Quantitative iTRAQ-based proteomic analysis of differentially expressed proteins in aging in human and monkey.

BACKGROUND: The underlying physiological mechanisms associated with aging are still complex and unclear. As a very important tissue of human body, the circulatory system also plays a very important role in the process of aging. In this study, we use the isobaric tags for relative and absolute quantification (iTRAQ) method to identify differentially expressed proteins in plasma for humans and monkeys between young and aged. Western blotting and behavioral experiment in mice were performed to validate the expression of the candidate protein. RESULTS: Between the young / the old humans and the young / the old monkeys 74 and 69 proteins were found to be differently expressed, respectively. For the human samples, these included 38 up-regulated proteins and 36 down-regulated proteins (a fold change ≥1.3 or ≤ 0.667, p value ≤0.05).For the monkey samples, 51 up-regulated proteins and 18 down-regulated proteins (a fold change ≥1.3 or ≤ 0.667, p value ≤0.05). KEGG pathway analysis revealed that phagosome, focal adhesion, ECM-receptor interaction and PI3K/AKT signaling pathway were the most common pathways involved in aging. We found only IGFBP4 protein that existed in up-regulated proteins in aged both for human and monkey. In addition, the differential expression of IGFBP4 was validated by western blot analysis and IGFBP4 treatment mimicked aging-related cognitive dysfunction in mice. CONCLUSIONS: This first, the integrated proteomics for the plasma protein of human and monkey reveal one protein-IGFBP4, which was validated by western blotting and behavioral analysis can promote the process of aging. And, iTRAQ analysis showed that proteolytic systems, and inflammatory responses plays an important role in the process of aging. These findings provide a basis for better understanding of the underlying mechanisms involved in aging.

Transvaginal natural orifice transluminal endoscopic surgery in the diagnosis of ascites of unknown origin.

BACKGROUND AND AIMS: Natural orifice transluminal endoscopic surgery (NOTES) has been established in animal models and human studies, but few clinical studies have investigated transvaginal NOTES in the diagnosis of unexplained refractory ascites. We aimed to assess the feasibility, efficacy, and safety of transvaginal NOTES for the diagnosis of unexplained ascites in female patients. METHODS: A prospective study was done involving 3 female patients with unexplained ascites. After general anesthesia and disinfection, a 1.0-cm incision was made in the posterior fornix of the vagina. A gastroscope was inserted into the abdominal cavity through the transvaginal incision and an artificial pneumoperitoneum was created; NOTES peritoneoscopy was performed to scrutinize the pathologic changes. Endoscopic biopsy specimens were obtained for pathologic examination. The transvaginal incision was closed by direct suturing. RESULTS: Transvaginal NOTES for diagnostic peritoneoscopy was successfully performed in 3 patients. The mean operative time was 61 minutes. The estimated blood loss was 5 to 10 mL. The pathologic diagnoses were tuberculosis for all patients, and the symptoms and ascites disappeared after antituberculosis therapy. During the 4-year follow-up, no clinically significant adverse events occurred in any patient after NOTES. No patient experienced an annex inflammation, vaginitis, dyspareunia, or sexual dysfunction. All patients were comfortable and satisfied with the nonscarring surgical procedure. CONCLUSIONS: Transvaginal NOTES for the diagnosis of unexplained ascites is feasible, effective, and safe. This method had no long-term effect on female sexual function and is particularly suitable for women who have special aesthetic requirements. (Clinical trial registration number: ChiCTR-TRC-10001053.).

Influence of carboplatin on the proliferation and apoptosis of ovarian cancer cells through mTOR/p70s6k signaling pathway.

PURPOSE: To investigate the influence of carboplatin on the proliferation and apoptosis of ovarian cancer cells through mTOR/P70S6K signaling pathway. METHODS: The mRNA and protein expressions were detected via Western blotting and RT-PCR to study whether the mTOR/p70S6K signaling pathway was activated in OVCAR-3 and Caov-3 ovarian cancer cell lines. After cells were treated with different concentrations of carboplatin, the mRNA and protein expressions of mTOR, p70S6K and 4E-BP1 were detected via RT-PCR and Western blotting. OVCAR-3 cells were treated with 20 and 50 µM carboplatin for 4 hrs, and then apoptosis was analyzed and assessed. OVCAR-3 cells were treated with different concentrations of carboplatin (20, 50, 100, 150 and 200 µM) for 24 and 48 hrs, respectively. RESULTS: The mTOR signaling pathway was activated in OVCAR-3 and Caov-3 ovarian cancer cell lines. The mRNA level of mTOR in Caov-3 cells was higher, but that of p70S6K was lower. Carboplatin significantly reduced the mRNA expression of mTOR (p<0.01), whereas the mRNA expressions of p70S6K and 4E-BP1 in carboplatin-treated cells were increased in a dose-dependent manner (p<0.01). Carboplatin inhibited the mTOR protein expression in a dose-dependent manner (p<0.01). The proliferation of OVCAR-3 cells exposed to carboplatin was reduced compared with that of untreated cells (p<0.01), and the inhibitory effect of carboplatin on the proliferation of OVCAR-3 cells was time- and dose-dependent. CONCLUSION: The mTOR/p70S6K pathway was activated in ovarian cancer. Carboplatin could rapidly inhibit the expression of mTOR, and the phosphorylation of its major downstream effectors p70S6K and 4E-binding protein 1 (4E-BP1) arrested cells in G0/G1 phase and induced ovarian cancer cell apoptosis.

Promoter DNA methylation is associated with KLF11 expression in epithelial ovarian cancer.

As a transforming growth factor-ß (TGF-ß)-inducible gene, the expression of Krüppel-like transcription factor 11 (KLF11) is altered in several types of cancer. In the current study, through using human 9K CpG island array, KLF11 was identified as one of hypermethylated genes in RAS-transformed ovarian T29H cells. Methylation of the KLF11 promoter was also observed in ovarian cancer tissue samples accompanied by significantly reduced KLF11 gene expression. Interestingly, the expression of SMAD2, SMAD3, and SMAD7 genes was reduced in the tumour, whilst no change was found in TGF-ß expression. Our data suggest a relationship between promoter DNA methylation and KLF11 gene expression in ovarian cancer tumorigenesis.

Ras-induced epigenetic inactivation of the RRAD (Ras-related associated with diabetes) gene promotes glucose uptake in a human ovarian cancer model.

RRAD (Ras-related associated with diabetes) is a small Ras-related GTPase that is frequently inactivated by DNA methylation of the CpG island in its promoter region in cancer tissues. However, the role of the methylation-induced RRAD inactivation in tumorigenesis remains unclear. In this study, the Ras-regulated transcriptome and epigenome were profiled by comparing T29H (a Ras(V12)-transformed human ovarian epithelial cell line) with T29 (an immortalized but non-transformed cell line) through reduced representation bisulfite sequencing and digital gene expression. We found that Ras(V12)-mediated oncogenic transformation was accompanied by RRAD promoter hypermethylation and a concomitant loss of RRAD expression. In addition, we found that the RRAD promoter was hypermethylated, and its transcription was reduced in ovarian cancer versus normal ovarian tissues. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine resulted in demethylation in the RRAD promoter and restored RRAD expression in T29H cells. Additionally, treatment with farnesyltransferase inhibitor FTI277 resulted in restored RRAD expression and inhibited DNA methytransferase expression and activity in T29H cells. By employing knockdown and overexpression techniques in T29 and T29H, respectively, we found that RRAD inhibited glucose uptake and lactate production by repressing the expression of glucose transporters. Finally, RRAD overexpression in T29H cells inhibited tumor formation in nude mice, suggesting that RRAD is a tumor suppressor gene. Our results indicate that Ras(V12)-mediated oncogenic transformation induces RRAD epigenetic inactivation, which in turn promotes glucose uptake and may contribute to ovarian cancer tumorigenesis.

A retrospective study of the effects of uterine manipulators on prognosis in patients with cervical cancer.

OBJECTIVE: This retrospective study investigated the effects of uterine manipulator use during minimally invasive radical hysterectomy on prognosis in patients with cervical cancer. METHODS: We collected clinical data on 762 patients with stage IA2 to IIB cervical cancer who underwent radical hysterectomy and pelvic lymphadenectomy in Chinese PLA General Hospital from 2009 to 2019. Kaplan-Meier analysis and log-rank tests were used to compare the 5-year overall survival rates between patients treated with and without a uterine manipulator. RESULTS: Patient demographics did not differ between the two groups. In addition, the incidence of lymphovascular space invasion, tumor size, pathologic types, the International Federation of Gynecology and Obstetrics stage, the histologic grade, and the rate of lymphatic metastases did not differ between the groups. Meanwhile, perioperative clinical indicators were similar in the groups. Furthermore, no significant differences in 5-year survival rates and survival curves were recorded between the groups among both all patients (84.5% vs. 85.6%) and early-stage patients (89.1% vs. 89.2%). CONCLUSIONS: The use of uterine manipulators during minimally invasive radical hysterectomy for cervical cancer did not affect clinicopathological markers or increase the risk of death.

Overexpression of the halophyte Kalidium foliatum H⁺-pyrophosphatase gene confers salt and drought tolerance in Arabidopsis thaliana.

According to sequences of H(+)-pyrophosphatase genes from GenBank, a new H(+)-pyrophosphatase gene (KfVP1) from the halophyte Kalidium foliatum, a very salt-tolerant shrub that is highly succulent, was obtained by using reverse transcription PCR and rapid amplification of cDNA ends methods. The obtained KfVP1 cDNA contained a 2295 bp ORF and a 242 bp 3'-untranslated region. It encoded 764 amino acids with a calculated molecular mass of 79.78 kDa. The deduced amino acid sequence showed high identity to those of H(+)-PPase of some Chenopodiaceae plant species. Semi-quantitative PCR results revealed that transcription of KfVP1 in K. foliatum was induced by NaCl, ABA and PEG stress. Transgenic lines of A. thaliana with 35S::KfVP1 were generated. Three transgenic lines grew more vigorous than the wild type (ecotype Col-0) under salt and drought stress. Moreover, the transgenic plants accumulated more Na(+) in the leaves compared to wild type plants. These results demonstrated that KfVP1 from K. foliatum may be a functional tonoplast H(+)-pyrophosphatase in contributing to salt and drought tolerance.

Efficacy and safety of MIL60 compared with bevacizumab in advanced or recurrent non-squamous non-small cell lung cancer: a phase 3 randomized, double-blind study.

BACKGROUND: We compared the efficacy, safety, and immunogenicity of MIL60 with reference bevacizumab as first-line treatment in patients with advanced or recurrent non-squamous non-small cell lung cancer (NSCLC) in this phase 3, randomized, double-blind study. METHODS: Patients with untreated advanced or recurrent NSCLC were randomized (1:1 ratio) to receive either MIL60 or bevacizumab in combination with paclitaxel/carboplatin. Patients with non-progressive disease continued maintenance single-agent MIL60 until disease progression, or intolerable toxicity. The primary endpoint was the 12-week objective response rates (ORR12) by independent review committee (IRC) using RECIST 1.1. Bioequivalence was established if the ORR ratio located between 0.75 and 1/0.75. The trial was registered with clinicaltrials.gov (NCT03196986). FINDINGS: Between Aug 23, 2017, and May 8, 2019, 517 patients were randomly assigned to MIL60 group (n=257) and bevacizumab group (n=260). In the full analysis set (FAS) population including all randomized and evaluable patients who received at least one dose of MIL60 or bevacizumab, the ORR12 in MIL60 group and bevacizumab group were 48.6% and 43.1%, respectively. The ORR ratio of these two groups were 1.14 (90% CI 0.97-1.33), which fell within the pre-specified equivalence boundaries (0.75-1/0.75). The median DOR was 5.7 months (95% CI 4.5-6.2) for MIL60 and 5.6 months (95% CI 4.3-6.4) for bevacizumab. No significant difference was noted in median PFS (7.2 vs. 8.1 months; HR 1.01, 95% CI 0.78-1.30, p=0.9606) and OS (19.3 vs. 16.3 months; HR 0.81, 95% CI 0.64-1.02, p=0.0755). Safety and tolerability profiles were similar between the two groups. No patient detected positive for Anti-drug antibody (ADA). INTERPRETATION: The efficacy, safety and immunogenicity of MIL60 were similar with bevacizumab, providing an alternative treatment option for advanced or recurrent non-squamous NSCLC. FUNDING: This study was sponsored by Betta Pharmaceutical Co., Ltd.

Flavonoid biosynthesis controls fiber color in naturally colored cotton.

The existence of only natural brown and green cotton fibers (BCF and GCF, respectively), as well as poor fiber quality, limits the use of naturally colored cotton (Gossypium hirsutum L.). A better understanding of fiber pigment regulation is needed to surmount these obstacles. In this work, transcriptome analysis and quantitative reverse transcription PCR revealed that 13 and 9 phenylpropanoid (metabolic) pathway genes were enriched during pigment synthesis, while the differential expression of phenylpropanoid (metabolic) and flavonoid metabolic pathway genes occurred among BCF, GCF, and white cotton fibers (WCF). Silencing the chalcone flavanone isomerase gene in a BCF line resulted in three fiber phenotypes among offspring of the RNAi lines: BCF, almost WCF, and GCF. The lines with almost WCF suppressed chalcone flavanone isomerase, while the lines with GCF highly expressed the glucosyl transferase (3GT) gene. Overexpression of the Gh3GT or Arabidopsis thaliana 3GT gene in BCF lines resulted in GCF. Additionally, the phenylpropanoid and flavonoid metabolites of BCF and GCF were significantly higher than those of WCF as assessed by a metabolomics analysis. Thus, the flavonoid biosynthetic pathway controls both brown and green pigmentation processes. Like natural colored fibers, the transgenic colored fibers were weaker and shorter than WCF. This study shows the potential of flavonoid pathway modifications to alter cotton fibers' color and quality.

Promoter Hypermethylation of FANCF and Susceptibility and Prognosis of Epithelial Ovarian Cancer.

OBJECTIVE: To assess the 5' CpG island methylation of Fanconi anemia, complementation group F (FANCF) gene in epithelial ovarian cancer (EOC) tissues and normal ovarian tissues and to investigate the relationship between FANCF methylation and clinicopathologic features and prognosis of EOC. METHODS: The experiment was performed with 112 EOC tissue samples (case group) and 60 normal ovarian tissues (control group). With methylation-specific polymerase chain reaction (MSP), FANCF methylation status of cases and controls was assessed. And the association between FANCF methylation and the clinicopathological features of EOC was investigated with univariate survival analysis and Cox regression model analysis. RESULTS: The methylation-positive rate of the case group was significantly higher than that of the control group (P = 0.015). The FANCF promoter methylation rates showed significant differences in the comparisons stratified by age, International Federation of Gynecology and Obstetrics (FIGO) staging, histopathological classification, and lymph node metastasis (all P < .05). Univariate survival analysis showed there were significant differences in mean survival time between the groups based on FIGO staging, histopathological classification, lymph node metastasis, and FANCF methylation (all P < .05). Cox regression model analysis suggested that FIGO staging and FANCF methylation were independent risk factors for EOC prognosis. CONCLUSION: CpG island methylation of FANCF gene promoter region is strongly associated with the susceptibility and clinicopathologic features of EOC. The FIGO staging and FANCF methylation are independent risk factors for EOC prognosis.

Changes in epidemiology and clinical characteristics of cervical cancer over the past 50 years.

OBJECTIVE: To evaluate the changes in the epidemiology and clinical characteristics of cervical cancer over the past 50 years, and explore appropriate treatment corresponding to these changes. METHODS: The clinical and pathological data of 1557 patients with invasive cervical cancer treated between January, 1955 and December, 2004 were retrospectively analyzed. RESULTS AND CONCLUSIONS: The average age of cervical cancer onset gradually decreased over the past 50 years, from 56.27+/-8.45 in 1955-1964 to 43.81+/-8.9 years in 1995-2004, whereas the ratio of young (< or =35 years old) patients rose from 3.42% to 24.91%. The ratio of early clinical stage (stages I-II) and non-squamous cancer also steadily increased (P<0.05, respectively). The tumor stage, pathological type and rate of lymph node metastasis were all significantly different among different age groups (P<0.05). In particular, the young (< or =35 years old) group had evidently higher ratios of non-squamous and advanced stage (III-IV) cancers with a higher rate of lymphatic metastasis in comparison with other age groups (P<0.01, respectively). Because of the changes in epidemiology and clinical characteristics of cervical cancer, it is necessary to modify the conventional treatment regimens and explore reasonable therapy corresponding to these changes. Preservation of reproductive endocrine function ought to be fully considered in cervical cancer treatment in women at childbearing age. Neoadjuvant intraarterial chemotherapy is an useful method for cervical cancer treatment at present.

A clinical Comparison of Lobaplatin or Cisplatin with Mitomycine and Vincristine in Treating Patients with Cervical Squamous Carcinoma.

BACKGROUND: The research was to compare the efficacy and side effects of cisplatin or lobaplatin in combination with mitomycine (MMC) and vincristine in treating patients with cervical squamous carcinoma. MATERIALS AND METHODS: Cervical squamous carcinoma patients who were pathologically diagnosed with stage Ib-IIb from April 2012 to May 2013 in the general hospital of Chinese People's Libration Amy were enrolled. All patients were confirmed without prior treatment and were randomly divided into two groups, Group A and B. Efficacy and side effects were evaluated after one cycle of chemotherapy. RESULTS: Group A (n=42) were treated with Loubo® (Lobaplatin) 50mg/m2, MMC 16mg/m2 and Vincristine 2mg/m2 every 21 days. Group B (n=44) were treated with Cisplatin 100mg/m2, MMC 16mg/m2 and Vincristine 2mg/m2 every 21 days. All 86 patients completed one cycle of chemotherapy with cisplatin or lobaplatin in combination with MMC and vincristine. No difference was observed regardiing short-term effect between two groups. Main side effects were bone marrow suppression and gastrointestinal reactions including decrease of white blood cells, platelet and nausea/vomiting. Grade III-VI liver and kidney impairment was not reported in two groups. In group A the incidence of uterine artery spasm in the process of drug delivery was significantly lower than the group B. CONCLUSIONS: Cisplatin or lobaplatin with MMC and Vincristine in the interventional treatment of cervical squamous carcinoma were effective, especially after uterine artery perfusion chemotherapy at tumor reduction and tumor downstaging period. The adverse reactions of concurrent chemotherapy are tolerable, and low physical and mental pressure even more less stimulation of vascular in treatment with lobaplatin. However, the long-term effects of this treatment need further observation.

Risk factors for candida infection of the genital tract in the tropics.

OBJECTIVE: To investigate the risk factors associated with candida infection of the genital tract in the tropics. METHODS: We performed questionnaire survey and experiments at the Hainan branch of General Hospital of People's Liberation Army, Hainan General Hospital and Sanya Maternity and Child Health Care Hospital in 2013. Controls were without Candida infection of genital tract, and cases had from Candida infection. RESULTS: We recruited 689 cases and 652 controls. The average age of cases with Candida infection of the genital tract was higher than that of controls. In the multivariate modeling, marriage (adjusted odds ratio: 2.49, 95% confidential interval: 1.09-5.67) and vaginal lavage (adjusted odds ratio: 4.41, 95% confidential interval: 1.13-5.14) were significantly associated with Candida infection of genital tract in tropics. CONCLUSION: Candida infection was related with age. Marriage and Vaginal lavage were significant risk factors. Attention should be paid to health education for the prevention of these infections.

Development of serous ovarian cancer is associated with the expression of homologous recombination pathway proteins.

To investigate the expressions of key markers in the homologous recombination (HR) pathway and the correlation with clinicopathological parameters in serous ovarian cancer (SOC). We analyzed the protein expression of MRE11, MDC1, ATM, ATR and BRCA1 by immunohistochemistry (IHC) in 97 SOC samples, and correlated with clinical parameters including age, tumor grades, clinical stage, status of menstruation and chemotherapy. Low expression of MRE11 and MDC1 was detected in 14.4 % and 3.1 % of the patient samples, and negative expression of ATM, ATR and BRCA1 was found in 11.3 %, 6.3 % and 29.9 % of the patient samples, respectively. ATR deficiency was significantly associated with menopause (P = 0.025), strong expression of ATM (P = 0.017) and MRE11 (P = 0.040) with pre-menopausal SOC, strong expression of MRE11 (P = 0.016) with low tumor grade, and strong expression of BRCA1 (P = 0.015) with early clinical stage. In addition, low expression of MRE11 was strongly associated with negativity of ATR (P < 0.001) and BRCA1 (P = 0.004) Furthermore, ATR deficiency was associated with low expression of ATM (P = 0.028) and loss expression of BRCA1 (P = 0.009). Our results suggest that reduced expression or loss of proteins in HR pathway is associated with SOC development. Abnormality of MRE11 and BRCA1 are strongly associated with late clinical stage in SOC patients.

TALEN-mediated gene mutagenesis in rhesus and cynomolgus monkeys.

Recent advances in gene editing technology have introduced the potential for application of mutagenesis approaches in nonhuman primates to model human development and disease. Here we report successful TALEN-mediated mutagenesis of an X-linked, Rett syndrome (RTT) gene, methyl-CpG binding protein 2 (MECP2), in both rhesus and cynomolgus monkeys. Microinjection of MECP2-targeting TALEN plasmids into rhesus and cynomolgus zygotes leads to effective gene editing of MECP2 with no detected off-target mutagenesis. Male rhesus (2) and cynomolgous (1) fetuses carrying MECP2 mutations in various tissues including testes were miscarried during midgestation, consistent with RTT-linked male embryonic lethality in humans. One live delivery of a female cynomolgus monkey occurred after 162 days of gestation, with abundant MECP2 mutations in peripheral tissues. We conclude that TALEN-mediated mutagenesis can be an effective tool for genetic modeling of human disease in nonhuman primates.

[Expressions of heat shock protein in cervical cancer and precancerosis].

AIM: To investigate the expressions of main subtypes of heat shock protein (HSP) in cervical cancer and precancerosis tissues. METHODS: According to the pathological diagnosis, 478 cases of cervical biopsy specimen were divided into invasive carcinoma of cervix group (63 cases), cervical intraepithelial neoplasia group (CIN, 106 cases), chronic cervicitis group (293 cases) and normal uterine cervix (16 cases).The expression levels of HSP70, HSP90a and HSP90beta3 mRNA were detected by quantitative RT-PCR with specific complex cRNA as internal control. RESULTS: (1) The expressions of HSP70, HSP90a and HSP90beta mRNA were significantly down-trended stepwise in invasive carcinoma of cervix, CIN, chronic cervicitis and normal cervix tissue (P < 0.01, respectively). (2) In the invasive carcinoma of cervix group, the expression level of HSP90beta mRNA was higher in advanced stage (FIGO II b) compared with incipient(FIGO-II a) carcinoma of the cervix (P < 0.05). (3) The expressions of HSP70 and HSP90 mRNA were each higher in poorly differentiated tumor than in well-differentiated tumor (P < 0.05, respectively). (4) The expression levels of all three HSP mRNA had no significant differences, it was observed with different histological types of cervical cancer (P > 0.05). CONCLUSION: Heat shock protein may play some important roles in malignant transformation of cervix cell and aggravation of cervix cancer. HSP70 and HSP90alpha may promote cancer cell transition and proliferation, and HSP90beta may participate in cell differentiation.