MALDI-TOF-MS analysis in low molecular weight serum peptidome biomarkers for NSCLC.

OBJECTS: Lung cancer is one of the leading causes of death from cancer in the world. Screening new serum biomarkers is important for the early detection of lung cancer. The purpose of this study was to investigate the serum peptide model between non-small cell lung cancer (NSCLC) patients and healthy controls, as well as between paired pre- and postoperative NSCLC patients, and to find the low molecular weight (LMW) potential tumor markers for NSCLC. METHODS: 56 serum samples from NSCLC patients, 56 controls, and 20 matched pre- and postoperative patients were analyzed using magnetic-bead (MB)-based purification technique combined with MALDI-TOF-MS. To distinguish NSCLC from cancer-free controls, three models were established. Finally, comparing the three groups of serum protein fingerprints, nano-liquid chromatography-electrospray ionization tandem mass spectrometry was used to further identify the differential peptides. RESULTS: Among the three models constructed, the GA model had the best diagnostic efficacy. Five differential peaks were screened by combining the case group, healthy controls, and postoperative group analysis, which were up-regulated in the case group and showed a tendency to return to healthy control values after surgery. The protein matching the mass spectrometry peak m/z 2953.73 was identified as fibrinogen α chain. CONCLUSION: This study shows that the application of MALDI-TOF-MS is a promising approach for the identification of potential serum biomarkers for NSCLC, which is potentially valuable for establishing a new diagnostic method for lung cancer. In addition, we found that fibrinogen α chain may be an auxiliary diagnostic indicator for NSCLC.

Diagnostic accuracy of MALDI-TOF mass spectrometry for non-small cell lung cancer: a meta-analysis.

Objective: To assess the overall accuracy of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in identifying non-small cell lung cancer (NSCLC).Methods: A comprehensive search of PubMed, EMBASE and CNKI databases as well as the reference lists from relevant articles was performed prior to July 2017. Two authors independently screened articles based on inclusion and exclusion criteria and assessed the quality of each study using the Quality Assessment of Diagnostic Accuracy Studies 2 (QADAS-2) tool. Meta-disc 1.4 and Stata12.0 software programs were used for the statistical analysis.Results: Eleven eligible articles comprising 16 studies and representing 935 subjects were included in this meta-analysis. The pooled sensitivity and specificity were 0.84 (95% CI: 0.80-0.87) and 0.77 (95% CI: 0.74-0.80), respectively. The overall diagnostic performance as measured by the area under the curve (AUC) for the summary receiver-operating characteristic (SROC) curve was 0.9380.Conclusions: MALDI-TOF MS has a high diagnostic accuracy for NSCLC.

Detection and Identification of Serum Peptides Biomarker in Papillary Thyroid Cancer.

BACKGROUND Papillary thyroid cancer (PTC) is currently the most commonly diagnosed endocrine malignancy. In addition, the sex- and age-adjusted incidence of PTC has exhibited a greater increase over the last 2 decades than in many other malignancies. Thus, discovering noninvasive specific serum biomarker to distinguish PTC from cancer-free controls in its early stages remains an important goal. MATERIAL AND METHODS Serum samples from 88 PTC patients and 80 cancer-free controls were randomly allocated into training or validation sets. Serum peptide profiling was performed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) after using weak cation exchange magnetic beads (WCX-MB), and the results were evaluated by use of ClinProTools™ Software. To distinguish PTC from cancer-free controls, quick classifier (QC), supervised neural network (SNN), and genetic algorithm (GA) models were established. The models were blindly validated to verify their diagnostic capabilities. The most discriminative peaks were subsequently identified with a nano-liquid chromatography-electrospray ionization-tandem mass spectrometry system. RESULTS Six peptide ions were identified as the most discriminative peaks between the PTC and cancer-free control samples. The QC model exhibited satisfactory sensitivity and specificity among the 3 models that were validated. Two peaks, at m/z 2671.17 and m/z 1464.68, were identified as fragments of the alpha chain of fibrinogen, while a peak at m/z 1738.92 was a fragment of complement component 4A/B. CONCLUSIONS MS combined with ClinProTools™ software was able to detect peptide biomarkers in PTC patients. In addition, the constructed classification models provided a serum peptidome pattern for distinguishing PTC from cancer-free controls. Both fibrinogen a and complement C4A/B were identified as potential markers for diagnosis of PTC.

Elevated expression of TANK-binding kinase 1 enhances tamoxifen resistance in breast cancer.

Resistance to antiestrogens is one of the major challenges in breast cancer treatment. Although phosphorylation of estrogen receptor α (ERα) is an important factor in endocrine resistance, the contributions of specific kinases in endocrine resistance are still not fully understood. Here, we report that an important innate immune response kinase, the IκB kinase-related TANK-binding kinase 1 (TBK1), is a crucial determinant of resistance to tamoxifen therapies. We show that TBK1 increases ERα transcriptional activity through phosphorylation modification of ERα at the Ser-305 site. Ectopic TBK1 expression impairs the responsiveness of breast cancer cells to tamoxifen. By studying the specimens from patients with breast cancer, we find a strong positive correlation of TBK1 with ERα, ERα Ser-305, and cyclin D1. Notably, patients with tumors highly expressing TBK1 respond poorly to tamoxifen treatment and show high potential for relapse. Therefore, our findings suggest that TBK1 contributes to tamoxifen resistance in breast cancer via phosphorylation modification of ERα.

Diagnostic value of total plasma lysophosphatidic acid in ovarian cancer: a meta-analysis.

OBJECTIVE: This study aimed to assess the diagnostic value of lysophosphatidic acid (LPA) in ovarian cancer. METHODS: A systematic review of related studies was performed; sensitivity, specificity, and other measures about the accuracy of serum LPA in the diagnosis of ovarian cancer were pooled using random-effects models. Summary receiver operating characteristic curve analysis was used to summarize the overall test performance. RESULTS: Six studies involving 363 patients with ovarian cancer and 273 healthy control women met the inclusion criteria. The summary estimates for LPA in diagnosing ovarian cancer in the included studies were as follows: sensitivity, 0.94 [95% confidence interval (CI), 0.91-0.96]; specificity, 0.88 (95% CI, 0.83-0.91); and diagnostic odds ratio, 141.59 (95% CI, 52.1-384.63). The area under the curve and Q value for summary receiver operating characteristic curves were 0.97 and 0.92, respectively. CONCLUSIONS: The LPA assay showed high accuracy and sensitivity for the diagnosis of ovarian cancer. The present study was limited by the small number of available studies and sample size; therefore, additional studies with a better design and larger samples are needed to further assess the diagnostic accuracy of LPA.

Lack of association between IL-4 -588C>T polymorphism and NHL susceptibility.

The relationship of non-Hodgkin's lymphoma (NHL) with the presence of interleukin-4 genetic polymorphism -588C>T has been reported with inconsistent results. The objective of this study was to quantitatively evaluate the association between -588C>T polymorphism and NHL susceptibility. Two investigators independently searched Medline and the Cochrane Library up to September 20, 2013. Pooled odds ratio and 95% confidence interval were calculated using a fixed or random effects model. Statistical analysis was performed with Review Manage 5.0 and Stata 11. Of the six case-control studies selected for this meta-analysis, a total of 1,909 NHL cases and 1,834 controls were included. The combined results based on all studies suggested that -588C>T was not associated with NHL risk under all genetic models. When stratifying for race, no noteworthy associations were observed in mixed populations or Caucasians. This meta-analysis suggests that IL-4 -588C>T polymorphism might not be a risk factor for NHL risk. However, further well-designed studies are required to confirm our findings.

Hepatitis C virus infection and the risk of Sjögren or sicca syndrome: a meta-analysis.

Previous studies have suggested an association between hepatitis C virus (HCV) infection and the development of Sjögren's syndrome (SS), also known as sicca syndrome. The main objective of this study was to summarize the existing evidence and quantitatively evaluate the association between hepatitis C virus infection and SS/sicca syndrome by performing a meta-analysis of observational studies. MEDLINE and PubMed (January 1980-August 2013) were searched to identify relevant studies in English. Outcomes were calculated and are reported as odds risk (OR) and 95% CIs based on a random-effects model. Heterogeneity was assessed with I(2) statistics. Quality assessment was performed with the Newcastle-Ottawa scale. Based on meta-analysis of five cross-sectional and five cohort studies, a significant positive relationship between HCV infection and development of SS/sicca syndrome was found, the pooled random effects OR being 3.31 (95% CI, 1.46-7.48; P < 0.001). In subset analyses, the studies that used European diagnostic criteria showed a higher summary OR than did studies that adopted other diagnostic criteria. When the data were stratified by source of controls, significant associations were also observed when healthy people (OR = 9.44; 95% CI = 2.67-33.40; P = 0.204) or subjects with hepatitis B virus infection (OR = 6.57; 95% CI = 1.21-35.57; P = 0.5) were used as controls, but not when the controls were hospital-based (OR = 0.99; 95% CI = 0.61-1.61; P = 0.169). In summary, the findings suggest that HCV infection is associated with SS/sicca syndrome. The observed increased risk in studies in which European diagnostic criteria and healthy controls were used and the decreased risk in studies with hospital-based controls may be attributable to selection bias or other unknown factors.

Serum peptide pattern that differentially diagnoses hepatitis B virus-related hepatocellular carcinoma from liver cirrhosis.

BACKGROUND: Although alpha-fetoprotein (AFP) is a useful serologic marker of hepatocellular carcinoma (HCC), it is not sufficiently sensitive to differentiate HCC and liver cirrhosis (LC) caused by hepatitis B virus (HBV) infection. AIMS: The aim is to discover novel noninvasive specific serum biomarkers for the differential diagnosis of HBV-related HCC and LC. METHODS: With a highly optimized peptide extraction and matrix-assisted laser desorption/ionization time of flight/time of flight mass spectrometric approach, we investigated serum peptide profiles of 80 HCC and 67 LC patients. Three supervised machine learning methods were employed to construct classifiers. Receiver operator curves were plotted to evaluate the performance of classifiers. RESULTS: With a support vector machine-based strategy, we picked nine peaks with m/z ratios of 819.49, 1076.14, 1341.72, 2551.44, 3156.44, 3812.88, 4184.26, 4465.92, and 4776.41 to construct the classifier. We proposed a novel method for distinguishing HCC from cirrhosis, based on a multilayer perceptron (MLP) method. We obtained a sensitivity of 90.0%, specificity of 79.4%, and overall accuracy of 85.1% on an independent test set. The combination of the MLP model and serum AFP level outperformed serum AFP marker alone in distinguishing HCC patients from LC patients. In this experience, sensitivity increased from 62.5% to 87.5%, and specificity increased from 79.4% to 88.2%. CONCLUSIONS: Our results indicate that the MLP model is a novel and useful serum peptide pattern for distinguishing HCC and LC. The peptidome signature alone or together with serum AFP determination may be a more effective method for early diagnosis of HCC in patients with HBV-related LC.

[Characteristics of complex chromosomal rearrangement in Chinese male carriers and its impact on male fertility].

OBJECTIVE: To analyze the characteristics of complex chromosomal rearrangement (CCR) in Chinese male carriers and its influence on male fertility. METHODS: Using the G band technique, we conducted karyotype analysis on the peripheral blood lymphocytes of 1,625 Chinese males with reproductive problems. We also searched CNKI and Wanfang database for CCR-related literature published between January 1984 and November 2013, followed by statistical analysis on the CCR characteristics and reproduction-related data of the CCR carriers. RESULTS: Two CCR carriers were found among the 1,625 males and another 47 cases identified from the databases. Among the 49 CCR carriers, there were 17 three-way exchange cases (34.7%), 17 double two-way exchange cases (34.7%), and 15 exceptional cases (30.6%), with no statistically significant differences in the incidence of the three types (P > 0.05). Azoospermia- or oligospermia-induced infertility was found in 19 (38.8% ) of the CCR carriers. A total of 87 pregnancies were achieved in the other 30 (61.2%), among which spontaneous abortion occurred in 75.9% (66/87), dead fetus and malformed infant death in 9.2% (8/87), and phenotypically normal offspring in 14.9% (13/87). Recurrent abortion was associated frequently with breakpoints on CCR-involved chromosomes 6, 7, 8, 11, and 16, while dyszoospermia mostly with breakpoints on CCR-involved chromosomes 10 and 14. The breaking occurred more than 3 times at 1p22, 1q25, 2q31, 5p13, 5q35, 6q23, 8q13, and 20p13. Moreo- ver, the breakpoints at 2q31, 5q35, and 8q13 were particularly related to recurrent abortion, while that at 1p22 only to dyszoospermia. CONCLUSION: CCR is extremely rare. Male CCR carriers are often identified through reproductive problems and have high risks of infertility and abnormal pregnancy and a very low rate of normal newborns. In addition, chromosomes and breakpoints involved in CCR may affect the fertility of male CCR carriers, and some particular chromosomal breakpoints may play a key role in gametogenesis.

Correlation between KLK6 expression and the clinicopathological features of glioma.

AIM OF THE STUDY: We measured the impact of changing KLK6 expression levels on the pathological grade of gliomas and on proliferation rate, cell cycle progression, and apoptosis in the U251 glioblastoma cell line. MATERIAL AND METHODS: The expression of KLK6 in 35 brain glioma tissues and adjacent noncancerous tissues was measured using real-time quantitative polymerase chain reaction (PCR) and the relationship between KLK6 expression and pathological grades was analysed. RESULTS: The KLK6 expression in U251 cells was silenced by a specific siRNA, and the effects on proliferation, the cell cycle, and apoptosis were compared to wild type cells. Expression of KLK6 was downregulated in gliomas relative to matched noncancerous tissue. There was no obvious relationship between patient sex, pathological grade, or tumour classification and the expression of KLK6. In the U251 cell line, cell proliferation was enhanced and the fractions of cells in the G2 and S phases were increased by siRNA-mediated KLK6 silencing. CONCLUSIONS: Expression of KLK6 inhibits tumour growth. Decreased KLK6 expression may be a possible risk factor for glioma.

Targeting insulin resistance in type 2 diabetes via immune modulation of cord blood-derived multipotent stem cells (CB-SCs) in stem cell educator therapy: phase I/II clinical trial.

BACKGROUND: The prevalence of type 2 diabetes (T2D) is increasing worldwide and creating a significant burden on health systems, highlighting the need for the development of innovative therapeutic approaches to overcome immune dysfunction, which is likely a key factor in the development of insulin resistance in T2D. It suggests that immune modulation may be a useful tool in treating the disease. METHODS: In an open-label, phase 1/phase 2 study, patients (N=36) with long-standing T2D were divided into three groups (Group A, oral medications, n=18; Group B, oral medications+insulin injections, n=11; Group C having impaired ß-cell function with oral medications+insulin injections, n=7). All patients received one treatment with the Stem Cell Educator therapy in which a patient's blood is circulated through a closed-loop system that separates mononuclear cells from the whole blood, briefly co-cultures them with adherent cord blood-derived multipotent stem cells (CB-SCs), and returns the educated autologous cells to the patient's circulation. RESULTS: Clinical findings indicate that T2D patients achieve improved metabolic control and reduced inflammation markers after receiving Stem Cell Educator therapy. Median glycated hemoglobin (HbA1C) in Group A and B was significantly reduced from 8.61%±1.12 at baseline to 7.25%±0.58 at 12 weeks (P=2.62E-06), and 7.33%±1.02 at one year post-treatment (P=0.0002). Homeostasis model assessment (HOMA) of insulin resistance (HOMA-IR) demonstrated that insulin sensitivity was improved post-treatment. Notably, the islet beta-cell function in Group C subjects was markedly recovered, as demonstrated by the restoration of C-peptide levels. Mechanistic studies revealed that Stem Cell Educator therapy reverses immune dysfunctions through immune modulation on monocytes and balancing Th1/Th2/Th3 cytokine production. CONCLUSIONS: Clinical data from the current phase 1/phase 2 study demonstrate that Stem Cell Educator therapy is a safe approach that produces lasting improvement in metabolic control for individuals with moderate or severe T2D who receive a single treatment. In addition, this approach does not appear to have the safety and ethical concerns associated with conventional stem cell-based approaches. TRIAL REGISTRATION: ClinicalTrials.gov number, NCT01415726.

Red blood cell distribution width is a potential prognostic index for liver disease.

BACKGROUND: Red blood cell distribution width (RDW) is increased in liver disease. Its clinical significance, however, remains largely unknown. The aim of this study was to identify whether RDW was a prognostic index for liver disease. METHODS: We studied, retrospectively, 33 patients with non-cirrhotic HBV chronic hepatitis, 125 patients with liver cirrhosis after HBV infection, 81 newly diagnosed primary hepatocellular carcinoma (pHCC) patients, 17 alcoholic liver cirrhosis patients and 42 patients with primary biliary cirrhosis (PBC). Sixty-six healthy individuals represented the control cohort. We analyzed the relationship between RDW on admission and clinical features. The association between RDW and hospitalization outcome was estimated by receiver operating curve (ROC) analysis and a multivariable logistic regression model. RESULTS: Increased RDW was observed in liver disease patients. RDW was positively correlated with serum bilirubin and creatinine levels, prothrombin time, and negatively correlated with platelet counts and serum albumin concentration. A subgroup analysis, considering the different etiologies, revealed similar findings. Among the patients with liver cirrhosis, RDW increased with worsening of Child-Pugh grade. In patients with PBC, RDW positively correlated with Mayo risk score. Increased RDW was associated with worse hospital outcome, as shown by the AUC [95% confidence interval (CI)] of 0.76 (0.67-0.84). RDW above 15.15% was independently associated with poor hospital outcome after adjustment for serum bilirubin, platelet count, prothrombin time, albumin and age, with the odds ratio (95% CI) of 13.29 (1.67-105.68). CONCLUSIONS: RDW is a potential prognostic index for liver disease.

Reversal of type 1 diabetes via islet ß cell regeneration following immune modulation by cord blood-derived multipotent stem cells.

BACKGROUND: Inability to control autoimmunity is the primary barrier to developing a cure for type 1 diabetes (T1D). Evidence that human cord blood-derived multipotent stem cells (CB-SCs) can control autoimmune responses by altering regulatory T cells (Tregs) and human islet ß cell-specific T cell clones offers promise for a new approach to overcome the autoimmunity underlying T1D. METHODS: We developed a procedure for Stem Cell Educator therapy in which a patient's blood is circulated through a closed-loop system that separates lymphocytes from the whole blood and briefly co-cultures them with adherent CB-SCs before returning them to the patient's circulation. In an open-label, phase1/phase 2 study, patients (n=15) with T1D received one treatment with the Stem Cell Educator. Median age was 29 years (range: 15 to 41), and median diabetic history was 8 years (range: 1 to 21). RESULTS: Stem Cell Educator therapy was well tolerated in all participants with minimal pain from two venipunctures and no adverse events. Stem Cell Educator therapy can markedly improve C-peptide levels, reduce the median glycated hemoglobin A1C (HbA1C) values, and decrease the median daily dose of insulin in patients with some residual ß cell function (n=6) and patients with no residual pancreatic islet ß cell function (n=6). Treatment also produced an increase in basal and glucose-stimulated C-peptide levels through 40 weeks. However, participants in the Control Group (n=3) did not exhibit significant change at any follow-up. Individuals who received Stem Cell Educator therapy exhibited increased expression of co-stimulating molecules (specifically, CD28 and ICOS), increases in the number of CD4+CD25+Foxp3+ Tregs, and restoration of Th1/Th2/Th3 cytokine balance. CONCLUSIONS: Stem Cell Educator therapy is safe, and in individuals with moderate or severe T1D, a single treatment produces lasting improvement in metabolic control. Initial results indicate Stem Cell Educator therapy reverses autoimmunity and promotes regeneration of islet ß cells. Successful immune modulation by CB-SCs and the resulting clinical improvement in patient status may have important implications for other autoimmune and inflammation-related diseases without the safety and ethical concerns associated with conventional stem cell-based approaches. TRIAL REGISTRATION: ClinicalTrials.gov number, NCT01350219.

UrineCART, a machine learning method for establishment of review rules based on UF-1000i flow cytometry and dipstick or reflectance photometer.

BACKGROUND: Automated systems have been broadly used in the counting of particles in urine, while manual microscopic analyses are still required for confirming components of urine sediments, especially pathologic casts and other unknown particles. Good review rules can reduce the number of manual urine microscopy examinations safely, thereby increasing productivity. Although several methods have been proposed,establishment of microscopic review rules for fl ow cytometer remains challenging. METHODS: A total of 3014 urine samples from outpatient and inpatient were examined using UF-1000i flow cytometry,Urisys-2400 dipstick and RS 2003 urine sediment workstation,respectively. Based on the results above, three supervised machine learning methods were employed to construct classifiers for screening urine samples. RESULTS: Here, we propose a novel method for construction of microscopic review rules, termed UrineCART, which was based on a classification and regression tree (CART) method.With a cut-off value of 0.0745 for Urine CART, we obtained a sensitivity of 92.0 % , a specificity of 81.5 % and a total review rate of 32.4 % on an independent test set. Comparisons with the existing methods showed that Urine CART gave the acceptable sensitivity and lower total review rate. CONCLUSIONS: An algorithm based on machine learning methods for review criteria can be achieved via systematic comparison of UF-1000i flow cytometry and microscopy.Using Urine CART, our microscopic review rate can be reduced to around 30 % , while decreasing significant losses in urinalysis.

IL-21 Enhances the Immune Protection Induced by the Vibrio vulnificus Hemolysin A Protein.

We previously reported that the Vibrio vulnificus hemolysin A (VvhA) protein elicited good immune protection and could effectively control V. vulnificus infection in mice. However, its molecular mechanism remains unknown. We hypothesized that hemolysin A induces an immunoprotective response via IL-21 regulation. To demonstrate this, IL-21 expression in mice was regulated by injecting either specific antibodies or rIL-21, and the immune response was evaluated by flow cytometry. Our results suggested that IL-21 enhances immune protection by inducing a T follicular helper cell and germinal center B cell response. We used RNA-seq to explore molecular mechanisms and identified 10 upregulated and 32 downregulated genes involved in IL-21-upregulated protection. Gene Ontology analysis and pathway analysis of the differentially expressed genes were also performed. Our findings indicate that IL-21 can enhance the immune protection effect of the VvhA protein and may serve as a novel strategy for enhancing the immune protection effect of protein vaccines.

Development of a colloidal gold-based immunochromatographic test strip for the rapid, on-site detection of Pseudomonas aeruginosa in clinical samples.

BACKGROUND: Current procedures for the detection of Pseudomonas aeruginosa require sophisticated equipment, skilled technicians, and a great deal of time. Immunochromatography assays (ICA) are simple and rapid diagnostic procedures that can be performed and interpreted on the spot or at the bedside without a machine. METHODS: We developed a rapid, 1-step immunochromatographic test strip that is well suited to the on-site detection of P. aeruginosa with high sensitivity and specificity. In brief, a monoclonal antibody targeting the outer membrane protein F (OprF) of P. aeruginosa, 3C3B5, was conjugated to colloidal gold and used as a detection antibody. An OprF polyclonal antibody was developed as the capture antibody. Eighty-three clinical samples were examined for P. aeruginosa by rapid 1-step ICA and compared with Multiplex-polymerase chain reaction (M-PCR). RESULTS: The detection limit of this method is 5 × 10(5) CFU/ml for P. aeruginosa and 10 ng/ml for the OprF protein. The immunochromatographic strip test demonstrated a slightly lower sensitivity (84.8%), but a similar specificity (100%), to multi-PCR, which is an accurate method for the detection of P. aeruginosa in the laboratory. We observed no cross-reactivity with non-P. aeruginosa bacterial microbes. The detection of P. aeruginosa by the ICA strip can be completed within 5-10 min and is at least 10-fold faster than M-PCR. CONCLUSIONS: The ICA test strip developed in this study has proved to be a rapid, simple, effective and economical method for the detection of P. aeruginosa infection in clinical samples. To our knowledge, this is the first report of an ICA method being used to detect P. aeruginosa.

RNA interference-mediated silencing of UBCH10 gene inhibits colorectal cancer cell growth in vitro and in vivo.

1. UbcH10 is the cancer-related E2 ubiquitin-conjugating enzyme, and its overexpression has been demonstrated in a variety of malignancies. The aim of the present study is to silence UbcH10 gene by RNA interference (RNAi) and to observe its inhibitory effect on the colorectal cancer cell growth in vitro and in vivo. 2. We constructed the expression vector pGPU6/GFP/Neo/UbcH10-RNAi (pUbcH10-RNAi), which contained a UbcH10 short hairpin RNA expression cassette. Then the UbcH10 gene silencing cell lines LoVo/UbcH10-RNAi and HT-29/UbcH10-RNAi were established. Reverse transcription-polymerase chain reaction and western blot analysis were used to evaluate the expression of the UbcH10 gene. Cell Counting Kit-8 was used to assess properties of tumour cell growth in vitro. Flow cytometry was used to detect the effect of pUbcH10-RNAi on the cell cycle of colorectal cancer cells. Furthermore, the anti-tumour effects of pUbcH10-RNAi were evaluated in vivo in a nude mouse xenografts model. 3. Results demonstrated that UbcH10 gene expression was significantly decreased in pUbcH10-RNAi treated cells. Colorectal cancer cells growth was markedly suppressed in the pUbcH10-RNAi group compared with control conditions and colorectal cancer cells were arrested in the G2-M phase. In vivo, the downregulation of UbcH10 gene expression by pUbcH10-RNAi also inhibited tumour growth in a nude mice xenograft model. 4. Our study suggests that RNA interference-mediated silencing of UbcH10 gene has anti-tumour activity on colorectal cancer and might have therapeutic potential for the treatment of colorectal cancer.

Chinese Expert Consensus on the Nucleic Acid Detection of SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) has already become a pandemic wherein the infection's timely diagnosis has proven beneficial to patient treatment and disease control. Nucleic acid detection has been the primary laboratory diagnostic method for the detection of SARS-CoV-2. To ensure laboratory staff safety and quality nucleic acid testing, the Chinese Society of Laboratory Medicine formulated this consensus, based on the Chinese National Recommendations and previous literature for nucleic acid detection. A working group comprises 34 hospital professionals experience with real-time polymerase chain reactions (PCR) testing for SARS-CoV-2 drafted guidance statements during online discussions. A modified Delphi methodology was used in forming a consensus among a wider group of hospital professionals with SARS-CoV-2 detection experience. Guidance statements were developed for four categories: (I) specimen type, priority, collecting, transportation and receiving; (II) nucleic acid isolation and amplification; (III) quality control; (IV) biosafety management and decontamination. The modified Delphi voting process included a total of 29 guidance statements and final agreement. Consensus was reached after two rounds of voting. Recommendations were established for the detection of SARS-CoV-2 using real time PCR testing based on evidence and group consensus. The manuscript was evaluated against The Appraisal of Guidelines for Research & Evaluation Instrument (AGREE II) and was developed to aid medical laboratory staff in the detection of the ribonucleic acid (RNA) of SARS-CoV-2.

[Correlation of hK6 expression with clinicopathological features and prognosis in epithelial ovarian cancer].

OBJECTIVE: To approach the relationship between the expression of hK6 in ovarian neoplasm and clinicopathological variables and prognosis in ovarian cancer patients for finding a new tumor marker of the ovarian cancer. METHODS: The expression of hK6 was detected by immunohistochemistry in 19 cases of benign, 11 cases of borderline and 45 cases of malignant ovarian neoplasms and statistically analyzed whether its expression correlate with clinicopathological variables and prognosis in patients with ovarian cancer. RESULTS: The expression of hK6 in ovarian cancer tissues (60.0%) was significantly higher than that in the benign (15.8%) and borderline (27.3%) ovarian neoplasm tissues (P < 0.01). The expression of hK6 in higher-grade ovarian cancer tissues (68.4% ) was higher than that in low-grade ones (14.3%, P < 0.05). The expression of hK6 in late-stage (stage III, 76.7%) was significantly higher than that in early-stage (stage I or II, 26.7%, P < 0.01). The expression of hK6 was significantly higher in patients with lymph node metastasis (77.8%) than that in patients without (33.3%, P < 0.01). The expression of hK6 in the cancer tissues in the patients died, or with reccurence or metastasis within 3 years after surgery was higher (75.0%) than that in the patients with stable disease (42.9%, P < 0.05). CONCLUSION: The expression of hK6 in ovarian cancer was higher than that in benign and borderline ovarian neoplasms. The expression of hK6 is higher in the ovarian cancer of late stage, higher-grade, with lymph node metastasis and is associated with a poorer prognosis. hK6 may become a new markers in prediction of prognosis of the patients with ovarian tumors.

Direct Detection of Vibrio vulnificus, Vibrio parahaemolyticus, and Vibrio alginolyticus from Clinical and Environmental Samples by a Multiplex Touchdown Polymerase Chain Reaction Assay.

BACKGROUND: Vibrio vulnificus, V. alginolyticus, and V. parahaemolyticus are commonly and opportunistically pathogenic to humans. METHODS: In this study, a novel multiple touchdown polymerase chain reaction method (MT-PCR) was developed to benefit rapid and simultaneous detection of the presence of the three Vibrio species from the enriched clinical and environmental samples. RESULTS: The method showed a sensitivity of 104 colony forming units (CFU)/mL for V. vulnificus, 103 CFU/mL for V. parahaemolyticus and V. alginolyticus, and a specificity of 100% for all the three Vibrio species. All strains of the three Vibrio species were detected in the spiked samples artificially contaminated with reference strains and were identified directly from the enriched clinical and environmental samples within three hours by this MT-PCR assay. All the corresponding bacteria were isolated from these enriched samples in 48 hours by standard microbiologic procedures. CONCLUSIONS: This MT-PCR method, which can detect V. vulnificus, V. parahaemolyticus, and V. alginolyticus directly and simultaneously, was rapid, sensitive, specific, and can be used in clinical diagnostics, food industry studies, and risk assessment of environment.