Highly selective four-component domino multi-cyclizations for the synthesis of new fused acridines and azaheterocyclic skeletons have been established by mixing common reactants in isobutyric acid under microwave irradiation. The reactions proceeded at fast rates, and were conducted to completion within 20–30 min. Up to seven new chemical bonds, four rings and four stereocenters were assembled in a convenient one-pot operation. The resulting hexacyclic and pentacyclic fused acridines and their stereochemistry have been fully characterized and determined by X-ray structural analysis.
Antimicrobial peptides (AMPs) can cause lysis of target bacteria by directly inserting themselves into the lipid bilayer. This killing mechanism confounds the identification of the intracellular targets of AMPs. To circumvent this, we used a shuttle vector containing the inducible expression of a human cathelicidin-related AMP, LL-37, to examine its effect on Escherichia coli TOP10 under aerobic and anaerobic growth conditions. Induction of LL-37 caused growth inhibition and alteration in cell morphology to a filamentous phenotype. Further examination of the E. coli cell division protein FtsZ revealed that LL-37 did not interact with FtsZ. Moreover, intracellular expression of LL-37 results in the enhanced production of reactive oxygen species (ROS), causing lethal membrane depolarization under aerobic conditions. Additionally, the membrane permeability was increased after intracellular expression of LL37 under both aerobic and anaerobic conditions. Transcriptomic analysis revealed that intracellular LL-37 mainly affected the expression of genes related to energy production and carbohydrate metabolism. More specifically, genes related to oxidative phosphorylation under both aerobic and anaerobic growth conditions were affected. Collectively, our current study demonstrates that intracellular expression of LL-37 in E. coli can inhibit growth under aerobic and anaerobic conditions. While we confirmed that the generation of ROS is a bactericidal mechanism for LL-37 under aerobic growth conditions, we also found that the intracellular accumulation of cationic LL-37 influences the redox and ion status of the cells under both growth conditions. These data suggest that there is a new AMP-mediated bacterial killing mechanism that targets energy metabolism.
Catechins are the most important bioactive compounds in tea, and have been demonstrated to possess a wide variety of pharmacological activities. To characterize quantitative trait loci (QTLs) for catechins content in the tender shoots of tea plant, we constructed a moderately saturated genetic map using 406 simple sequence repeat (SSR) markers, based on a pseudo-testcross population of 183 individuals derived from an intraspecific cross of two Camellia sinensis varieties with diverse catechins composition. The map consisted of fifteen linkage groups (LGs), corresponding to the haploid chromosome number of tea plant (2n = 2x = 30). The total map length was 1,143.5 cM, with an average locus spacing of 2.9 cM. A total of 25 QTLs associated with catechins content were identified over two measurement years. Of these, nine stable QTLs were validated across years, and clustered into four main chromosome regions on LG03, LG11, LG12 and LG15. The population variability explained by each QTL was predominantly at moderate-to-high levels and ranged from 2.4% to 71.0%, with an average of 17.7%. The total number of QTL for each trait varied from four to eight, while the total population variability explained by all QTLs for a trait ranged between 38.4% and 79.7%. This is the first report on the identification of QTL for catechins content in tea plant. The results of this study provide a foundation for further cloning and functional characterization of catechin QTLs for utilization in improvement of tea plant.
The effect of trientine hydrochloride (TRIEN), a copper-selective chelating agent, on retinal inflammation induced by photocoagulation laser treatment was studied. Nine Long-Evans rats were treated with TRIEN (0.5 mmol/kg per day, intraperitoneal injection) for 9 days. On day 8, each animal underwent unilateral photocoagulation laser treatment with an argon dye laser. On day 9, animals were killed and the eyes processed for immunohistochemistry and light microscopy. In the TRIEN-treated group, retinal thickness and number of macrophages (ED-1) were both significantly lower than in the saline-treated, control group exposed to laser photocoagulation. The results support the hypothesis that selective copper chelation prior to laser treatment may inhibit ocular inflammation. The results suggest that pretreatment with a selective copper-chelating compound can minimize retinal inflammation secondary to laser photocoagulation treatment, which may improve overall outcome of photocoagulation treatment for diabetic retinopathy.
PMID: 18566852 CAMSID: cams4168
Inflammation; Trientine; Immunohistochemistry; Laser therapy; Retinal edema; Diabetic retinopathy
Alzheimer’s disease (AD) is the most common type of presenile and senile dementia. The human β-amyloid precursor cleavage enzyme (BACE-1) is a key enzyme responsible for amyloid plaque production, which implicates the progress and symptoms of AD. Here we assessed the anti-BACE-1 and behavioral activities of curcuminoids from rhizomes of Curcuma longa (Zingiberaceae), diarylalkyls curcumin (CCN), demethoxycurcumin (DMCCN), and bisdemethoxycurcumin (BDMCCN) against AD Drosophila melanogaster models.
Neuro-protective ability of the curcuminoids was assessed using Drosophila melanogaster model system overexpressing BACE-1 and its substrate APP in compound eyes and entire neurons. Feeding and climbing activity, lifespan, and morphostructural changes in fly eyes also were evaluated.
BDMCCN has the strongest inhibitory activity toward BACE-1 with 17 μM IC50, which was 20 and 13 times lower than those of CCN and DMCCN respectively. Overexpression of APP/BACE-1 resulted in the progressive and measurable defects in morphology of eyes and locomotion. Remarkably, supplementing diet with either 1 mM BDMCCN or 1 mM CCN rescued APP/BACE1-expressing flies and kept them from developing both morphological and behavioral defects. Our results suggest that structural characteristics, such as degrees of saturation, types of carbon skeleton and functional group, and hydrophobicity appear to play a role in determining inhibitory potency of curcuminoids on BACE-1.
Further studies will warrant possible applications of curcuminoids as therapeutic BACE-1 blockers.
Alzheimer’s disease; Drosophila melanogaster; Curcuma longa; Curcuminoids; BACE-1; Structure–activity relationship
Cryptorchidism is a common congenital birth defect in human beings with the possible complication of infertility. An in vitro model of cryptorchidism might be valuable due to the inaccessibility of human embryos for research purposes. In this study, we reprogrammed urine cells containing genetic variations in insulin-like factor 3, zinc finger (ZNF) 214, and ZNF215 from a cryptorchid patient by introducing human OCT4, SOX2, C-MYC, and KLF4 with lentivirus. The cells were then replated on irradiated mouse embryonic fibroblasts and cultured with the human embryonic stem (ES) cell medium. The compact colonies with well-defined borders were manually picked, and 2 induced pluripotent cell lines were fully characterized. Our results demonstrated that these 2 cell lines were similar to human ES cells in morphological appearance, marker expression, and epigenetic status of the pluripotent cell-specific gene, OCT4. These cells could be differentiated into cells of all 3 germ layers in teratomas and in vitro, including into the VASA-positive germ cell lineage. Both parental urine cells and the reprogrammed cells possessed the normal karyotype and the same short tandem repeat loci, indicating that these 2 cell population share the same genetic identity. This establishment and characterization of human urine-derived cryptorchid-specific induced pluripotent stem cells could present a good human genetic system for future studies investigating the molecular mechanism of cryptorchidism.
PLGA microparticles loaded with three different fluorescent model drugs, fluorescein sodium (hydrophilic), sulforhodamine (amphoteric), and boron-dipyrromethene (BODIPY® 493/503, lipophilic), were prepared by the solvent evaporation technique. Due to varying hydrophilicity, the diameters of the microparticles ranged between 4.1 and 4.7 μm. According to fluorimetric analysis, the loading varied from 0.06 to 2.25 μg of the model drug per mg PLGA. In terms of the release profile, the fluorescein sodium-entrapped formulation exhibited thermo-responsive release kinetics. In the case of sulforhodamine- and BODIPY® 493/503-loaded particles, almost no release was observed, neither at 4°C nor 37°C during the first 50 hours. Furthermore, to estimate the bioadhesive properties of such drug delivery systems, the surface of the loaded particles was grafted with wheat germ agglutinin by applying the carbodiimide method. Cytoadhesion studies with Caco-2 monolayers revealed an up to 1.9-fold and 3.6-fold increase in the bioadhesion of the lectin-functionalized, model drug-loaded particles as compared to the albumin- and non-grafted microcarriers, respectively. All in all, the results clearly indicated that the lipophilicity of the polymer matching that of the drug favored entrapment, whereas mismatching impeded loading into the PLGA-microparticles. Even in the case of low loading, these delivery systems might be useful for the fluorescent detections and microscopic imaging of cellular interactions due to their fluorescent properties and lack of dye leakage. Moreover, lectin grafting can mediate bioadhesive properties to such particulate drug carriers which could be a promising approach to improve drug delivery.
Caco-2; Cytoadhesion; Microparticles; PLGA; Wheat Germ Agglutinin (WGA)
Programmed death-1 (PD-1) expression was investigated in CD4+ and CD8+ T cells from hepatitis B virus (HBV)-infected patients at the chronic hepatitis B (CHB) infection, liver cirrhosis (LC), and hepatocellular carcinoma (HCC) stages.
PD-1 expression in circulating CD4+ and CD8+ T cells was detected by flow cytometry. The correlations between PD-1 expression and HBV viral load, alanine aminotransaminase (ALT) levels and aspartate aminotransferase (AST) levels were analyzed using GraphPad Prism 5.0.
PD-1 expression in CD4+ and CD8+ T cells was significantly increased in both the CHB group and advanced-stage group (LC plus HCC). In the CHB group, PD-1 expression in both CD4+ and CD8+ T cells was positively correlated with the HBV viral load, ALT, and AST levels. However, in the LC plus HCC group, significant correlations between PD-1 expression and the clinical parameters were nearly absent.
PD-1 expression in peripheral CD4+ and CD8+ T cells is dynamic, changes with HBV infection progression, and is related to HBV viral load and liver function, especially in CHB. PD-1 expression could be utilized as a potential clinical indicator to determine the extent of virus replication and liver injury.
Programmed death-1; Hepatitis B virus; Hepatitis B, chronic; Liver cirrhosis; Carcinoma, hepatocellular
Objective: A study in a Caucasian population has identified two single-nucleotide polymorphisms (SNPs) in ZNF533, one in DOCK4, and two in IMMP2L, which were all significantly associated with autism. They are located in AUTS1 and AUTS5, which have been identified as autism susceptibility loci in several genome-wide screens. The present study aimed to investigate whether ZNF533, DOCK4, and IMMP2L genes are also associated with autism in a northeastern Chinese Han population. Methods: We performed a similar association study using families with three individuals (one autistic child and two unaffected parents). A family-based transmission disequilibrium test (TDT) was used to analyze the results. Results: There were significant associations between autism and the two SNPs of ZNF533 gene (rs11885327: χ
2=4.5200, P=0.0335; rs1964081: χ
2=4.2610, P=0.0390) and the SNP of DOCK4 gene (rs2217262: χ
2=5.3430, P=0.0208). Conclusions: Our data suggest that ZNF533 and DOCK4 genes are linked to a predisposition to autism in the northeastern Chinese Han population.
Autism; ZNF533; DOCK4; IMMP2L; Northeastern Chinese Han population; Single-nucleotide polymorphism
Association of hepatitis B virus (HBV) genotype C with hepatocellular carcinoma (HCC) development remains controversial. HBV basal core promoter (BCP) double mutations (T1762A1764) are very strong confounding factors of genotypes B and C in HCC development.
To investigate the association of HBV genotype C with HCC development after controlling for BCP double mutations.
Materials and methods:
Four hundred and two serum samples from patients with HCC, liver cirrhosis (LC) and chronic hepatitis (CH) and also from asymptomatic HBsAg carriers were analyzed.
Genotypes B (31.1%), C (62.8%), and I (6.1%) were detected. With the severity of liver disease the prevalence of genotype B decreased, but genotype C increased. No trend was found for genotype I. The prevalence of BCP double mutations in genotypes C and I viruses was significantly higher than genotype B. BCP double mutations are risk factors for CH, LC and HCC. Genotype C was not identified as a particular risk factor for HCC prior to the stratification analysis but after that genotype C viruses with BCP double mutations were found to be a particular risk factor for HCC (P = 0.008, OR = 17.19 [95% CI: 2.10 - 140.41]), but those with the wild-type BCP were not. In the interaction analysis, genotype C and BCP double mutations were found to have a synergistic effect on HCC development (P < 0.0001, OR = 52.56 [95% CI: 11.49-240.52]).
The effect of HBV genotype C on the development of HCC differs between wild-type viruses and those with BCP double mutations, suggesting that not all individuals infected with genotype C HBV are at increased risk of HCC.
Hepatitis B Virus; Genotypes; Basal Core Promoter Double Mutations; Hepatocellular Carcinoma
AIM: To investigate H2O2-induced promotion proliferation and malignant transformation in WB-F344 cells and anti-tumor effects of ursolic acid (UA) and oleanolic acid (OA).
METHODS: WB-F344 cells were continuously exposed to 7 x 10-7 mol/L H2O2 for 21 d. Observations of cell morphology, colony formation rates, flow cytometric analysis of cell cycle changes and aneuploidy formation indicated that H2O2 was able to induce malignant transformation of WB-F344 cells. We treated malignantly transformed WB-F344 cells with 4 μmol/L OA or 8 μmol/L UA for 72 h and analyzed the cell cycle distribution by flow cytometry.
RESULTS: MTT assay showed that 7 x 10-7 mol/L H2O2 decreased G1 phase subpopulation from 73.8% to 49.6% compared with the control group, and increased S phase subpopulation from 14.5% to 31.8% (P < 0.05 vs control group). Cell morphology showed that nucleus to cytoplasm ratio increased, many mitotic cells, prokaryotes and even tumor giant cells were shown in H2O2-induced WB-F344 cells. Fluorescence activated cell sorting analysis showed that WB-F344 cell aneuploidy increased to 12% following H2O2 treatment. Flow cytometric analysis of the transformed WB-F344 cells following treatment with OA (4 μmol/L) and UA (8 μmol/L) showed that OA increased G1 subpopulation to 68.6%, compared to 49.7% in unexposed cells. UA increased G1 subpopulation to 67.4% compared to 49.7% in unexposed cells (P < 0.05 vs H2O2 model group).
CONCLUSION: H2O2 causes the malignant transformation of WB-F344 cells. OA and UA exert anti-tumor effects by inhibiting the proliferation in malignantly transformed WB-F344 cells.
Oxidative stress; Hepatocarcinogenesis; Malignant transformation; Oleanolic acid; Ursolic acid
Vascular endothelial growth factor (VEGF)-D has been shown to promote lymph node metastasis in several cancers. Although generally overexpressed in ovarian carcinoma, its role in nodal dissemination of this cancer is unclear. To clarify the role of VEGF-D and the underlying molecular mechanisms, we investigated the function of VEGF-D using a mouse xenograft model of ovarian cancer.
Human ovarian serous adenocarcinoma SKOV3 cells were transfected with VEGF-D recombinant plasmid DNA, or with control vectors. The cells were injected subcutaneously into the footpads of nude mice. Tumor growth was evaluated weekly. Draining lymphatics were observed grossly with Evan’s blue lymphangiography. Tumoral lymphatics were delineated with both Evan’s blue and LYVE-1 immunostaining. Tumor metastases to lymph nodes were evaluated by H&E and CA125/CD40 staining. Expression of VEGF-D in primary tumors and levels of CA125 in involved lymph nodes were examined by immunohistochemistry. Tumor cell apoptosis was analyzed by Hoechst dyeing.
Mice bearing VEGF-D overexpressing xenografts showed a significantly higher rate of lymph node metastasis and markedly greater tumor volume compared with the controls. The functional lymphatic vessels were denser and enlarged in marginal and central tumor portions. Additionally, higher CA125 expression was observed in the involved lymph nodes. Mice bearing VEGF-D overexpressing xenografts also exhibited a markedly lower apoptotic index compared with the controls.
Our data demonstrate the important role of VEGF-D in promoting lymph node metastasis by increasing tumor lymphangiogenesis, stimulating draining lymphatic vessel formation, and enhancing tumor invasiveness. Our findings show that VEGF-D can be a promising therapeutic target for ovarian cancer.
Ovarian carcinoma; VEGF-D; Metastasis; Lymphangiogenesis; Xenograft
To illustrate the ethical challenges that arose from investigating a novel treatment procedure, transcranial direct current stimulation (tDCS), in a research participant with aphasia.
First, we reviewed the current evidence supporting the use of tDCS in aphasia research, highlighting methodological gaps in our knowledge of tDCS. Second, we examined the case of Mr. C, a person with chronic aphasia who participated in a research protocol investigating the impact of tDCS on aphasia treatment.
We describe the procedures that he underwent and the resulting behavioral and neurophysiological outcomes bed. Finally, we share the steps that were taken to balance beneficence and nonmaleficence, and to ensure Mr. C’s autonomy. Conclusion: Researchers must consider not only the scientific integrity of their studies, but also potential ethical issues and consequences to the research participants.
aphasia; brain stimulation; ethics; functional MRI; rehabilitation
Insulin resistance plays an important role in the development of type 2 diabetes mellitus (T2DM) and hypertension. The purpose of the present study was to evaluate the association between four insulin resistance genes (ADIPOQ, LEPR, RETN, and TRIB3) and both T2DM and hypertension. A total of 768 Han Chinese subjects were recruited into this study, including 188 cases who had T2DM alone, 223 cases who had hypertension alone, 181 cases with both T2DM and hypertension, and 176 control subjects with neither T2DM nor hypertension. Twenty-three tag SNPs in four insulin resistance genes were genotyped and analyzed for association with T2DM and hypertension. One intron SNP (rs13306519) in LEPR and one 3′UTR SNP (rs1063537) in ADIPOQ demonstrated a significant association with T2DM (P = 0.024 and 0.014 respectively). Another intron SNP (rs12037879) in LEPR and a promoter region SNP (rs266729) in ADIPOQ were significantly associated with hypertension (P = 0.041 and 0.042, respectively). These associations survived the permutation test (P = 0.023, 0.018, 0.026, and 0.035, respectively). These associations were still found to be significant in the additive model after adjusting for potential confounding factors including age, sex, BMI, HDL, LDL, total cholesterol, and triglyceride levels (P = 0.024, 0.016, 0.04, and 0.043, respectively). No other gene variants were found to be significantly associated with T2DM or hypertension (P > 0.05). None of the studied gene variants were found to be significantly associated with T2DM+ hypertension (P > 0.05). A significant interaction was observed between two SNPs rs13306519 in LEPR and rs266729 in ADIPOQ for T2DM (P_int = 0.012, OR_int = 2.67) and hypertension (P_int = 0.0041, OR_int = 2.23). These findings suggest that variants in ADIPOQ and LEPR are risk factors for T2DM and hypertension in the Chinese population and that variants in RETN and TRIB3 are not major risk factors for these diseases.
Type 2 diabetes mellitus; Hypertension; Polymorphisms; ADIPOQ; LEPR
Endoscopic retrograde cholangiopancreatography (ERCP) is efficacious in patients who have undergone Billroth II gastroenterostomies, but the success rate decreases in patients who also have experienced Braun anastomoses. There are currently no reports describing the preferred enterography route for cannulation in these patients. We first review the patient’s previous surgery records, which most often indicate that the efferent loop is at the greater curvature of the stomach. We recommend extending the duodenoscope along the greater curvature of the stomach and then advancing it through the “lower entrance” at the site of the gastrojejunal anastomosis, along the efferent loop, and through the “middle entrance” at the site of the Braun anastomosis to reach the papilla of Vater. Ten patients who had each undergone Billroth II gastroenterostomy and Braun anastomosis between January 2009 and December 2011 were included in our study. The overall success rate of enterography was 90% for the patients who had undergone Billroth II gastroenterostomy and Braun anastomosis, and the therapeutic success rate was 80%. We believe that this enterography route for ERCP is optimal for a patient who has had Billroth II gastroenterostomy and Braun anastomosis and helps to increase the success rate of the procedure.
Optimal enterography route; Endoscopic retrograde cholangiopancreatography; Billroth II; Gastroenterostomy; Braun anastomosis
Hepatitis B virus (HBV) infection significantly contributes to the onset of liver disease and hepatocellular carcinoma. Understanding the pathogenesis of HBV infection susceptibility could help us to control HBV infection effectively.
This study investigated single nucleotide polymorphisms (SNPs) of the tripartite motif-containing 22 (TRIM22) gene associated with HBV infection outcome.
Patients and Methods:
A total of 765 Chinese Han subjects were enrolled: 293 patients were presented with chronic hepatitis B (CHB), 224 were asymptomatic HBV carriers, 248 had self-limited HBV infection, and all of them were recruited for TRIM22 SNPs genotyping. RING and SPRY domains of TRIM22 gene were DNA-sequenced, and HBV serum markers and HBV DNA were measured quantitatively in all subjects.
243 (31.76%) of 765 Chinese Han patients showed genetic variation in the TRIM22 gene. TRIM22 SNPs were mainly in RING area -364T/C site, accounting for 98.35% of the population. There were no significant differences (P > 0.05) in the RING domain -364T/C SNP and allele frequencies between patients with chronic hepatitis and asymptomatic HBV carriers. The CC genotype of TRIM22 gene RING domain -364T/C locus (rs10838543) was associated with chronic HBV infection (OR = 2.30, 95% CI = 1.24-3.97, P = 0.0012; OR = 2.26, 95% CI = 1.08-3.74, P = 0.002) and a mutant allele C carrier of the TRIM22 gene was associated with HBV chronic infection (OR = 1.97, 95% CI = 1.10-3.75, P = 0.0049; OR = 2.12, 95% CI = 1.17-3.89, P = 0.0038).
TRIM22 gene RING domain -364T/C polymorphism is associated with chronic HBV infection in Chinese Han population.
Hepatitis B virus; TRIM22 Protein, Human; Polymorphism, Single Nucleotide
Community-acquired pneumonia in children is common in China. To understand current clinical characteristics and practice, we conducted a cross-sectional study to analyze quality of care on childhood pneumonia in eight eastern cities in China.
Consecutive hospital records between January 1, 2010 and December 31, 2010 were collected from 13 traditional Chinese medicine (TCM) and western medicine (WM) hospitals in February, May, August, and November (25 cases per season, 100 cases over the year), respectively. A predesigned case report form was used to extract data from the hospital medical records.
A total of 1298 cases were collected and analyzed. Symptoms and signs upon admission at TCM and WM hospitals were cough (99.3% vs. 98.6%), rales (84.8% vs. 75.0%), phlegm (83.3% vs. 49.1%), and fever (74.9% vs. 84.0%) in frequency. Patients admitted to WM hospitals had symptoms and signs for a longer period prior to admission than patients admitted to TCM hospitals. Testing to identify etiologic agents was performed in 1140 cases (88.4%). Intravenous antibiotics were administered in 99.3% (595/598) of cases in TCM hospitals and in 98.6% (699/700) of cases in WM hospitals. Besides, Chinese herbal extract injection was used more frequently in TCM hospitals (491 cases, 82.1%) than in WM hospitals (212 cases, 30.3%) (p < 0.01). At discharge, 818 cases (63.0%) were clinically cured, with a significant difference between the cure rates in TCM (87.6%) and WM hospitals (42.0%) (OR = 9.8, 95% confidence interval (CI): 7.3 ~ 12.9, p < 0.01). Pathogen and previous medical history were more likely associated with the disappearance of rales (OR = 7.2, 95% CI: 4.8 ~ 10.9). Adverse effects were not reported from the medical records.
Intravenous use of antibiotics is highly prevalent in children with community-acquired pneumonia regardless of aetiology. There was difference between TCM and WM hospitals with regard to symptom profile and the use of antibiotics. Intravenous use of herbal injection was higher in TCM hospitals than in WM hospitals. Most of the cases were diagnosed based on clinical signs and symptoms without sufficient confirmation of aetiology. Audit of current practice is urgently needed to improve care.
Childhood pneumonia; Community-acquired; Clinical characteristics; Treatment; Cross-sectional study; Chinese population
Trace elements have been recognized to play an important role in the development of Parkinson’s disease (PD). However, it is difficult to precisely identify the relationship between these elements and the progression of PD because of an insufficient number of patients. In this study, quantifications of selenium (Se), copper (Cu), iron (Fe) and zinc (Zn) by atomic absorption spectrophotometry were performed in plasma from 238 PD patients and 302 controls recruited from eastern China, which is so far the largest cohort of PD patients and controls for measuring plasma levels of these elements. We found that plasma Se and Fe concentrations were significantly increased whereas Cu and Zn concentrations decreased in PD patients as compared with controls. Meanwhile, these four elements displayed differential changes with regard to age. Linear and logistic regression analyses revealed that both Fe and Zn were negatively correlated with age in PD patients. Association analysis suggests that lower plasma Se and Fe levels may reduce the risk for PD, whereas lower plasma Zn is probably a PD risk factor. Finally, a model was generated to predict PD patients based on the plasma concentrations of these four trace elements as well as other features such as sex and age, which achieved an accuracy of 80.97±1.34% using 10-fold cross-validation. In summary, our data provide new insights into the roles of Se, Cu, Fe and Zn in PD progression.
Identifying Ca2+-binding sites in proteins is the first step towards understanding the molecular basis of diseases related to Ca2+-binding proteins. Currently, these sites are identified in structures either through X-ray crystallography or NMR analysis. However, Ca2+-binding sites are not always visible in X-ray structures due to flexibility in the binding region or low occupancy in a Ca2+-binding site. Similarly, both Ca2+ and its ligand oxygens are not directly observed in NMR structures. To improve our ability to predict Ca2+-binding sites in both X-ray and NMR structures, we report a new graph theory algorithm (MUGC) to predict Ca2+-binding sites. Using carbon atoms covalently bonded to the chelating oxygen atoms, and without explicit reference to side-chain oxygen ligand coordinates, MUGC is able to achieve 94% sensitivity with 76% selectivity on a dataset of X-ray structures comprised of 43 Ca2+-binding proteins. Additionally, prediction of Ca2+-binding sites in NMR structures were obtained by MUGC using a different set of parameters determined by analysis of both Ca2+-constrained and unconstrained Ca2+-loaded structures derived from NMR data. MUGC identified 20 out of 21 Ca2+-binding sites in NMR structures inferred without the use of Ca2+ constraints. MUGC predictions are also highly-selective for Ca2+-binding sites as analyses of binding sites for Mg2+, Zn2+, and Pb2+ were not identified as Ca2+-binding sites. These results indicate that the geometric arrangement of the second-shell carbon cluster is sufficient for both accurate identification of Ca2+-binding sites in NMR and X-ray structures, and for selective differentiation between Ca2+ and other relevant divalent cations.
Ca2+-binding proteins; graph theory; carbon clusters; side-chain center of mass; NMR
A retrospective study was conducted to evaluate the advantages of anterolateral thigh (ALT) flap in tongue reconstruction.
From September 2008 to February 2012, patients receiving ALT flap tongue reconstruction were included in the study. Patients undergoing ALT flap were compared with those undergoing similar surgery with radial forearm flap (RFF). The medical records of the included patients were reviewed, and a questionnaire was used to assess acceptability of the surgery.
All flaps (both ALT and RFF) were successful In the ALT group, most patients were satisfied with the appearance of the reconstructed tongue and the intelligibility of their speech, and there were fewer complications with this technique compared with the RFF.
The ALT flap is an ideal method for tongue reconstruction. The thickness and volume of the ALT flap can be adjusted based on the individual extent of the defect, and it can not only provide bulk but also ensure mobility, and it has other advantages also, including a long pedicle and low donor site morbidity.
Anterolateral thigh flap; Functional outcome; Tongue reconstruction; Microsurgery
Equine lentivirus receptor 1 (ELR1) has been identified as the sole receptor for equine infectious anemia virus (EIAV) and is a member of the tumor necrosis factor receptor (TNFR) superfamily. In addition to the previously described membrane-associated form of ELR1, two other major alternative splicing variant mRNAs were identified in equine monocyte-derived macrophages (eMDMs). One major spliced species (ELR1-IN) contained an insertion of 153 nt, which resulted in a premature stop codon situated 561 nt upstream of the predicted membrane spanning domain. The other major species (ELR1-DE) has a deletion of 109 nt that causes a shift of the open reading frame and generates a stop codon 312 nt downstream. Because ELR1-DE presumably encodes a peptide of a mere 23 residues, only ELR1-IN was further analyzed. The expression of a soluble form of ELR1 (sELR1) by ELR1-IN was confirmed by Western blot and immunofluorescence analyses. Similar to ELR1, the transcription level of ELR1-IN varied among individual horses and at different time points in the same individuals. The ratio of ELR1-IN mRNA species to ELR1 mRNA was approximately 1∶2.5. Pre-incubation of the recombinant sELR1 with EIAV significantly inhibited EIAV infection in equine macrophages, the primary in vivo target cell of the virus. Fetal equine dermal (FED) cells are susceptible to EIAV in vitro, and the replication of EIAV in FED cells transiently transfected with ELR1-IN was markedly reduced when compared with replication in cells transfected with the empty vector. Finally, the expression levels of both forms of the EIAV receptor were significantly regulated by infection with this virus. Taken together, our data indicate that sELR1 acts as a secreted cellular factor that inhibits EIAV infection in host cells.
2’,3’-cyclic nucleotide 3’-phosphodiesterase (CNP) is a member of the interferon-stimulated genes, which includes isoforms CNP1 and CNP2. CNP1 is locally expressed in the myelin sheath but CNP2 is additionally expressed at low levels outside the nervous system. CNPs regulate multiple cellular functions and suppress protein production by association with polyadenylation of mRNA. Polyadenylation of Hepatitis B virus (HBV) RNAs is crucial for HBV replication. Whether CNPs interact with polyadenylation signal of HBV RNAs and interfere HBV replication is unknown. In this study, we evaluated expressions of CNP isoforms in hepatoma cell lines and their effects on HBV replication. We found that CNP2 is moderately expressed and gently responded to interferon treatment in HepG2, but not in Huh7 cells. The CNP1 and CNP2 potently inhibited HBV production by blocking viral proteins synthesis and reducing viral RNAs, respectively. In chronic hepatitis B patients, CNP was expressed in most of HBV-infected hepatocytes of liver specimens. Knockdown of CNP expression moderately improved viral production in the HepG2.2.15 cells treated with IFN-α. In conclusion, CNP might be a mediator of interferon-induced response against HBV.
To investigate the magnetic resonance imaging (MRI) characteristics of ovarian Sertoli-Leydig cell tumors (SLCT).
The clinical, MRI and pathological findings of five cases of SLCT were reviewed retrospectively. MRI appearances of tumors including laterality, shape and size, architecture, wall, septa and vegetation, signal intensity and contrast-enhancement pattern were evaluated and correlated with pathological findings.
Two tumors were solid which appeared as low signal intensity on T1-weighted imaging (T1WI) and moderate on T2-weighted imaging (T2WI) with multiple small cysts in one of them. The remaining three SLCT were multilocular cystic with the irregularly thickened wall and septa, and with solid area and mural nodules in one of them. The cystic components had the same signal intensity as urine. All the solid components were intensely enhanced after administration of contrast medium. All five tumors were pathologically intermediate differentiation and at FIGO stage I.
SLCT demonstrate variable MRI morphological appearances. However, the irregularly thickened wall and septa, the moderate T2WI signal intensity and obvious enhancement in the solid components are three MRI features.
Ovary; Sertoli-Leydig cell tumors; Magnetic resonance imaging; Pathology
Our previous studies have demonstrated that the urotensin (UII) and its receptor are up-regulated in the skeletal muscle of mice with type II diabetes mellitus (T2DM), but the significance of UII in skeletal muscle insulin resistance remains unknown. The purpose of this study was to investigate the effect of UII on NADPH oxidase and glucose transport signaling pathways in the skeletal muscle of mice with T2DM and in C2C12 mouse myotube cells. KK/upj-AY/J mice (KK) mice were divided into the following groups: KK group, with saline treatment for 2 weeks; KK+ urantide group, with daily 30 µg/kg body weight injections over the same time period of urantide, a potent urotensin II antagonist peptide; Non-diabetic C57BL/6J mice were used as normal controls. After urantide treatment, mice were subjected to an intraperitoneal glucose tolerance test, in addition to measurements of the levels of ROS, NADPH oxidase and the phosphorylated AKT, PKC and ERK. C2C12 cells were incubated with serum-free DMEM for 24 hours before conducting the experiments, and then administrated with 100 nM UII for 2 hours or 24 hours. Urantide treatment improved glucose tolerance, decreased the translocation of the NADPH subunits p40-phox and p47-phox, and increased levels of the phosphorylated PKC, AKT and ERK. In contrast, UII treatment increased ROS production and p47-phox and p67-phox translocation, and decreased the phosphorylated AKT, ERK1/2 and p38MAPK; Apocynin abrogated this effect. In conclusion, UII increased ROS production by NADPH oxidase, leading to the inhibition of signaling pathways involving glucose transport, such as AKT/PKC/ERK. Our data imply a role for UII at the molecular level in glucose homeostasis, and possibly in skeletal muscle insulin resistance in T2DM.
Nanocarriers represent an attractive means of drug delivery, but their biosafety must be established before their use in clinical research.
Four kinds of amphiphilic polymeric (PEG-PG-PCL, PEEP-PCL, PEG-PCL and PEG-DSPE) micelles with similar hydrophilic or hydrophobic structure were prepared and their in vitro and in vivo safety were evaluated and compared.
In vitro nanotoxicity evaluations included assessments of cell morphology, cell volume, inflammatory effects, cytotoxicity, apoptosis and membrane fluidity. An umbilical vein cell line (Eahy.926) and a kind of macrophages (J774.A1) were used as cell models considering that intravenous route is dominant for micelle delivery systems. In vivo analyses included complete blood count, lymphocyte subset analysis, detection of plasma inflammatory factors and histological observations of major organs after intravenous administration to KM mice.
All the micelles enhanced inflammatory molecules in J774.A1 cells, likely resulting from the increased ROS levels. PEG-PG-PCL and PEEP-PCL micelles were found to increase the J774.A1 cell volume. This likely correlated with the size of PEG-PG-PCL micelles and the polyphosphoester structure in PEEP-PCL. PEG-DSPE micelles inhibited the growth of Eahy.926 cells via inducing apoptosis. This might relate to the structure of DSPE, which is a type of phospholipid and has good affinity with cell membrane. No evidence was found for cell membrane changes after treatment with these micelles for 24 h. In the in vivo study, during 8 days of 4 time injection, each of the four nanocarriers altered the hematic phase differently without changes in inflammatory factors or pathological changes in target organs.
These results demonstrate that the micelles investigated exhibit diverse nanotoxicity correlated with their structures, their biosafety is different in different cell model, and there is no in vitro and in vivo correlation found. We believe that this study will certainly provide more scientific understandings on the nanotoxicity of amphiphilic polymeric micelles.
Nanotoxicity; Amphiphilic polymeric micelles; J774.A1 cells; Eahy.926 cells; KM mice