Pyruvate kinase M in germ cells is essential for sperm motility and male fertility but not spermatogenesis.

ABSTRACT: Male germ cells employ specific metabolic pathways throughout their developmental stages. In a previous study, we discovered heightened expression of pyruvate kinase M (PKM), a pivotal glycolytic enzyme, in spermatogonia and spermatids. To gain deeper insights into PKM's roles in spermatogenesis, sperm function, and male fertility, we engineered a conditional-knockout mouse model (Pkm-vKO mice) to selectively disrupt the Pkm gene within germ cells. Despite maintaining regular testicular histology and sperm morphology, the male Pkm-vKO mice were infertility, characterized by significant impairments in sperm motility and adenosine triphosphate (ATP) generation. In addition, Pkm-null spermatozoa exhibited similar deficits in protein tyrosine phosphorylation linked to capacitation, as well as compromised performance in in vitro fertilization experiments. To conclude, PKM's presence is not obligatory for the entirety of spermatogenesis in male germ cells; however, it emerges as a critical factor influencing sperm motility and overall male fertility.

Tumour follower cells: A novel driver of leader cells in collective invasion (Review).

Collective cellular invasion in malignant tumours is typically characterized by the cooperative migration of multiple cells in close proximity to each other. Follower cells are led away from the tumour by specialized leader cells, and both cell populations play a crucial role in collective invasion. Follower cells form the main body of the migration system and depend on intercellular contact for migration, whereas leader cells indicate the direction for the entire cell population. Although collective invasion can occur in epithelial and non­epithelial malignant neoplasms, such as medulloblastoma and rhabdomyosarcoma, the present review mainly provided an extensive analysis of epithelial tumours. In the present review, the cooperative mechanisms of contact inhibition locomotion between follower and leader cells, where follower cells coordinate and direct collective movement through physical (mechanical) and chemical (signalling) interactions, is summarised. In addition, the molecular mechanisms of follower cell invasion and metastasis during remodelling and degradation of the extracellular matrix and how chemotaxis and lateral inhibition mediate follower cell behaviour were analysed. It was also demonstrated that follower cells exhibit genetic and metabolic heterogeneity during invasion, unlike leader cells.

Aluminum-catalyzed tunable halodefluorination of trifluoromethyl- and difluoroalkyl-substituted olefins.

Herein, we report unprecedented aluminum-catalyzed halodefluorination reactions of trifluoromethyl- and difluoroalkyl-substituted olefins with bromo- or chlorotrimethylsilane. The interesting feature of these reactions is that one, two, or three fluorine atoms can be selectively replaced with bromine or chlorine atoms by modification of the reaction conditions. The generated products can undergo a variety of subsequent transformations, thus constituting a valuable stock of building blocks for installing fluorine-containing olefin motifs in other molecules.

Long noncoding RNAs in cancer-immunity cycle.

The imbalance of immune status in cancer microenvironment plays an important role in the development and progression of cancer. Immunotherapy based on this has become an important field of cancer research in recent years. Many studies on long noncoding RNA (lncRNA) in cancer have focus on its regulation in cancer development and metastasis. Recent studies have suggested that lncRNAs play crucial roles in different phases of cancer immunity, including antigen releasing, antigen presentation, immune activation, immune cells migration, infiltrating into cancer tissues, and killing cancer cells. The functional studies of lncRNAs in cancer immuntity revealed the complicated molecular mechanisms in cancer immunity from a new point of view, which may provide novel potential targets for cancer immunotherapies. Based on the classical cancer-immunity cycle theory, we review the recent studies on the functions and mechanisms of immune-related lncRNAs in different stages of cancer immunity, to summarize the relationship between lncRNAs, and cancer immunity and to provide a framework for further research.

Reproductive outcomes in women with prior cesarean section undergoing in vitro fertilization: A retrospective case-control study.

The impact of prior cesarean section (CS) on the pregnancy and neonatal outcomes of in vitro fertilization and embryo transfer (IVF-ET) was investigated. A retrospective analysis was performed on 144 patients with prior CS between January 2013 and December 2015. The pregnancy, delivery, and neonatal outcomes of patients who had previous CS delivery and received IVF-ET were analyzed. The control group comprised 166 patients who had only previous vaginal delivery (VD) and received IVF-ET during the same period. The results showed that the basal follicle stimulating hormone level, estradiol level on human chorionic gonadotropin (hCG) day, gonadotrophin dosage, duration of stimulation, retrieved oocytes, fertilization rate, high-quality embryo rate, multiple birth rate, abortion rate and ectopic pregnancy rate had no significant difference between the two groups (P>0.05). The pregnancy rate (40.28% vs. 54.22%) and implantation rate (24.01% vs. 34.67%) were significantly lower (P<0.05), and the ratio of embryo difficulty transfer (9/144 vs. 0/166) was significantly higher in CS group than in VD group. The risk of pernicious placenta previa and postpartum hemorrhage in twin deliveries was significantly increased in CS group as compared with that in VD group (P<0.05), and gestational age and neonatal birth weight were significantly reduced in twin deliveries as compared with singleton deliveries in both groups (P<0.05). It was suggested that the existence of CS scar may impact embryo implantation and clinical pregnancy outcome, and increase the difficulty of ET. We should limit the number of transfer embryos to avoid multiple pregnancies and strengthen gestational supervision in patients with cesarean scar.

Chimeric-antigen receptor T (CAR-T) cell therapy for solid tumors: challenges and opportunities.

Chimeric antigen receptor (CAR)-engineered T cells (CAR-T cells) have been shown to have unprecedented efficacy in B cell malignancies, most notably in B cell acute lymphoblastic leukemia (B-ALL) with up to a 90% complete remission rate using anti-CD19 CAR-T cells. However, CAR T-cell therapy for solid tumors currently is faced with numerous challenges such as physical barriers, the immunosuppressive tumor microenvironment and the specificity and safety. The clinical results in solid tumors have been much less encouraging, with multiple cases of toxicity and a lack of therapeutic response. In this review, we will discuss the current stats and challenges of CAR-T cell therapy for solid tumors, and propose possibl e solutions and future perspectives.

QuEChERS followed by dispersive liquid-liquid microextraction based on solidification of floating organic droplet method for organochlorine pesticides analysis in fish.

A fast, sensitive and environmental-friendly method was developed to determine 13 organochlorine pesticides in fish tissue through gas chromatography with electron capture detection. Sample was extracted initially by acetonitrile and then concentrated through dispersive liquid-liquid microextraction based on solidification of floating organic droplet (DLLME-SFO). Dispersive solid phase extraction (d-SPE) was not a separate step but integrated to DLLME-SFO procedure. In order to separate the d-SPE sorbent, water and organic phase well, a modification to the glass centrifuge tube was made to achieving high centrifugal speed. The optimized method was validated with recoveries ranging from 88.1% to 121.2% (with relative standard deviations <15%) at three spiked levels for all of the pesticides. Good linearity was achieved at seven concentration levels from 6.25 to 625µgL-1, which corresponded to 0.001-0.100mgkg-1 in the sample. The limits of quantification and the limits of detection for 13 OCPs ranged from 1.94×10-3 to 4.93×10-3mgkg-1 and 6.50×10-4 to 1.58×10-3mgkg-1, respectively. The method was successfully applied to analyze real fish samples.

Trends and Prospects of Stem Cell Research in China.

Great progresses have been made in fundamental and clinical stem cell research in China in recent years. The official policy on stem cells, which was announced in 2015, seems as the spring of stem cell therapy in China. However, the regulation, governance, and management of clinical expectations are still challenging. This review summarized the current stem cell research and development in the field, as well as its rapidly evolving commercial, regulatory and ethical environment in China. As expected, the prospects of stem cells in China look prospective.

Methylophiopogonanone A suppresses ischemia/reperfusion-induced myocardial apoptosis in mice via activating PI3K/Akt/eNOS signaling pathway.

AIM: The dried tuber root of Ophiopogon japonicus has been used in the traditional Chinese medicine for treatment of myocardial ischemia and thrombosis. In this study we investigated the effects of methylophiopogonanone A (MO-A), a major homoisoflavonoid in Ophiopogon japonicus, on myocardial ischemia/reperfusion (I/R) injury. METHODS: Mice were pretreated with MO-A (10 mg·kg(-1)·d(-1), po) for 2 weeks and then subjected to transient occlusion of the left anterior descending coronary artery. Cardiac function was evaluated, and the infarct size and apoptosis index were assessed. The mechanisms underlying the cardio-protection of MO-A were analyzed in H9C2 rat cardiomyocytes subjected to hypoxia/reoxygenation (H/R). The cell viability and apoptosis were evaluated; apoptotic and relevant signaling proteins were analyzed. NO levels in the culture medium were assessed. RESULTS: In I/R mice, pretreatment with MO-A significantly reduced the infarct size (by 60.7%) and myocardial apoptosis (by 56.8%), and improved cardiac function. In H9C2 cells subjected to H/R, pretreatment with MO-A (10 µmol/L) significantly decreased apoptosis and cleaved caspase-3 expression, elevated the Bcl-2/Bax ratio and restored NO production. Furthermore, pretreatment with MO-A markedly increased the activation of PI3K/Akt/eNOS pathway in H9C2 cells subjected to H/R, and the protective effects of MO-A were abolished in the presence of the PI3K inhibitor wortmannin (100 nmol/L). CONCLUSION: MO-A attenuates I/R-induced myocardial apoptosis in mice via activating the PI3K/Akt/eNOS signaling pathway.

Flavone synthases from Lonicera japonica and L. macranthoides reveal differential flavone accumulation.

Flavones are important secondary metabolites found in many plants. In Lonicera species, flavones contribute both physiological and pharmaceutical properties. However, flavone synthase (FNS), the key enzyme responsible for flavone biosynthesis, has not yet been characterized in Lonicera species. In this study, FNSII genes were identified from Lonicera japonica Thunb. and L. macranthoides Hand.-Mazz. In the presence of NADPH, the recombinant cytochrome P450 proteins encoded by LjFNSII-1.1, LjFNSII-2.1, and LmFNSII-1.1 converted eriodictyol, naringenin, and liquiritigenin to the corresponding flavones directly. The different catalytic properties between LjFNSII-2.1 and LjFNSII-1.1 were caused by a single amino acid substitution at position 242 (glutamic acid to lysine). A methionine at position 206 and a leucine at position 381 contributed considerably to the high catalytic activity of LjFNSII-1.1. In addition, LjFNSII-1.1&2.1 and LmFNSII-1.1 also biosynthesize flavones that were further modified by O-glycosylation in transgenic tobacco. The expression levels of the FNSII genes were consistent with flavone accumulation patterns in flower buds. Our findings suggested that the weak catalytic activity of LmFNSII-1.1 and the relatively low expression of LmFNSII-1.1 in flowers might be responsible for the low levels of flavone accumulation in flower buds of L. macranthoides.

MicroRNA-related single-nucleotide polymorphism of XPO5 is strongly correlated with the prognosis and chemotherapy response in advanced non-small-cell lung cancer patients.

This study was performed to investigate if the microRNA-related single-nucleotide polymorphisms (miR-SNPs) of XPO5 gene predicted the prognosis and pathological features of advanced non-small-cell lung cancer patients receiving chemotherapy. A total of 131 advanced non-small-cell lung cancer (NSCLC) patients were recruited. MicroRNA (miRNA) binding site prediction software was adopted for the prediction and screening of SNPs in XPO5 and miRNA binding regions. Polymerase chain reaction (PCR) amplification was further performed. Time-dependent survival-free curves were constructed using the Kaplan-Meier technique. Univariate and the multivariate survival analyses were conducted for confirmation of prognostic factor for advanced NSCLC patients receiving chemotherapy. There were no significant differences of SNP distribution frequencies between groups, without statistical significance (P > 0.05). Included clinical pathological features and chemotherapy regimens showed no apparent statistical significance in influencing the curative effect of chemotherapy in advanced NSCLC patients (all P > 0.05). While the objective response rate (ORR) in patients who carried AA and AC genotype was 35.48 and 51.22 %, respectively, with statistically significant difference (P < 0.05). Univariate survival analysis indicated that patients who carried AA genotype showed a significantly lower 5-year survival rate to those who carried AC genotype (P < 0.05). And, considering pathological features, statistical significance was found in patients with different pathological types, lymph node metastasis, differentiation degree, T staging, and pathological staging (all P < 0.05). Multivariate analysis results indicated that the SNP sites of rs11077 might be an independent prognostic factor of advanced NSCLC patients receiving chemotherapy (risk ratio [RR] = 0.346; 95 % confidence interval [95 % CI] = 0.174-0.685, P = 0.002). Other clinical features were all considered to have no apparent effect in influencing the prognostic outcomes of advanced NSCLC patients receiving chemotherapy except lymph node metastasis (P < 0.05). miR-SNP rs11077 of XPO5 may be independently connected with the prognosis and chemotherapy response of advanced NSCLC patients, and patients with AC genotype have relatively improved prognostic outcomes and better curative effect of chemotherapy than those with AA allele of XPO5. Further, lymph node metastasis may be also involved in influencing the prognosis of advanced NSCLC patients.

MicroRNA-185 regulates expression of lipid metabolism genes and improves insulin sensitivity in mice with non-alcoholic fatty liver disease.

AIM: To assess the regulatory effect of microRNA-185 (miR-185) on lipid metabolism and the insulin signalling pathway in human HepG2 hepatocytes and a high-fat diet mouse model. METHODS: Quantitative reverse transcription-polymerase chain reaction was used to assess the mRNA levels of lipogenic genes after loss or gain of miR-185. In addition, the amounts of insulin signalling intermediates were determined after transfection of HepG2 cells with pre-miR-185. RESULTS: MiR-185 levels decreased in a time- and dose-dependent manner in response to palmitic acid in human HepG2 hepatocytes. Transfection of HepG2 cells with miR-185 significantly decreased the mRNA levels of fatty acid synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, sterol-regulatory element binding protein-2, and sterol-regulatory element binding protein-1c, whereas inhibition of miR-185 using an anti-miR-185 oligonucleotide produced the opposite effect in HepG2 cells. In a high-fat diet mouse model, the accumulation of lipids was significantly improved after treatment with miR-185, compared with control animals. Induction of miR-185 enhanced the insulin signalling pathway by up-regulating the insulin-receptor substrate-2. CONCLUSION: These findings suggest that miR-185 plays an important role in regulating fatty-acid metabolism and cholesterol homeostasis in hepatocytes, as well as in improving insulin sensitivity, both in vitro and in vivo.

Expression and clinical significance of the DNA repair enzyme MYH in esophageal squamous cell carcinoma.

MYH is an important enzyme in combating DNA oxidative stress in the occurrence and development of various types of tumors. To investigate the correlation between expression of the DNA repair enzyme MYH in esophageal squamous cell carcinoma and 8-oxoguanine (8-oxoG) oxidative damage, as well as the clinical significance of altered MYH expression, tissues from 175 esophageal carcinoma cases were investigated in the present study. MYH expression and 8-oxoG oxidative damage in squamous cell carcinoma and adjacent normal tissue were assessed by immunohistochemistry and Western blotting. In 82.9% (145/175) of the cases, MYH protein expression in esophageal squamous cell carcinoma was lower than that of adjacent normal tissue (t=4.24, P<0.001). Additionally, 8-oxoG staining was higher in the tumors than in the normal tissue. Lower expression of MYH in esophageal squamous cell carcinoma was associated with depth of invasion, venous invasion, TNM stage and lymph node metastasis (P<0.05). In conclusion, a lower MYH expression level in esophageal cell carcinoma tissue was inversely associated with more severe 8-oxoG oxidative damage, suggesting that changes in MYH activity correspond to increased DNA damage in tumor cells. The use of MYH expression as a postoperative index for esophageal squamous cell carcinoma may guide the formulation of individualized chemotherapy for patients after surgery.

CREB is a regulatory target for the protein kinase Akt/PKB in the differentiation of pancreatic ductal cells into islet ß-cells mediated by hepatocyte growth factor.

We have previously reported that the PI3K/Akt signaling pathway is involved in hepatocyte growth factor (HGF)-induced differentiation of adult rat pancreatic ductal epithelial cells (PDECs) into islet ß-cells in vitro. The transcription factor CREB is one of the downstream key effectors of the PI3K/Akt signaling pathway. Recent studies showing that CREB is required for the survival of certain cell types prompted us to examine whether CREB is a nuclear target for activation via the HGF-dependent Ser/Thr kinase Akt/PKB in the differentiation of pancreatic duct cell into islet ß-cells. In this study, we first attempted to examine whether HGF modulates the Akt-dependent activation of target gene CREB and then investigated whether CREB activity affects the differentiation of HGF-induced PDECs. Finally, we studied the role of CREB in modulating the expression of transcription factors in PDECs during the differentiation of HGF-induced PDECs. Our results demonstrated that CREB is a regulatory target for the protein kinase Akt/PKB in the differentiation of pancreatic ductal cells into islet ß-cells mediated by HGF.

[Synthesis and anti-tumor activity of oleanolic acid derivatives].

Structural modifications were performed with natural product of oleanolic acid to search for novel anticancer drugs. Ten oleanolic acid derivatives were designed and obtained by the reaction of oxidation, acylation or hydrolyzation, etc. The cytotoxic activity of derivatives was evaluated against HeLa, HepG2 and BGC-823 cells in vitro by MTT assay, gefitinib and etoposide used as a positive control. The results showed that compound 5a was particularly active to inhibit HepG2 cells growth, and anti-tumor activity of compound 7 on HeLa cells was significantly stronger than oleanolic acid. They are worthy to be studied further.

Impact of a laparoscopic resection on the quality of life in rectal cancer patients: results of 135 patients.

PURPOSE: To investigate the impact of a laparoscopic resection on the quality of life in rectal cancer patients. METHODS: This study included 135 patients (laparoscopic resection [LR] 65 cases and open resection [OR] 70 cases). The European Organization for Research and Treatment of Cancer QLQ-C30 and QLQ-CR38 questionnaires were used to measure the quality of life before the operation, then 1 week, 3 months, and 1 year after the operation. RESULTS: Eleven (16.9%) patients underwent a conversion from laparoscopic to open surgery. The incision length and blood loss both decreased significantly in the LR group in comparison to the OR group (P < 0.05). Recovery of the gastrointestinal function, bladder function, and ambulation was more rapid in the LR group (P < 0.05). The patients in the LR group reported better global health status (33.3 vs 25.0, P < 0.001), body image (77.8 vs 66.7, P = 0.008), and less pain (33.3 vs 50. 0, P = 0.009) 1 week after operation. Better body image was reported in the LR group even 1 year after the operation (P < 0.05). Fewer financial difficulties were reported by patients in the LR group (P < 0.001). No significant differences were found between two groups on other scales. CONCLUSIONS: This study showed that the quality of life benefits due to minimally invasive laparoscopic surgery were evident only in the immediate postoperative period. A laparoscopic rectal resection therefore provided only better cosmetic benefit over the longer term.

Mechanisms of hepatocyte growth factor-mediated signaling in differentiation of pancreatic ductal epithelial cells into insulin-producing cells.

Pancreatic ductal epithelial cells (PDECs) were induced to differentiate into insulin-producing cells by hepatocyte growth factor (HGF) in our previous study, but the mechanism through which this induction occurs is still unknown. HGF is a ligand that activates a tyrosine kinase encoded by the c-Met proto-oncogene. This activation is followed by indirect activation of multiple downstream signal transduction pathways (including MAPKs and the PI3K/AKT signaling pathways) that initiate various biological effects. Therefore, we speculated that the differentiation of PDECs is through either the MAPK signaling pathway or the PI3K/AKT signaling pathway. To test this hypothesis, isolated PDECs from adult rats were stimulated by adding HGF to their medium for 28days. Then, the expression levels of several protein kinases, including MAPKs (ERK1/2, p38, and JNK) and AKT, were determined by Western blotting to determine if specific protein kinases are activated in these pathways. Subsequently, re-isolated from adult rats and cultured PDECs were pre-treated with specific inhibitors of proteins shown to be activated in these signaling pathways; these cells were then induced to differentiate by the addition of HGF. The expression levels of protein kinases were determined by Western blotting, and the differentiation rate of insulin-positive cells was determined by flow cytometry. The change of PDEC differentiation rates were compared between the groups in which cells with or without inhibitors pretreatment to determine the specific signaling pathway(s) that may be involved in HGF-induced differentiation of PDECs. After isolating PDECs and stimulating them with HGF for 28days, the expression levels of phosphorylated ERK1/2 as well as total and phosphorylated AKT of cultured cells were significantly increased compared to the normal control group (p<0.05), suggesting that the signaling pathways involving ERK1/2 and Akt (MEK-ERK and PI3K-AKT) are activated during HGF-induced PDEC differentiation. MEK1/2 or PI3K inhibitors were separately added to the culture medium of PDECs pre-treated with HGF. These results show that compared to the HGF-treated group, the differentiation rate of insulin-positive cells was significantly decreased in the HGF/LY294002 (PI3K inhibitor) group (13.47+/-1.57% vs. 33.47+/-1.34%, p<0.05); however, the differentiation rate of insulin-positive cells was not significantly different in the HGF/PD98059 (MEK1/2 inhibitor) group. These data suggest that HGF induces PDECs to differentiate into insulin-producing cells through the PI3K/AKT signaling pathway.

Laparoscopic surgery for the curative treatment of rectal cancer: results of a Chinese three-center case-control study.

BACKGROUND: This study aimed to assess the efficacy and safety of laparoscopic resection (LR) for rectal cancer. METHODS: A case-control study involving three Chinese medical centers was conducted. Rectal cancer patients undergoing LR were compared with open resection (OR) cases simultaneously from January 2004 to December 2005. Data were collected, and basic characteristics, conversion rate, recovery, complications, adjuvant therapy, and recurrence rate were compared. Analysis was by intention to treat. RESULTS: A total of 335 rectal cancer procedures (115 LR and 220 OR) met the inclusion criteria. The patients' basic characteristics were similar in the two groups (p>0.05). Total mesorectal excision was performed for 85.59% of the patients (201/235), who received anal sphincter preservation. Compared with OR, LR had a shorter incision length, less blood loss, and less need for transfusion, but the operation time was longer (p<0.05). No significant differences were observed between the two groups in positive rates of longitudinal resection margins, numbers of harvested lymph nodes, complication rates during operation and postoperation, and perioperative reoperation and morbidity rates (p>0.05). Postoperative parenteral narcotics were used less in LR than in OR (47.8% vs 62.7%; chi(2)=6.867; p=0.009). The median time until first flatus; resumption of diet, defecation, micturition, and ambulation; and discharge were reduced in LR (p<0.05). Conversion from LR to OR was required by 11.3% of the patients (13/115). The intraoperative complication rate was 30.8% for the patients who underwent conversion. The operation time and postoperative complication rate were the same as for LR alone (p>0.05). The local recurrence rate was 3.7% for the LR group and 4.9% for the OR group (chi (2)=0.209; p=0.647) during the 20-month median follow-up period. CONCLUSIONS: The findings showed that LR for rectal cancer was safe and effective, resulting in faster recovery and a similar complication rate compared with OR. Conversion did not alter the patients' outcomes.

[Quality of life in patients with rectal cancer after laparoscopic colectomy].

OBJECTIVE: To assess the quality of life in patients with rectal cancer after laparoscopic colectomy. METHODS: From Sep.2004 to Dec. 2005, 51 patients with rectal cancer were recruited in this prospective, non-randomized study.Twenty-three patients underwent laparoscopic colectomy (LC), 28 patients had open colectomy (OC). EORTC QLQ-C30 and QLQ-CR38 questionnaire were applied to evaluate quality of life baseline, discharging and 3 months after operation. RESULTS: Before operation,the median score of role functioning in LC group was lower. However, LC group patients complained less financial difficulties. The differences were of statistical significance (P<0.05), but not of clinical significance. The median scores of other function domains and symptom domains were similar between two groups (P>0.05). Postoperatively, the most median scores of function domains and symptom domains between two groups were similar (P>0.05). The only score with statistically significant difference was the pain when patients left hospital (U=218.5, P=0.042). However, the difference was not of clinical significance, too. CONCLUSION: Only minimal benefits in short-term postoperative quality of life are found with laparoscopic colectomy in patients with rectal cancer compared with open colectomy.