[The research advances of graphene and its derivatives in regulating the fate of neural stem cells].

As a new class of nanomaterials, graphene and its derivatives have excellent mechanical, electrical and optical properties, which are widely used in various fields. In recent years, more and more scholars have linked it to stem cell research. Their effects on stem cell proliferation, differentiation can not be underestimated. Here we review the regulation of graphene and its derivatives on the fate of neural stem cells, hoping that more ophthalmologists will invest in this research and provide a new way for neurodegenerative diseases in ophthalmology.

Genotype and genetic variation of HCV infections with low-risk factors in Putian coastal regions, China.

Hepatitis C virus (HCV) infection is one of the leading causes of death and morbidity associated with liver disease. Risk factors identified for the transmission of HCV include contaminated blood products, intravenous drug use, body piercing, an infected mother at birth, sexual activity, and dental therapy, among others. However, the exact diversity of the HCV genotype and genetic variation among patients with low-risk factors is still unknown. In this study, we briefly described and analysed the genotype distribution and genetic variation of HCV infections with low-risk factors using molecular biology techniques. The results suggested that genotype 1b was predominant, followed by genotypes 2a and 1a. Genetic variations in the 5' UTR sequences of HCV were identified, including point mutations, deletions, and insertions. The frequency of genetic variations in 1b was higher than in 2a. This study provides considerable value for the prevention and treatment of liver disease caused by HCV among patients with low-risk factors and for the development of HCV diagnostic reagents and vaccines.

Analysis of the safety and efficacy of combined extracorporeal shock wave lithotripsy and percutaneous nephrolithotomy for the treatment of complex renal calculus.

OBJECTIVE: To investigate the safety and efficacy of extracorporeal shock wave lithotripsy (ESWL) combined with percutaneous nephrolithotomy (PCNL) for treatment of complex renal calculus. PATIENTS AND METHODS: Seventy-eight patients diagnosed with complex renal calculus and who accepted treatment in our hospital were consecutively selected. Patients were divided randomly into the observation group (n=40) treated by combined ESWL and PCNL and the control group (n=38) treated by PCNL. The effect of treatment between the two groups was compared. RESULTS: The stone-free rate at 3 months after surgery was higher in the observation group than in the control group. There were no differences in the rates of complications (including infection, hemorrhage, collection system perforation and laceration, peripheral organ impairment, and urination extravasation). There were gradual decreases of serum creatinine in the observation group at 4 weeks after extubation of the double J catheter and at 3 months after surgery, while there were no apparent decreases in the control group. The levels of cysteine protease inhibitor and neutrophil gelatinase-associated lipocalin in both groups increased at 4 weeks after extubation of the double J catheter, and decreased at 3 months after surgery. The decreases were more apparent in the observation group compared with the control group, and the differences were statistically significant (p<0.05). CONCLUSIONS: Combined use of ESWL and PCNL to treat complex renal calculus can improve the stone-free rate and renal function, and does not increase the complication rate. It is, therefore, safe and effective.

Overexpression of ß-arrestin2 induces G1-phase cell cycle arrest and suppresses tumorigenicity in renal cell carcinoma.

OBJECTIVE: The objective of this study was to investigate the role of ß-arrestin2 in the proliferation, migration, apoptosis, cell cycle and clone formation of renal cell carcinoma (RCC) cell lines and to explore the possible mechanism of ß-arrestin2 in RCC invasion and metastasis to find a new therapeutic target. MATERIALS AND METHODS: Cell proliferation, migration, apoptosis, cell cycle and clone formation were analyzed after RCC cell lines (786-0 and CaKi) and transfected with ß-arrestin2 overexpression plasmid. Using small interfering RNA (siRNA) interference technology abrogates ß-arrestin2 overexpression, and changes in cell proliferation, migration, apoptosis, cell cycle and clone formation were analyzed. The expression levels of total IkBa, IkBa phosphorylation (P-IkBa) and NFkB P65 in 786-0 cells were examined after transfection with ß-arrestin2 overexpression plasmid to explore the mechanism of ß-arrestin2. RESULTS: After transfection with ß-arrestin2 overexpression plasmid, the abilities of proliferation, migration, and cloning formation in 786-0 and CaKi cells decreased significantly, the apoptosis rate increased significantly, and the cell cycles were blocked in the G1 phase. After siRNA reduced the expression of ß-arrestin2, the abilities to proceed through cell proliferation, migration, apoptosis, the cell cycle and clone formation were enhanced. The P-IkBa level in 786-0 cells decreased significantly after transfection, while the expression of P-IkBa in the control group remained high. The expression of NFkB P65 was high in the control group and low in the transfection group. CONCLUSIONS: The overexpression of ß-arrestin2 can inhibit the growth of RCC cells in vitro, and ß-arrestin2 acts as a tumor suppressor gene in RCC. The main mechanism may directly suppress the phosphorylation of IkBa and indirectly suppress NFkB activation. Thus, ß-arrestin2 is expected to be an important marker of RCC prognosis and a new therapeutic target.

Urinary apolipoprotein M could be used as a biomarker of acute renal injury: an ischemia-reperfusion injury model of kidney in rat.

BACKGROUND: It has been well documented that apolipoprotein M (apoM) is principally expressed in hepatocytes as well as renal tubular epithelial cells. The importance of apoM in the kidney is unknown. In the present study we examined urinary any apoM after short-term ischemia-reperfusion injury (IRI) of kidney in a rat model. METHODS: The kidneys of 11 male Sprague-Dawley rats were rendered ischemic for 45 minutes followed by different intervals of reperfusion. Serum and urine apoM concentrations were determined using a dot-blot analysis with specific rabbit anti-human apoM antibodies that cross-react with rat apoM. Serum concentrations of blood urea nitrogen (BUN) and creatinine (Cr) were determined using standard clinical automated analyses. RESULTS: BUN was significantly elevated after 45 minutes of ischemia followed by 24 hours of reperfusion; serum Cr concentrations were also significantly increased at 6 and 24 hours of reperfusion. Interestingly, similar to BUN and Cr, serum apoM concentrations were significantly increased after ischemia for 45 minutes alone and after 2 hours of reperfusion. Urinary apoM concentrations were obviously increased after 2 h as well as 6 hours of reperfusion. CONCLUSION: apoM showed characteristics of an acute-phase reactive protein; its occurrence in urine may be considered to be a biomarker of acute renal injury.

Screening of differentially expressed genes of middle cerebral artery occlusion with DNA microarray.

OBJECTIVES: To screen differentially expressed genes of different days after cerebral artery occlusion and drug treatment, and identify related small drug molecules. MATERIALS AND METHODS: The gene expression profile GSE35338 of cerebral artery occlusion was downloaded from Gene Expression Omnibus database, including a total of 14 samples. 5 samples are 1 day after cerebral artery occlusion (control), 3 samples are 7 days after cerebral artery occlusion and 3 samples are under lipopolysaccharide (LPS) treatment. Differentially expressed genes (DEGs) between different days after cerebral artery occlusion were screened (p < 0.05, FDR < 0.05, |logFC| > 1). The DEGs were then entered into the CMAP database and related small drug molecules were retrieved, followed by calculation of co-expression score of the genes and construction of co-expression-drug network. FuncAssociate software and DAVID were used to obtain the functional clusters of genes with p-value < 0.05 and FDR < 0.05. RESULTS: Compared with the control group, 825, 1445, 218 DEGs and 4, 3, 2 most-related small drug molecules were respectively identified from 3, 7 days after cerebral artery occlusion and LPS treated group. Co-expression network was constructed and functional clusters were found to be 161, 146, and 6 in each group. CONCLUSIONS: Our study provides some underlying biomarkers for cerebral artery occlusion under varied conditions and potential small drug molecules for treatment of cerebral artery occlusion.

Electrical shielding box measurement of the negative hydrogen beam from Penning ion gauge ion source.

The cold-cathode Penning ion gauge (PIG) type ion source has been used for generation of negative hydrogen (H(-)) ions as the internal ion source of a compact cyclotron. A novel method called electrical shielding box dc beam measurement is described in this paper, and the beam intensity was measured under dc extraction inside an electrical shielding box. The results of the trajectory simulation and dc H(-) beam extraction measurement were presented. The effect of gas flow rate, magnetic field strength, arc current, and extraction voltage were also discussed. In conclusion, the dc H(-) beam current of about 4 mA from the PIG ion source with the puller voltage of 40 kV and arc current of 1.31 A was extrapolated from the measurement at low extraction dc voltages.

Diabetes care for older patients in America.

BACKGROUND: Underuse of diabetes care was common for older patients. This study examined whether patient or physician practice characteristics predict the likelihood of diabetes care. METHODS: We studied the 2006 and 2007 National Ambulatory Medical Care Survey data for a nationally-representative sample of 2912 visits by older patients with diabetes. We examined the patterns of diabetes care, including diagnostic testing (glucose, haemoglobinA1c, blood pressure and cholesterol) and patient counselling services (diet/nutrition, exercise). Multivariate analysis was performed to identify independent predictors of diabetes care, controlling for patient and physician practice characteristics. All analyses were adjusted for the complex survey design. RESULTS: Having a designated primary care physician and the availability of electronic medical record or on-site laboratory testing were associated with more effective diabetes care (p < 0.05). If physician compensation relied on the productivity, physicians were less likely to provide diabetes care services (odds ratio = 0.5). The patterns of patient counselling and diagnostic testing services were similar (odds ratio = 2.5 and 18.2 for men; odds ratio = 1.8 and 9.6 for women). Older patients with diabetes were substantially more likely to receive diagnostic testing services than patient counselling. CONCLUSION: A designated primary care physician is crucial for providing recommended diabetes care services for older patients. Strengthening structural capabilities of primary care practices and implementing patient-centred primary care initiatives in concert with health system reforms are necessary to deliver the co-ordinated diabetes care with maximised health outcomes.

Physiological and biochemical analysis of L. tredecimguttatus venom collected by electrical stimulation.

The L. tredecimguttatus venom was collected by electrical stimulation and systematically analyzed. Gel electrophoresis and RP-HPLC showed that the venom consisted primarily of proteins with molecular weights above 10 kDa, most of which were high-molecular-mass acidic proteins, with fewer proteins and peptides below 10 kDa. The most abundant proteins in the venom were concentrated at around 100 kDa, which included latrotoxins- the principal toxic components of the venom. Injection of the venom in mice and cockroaches P. americana gave rise to obvious poisoned symptoms, with LD50 values of 0.16 mg/kg and 1.87 microg/g, respectively. Electrophysiological experiments showed that the venom could block the neuromuscular transmission in isolated mouse phrenic nerve-hemidiaphragm and rat vas deferens preparations. The low-molecular-weight fraction (<10 kDa) of the venom had no effect on the transmission. Enzymatic analysis indicated that the venom possess activities of several kinds of hydrolases including hyaluronidase and proteases. These results demonstrated that L. tredecimguttatus venom was basically a large-protein-constituted venom and is one of the most poisonous spider venoms known in the world. The mammalian toxicity of the venom was based on its larger proteins rather than on smaller proteins and peptides, and its hydrolase activities might be involved in the latrodectism. The use of electrical stimulation method to collect the venom has the advantages of avoiding contamination and repeated use of the valuable L. tredecimguttatus venom resources.

Extraction and protein component analysis of venom from the dissected venom glands of Latrodectus tredecimguttatus.

Black widow spiders (genus Latrodectus) have attracted increasing attention due to frequently reported human injuries caused by them and the potential applications of biologically active components in their venoms. Although a number of studies have described the biological properties and structures of several venomous proteins such as latrotoxins, a comprehensive analysis of protein component of the venom from the spider is not available. We used combinative proteomic strategies to assess the protein components of the crude venom collected from Latrodectus tredecimguttatus by extracting the dissected venom glands. The experiments demonstrated that the crude venom of L. tredecimguttatus has a high abundance of acidic proteins with molecular masses greater than 15 kDa, and the content of proteins and peptides of below 15 kDa is low. 86 unique proteins were identified, part of which were contaminations of cellular components during the extraction, determined in comparison with venom obtained by electrostimulation. Except for members of latrotoxin family that were commonly considered as the primary toxic components of the venom, several other special enzymes and proteins were detected such as protease, phosphatase, lysozyme, inhibitory protein, and so on. These protein components, particularly the proteases, were speculated to play important roles in the action of L. tredecimguttatus venom.

Familial aggregation of lung cancer in a high incidence area in China.

To investigate whether lung cancer clusters in families in a high incidence county of China, an analysis was conducted using data on domestic fuel history and tobacco use for family members of 740 deceased lung cancer probands and 740 controls (probands' spouses). Lung cancer prevalence was compared among first-degree relatives of probands and of controls, taking into account various factors using logistic regression and generalised estimating equations. First-degree relatives of probands, compared with those of controls, showed an excess risk of lung cancer (odds ratio (OR)=2.05, 95% confidence interval (CI): 1.68-2.53). Overall, female relatives of probands had a greater risk than did their male counterparts, and the risk was 2.90-fold for parents of probands as compared with parents of spouses. Female relatives of probands had 2.67-fold greater risk than female controls. Lung cancer risk was particularly marked among mothers (OR=3.78, 95% CI: 2.03-7.12). Having two or more affected relatives was associated with a 2.69-5.40-fold risk increase. The risk elevation was also found for other cancers overall. Results confirm previous findings of a genetic predisposition to lung cancer, and also imply that lung cancer may share a genetic background with other cancers.

Structures of two natural product methyltransferases reveal the basis for substrate specificity in plant O-methyltransferases.

Chalcone O-methyltransferase (ChOMT) and isoflavone O-methyltransferase (IOMT) are S-adenosyl-l-methionine (SAM) dependent plant natural product methyltransferases involved in secondary metabolism in Medicago sativa (alfalfa). Here we report the crystal structure of ChOMT in complex with the product S-adenosyl-l-homocysteine and the substrate isoliquiritigenin (4,2',4'-trihydroxychalcone) refined to 1.8 A as well as the crystal structure of IOMT in complex with the products S-adenosyl-l-homocysteine and isoformononetin (4'-hydroxy-7-methoxyisoflavone) refined to 1.4 A. These two OMTs constitute the first plant methyltransferases to be structurally characterized and reveal a novel oligomerization domain and the molecular determinants for substrate selection. As such, this work provides a structural basis for understanding the substrate specificity of the diverse family of plant OMTs and facilitates the engineering of novel activities in this extensive class of natural product biosynthetic enzymes.

Genetic manipulation of isoflavone 7-O-methyltransferase enhances biosynthesis of 4'-O-methylated isoflavonoid phytoalexins and disease resistance in alfalfa.

4'-O-Methylation of an isoflavonoid intermediate is a key reaction in the biosynthesis of the phytoalexin medicarpin in legumes. However, isoflavone O-methyltransferase (IOMT) from alfalfa converts the isoflavone daidzein to 7-O-methyl daidzein (isoformononetin) in vitro as well as in vivo in unchallenged leaves of transgenic alfalfa ectopically expressing IOMT. In contrast, elicitation of IOMT-overexpressing plants with CuCl(2) or infecting these plants with Phoma medicaginis leads to greater accumulation of formononetin (4'-O-methyl daidzein) and medicarpin in the leaves than does elicitation or infection of control plants, and no isoformononetin is detected. Overexpression of IOMT results in increased induction of phenylpropanoid/isoflavonoid pathway gene transcripts after infection but has little effect on basal expression of these genes. IOMT-overexpressing plants display resistance to P. medicaginis. The apparently different regiospecificities of IOMT in vivo and in vitro are discussed in relation to potential metabolic channeling at the entry point into the isoflavonoid pathway.

Emerging tobacco hazards in China: 2. Early mortality results from a prospective study.

OBJECTIVE: To monitor the evolving epidemic of mortality from tobacco in China following the large increase in male cigarette use in recent decades. DESIGN: Prospective study of smoking and mortality starting with 224 500 interviewees who should eventually be followed for some decades. SETTING: 45 nationally representative small urban or rural areas distributed across China. SUBJECTS: Male population aged 40 or over in 1991, of whom about 80% were interviewed about smoking, drinking, and medical history. MAIN OUTCOME MEASURE: Cause specific mortality, initially to 1995 but later to continue, with smoker versus non-smoker risk ratios standardised for area, age, and use of alcohol. RESULTS: 74% were smokers (73% current, only 1% former), but few of this generation would have smoked substantial numbers of cigarettes since early adult life. Overall mortality is increased among smokers (risk ratio 1.19; 95% confidence interval 1.13 to 1.25, P<0.0001). Almost all the increased mortality involved neoplastic, respiratory, or vascular disease. The overall risk ratios currently associated with smoking are less extreme in rural areas (1.26, 1.12, or 1.02 respectively for smokers who started before age 20, at 20-24, or at older ages) than in urban areas (1.73, 1.40, or 1.16 respectively). CONCLUSION: This prospective study and the accompanying retrospective study show that by 1990 smoking was already causing about 12% of Chinese male mortality in middle age. This proportion is predicted to rise to about 33% by 2030. Long term continuation of the prospective study (with periodic resurveys) can monitor the evolution of this epidemic.

Prospects for the metabolic engineering of bioactive flavonoids and related phenylpropanoid compounds.

The successful engineering of complex metabolic pathways will require, in addition to availability of cloned genes and promoters, knowledge of the regulatory mechanisms that control metabolic flux into the pathway including post-translational phenomena such as metabolite channeling. We are interested in modifying pathways for the synthesis of isoflavonoids and other bioactive phenylpropanoid compounds in transgenic plants. We describe studies on flux control utilizing transgenic tobacco plants that under- and over-express key biosynthetic enzymes, and outline experimental approaches for the molecular dissection of potential metabolic channels in the synthesis of antimicrobial flavonoid derivatives in alfalfa and other species.

Stress responses in alfalfa (Medicago sativa L). XXII. cDNA cloning and characterization of an elicitor-inducible isoflavone 7-O-methyltransferase.

Medicarpin, the major phytoalexin in alfalfa, is synthesized via the isoflavonoid branch of phenylpropanoid metabolism. The methyl group at the 9 position of medicarpin is generally accepted to arise via the methylation of the 4' position (B-ring) of daidzein. Surprisingly, the isoflavone-O-methyltransferase (IOMT), which is induced along with other enzymes involved in medicarpin biosynthesis, methylates the A-ring 7-hydroxyl group of daidzein in vitro, a reaction that probably does not occur in vivo. Utilizing internal amino acid sequence information from purified alfalfa IOMT, we have isolated three full-length IOMT cDNA clones. A search of the protein databases revealed sequence similarities to O-methyltransferases from various sources. The highest match (50.5% identity) was found between IOMT8 and 6a-hydroxymaackiain 3-O-methyltransferase from Pisum sativum. The molecular weight of alfalfa IOMT expressed in Escherichia coli was similar to that of purified IOMT from alfalfa cell cultures (41 kDa by SDS-PAGE). The recombinant enzyme catalyzed the O-methylation of A-ring hydroxyl group(s) of isoflavones, and could also methylate the pterocarpan (+) 6a-hydroxymaackiain. Alfalfa contains multiple IOMT genes, and closely related sequences are present in the genomes of chickpea and cowpea, species that also produce B-ring methylated isoflavonoids in vivo. Northern blot analysis indicated that IOMT transcripts are rapidly induced following elicitation, prior to the increase in IOMT activity and medicarpin accumulation. The possible role of the isoflavone 7-OMT in the synthesis of formononetin in vivo is discussed.

Second-site reversion of a dysfunctional mutation in a conserved region of the tobacco mosaic tobamovirus movement protein.

The N-terminal two-thirds of tobamovirus movement proteins (MPs) contain two well conserved regions. Within region I (amino acids 56-96) is an area predicted by computer analysis to have loop secondary structure (amino acids 76-87). A single or two double amino acid mutations were introduced into the loop in region I of the TMV MP to destabilize the structure. The three mutant MPs were defective in movement function. The single amino acid mutation resulted in a Pro81-->Ser substitution. The mutant virus, TP81S, containing the Pro81-->Ser substitution, was propagated on a transgenic line of Nicotiana tabacum that expresses the sunn-hemp mosaic tobamovirus MP. Inoculation of virus progeny from the transgenic plants onto hypersensitive N. tabacum indicated the presence of infectious virus at a low frequency. Necrotic lesions were detected at 4 days postinoculation, 2 days later than those induced by wild-type TMV. Inoculation of virus extracted from necrotic lesions onto N. tabacum resulted in a delayed and attenuated systemic infection relative to that induced by TMV, indicating that a second-site mutation restored movement function rather than a reversion of the original mutation. Sequence analysis revealed that the revertant MP gene had two additional amino acid substitutions, a Thr104-->Ile and a Arg167-->Lys. Introduction of the amino acid substitutions individually or in combination into the MP of TP81S indicated that both substitutions were required for the revertant phenotype. The data indicate that structure within region I is important in maintaining an active conformation for functional MP, that changes outside region I can compensate for alterations within the region, and suggest that region I may interact with a distal portion of the protein.

Affinity chromatography, substrate/product specificity, and amino acid sequence analysis of an isoflavone O-methyltransferase from alfalfa (Medicago sativa L.).

Isoflavone O-methyltransferase (IOMT) is a key enzyme in the biosynthesis of the phytoalexin medicarpin in alfalfa. In vivo, the B-ring 4'-hydroxyl group of the isoflavone daidzein is methylated. Surprisingly, the O-methyltransferase activity measured in vitro preferentially methylates the A-ring 7-hydroxyl group, a reaction that probably does not occur in vivo. To resolve this anomaly, we are attempting to clone the alfalfa IOMT. A substrate-based affinity chromatographic system was developed to purify the enzyme (molecular weight 41 kDa) to near homogeneity. Four internal peptide sequences were obtained from the purified protein, one of which has high (72%) sequence identity to a region of a catechol O-methyltransferase from barley. All four internal peptides, respectively, have about 55% amino acid sequence identity to four regions of 6alpha-hydroxymaackiain 3-O-methyltransferase from Pisum sativum, but have no sequence identity to alfalfa caffeic acid 3-O-methyltransferase or chalcone 2'-O-methyltransferase. The purified IOMT has substrate specificity toward isoflavones with a free 7-hydroxyl group, but can also methylate the 5-hydroxyl group of genistein.

Influence of heterologous tobamovirus movement protein and chimeric-movement protein genes on cell-to-cell and long-distance movement.

Sunn-hemp mosaic tobamovirus (SHMV) moves slowly from cell to cell in Nicotiana tabacum cv. Xanthi, but fails to move long distance. To determine the role of the SHMV movement protein (MP) in cell-to-cell and long-distance movement in tobacco, the SHMV MP gene was inserted into a TMV-cDNA clone that had approximately the 5'-half of the endogenous MP gene deleted. RNA transcripts inoculated onto tobacco induced systemic infections by 8 days postinoculation. Sequence analysis of the MP genes from purified virus isolated from systemically infected leaf tissue indicated that chimeric SHMV/TMV MP genes had been generated through RNA-RNA recombination within the 3'-termini of the MP gene sequences. When exchanged for the MP gene of TMV, three of four chimeric MP genes analyzed provided long-distance movement function for the hybrid viruses in tobacco. Two of the three hybrid viruses that moved long distance showed enhanced cell-to-cell movement relative to a recombinant TMV that expressed the intact SHMV MP gene. These observations suggest that the C-terminus of the TMV MP contains a determinant that can influence cell-to-cell movement in tobacco. A recombinant virus, TLSM, that expressed the intact SHMV MP gene exhibited cell-to-cell movement that was intermediate to SHMV and TMV, but failed to produce coat protein and was defective in long-distance movement. To further examine the role of the SHMV MP gene in long-distance movement, transgenic N. tabacum cv. Xanthi that expressed the wild-type SHMV MP gene were generated and found to facilitate rapid and efficient long-distance movement of a TMV mutant that contained a dysfunctional MP gene. Therefore, the inability of SHMV to systemically infect tobacco is a function of virus components and sequences other than those encoded by the SHMV MP gene.