Agrobacterium tumefaciens-mediated transformation for edible mushrooms has been previously established. However, the enhancement of heterologous protein production and the expression of multi-target genes remains a challenge. In this study, heterologous protein expression in the enoki mushroom Flammulina velutipes was notably enhanced using 2A peptide-mediated cleavage to co-express multiple copies of single gene. The polycistronic expression vectors were constructed by connecting multi copies of the enhanced green fluorescent protein (egfp) gene using 2A peptides derived from porcine teschovirus-1. The P2A peptides properly self-cleaved as shown by the formation of the transformants with antibiotic resistant capacity and exciting green fluorescence levels after introducing the vectors into F. velutipes mycelia. The results of western blot analysis, epifluorescent microscopy and EGFP production showed that heterologous protein expression in F. velutipes using the polycistronic strategy increased proportionally as the gene copy number increased from one to three copies. In contrast, much lower EGFP levels were detected in the F. velutipes transformants harboring four copies of the egfp gene due to mRNA instability. The polycistronic strategy using 2A peptide-mediated cleavage developed in this study can not only be used to express single gene in multiple copies, but also to express multiple genes in a single reading frame. It is a promising strategy for the application of mushroom molecular pharming.
Little is known on the effectiveness of influenza vaccine in ESRD patients. This study compared the incidence of hospitalization, morbidity, and mortality in end-stage renal disease (ESRD) patients undergoing hemodialysis (HD) between cohorts with and without influenza vaccination.
We used the insurance claims data from 1998 to 2009 in Taiwan to determine the incidence of these events within one year after influenza vaccination in the vaccine (N = 831) and the non-vaccine (N = 3187) cohorts. The vaccine cohort to the non-vaccine cohort incidence rate ratio and hazard ratio (HR) of morbidities and mortality were measured.
The age-specific analysis showed that the elderly in the vaccine cohort had lower hospitalization rate (100.8 vs. 133.9 per 100 person-years), contributing to an overall HR of 0.81 (95% confidence interval (CI) 0.72–0.90). The vaccine cohort also had an adjusted HR of 0.85 [95% CI 0.75–0.96] for heart disease. The corresponding incidence of pneumonia and influenza was 22.4 versus 17.2 per 100 person-years, but with an adjusted HR of 0.80 (95% CI 0.64–1.02). The vaccine cohort had lowered risks than the non-vaccine cohort for intensive care unit (ICU) admission (adjusted HR 0.20, 95% CI 0.12–0.33) and mortality (adjusted HR 0.50, 95% CI 0.41–0.60). The time-dependent Cox model revealed an overall adjusted HR for mortality of 0.30 (95% CI 0.26–0.35) after counting vaccination for multi-years.
ESRD patients with HD receiving the influenza vaccination could have reduced risks of pneumonia/influenza and other morbidities, ICU stay, hospitalization and death, particularly for the elderly.
Endometriosis is a common gynecological condition with complex etiology defined by the presence of endometrial glands and stroma outside the womb. Endometriosis is a common cause of both cyclic and chronic pelvic pain, reduced fertility, and reduced quality-of-life. Diagnosis and treatment of endometriosis is, on average, delayed by 7–10 years from the onset of symptoms. Absence of a timely and non-invasive diagnostic tool is presently the greatest barrier to the identification and treatment of endometriosis. Twin and family studies have documented an increased relative risk in families. To identify genetic factors that contribute to endometriosis we conducted a two-stage genome-wide association study (GWAS) of a European cohort including 2,019 surgically confirmed endometriosis cases and 14,471 controls. Three of the SNPs we identify associated at P<5×10−8 in our combined analysis belong to two loci: LINC00339-WNT4 on 1p36.12 (rs2235529; P = 8.65×10−9, OR = 1.29, CI = 1.18–1.40) and RND3-RBM43 on 2q23.3 (rs1519761; P = 4.70×10−8, OR = 1.20, Cl = 1.13–1.29, and rs6757804; P = 4.05×10−8, OR = 1.20, Cl = 1.13–1.29). Using an adjusted Bonferoni significance threshold of 4.51×10−7 we identify two additional loci in our meta-analysis that associate with endometriosis:, RNF144B-ID4 on 6p22.3 (rs6907340; P = 2.19×10−7, OR = 1.20, Cl = 1.12–1.28), and HNRNPA3P1-LOC100130539 on 10q11.21 (rs10508881; P = 4.08×10−7, OR = 1.19, Cl = 1.11–1.27). Consistent with previously suggested associations to WNT4 our study implicate a 150 kb region around WNT4 that also include LINC00339 and CDC42. A univariate analysis of documented infertility, age at menarche, and family history did not show allelic association with these SNP markers. Clinical data from patients in our study reveal an average delay in diagnosis of 8.4 years and confirm a strong correlation between endometriosis severity and infertility (n = 1182, P<0.001, OR = 2.18). This GWAS of endometriosis was conducted with high diagnostic certainty in cases, and with stringent handling of population substructure. Our findings broaden the understanding of the genetic factors that play a role in endometriosis.
Domain swapping is a mechanism for forming protein dimers and oligomers with high specificity. It is distinct from other forms of oligomerization in that the binding interface is formed by reciprocal exchange of polypeptide segments. Swapping plays a physiological role in protein-protein recognition and it can also potentially be exploited as a mechanism for controlled self assembly. Here, we demonstrate that domain-swapped interfaces can be engineered by inserting one protein into a surface loop of another protein. The key to facilitating a domain swap is to destabilize the protein when it is monomeric but not when it is oligomeric. We achieve this condition by employing the ‘mutually exclusive folding’ design to apply conformational stress to the monomeric state. Ubiquitin is inserted into one of six surface loops of barnase. The 38 Å amino-to-carboxy terminal distance of ubiquitin stresses the barnase monomer, causing it to split at the point of insertion. The 2.2 Å X-ray structure of one insertion variant reveals that strain is relieved by intermolecular folding with an identically-unfolded barnase domain, resulting in a domain-swapped polymer. All six constructs oligomerize suggesting that inserting ubiquitin into each surface loop of barnase results in a similar domain-swapping event. Binding affinity can be tuned by varying the length of the peptide linkers used to join the two proteins, which modulates the extent of stress. Engineered, swapped proteins have the potential to be used to fabricate ‘smart’ biomaterials, or as binding modules from which to assemble heterologous, multi-subunit protein complexes.
There are many discussions about dyslexia based on studies conducted in western countries, and some risk factors to dyslexia, such as gender and home literacy environment, have been widely accepted based on these studies. However, to our knowledge, there are few studies focusing on the risk factors of dyslexia in China. Therefore, the aim of our study was to investigate the prevalence of dyslexia and its potential risk factors.
A cross-sectional study was conducted in Qianjiang, a city in Hubei province, China. Two stages sampling strategy was applied to randomly selected 5 districts and 9 primary schools in Qianjiang. In total, 6,350 students participated in this study and there were 5,063 valid student questionnaires obtained for the final analyses. Additional questionnaires (such as Dyslexia Checklist for Chinese Children and Pupil Rating Scale) were used to identify dyslexic children. The chi-square test and multivariate logistic regression were employed to reveal the potential risk factors to dyslexia.
Our study revealed that the prevalence of dyslexia was 3.9% in Qianjiang city, which is a middle-sized city in China. Among dyslexic children, the gender ratio (boys to girls) was nearly 3∶1. According to the P-value in the multivariate logistic regression, the gender (P<0.01), mother's education level (P<0.01), and learning habits (P<0.01) (active learning, scheduled reading time) were associated with dyslexia.
The prevalence rate of dyslexic children in middle-sized cities is 3.9%. The potential risk factors of dyslexic children revealed in this study will have a great impact on detecting and treating dyslexic children in China as early as possible, although more studies are still needed to further investigate the risk factors of dyslexic children in China.
We investigated serological correlates of protection against Neisseria meningitidis serogroup A (NmA) in Burkina Faso before the introduction of NmA conjugate vaccine.
We collected blood from a representative sample (N = 1022) of Bobo-Dioulasso residents. Sera were evaluated for serum bactericidal antibody (SBA) activity against NmA strains of immunotype L11 (F8238) and L10 (3125) and NmA-specific IgG. Seroprevalence was compared to the age-specific NmA meningitis incidence in Bobo-Dioulasso during March 2007–February 2008. Meningococcal carriage was evaluated in a subset (N = 538). Geometric mean titres (GMT)/concentrations (GMC) of SBA and NmA-specific IgG increased with age, peaking around age 20 years. Overall, 70% of our sample had NmA-specific IgG ≥2 ug/mL. Meningitis incidence was highest in those aged <6 months and 5–19 years. No NmA carriers were found. Compared to the reference strain SBA, GMTs were higher against a locally isolated strain and around 40-fold lower against Dutch strain 3125.
This study provides estimates of natural immunity to NmA, according to a variety of antibody measures, which will be helpful in ascertaining antibody persistence after MenAfriVac™ introduction. Age-specific seroprevalence of reference strain SBA titres most likely reflects exposure to meningococci and consecutive reactive immunity. We could not define any serological correlate of protection.
Diabetes mellitus complicates pregnancies, leading to diseases in adult life in the offspring. Asymmetric dimethylarginine (ADMA) is increased in diabetes mellitus, kidney disease, and hypertension. We tested whether maternal diabetes causes increased ADMA in rats, resulting in kidney disease and hypertension in the adult offspring, and whether these can be prevented by maternal citrulline supplementation. Newborn female and pregnant Sprague-Dawley rats were injected with streptozotocin (STZ), which made up the nSTZ and STZ models, respectively. For the STZ model, 4 groups of male offspring were killed at age 3 months: the control, STZ, and Cit and STZ+Cit (control and STZ rats treated with 0.25% l-citrulline solution, respectively) groups. The nSTZ rats had lower nephron numbers. The renal level of ADMA was higher in the nSTZ rats than in controls. The STZ group developed kidney injury, renal hypertrophy, and elevated blood pressure at the age of 12 weeks. These conditions were found to be associated with increased ADMA levels, decreased nitric oxide (NO) production, and decreased dimethylarginine dimethylaminohydrolase (DDAH) activity in the kidney. In addition, ADMA caused a nephron deficit in cultured rat metanephroi. Maternal citrulline supplementation prevented hypertension and kidney injury, increased the renal DDAH-2 protein level, and restored the levels of ADMA and NO in the STZ+Cit group. Reduced nephron number and increased ADMA contribute to adult kidney disease and hypertension in offspring of mothers with STZ-induced diabetes. Manipulation of the ADMA-NO pathway by citrulline supplementation may be a potential approach to prevent these conditions.
Coxsackievirus A9 (CA9) was one of the most prevalent serotype of enteroviral infections in Taiwan in 2011. After several patient series were reported in the 1960s and 1970s, few studies have focused on the clinical manifestations of CA9 infections. Our study explores and deepens the current understanding of CA9.
We analyzed the clinical presentations of 100 culture-proven CA9-infected patients in 2011 by reviewing their medical records and depicted the CA9 phylogenetic tree.
Of the 100 patients with culture-proven CA9 infections, the mean (SD) age was 4.6 (3.4) years and the male to female ratio was 1.9. For clinical manifestations, 96 patients (96%) had fever and the mean (SD) duration of fever was 5.9 (3.4) days. Sixty one patients (61%) developed a skin rash, and the predominant pattern was a generalized non-itchy maculopapular rash without vesicular changes. While most patients showed injected throat, oral ulcers were found in only 19 cases (19%), among whom, 6 were diagnosed as herpangina. Complicated cases included: aseptic meningitis (n=8), bronchopneumonia (n=6), acute cerebellitis (n=1), and polio-like syndrome (n=1). Phylogenetic analysis for current CA9 strains is closest to the CA9 isolate 27-YN-2008 from the border area of mainland China and Myanmar.
The most common feature of CA9 during the 2011 epidemic in Taiwan is generalized febrile exanthema rather than herpangina or hand, foot, and mouth disease. Given that prolonged fever and some complications are possible, caution should be advised in assessing patients as well as in predicting the clinical course.
Coxsackievirus A9; Enterovirus; Viral exanthema; Phylogenetic tree
Fatty acid profiling has been widely used in the bacteria species identification, we hypothesized that fatty acid characteristics might discriminate the Panax herbs according to species. To test the hypothesis, fatty acids of Panax species, including Panax ginseng, Panax notoginseng and Panax quinquefolius, were characterized and compared using gas chromatography–mass spectrometry (GC-MS) followed by multivariate statistical analysis.
The content of investigated 11 fatty acids, including myristic acid, pentadecanoic acid, palmitic acid, palmitoleic acid, heptadecanoic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, arachidic acid and eicosadienoic acid, obviously varied among three species, suggesting each species has its own fatty acid pattern. Principal component analysis and hierarchical clustering analysis according to the absolute and relative contents of fatty acids, showed that 30 tested samples could be clearly differentiated according to the species.
These findings demonstrated that GC-MS-based fatty acid profiling coupled with multivariate statistical analysis provides reliable platform to classify these three Panax species, which is helpful for ensuring their safety and efficacy.
Panax species; Fatty acids; Gas chromatography–mass spectrometry; Multivariate statistical analysis; Principal component analysis; Hierarchical cluster analysis
Increased incidence of adenovirus infection in children was noticed since September 2010 in Taiwan and severe cases requiring intensive care were noted later. We did this study to find the clinical characteristics and risk factors associated with severe adenovirus infection.
Patients and Methods
We collected cases of severe adenovirus infection between November 2010 and June 2011 to analyze their clinical characteristics in two medical centers in northern Taiwan. Severe adenovirus infection was defined as laboratory-confirmed adenovirus cases with required intensive care. Hexon gene sequencing was performed for molecular genotyping.
45 patients were included, 22 cases (49%) were infected with serotype 7, 19 (42%) with serotype 3, and 4 with serotype 2. The median age (range) was 2.75 years (0.08–15.43 years); 87% were below 5 years. Male to female ratio was 1.65 (28 to 17). Of these patients, 56% had underlying neurological diseases, 50% experienced fever higher than 40°C and 69% suffered fever longer than one week. The clinical diagnosis included pneumonia in 40 (89%) patients, bronchopneumonia in 5 (11%), and encephalitis in 7 (16%). At least 22 patients had pleural effusion. They had complications of respiratory failure (53%), acute respiratory distress syndrome (24%), hypotension (40%), and 6 (13%) patients needed extracorporeal membranous oxygenation. Ten (22%) patients died, all with underlying major systemic diseases and 7 (70%) infected with serotype 7.
Adenovirus serotype 7 and 3 can cause severe disease–even death–in children, especially those with underlying neurological diseases. Patients infected with adenovirus serotype 7 tended to have a higher case-fatality rate.
There is increasing evidence on complex interaction between the nervous and immune systems in patients with cerebral infarction. This study was conducted to evaluate cytotoxic function of CD8+ T lymphocytes isolated from patients with acute severe cerebral infarction. In order to determine role of immune system in stroke, peripheral blood mononuclear cells (PBMCs) were taken and cytotoxic function of CD8+ T lymphocytes were induced by virus peptides and cells were analyzed on a four-color flow cytometer. Expression of CD107a, intracellular expression of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), and cell proliferation assay were analyzed by using carboxyl fluorescein diacetate succinimidyl ester (CFSE).
A total of 30 patients with cerebral infarction and 30 healthy volunteers with an average age 57 (range, 49 to 71) years, were evaluated. The PBMCs were separated from blood samples of both, patients with cerebral infarction 6 hours after onset of stroke and healthy volunteers. After stimulation with virus peptides, CD107a expression and intracellular production of IFN-γ and TNF-α was decreased in patients with cerebral infarction as compared to healthy volunteers (p < 0.01). Degranulation analysis reported decreased expression of CD107a + in patient group as compared to healthy group, p <0.01. A mild decrease in intracellular expression of IFN-γ and TNF-α was also shown in patients without stimulation of virus peptides (p < 0.05). However, proliferation of CD8+ T lymphocytes in patients with acute severe cerebral infarction was not decreased.
The study results indicated that cytotoxic function of CD8+ T lymphocytes were suppressed in patients with acute severe cerebral infarction. This could possibly be associated with complicated infectious diseases and neuroprotective mechanism.
CD8+ T lymphocytes; Cerebral infarction; Cytotoxic function
In this study, 1R,2R-dicamphanoyl-3,3-dimethydihydropyrano[2,3-c]xanthen-7(1H)-one (DCX) derivatives were designed and synthesized as novel anti-HIV agents against both wild-type and nonnucleoside reverse transcriptase (RT) inhibitor-resistant HIV-1 (RTMDR-1) strains. Twenty-four DCX analogs (6-29) were synthesized and evaluated against the non-drug-resistant HIV-1 NL4-3 strain, and selected analogs were also screened for their ability to inhibit the RTMDR-1 strain. Compared with the control 2-ethyl-3′,4′-di-O-(-)-camphanoyl-2′,2′-dimethyldihydropyrano[2,3-f]chromone (2-EDCP, 2), one of the best anti-HIV coumarin derivatives in our prior study, three DCX compounds (7, 12, and 22) showed better activity against both HIV strains with an EC50 range of 0.062 – 0.081 μM, and five additional compounds (8, 11, 16, 18, and 21) exhibited comparable anti-HIV potency. Six DCX analogs (7, 11-12, 18, and 21-22) also showed enhanced selectivity index (SI) values in comparison to the control. Structure-activity relationship (SAR) information suggested that the extended conjugated system of the pyranoxanthone skeleton facilitates the interaction of the small DCX molecule within the viral binding pocket, consequently leading to enhanced anti-HIV activity and selectivity. Compared to DCP compounds, DCX analogs are a more promising new class of anti-HIV agents.
1R,2R-dicamphanoyl-3,3-dimethydihydropyrano[2,3-c]xanthen-7(1H)-one (DCX); Anti-HIV activity; Structure-activity relationship (SAR)
AIM: To identify genes potentially involved in Helicobacter pylori (H. pylori)-induced gastric carcinogenesis.
METHODS: GES-1 cells were co-cultured with H. pylori strains isolated from patients with gastric carcinoma (GC, n = 10) or chronic gastritis (CG, n = 10) for in vitro proliferation and apoptosis assays to identify the most and least virulent strains. These two strains were cagA-genotyped and used for further in vivo carcinogenic virulence assays by infecting Mongolian gerbils for 52 wk, respectively; a broth free of H. pylori was lavaged as control. Genomic profiles of GES-1 cells co-cultured with the most and least virulent strains were determined by microarray analysis. The most differentially expressed genes were further verified using quantitative real-time polymerase chain reaction in GES-1 cells infected with the most and least virulent strains, and by immunohistochemistry in H. pylori positive CG, precancerous diseases, and GC biopsy specimens in an independent experiment.
RESULTS: GC-derived H. pylori strains induced a potent proliferative effect in GES-1 cells in co-culture, whereas CG-derived strains did not. The most (from a GC patient) and least (from a CG patient) virulent strains were cagA-positive and negative, respectively. At week 52, CG, atrophy, metaplasia, dysplasia, and GC were observed in 90.0%, 80.0%, 80.0%, 90%, and 60.0%, respectively, of the animals lavaged with the most virulent strain. However, only mild CG was observed in 90% of the animals lavaged with the least virulent strain. On microarray analysis, 800 differentially expressed genes (49 up- and 751 down-regulated), involving those associated with cell cycle regulation, cell apoptosis, cytoskeleton, immune response, and substance and energy metabolisms, were identified in cells co-cultured with the most virulent strain as compared with those co-cultured with the least virulent strain. The six most differentially expressed genes (with a betweenness centrality of 0.1-0.2) were identified among the significant differential gene profile network, including JUN, KRAS, BRCA1, SMAD2, TRAF1, and HDAC6. Quantitative real-time polymerase chain reaction analyses verified that HDAC6 and TRFA1 mRNA expressions were significantly more up-regulated in GES-1 cells co-cultured with the most virulent strain than in those co-cultured with the least virulent strain. Immunohistochemistry of gastric mucosal specimens from H. pylori-positive patients with CG, intestinal metaplasia (IM), dysplasia, and GC showed that moderately positive and strongly positive HDAC6 expression was detected in 21.7% of CG patients, 30.0% of IM patients, 54.5% of dysplasia patients, and 77.8% of GC patients (P < 0.001). The up-regulation of TRAF1 expressions was detected in 34.8%, 53.3%, 72.7%, and 88.9% specimens of CG, IM, dysplasia, and GC, respectively (P < 0.001).
CONCLUSION: The overexpression of HDAC6 and TRAF1 in GES-1 cells co-cultured with the GC-derived strain and in H. pylori-positive dysplasia and GC suggests that HDAC6 and TRAF1 may be involved in H. pylori-induced gastric carcinogenesis.
Helicobacter pylori; Gastric carcinoma; Proliferation; Genomic profiles
Activation of p53 effectively inhibits tumor angiogenesis that is necessary for tumor growth and metastasis. Reactivation of the p53 by small molecules has emerged as a promising new strategy for cancer therapy. Several classes of small-molecules that activate the p53 pathway have been discovered using various approaches. Here, we identified harmine (β-carboline alkaloid) as a novel activator of p53 signaling involved in inhibition of angiogenesis and tumor growth. Harmine induced p53 phosphorylation and disrupted the p53-MDM2 interaction. Harmine also prevented p53 degradation in the presence of cycloheximide and activated nuclear accumulation of p53 followed by increasing its transcriptional activity in endothelial cells. Moreover, harmine not only induced endothelial cell cycle arrest and apoptosis, but also suppressed endothelial cell migration and tube formation as well as induction of neovascularity in a mouse corneal micropocket assay. Finally, harmine inhibited tumor growth by reducing tumor angiogenesis, as demonstrated by a xenograft tumor model. Our results suggested a novel mechanism and bioactivity of harmine, which inhibited tumor growth by activating the p53 signaling pathway and blocking angiogenesis in endothelial cells.
People worldwide depend upon daily fish consumption as a major source of protein and other nutrients. Fish are high in nutrients essential for normal brain development, but they also contain methylmercury (MeHg), a neurotoxicant. Our studies in a population consuming fish daily have indicated no consistent pattern of adverse associations between prenatal MeHg and children’s development. For some endpoints we found performance improved with increasing prenatal exposure to MeHg. Follow up studies indicate this association is related to the beneficial nutrients present in fish.
To determine if the absence of adverse outcomes and the presence of beneficial associations between prenatal MeHg and developmental outcomes previously reported persists into adolescence.
This study was conducted on the Main Cohort of the Seychelles Child Development Study (SCDS). We examined the association between prenatal MeHg exposure and subjects’ performance at 17 years of age on 27 endpoints. The test battery included the Wisconsin Card Sorting Test (WCST), the California Verbal Learning Test (CVLT), the Woodcock-Johnson (W-J-II) Achievement Test, subtests of the Cambridge Neuropsychological Test Automated Battery (CANTAB), and measures of problematic behaviors. Analyses for all endpoints were adjusted for postnatal MeHg, sex, socioeconomic status, maternal IQ, and child’s age at testing and the child’s IQ was added for problematic behavioral endpoints.
Mean prenatal MeHg exposure was 6.9 ppm. There was no association between prenatal MeHg and 21 endpoints. Increasing prenatal MeHg was associated with better scores on four endpoints (higher W-J-II math calculation scores, reduced numbers of trials on the Intra-Extradimensional Shift Set of the CANTAB, fewer reports of substance use and incidents of and referrals for problematic behaviors in school. Increasing prenatal MeHg was adversely associated with one level of referrals to a school counselor.
At age 17 years there was no consistent pattern of adverse associations present between prenatal MeHg exposure and detailed domain specific neurocognitive and behavioral testing. There continues to be evidence of improved performance on some endpoints as prenatal MeHg exposure increases in the range studied, a finding that appears to reflect the role of beneficial nutrients present in fish as demonstrated previously in younger subjects. These findings suggest that ocean fish consumption during pregnancy is important for the health and development of children and that the benefits are long lasting.
The occurrence of dengue haemorrhagic fever (DHF) is thought to result from a complex interplay between the virus, host genetics and host immune factors. Existing published data are not consistent, in part related to relatively small sample sizes. We set out to determine possible associations between dengue virus (DEN-V) NS3 specific T cells and cytokine and chemokine levels and the pathogenesis of severe disease in a large cohort of individuals with DHF.
By using ex vivo IFNγ ELISpot assays we determined DENV-NS3 specific responses in patients with varying severity of DHF. Other cytokines produced by DENV-NS3 specific T cells were determined by using multiple bead array analysis (MBAA). We also determined the serum cytokine levels using MBAA, lymphocyte subsets and Annexin V expression of lymphocytes in patients with varying severity of DHF. Of the 112 DHF patients studied, 29 developed shock. Serum IL-10 and IP-10 levels positively and significantly correlated with T cell apoptosis while IL-10 levels inversely correlated with T cell numbers. In contrast, TGFß showed a very significant (P<0.0001) and positive correlation (Spearman’s R = 0.65) with the platelet counts, consistent with platelet release. We found that whilst patients with severe dengue had lower total T cell numbers, the DV-NS3 specific T cells persisted and produced high levels of IFNγ but not TNFα, IL-3, IL-13, IL-2, IL-10 or IL-17.
Our data suggest that serum IL-10, TNFα and TGFβ differentially associate with dengue disease severity.
The peanut (Arachis hypogaea) is an important oil crop. Breeding for high oil content is becoming increasingly important. Wild Arachis species have been reported to harbor genes for many valuable traits that may enable the improvement of cultivated Arachis hypogaea, such as resistance to pests and disease. However, only limited information is available on variation in oil content. In the present study, a collection of 72 wild Arachis accessions representing 19 species and 3 cultivated peanut accessions were genotyped using 136 genome-wide SSR markers and phenotyped for oil content over three growing seasons. The wild Arachis accessions showed abundant diversity across the 19 species. A. duranensis exhibited the highest diversity, with a Shannon-Weaver diversity index of 0.35. A total of 129 unique alleles were detected in the species studied. A. rigonii exhibited the largest number of unique alleles (75), indicating that this species is highly differentiated. AMOVA and genetic distance analyses confirmed the genetic differentiation between the wild Arachis species. The majority of SSR alleles were detected exclusively in the wild species and not in A. hypogaea, indicating that directional selection or the hitchhiking effect has played an important role in the domestication of the cultivated peanut. The 75 accessions were grouped into three clusters based on population structure and phylogenic analysis, consistent with their taxonomic sections, species and genome types. A. villosa and A. batizocoi were grouped with A. hypogaea, suggesting the close relationship between these two diploid wild species and the cultivated peanut. Considerable phenotypic variation in oil content was observed among different sections and species. Nine alleles were identified as associated with oil content based on association analysis, of these, three alleles were associated with higher oil content but were absent in the cultivated peanut. The results demonstrated that there is great potential to increase the oil content in A. hypogaea by using the wild Arachis germplasm.
Giving cigarettes as gifts is a common practice in China, but there have been few systematic studies of this practice. The present study was designed to estimate the incidence of receiving cigarettes as gifts, correlates of this practice, and its impact on brand selection in a representative sample of urban adult smokers in China.
Data were analyzed from Wave 2 of the International Tobacco Control (ITC) China Survey, where 4843 adult urban smokers were interviewed in six major Chinese cities between October 2007 and January 2008. The incidence of most recent cigarette acquisition due to gifting and the prevalence of preferred brand selection due to having received it as a gift were estimated. Bivariate and adjusted logistic regression models were estimated to identify factors associated with these two outcomes.
The incidence of receiving cigarettes as a gift at most recent cigarette acquisition was 3.5%. Smokers who received these gifted cigarettes were more likely to be female, older, have higher educational attainment, live in Beijing, and smoke fewer cigarettes per day. The prevalence of choosing one’s preferred brand due to having received it as a gift was 7.0%, and this was more likely among smokers who lived in Beijing and Guangzhou, had lower educational attainment, smoked less frequently, and had smoked their preferred brand for less than one year.
The 3.5% incidence of one’s most recent cigarette acquisition due to gifting is consistent with prevalence estimates based on longer reference periods and translates into the average smoker receiving a gift of cigarettes approximately five times a year. Gifting also appears to have a significant influence on brand preference. Tobacco control interventions in China may need to denormalize the practice of giving cigarettes as gifts in order to decrease the social acceptability of smoking.
Tobacco; Cigarette gifting; Preferred cigarette brand
Adenovirus type 7 caused a high proportion of severe infections.
In 2011, a large community outbreak of human adenovirus (HAdV) in Taiwan was detected by a nationwide surveillance system. The epidemic lasted from week 11 through week 41 of 2011 (March 14–October 16, 2011). Although HAdV-3 was the predominant strain detected (74%), an abrupt increase in the percentage of infections caused by HAdV-7 occurred, from 0.3% in 2008–2010 to 10% in 2011. Clinical information was collected for 202 inpatients infected with HAdV; 31 (15.2%) had severe infection that required intensive care, and 7 of those patients died. HAdV-7 accounted for 10%, 12%, and 41% of infections among outpatients, inpatients with nonsevere infection, and inpatients with severe infection, respectively (p<0.01). The HAdV-7 strain detected in this outbreak is identical to a strain recently reported in the People’s Republic of China (HAdV7-HZ/SHX/CHN/2009). Absence of circulating HAdV-7 in previous years and introduction of an emerging strain are 2 factors that caused this outbreak.
adenovirus; surveillance; pneumonia; outbreak; viruses; Taiwan; severe infection; adenovirus type 7; HAdV; human adenovirus
Evaluation of analgesics in large animals is a necessary step in the development of better pain medications or gene therapy prior to clinical trials. However, chronic neuropathic pain models in large animals are limited. To address this deficiency, we developed a neuropathic pain model in sheep, which shares many anatomical similarities in spine dimensions and cerebrospinal fluid volume as humans.
A neuropathic pain state was induced in sheep by tight ligation and axotomy of the common peroneal nerve. The analgesic effect of intrathecal (IT) morphine was investigated. Interspecies comparison was conducted by analyzing the ceiling doses of IT morphine for humans, sheep, and rats.
Peroneal nerve injury (PNI) produced an 86% decrease in von-Frey filament-evoked withdrawal threshold on postsurgery day 3 and the decrease lasted for the 8-week test period. Compared to the pre-injury, sham, and contralateral hindlimb, the IT morphine dose that produces 50% of maximum analgesia (ED50) for injured PNI hindlimb was 1.8-fold larger and Emax, the dose that produces maximal analgesia, was 6.1-fold lower. The sheep model closely predicts human IT morphine ceiling dose by allometric scaling. This is in contrast to the approximately 10-fold lower morphine ceiling dose predicted by the rat spinal nerve ligated or spared nerve injury models.
PNI sheep model has a fast onset and shows stable and long-lasting pain behavioral characteristics. Since the antinociceptive properties of IT morphine are similar to those observed in humans, the PNI sheep model will be a useful tool for the development of analgesics. Its large size and consistent chronic pain behavior will facilitate the development and evaluation of surgical intervention and gene therapy. The PNI sheep pain model provides us with the opportunity for multi-species testing, which will improve the success of clinical trials.
interspecies drug scaling; neuropathic pain model; ovine; ovine pain model
AIM: To study the effect of H2 gas on liver injury in massive hepatectomy using the Intermittent Pringle maneuver in swine.
METHODS: Male Bama pigs (n = 14) treated with ketamine hydrochloride and Sumianxin II as induction drugs followed by inhalation anesthesia with 2% isoflurane, underwent 70% hepatotectomy with loss of bleeding less than 50 mL, and with hepatic pedicle occlusion for 20 min, were divided into two groups: Hydrogen-group (n = 7), the pigs with inhalation of 2% hydrogen by the tracheal intubation during major hepatotectomy; Contrast-group (n = 7), underwent 70% hepatotectomy without inhalation of hydrogen. Hemodynamic changes and plasma concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hyaluronic acid (HA), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and malondialdehyde (MDA) in liver tissue were measured at pre-operation, post-hepatotectomy (PH) 1 h and 3 h. The apoptosis and proliferating cell nuclear antigen (PCNA) expression in liver remnant were evaluated at PH 3 h. Then we compared the two groups by these marks to evaluate the effect of the hydrogen in the liver injury during major hepatotectomy with the Pringle Maneuver in the swine.
RESULTS: There were no significant differences in body weight, blood loss and removal liver weight between the two groups. There was no significant difference in changes of portal vein pressure between two groups at pre-operation, PH 30 min, but in hydrogen gas treated-group it slightly decrease and lower than its in Contrast-group at PH 3 h, although there were no significant difference (P = 0.655). ALT and AST in Hydrogen-group was significantly lower comparing to Contrast-group (P = 0.036, P = 0.011, vs P = 0.032, P = 0.013) at PH 1 h and 3 h, although the two groups all increased. The MDA level increased between the two group at PH 1 h and 3 h. In the hydrogen gas treated-group, the MDA level was not significantly significant at pre-operation and significantly low at PH 1 h and 3 h comparing to Contrast-group (P = 0.0005, P = 0.0004). In Hydrogen-group, the HA level was also significantly low to Contrast-group (P = 0.0005, P = 0.0005) although the two groups all increased at PH 1 h and 3 h. The expression of cluster of differentiation molecule 31 molecules Hydrogen-group was low to Contrast-group. However, PCNA index (%) was not statistically significant between the two groups (P = 0.802). Microphotometric evaluation of apoptotic index (AI) in terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling-stained tissue after hepatotectomy for 3h, the AI% level in the hydrogen was significantly low to Contrast-group (P = 0.012). There were no significant difference between Hydrogen-group and Contrast-group at pre-operation (P = 0.653, P = 0.423), but after massive hepatotectomy, the TNF-α and IL-6 levels increase, and its in Hydrogen-group was significantly low compared with Contrast-group (P = 0.022, P = 0.013, vs P = 0.016, P = 0.012), respectively. Hydrogen-gas inhalation reduce levels of these markers and relieved morphological liver injury and apoptosis.
CONCLUSION: H2 gas attenuates markedly ischemia and portal hyperperfusion injury in pigs with massive hepatotectomy, possibly by the reduction of inflammation and oxidative stress, maybe a potential agent for treatment in clinic.
Massive hepatotectomy; Hydrogen gas; Anti-oxidant; Hyperperfusion; Malondialdehyde; Oxidative stress
In this study, 22 new betulinic acid (BA) derivatives were synthesized and tested for their inhibition of the chymotrypsin-like activity of 20S proteasome. From the SAR study, we concluded that the C-3 and C-30 positions are the pharmacophores for increasing the proteasome inhibition effects, and larger lipophilic or aromatic side chains are favored at these positions. Among the BA derivatives tested, compounds 13, 20, and 21 showed the best proteasome inhibition activity with IC50 values of 1.42, 1.56, and 1.80 µM, respectively, which are three- to four-fold more potent than the proteasome inhibition controls LLM-F and lactacystin.
Hepatocyte Nuclear Factor (HNF)4α is a central regulator of gene expression in cell types that play a critical role in metabolic homeostasis, including hepatocytes, enterocytes, and pancreatic β-cells. Although fatty acids were found to occupy the HNF4α ligand-binding pocket and proposed to act as ligands, there is controversy about both the nature of HNF4α ligands as well as the physiological role of the binding. Here, we report the discovery of potent synthetic HNF4α antagonists through a high-throughput screen for effectors of the human insulin promoter. These molecules bound to HNF4α with high affinity and modulated the expression of known HNF4α target genes. Notably, they were found to be selectively cytotoxic to cancer cell lines in vitro and in vivo, although in vivo potency was limited by suboptimal pharmacokinetic properties. The discovery of bioactive modulators for HNF4α raises the possibility that diseases involving HNF4α, such as diabetes and cancer, might be amenable to pharmacologic intervention by modulation of HNF4α activity.
Multi-drug resistance to chemotherapeutic agents is a major cause of treatment failure in breast cancer. In this study, we investigated the effects of emodin on reversing the multi-drug resistance, examined the ERCC1 protein expression in breast cancer cell line, and explored the relationship between reversal of multi-drug resistance and ERCC1 protein expression.
MTT assay was conducted to test the cytotoxicity of adriamycin and cisplatin to MCF-7/Adr cells with and without emodin pretreatment, and Western blot was performed to examine the ERCC1 protein expression.
MCF-7/Adr cells had 21-fold and 11-fold baseline resistances to adriamycin and cisplatin, respectively. When emodin was added to the cell culture at the concentration of 10 μg/ml, the drug resistance was reduced from 21 folds to 2.86 folds for adriamycin, and from 11 folds to 1.79 folds for cisplatin. MCF-7/Adr cells treated with two concentrations (10μg/mL and 20μg/mL) of emodin, after 2, 4, 6, 10 days, the trend of ERCC1 expression was gradually decreased and the reduction was more obvious comparatively at the concentration of 20μg/mL.
Emodin could reverse the multi-drug resistance in MCF-7/Adr cells and down-regulate ERCC1 protein expression.
HIV-1 envelope glycoproteins are the key viral proteins that mediate HIV-1 entry and cell–cell fusion. In contrast to HIV-1 entry, the mechanism of HIV-1 Env-mediated cell–cell fusion is relatively unclear. This study demonstrated that dynasore, a dynamin inhibitor, suppressed HIV-1 Env-mediated cell–cell fusion. Dynasore sensitivity of HIV-1 Env-mediated cell–cell fusion varied depending on the viral strains. Results from testing a panel of gp41 cytoplasmic tail truncation mutants suggested that the gp41 cytoplasmic tail might play a role in dynasore sensitivity. HIV-1 Env-mediated cell–cell fusion could also be suppressed by a dynamin dominant-negative mutant DNM2(K44A). In summary, these results suggested that dynamin 2 might play a role in HIV-1 Env-mediated cell–cell fusion.