High Throughput Isolation and Data Independent Acquisition Mass Spectrometry (DIA-MS) of Urinary Extracellular Vesicles to Improve Prostate Cancer Diagnosis.

Proteomic profiling of extracellular vesicles (EVs) represents a promising approach for early detection and therapeutic monitoring of diseases such as cancer. The focus of this study was to apply robust EV isolation and subsequent data-independent acquisition mass spectrometry (DIA-MS) for urinary EV proteomics of prostate cancer and prostate inflammation patients. Urinary EVs were isolated by functionalized magnetic beads through chemical affinity on an automatic station, and EV proteins were analyzed by integrating three library-base analyses (Direct-DIA, GPF-DIA, and Fractionated DDA-base DIA) to improve the coverage and quantitation. We assessed the levels of urinary EV-associated proteins based on 40 samples consisting of 20 cases and 20 controls, where 18 EV proteins were identified to be differentiated in prostate cancer outcome, of which three (i.e., SERPINA3, LRG1, and SCGB3A1) were shown to be consistently upregulated. We also observed 6 out of the 18 (33%) EV proteins that had been developed as drug targets, while some of them showed protein-protein interactions. Moreover, the potential mechanistic pathways of 18 significantly different EV proteins were enriched in metabolic, immune, and inflammatory activities. These results showed consistency in an independent cohort with 20 participants. Using a random forest algorithm for classification assessment, including the identified EV proteins, we found that SERPINA3, LRG1, or SCGB3A1 add predictable value in addition to age, prostate size, body mass index (BMI), and prostate-specific antigen (PSA). In summary, the current study demonstrates a translational workflow to identify EV proteins as molecular markers to improve the clinical diagnosis of prostate cancer.

microRNA-375 inhibits the malignant behaviors of hepatic carcinoma cells by targeting NCAPG2.

Hepatocellular carcinoma (HCC) is a major cause of cancer-related deaths worldwide. Emerging evidence has revealed the vital functions of microRNAs (miRNAs) in cancer malignant progressions. miR-375 has been verified to serve as an antioncogene in tumorigenesis and a potential therapeutic target in various types of cancer. In this study, we aimed to determine the role of miR-375 in the regulation of chemoresistance and metastasis of HCC. Differentially expressed miR-375 and NCAPG2 were externally validated using expression data from The Cancer Genome Atlas (TCGA) database. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression levels of miR-375 in HCC tissues and cell lines. miR-375 mimics and NCAPG2-overexpression were transfected into HepG2 and Huh7 cells to establish miR-375 overexpression models. Cell Counting Kit-8, Transwell, and flow cytometry experiments were conducted to monitor cell proliferation, migration, and apoptosis. The targeting relationship between miR-375 and non-SMC condensin II complex subunit G 2 (NCAPG2) was determined by qRT-PCR, western blot, and luciferase reporter gene assay. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted using Gene Set Enrichment Analysis (GSEA). The pathway enrichment analysis was used to predict the potential pathways for further study. miR-375 was significantly downregulated in HCC tissues and cells compared to adjacent tissue and normal hepatocyte cell line respectively while NCAPG2 was upregulated. The targeting relationship was verified by luciferase reporting assay, and miR-375 could target the 3'UTR of NCAPG2 mRNA and effectively suppress NCAPG2 protein expression. Replenishing of miR-375 significantly repressed HCC cell proliferation and migration, and induced cell apoptosis. Overexpression of NCAPG2 recovered those biological abilities in miR-375 overexpressed cells. Collective data suggested that miR-375 served as a tumor suppressor via regulating NCAPG2. Replenishing of miR-375 or knockout of NCAPG2 could be therapeutically exploited for HCC.

Prognostic value of splenic volume in hepatocellular carcinoma patients receiving transarterial chemoembolization.

BACKGROUND: Liver function is a key determinant for the survival of hepatocellular carcinoma (HCC) patients receiving transarterial chemoembolization (TACE). However, establishing robust prognostic indicators for liver insufficiencies and patient survival remains an unmet demand. This retrospective study evaluated the prognostic value of splenic volume (SV) in HCC patients undergoing TACE. METHODS: A total of 67 HCC patients who underwent at least two consecutive TACE procedures were retrospectively included in this study. Comprehensive clinical information and follow-up data were collected, and the SV was measured based on dynamic contrast enhanced images. Risk factors of SV enlargement were assessed. The prognostic value of SV on survival was analyzed and compared with Child-Pugh (CP) classification and albumin-bilirubin (ALBI) grade. RESULTS: The baseline SV was 299.74±143.63 cm3, and showed a moderate and statistically significant correlation with CP classification (R=0.31, P<0.05). The SV increased remarkably after the first and second TACE procedures (330.16±155.38 cm3, P<0.01, and 355.63±164.26 cm3, P<0.01, respectively). In survival analysis, the optimal cut-off value of SV was determined as 373 cm3 using X-tile software, and the patients were divided into the small SV group and the large SV groups accordingly. Based on the pre-TACE SV, the median overall survival (mOS) for patients in the small SV group and the large SV group was 458 days and 249 days, respectively (P<0.05). After the first and second TACE, the mOS in the small SV group and the large SV group were 454 vs. 266 days (P<0.05) and 526 vs. 266 days (P<0.05), respectively. No prognostic value of CP classification and ALBI grade was identified for these patients. Furthermore, there were no significant differences between the small and large SV groups in age, tumor stage, and ALBI grade, except for CP classification (P<0.05). CONCLUSIONS: SV was correlated with CP classification and was a robust predictor for HCC patients undergoing TACE treatment.

[Changes of semen parameters in Chinese fertile men in the past 25 years].

OBJECTIVE: To analyze the changes of seminal parameters in Chinese fertile men during the past 25 years. METHODS: We collected semen samples from 5,834 fertile men in 14 different provinces (including Beijing) between 1980 and 2005 and retrospectively studied their seminal parameters, abstinence durations and total testis volumes by meta-analysis. RESULTS: Compared with the first 15 years, a significant decrease was observed in both sperm density and total number of sperm per ejaculate in the semen samples collected between 1996-2000 (P < 0.0001 and P < 0.05), but not obvious in those between 1996-2000 and after 2005 (P > 0.05). As for sperm motility, no time-related changes were noted (P > 0.05) except a reduction with the increase of age. CONCLUSION: There was a decline in sperm density and total number of sperm per ejaculate in Chinese fertile men over the past 25 years, although not significant in the latter 10 years since 1996, but with no time-related changes in sperm motility.

Quantitative (stereological) study of incomplete spermatogenic suppression induced by testosterone undecanoate injection in rats.

AIM: To evaluate the key lesions in spermatogenesis suppressed partially by testosterone undecanoate (TU) treatment. METHODS: Adult male SD rats were treated with vehicle or TU (19 mg/kg) injection (i.m.) every 15 days for 130 days. The numbers of all types of cells (nuclei) in the seminiferous tubules and the interstitial tissue were estimated using a contemporary stereological tool, the optical disector. RESULTS: In response to TU treatment, the numbers of non-type B spermatogonia, type B spermatogonia and late elongated spermatids per testis were reduced to 51 %, 66 % and 14 % of the controls, respectively. The conversion ratios from type B spermatogonia to early spermatocytes and pachytene spermatocytes were not significantly affected and the ratios to the later germ cell types fell to 51 % - 65 % of the controls. Less than 1.0 % of immature round spermatids were seen sloughing into the tubule lumen, 4.0 % of elongated spermatids retained in the seminiferous epithelium, and about half of the elongated spermatid nuclei appreciably malformed. Leydig cells were atrophied but their number and the peritubular myoid cell number per testis were unchanged. CONCLUSION: Double inhibition of spermatogenesis (i.e. inhibition at spermiation and spermatogonial conversion to type B spermatogonia), a scenario seen in the monkey and human following gonadotrophin withdrawal, was not sufficiently effective for a complete spermatogenic suppression in the rat after TU treatment, probably due to ineffective inhibition of the Leydig cell population and therefore the intra-testicular testosterone levels.

[Effect of androgen on erythropoietin in patients with hypogonadism].

OBJECTIVES: To observe the change of erythropoietin (EPO) in patients of hypogonadism who received androgen replacement treatment and explore the mechanism of androgen-induced increase of red blood cells and haemoglobin. METHODS: Eight patients with Klinefelter's syndrome, divided into two groups, received TU intramuscular injections of 500 mg or 1000 mg dose, respectively. After three months, seven patients received the second injection of crossover dose. Testosterone levels in serum were measured with RIA before and after the injections treatment. RBC count, impacted volume of blood cells and haemoglobin concentration were measured before treatment and 4, 8 weeks after treatment. At the same interval, EPO levels were measured with ELISA method. RESULTS: Development of the secondary sex characters was improved in all patients after the TU injection. Serum testosterone levels raised significantly and reached the peak one week after the injections. Effective level of testosterone lasted for over 6 weeks. RBC count, impacted volume of blood cells and haemoglobin increased at different degrees after TU injections, but these changes were not significant in statistic(P < 0.05). The increased levels remained for 8 weeks. EPO levels were elevated significantly (P < 0.01 or 0.05) after the TU injection(Pbat > 0.05). The second injection could still make the EPO level go up. CONCLUSIONS: Androgen replacement treatment can increase the EPO levels in patients of hypogonadism, which is one of the mechanism of RBC production increase.

A multicenter contraceptive efficacy study of injectable testosterone undecanoate in healthy Chinese men.

This report describes a Phase II, multicenter, contraceptive efficacy clinical trial using monthly injections of testosterone undecanoate (TU) alone at a dose of 500 mg in healthy Chinese men. Three hundred eight healthy men were recruited in six centers distributed throughout China. Volunteers underwent a control period with no treatment, then a 12-month treatment period including a 6-month suppression phase followed by a 6-month efficacy phase and a 12-month recovery period. During the suppression phase, an initial loading dose of 1000 mg TU, followed by 500 TU at monthly intervals were given until azoospermia or severe oligozoospermia was achieved, up to a maximum of six injections. During the efficacy phase, 500 mg TU were administered at monthly intervals for 6 months. Nine of 308 men did not achieve azoospermia or severe oligozoospermia (<3 x 10(6)/ml) within the 6-month suppression phase. This gave a methodological failure rate of 2.9/100 couple years (95% confidence interval of 1.0-4.8/100 couple years). Two hundred ninety-six men entered the efficacy phase. The continuation rate during the efficacy phase was 95/100 couple years. There were no pregnancies caused by men who achieved azoospermia or severe oligozoospermia. Reappearance of sperm occurred in six men during the efficacy phase, and one pregnancy was attributed to sperm rebound. This gave a secondary failure rate of 2.3/100 couple years (95% confidence interval of 0.5-4.2/100 couple years). Thus, the total failure rate was 5.2%, and total efficacy was 94.8%. Spermatogenesis in all subjects returned to the normal reference range within the recovery period. The mean serum testosterone concentration increased 131%, and the mean serum LH and FSH concentrations decreased 72% and 70%, respectively, after TU injections during the treatment period. The mean level of serum high density lipoprotein cholesterol decreased (14%), and the mean hematocrit increased 6% compared with baseline. No serious adverse events and no significant changes in serum chemistry occurred during the study. The results showed that monthly TU injection at a dose of 500 mg after an initial loading dose of 1000 mg can effectively, safely, and reversibly suppress spermatogenesis in healthy Chinese men without serious adverse effects.