Tumour-suppressive microRNA-24-1 inhibits cancer cell proliferation through targeting FOXM1 in bladder cancer

Here, we found that microRNA-24-1 (miR-24-1) is significantly reduced in bladder cancer (BC) tissues, suggesting that it functions as a tumour suppressor. Restoration of mature miR-24-1 inhibits cancer cell proliferation and induces apoptosis. Forkhead box protein M1 (FOXM1) is a direct target gene of miR-24-1, as shown by genome-wide gene expression analysis and luciferase reporter assay. Overexpressed FOXM1 is confirmed in BC clinical specimens, and silencing of FOXM1 induces apoptosis in cancer cell lines. Our data demonstrate that the miR-24-1–FOXM1 axis contributes to cancer cell proliferation in BC, and elucidation of downstream signalling will provide new insights into the molecular mechanisms of BC oncogenesis.     

Molecular characterization of Indian Sugarcane streak mosaic virus isolates reveals recombination and negative selection in the P1 gene

Sugarcane streak mosaic virus (SCSMV), a member of the genus Poacevirus is an important viral pathogen affecting sugarcane production in India. The P1 gene of ten Indian isolates was sequenced and compared with previously reported SCSMV isolates. Comparative sequence analysis revealed a high level of diversity in the P1 gene (83–98% nucleotide sequence identity; 87–100% amino acid sequence identity), and the Indian SCSMV isolates were found to be the most variable (up to 9% diversity at the amino acid level). Phylogenetic tree analysis showed clustering of 17 SCSMV isolates into two groups: group I included isolates from India (except SCSMV-TPT) and Pakistan, and group II consisted of isolates from Japan, Indonesia, Thailand and SCSMV-TPT. The results obtained from phylogenetic study were further supported by the different in silico analysis viz. SNPs (single nucleotide polymorphism), INDELs (insertion and deletion) and evolutionary distance analysis. A significant proportion of recombination sites were observed at the N terminal region of P1 gene. Analysis of selection pressure indicated that the P1 gene of the Indian SCSMV isolates is under strong negative or purifying selection. It is likely that recombination identified in Indian SCSMV isolates, along with strong purifying selection, enhances the speed of elimination of deleterious mutations in the P1 gene. The evolutionary processes (recombination and selection pressure) together contributed to the observed genetic diversity and population structure of Indian SCSMV isolates.     

Molecular evolution of VP3, VP1, 3Cpro and 3Dpol coding regions in coxsackievirus group A type 24 variant isolates from acute hemorrhagic conjunctivitis in 2011 in Okinawa,Japan

A large acute hemorrhagic conjunctivitis (AHC) outbreak occurred in 2011 in Okinawa Prefecture in Japan. Ten strains of coxsackievirus group A type 24 variant (CA24v) were isolated from patients with AHC and full sequence analysis of the VP3, VP1, 3C^pro and 3D^pol coding regions performed. To assess time‐scale evolution, phylogenetic analysis was performed using the Bayesian Markov chain Monte Carlo method. In addition, similarity plots were constructed and pairwise distance (p‐distance) and positive pressure analyses performed. A phylogenetic tree based on the VP1 coding region showed that the present strains belong to genotype 4 (G4). In addition, the present strains could have divided in about 2010 from the same lineages detected in other countries such as China, India and Australia. The mean rates of molecular evolution of four coding regions were estimated at about 6.15 to 7.86 × 10^?3 substitutions/site/year. Similarity plot analyses suggested that nucleotide similarities between the present strains and a prototype strain (EH24/70 strain) were 0.77–0.94. The p‐distance of the present strains was relatively short (<0.01). Only one positive selected site (L25H) was identified in the VP1 protein. These findings suggest that the present CA24v strains causing AHC are genetically related to other AHC strains with rapid evolution and emerged in around 2010.

     

Long-range effects of tag sequence on marginally stabilized structure in HIV-1 p24 capsid protein monitored using NMR

N-terminal domain of HIV-1 p24 capsid protein is a globular fold composed of seven helices and two β-strands with a flexible structure including the α4–5 loop and both N- and C-terminal ends. However, the protein shows a high tendency (48%) for an intrinsically disordered structure based on the PONDR VL-XT prediction from the primary sequence. To assess the possibility of marginally stabilized structure under physiological conditions, the N-terminal domain of p24 was destabilized by the addition of an artificial flexible tag to either N- or C-terminal ends, and it was analyzed using T₁, T₂, hetero-nuclear NOE, and amide-proton exchange experiments. When the C-terminal tag (12 residues) was attached, the regions of the α3–4 loop and helix 6 as well as the α4–5 loop attained the flexible structures. Furthermore, in the protein containing the N-terminal tag (27 residues), helix 4 in addition to the above-mentioned area including α3–4 and α4–5 loops as well as helix 6 exhibited highly disordered structures. Thus, the long-range effects of the existence of tag sequence was observed in the stepwise manner of the appearance of disordered structures (step 1: α4–5 loop, step 2: α3–4 loop and helix 6, and step 3: helix 4). Furthermore, the disordered regions in tagged proteins were consistent with the PONDR VL-XT disordered prediction. The dynamic structure located in the middle part (α3–4 loop to helix 6) of the protein shown in this study may be related to the assembly of the viral particle.     

多种无创检查组合在冠心病诊断中的价值

目的：评价多种无创辅助检查组合对冠状动脉粥样硬化性心脏病（冠心病）辅助诊断价值,筛选有效的冠心病确诊和排除指标,初步确定优化的冠心病早期诊断策略。方法：回顾性分析6419例冠心病患者多项无创辅助检查结果（包括静息心电图、24小时动态心电图、负荷心电图、负荷核素心肌显像、16或64排CT冠状动脉成像）,以冠状动脉造影阳性（至少一支主要冠状动脉或其主要分支的内径有≥50％的狭窄）为金标准,观察各种无创辅助检查组合对冠心病诊断的特异性、敏感性、误诊率、漏诊率、阳性预测值和阴性预测值。结果：多项无创辅助检查组合在冠心病的诊断中敏感性56．02-87．43％,特异36．13-87．61％,阳性预测值58．83．97．16％,阴性预测值30．21．73．36％,非介入手段中,敏感性和阴性预测值以动态心电图联合核素心肌灌注显像组最高,特异性和阳性预测值以动态心电图联合冠脉CT成像组最高。结论：辅助检查组合可作为无创性诊断、评价冠心病的重要方法,动态心电图可作为各级别医院冠心病筛查的基本及重要手段。     

Induction of apoptosis and necroptosis by 24(S)-hydroxycholesterol is dependent on activity of acyl-CoA:cholesterol acyltransferase 1

24(S)-hydroxycholesterol (24S-OHC), which is enzymatically produced in the brain, has an important role in maintaining brain cholesterol homeostasis. We have previously reported that 24S-OHC induces necroptosis in human neuroblastoma SH-SY5Y cells. In the present study, we investigated the mechanisms by which 24S-OHC-induced cell death occurs. We found that lipid droplets formed at the early stages in the treatment of SH-SY5Y cells with 24S-OHC. These lipid droplets could be almost completely eliminated by treatment with a specific inhibitor or by siRNA knockdown of acyl-CoA:cholesterol acyltransferase 1 (ACAT1). In association with disappearance of lipid droplets, cell viability was recovered by treatment with the inhibitor or siRNA for ACAT1. Using gas chromatography–mass spectrometry, we confirmed that 24S-OHC-treated cells exhibited accumulation of 24S-OHC esters but not of cholesteryl esters and confirmed that accumulation of 24S-OHC esters was reduced when ACAT1 was inhibited. 24S-OHC induced apoptosis in T-lymphoma Jurkat cells, which endogenously expressed caspase-8, but did not induce apoptosis in SH-SY5Y cells, which expressed no caspase-8. In Jurkat cells treated with the pan-caspase inhibitor ZVAD and in caspase-8-deficient Jurkat cells, 24S-OHC was found to induce caspase-independent cell death, and this was partially but significantly inhibited by Necrostatin-1. Similarly, knockdown of receptor-interacting protein kinase 3, which is one of the essential kinases for necroptosis, significantly suppressed 24S-OHC-induced cell death in Jurkat cells treated with ZVAD. These results suggest that 24S-OHC can induce apoptosis or necroptosis, which of the two is induced being determined by caspase activity. Regardless of the presence or absence of ZVAD, 24S-OHC treatment induced the formation of lipid droplets and cell death in Jurkat cells, and this was suppressed by treatment with ACAT1 inhibitor. Collectively, these results suggest that it is ACAT1-catalyzed 24S-OHC esterification and the resulting lipid droplet formation that is the initial key event which is responsible for 24S-OHC-induced cell death.     

Reducing dietary protein in dairy cow diets: implications for nitrogen utilization,milk production,welfare and fertility

In light of increasing global protein prices and with the need to reduce environmental impact of contemporary systems of milk production, the current review seeks to assess the feasibility of reducing levels of dietary CP in dairy cow diets. At CP levels between 140 and 220 g/kg DM there is a strong positive relationship between CP concentration and dry matter intake (DMI). However, such effects are modest and reductions in DMI when dietary CP is below 180 g/kg DM can be at least partially offset by improving the digestibility and amino acid profile of the undegradable protein (UDP) component of the diet or by increasing rumen fermentable energy. Level and balance of intestinally absorbable amino acids, in particular methionine and lysine, may become limiting at lower CP concentrations. In general the amino acid composition of microbial protein is superior to that of UDP, so that dietary strategies that aim to promote microbial protein synthesis in the rumen may go some way to correcting for amino acid imbalances in low CP diets. For example, reducing the level of NDF, while increasing the proportion of starch, can lead to improvements in nitrogen (N) utilisation as great as that achieved by reducing dietary CP to below 150 g/kg. A systematic review and meta-analysis of responses to rumen protected forms of methionine and lysine was conducted for early/mid lactation cows fed diets containing ⩽150 g CP/kg DM. This analysis revealed a small but significant (P=0.002) increase in milk protein yield when cows were supplemented with these rumen protected amino acids. Variation in milk and milk protein yield responses between studies was not random but due to differences in diet composition between studies. Cows fed low CP diets can respond to supplemental methionine and lysine so long as DMI is not limiting, metabolisable protein (MP) is not grossly deficient and other amino acids such as histidine and leucine do not become rate limiting. Whereas excess dietary protein can impair reproduction and can contribute to lameness, there is no evidence to indicate that reducing dietary CP levels to around 140 to 150 g CP/kg DM will have any detrimental effect on either cow fertility or health. Contemporary models that estimate MP requirements of dairy cows may require refinement and further validation in order to predict responses with low CP diets.     

The naïve effector cells of collagen type I during acute experimental pancreatitis are acinar cells and not pancreatic stellate cells

Objective

The purpose of this study was to investigate the expression of collagen type I and the mRNA level of its regulatory factor, TGF-β1, in tissue samples of acute pancreatitis and to determine the significance of collagen type I in predisposition to pancreatic fibrosis during acute pancreatitis.

Methods

Sprague–Dawley rats were divided into an experimental group (30 rats) and a control group (12 rats). The rats in the experimental group were intraperitoneally injected with cerulein to induce acute pancreatitis. The distribution and expression of collagen type I in the pancreatic tissues were examined by immunohistochemical staining. The mRNA level of TGF-β1 was determined by real-time polymerase chain reaction (PCR).

Results

(1) Collagen type I was localized in the cytoplasm of pancreatic acinar cells. With pancreatitis progressed, strong positive staining for collagen type I covered whole pancreatic lobules, whereas, the islet tissue, interlobular area, and pancreatic necrotic area were negative for collagen type I. (2) The level of TGF-β1 mRNA in rats from the experimental group increased gradually the establishment of acute pancreatitis, and was significantly higher than that in the control group at every time point.

Conclusions

(1) During acute pancreatitis, pancreatic acinar cells, not pancreatic stellate cells as traditionally believed, were the naïve effector cells of collagen type I. (2) TGF-β1 played a key role in regulating collagen I expression during acute pancreatitis.     

Sigma 1 Receptor plays a prominent role in IL-24-induced cancer-specific apoptosis

Interleukin-24 (IL-24), a member of the IL-10 cytokine family, is an immunomodulatory cytokine that also displays broad cancer-specific suppressor effects. The tumor suppressor activities of IL-24 include inhibition of angiogenesis, sensitization to chemotherapy, and cancer-specific apoptosis. We show that Sigma 1 Receptor (S1R), a ligand-regulated protein chaperone contributes to IL-24 induction of apoptosis. IL-24 generated from an adenovirus expressing IL-24 (Ad.IL-24) induces cancer-specific apoptosis by inducing an endoplasmic reticulum (ER) stress, reactive oxygen species production, and calcium mobilization. The present studies reveals that S1R is required for Ad.IL-24-induced cell death. We provide several lines of evidence to confirm a physical and functional interaction between IL-24 and S1R including: (a) S1R and IL-24 co-localize, as judged by immunocytochemical analysis studies; (b) S1R and IL-24 co-immunoprecipitate using either S1R or IL-24 antibody; (c) S1R agonist (+)-SKF10047 inhibits apoptosis by Ad.IL-24; (d) (+)-SKF10047-mediated inhibition of Ad.IL-24 results in: diminished ER stress protein expression; (e) Calcium mobilization; and (f) ROS production. Collectively, these data demonstrate that S1R interacts with IL-24 and suggest that IL-24:S1R interaction determines apoptosis induction by Ad.IL-24. These studies define Sigma 1 Receptor as a key initial mediator of IL-24 induction of cancer-specific killing. These findings have important implications for our understanding of IL-24 as a tumor suppressor protein as well as an immune modulating cytokine.