Clinical Value and Mechanism Exploration of Serum miR-379 in Obesity-Polycystic Ovary Syndrome.

Objective: As a common endocrine and metabolic disorder, polycystic ovary syndrome (PCOS) is mostly associated with an obese phenotype. The present research focuses on the clinical significance of miR-379 in obesity-PCOS and attempts to elucidate its potential mechanisms. Methods: Healthy individuals (n = 46), obesity-PCOS (n = 92), and non-obesity PCOS (n = 31) subjects were enrolled. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to examine the level of serum miR-379. The receiver operating characteristic (ROC) curve and logistic regressions were applied to reveal the diagnostic significance. Dual luciferase reporters were performed to validate the targeting relationships. And cell count kit (CCK-8) assay was used to detect cell proliferation. Results: Serum miR-379 was highly expressed in PCOS patients (P < 0.05), in especially obesity-PCOS patients. Higher miR-379 was associated with greater body mass index (BMI), higher bioavailable testosterone (bT), and greater insulin resistance (IR). Additionally, miR-379 was an independent risk factor for the development of obesity-PCOS. The sensitivity of miR-379 in identifying patients with obesity-PCOS from healthy or non-obesity-PCSO patients was 81.52% and 72.83%, and the specificity was 86.96% and 80.65%. Semaphorin 3 A (SEMA3A) was identified as a target of miR-379 and was reduced in the patients with obesity PCOS (P < 0.05). Inhibition of miR-375 reduced KGN proliferation, but this reduction was partially restored by silencing of SEMA3A (P < 0.05). Conclusion: Elevated miR-379 assists the diagnosis of obesity-PCOS and regulates the proliferation of KGN by targeting SEMA3A engaged in disease development.

Adjuvants to prevent reformation of adhesions following adhesiolysis for Asherman syndrome: a systematic review and meta-analysis.

Two international guidelines published on the management of Asherman syndrome (AS) have made recommendations on various adjuvant methods to prevent intrauterine reformation. Nevertheless, the effectiveness of these methods when used in primary or secondary prevention settings is different. Our aim is to assess the effectiveness of various adjuvant methods for the secondary prevention of intrauterine adhesions (IUAs). Articles were considered eligible if they included subjects with AS before surgery and compared a chosen method with either a control or a comparison group (using another method). The primary outcome was the IUA reformation rate at follow-up hysteroscopy. A total of 29 studies [15 randomised controlled trials (RCTs) and 14 cohort studies] were included. Adhesion reformation with various methods to prevented IUA reformation when compared with controls were: second-look hysteroscopy: [risk ratio (RR): 0.21, 95% confidence interval (CI): 0.05-0.90 (p = 0.02)]; intrauterine contraceptive device: RR: 0.64, 95% CI: 0.36-1.12 (p = 0.12); continuous intrauterine balloon: RR: 0.18, 95% CI: 0.05-0.68 (p = 0.01); intermittent intrauterine balloon: RR: 0.50, 95% CI: 0.31-0.80 (p = 0.004); anti-adhesion gel: RR: 0.80, 95% CI: 0.58-1.10 (p = 0.17); amnion graft: RR: 0.63, 95% CI: 0.44-0.91 (p = 0.01).

Validation of two risk assessment models for venous thromboembolism in patients undergoing gynecologic surgery.

BACKGROUND: According to published guidelines, gynecologic surgical patients should be stratified into different risk level groups to receive prophylaxis for venous thromboembolism (VTE), but the applicability of available risk assessment models (RAMs) in common gynecologic surgical patients remained to be confirmed. We aimed to validate the use of the Caprini RAM and gynecologic Caprini (G-Caprini) RAM for assessing postoperative VTE risk in gynecologic surgical patients. METHODS: The database of a randomized controlled trial (RCT) was used to select patients who underwent gynecologic surgeries for benignant and malignant diseases in five institutions in China between 2011 and 2018. The Caprini RAM version recommended by the American College of Chest Physicians (ACCP) was adopted. Participants were divided into four risk levels based on the Caprini and G-Caprini scores. For each risk level group, the incidence of VTE was calculated. The correlation between VET incidence and risk levels was assessed by Spearman's rank correlation coefficient (RS) value. RESULTS: As a result, 800 patients in the data base were analyzed with an overall VTE incidence of 5.8%. Caprini RAM showed that the percentage of patients at very low risk, low risk, moderate risk, and high risk were 0%, 4.3%, 44.4%, and 51.4%, respectively, and the VTE incidence was 2.9%, 2.3%, and 9.0%, respectively. RS value between the risk stratification and VTE incidence was 0.500 (P=0.667). G-Caprini RAM showed that the percentage of patients at very low risk, low risk, moderate risk, and high risk were 7.8%, 28.0%, 32.0%, and 32.3%, respectively, and the VTE incidence was 0.0%, 2.9%, 2.3%, and 9.0%, respectively. RS value between the risk stratification and VTE incidence was 1.000 (P<0.01). CONCLUSIONS: The G-Caprini RAM was as suitable as the Caprini RAM for VTE risk assessment in gynecologic surgical patients. The gynecologic model has the advantages of ease of use and more accurate identification of low-risk groups.

Overexpression of Nuclear Enriched Autosomal Transcript 1 Facilitates Cell Proliferation, Migration Invasion, and Suppresses Apoptosis in Endometrial Cancer by Targeting MicroRNA-202-3p/T Cell Immunoglobulin and Mucin Domain 4 Axis.

Background: Endometrial cancer (EC) is an intractable gynecological cancer with increasing incidence and mortality worldwide. Accumulating studies indicated that long noncoding RNA nuclear enriched autosomal transcript 1 (NEAT1) was a novel oncogene implicated in a variety of cancers. However, whether NEAT1 could accelerate cell growth in EC is unclear. Materials and Methods: NEAT1, microRNA (miR)-202-3p, and T cell immunoglobulin and mucin domain 4 (TIMD4) levels were detected by quantitative real-time polymerase chain reaction. Cell proliferation and apoptosis were examined by cell counting kit-8 and flow cytometry. Transwell assay was employed for the evaluation of cell migration and invasion. The relationship between miR-202-3p and NEAT1 or TIMD4 was determined by luciferase reporter system. TIMD4 protein expression was assessed by Western blot assay. Results: NEAT1 was upregulated, whereas miR-202-3p was downregulated in EC tumors and cells. Depletion of NEAT1 restrained EC cell proliferation, migration, invasion, and improved apoptosis. MiR-202-3p was targeted by NEAT1 and could bind to TIMD4. Subsequently, it is observed that miR-202-3p inhibitor neutralized NEAT1 silencing mediated suppression on EC cell progression. Meanwhile, TIMD4 rescued miR-202-3p induced inhibition on cell progression in EC. Furthermore, it was obvious that NEAT1 facilitated TIMD4 expression by absorbing miR-202-3p in EC. Conclusions: Upregulation of NEAT1 accelerated EC cell progression through sponging miR-202-3p to facilitate TIMD4 expression, providing potential novel treatment method for EC.

Evaluation of folate receptor-mediated tumor detection as a triage tool in cervical cancer screening.

OBJECTIVE: To evaluate the efficacy of folate receptor-mediated tumor detection (FRD) for identifying high-grade intraepithelial squamous lesions (HSIL) in the triage of women who are positive for human papillomavirus (HPV), and those with cytology findings of atypical squamous cells of undetermined significance (ASCUS). METHOD: A secondary analysis of prospectively collected data from 1504 women who had abnormal results during primary cervical cancer screening at 13 hospitals in Beijing, China, between November 2014 and August 2015. The detection accuracy of FRD was evaluated among HPV-positive women and women with ASCUS referred for colposcopy examination. RESULTS: Among 1338 women with HPV, the percentage coincidence with pathology findings was higher for FRD (66.7%) than for cytology of ASCUS or higher (51.5%). The rate of colposcopy referral for cytology and FRD as a triage tool was 969 (72.4%) and 736 (55.0%), respectively. Thus, 233 (17.4%) fewer women would be referred for colposcopy by FRD. Among 476 women with cytology of ASCUS, the percentage coincidence with pathology findings was higher for FRD (63.4%) than for HPV (35.9%). CONCLUSION: FRD was found to be a promising triage tool for women who are HPV-positive and those with cytology findings of ASCUS.

PGC-1α protects against oxidized low-density lipoprotein and luteinizing hormone-induced granulosa cells injury through ROS-p38 pathway.

Obese women with polycystic ovary syndrome (PCOS) often suffer from ovulation failure, which may be driven by granulosa cells (GCs) injury caused by increased levels of circulating oxidized low-density lipoprotein (ox-LDL) and luteinizing hormone (LH). PGC-1α may play an important role in this pathophysiological processes. However, the effect and the potential mechanism of PGC-1α on GCs injury evoked by obese PCOS is fully unclear. To investigate the protective effect and the potential mechanism of PGC-1α on GCs injury evoked by ox-LDL + LH stimulation. Patients with PCOS and women of normal reproductive age who undergoing egg retrievals and consenting for this research were collected. Those women were divided into normal-weight non-PCOS group, obese non-PCOS group, normal-weight PCOS group and obese PCOS group according to the body mass index (BMI) and PCOS diagnosis. Follicular fluid was collected and primary GCs were isolated. The levels of LH and ox-LDL in follicular fluid in the four groups were measured. And, the expressions of PGC-1α, cell apoptosis and ROS generation in primary GCs in the four groups were evaluated. After GCs from women of normal reproductive age at normal-weight pre-treated with adenovirus encoding PGC-1α (Ad-PGC-1α) prior to ox-LDL + LH treatment in vitro, the cell viability, apoptosis, apoptosis-related proteins expressions and ROS generation were evaluated by CCK-8 assay, AnnexinV/PI double staining, Western blot and H2DCF-DA staining, respectively. The expression of PGC-1α was significantly decreased, whereas the cell apoptosis and ROS generation were significantly increased in GCs of PCOS group, especially obese PCOS group. Our data also revealed that over-expression of PGC-1α in GCs from women of normal reproductive age at normal-weight markedly inhibited cell injury, ROS generation and p38 activation, accompanied by increased Bcl-2 expression, decreased Bax and cleaved caspase-3 expressions induced by ox-LDL + LH stimulation. Ox-LDL + LH-induced cell apoptosis was abrogated by attenuation of ROS generation or p38 activation. Attenuation of ROS generation reversed ox-LDL + LH-induced p38 activation, however, p38 inhibitors had an effect on ROS generation. Our findings suggested that PGC-1α protected against ox-LDL + LH-induced GCs injury through inhibiting cell apoptosis. And, the mechanism may be related to the inhibition of ROS-initiated p38 pathway. Our data indicated that PGC-1α may be a potential therapeutic target for obese PCOS.

Evaluation of Folate Receptor-Mediated Cervical Dyeing as a Method for Detection of Cervical Lesions.

OBJECTIVES: The aim of the study was to evaluate the performance of a folate receptor-mediated tumor detection (FRD) assay for detection of cervical high-grade lesions. MATERIALS AND METHOD: A total of 1504 patients with abnormal cytology and/or positive human papillomavirus (HPV) testing during primary screening from November 2014 to August 2015 were enrolled. The patients were recruited from the Peking University People's Hospital and 12 other hospitals. Folate receptor-mediated tumor detection was applied in all the patients before colposcopy to compare the detection rate, sensitivity, specificity, positive predictive value, negative predictive value, and coincidence rate with HPV and cytology tests according to the pathologic diagnosis. RESULTS: In the total of 1504 patients, 503 patients were negative for intraepithelial lesion or malignancy, 440 patients were cervical intraepithelial neoplasia (CIN) 1, 254 patients were CIN 2, 257 patients were CIN 3, 46 patients were squamous cell carcinoma, and 4 patients were adenocarcinoma in situ. The sensitivity of FRD was 77.72%, which was less than cytology (80.39%) and HPV testing (95.54%). The specificity of FRD was 60.02%, which was greater than cytology (30.12%) and HPV testing (14.95%). The coincidence rate of FRD to the pathologic diagnosis (66.62%) was also significantly greater than atypical squamous cells of undetermined significance cytology and above (48.87%) and HPV testing (45.01%, p < .0001). The detection rate of FRD for all grades of lesions increased with the severity of lesions. CONCLUSIONS: Folate receptor-mediated tumor detection has a slightly lower sensitivity and a higher specificity than cytology and HPV testing for detection of CIN 2+. Simplicity of FRD requires less professional skill. Folate receptor-mediated tumor detection could be a candidate test for cervical cancer screening especially in low- and middle-income countries. However, FRD still needs more clinical trial data to demonstrate its ability in general screening population.

Expression and role of hScrib in endometrium, endometriosis, and endometrial adenocarcinoma.

To explore the role of hScrib in the pathogenesis of endometriosis.This was a retrospective study of 240 women in our hospital between January 2014 and January 2017. The expression of hScrib in endometrium (EM), endometriosis (EMs), and endometrial adenocarcinoma (EC) was investigated, and compared the differences among them. Serum levels, protein expressions, localizations, and correlations of hScrib and E-cadherin were determined.The levels of serum soluble hScrib and E-cadherin were significantly highest in EC, followed by EMs, and healthy women (P < .05). hScrib protein content was opposite result in 3 tissues (P < .05), and was negatively correlated with r-AFS stage in EMs. The location changed from membrane to cytoplasm. Co-localization of hScrib with E-cadherin was found at extensive cell-cell boundaries in EMs.hScrib and E-cadherin may be as new diagnostic markers of endometriosis. Low expression of hScrib leads to the loss of cell polarity and stability. Also, hScrib may induce EMT through regulating E-cadherin, might play an important role in pathogenesis of endometriosis.

Effect of twin pregnancy chorionic properties on maternal and fetal outcomes.

OBJECTIVE: This study aimed to determine the effect of twin pregnancy chorionic properties on pregnancy complications and fetal outcomes. MATERIALS AND METHODS: A total of 559 subjects with gemellary pregnancy were included in the retrospective analysis, and clinical data, such as monitoring data during pregnancy and maternal and fetal outcomes, were recorded in detail. Based on the ultrasound results and methods of the postpartum pathologic examination of the placental membranes, the subjects were divided into the twin group with monochorionic diamnion (MCDA group, n = 198) and twin group with dichorionic diamnion (DCDA group, n = 361). The relationships of different chorionic properties and maternal and fetal outcomes were determined by comparing the maternal complications and fetal outcomes. RESULTS: The occurrence rate of gemellary pregnancy was 2.97% and that of monochorionic twin pregnancy was 34.8%. The MCDA group showed a higher incidence of pregnancy-induced hypertension, gestational diabetes mellitus, polyhydramnios, premature rupture of membranes, and abruptio placenta and a lower incidence of severe postpartum hemorrhage than the DCDA group. However, the incidence of preterm birth was significantly different (57.6% vs. 45.7%, P < 0.05). Significant differences were also detected in the incidence of fetal loss, complicated twins, neonatal asphyxia, and perinatal death between the two groups (P < 0.05). CONCLUSION: The incidence of maternal complication (such as pregnancy-induced hypertension, gestational diabetes mellitus, polyhydramnios, premature rupture of membranes, and abruptio placenta and severe postpartum hemorrhage) in the two groups was not significantly different; however, the fetal outcomes in the MCDA group were inferior to those in the DCDA group. The fetal outcomes may be improved by determining the chorionic properties in early pregnancy by using ultrasound and consequently planning for pregnancy monitoring and intervention.

Different combination strategies for prophylaxis of venous thromboembolism in patients: A prospective multicenter randomized controlled study.

The aim was to evaluate the efficacy and safety of different combination strategies for prophylaxis of venous thromboembolism (VTE) after gynecologic surgery in patients at different levels of risk. This was a prospective multicenter randomized controlled study, in which 625 women who would undergo pelvic surgery for gynecologic diseases were stratified into three risk groups and then randomized into four groups to receive graduated compression stockings (GCS) alone (group A), GCS + low molecular weight heparin (LMWH) (group B), GCS + intermittent pneumatic compression (IPC) (group C), and GCS + IPC + LMWH (group C), respectively. The overall incidence of DVT was 5.1%. Group A had the highest incidence of DVT (8.8%), followed by group C (5.2%), group B (3.8%), and group D (2.6%). There was a significant difference in the incidence of DVT between groups A and D. The incidence of DVT was significantly lower in LMWH-treated patients (group B + group D) than in non-LMWH-treated patients (group A + group C). In conclusion, combination prophylaxis, especially LMWH-containing strategies, is better than monoprophylaxis in reducing VTE after gynecologic surgery. Risk-stratified prophylactic strategies should be implemented in patients undergoing gynecologic surgery, with LMWH-containing strategies being recommended for high-risk and very-high-risk patients.

Proteomic analysis of FUS interacting proteins provides insights into FUS function and its role in ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease. Mutations in the Fused in Sarcoma/Translocated in Liposarcoma (FUS/TLS) gene cause a subset of familial ALS cases and are also implicated in sporadic ALS. FUS is typically localized to the nucleus. The ALS-related FUS mutations cause cytoplasmic mis-localization and the formation of stress granule-like structures. Abnormal cytoplasmic FUS localization was also found in a subset of frontotemporal dementia (FTLD) cases without FUS mutations. To better understand the function of FUS, we performed wild-type and mutant FUS pull-downs followed by proteomic identification of the interacting proteins. The FUS interacting partners we identified are involved in multiple pathways, including chromosomal organization, transcription, RNA splicing, RNA transport, localized translation, and stress response. FUS interacted with hnRNPA1 and Matrin-3, RNA binding proteins whose mutations were also reported to cause familial ALS, suggesting that hnRNPA1 and Matrin-3 may play common pathogenic roles with FUS. The FUS interactions displayed varied RNA dependence. Numerous FUS interacting partners that we identified are components of exosomes. We found that FUS itself was present in exosomes, suggesting that the secretion of FUS might contribute to the cell-to-cell spreading of FUS pathology. FUS interacting proteins were sequestered into the cytoplasmic mutant FUS inclusions that could lead to their mis-regulation or loss of function, contributing to ALS pathogenesis. Our results provide insights into the physiological functions of FUS as well as important pathways where mutant FUS can interfere with cellular processes and potentially contribute to the pathogenesis of ALS.

Aberrant expression profile of translationally controlled tumor protein and tumor-suppressive microRNAs in cervical cancer.

PURPOSE: Invasive and recurrent cervical cancer accounts for major mortality among women. The activity of biomarkers in cervical cancer varies with different pathological stages. The purpose of the present study was to evaluate the expression of 2 biomarkers in cervical cancer and their possible contribution to novel therapeutic strategies. METHODS: In this study, we assessed the expression of translationally controlled tumor protein (TCTP) using immunohistochemistry and Western blot analysis. The expression pattern of miR-143 was also evaluated using Northern blot analysis. RESULTS: HeLa cells and mice were used for tumor induction. A group of mice injected with HeLa cells and incubated for 6 weeks developed initial tumor, while a different group of mice injected with HeLa cells and incubated for about 10 weeks developed advanced stage cervical cancer. Histological analysis revealed higher proliferation of cells resulting in complex forms of tumor in advanced cervical cancer, whereas cell clustering was not found to be initiated in the initial stage. The results of immunohistochemistry and Western blot analysis indicated less variation in the expression of TCTP, but significant difference was observed in advanced stage. Expression of Bax apoptotic protein was higher in the initial stage of the tumor than in the advanced cervical cancer. Similar pattern of marginal downregulation of miR-143 was observed between control and initial tumor stages, but striking reduction in miR-143 expression was observed in advanced stages of tumor development. CONCLUSION: The results of this study reveal a new aspect of altered expression of biomarkers in different pathological stages that could help identify novel therapeutic strategies for cervical cancer treatment.

Expression of glucose transporter protein 1 and p63 in serous ovarian tumor.

AIM: It has been shown that glycolytic metabolism is increased in malignant cells. Cancer cell growth is an energy-related process supported by an increased glucose metabolism. In addition, p63, a known homolog of p53, is expressed predominantly in basal cell and squamous cell carcinomas. The purpose of this study was to evaluate the expression of glucose transporter protein 1 (GLUT1) and p63 in patients with serous ovarian tumor (benign, borderline and malignant) and study their close relationship with the malignant transformation of serous ovarian tumors. METHODS: Two hundred formalin-fixed, paraffin-embedded sections were immunostained with rabbit anti-GLUT1 polyclonal antibody and mouse anti-p63 monoclonal antibody using the streptavidin-biotin method. The samples were as follows: 40 normal ovarian tissues, 40 serous cystadenomas, 40 borderline serous cystadenomas and 80 serous cystadenocarcinomas were stained. RESULT: Normal ovarian tissues showed completely negative staining for GLUT1 and p63. However, from benign serious cystadenomas, borderline cystadenomas to cystadenocarcinomas, the expression of GLUT1 and p63 grew stronger (P < 0.05). Moreover, the intensity staining of GLUT1 maintained a significant association with the expression of p63 (P < 0.05). In χ²-test analysis, expression of borderline cystadenomas and cystadenocarcinomas, intraperitoneal implants, ascites, lymph node status and International Federation of Gynecology and Obstetrics (FIGO) stage and GLUT1 expression levels have an appalling significance (P < 0.05), while FIGO stage, intraperitoneal implants and lymph node status except patient age and ascites have a statistical significance with the expression of p63 levels (P < 0.05). CONCLUSION: Our findings show a progressive increase in the expression of GLUT1 and p63 from the benign serous cystadenomas, borderline cystadenomas to cystadenocarcinomas. Overexpression of GLUT1 and p63 are associated with the histology FIGO stage and metastasis of the tumors. These data suggested that the expression of GLUT1 and p63 may be closely related to the malignant transformation of serous ovarian tumors. However, the relative importance of GLUT1 and p63 in ovarian serous tumor development and tumorigenesis remains mostly unclear and awaits further investigation.

Activation of protease-activated receptor-2 disrupts vaginal epithelial barrier function.

The epithelial cell barrier function is a critical factor in the maintenance of the homeostasis of the vaginal mucosa. This study elucidates one of the mast cell-derived chemical mediators, tryptase, on compromising the vaginal epithelial barrier function. The results showed that human vaginal cell line, VK2, express PAR2. Activation of PAR2 increases the expression of ADAM10 in VK2 cells, interferes with the endosome/lysosome fusion, and compromises the epithelial barrier function. We conclude that the activation of PAR2 on VK2 cells increases the expression of ADAM10, and compromises the VK2 monolayer barrier function.

Investigation on delivery analgesia effect of combined spinal epidural anesthesia plus Doula and safety of mother and baby.

OBJECTIVE: To explore the effect of patient-controlled lumbar epidural combined anesthesia with Doula for labor analgesia with ropivacaine and sufentanil, and its influence on the progress of labor, and outcomes of mother and infant. MATERIALS AND METHODS: Two hundred parturients that requested labor analgesia were randomly selected by patient-controlled lumbar epidural combined anesthesia with Doula as the observation group, meanwhile another 200 parturients were selected as the control group without any analgesic measurements. Labor pain score, labor duration, blood gas analysis results, the incidence of cesarean section, neonatal asphyxia, and postpartum hemorrhage were compared between the two groups. RESULTS: Compared with the control group, labor analgesic effect was remarkable, the cesarean section rate was significantly reduced in observation group, and the difference was statistically significant (p < 0.05), but with respect to the duration of labor, maternal, postpartum hemorrhage, and neonatal asphyxia, there was no statistical significance between the two groups (p > 0.5). In the observation group regarding maternal and neonatal blood gas analysis results, PO2 was higher and PCO2 was lower than those in the control group. The differences were statistically significant (p < 0.05). CONCLUSION: Labor analgesia by patient-controlled lumbar epidural combined anesthesia accompanied with Doula with ropivacaine and sufentanil is effective, safe, reliable, has no adverse effects, and reduces cesarean section rate.

Nuclear localization sequence of FUS and induction of stress granules by ALS mutants.

Mutations in fused in sarcoma (FUS) have been reported to cause a subset of familial amyotrophic lateral sclerosis (ALS) cases. Wild-type FUS is mostly localized in the nuclei of neurons, but the ALS mutants are partly mislocalized in the cytoplasm and can form inclusions. We demonstrate that the C-terminal 32 amino acid residues of FUS constitute an effective nuclear localization sequence (NLS) as it targeted beta-galactosidase (LacZ, 116 kDa) to the nucleus. Deletion of or the ALS mutations within the NLS caused cytoplasmic mislocalization of FUS. Moreover, we identified the poly-A binding protein (PABP1), a stress granule marker, as an interacting partner of FUS. Large PABP1-positive cytoplasmic foci (i.e. stress granules) colocalized with the mutant FUS inclusions but were absent in wild-type FUS-expressing cells. Processing bodies, which are functionally related to stress granules, were adjacent to but not colocalized with the mutant FUS inclusions. Our results suggest that the ALS mutations in FUS NLS can impair FUS nuclear localization, induce cytoplasmic inclusions and stress granules, and potentially perturb RNA metabolism.

Fractionation and evaluation of radical-scavenging peptides from in vitro digests of buckwheat protein.

Buckwheat protein (BWP) isolate was subjected to a two-stage in vitro digestion (1 h pepsin followed by 2 h pancreatin at 37 °C). The antioxidant potential of the BWP digests was compared by assessing their capacity to scavenge 2,2'-azinobis (3-ethylbenzothiszoline-6-sulphonic acid) (ABTS(+•)) and hydroxyl ((•)OH) radicals. The 2-h pancreatin digest, which demonstrated the strongest activity against both radicals, was subjected to Sephadex G-25 gel filtration. Of the six fractions collected, fractions IV (456 Da) and VI (362 Da) showed the highest ABTS(+•) scavenging activity and were 23-27% superior to mixed BWP digest (P < 0.05). Fraction VI was most effective in neutralizing (•)OH and was 86 and 24% more efficient (P < 0.05) than mixed BWP digest and fraction IV, respectively. LC-MS/MS identified Trp-Pro-Leu, Val-Pro-Trp, and Val-Phe-Pro-Trp (IV), Pro-Trp (V) and tryptophan (VI) to be the prominant peptides/amino acid in these fractions.

Prohibitin regulates TGF-beta induced apoptosis as a downstream effector of Smad-dependent and -independent signaling.

BACKGROUND: Prohibitin (PHB), a protein located on the inner mitochondrial membrane and nuclei, is an intracellular effector of transforming growth factor-beta (TGF-beta) signaling in prostate cancer cells. This study investigated the involvement of PHB in the apoptosis and survival outcomes of human prostate cancer cell to TGF-beta. shRNA PHB loss of function in prostate cancer cells led to enhanced apoptotic response to TGF-beta via Smad-dependent mechanism. METHOD: TGF-beta activation of Raf-Erk intracellular signaling, led to PHB phosphorylation, decreased inner mitochondrial permeability, and increased cell survival. Calcein-based immunofluorescence studies revealed the functional involvement of PHB in maintaining inner mitochondrial membrane permeability as an integral component of TGF-beta induced apoptosis in prostate cancer cells. RESULTS: These finding indicates that induction of TGF-beta apoptosis is mediated by Smad-dependent and Smad-independent signaling (MAPK) converging at PHB as a downstream effector regulating inner mitochondrial permeability. Putative PHB associated proteins were identified by subjecting TGF-beta treated cells to immunoprecipitation with anti-PHB, and mass spectrometry. A screen for the kinase specific phosphorylation sites of PHB revealed three protein kinase (PKC) binding sites. CONCLUSION: Our results demonstrate that TGF-beta led to upregulation of the PKC inhibitor 14-3-3 protein and promoted its association with PHB, while PHB association with PKC-delta, was inhibited by the MEK1 inhibitor, documenting a critical interdependence between the MEK-ERK signaling and prohibitin phosphorylation. These findings suggest a dual role for PHB as a downstream determinant of the cellular response to TGF-beta via Smad-dependent pathway (apoptosis) and MAPK intracellular signaling (survival).

RABA (reductive alkylation by acetone): a novel stable isotope labeling approach for quantitative proteomics.

Quantitative proteomics is challenging and various stable isotope based approaches have been developed to meet the challenge. Hereby we describe a simple, efficient, reliable, and inexpensive method named reductive alkylation by acetone (RABA) to introduce stable isotopes to peptides for quantitative analysis. The RABA method leads to alkylation of N-terminal and lysine amino groups with isopropyl moiety. Using unlabeled (d(0)) and deuterium labeled (d(6)) acetone, a 6 Da mass split is introduced to each isopropyl modification between the light and heavy isotope labeled peptides, which is ideally suited for quantitative analysis. The reaction specificity, stoichiometry, labeling efficiency, and linear range of the RABA method have been thoroughly evaluated in this study using standard peptides, tryptic digest of proteins, as well as human cell lysate. Reliable quantitative results have been consistently obtained in all experiments. We also applied the RABA method to quantitative analysis of proteins in spinal cords of transgenic mouse models of amyotrophic lateral sclerosis. Highly homologous proteins (transgenic human SOD1 and endogenous mouse SOD1) were distinguished and quantified using the method developed in this study. In addition, the quantitative results using the RABA approach were independently validated by Western blot.

Proteomic characterization of lipid raft proteins in amyotrophic lateral sclerosis mouse spinal cord.

Familial amyotrophic lateral sclerosis (ALS) has been linked to mutations in the copper/zinc superoxide dismutase (SOD1) gene. The mutant SOD1 protein exhibits a toxic gain-of-function that adversely affects the function of neurons. However, the mechanism by which mutant SOD1 initiates ALS is unclear. Lipid rafts are specialized microdomains of the plasma membrane that act as platforms for the organization and interaction of proteins involved in multiple functions, including vesicular trafficking, neurotransmitter signaling, and cytoskeletal rearrangements. In this article, we report a proteomic analysis using a widely used ALS mouse model to identify differences in spinal cord lipid raft proteomes between mice overexpressing wild-type (WT) and G93A mutant SOD1. In total, 413 and 421 proteins were identified in the lipid rafts isolated from WT and G93A mice, respectively. Further quantitative analysis revealed a consortium of proteins with altered levels between the WT and G93A samples. Functional classification of the 67 altered proteins revealed that the three most affected subsets of proteins were involved in: vesicular transport, and neurotransmitter synthesis and release; cytoskeletal organization and linkage to the plasma membrane; and metabolism. Other protein changes were correlated with alterations in: microglia activation and inflammation; astrocyte and oligodendrocyte function; cell signaling; cellular stress response and apoptosis; and neuronal ion channels and neurotransmitter receptor functions. Changes of selected proteins were independently validated by immunoblotting and immunohistochemistry. The significance of the lipid raft protein changes in motor neuron function and degeneration in ALS is discussed, particularly for proteins involved in vesicular trafficking and neurotransmitter signaling, and the dynamics and regulation of the plasma membrane-anchored cytoskeleton.