[Cluster classification and clinical prognostic modeling based on m6A RNA methylation regulators in liver cancer].

Objective: Cluster classification based on m6A methylation regulators and construct prognostic evaluation model. Methods: Utilizing consensus cluster to classify the liver cancer samples form TCGA based on the expression of 13 m6A methylation regulators, and verify the function and prognostic significance of the clustered subtypes. Marker genes were further screened to construct a risk prediction model for evaluating the prognosis of liver cancer patients. Results: The two clustered subtypes based on m6A methylation regulators showed significant differences in the prognosis value of liver cancer patients (P=0.048), and 38 prognostic markers related to m6A methylation in liver cancer were screened from the subgroup with poor prognosis. Two m6A regulatory genes, YTHDF1 and YTHDF2, are proved with adverse prognosis by univariate cox analysis (P<0.05, Hazard ratio>1). We used Lasso regression method to build risk assessment model and effectively predicted the prognosis status of liver cancer patients within 4 years (4-year AUC=0.685, 3-year AUC=0.669). Moreover, the assessment model was validated in another dataset of Asia liver cancer patients. Conclusion: The study provided ideas for studying m6A methylation in liver cancer, and the risk prediction model can be used to evaluate the short-term prognosis of liver cancer patients.

Clinical-grade Garcinia cambogia extract dissolves calcium oxalate crystals in Drosophila kidney stone models.

OBJECTIVE: Kidney stone formers have a high rate of stone recurrence after kidney stone removal surgery and there is no effective medication for treatment. Hydroxycitric acid (HCA), which is the major component of Garcinia cambogia extract, can dissolve calcium oxalate crystals in vitro, suggesting that Garcinia cambogia could be used to treat calcium oxalate kidney stone. In this study, we used the Drosophila kidney disease model to evaluate the effect of Garcinia cambogia on the prevention and removal of calcium oxalate stones in vivo. MATERIALS AND METHODS: Flies were reared in fly food containing different concentrations of GCE for one week. The effect of GCE on preventing the formation of calcium oxalate stone was examined. WT and v-ATPase gene RNAi knockdown flies were reared in fly food with 0.3% NaOx for one week, then fed different concentrations of GCE for one week. The effect of GCE on the removal of calcium oxalate stone was examined. RESULTS: Garcinia cambogia extract dissolves calcium oxalate crystals from Malpighian tubules in both genetic and non-genetic Drosophila kidney stone models compared to citric acid. Hydroxycitric acid also directly dissolves calcium oxalate crystals in Drosophila Malpighian tubules ex vivo. CONCLUSIONS: Garcinia cambogia extract removes calcium oxalate kidney stones from Drosophila Malpighian tubules via directly dissolving calcium oxalate stones by HCA. Our study strongly suggests that clinical-grade Garcinia cambogia extract could be used to treat patients with nephrolithiasis in the future.

Retigabine attenuates focal cerebral ischemic injury through inhibiting mitochondria-dependent apoptotic pathway.

OBJECTIVE: We explored the protective effect of retigabine (RTG) on focal cerebral ischemic injury and the potential molecular mechanism. MATERIALS AND METHODS: A mouse model of middle cerebral artery occlusion (MCAO) was established to induce cerebral ischemic injury. Blood samples were collected for the measurement of malondialdehyde (MDA), superoxide dismutase (SOD) and reduced glutathione (GSH). The brain infarct volume was stained by triphenyltetrazolium chloride. The cell apoptosis was observed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining. The expression of B-cell lymphoma-2 (Bcl-2), BCL2-Associated X (Bax), cleaved caspase 3, p-p38 and p-JNK, were determined by Western blot. RESULTS: RTG treatment reduced the MCAO-induced increase in brain infarct volume and neurological deficit scores. RTG treatment reduced the level of MDA and increased the activity of SOD and GSH. RTG treatment also decreased the Bax/Bcl-2 ratio and cleaved caspase 3 expression in the ischemic tissues. Further, RTG treatment decreased the phosphorylation levels of p38 and JNK in the ischemic tissues. CONCLUSIONS: RTG attenuated cerebral ischemic injury through reducing oxidative stress and mitochondria-mediated apoptosis via inhibiting p38 and JNK phosphorylation.

The effect of leflunomide on the transplanted endometriosis lesions in SD rats.

OBJECTIVE: To investigate the effects of the leflunomide (LEF) on the size of the transplanted endometriosis (EMS) lesions and trans- forming growth factor (TGF) -ß1gray level in SD rats. MATERIALS AND METHODS: EMS was surgically induced in rats by autologous trans- plantation and the focal volume was also measured. The rats were divided into three groups: group A: normal SD rats, group B: rats irrigated by one ml-kg⁻¹d⁻¹ saline for three weeks, and group C: rats irrigated by 35 mg-kg⁻¹d⁻¹ LEF for three weeks. The rats were then sacrificed and measured their focal volume and TGF-ß1 gray value with immunohistochemical method. RESULTS: The sizes of the focal volume in group C were significantly reduced compared to the rats before feeding, and the volume in group C was smaller than group B after feeding and so was the TGF-ß1. CONCLUSION: LEF could be a new therapeutic drug for EMS.

High-precision evaluation of Wigner's d matrix by exact diagonalization.

The precise calculations of Wigner's d matrix are important in various research fields. Due to the presence of large numbers, direct calculations of the matrix using Wigner's formula suffer from a loss of precision. We present a simple method to avoid this problem by expanding the d matrix into a complex Fourier series and calculate the Fourier coefficients by exactly diagonalizing the angular momentum operator J(y) in the eigenbasis of J(z). This method allows us to compute the d matrix and its various derivatives for spins up to a few thousand. The precision of the d matrix from our method is about 10(-14) for spins up to 100.

Adipose afferent reflex response to insulin is mediated by melanocortin 4 type receptors in the paraventricular nucleus in insulin resistance rats.

BACKGROUND: Adipose afferent reflex (AAR) contributes to sympathetic activation and hypertension. Paraventricular nucleus (PVN) plays an important role in AAR and sympathetic outflow. The aim of the present study was to determine whether PVN mediates AAR response to insulin in a rat model of insulin resistance (IR). METHODS: Male Sprague-Dawley rats were randomly divided into Control and IR groups. Insulin resistance was induced by supplementing fructose (125 g L(-1) , 12 weeks) in the drinking water. Renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded in anesthetized rats. AAR was evaluated by the RSNA and MAP responses to injection of capsaicin into four sites of right inguinal white adipose tissue. RESULTS: Rats in IR group showed a rise in plasma noradrenaline (NE), glucose, insulin and triglyceride levels, left ventricular weight, systolic blood pressure, homeostasis model assessment of insulin resistance (HOMA-IR) and PVN glucose and insulin levels, melanocortin 4 type receptors (MC4Rs) protein expression, but not MC3Rs and insulin receptors. Compared with Control group, AAR in IR group was significantly enhanced, which contributed to the elevation of NE level; and insulin microinjection into the PVN or the third ventricle significantly strengthened AAR, which was attenuated by pre-treatment with MC4Rs antagonist HS024 and anti-insulin affibody, respectively, but not insulin receptors antagonist S961. CONCLUSION: The enhanced AAR participates in sympathetic activation in IR, which can be strengthened by PVN insulin. PVN MC4Rs mediate the AAR response to insulin in IR, but not MC3Rs and insulin receptors.

Superoxide anions in the paraventricular nucleus mediate cardiac sympathetic afferent reflex in insulin resistance rats.

AIM: Cardiac sympathetic afferent reflex (CSAR) participates in sympathetic over-excitation. Superoxide anions and angiotensin II (Ang II) mechanisms are associated with sympathetic outflow and CSAR in the paraventricular nucleus (PVN). This study was designed to investigate whether PVN superoxide anions mediate CSAR and Ang II-induced CSAR enhancement response in fructose-induced insulin resistance (IR) rats. METHODS: CSAR was evaluated with the changes of renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) responses to the epicardial application of capsaicin (CAP) in anaesthetized rats. RESULTS: Compared with Control rats, IR rats showed that CSAR, PVN NAD(P)H oxidase activity, superoxide anions, malondialdehyde (MDA), Ang II and AT1 receptor levels were significantly increased, whereas PVN superoxide dismutase (SOD) and catalase (CAT) activities were decreased. In Control and IR rats, PVN microinjection of superoxide anions scavengers tempol, tiron and PEG-SOD (an analogue of endogenous superoxide dismutase) or inhibition of PVN NAD(P)H oxidase with apocynin caused significant reduction of CSAR, respectively, but DETC (a superoxide dismutase inhibitor) strengthened the CSAR. PVN pre-treatment with tempol abolished, whereas DETC potentiated, Ang II-induced CSAR enhancement response. Moreover, PVN pre-treatment with tempol or losartan prevented superoxide anions increase caused by Ang II in IR rats. CONCLUSION: PVN superoxide anions mediate CSAR and Ang II-induced CSAR response in IR rats. In IR state, increased NAD(P)H oxidase activity and decreased SOD and CAT activities in the PVN promote superoxide anions increase to involve in CSAR enhancement. Ang II may increase NAD(P)H oxidase activity via AT1 receptor to induce superoxide anion production.

Image analysis for monitoring the barley tempeh fermentation process.

AIMS: To develop a fast, accurate, objective and nondestructive method for monitoring barley tempeh fermentation. METHODS AND RESULTS: Barley tempeh is a food made from pearled barley grains fermented with Rhizopus oligosporus. Rhizopus oligosporus growth is important for tempeh quality, but quantifying its growth is difficult and laborious. A system was developed for analysing digital images of fermentation stages using two image processing methods. The first employed statistical measures sensitive to image colour and surface structure, and these statistical measures were highly correlated (r=0.92, n=75, P<0.001) with ergosterol content of tempeh fermented with R. oligosporus and lactic acid bacteria (LAB). In the second method, an image-processing algorithm optimized to changes in images of final tempeh products was developed to measure number of visible barley grains. A threshold of 5 visible grains per Petri dish indicated complete tempeh fermentation. When images of tempeh cakes fermented with different inoculation levels of R. oligosporus were analysed the results from the two image processing methods were in good agreement. CONCLUSION: Image processing proved suitable for monitoring barley tempeh fermentation. The method avoids sampling, is nonintrusive, and only requires a digital camera with good resolution and image analysis software. SIGNIFICANCE AND IMPACT OF THE STUDY: The system provides a rapid visualization of tempeh product maturation and qualities during fermentation. Automated online monitoring of tempeh fermentation by coupling automated image acquisition with image processing software could be further developed for process control.

Cytotoxicity and altered c-myc gene expression by medical polyacrylamide hydrogel.

Medical Polyacrylamide Hydrogel (PAMG)has been used in plastic and aesthetic surgery for years. However, its safety is still in doubt in many countries. In the current research, first an approach, using high performance liquid chromatography (HPLC), to determine the amount of residual acrylamide monomer (AM) in the PAMG was presented. Then the cytotoxicity of PAMG was investigated using cell counting and methyl thiazolyl tetrazolium (MTT) assay. To explore the mechanism of this toxicity, normal human fibroblasts cultured in medium extracts were analyzed. Membrane changes and other related parameters were investigated using flow cytometry (FCM). Real time fluorescent polymerase chain reaction (real time PCR) was also introduced to determine the biological response of the fibroblasts. During this process, three representative genes (p53, beta-actin, and c-myc, which are tumor suppressor genes, housekeeping genes, and proto-oncogenes respectively) were selected for examination. Results indicated that a method based on HPLC is practical and simple for determining AM in PAMG. The detection limits can reach the desired ppb level, and so it can fully meet the requirements of the studies of PAMG. Polyacylamide Hydrogel inhibits the growth of human fibroblasts and may cause the apoptosis of human fibroblasts. Moreover, it can alter physical parameters such as the size and the granularity of these cells. Furthermore, these three genes have a relatively typical amplification plot and highly related, wide-range standard curves, and so this reaction system is definitely suitable for the semiquantification of these genes. PAMG induces the increase of the message ribonucleic acid (mRNA) expression of c-myc, while the p53 and beta-actin remain even. This change is not related to the concentration of AM in the gel and may be incited by other components in the extract of PMAG.

Supracondylar extension fracture of the humerus in children. Manipulative reduction, immobilisation and fixation using a U-shaped plaster slab with the elbow in full extension.

We present a method of manipulative reduction, immobilisation and fixation using a U-shaped plaster with the elbow in extension for extension-type supracondylar fractures of the humerus in children. When the elbow is in full extension, both the extensor and the flexor muscles are neutralised during manipulative reduction and the carrying angle can be easily assessed thus preventing cubitus varus, the most common complication. In order to evaluate the efficiency of this method, we compared the clinical results of the new method with those of conventional treatment. In a group of 95 children who sustained an extension-type supracondylar fracture of the humerus, 49 were treated by the new method and 46 by the conventional method, reduction and immobilisation in a plaster slab with the elbow in flexion. Reduction and immobilisation were easily achieved and reliably maintained by one manipulation for all the children treated by the new method. In 12 children treated by the conventional method, the initial reduction failed and in seven secondary displacement of the distal fragment occurred during the period of immobilisation in plaster. All required a second or third manipulation. Of the 46 children, 28 (60.9%) had developed cubitus varus at a mean follow-up of 4.6 years when treated by the conventional method. None of the children treated by the new method developed cubitus varus. The mean score, according to the Hospital for Special Surgery (HSS) elbow scoring system, was 91 points using the new method and 78 with the conventional method. The results were statistically significant with regard to the incidence of cubitus varus and the elbow score (p < 0.01) suggesting that the new method is reliable and gives a satisfactory outcome.

A novel AvaI polymorphism within exon 5 of the rhodopsin gene.

We identified a novel AvaI polymorphism within 3' non-coding region within exon 5 of the human rhodopsin gene and determined the allele frequency in a Japanese population. The polymorphism was found to be due to A/G transversion at nucleotide 5510 of the gene.

Inhibition of hepatitis B virus in vitro by antisense oligonucleotides.

A series of antisense phosphorothioate oligodeoxynucleotides against hepatitis B virus (HBV) were synthesized and evaluated for their antiviral effect in Hep-G2 cells transfected with HBV genome. The inhibitory effect of the tested antisense oligonucleotides was sequence-specific, dose-and time-dependent, and synergistic for certain combinations. In virus-inhibitory concentrations the oligonucleotides were harmless to 2.2.15 cells. The most effective antisense oligonucleotides were found directed against the HBV mRNA transcribed from the cap site of SP II promoter, the portion of polyadenylation signal and the initiation region of gene S, with an inhibition of the HBsAg and HBeAg production by 85-95% and 50- 60%, respectively. To our surprise, antisense oligonucleotides directed against three key sites of HBV X gene blocked the expression of HBsAg, HBeAg and HBxAg. This fact might be related to the trans-activation of HBV X protein. Using radioisotope labelling, we demonstrated that Lipofectin promoted the cellular uptake and antiviral effect of antisense oligomers in 2.2.15 cells. These results suggest a therapeutic potential of antisense oligonucleotides in the treatment of patients chronically infected with HBV.

Specific inhibition of hepatitis b virus gene expression by an antisense oligonucleotide in vitro.

It was previously shown that a number of antisense oligonucleotides against hepatitis B virus (HBV) mRNAs were highly effective in inhibition of HBV gene expression (Yao et al., 1995). Here, using radioisotope techniques, we report a specific inhibition of HBV surface antigen (HBsAg) production in vitro by 2.2.15 cells (Hep-G2 cells transfected with HBV genome) by the antisense oligonucleotide 15-S-asON, a 15-mer phosphorothioate analogue complementary to the cap site of the SPII promoter of HBV mRNA, at a concentration of 2-5 mumol/l. After 24 and 48 hrs of incubation of cells with 15-S-asON, the intracellular concentration of the latter rose to 69.4 and 75.8 nmol/l, respectively, and the HBsAg level assayed by ELISA was reduced by 50.0% and 70.6%, respectively. These results were checked by use of the radioimmunoprecipitation method: 2.2.15 cells exposed to 15-S-asON and labelled with [35S]-methionine for 48 hrs showed a decrease of the HBsAg level by 81.26% but almost none of the total proteins. No cytotoxicity of the 15-S-asON was observed with regard to the cell morphology and growth. These results indicate that the tested antisense oligonucleotide specifically inhibits the HBV gene expression.