New insights into the dihydro-mureidomycin biosynthesis controlled by two unusual proteins in Streptomyces roseosporus.

BACKGROUND: Uridyl peptide compounds are renowned as a subclass of nucleoside antibiotics for their highly specific antibacterial activity against Gram-negative bacteria and the unique target of action. We previously activated the biosynthetic gene cluster of a uridyl peptide antibiotic, mureidomycin, in Streptomyces roseosporus NRRL 15998 by introducing an exogenous positive regulator gene ssaA, and the generated strain was designated as Sr-hA. This study aims to further explore mureidomycin analogs from Sr-hA as well as the collaborative roles of two wide-spread genes, SSGG-02980 and SSGG-03002 encoding putative nuclease/phosphatase and oxidoreductase respectively, in mureidomycin diversification. RESULTS: In order to understand how SSGG-02980 and SSGG-03002 contribute to mureidomycin biosynthesis, the gene disruption mutants and complementary strains were constructed. Mass spectrometry analyses revealed that two series of pairwise mureidomycin analogs were synthesized in Sr-hA with a two-dalton difference in molecular weight for each pair. By disruption of SSGG-03002, only mureidomycins with lower molecular weight (MRDs, 1-6) could be specifically accumulated in the mutant (∆03002-hA), whereas the other series of products with molecular weight plus 2 Da (rMRDs, 1'-6') became dominant in SSGG-02980 disruption mutant (∆02980-hA). Further comprehensive NMR analyses were performed to elucidate the structures, and three MRDs (3, 4, 5) with unsaturated double bond at C5-C6 of uracil group were characterized from ∆03002-hA. In contrast, the paired rMRDs analogs (3', 4', 5') from ∆SSGG-02980 corresponding to 3, 4 and 5 were shown to contain a single bond at this position. The results verified that SSGG-03002 participates in the reduction of uracil ring, whereas SSGG-02980 antagonizes the effect of SSGG-03002, which has been rarely recognized for a phosphatase. CONCLUSIONS: Overall, this study revealed the key roles of two wide-spread families of enzymes in Streptomyces. Of them, oxidoreductase, SSGG-03002, is involved in dihydro-mureidomycin biosynthesis of S. roseosporus, whereas nuclease/phosphatase, SSGG-02980, has an adverse effect on SSGG-03002. This kind of unusual regulation model between nuclease/phosphatase and oxidoreductase is unprecedented, providing new insights into the biosynthesis of mureidomycins in Streptomyces. The findings would be of significance for structural diversification of more uridyl peptide antibiotics against Gram-negative bacteria.

iNOS aggravates pressure overload-induced cardiac dysfunction via activation of the cytosolic-mtDNA-mediated cGAS-STING pathway.

Background: Sterile inflammation contributes to the pathogenesis of cardiac dysfunction caused by various conditions including pressure overload in hypertension. Mitochondrial DNA (mtDNA) released from damaged mitochondria has been implicated in cardiac inflammation. However, the upstream mechanisms governing mtDNA release and how mtDNA activates sterile inflammation in pressure-overloaded hearts remain largely unknown. Here, we investigated the role of inducible NO synthase (iNOS) on pressure overload-induced cytosolic accumulation of mtDNA and whether mtDNA activated inflammation through the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. Methods: To investigate whether the cGAS-STING cascade was involved in sterile inflammation and cardiac dysfunction upon pressure overload, cardiomyocyte-specific STING depletion mice and mice injected with adeno-associated virus-9 (AAV-9) to suppress the cGAS-STING cascade in the heart were subjected to transverse aortic constriction (TAC). iNOS null mice were used to determine the role of iNOS in cGAS-STING pathway activation in pressure-stressed hearts. Results: iNOS knockout abrogated mtDNA release and alleviated cardiac sterile inflammation resulting in improved cardiac function. Conversely, activating the cGAS-STING pathway blunted the protective effects of iNOS knockout. Moreover, iNOS activated the cGAS-STING pathway in isolated myocytes and this was prevented by depleting cytosolic mtDNA. In addition, disruption of the cGAS-STING pathway suppressed inflammatory cytokine transcription and modulated M1/M2 macrophage polarization, and thus mitigated cardiac remodeling and improved heart function. Finally, increased iNOS expression along with cytosolic mtDNA accumulation and cGAS-STING activation were also seen in human hypertensive hearts. Conclusion: Our findings demonstrate that mtDNA is released into the cytosol and triggers sterile inflammation through the cGAS-STING pathway leading to cardiac dysfunction after pressure overload. iNOS controls mtDNA release and subsequent cGAS activation in pressure-stressed hearts.

Evaluation of parametrial infiltration in patients with IB-IIB cervical cancer by a radiomics model integrating features from tumoral and peritumoral regions in 18 F-fluorodeoxy glucose positron emission tomography/MR images.

Parametrial infiltration (PMI) is an essential factor in staging and planning treatment of cervical cancer. The purpose of this study was to develop a radiomics model for accessing PMI in patients with IB-IIB cervical cancer using features from 18 F-fluorodeoxy glucose (18 F-FDG) positron emission tomography (PET)/MR images. In this retrospective study, 66 patients with International Federation of Gynecology and Obstetrics stage IB-IIB cervical cancer (22 with PMI and 44 without PMI) who underwent 18 F-FDG PET/MRI were divided into a training dataset (n = 46) and a testing dataset (n = 20). Features were extracted from both the tumoral and peritumoral regions in 18 F-FDG PET/MR images. Single-modality and multimodality radiomics models were developed with random forest to predict PMI. The performance of the models was evaluated with F1 score, accuracy, and area under the curve (AUC). The Kappa test was used to observe the differences between PMI evaluated by radiomics-based models and pathological results. The intraclass correlation coefficient for features extracted from each region of interest (ROI) was measured. Three-fold crossvalidation was conducted to confirm the diagnostic ability of the features. The radiomics models developed by features from the tumoral region in T2 -weighted images (F1 score = 0.400, accuracy = 0.700, AUC = 0.708, Kappa = 0.211, p = 0.329) and the peritumoral region in PET images (F1 score = 0.533, accuracy = 0.650, AUC = 0.714, Kappa = 0.271, p = 0.202) achieved the best performances in the testing dataset among the four single-ROI radiomics models. The combined model using features from the tumoral region in T2 -weighted images and the peritumoral region in PET images achieved the best performance (F1 score = 0.727, accuracy = 0.850, AUC = 0.774, Kappa = 0.625, p < 0.05). The results suggest that 18 F-FDG PET/MRI can provide complementary information regarding cervical cancer. The radiomics-based method integrating features from the tumoral and peritumoral regions in 18 F-FDG PET/MR images gave a superior performance for evaluating PMI.

The tRNA-Cys-GCA Derived tsRNAs Suppress Tumor Progression of Gliomas via Regulating VAV2.

The tsRNAs (tRNA-derived small RNAs) are new types of small noncoding RNAs derived from tRNAs. Gliomas are well-known malignant brain tumors. The study focused on tsRNA characterizations within gliomas. Datasets processing, bioinformatics analyses, and visualizations were performed with the packages of Python and R. Cell proliferations were demonstrated via CCK8 assays and colony formation assays, and in vivo xenograft experiments. Dual-luciferase reporter assay was performed to confirm the binding of tsRNA with its targets. Via using bioinformatics approaches, the hundreds of tsRNAs with available expression abundance were identified in gliomas dataset, most of them derived from D-loop or T-loop fragments of tRNAs. Among tsRNAs derived from tRNA-Cys-GCA, tRFdb-3003a and tRFdb-3003b (tRFdb-3003a/b) were remarkably down-regulated in gliomas. The survival outcome of gliomas patients with low tRFdb-3003a/b expressions was notably worse than that of high-expression patients. In glioma cells, tRFdb-3003a could suppress cells proliferation and colony formation ability. In vivo, tRFdb-3003a suppressed the tumor growth of xenograft gliomas. Enrichment analyses displayed the tRFdb-3003a-related mRNAs were enriched in the specific GO terms, spliceosome and autophagy pathways, and three GSEA molecular signatures. Mechanically, 3'-UTR regions of VAV2 mRNA were predicted to contain the binding positions of tRFdb-3003a/b, tRFdb-3003a and tRFdb-3003b was effective to reduce the relative luciferase activity of cells with VAV2 wild-type reporter. Overexpression of tRFdb-3003a/b could down-regulated the expression levels of VAV2 protein and mRNA in glioma cells. The tRNA-Cys-GCA derived tRFdb-3003a and tRFdb-3003b might act as key player in tumor progressions of gliomas; tRFdb-3003a/b might directly bind to VAV2 and regulate VAV2 expressions in gliomas.

Multiplexed immunofluorescence identifies high stromal CD68+PD-L1+ macrophages as a predictor of improved survival in triple negative breast cancer.

Triple negative breast cancer (TNBC) comprises 10-15% of all breast cancers and has a poor prognosis with a high risk of recurrence within 5 years. PD-L1 is an important biomarker for patient selection for immunotherapy but its cellular expression and co-localization within the tumour immune microenvironment and associated prognostic value is not well defined. We aimed to characterise the phenotypes of immune cells expressing PD-L1 and determine their association with overall survival (OS) and breast cancer-specific survival (BCSS). Using tissue microarrays from a retrospective cohort of TNBC patients from St George Hospital, Sydney (n = 244), multiplexed immunofluorescence (mIF) was used to assess staining for CD3, CD8, CD20, CD68, PD-1, PD-L1, FOXP3 and pan-cytokeratin on the Vectra Polaris™ platform and analysed using QuPath. Cox multivariate analyses showed high CD68+PD-L1+ stromal cell counts were associated with improved prognosis for OS (HR 0.56, 95% CI 0.33-0.95, p = 0.030) and BCSS (HR 0.47, 95% CI 0.25-0.88, p = 0.018) in the whole cohort and in patients receiving chemotherapy, improving incrementally upon the predictive value of PD-L1+ alone for BCSS. These data suggest that CD68+PD-L1+ status can provide clinically useful prognostic information to identify sub-groups of patients with good or poor prognosis and guide treatment decisions in TNBC.

LsrR, the effector of AI-2 quorum sensing, is vital for the H2O2 stress response in mammary pathogenic Escherichia coli.

Mammary pathogenic Escherichia coli (MPEC) is an important causative agent of mastitis in dairy cows that results in reduced milk quality and production, and is responsible for severe economic losses in the dairy industry worldwide. Oxidative stress, as an imbalance between reactive oxygen species (ROS) and antioxidants, is a stress factor that is common in most bacterial habitats. The presence of ROS can damage cellular sites, including iron-sulfur clusters, cysteine and methionine protein residues, and DNA, and may cause bacterial cell death. Previous studies have reported that Autoinducer 2 (AI-2) can regulate E. coli antibiotic resistance and pathogenicity by mediating the intracellular receptor protein LsrR. This study explored the regulatory mechanism of LsrR on the H2O2 stress response in MPEC, showing that the transcript levels of lsrR significantly decreased under H2O2 stress conditions. The survival cell count of lsrR mutant XW10/pSTV28 was increased about 3080-fold when compared with that of the wild-type WT/pSTV28 in the presence of H2O2 and overexpression of lsrR (XW10/pUClsrR) resulted in a decrease in bacterial survival rates under these conditions. The ß-galactosidase reporter assays showed that mutation of lsrR led to a remarkable increase in expression of the promoters of ahpCF, katG and oxyR, while lsrR-overexpressing significantly reduced the expression of ahpCF and katG. The electrophoretic mobility shift assays confirmed that LsrR could directly bind to the promoter regions of ahpCF and katG. These results revealed the important role played by LsrR in the oxidative stress response of MPEC.

Prevalence and modifiable risk factors of degenerative valvular heart disease among elderly population in southern China.

OBJECTIVE: To investigate the prevalence and modifiable risk factors of degenerative valvular heart disease (DVHD) among elderly population in southern China. METHODS: A stratified multistage sampling method was used to recruit subjects. The contents of the survey included the questionnaire, laboratory examination, echocardiography, and other auxiliary examinations. The possible risk factors of DVHD were analyzed by logistic regression analysis. RESULTS: A total of 3538 subjects ≥ 65 years of age were enrolled. One thousand three hundred and seven subjects (36.9%) were diagnosed with DVHD. Degenerative was the most common etiology of VHD. Prevalence of DVHD increased with advancing age. The prevalence of DVHD differed by living region (χ 2 = 45.594, P < 0.001), educational level ( χ 2 = 50.557, P < 0.001), and occupation ( χ 2 = 36.961, P < 0.001). Risk factors associated with DVHD included age (two-fold increased risk for each 10-year increase in age), elevated level C-reactive protein (OR = 1.346, 95% CI: 1.100-1.646), elevated level low density lipoprotein (OR = 1.243, 95% CI: 1.064-1.451), coronary artery disease (OR = 1.651, 95% CI: 1.085-2.513), smoking (OR = 1.341, 95% CI: 1.132-1.589), and hypertension (OR = 1.414, 95% CI: 1.221-1.638). Other significant risk factors included reduced or elevated level red blood cell (OR = 1.347, 95% CI: 1.031-1.761; OR = 1.599, 95% CI: 1.097-2.331; respectively), elevated level platelets (OR = 1.891, 95% CI: 1.118-3.198), elevated level uric acid (OR = 1.282, 95% CI: 1.112-1.479), and stroke (OR: 1.738, 95% CI = 1.085-2.513). CONCLUSIONS: The survey characterized the baseline conditions of DVHD cohort of elderly population in Guangzhou city. The established and emerging risk factors for DVHD may represent challenges and opportunities for therapy.

Tubeimoside I promotes angiogenesis via activation of eNOS-VEGF signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Tubeimoside I (TBM) is a triterpenoid saponin purified from tubeimu (tuber of Bolbostemma paniculatum (Maxim.) Franquet). In traditional Chinese medicine, tubeimu had been used to treat acute mastitis, snake bites, detoxication, inflammatory diseases, and tumors for over 1000 years. AIM OF THE STUDY: This study aimed to investigate whether TBM could promote angiogenesis and how to promote angiogenesis. MATERIALS AND METHODS: In vivo, the pro-angiogenic effects of TBM were examined using the hindlimb ischemia model. After the ischemia operation, 1 mg/kg/day TBM was given via intraperitoneal injection for 28 days and the recovery of blood flow was monitored by Doppler scanner every 7 days. The capillary density in gastrocnemius muscle was detected by immunofluorescence. Expression of related proteins were determined by western blotting. In vitro, the pro-angiogenic effects of TBM on HUVECs were examined by Cell Counting Kit-8, scratch assay, endothelial cell tube formation assay and western blotting. RESULTS: TBM improved recovery from hindlimb ischemia in C57BL/6 mice. TBM promoted endothelial cell viability, migration and tube formation in HUVECs. TBM could activate eNOS-VEGF signaling pathway by enhancing expression of eNOS. And TBM's pro-angiogenesis effects could be abolished by L-NAME (an inhibitor of eNOS). CONCLUSIONS: TBM promoted angiogenesis via the activation of eNOS-VEGF signaling pathway and TBM could be a novel agent for therapeutic angiogenesis in ischemic diseases.

TILs Immunophenotype in Breast Cancer Predicts Local Failure and Overall Survival: Analysis in a Large Radiotherapy Trial with Long-Term Follow-Up.

AIM: To determine the prognostic significance of the immunophenotype of tumour-infiltrating lymphocytes (TILs) within a cohort of breast cancer patients with long-term follow-up. METHODS: Multiplexed immunofluorescence and automated image analysis were used to assess the expression of CD3, CD8, CD20, CD68, Fox P3, PD-1 and PD-L1 in a clinical trial of local excision and radiotherapy randomised to a cavity boost or not (n = 485, median follow-up 16 years). Kaplan-Meier and Cox multivariate analysis (MVA) methodology were used to ascertain relationships with local recurrence (LR), overall survival (OS) and disease-free survival (DFS). NanoString BC360 gene expression panel was applied to a subset of luminal patients to identify pathways associated with LR. RESULTS: LR was predicted by low CD8 in MVA in the whole cohort (HR 2.34, CI 1.4-4.02, p = 0.002) and luminal tumours (HR 2.19, CI 1.23-3.92, p = 0.008) with associations with increased stromal components, decreased Tregs (FoxP3), inflammatory chemokines and SOX2. Poor OS was associated with low CD20 in the whole cohort (HR 1.73, CI 1.2-2.4, p = 0.002) and luminal tumours on MVA and low PD-L1 in triple-negative cancer (HR 3.44, CI 1.5-7, p = 0.003). CONCLUSIONS: Immunophenotype adds further prognostic data to help further stratify risk of LR and OS even in TILs low-luminal tumours.

Progesterone promotes endothelial nitric oxide synthase expression through enhancing nuclear progesterone receptor-SP-1 formation.

Progesterone exerts antihypertensive actions partially by modulating endothelial nitric oxide synthase (eNOS) activity. Here, we aimed to investigate the effects and mechanisms of progesterone on eNOS expression. First, human umbilical vein endothelial cells (HUVECs) were exposed to progesterone and then the eNOS transcription factor specificity protein-1 (SP-1) and progesterone receptor (PRA/B) expression were assessed by Western blotting and qRT-PCR. The interaction between SP-1 and PRA/B was next determined through coimmunoprecipitation assay. The chromatin immunoprecipitation assay and luciferase assay were used to investigate the relationship of PRA/B, SP-1, and eNOS promoter. At last, rats were intraperitoneally injected with progesterone receptor antagonist RU-486, and then the expression of eNOS and vasodilation function in thoracic aorta and mesenteric artery were measured. The results showed that progesterone could increase eNOS expression in HUVECs. Further study showed that progesterone increased PRA-SP-1 complex formation and facilitated PRA/B and SP-1 binding to eNOS promoter. Mutating SP-1 or PR-binding motif on eNOS promoter abolished the effect of progesterone on eNOS gene transcription. We also observed that progesterone receptor antagonist RU-486 reduced eNOS expression and impaired vasodilation in rats. Those results suggest that progesterone modulates eNOS expression through promoting PRA-SP-1 complex formation, and progesterone antagonist attenuates eNOS expression, leading to the loss of vascular relaxation.NEW & NOTEWORTHY Progesterone directly upregulated endothelial nitric oxide synthase (eNOS) expression in human endothelial cells. Progesterone augmented eNOS promoter activity through a progesterone receptor A- and specificity protein-1-dependent manner. Antagonism of the progesterone receptor reduced eNOS expression and impaired vasodilation in rats.

Significance of Single-Nucleotide Variants in Long Intergenic Non-protein Coding RNAs.

Single-nucleotide variants (SNVs) are the most common genetic variants and universally present in the human genome. Genome-wide association studies (GWASs) have identified a great number of disease or trait-associated variants, many of which are located in non-coding regions. Long intergenic non-protein coding RNAs (lincRNAs) are the major subtype of long non-coding RNAs; lincRNAs play crucial roles in various disorders and cellular models via multiple mechanisms. With rapid growth in the number of the identified lincRNAs and genetic variants, there is great demand for an investigation of SNVs in lincRNAs. Hence, in this article, we mainly summarize the significant role of SNVs within human lincRNA regions. Some pivotal variants may serve as risk factors for the development of various disorders, especially cancer. They may also act as important regulatory signatures involved in the modulation of lincRNAs in a tissue- or disorder-specific manner. An increasing number of researches indicate that lincRNA variants would potentially provide additional options for genetic testing and disease risk assessment in the personalized medicine era.

CircRNA_0001449 disturbs phosphatidylinositol homeostasis and AKT activity by enhancing Osbpl5 translation in transient cerebral ischemia.

Phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] is a phosphorylated derivative of phosphatidylinositol 4-phosphate [PI(4)P] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], which recruit and activate AKT in the plasma membrane (PM) to promote cellular survival. ORP5 anchors at the endoplasmic reticulum (ER)-PM contact sites and acts as a PI(4)P and PI(4,5)P2/phosphatidylserine (PS) exchanger. Here, a lipidomics analysis of the sensorimotor cortex revealed that transient middle cerebral artery occlusion (tMCAO) disturbs the homeostasis of phosphatidylinositols (PIs) and PS between the PM and ER. Conditional knockout mice showed that ORP5 contributes to this abnormal distribution. Abolishing the ORP5 gene significantly inhibited apoptosis and autophagy. RNA sequencing and RNA pull down analyses confirmed a competing endogenous RNA pathway in which circ_0001449 sponges miR-124-3p and miR-32-5p to promote Osbpl5 translation. Our data showed that circRNA_0001449 regulates membrane homeostasis via ORP5 and is involved in the AKT survival pathway.

Caveolin-1 regulates the expression of miR-183 and inhibits the invasion and migration of invasive pituitary adenomas by affecting early growth response 1 (EGR1)/Krueppel-like factor 5 (KLF5) interaction.

BACKGROUND: This study aims to investigate the mechanism through which Caveolin-1 (CAV-1) regulates the expression of micro ribonucleic acid (miR)-183 in invasive pituitary adenoma (IPA) tissues and GH3 cells, and explore the effects of CAV-1 and miR-183 on the invasion and migration ability of GH3 cells. METHODS: Western blotting was used to detect the expression level of CAV-1, early growth response 1 (EGR1) and Krueppel-like factor 5 (KLF5). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression of miR-183. The mechanisms of interaction between CAV-1, EGR1, and KLF5 were studied by immunoprecipitation experiments. Transwell and cell scratch tests were used to determine the invasion and migration ability of GH3 cells. The dual-luciferase reporter gene experiment was used to detect the effects of EGR1 and KLF5 on miR-183 luciferase activity and verify the targeting relationship between miR-183 and ezrin. RESULTS: The expression of CAV-1 was up-regulated. However, following the knockdown of CAV-1, the invasion and migration ability of GH3 cells was significantly inhibited (P<0.05). The expression of miR-183 was down-regulated, but the expression level of miR-183 was markedly increased following the knockdown of CAV-1 (P<0.05). The knockdown of CAV-1 inhibited the nuclear ectopic of the EGR1 protein in GH3 cells. At the same time, the interaction between EGR1 and KLF5 in GH3 cells was significantly inhibited (P<0.05). The luciferase activity of miR-183 increased significantly after overexpression of KLF5 while overexpression of EGR1 and KLF5 had no significant effect on intracellular luciferase activity. Overexpression of miR-183 markedly inhibited the luciferase activity of wild-type EZR and the expression of the EZR protein in GH3 cells. Furthermore, the overexpression of miR-183 or the inhibition of EZR can reduce the invasion and migration ability of GH3 cells. The simultaneous overexpression or inhibition of miR-183 and EZR expression has no obvious effect on the invasion and migration ability of GH3 cells. CONCLUSIONS: CAV-1 up-regulates the expression of miR-183 by inhibiting the nuclear ectopic of EGR1 and the interaction between EGR1 and KLF5 in GH3 cells. Also, miR-183 negatively regulates the expression of EZR and inhibits the invasion and migration of GH3 cells.

Crystal Structure of Aeromonas hydrophila Cytoplasmic 5'-Methylthioadenosine/S-Adenosylhomocysteine Nucleosidase.

5'-Methylthioadenosine/S-adenosyl-l-homocysteine (MTA/SAH) nucleosidase (MTAN) is an important enzyme in a number of critical biological processes. Mammals do not express MtaN, making this enzyme an attractive antibacterial drug target. In pathogen Aeromonas hydrophila, two MtnN subfamily genes (MtaN-1 and MtaN-2) play important roles in the periplasm and cytosol, respectively. We previously reported structural and functional analyses of MtaN-1, but little is known regarding MtaN-2 due to the lack of a crystal structure. Here, we determined the crystal structure of cytosolic A. hydrophila MtaN-2 in complex with adenine (ADE), which is a cleavage product of adenosine. AhMtaN-1 and AhMtaN-2 exhibit a high degree of similarity in the α-ß-α sandwich fold of the core structural motif. However, there is a structural difference in the nonconserved extended loop between ß7 and α3 that is associated with the channel depth of the substrate-binding pocket and dimerization. The ADE molecules in the substrate-binding pockets of AhMtaN-1 and AhMtaN-2 are stabilized with π-π stacking by Trp199 and Phe152, respectively, and the hydrophobic residues surrounding the ribose-binding sites differ. A structural comparison of AhMtaN-2 with other MtaN proteins showed that MtnN subfamily proteins exhibit a unique substrate-binding surface and dimerization interface.

High risk human papilloma viruses (HPVs) are present in benign prostate tissues before development of HPV associated prostate cancer.

BACKGROUND: Although high risk HPVs are associated with an increased risk of prostate cancer it is not known if they have a causal role. The purpose of this study is to investigate the potential role of human papilloma viruses (HPVs) in prostate cancer. The aims are (i) to investigate the presence and confirm the identity of high risk HPVs in benign prostate tissues prior to the development of HPV positive prostate cancer in the same patients, and (ii) to determine if HPVs are biologically active. METHODS: We used polymerase chain reaction (PCR) to identify HPVs in specimens from 52 Australian men with benign prostate biopsies who 1 to 10 years later developed prostate cancer. Immunohistochemistry (IHC) was used to assess the expression of HPV E7 oncoproteins, cytokeratin and prostate specific antigen (PSA). We used RNASeq data from The Cancer Genome Atlas (TCGA) to identify possible HPV RNA sequences in prostate cancer. RESULTS: HPV screening using standard PCR was conducted on 28 of the 52 sets of benign and later prostate cancers. HPV L1 genes were identified in 13 (46%) benign and 8 (29%) of 28 later prostate cancers in the same patients. HPV E7 genes were identified in 23 (82%) benign and 19 (68%) of 28 subsequent prostate cancers in the same patients. The same HPV types were present in both the benign and subsequent prostate cancers in 9 sets of specimens. HPV type 16 was identified in 15% of benign and 3% of prostate cancers. HPV type 18 was identified in 26% of benign and 16% of prostate cancers. Small numbers of HPV types 45, 47, 76 and 115 were also identified. High confidence RNA-Seq evidence for high risk HPV types 16 and 18 was identified in 12 (2%) of the 502 TCGA prostate cancer transcriptomes. High risk HPV E7 oncoprotein was positively expressed in 23 (82%) of 28 benign prostate specimens but only in 8 (29%) of 28 of the later prostate cancer specimens. This difference is statistically significant (p = 0.001). Prostate specific antigen (PSA) was more highly expressed in 26 (50%) of 52 prostate cancer specimens as compared to prior benign prostate specimens in the same patients. CONCLUSIONS: High risk HPVs are present in benign prostate tissues prior to the development of HPV positive prostate cancer. There is a significantly higher expression of HPV E7 oncoproteins in benign prostate tissues as compared to late prostate cancer that subsequently developed in the same patients. This observation suggests that HPV oncogenic activity is an early phenomenon in a majority of prostate oncogenesis. TCGA RNA-Seq data suggests that HPV is biologically active in some prostate tumour samples.

Role of Nicotinamide N-Methyltransferase in Dorsal Striatum in Cocaine Place Preference.

Nicotinamide N-methyltransferase (NNMT) transfers the methyl from S-adenosyl-L-methionine (SAM) to nicotinamide (NA) to produce S-adenosyl-L-homocysteine (SAH) and 1-methylnicotinamide (MeN). NNMT has been implicated in a variety of diseases; however, the role of NNMT in drug addiction is largely unknown. Here, we found that the expression of Nnmt was significantly upregulated in the dorsal striatum (DS) of cocaine-conditioned mice. Cocaine significantly decreased SAM/SAH ratio levels in the DS, which was accompanied with the decreased activities of Rac1 and RhoA. Lentivirus-mediated knockdown of Nnmt in the dorsomedial striatum (DMS) attenuated cocaine conditioned place preference (CPP) reward, but increased striatal SAM/SAH ratio levels as well as Rac1 and RhoA activities. In addition, pharmacological inhibition of NNMT through intra-DMS infusion of MeN attenuated cocaine CPP and the activities of Rac1 and RhoA, but increased SAM/SAH ratio. These results suggest that NNMT-dependent transmethylation is involved in the activation of Rac1 and RhoA, which utilize SAM as a methyl donor cofactor. Co-immunoprecipitation assay using a RhoGDIα antibody indirectly captured Rac1 or RhoA that were bound to RhoGDIα. The results showed that cocaine increased the association of RhoGDIα with Rac1 or RhoA, whereas such effect was inhibited by Nnmt knockdown. Collectively, our findings show that NNMT regulates cocaine CPP through SAM-mediated modification of Rac1 and RhoA.

Mechanisms of PDGF siRNA-mediated inhibition of bone cancer pain in the spinal cord.

Patients with tumors that metastasize to bone frequently suffer from debilitating pain, and effective therapies for treating bone cancer are lacking. This study employed a novel strategy in which herpes simplex virus (HSV) carrying a small interfering RNA (siRNA) targeting platelet-derived growth factor (PDGF) was used to alleviate bone cancer pain. HSV carrying PDGF siRNA was established and intrathecally injected into the cavum subarachnoidale of animals suffering from bone cancer pain and animals in the negative group. Sensory function was assessed by measuring thermal and mechanical hyperalgesia. The mechanism by which PDGF regulates pain was also investigated by comparing the differential expression of pPDGFRα/ß and phosphorylated ERK and AKT. Thermal and mechanical hyperalgesia developed in the rats with bone cancer pain, and these effects were accompanied by bone destruction in the tibia. Intrathecal injection of PDGF siRNA and morphine reversed thermal and mechanical hyperalgesia in rats with bone cancer pain. In addition, we observed attenuated astrocyte hypertrophy, down-regulated pPDGFRα/ß levels, reduced levels of the neurochemical SP, a reduction in CGRP fibers and changes in pERK/ERK and pAKT/AKT ratios. These results demonstrate that PDGF siRNA can effectively treat pain induced by bone cancer by blocking the AKT-ERK signaling pathway.

The anti-angiogenic effect of dexamethasone in a murine hepatocellular carcinoma model by augmentation of gluconeogenesis pathway in malignant cells.

PURPOSE: Angiogenesis is a long-term complex process involving various protein factors in hepatocellular carcinoma (HCC). Dexamethasone (Dex), considered as a synthetic glucocorticoid drug in clinical therapy, has been reported to have the therapeutic efficacy against liver cancer by intervention of abnormal glycolysis. In this study, we investigated the anti-angiogenic effect of Dex in murine liver cancer and attempted to demonstrate the potential mechanism. METHODS: The malignant cells H22 were treated with Dex. Western blotting was used to explore the expression of PEPCK and G6Pase which were the two key enzymes that regulated gluconeogenesis. The supernatants from cultured H22 treated by Dex were collected and co-cultured with HUVECs. In vitro, migration assay, transwell assay and tube formation assay were performed to assess for migration, proliferation and tube formation abilities of HUVECs, respectively. In situ murine hepatoma model with green fluorescent protein markers (HepG2-GFP) was constructed to determine angiogenesis after treatment by Dex. RESULTS: PEPCK and G6Pase were almost deficient in H22 compared with normal liver cells NCTC-1469 (P < 0.01). After treated by Dex, the gluconeogenesis could be restored significantly (P < 0.01) in H22 cells. The supernatant of H22 treated by Dex inhibited the migration, tube formation and endothelial permeability in HUVECs (P < 0.05). In mouse tissue, PEPCK and G6Pase were highly expressed in Dex group than control groups (P < 0.01). 11ß-HSDs abnormally expressed in tumor also could be restored by Dex. Meanwhile, the density and total length of microvessels in Dex-treated group were less than those in HCC groups (P < 0.05). CONCLUSIONS: This study explored the therapeutic efficacy of Dex in murine HCC. Dex might inhibit tumor growth and angiogenesis by augmenting the gluconeogenesis pathway.

Detecting and segmenting cell nuclei in two-dimensional microscopy images.

INTRODUCTION: Cell nuclei are important indicators of cellular processes and diseases. Segmentation is an essential stage in systems for quantitative analysis of nuclei extracted from microscopy images. Given the wide variety of nuclei appearance in different organs and staining procedures, a plethora of methods have been described in the literature to improve the segmentation accuracy and robustness. MATERIALS AND METHODS: In this paper, we propose an unsupervised method for cell nuclei detection and segmentation in two-dimensional microscopy images. The nuclei in the image are detected automatically using a matching-based method. Next, edge maps are generated at multiple image blurring levels followed by edge selection performed in polar space. The nuclei contours are refined iteratively in the constructed edge pyramid. The validation study was conducted over two cell nuclei datasets with manual labeling, including 25 hematoxylin and eosin-stained liver histopathology images and 35 Papanicolaou-stained thyroid images. RESULTS: The nuclei detection accuracy was measured by miss rate, and the segmentation accuracy was evaluated by two types of error metrics. Overall, the nuclei detection efficiency of the proposed method is similar to the supervised template matching method. In comparison to four existing state-of-the-art segmentation methods, the proposed method performed the best with average segmentation error 10.34% and 0.33 measured by area error rate and normalized sum of distances (×10). CONCLUSION: Quantitative analysis showed that the method is automatic and accurate when segmenting cell nuclei from microscopy images with noisy background and has the potential to be used in clinic settings.

Unique post-translational oxime formation in the biosynthesis of the azolemycin complex of novel ribosomal peptides from Streptomyces sp. FXJ1.264.

Streptomycetes are a rich source of bioactive specialized metabolites, including several examples of the rapidly growing class of ribosomally-biosynthesized and post-translationally-modified peptide (RiPP) natural products. Here we report the discovery from Streptomyces sp. FXJ1.264 of azolemycins A-D, a complex of novel linear azole-containing peptides incorporating a unique oxime functional group. Bioinformatics analysis of the Streptomyces sp. FXJ1.264 draft genome sequence identified a cluster of genes that was hypothesized to be responsible for elaboration of the azolemycins from a ribosomally-biosynthesized precursor. Inactivation of genes within this cluster abolished azolemycin production, consistent with this hypothesis. Moreover, mutants lacking the azmE and azmF genes accumulated azolemycin derivatives lacking the O-methyl groups and an amino group in place of the N-terminal oxime (as well as proteolysed derivatives), respectively. Thus AzmE, a putative S-adenosyl methionine-dependent methyl transferase, is responsible for late-stage O-methylation reactions in azolemycin biosynthesis and AzmF, a putative flavin-dependent monooxygenase, catalyzes oxidation of the N-terminal amino group in an azolemycin precursor to the corresponding oxime. To the best of our knowledge, oxime formation is a hitherto unknown posttranslational modification in RiPP biosynthesis.