Combined analysis of polymorphism variants in hMTH1, hOGG1 and MUTYH genes on the risk of type 2 diabetes in the Chinese population

Reactive oxygen species are considered to play a role in the development of type 2 diabetes mellitus (T2DM) and its complications. 8-Oxoguanine, which is one of the major oxidation base lesions produced by reactive oxygen species, may cause G:C to T:A transversion mutations because it can mispair with adenine. hMTH1 (human mutT homolog 1), hOGG1 (human 8-oxoguanine glycosylase 1) and MUTYH (human mutY homolog) genes constitute the 8-oxoG repair pathway. In this study, we screened for the polymorphism variants Val83Met (c.247G>A, rs4866) in hMTH1; c.-53G>C (rs56387615), c.-23A>G (rs1801129) and c.-18G>T (rs1801126) in the 5′-UTR of hOGG1; and AluYb8 insertion in MUTYH (AluYb8MUTYH, rs10527342) and investigated their synergistic effect on the risk of T2DM in the Chinese population. The genotypes were determined by electrophoresis, a high-resolution melting technique and sequencing of PCR products. Our results showed that the c.247G>A variant in the hMTH1 gene increased the risk of T2DM in > 55 years of age groups (OR = 1.579; 95%CI: 1.029–2.421). The set of c.-53G>C, c.-23A>G and c.-18G>T variants detected in the 5′-UTR of the hOGG1 gene and the AluYb8 insertion in the MUTYH gene were each associated with an increased risk of T2DM (OR = 1.507, 95%CI: 1.122–2.024; OR = 1.229, 95%CI: 1.030–1.466, respectively). Combined analysis of the variations among the three genes suggested that the c.247G>A variant in hMTH1 combined with AluYb8MUTYH variant had a synergistic effect on increasing the risk of T2DM (OR = 1.635; 95%CI: 1.147–2.330). This synergy was also observed between the variants in the 5′-UTR of the hOGG1 and the AluYb8MUTYH variant (OR = 1.804; 95%CI: 1.254–2.595). Our results suggest, for the first time, the combined effects of AluYb8MUTYH with either hMTH1 c.247G>A or variants in the 5′-UTR of the hOGG1 on the risk of T2DM.     

MicroRNA-21 regulates T-cell apoptosis by directly targeting the tumor suppressor gene Tipe2

MicroRNAs (MiRs) are short noncoding RNAs that can regulate gene expression. It has been reported that miR-21 suppresses apoptosis in activated T cells, but the molecular mechanism remains undefined. Tumor suppressor Tipe2 (or tumor necrosis factor-α-induced protein 8 (TNFAIP8)-like 2 (TNFAIP8L2)) is a newly identified anti-inflammatory protein of the TNFAIP8 family that is essential for maintaining immune homeostasis. We report here that miR-21 is a direct target of nuclear factor-κB and could regulate Tipe2 expression in a Tipe2 coding region-dependent manner. In activated T cells and macrophages, Tipe2 expression was markedly downregulated, whereas miR-21 expression was upregulated. Importantly, Tipe2-deficient T cells were significantly less sensitive to apoptosis. Conversely, overexpression of Tipe2 in EL-4 T cells increased their susceptibility to activation-induced apoptosis. Therefore, Tipe2 provides a molecular bridge between miR-21 and cell apoptosis; miR-21 suppresses apoptosis in activated T cells at least in part through directly targeting tumor suppressor gene Tipe2.     

Evidence of expression variation and allelic imbalance in Crohn's disease susceptibility genes NOD2 and ATG16L1 in human dendritic cells

Human dendritic cells (DCs) play an important role in induction and progression of Crohn's disease (CD). Accumulating evidence suggests that viral infection is required to trigger CD pathogenesis in genetically predisposed individuals. NOD2 and ATG16L1 are among the major CD susceptibility genes implicated in impaired immune response to bacterial infection. In this study, we investigated gene expression and allelic imbalance (AI) of NOD2 and ATG16L1 using common variants in human monocyte-derived DCs. Significant AI was observed in ~ 40% and ~ 70% of NOD2 and ATG16L1 heterozygotes, respectively (p < 0.05). AI of NOD2 was inversely associated with its expression level (p = 0.015). No correlation was detected between gene expression and AI for ATG16L1. When infected with Newcastle Disease Virus (NDV), NOD2 expression in DCs was induced about four-fold (p < 0.001), whereas ATG16L1 expression was not affected (p = 0.88). In addition, NDV infection tended to lower the variance in AI among DC populations for the NOD2 gene (p = 0.05), but not the ATG16L1 gene (p = 0.32). Findings of a simulation study, aimed to verify whether the observed variation in gene expression and AI is a result of sample-to-sample variability or experimental measurement error, suggested that NOD2 AI is likely to result from a deterministic event at a single cell level. Overall, our results present initial evidence that AI of the NOD2 and ATG16L1 genes exists in populations of human DCs. In addition, our findings suggest that viral infection may regulate NOD2 expression.     

Molecular characterization and transcriptional analysis of non-mammalian type Toll like receptor (TLR21) from rock bream (Oplegnathus fasciatus)

Toll-like receptors (TLRs) are a large family of pattern recognition receptors, which are involved in triggering host immune responses against various pathogens by detecting their evolutionarily conserved pathogen associated molecular patterns (PAMPs). TLR21 is a non-mammalian type TLR, which recognizes unmethylated CpG DNA, and is considered as a functional homolog of mammalian TLR9. In this study, we attempted to identify and characterize a novel TLR21 counterpart from rock bream (Oplegnathus fasciatus) designated as RbTLR21, at molecular level. The complete coding sequence of RbTLR21 was 2919 bp in length, which encodes a polypeptide of 973 amino acids with a predicted molecular mass of 112 kDa and a theoretical isoelectric point of 8.6. The structure of the deduced RbTLR21 protein is similar to that of the members of typical TLR family, and includes the ectodomain, which consists of 16 leucine rich repeats (LRRs), a transmembrane domain, and a cytoplasmic Toll/interleukin-1 receptor (TIR) domain. According to the pairwise sequence analysis data, RbTLR21 was homologous to that of the orange-spotted grouper (Epinephelus coioides) with 76.9% amino acid identity. Furthermore, our phylogenetic analysis revealed that RbTLR21 is closely related to E. coioides TLR21. The RbTLR21 was ubiquitously expressed in all the tissues tested, but the highest expression was found in spleen. Additionally, upon stimulation with Streptococcus iniae, rock bream iridovirus (RBIV), and Edwardsiella tarda, RbTLR21 mRNA was significantly up-regulated in spleen tissues. Collectively, our findings suggest that RbTLR21 is indeed an ortholog of the TLR21 family and may be important in mounting host immune responses against pathogenic infections.     

In vitro and in vivo pharmacology of CP-945,598, a potent and selective cannabinoid CB1 receptor antagonist for the management of obesity

Cannabinoid CB₁ receptor antagonists exhibit pharmacologic properties favorable for the treatment of metabolic disease. CP-945,598 (1-[9-(4-chlorophenyl)-8-(2-chlorophenyl)-9H-purin-6-yl]-4-ethylamino piperidine-4-carboxylic acid amide hydrochloride) is a recently discovered selective, high affinity, competitive CB₁ receptor antagonist that inhibits both basal and cannabinoid agonist-mediated CB₁ receptor signaling in vitro and in vivo. CP-945,598 exhibits sub-nanomolar potency at human CB₁ receptors in both binding (K_i = 0.7 nM) and functional assays (K_i = 0.2 nM). The compound has low affinity (K_i = 7600 nM) for human CB₂ receptors. In vivo, CP-945,598 reverses four cannabinoid agonist-mediated CNS-driven responses (hypo-locomotion, hypothermia, analgesia, and catalepsy) to a synthetic cannabinoid receptor agonist. CP-945,598 exhibits dose and concentration-dependent anorectic activity in two models of acute food intake in rodents, fast-induced re-feeding and spontaneous, nocturnal feeding. CP-945,598 also acutely stimulates energy expenditure in rats and decreases the respiratory quotient indicating a metabolic switch to increased fat oxidation. CP-945,598 at 10 mg/kg promoted a 9%, vehicle adjusted weight loss in a 10 day weight loss study in diet-induced obese mice. Concentration/effect relationships combined with ex vivo brain CB₁ receptor occupancy data were used to evaluate efficacy in behavioral, food intake, and energy expenditure studies. Together, these in vitro, ex vivo, and in vivo data indicate that CP-945,598 is a novel CB₁ receptor competitive antagonist that may further our understanding of the endocannabinoid system.     

Characterization of functional intermediates of endoglucanase from Aspergillus aculeatus during urea and guanidine hydrochloride unfolding

Low concentrations of urea and GuHCl (2 M) enhanced the activity of endoglucanase (EC 3.1.2.4) from Aspergillus aculeatus by 2.3- and 1.9-fold, respectively. The K_m values for controls, in the presence of 2 M urea and GuHCl, were found to be 2.4 ± 0.2 × 10⁻⁸ mol L⁻¹, 1.4 ± 0.2 × 10⁻⁸ mol L⁻¹, and 1.6 ± 0.2 × 10⁻⁸ mol L⁻¹, respectively. The dissociation constant (K_d) showed changes in the affinity of the enzyme for the substrate with increases in the K_cat suggesting an increased turnover number in the presence of urea and GuHCl. Fluorescence studies showed changes in the microenvironment of the protein. The increase in the activity of this intermediate state was due to conformational changes accompanied by increased flexibility at the active site.     

Engineered biosynthesis of bacteriochlorophyll b in Rhodobacter sphaeroides

Bacteriochlorophyll b has the most red-shifted absorbance maximum of all naturally occurring photopigments. It has a characteristic ethylidene group at the C8 position in place of the more common ethyl group, the product of a C8-vinyl reductase, which is carried by the majority of chlorophylls and bacteriochlorophylls used in photosynthesis. The subsequent and first step exclusive to bacteriochlorophyll biosynthesis, the reduction of the C7 = C8 bond, is catalyzed by chlorophyllide oxidoreductase. It has been demonstrated that the enzyme from bacteriochlorophyll a-utilizing bacteria can catalyze the formation of compounds carrying an ethyl group at C8 from both ethyl- and vinyl-carrying substrates, indicating a surprising additional C8-vinyl reductase function, while the enzyme from organisms producing BChl b could only catalyze C7 = C8 reduction with a vinyl substrate, but this product carried an ethylidene group at the C8 position. We have replaced the native chlorophyllide oxidoreductase-encoding genes of Rhodobacter sphaeroides with those from Blastochloris viridis, but the switch from bacteriochlorophyll a to b biosynthesis is only detected when the native conventional C8-vinyl reductase is absent. We propose a non-enzymatic mechanism for ethylidene group formation based on the absence of cellular C8-vinyl reductase activity.     

Colonization of Aspergillus japonicus on synthetic materials and application to the production of fructooligosaccharides

The ability of Aspergillus japonicus ATCC 20236 to colonize different synthetic materials (polyurethane foam, stainless steel sponge, vegetal fiber, pumice stones, zeolites, and foam glass) and to produce fructooligosaccharides (FOS) from sucrose (165 g/L) is described. Cells were immobilized in situ by absorption, through direct contact with the carrier particles at the beginning of fermentation. Vegetal fiber was the best immobilization carrier as A. japonicus grew well on it (1.25 g/g carrier), producing 116.3 g/L FOS (56.3 g/L 1-kestose, 46.9 g/L 1-nystose, and 13.1 g/L 1-β-fructofuranosyl nystose) with 69% yield (78% based only in the consumed sucrose amount), giving also elevated activity of the β-fructofuranosidase enzyme (42.9 U/mL). In addition, no loss of material integrity, over a 2 day-period, was found. The fungus also immobilized well on stainless steel sponge (1.13 g/g carrier), but in lesser extents on polyurethane foam, zeolites, and pumice stones (0.48, 0.19, and 0.13 g/g carrier, respectively), while on foam glass no cell adhesion was observed. When compared with the FOS and β-fructofuranosidase production by free A. japonicus, the results achieved using cells immobilized on vegetal fiber were closely similar. It was thus concluded that A. japonicus immobilized on vegetal fiber is a potential alternative for high production of FOS at industrial scale.     

Effects of CO2 concentration on growth of filamentous algae and Littorella uniflora in a Danish softwater lake

An experiment was conducted from May to November in Lake Hampen, Denmark, to study the effect of higher CO₂ concentration on the biomass of filamentous algae. Three enclosures (1.5 m diameter) were enriched with free CO₂ to ∼10 times atmospheric equilibrium (∼170 μM) and three enclosures were kept at atmospheric equilibrium (∼17 μM). The isoetid Littorella uniflora dominated the vegetation in the enclosures. Low concentrations of nitrate and phosphate in the water were observed, especially in the summer months. During the summer, a high biomass of filamentous algae (dominated by Zygnema sp.) developed in both types of enclosures (18–58 g dry wt. m⁻² in July and August), but the biomass of algae was significantly higher (1.9–38 times) in the CO₂ enriched enclosures than in enclosures with low CO₂ concentration. L. uniflora biomass, especially leaf biomass, also showed a significant positive response to increased CO₂ concentration (75.0 ± 10.4 and 133.3 ± 42.5 g dry wt. m⁻² at low and high CO₂ concentrations, respectively) even though the massive filamentous algal growth decreased the light intensity. Both filamentous algae (in August) and L. uniflora showed lower tissue concentrations of N and P at high CO₂ concentration.     

Impacts and interactions of PDGFRB, MMP-3, TIMP-2, and RNF213 polymorphisms on the risk of Moyamoya disease in Han Chinese human subjects

Polymorphisms of PDGFRB, MMP-3, TIMP-2, RNF213, TGFB1, Raptor and eNOS genes have been associated with Moyamoya disease (MMD) separately in studies, but their interactions on MMD have never been evaluated in one study. This study enrolled 96 MMD patients and 96 controls to evaluate the contributions and interactions of these polymorphisms on MMD in Chinese Hans. After genotyping, five polymorphisms loci were deemed suitable for analysis, rs3828610 in PDGFRB, rs3025058 in MMP-3, rs8179090 in TIMP-2, rs112735431 and rs148731719 in RNF213. Interactions of different loci on MMD were evaluated by multifactor dimensionality reduction (MDR) method. Significantly higher frequencies of A allele and G/A genotype of rs112735431 in RNF213 were observed in MMD patients compared with controls (P = 0.011; P = 0.018, respectively). In the dominant model, G/A genotype of rs112735431 was associated with increased risk of MMD (P = 0.018). A higher frequency of G allele and G/G genotype of rs148731719 in RNF213 gene in patient than control group (P < 0.001; P < 0.01, respectively) was also detected. No significant association between MMD and other three loci (P > 0.05) was detected. MDR analysis failed to detect any significant interaction among these five loci in the occurrence of MMD (P > 0.05), but the combination of three loci (rs112735431 in RNF213, rs3828610 in PDGFRB, rs3025058 in MMP-3) could have the maximum testing accuracy (57.29%) and cross-validation consistency (10/10). The results indicated that RNF213 rs112735431 and rs148731719 may exert a significant influence on MMD occurrence. Compared with this overwhelming effect, the influences of PDGFRB, MMP-3, and TIMP-2 on MMD may be unremarkable in Chinese Hans. There may be no prominent interaction among these five gene polymorphisms on the occurrence of MMD.     

Effect of genistein-8-C-glucoside from Lupinus luteus on DNA damage assessed using the comet assay in vitro

Genistein-8-C-glucoside (G8CG) belongs to natural isoflavones phytoestrogens, which are a subclass of flavonoids, a large group of polyphenolic compounds widely distributed in plants, with possible anticarcinogenic effects in various in vitro systems and in vivo animal models. We used glycosylated genistein (genistein-8-C-glucoside) from flowers of lupine (Lupinus luteus L.) to study its cytotoxic and genotoxic effects on mouse embryonic fibroblast (line NIH 3T3). The MTT assay to assess cytotoxicity and comet assay for the detection of DNA damage were used. The cells were exposed to various concentrations of genistein-8-C-glucoside (2.5-110 μM) and hydrogen peroxide (5-90 μM). The effect of G8CG alone or in combination with H₂O₂ was determined. G8CG at concentrations >20 μM significantly reduced cell viability and induced DNA damage. In contrast, lower concentrations of (2.5-10 μM) G8CG showed antioxidant properties against H₂O₂-induced DNA damage with no associated toxicity.     

Characterization of Oncorhynchus mykiss 5-hydroxyisourate hydrolase/transthyretin superfamily: Evolutionary and functional analyses

Teleosts have highly diverged genomes that resulted from whole genome duplication, which leads to an extensive diversity of paralogous genes. Transthyretin (TTR), an extracellular thyroid hormone (TH) binding protein, is thought to have evolved from an ancestral 5-hydroxyisourate hydrolase (HIUHase) by gene duplication at some stage of chordate evolution. To characterize the functions of proteins that arose from duplicated genes in teleosts, we investigated the phylogenetic relationship of teleost HIUHase and TTR aa sequences, the expression levels of Oncorhynchus mykiss HIUHase and TTR mRNA in various tissues and the biological activities of the O. mykiss re-HIUHase and re-TTR. Phylogenetic analysis of the teleost aa sequences revealed the presence of two HIUHase subfamilies, HIUHase 1 (which has an N-terminal peroxisomal targeting signal-2 [PTS2]) and HIUHase 2 (which does not have an N-terminal PTS2), and one TTR family. The tissue distributions of HIUHase 1 and TTR mRNA were similar in juvenile O. mykiss and the mRNA levels were highest in the liver. The O. mykiss re-HIUHase and re-TTR proteins were both 40–50 kDa homotetramers consisting of 14–15 kDa subunits, with 30% identity. HIUHase had 5-hydroxyisourate (5-HIU) hydrolysis activity with Zn^2 + sensitivity, whereas TTR had ligand binding activity with a preference for THs and several environmental chemicals, such as halogenated phenols. Our results suggest that O. mykiss HIUHase and TTR have diverged from a common ancestral HIHUase with no functional complementation.     

Genetic structure,polymorphism identification of LGP2 gene and their relationship with the resistance/susceptibility to GCRV in grass carp, Ctenopharyngodon idella

Laboratory of genetics and physiology 2 (LGP2) is an actual detector and regulator during RNA viral infection in innate immunity. In this study, 5′-flanking region and all introns of LGP2 in grass carp (Ctenopharyngodon idella) were excavated. The genomic CiLGP2 (C. idella LGP2) was 8062 bp in length, with a 364 bp 5′-flanking region, twelve exons and eleven introns. Besides, the promoter activity of the upstream region before initiator codon was identified. By sequencing, six single nucleotide polymorphisms (SNPs) and one 20-bp insertion/deletion polymorphism were detected in CiLGP2. With a challenge experiment, the genotype and allele distributions of these seven polymorphisms were examined. Analytic result revealed only the − 1392 C/G, 494 A/T and 4403 C/T loci were significantly associated with the resistance/susceptibility to grass carp reovirus (GCRV) (P < 0.05). To further identify these correlations, another independent challenge test was performed. The analytic result based on the cumulative mortality demonstrated that the stock in − 1392 GG genotype was more susceptible to GCRV than that in CC genotype, while the stocks in 494 TT genotype and 4403 TT genotype were more resistant to GCRV than that in AA and CC genotype stocks, respectively (P < 0.05). Those significant SNPs might be potential gene markers for the future molecular selection of C. idella strains that are resistant to GCRV.     

CFTR genotype and clinical outcomes of adult patients carried as cystic fibrosis disease

Background

There are nearly 2000 cystic fibrosis transmembrane regulator (CFTR) mutations that cause cystic fibrosis (CF). These mutations are classified into six classes; on the one hand, the first three classes cause severe disease involvement in early childhood, on the other hand, the Class IV, V and VI mutations cause minor severe disease in the same age. Nowadays, with therapeutic advances in CF management and competence of pediatricians, physicians of adults have to deal with two groups of CF patients: (i) adults diagnosed in childhood with severe mutations and (ii) adults who initiated symptoms in adulthood and with Class IV, V and VI mutations. The aim of this study was to analyze adults from a clinical center, treated as CF disease, screening the CFTR genotype and evaluating the clinical characteristics.

Methods

Thirty patients followed as CF disease at the University Hospital were enrolled. After a complete molecular CFTR negative screening and sweat test levels between 40 and 59 mEq/L, five patients were characterized as non-CF disease and were excluded. Molecular screening was performed by CFTR gene sequencing/MLPA or by specific mutation screening. Clinical data was obtained from medical records. The patients were divided into three groups: (1) patients with Class I, II and III mutations in two CFTR alleles; (2) genotype with at least one allele of Class IV, V or VI CFTR mutations and, (3) non-identified CFTR mutation + one patient with one allele with CFTR mutation screened (Class I).

Results

There was an association of CFTR class mutation and sodium/chloride concentration in the sweat test (sodium: p = 0.040; chloride: p = 0.016), onset of digestive symptoms (p = 0.012), lung function parameter (SpO₂ — p = 0.016), Bhalla score (p = 0.021), age at diagnosis (p = 0.008) and CF-related diabetes (p = 0.029). There was an association between Pseudomonas aeruginosa chronic colonization (as clinical marker for the lung disease status) and lung impairment (FEV₁% — p = 0.027; Bhalla score — p = 0.021), CF-related diabetes (p = 0.040), chloride concentration in the sweat test (p = 0.040) and chronic infection by microorganisms (Staphylococcus aureus — p = 0.039; mucoid P. aeruginosa — p = 0.001). There is no positive association with the status of other clinical markers and the CFTR genotype groups. For clinical association with pancreatic insufficiency (as clinical marker for digestive symptoms), no association was related.

Conclusion

The adults with CF diagnosed by sweat test have specific clinical and genotypic characteristics, being a population that should be studied to cause better future management. Some patients treated as CF disease by clinical symptoms, showed no disease, taking into account the sweat test and complete exon sequencing/MLPA screening.