Escherichia coli phage phi2013: genomic analysis and receptor identification.

Escherichia coli is an important foodborne pathogen that can cause severe human disease. Here, we report the isolation and characterization of the lytic virus phi2013, which is specific for Escherichia coli laboratory strains. Transmission electron microscopy showed that phage phi2013 has an icosahedral head and a long, fragile, noncontractile tail, exhibiting the typical form of a siphovirus. Evidence revealed that the phi2013 genome is a linear double-stranded DNA molecule of 49,833 bp with 79 predicted genes without any known antibiotic resistance genes, virulence factor genes, or integrase genes. Moreover, the conserved outer membrane protein FhuA, which is present in members of several genera of the family Enterobacteriaceae, was identified as the receptor of phage phi2013. To evaluate the potential of phage phi2013 as a biocontrol agent for controlling E. coli contamination, it was tested in several foods, including sterilized milk, ready-to-eat beef, and crisphead lettuce. The data showed that phage phi2013 can efficiently inhibit E. coli growth in the tested foods at 4°C and 25°C. We therefore conclude that phage phi2013 or cocktails containing phi2013 may be used as an antimicrobial agent in extending the shelf-life of food products by effectively controlling the growth of E. coli.

Heterozygous/dispermic complete mole confers a significantly higher risk for post-molar gestational trophoblastic disease.

Hydatidiform moles are classified at the genetic level as androgenetic complete mole and diandric-monogynic partial mole. Conflicting data exist whether heterozygous complete moles are more aggressive clinically than homozygous complete moles. We investigated clinical outcome in a large cohort of hydatidiform moles in Chinese patients with an emphasis on genotypical correlation with post-molar gestational trophoblastic disease. Consecutive products of conceptions undergoing DNA genotyping and p57 immunohistochemistry to rule out molar gestations were included from a 5-year period at Beijing Obstetrics and Gynecology Hospital. Patient demographics and clinical follow-up information were obtained. Post-molar gestational trophoblastic disease or gestational trophoblastic neoplasia was determined by the 2002 WHO/FIGO criteria. A total of 1245 products of conceptions were classified based on genotyping results into 219 complete moles, 250 partial moles, and 776 non-molar gestations. Among 219 complete moles, 186 were homozygous/monospermic and 33 were heterozygous/dispermic. Among 250 partial moles, 246 were triploid dispermic, 2 were triploid monospermic, and 2 were tetraploid heterozygous partial moles. Among 776 non-molar gestations, 644 were diploid without chromosomal aneuploidies detectable by STR genotyping and 132 had various genetic abnormalities including 122 cases of various trisomies, 2 triploid digynic-monoandric non-molar gestations, 7 cases of possible chromosomal monosomy or uniparental disomy. Successful follow-up was achieved in 165 complete moles: post-molar gestational trophoblastic disease developed in 11.6% (16/138 cases) of homozygous complete moles and 37.0% (10/27 cases) of heterozygous complete moles. The difference between the two groups was highly significant (p = 0.0009, chi-square). None of the 218 partial moles and 367 non-molar gestations developed post-molar gestational trophoblastic disease. In conclusion, heterozygous/dispermic complete moles are clinically more aggressive with a significantly higher risk for development of post-molar gestational trophoblastic disease compared with homozygous/monospermic complete moles. Therefore, precise genotyping classification of complete moles is important for clinical prognosis and patient management.

Effects of inhibiting the PI3K/Akt/mTOR signaling pathway on the pain of sciatic endometriosis in a rat model.

This study investigated the relationship between the pain of sciatic endometriosis and the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway. Adult female Sprague-Dawley rats successfully received sciatic endometriosis induction. Mechanical paw withdrawal threshold and paw withdrawal latency were recorded to assess the mechanical hypersensitivity and thermal hyperalgesia. Quantitative real-time PCR, Western blotting, and enzyme-linked immunosorbent assays were used to detect PI3K, Akt, and mTOR expressions and their phosphorylation as well as the expressions of substance P, calcitonin gene-related peptide (CGRP), and nerve growth factor (NGF). Mechanical paw withdrawal threshold and paw withdrawal latency significantly decreased after sciatic endometriosis induction in rats; this decrease was ameliorated by inhibiting the PI3K/Akt/mTOR signaling pathway using LY294002. Compared with controls, rats with sciatic endometriosis showed increased PI3K, Akt, and mTOR expressions and elevated p-PI3K, p-Akt, and p-mTOR protein expressions. Higher NGF, substance P, and CGRP expressions were also found in the superficial dorsal horn of the spinal cord in rats with sciatic endometriosis than in control rats 21 days after surgery. Following the injection of LY294002 into rats with sciatic endometriosis, there was a significant decrease in the expressions of NGF, substance P, and CGRP. In conclusion, the inhibition of the PI3K/Akt/mTOR signaling pathway may alleviate endometriosis-associated sciatic nerve pain in a rat model of sciatic endometriosis.

Restoration of microRNA-708 sensitizes ovarian cancer cells to cisplatin via IGF2BP1/Akt pathway.

A previous study has shown that microRNA-708 (miR-708) functions as a metastasis suppressor in ovarian cancer. In this study, we aimed to explore its implication in regulating cisplatin sensitivity in ovarian cancer cells. To this end, ovarian cancer cells were transfected with miR-708-expressing plasmids or vector before treatment with different concentrations of cisplatin for 48 h. The 50% inhibitory concentration (IC50 ) value was calculated. Apoptosis was analyzed by measuring caspase-3 activity. The target gene mediating the function of miR-708 was identified. Ectopic expression of miR-708 sensitized SKOV3 and A2780 cells to cisplatin, decreasing the IC50 value by two- to threefold. miR-708 overexpression significantly augmented cisplatin-induced apoptosis in ovarian cancer cells, which was coupled with increased caspase-3 activity by two- to fourfold. Similarly, overexpression of miR-708 increased the sensitivity of cisplatin-resistant SKOV3/DDP and A2780/DDP cells to cisplatin-induced toxicity, reducing the IC50 by three- and fivefold, respectively. Delivery of miR-708 enhanced cisplatin-induced elevation in caspase-3 activity in both cisplatin-resistant and parental ovarian cancer cells. Mechanistically, miR-708 downregulated the expression of insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) and suppressed Akt phosphorylation. Silencing of IGF2BP1 markedly blocked the phosphorylation of Akt. Overexpression of IGF2BP1 restored cisplatin resistance and Akt phosphorylation in miR-708-overexpressing ovarian cancer cells. Collectively, miR-708 increases the susceptibility of ovarian cancer cells to cisplatin by targeting IGF2BP1 and inhibiting Akt signaling. Delivery of miR-708 may represent a promising strategy for improving cisplatin chemotherapy.

Quorum sensing influences phage infection efficiency via affecting cell population and physiological state.

Bacterial growth phase has been reported affecting phage infection. To underpin the related mechanism, infection efficiency of Pseudomonas aeruginosa phage K5 is characterized. When infecting the logarithmic cells, phage K5 produced significantly more infection centers than the stationary cells, well concordant with the viable cell ratio in the different growth phases. Additionally, the burst size decreased dramatically in the stationary cells, implying that the physiological state of the viable cells contributed to the productivity of phage K5, and it was consistent with the expression variation of the phage RNA polymerase. Quorum sensing inhibitor penicillic acid was applied and could significantly improve the viable cell proportion and the infection center numbers, but had less effect on the corresponding burst sizes. Moreover, the effect of penicillic acid and the quorum sensing regulator mutants on the production of phage C11 was also analyzed. Taken together, our data suggest that quorum sensing is involved in the defense of phage K5 infection by influencing the viable cell population and their physiological state, and it is an efficient and intrinsic pathway allowing bacteria to resist phage attacks in natural environment.

[Diagnostic utility of STR genotyping for partial hydatidiform moles].

OBJECTIVE: To investigate the clinical utility of short tandem repeats(STR) genotyping technique for diagnosis of partial hydatidiform moles (PHM). METHODS: Ten cases with the original diagnosis of PHM and six cases diagnosed as "favour PHM" or "abnormal villous, PHM not excluded" were selected for the study. The clinical information and follow-up data were reviewed. Histopathologic features were evaluated along with p57 immunohistochemistry. After DNA extraction from each sample, genotyping was performed by AmpFlSTR(®) Identifiler™ PCR kit to amplify 15 STR polymorphism loci plus the amelogenin gender-determining in a single robust PCR. RESULTS: The age of patients ranged from 18 to 49 years (mean=29 years, median=29 years). Two villous populations (7/16), irregular villous contour (13/16), at least moderate trophoblastic hyperplasia (2/16), cistern formation (8/16), syncytiotrophoblastic knuckles (14/16), trophoblastic pseudoinclusions (6/16) and nucleated fetal red blood cells (8/16) were presented in these cases. Of the cases in the study, STR genotyping identified 4 monospermic complete hydatidiform moles (MCM), 3 dispermic partial hydatidiform moles (DPM) and 9 hydropic abortions (HA). The misdiagnosis rate was 13/16 only relied on morphology evaluation. Immunostaining of p57 showed 3/4 of MCM were focally positive (<5%-20%+), 1/4 of MCM were diffusely positive (70%+), 3/3 of DPM were diffusely positive (≥50%+), 7/9 of HA were diffusely positive (≥50%+), and 2/9 of HA were focally positive (10%+). CONCLUSIONS: Combination of histomorphologic evaluation and p57 immunostaining is insufficient for a definitive diagnosis of PHM. STR genotyping offers an accurate diagnosis of PHM.

Psychrophilic phage phiGM22-3 efficiently controls Pseudomonas fluorescens contamination in cold-stored milk.

Pseudomonas fluorescens is a common spoilage causing microbe found in milk. Antibiotic preservatives may cause emergence of multidrug resistance, posing food safety related risks to public health. Phage treatment may be used as an alternative to antibiotics in controlling P. fluorescens contaminations. Here we reported that P. fluorescens phage phiGM22-3 reproduced rapidly over a broad temperature range of 4 through 30°C, and the optimum growth of phiGM22-3 occurred at 10°C, indicating that it was a psychrophilic virus. Genome analysis revealed that phiGM22-3 has a genome of 42,662 bp with an identical terminal direct repeat sequence of 328 bp and encodes 58 predicted proteins. Evidence revealed that phiGM22-3 recognized lipopolysaccharides (LPS) as receptor for infection. Additionally, two phage mutants phiMX2 and phiMX8 with different host ranges were identified in the phiGM22-3 population. Phage killing efficiency of P. fluorescens cells artificially inoculated in milk was evaluated. Phage phiGM22-3 and the cocktails containing phiMX2 and phiMX8 can lyse almost 100% bacterial cells at 4°C within 24 h. Taken together, our data indicated that the psychrophilic virus phiGM22-3 and its two mutants can efficiently inhibit bacteria growth at 4°C, showing a great potential to be used as alternatives to conventional antibiotics against P. fluorescens in refrigerated foods.

Variants of a putative baseplate wedge protein extend the host range of Pseudomonas phage K8.

BACKGROUND: Narrow host range is a major limitation for phage applications, but phages can evolve expanded host range through adaptations in the receptor-binding proteins. RESULTS: Here, we report that Pseudomonas phage K8 can evolve broader host range and higher killing efficiency at the cost of virion stability. Phage K8 host range mutant K8-T239A carries a mutant version of the putative baseplate wedge protein GP075, termed GP075m. While phage K8 adsorbs to hosts via the O-specific antigen of bacterial LPS, phage K8-T239A uses GP075m to also bind the bacterial core oligosaccharide, enabling infection of bacterial strains resistant to K8 infection due to modified O-specific antigens. This mutation in GP075 also alters inter-protein interactions among phage proteins, and reduces the stability of phage particles to environmental stressors like heat, acidity, and alkalinity. We find that a variety of mutations in gp075 are widespread in K8 populations, and that the gp075-like genes are widely distributed among the domains of life. CONCLUSION: Our data show that a typical life history tradeoff occurs between the stability and the host range in the evolution of phage K8. Reservoirs of viral gene variants may be widely present in phage communities, allowing phages to rapidly adapt to any emerging environmental stressors. Video Abstract.

Anti-nociceptive and anti-inflammatory effects of sulforaphane on sciatic endometriosis in a rat model.

Endometriosis of sciatic nerve is a common gynecological disease. Here we aimed to study the anti-inflammatory and anti-nociceptive role of sulforaphane on sciatic nerve endometriosis. The sciatic nerve endometriosis rat model was constructed by autologous implantation of uterine tissue. Sulforaphane was administered intraperitoneally at the dose of 5, 15, 30 and 60 mg/kg/day for 28 days. Behavioral testing was performed at day 7, 14, 21 and 28. At day 28, rats were sacrificed, followed by collecting superficial dorsal horn tissues and lesions. Quantitative real-time PCR and Western blot were performed to assess COX2, Keap1, Nrf2 expression in collected tissues. Enzyme-linked immunosorbent assay was conducted to assess the expression of pro-inflammatory cytokines. Sulforaphane alleviated pain of sciatic endometriosis as evidenced by the increase in paw withdrawal threshold and paw withdrawal latency. Sulforaphane also inhibited ectopic endometrial tissue growth in sciatic endometriosis rat, shown as the shrinkage of lesion size and decreased VEGF levels. IL6, IL-1ß and TNF-α levels were decreased by sulforaphane. Sulforaphane induced DOX2 and INOS suppression and Keap1 and Nrf2 upregulation. Sulforaphane alleviates pain induced by sciatic endometriosis, which is mediated by inhibiting inflammation.

Ginsenoside Rf alleviates dysmenorrhea and inflammation through the BDNF-TrkB-CREB pathway in a rat model of endometriosis.

To investigate the effects and the underlying mechanisms of ginsenoside Rf in a surgically induced rat endometriosis model, endometriosis was constructed through homologous transplantation and the Wistar rats were further randomly classified into the sham group, the estradiol valerate (E2V) control group, the endometriosis group, and the ginsenoside Rf groups (1.0, 2.0 and 4.0 mg kg-1, respectively). After 7 days of treatment, the implant volume and writhing responses were recorded. Vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF)-α were analyzed using enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR) assay. Brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinases (TrkB), and phosphate-c-AMP-responsive element binding protein (pCREB) were further measured. Compared with the endometriosis group, ginsenoside Rf could decrease the volume of the endometriotic implants and writhing responses. Furthermore, the expression levels of VEGF and inflammation-related iNOS, IL-6, IL-1ß, and TNF-α were significantly down-regulated in the ginsenoside Rf groups in a dose-dependent manner. The results also showed that ginsenoside Rf could decrease the expression of BDNF, TrkB, and pCREB in the endometriotic implants. The alleviation of endometriosis-associated dysmenorrhea and inflammation by ginsenoside Rf may be partially mediated by the BDNF-TrkB-CREB pathway.

18F-FDG-PET-based Radiomics signature predicts MGMT promoter methylation status in primary diffuse glioma.

BACKGROUND: The methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) promoter has emerged as a favorable independent prognostic and predictive biomarker in glioma. This study aimed to build a radiomics signature based on 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) for noninvasive measurement of the MGMT promoter methylation status in glioma. METHODS: One hundred and seven pathology-confirmed primary diffuse glioma patients were retrospectively included and randomly assigned to the primary (n = 71) or validation cohort (n = 36). The MGMT promoter methylation status was measured by pyrosequencing. A total of 1561 radiomics features were extracted from the three-dimensional region of interest (ROI) on the standard uptake value (SUV) maps that were generated from the original 18F-FDG PET data. A radiomics signature, a clinical signature and a fusion signature that combined the clinical and radiomics features together were generated. The performance of the three signatures was evaluated by receiver operating characteristic (ROC) curve analysis, and the patient prognosis was stratified based on the MGMT promoter methylation status and the signature with the best performance. RESULTS: Five radiomics features were selected to construct the radiomics signature, and displayed the best performance with area under the receiver operating characteristic (ROC) curve (AUC) reaching 0.94 and 0.86 in the primary and validation cohorts, respectively, which outweigh the performances of clinical signature and fusion signature. With a median follow-up time of 32.4 months, the radiomics signature stratified the glioma patients into two risk groups with significantly different prognoses (p = 0.04). CONCLUSIONS: 18F-FDG-PET-based radiomics is a promising approach for preoperatively evaluating the MGMT promoter methylation status in glioma and predicting the prognosis of glioma patients noninvasively.

Crocin improves endometriosis by inhibiting cell proliferation and the release of inflammatory factors.

Endometriosis is one of the most common diseases in women. Inflammation and angiogenesis have been shown to be important in pathogenesis of endometriosis. Crocin is known as an anti-inflammatory, anti- proliferation substance. This study was designed to assess the potential effects of crocin on endometriosis. We established the mice model of endometriosis and administrated crocin to the mice. We monitored the endometriotic lesion growth, PCNA and VEGF expression in the lesion. We tested the serum levels of inflammatory cytokines in crocin-treated endometriosis mice. Finally we tested the effect of crocin on endothelial cell apoptosis and proliferation, and cytokine production in LPS-stimulated human monocyte. Crocin inhibited lesion growth in endometriosis mice and prevented PCNA and VEGF expression in the lesions. Crocin decreased the levels of inflammatory cytokines including INF-γ, TNF-α, VEGF and IL-6 in serum. Crocin inhibited endothelial cells proliferation but did not cause apoptosis in endothelia cells. Crocin inhibited cytokine production in LPS-stimulated THP-1 cells in vitro. Crocin protected endometriosis by inhibiting endothelial cells proliferation and preventing inflammatory cytokines production.

MiR-96 enhances cellular proliferation and tumorigenicity of human cervical carcinoma cells through PTPN9.

Up to date, the cervical cancer remains to be one of the leading gynecological malignancies worldwide. MicroRNAs (miRNAs) play critical roles in the process of tumor initiation and progression. However, miR-96 has rarely been investigated in human cervical carcinoma. We aimed to investigate the biological function and underlying molecular mechanism of miR-96 in human cervical carcinoma. MiR-96 levels were determined by qRT-PCR. Protein tyrosine phosphatase, non-receptor type 9 (PTPN9) mRNA and protein levels were investigated by qRT-PCR and western blotting. The cellular proliferation in cervical cells was monitored by CyQuant assay. Soft agar assay was employed to determine the tumorigenicity. 3' UTR luciferase assay was used to validate the target gene of miR-96. SPSS was used to analyze statistical significance in different treatment. MiR-96 was dramatically upregulated in human cervical tumor tissues. Overexpression of miR-96 was found to significantly promote the cellular proliferation and tumorigenicity of cervical cells. Furthermore, we showed that PTPN9 was a direct target gene of miR-96 and had opposite effect to those of miR-96 on cervical cells. MiR-96 may promote the cellular proliferation and tumorigenicity of cervical cells by silencing PTPN9. Our study highlights an importantly regulatory role of miR-96 and suggests that an appropriate manipulation of miR-96 may be a new treatment of human cervical carcinoma in the future.

Somatic FGFR3 Mutations Distinguish a Subgroup of Muscle-Invasive Bladder Cancers with Response to Neoadjuvant Chemotherapy.

The administration of neoadjuvant chemotherapy (NAC) preceding radical cystectomy benefits overall survival for patients with muscle-invasive bladder cancer (MIBC). However, the relationship between the genetic profiling of MIBC and NAC response remains unclear. Here, a mutation panel of six cancer-associated genes (TSC1, FGFR3, TERT, TP53, PIK3CA and ERBB2) and an immunohistochemistry (IHC) panel containing eight bladder cancer (BC) biomarkers (EGFR, RRM1, PD-L1, BRCA1, TUBB3, ERCC, ERCC1, aberrantly glycosylated integrin α3ß1 (AG) and CK5/6) were developed. BC samples from patients who showed a pathologic response (n = 39) and non-response (n = 13) were applied to the panel analysis. ERBB2, FGFR3 and PIK3CA exclusively altered in the responders group (19/39, 48.7%), in which FGFR3 mutations were significantly enriched in patients with a response in the cohort (14/39, 35.9%; P = 0.01). Additionally, strong expression of ERCC1 was associated with a pathologic response (P = 0.01). However, positive lymph node metastasis (P < 0.01) and lymph-vascular invasion (LVI) (P = 0.03) were correlated with a non-response. Overall, the data show that FGFR3 mutations and elevated expression of ERCC1 in MIBCs are potential predictive biomarkers of the response to NAC.

Feature study of hysterical blindness EEG based on FastICA with combined-channel information.

BACKGROUND: An appropriate feature study of hysteria electroencephalograms (EEG) would provide new insights into neural mechanisms of the disease, and also make improvements in patient diagnosis and management. OBJECTIVE: The objective of this paper is to provide an explanation for what causes a particular visual loss, by associating the features of hysterical blindness EEG with brain function. METHODS: An idea for the novel feature extraction for hysterical blindness EEG, utilizing combined-channel information, was applied in this paper. After channels had been combined, the sliding-window-FastICA was applied to process the combined normal EEG and hysteria EEG, respectively. Kurtosis features were calculated from the processed signals. As the comparison feature, the power spectral density of normal and hysteria EEG were computed. RESULTS: According to the feature analysis results, a region of brain dysfunction was located at the occipital lobe, O1 and O2. Furthermore, new abnormality was found at the parietal lobe, C3, C4, P3, and P4, that provided us with a new perspective for understanding hysterical blindness. CONCLUSIONS: Indicated by the kurtosis results which were consistent with brain function and the clinical diagnosis, our method was found to be a useful tool to capture features in hysterical blindness EEG.