored. The aim of our study is to evaluate and compare the prognostic values of the C-reactive protein to albumin ratio (CAR), Glasgow prognostic score (GPS), modified Glasgow prognostic score (mGPS) and high-sensitivity modified Glasgow prognostic score (HS-mGPS) in patients with GBC. 144 GBC patients who received curative surgery in our hospital from January 2010 to May 2017 were enrolled in this research. The Kaplan-Meier analysis showed that the median OS of the patients in the high CAR group was significantly shorter than the patients in the low group (p < 0.001), and higher scores of GPS, mGPS and HS-mGPS were also associated with decreased OS, respectively. However, according to the Receiver Operating Characteristic (ROC) curve, the CAR was superior to the other prognostic scores in determining the prognosis for the GBC patients. In the multivariate analysis, CAR was verified as an independent risk factor for poor prognosis, together with tumor differentiation, T stage and postoperative complications. All in all, compared to the other three CRP-albumin-related prognostic predictors, CRA is a better indicator in predicting poor long-term outcomes in GBC patients after radical surgery.

Nomogram Based on Systemic Immune Inflammation Index and Prognostic Nutrition Index Predicts Recurrence of Hepatocellular Carcinoma After Surgery.

BACKGROUND: Surgery is a potential cure for hepatocellular carcinoma (HCC), but its postoperative recurrence rate is high, its prognosis is poor, and reliable predictive indicators are lacking. This study was conducted to develop a simple, practical, and effective predictive model. MATERIALS AND METHODS: Preoperative clinical and postoperative pathological data on patients with HCC undergoing partial hepatectomies at the Third Affiliated Hospital of Soochow University from January 2010 to December 2015 were retrospectively analyzed, and a nomogram was constructed. The model performance was evaluated using C-indexes, receiver operating characteristic curves, and calibration curves. The results were verified from validation cohort data collected at the same center from January 2016 to January 2017 and compared with the traditional staging systems. RESULTS: Three hundred three patients were enrolled in this study: 238 in the training cohort and 65 in the validation cohort. From the univariate and multivariate Cox regression analyses in the training cohort, six independent risk factors, i.e., age, alpha-fetoprotein (AFP), tumor size, satellite nodules, systemic immune inflammation index (SII), and prognostic nutritional index (PNI), were filtered and included in the nomogram. The C-index was 0.701 [95% confidence interval (CI): 0.654-0.748] in the training cohort and 0.705 (95% CI: 0.619-0.791) in the validation cohort. The areas under the curve for the 1- and 3-year recurrence-free survival were 0.706 and 0.716 in the training cohort and 0.686 and 0.743 in the validation cohort, respectively. The calibration curves showed good agreement. Compared with traditional American Joint Committee on Cancer 8th edition (AJCC8th) and Barcelona Clinic Liver Cancer (BCLC) staging systems, our nomogram showed better predictive ability. CONCLUSION: Our nomogram is simple, practical, and reliable. According to our nomogram, predicting the risk of recurrence and stratifying HCC patient management will yield the greatest survival benefit for patients.

Unconservative_15_2570409 suppresses progesterone receptor expression in the granulosa cells of Hu sheep.

Female fertility potential depends on the number of mature follicles; however, the underlying molecular mechanisms remain unclear. Based on previously generated miRNA and mRNA sequencing data of healthy ovarian follicles (>5 mm in diameter) isolated from Hu sheep with different prolificacy, we investigated the roles of a novel miRNA (unconservative_15_2570409) and the progesterone receptor (PGR) gene in follicular development. During the periovulatory phase, the expression of unconservative_15_2570409 and PGR was lower and higher, respectively, in the >5 mm follicles of high prolificacy (HP) ewes than in those of low prolificacy (LP) ewes and in the >3 mm follicles than in the smaller follicles of the HP ewes. Subsequently, the granulosa cells (GCs) of Hu sheep were used as an in vitro model. PGR overexpression significantly increased the mRNA expression of steroidogenic acute regulatory protein (StAR), 3-beta-hydroxysteroid dehydrogenase/isomerase (3ß-HSD), and cytochrome P450 family 19 subfamily A member 1 (CYP19A1), which promoted the secretion of progesterone (P4) and estradiol (E2). PGR knockdown significantly downregulated the levels of StAR and 3ß-HSD mRNA and decreased the production of P4, whereas no effects on CYP19A1 mRNA expression and E2 levels were observed. We also found the negative regulatory effect of unconservative_15_2570409 on the mRNA and protein expression of PGR by targeting the 3'-untranslated region. The regulation of PGR levels resulted in a corresponding change in the ADAMTS1, PPAR-γ, and CTSL gene transcripts, which are important for follicular maturation and ovulation. Additionally, PGR, ADAMTS1, and PPAR-γ were predominantly localized in the GCs. Collectively, our results suggest that by regulating PGR expression and consequently affecting the expression of target genes and steroidogenesis, unconservative_15_2570409 plays a role in follicular development during the periovulatory stage, which provides novel insights into the molecular mechanisms affecting Hu sheep prolificacy.

Effects of spirulina supplementation on lipid metabolism disorder, oxidative stress caused by high-energy dietary in Hu sheep.

The aim of this study was to investigate the effect of spirulina supplementation in a high-energy (HE) diet on lipid metabolism, oxidative status and immunity in Hu lambs. The lambs were assigned to two groups receiving either a standard diet (ST) or a HE diet. Each group was divided into three subgroups: no spirulina supplementation (control), 1% spirulina supplementation, or 3% spirulina supplementation. The body fat, serum cholesterol, triacylglycerol and oxidative stress increased in lambs fed the HE diet. However, 3% spirulina supplementation in the HE diet reduced above parameters and enhanced antioxidant capacity, including increased SOD activity and T-AOC content in serum and Longissimus thoracis et lumborum (LTL). Additionally, lambs receiving 3% spirulina supplementation showed an improvement in immunity-related parameters, including increased IgG concentration in serum and red and white blood cell counts. In conclusion, 3% spirulina supplementation in HE diet ameliorated lipid metabolic disorder and oxidative stress caused by a HE diet.

Multi-modal neuroimaging feature selection with consistent metric constraint for diagnosis of Alzheimer's disease.

The accurate diagnosis of Alzheimer's disease (AD) and its early stage, e.g., mild cognitive impairment (MCI), is essential for timely treatment or possible intervention to slow down AD progression. Recent studies have demonstrated that multiple neuroimaging and biological measures contain complementary information for diagnosis and prognosis. Therefore, information fusion strategies with multi-modal neuroimaging data, such as voxel-based measures extracted from structural MRI (VBM-MRI) and fluorodeoxyglucose positron emission tomography (FDG-PET), have shown their effectiveness for AD diagnosis. However, most existing methods are proposed to simply integrate the multi-modal data, but do not make full use of structure information across the different modalities. In this paper, we propose a novel multi-modal neuroimaging feature selection method with consistent metric constraint (MFCC) for AD analysis. First, the similarity is calculated for each modality (i.e. VBM-MRI or FDG-PET) individually by random forest strategy, which can extract pairwise similarity measures for multiple modalities. Then the group sparsity regularization term and the sample similarity constraint regularization term are used to constrain the objective function to conduct feature selection from multiple modalities. Finally, the multi-kernel support vector machine (MK-SVM) is used to fuse the features selected from different models for final classification. The experimental results on the Alzheimer's Disease Neuroimaging Initiative (ADNI) show that the proposed method has better classification performance than the start-of-the-art multimodality-based methods. Specifically, we achieved higher accuracy and area under the curve (AUC) for AD versus normal controls (NC), MCI versus NC, and MCI converters (MCI-C) versus MCI non-converters (MCI-NC) on ADNI datasets. Therefore, the proposed model not only outperforms the traditional method in terms of AD/MCI classification, but also discovers the characteristics associated with the disease, demonstrating its promise for improving disease-related mechanistic understanding.