[shRNA-Mediated Suppression of γ-Synuclein Leading to Downregulation of p38/ERK/JNK Phosphorylation and Cell Cycle Arrest in Endometrial Cancer Cells].

In this study, we explored the effects of treating human endometrial cancer cells with γ-synuclein-specific short hairpin RNA (shRNA) and elucidated the associated mechanisms in vitro and in vivo through the p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) signaling pathways. Cell proliferation and migration were assessed using CCK8, Transwell, and scratch wound healing assays. Flow cytometry and laser scanning confocal microscopy were used to detect cell cycle changes. Relative levels of phosphorylated and non-phosphorylated (p) p38, ERK1/2 and JNK1/2/3 were determined in vitro and in vivo using simple western blotting assays. Cell proliferation in the experimental group decreased significantly and cells transfected with shRNA showed reduced migration rates (P < 0.05). p-p38, p-ERK1/2, and p-JNK1/2/3 levels were downregulated in the experimental group in vitro and in vivo. Tumor volumes and weights in the experimental group were significantly lower (P < 0.05). Tumor formation time in the negative control group was significantly shorter (P < 0.05). Flow cytometry showed that the number of cells in the G1 and mitotic phases increased and that in the S phase decreased after SNCG silencing (P < 0.05). Confocal microscopy showed that the percentage of cells in the mitotic phase increased after SNCG gene silencing (P < 0.05). We conclude that shRNA-mediated suppression of γ-synuclein decreased the proliferation, migration, and tumorigenicity of endometrial cancer cells via downregulation of p38, ERK, and JNK phosphorylation. High SNCG expression is closely related to the growth cycle of endometrial cancer cells.

Blood-derived amyloid-ß protein induces Alzheimer's disease pathologies.

The amyloid-ß protein (Aß) protein plays a pivotal role in the pathogenesis of Alzheimer's disease (AD). It is believed that Aß deposited in the brain originates from the brain tissue itself. However, Aß is generated in both brain and peripheral tissues. Whether circulating Aß contributes to brain AD-type pathologies remains largely unknown. In this study, using a model of parabiosis between APPswe/PS1dE9 transgenic AD mice and their wild-type littermates, we observed that the human Aß originated from transgenic AD model mice entered the circulation and accumulated in the brains of wild-type mice, and formed cerebral amyloid angiopathy and Aß plaques after a 12-month period of parabiosis. AD-type pathologies related to the Aß accumulation including tau hyperphosphorylation, neurodegeneration, neuroinflammation and microhemorrhage were found in the brains of the parabiotic wild-type mice. More importantly, hippocampal CA1 long-term potentiation was markedly impaired in parabiotic wild-type mice. To the best of our knowledge, our study is the first to reveal that blood-derived Aß can enter the brain, form the Aß-related pathologies and induce functional deficits of neurons. Our study provides novel insight into AD pathogenesis and provides evidence that supports the development of therapies for AD by targeting Aß metabolism in both the brain and the periphery.

Heteronemin Induces Anti-Proliferation in Cholangiocarcinoma Cells via Inhibiting TGF-ß Pathway.

A marine sesterterpenoid-type natural product, heteronemin, retains anticancer effects. In the current study, we investigate the antitumor mechanism of heteronemin in cholangiocarcinoma cells and further explore its molecular targets. Initially, heteronemin exhibited potent cytotoxic effects against cholangiocarcinoma HuccT1 and SSP-25 cells. In vitro, heteronemin altered the abilities of cell adhesion and cell migration in HuccT1 and SSP-25 cell lines. It repressed messenger ribonucleic acid (mRNA) expression levels of transforming growth factor (TGF)-ß, mothers against decapentaplegic homolog (SMAD) and Myc, whose protein products play important roles in regulating cell growth, angiogenesis, and metastasis. In addition, heteronemin altered several signaling pathways. The results indicate that heteronemin was able to modulate cell adhesion, the expression of extracellular matrix (ECM) receptors, the TGF-ß pathway, cell motility, the membrane integration, metastasis response, matrix metalloproteinase (MMP) remodeling, the regulation of metabolism, sprouting angiogenesis, transcription factors, and vasculogenesis in cholangiocarcinoma cell lines. The results also suggest that it activated multiple signal transduction pathways to induce an anti-proliferation effect and anti-metastasis in cholangiocarcinoma. In conclusion, heteronemin may be used as a potential medicine for anticancer therapy.

A simulation impact evaluation of a cigarette excise tax increase on licit and illicit cigarette consumption and tax revenue in 36 European countries.

OBJECTIVES: To assess the impact of a simulated 10% tax-induced cigarette price increase on licit and illicit consumption and tax revenues in 36 European countries. METHODS: Employing panel data for licit and illicit cigarette consumption, fixed effects regression models were applied for different income clusters. RESULTS: Total cigarette consumption dropped by about 3.1% as a result of the simulated tax-induced price increase. Annual illicit cigarette consumption increased by 1.52%, (95% confidence interval: 0.21, 2.83), while annual licit cigarette consumption decreased by 4.61% (95% confidence interval: -6.51, -2.72) in the observed 36 European countries. With total consumption decreasing by about 8%, the Czech Republic, Latvia, Lithuania, Poland and Slovakia were affected the most by the price hike. More specifically, licit consumption in these countries decreased by 18.43% (95% confidence interval: -19.91, -16.95) while illicit use increased by 10.99% (95% confidence interval: 6.01, 15.96). Moreover, the overall annual tobacco tax revenue increased by US$14.69 billion in the simulation. CONCLUSION: Results of the study suggest that European policy makers continue to implement tobacco taxation policies to control smoking prevalence and national health care expenditures. At the same time, efforts to kerb contraband activities along EU Eastern borders should be intensified.

NUDT15 gene polymorphism related to mercaptopurine intolerance in Taiwan Chinese children with acute lymphoblastic leukemia.

A recent study identified a variant of the NUDT15 gene (rs116855232 C>T) associated with intolerance to thiopurine in Korean patients with Crohn's disease. This study prompted us to substantiate the finding in a Taiwanese population. Four hundred and four children with acute lymphoblastic leukemia (ALL), and 100 adults with chronic immune thrombocytopenic purpura or localized lymphoma having normal bone marrow were examined. Two candidate gene approaches, pyrosequencing for NUDT15 and TaqMan assay for thiopurine methyltransferase (TPMT) genotyping (rs1142345 A>G), were performed. We showed a risk allele frequency of NUDT15 of 11.6% in children with ALL and 15.5% in adults. By contrast, the risk allele frequency of TPMT was only 1.6% in children with ALL and 0.5% in adults. The high frequency of risk variant for NUDT15, but not the very low frequency of risk variant for TPMT, was closely associated with the intolerance to mercaptopurine in children with ALL in Taiwan, contrast to that of European descent. In regard to NUDT15 polymorphism, the maximal tolerable daily doses of mercaptopurine in homozygotes, heterozygotes and wild-type groups were 9.4 mg m-2, 30.7 mg m-2 and 44.1 mg m-2, respectively. The outcomes did not differ significantly among the different genotypes.

Factors associated with failure to achieve a glycated haemoglobin target of <8.0% in the Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial.

The aim of this study was to identify the clinical features of participants in the standard therapy arm of the Action to Control Cardiovascular Risk in Diabetes (ACCORD) glycaemia trial who failed to reach the glycated haemoglobin (HbA1c) target. We analysed 4685 participants in the standard therapy arm, comparing participants who reached the HbA1c target of <8.0% with those whose HbA1c level was ≥8.0% 12 months after randomization. Baseline and 12-month clinical characteristics were compared. At 12 months after randomization, 3194 participants had HbA1c <8.0% and 1491 had HbA1c ≥8.0%. Black race [odds ratio (OR) 0.74, 95% confidence interval (CI) 0.61-0.89; p = 0.002], severe hypoglycaemia (OR 0.57, CI 0.37-0.89; p = 0.014) and insulin use (OR 0.51, CI 0.40-0.65; p < 0.001) were associated with failure to reach HbA1c goal at 12 months in the adjusted model. Even with free medications, free visits with clinicians and aggressive titration of medications, >30% of participants in the standard arm of the ACCORD trial had an HbA1c ≥8.0% at 1 year. Participants who were black, had severe hypoglycaemia and were on insulin were more likely to have an above-target HbA1c concentration after 12 months on the standard protocol.

Tumor-specific suicide gene therapy for hepatocellular carcinoma by transcriptionally targeted retroviral replicating vectors.

Replicating virus vectors are attractive tools for anticancer gene therapy, but the potential for adverse events due to uncontrolled spread of the vectors has been a major concern. To design a tumor-specific retroviral replicating vector (RRV), we replaced the U3 region of the RRV ACE-GFP with a regulatory sequence consisting of the hepatitis B virus enhancer II (EII) and human α-fetoprotein (AFP) core promoter to produce ACE-GFP-EIIAFP, a hepatocellular carcinoma (HCC)-targeting RRV. Similar to ACE-GFP, ACE-GFP-EIIAFP exhibited robust green fluorescent protein (GFP) expression in HCC cells and, most importantly, it exhibited HCC-specific replication and did not replicate in non-HCC tumor cells or normal liver cells. We sequenced the promoter region of ACE-GFP-EIIAFP collected from serial infection cycles to examine the genomic stability of the vector during its replicative spread, and found that the vector could retain the hybrid promoter in the genome for at least six infection cycles. In vitro studies revealed that ACE-CD-EIIAFP and ACE-PNP-EIIAFP, which express the yeast cytosine deaminase and Escherichia coli purine nucleoside phosphorylase, respectively, exert a highly potent cytotoxic effect on HCC cells in the presence of their respective prodrugs. In vivo, ACE-CD-EIIAFP-mediated suicide gene therapy efficiently suppressed HCC tumor growth and no detectable RRV signal was observed in extratumoral tissues. These results suggest that the tumor-specific, suicide-gene-encoding RRV may fulfill the promise of retroviral gene therapy for cancer.

Further resolving the phylogeny of Myxogastria (slime molds) based on COI and SSU rRNA genes.

To date, molecular systematics of Myxogastria has been based primarily on small subunit ribosomal RNA (SSU rRNA) and elongation factor 1-alpha (EF-1α) genes. To establish a natural classification system for the organisms, we examined phylogenetic relationships among myxogastrian species using cytochrome c oxidase subunit I (COL) and SSU rRNA genes. Twenty new sequences were obtained, including 10 COI and 10 SSU rRNA sequences, were compared with sequences of related species from GenBank in order to construct phylogenic trees. The analysis of the two data sets supported the modern phylogeny of myxogastria: orders Liceida and Trichiida formed a sister group at the most basal clade, while orders Stemonitida and Physarida formed a close group, and order Echinostelida was a sister group to Stemonitida and Physarida. However, the partial COI sequences were too conserved to resolve of the branches in Stemonitida and Physarida. In addition, we also deemed the specific edited mRNA events of COI sequences in myxogastrian species.

MKL1-184C>T gene polymorphism is associated with coronary artery disease in the Chinese Han population.

We investigated genetic susceptibility to coronary artery disease (CAD) by studying the association of MKL1 gene polymorphisms with CAD in the Chinese Han population. We performed a case-control study with 476 unrelated CAD patients and 325 non-CAD controls. All SNPs were genotyped with a TaqMan SNP genotyping assay. The distribution of MKL1-184C>T gene polymorphism in each group was in Hardy-Weinberg equilibrium. The frequency of the MKL1 T allele in the CAD group was significantly higher than in the control group (38.6 vs 30.8%). After logistic regression models adjusted for CAD risk factors, the risk of CAD among CT genotypes was 1.765 times higher than among the CC genotypes [odds ration (OR) = 1.765, 95% confidence interval (CI) = 1.246-2.5], and for TT genotypes it was 1.806 times higher than for the CC genotypes (OR = 1.806, 95%CI = 1.203-2.71). In summary, genotypes with at least one T allele (CT or TT genotypes) had a significantly increased CAD risk than the CC genotypes, with a ratio of 1.78 to 1 (OR = 1.780, 95%CI = 1.311-2.418). There was a close association between -184 T allele and 3VD (OR = 1.614, 95%CI = 1.259-2.07, P < 0.05). We conclude that the -184C>T of MKL1 is an important susceptibility factor for CAD in the Han Chinese in Henan Province. Homozygosity for the T allele is not only associated with an increased risk for CAD, it is also correlated with severity of stenosis in the Chinese Han population.

Consumption of a Taiwanese cafeteria diet induces metabolic disorders and fecal flora changes in obese rats.

OBJECTIVES: Among diet-induced obesity animal models, the cafeteria diet, which contains human junk food and processed foods, is a popular experimental animal diets in Western countries. Consumption of a cafeteria diet can lead to the development of obesity and non-alcoholic liver disease in as soon as 2 mo, which more accurately reflects human eating patterns. The aim of this study was to establish a Taiwanese cafeteria diet and compare it with a traditional lard-based, 60% high-fat diet in a 12-wk animal model. METHODS: Six-wk-old male Wistar rats were assigned to the following three groups: control diet (C; LabDiet 5001); high-fat diet (HFD; 60% HFD); and the Taiwanese cafeteria diet (CAF). RESULTS: At the end of the study, weight gain and steatosis were observed in the HF and CAF groups. Compared with the HFD group, rats in the CAF group showed significantly higher plasma triacylglycerol concentrations and insulin resistance, which may have been correlated with increased inflammatory responses. Significantly lower hepatic sterol regulatory element-binding protein-1c and insulin receptor substrate-1 protein expressions were observed in the CAF group compared with the HFD group. Additionally, disruption of the microbiotic composition followed by increased obesity-related bacteria was observed in the CAF group. CONCLUSIONS: The present study confirmed that the Taiwanese cafeteria diet-induced rat model provided a potential platform for investigating obesity-related diseases.

Insulin resistance induced by zymosan as a new animal model in mice.

Insulin resistance (IR) is known as a main problem in diabetic disorders. Some animal models for research in IR have been mentioned. Each model shows merit with some disadvantages. Thus, a new animal model for IR is required. The present study used zymosan, a mixture of cell-wall particles from the yeast named Saccharomyces cerevisiae, to establish a new model of IR in mice. Also, we compared the difference of this model with fructose-rich chow-induced model and found some merits of this model. Moreover, we identified that this model induced by zymosan is reversible and IR can be reversed gradually after termination of treatment. Taken together, we suggest zymosan as a useful agent to induce IR through inflammatory pathway in mice.

Characterization of the specificity of imidazoline I-1 receptor antibody for subtype of imidazoline receptors in vitro.

Specific antibodies are essential in the study of receptor protein. Gene matching shows that Nischarin (NISCH) is a mouse homologue of human imidazoline receptor antisera-selective (IRAS) protein, a viable candidate for imidazoline I-1 receptor. However, selectivity of this antibody against imidazoline I-2 or imidazoline I-3 receptors remained obscure. At first, an intracerebroventricular (ICV) injection of anti-NISCH antibody blocked the blood pressure lowering action of rilmenidine (I-1 receptor agonist) in spontaneous hypertensive rat (SHR). However, the same injection of anti-NISCH antibody showed no effect in SHR treated with clonidine (α2 agonist). In order to clarify the selectivity of anti-NISCH antibody for each subtype of imidazoline receptors, this anti-NISCH antibody was subjected to the lysate of organs isolated from Wistar rats including cortex, hippocampus, cerebellum, and brain stem as central nervous tissues, and heart, liver, pancreas, skeletal muscle, kidney, prostate, and bladder as peripheral tissues. The results show that anti-NISCH antibody positively reacted with all tissues including heart, pancreas, skeletal muscle, kidney and bladder by Western blot analysis. Also, the blotting spots for anti-NISCH antibody show a concentration-dependent manner. Moreover, anti-NISCH antibody blocked the action of glucose uptake induced by 2-BFI (I-2 receptor agonist) in L6 cells. Taken together, the obtained data suggest that anti-NISCH antibody can be used not only for imidazoline I-1 receptor but also for I-2 and I-3 subtypes in immunoassays.

Low-temperature water dynamics in an aqueous methanol solution.

An aqueous methanol solution (x(MeOH) = 0.30) has been studied by quasielastic neutron scattering. The single-particle water dynamics were effectively isolated by employing deuterated methanol. A smooth dynamic transition to a sub-Arrhenius temperature dependence has been observed in the relaxation times. We associate this behavior with the formation of small crystallites in the system. These findings are compared with molecular dynamics simulations and previous nuclear magnetic resonance measurements. We discuss possible dynamic signatures of structuring in the mixture.

Noninvasive assessment of liver fibrosis via spleen stiffness measurement using acoustic radiation force impulse sonoelastography in patients with chronic hepatitis B or C.

Portal hypertension and splenomegaly are common in patients with cirrhosis. However, there is limited previous in vivo research on the correlation between spleen stiffness and stages of liver fibrosis. This study aimed to evaluate the diagnostic value of spleen stiffness measurement (SSM), using acoustic radiation force impulse (ARFI) technology, for liver fibrosis assessment. Eligible patients with chronic hepatitis B or C (n = 163) underwent concurrent liver stiffness measurement (LSM), SSM and percutaneous liver biopsy. Receiver operating characteristic curves estimated the diagnostic performance of SSM, with multiple linear regression models for LSM and SSM determining the significance of explanatory factors. Results indicated significant correlation between LSM and SSM (R(2) = 0.574, P < 0.0001). Using SSM to classify METAVIR fibrosis (METAVIR F) scores, the areas under curves were 0.839 (95% CI: 0.780-0.898) for METAVIR F1 vs F2-4, 0.936 (95% CI: 0.898-0.975) for F1-2 vs F3-4 and 0.932 (95% CI: 0.893-0.971) for F1-3 vs F4, all P < 0.001. Multiple linear regression models identified BMI, spleen stiffness, METAVIR F3 and F4, serum alanine aminotransferase, international normalized ratio of prothrombin time, sodium and platelet count as significant independent explanatory factors for liver stiffness (adjusted R(2) = 0.724, P < 0.001). Male gender, liver stiffness, METAVIR F2, F3 and F4 also significantly and independently explained spleen stiffness (adjusted R(2) = 0.647, P < 0.001). ARFI SSM is potentially useful as a single or adjunct predictor of stages of liver fibrosis.

Overexpression of zinc-finger protein 677 inhibits proliferation and invasion by and induces apoptosis in clear cell renal cell carcinoma.

Recent studies have demonstrated that zinc-finger protein 677 (ZNF677) acts as a tumor suppressor gene in cancer. However, the expression and function of ZNF677 in clear cell renal cell carcinoma (ccRCC) are still unclear. In this study, we used bioinformatics analysis and in vitro experiments to investigate the expression of ZNF677 in ccRCC tissues and the malignant biological behavior of ZNF677 in 786-0 cells. We demonstrated that ZNF677 is hypermethylated in ccRCC and is associated with clinicopathological features. The results of the functional assays indicate that ZNF677 inhibits tumor cell proliferation and invasion and induces apoptosis. Further prognostic analysis indicated that low expression of ZNF677 is associated with shorter overall survival. Additionally, ZNF677 overexpression suppressed the invasion and epithelial-mesenchymal transition of 786-0 cells by inactivating the PI3K/AKT signaling pathway. This is the first report to evaluate the influence of ZNF677 on ccRCC cells malignant biological behavior. The results indicate that high expression of ZNF677 could be considered as a favorable prognostic indicator for ccRCC.

T helper cells promote disease progression of osteoarthritis by inducing macrophage inflammatory protein-1γ.

OBJECTIVE: Immune cells are involved in the pathogenesis of osteoarthritis (OA). We examined the effects of T helper (Th) cells, which induce the expression of macrophage inflammatory protein (MIP-1γ), on the progression of OA. DESIGN: Using anterior cruciate ligament-transection (ACLT), we induced OA in one hind-leg knee joint of B6 mice. The CD4(+) T cells from splenocytes and synovium were flow-cytometrically and immunochemically evaluated, respectively. The knee joints were histologically assessed for manifestations of OA. MIP-1γ levels and nuclear factor-κB (NF-κB) in the knee joints were measured using enzyme-linked immunosorbent and immunoblotting assays, respectively; osteoclastogenesis was detected by tartrate-resistant acid phosphatase (TRAP) staining. The inflammatory responses and MIP-1γ expression were examined using immunohistochemistry. RESULTS: The number of CD4(+) T cells and the expression of interferon-γ (IFN-γ) increased during OA onset (30 days after ACLT) and then decreased at a later stage of OA (90 days after ACLT). Tissue damage induced by CD4(+) T cells was evident at the later stage. The activation of CD4(+) T cells induced the expression of MIP-1γ and NF-κB. The expression of MIP-1γ can be detected in synovium which CD4(+) T cells were infiltrated. The increased MIP-1γ expression caused an increase in the number of osteoclasts in joints. The regulation of CD4(+) T cells was accompanied by increased macrophage infiltration and matrix metalloproteinase (MMP)-9 expression. Histopathological examinations revealed that CD4(+) T cell knockout (CD4(-/-)) mice had less expression of MIP-1γ and slower cartilage degeneration than control mice had. CONCLUSIONS: CD4(+) T cells were activated during the onset of OA, but cartilage damage was more prominent at a later stage. CD4(+) T cells were involved in the pathogenesis of OA: they induced MIP-1γ expression and subsequent osteoclast formation.

The role of the dynamic crossover temperature and the arrest in glass-forming fluids.

We discuss the role of the dynamic glass-forming fragile-to-strong crossover (FSC) in supercooled liquids. In the FSC, significant dynamic changes such as the decoupling (the violation of the Stokes-Einstein relation) of homologous transport parameters, e.g., the density relaxation time τ and the viscosity η, occur at a characteristic temperature T(c). We study the FSC using a scaling law approach. In particular, we use both forms of the mode-coupling theory (MCT): the original (ideal) and the extended form, which explicitly describes energy hopping processes. We demonstrate that T(c) plays the most important physical role in understanding dynamic arrest processes.

NMR evidence of a sharp change in a measure of local order in deeply supercooled confined water.

Using NMR, we measure the proton chemical shift delta, of supercooled nanoconfined water in the temperature range 195 K < T < 350 K. Because delta is directly connected to the magnetic shielding tensor, we discuss the data in terms of the local hydrogen bond geometry and order. We argue that the derivative -( partial differential ln delta/ partial differentialT)(P) should behave roughly as the constant pressure specific heat C(P)(T), and we confirm this argument by detailed comparisons with literature values of C(P)(T) in the range 290-370 K. We find that -( partial differential ln delta/ partial differentialT)(P) displays a pronounced maximum upon crossing the locus of maximum correlation length at approximately 240 K, consistent with the liquid-liquid critical point hypothesis for water, which predicts that C(P)(T) displays a maximum on crossing the Widom line.

Complicated features in a young child with influenza B virus pneumonia and co-infection with Stenotrophomonas maltophilia.

A 3.5-year-old child with influenza B virus pneumonia developed pneumomediastinum and subcutaneous emphysema on the 3rd day of illness. Bronchoscopy demonstrated obstruction of the left main bronchus by mucopurulent sputum. Culture of the broncho-alveolar lavage yielded Stenotrophomonas maltophilia. After the respiratory complications resolved (11 days), the patient developed neurological symptoms and was diagnosed as acute disseminated encephalomyelitis (ADEM). Stenotrophomonas maltophilia was probably a factor in the development of pneumomediastinum. To our knowledge, this is the first case report of influenza virus infection with Stenotrophomonas maltophilia co-infection associated with spontaneous pneumomediastinum.