Terrirubrum flagellatum gen. nov., sp. nov. of Terrirubraceae fam. nov. and Lichenibacterium dinghuense sp. nov. from forest soil and proposal of Rhodoblastaceae fam. nov.

Two Gram-negative, aerobic, rod-shaped bacterial strains, 7MK25T and 6Y81T, were isolated from forest soil of Dinghushan Biosphere Reserve, Guangdong Province, PR China. Based on the results of 16S rRNA gene sequence analysis, strain 7MK25T showed the highest similarity (93.6 %) to Methyloferula stellata AR4T, followed by Bosea thiooxidans DSM 9653T (93.3 %). Strain 6Y81T had the highest similarity of 97.9 % to Lichenibacterium minor RmlP026T, followed by Lichenibacterium ramalinae RmlP001T (97.2 %). Phylogenomic analysis using the UBCG and PhyloPhlAn methods consistently showed that strain 7MK25T formed a sister clade to Boseaceae, while strain 6Y81T formed an independent clade within the genus Lichenibacterium, both in the order Hyphomicrobiales. The digital DNA-DNA hybridization and average nucleotide identity values between strains 7MK25T, 6Y81T and their close relatives were in the ranges of 19.1-29.9 % and 72.5-85.5 %, respectively. The major fatty acids of 7MK25T were summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c), C19 : 0 cyclo ω8c, C16 : 0 and C17 : 0 cyclo, while those of 6Y81T were summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c), C16 : 0 and C16 : 0 3-OH. Strains 7MK25T and 6Y81T took diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine as their dominant polar lipids, and Q-10 as their major respiratory quinone. On the basis of phenotypic and phylogenetic data, strain 7MK25T is proposed to represent a novel species of a novel genus with name Terrirubrum flagellatum gen. nov., sp. nov., within a novel family Terrirubraceae fam. nov., with 7MK25T (=KCTC 62738T=GDMCC 1.1452T) as its type strain. Strain 6Y81T represents a novel species in the genus Lichenibacterium, for which the name Lichenibacterium dinghuense sp. nov. (type strain 6Y81T=KACC 21 727T=GDMCC 1.2176T) is proposed. Rhodoblastaceae fam. nov. with Rhodoblastus as the type genus is also proposed to solve the non-monophylectic problem of the family Roseiarcaceae.

Trinickia violacea sp. nov. and Trinickia terrae sp. nov., isolated from forest soil.

Two Gram-stain negative, aerobic and rod-shaped bacterial strains, DHOD12T and 7GSK02T, were isolated from forest soil of Dinghushan Biosphere Reserve, Guangdong Province, PR China. Strain DHOD12T grew at 4-42 °C (optimum, 28-33 °C), pH 4.0-8.5 (optimum, pH 5.5-6.5) and in the presence of 0-1.5 % (w/v; optimum, 0-0.5 %)NaCl; while strain 7GSK02T grew at 12-42 °C (optimum, 28-33 °C), pH 4.0-8.5 (optimum, pH 5.0-6.0) and in the presence of 0-0.5 % (w/v; optimum, 0 %) NaCl. Strains DHOD12T and 7GSK02T had the highest 16S rRNA sequence similarities of 98.0 and 98.3 % with the same species Trinickia mobilis DHG64T, respectively, and 98.4 % between themselves. In the 16S rRNA phylogeny, they formed a clade that was sister to a major cluster consisting of all described Trinickia species. Phylogenomic analyses with the UBCG and PhyloPhlAn methods consistently showed that strains DHOD12T and 7GSK02T formed a clade with T. mobilis DHG64T that was a sister of a cluster containing the remainder of the Trinickia species. The DNA G+C contents of strains DHOD12T and 7GSK02T were 63.1 and 64.6 mol%, respectively. Digital DNA-DNA hybridization and average nucleotide identity values of strains DHOD12T, 7GSK02T and their closely related strains were in the ranges of 21.6-31.4 % and 77.1-86.9 %, respectively. These two strains had the same major respiratory quinone, ubiquinone-8, and both had C16 : 0, C17 : 0 cyclo and summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c) as their major fatty acids. Their major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Genomic analysis indicated that the two strains could have the potential to degrade aromatic compounds like other Trinickia species. On the basis of phenotypic and phylogenetic results, strains DHOD12T and 7GSK02T represent two novel species of the genus Trinickia, for which the names Trinickia violacea sp. nov. (type strain DHOD12T=LMG 30258T=CGMCC 1.15436T) and Trinickia terrae sp. nov. (type strain 7GSK02T=CGMCC 1.15432T=KCTC 62468T) are proposed.

Paraburkholderia flagellata sp. nov. and Paraburkholderia adhaesiva sp. nov., two novel species isolated from forest soil in Dinghushan Biosphere Reserve in Guangdong, China.

Two Gram-stain-negative, aerobic, motile and short rod strains, designated 4D117T and ZD32-2T, were isolated from the forest soils. Strains 4D117T and ZD32-2T grew optimally at pH 4.0-6.5, 20-33 °C and pH 4.5-7.0, 33 °C, respectively, and both at 0.5% (w/v) NaCl concentration. Strains 4D117T and ZD32-2T shared the highest 16S rRNA gene sequence similarity with P. acidiphila 7Q-K02T (99.1%) and P. ferrariae NBRC 106233T (98.7%), respectively. The genome size and G + C contents of strains 4D117T and ZD32-2T were 9,002,095 bp, 62.9% and 6,974,420 bp, 61.7%, respectively. The dDDH and ANI values between strains 4D117T, ZD32-2T and closely related Paraburkholderia species were in the ranges of 21.9-51.6% and 82.9-94.4%, and 81.7% and 25.4% between themself, respectively. Functional genomic analysis showed both strains were capable of degrading contaminants, such as benzoate, anthranilic acid and catechol for 4D117T, and benzene and catechol for ZD32-2T, indicating that they may have potentials for soil pollutant treatment. The main polar lipids of strains 4D117T and ZD32-2T were phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol. Strain 4D117T contained C16:0, C19:0 cyclo ω8c and C18:1 ω7c and/or C18:1 ω6c, while strain ZD32-2T had C16:0 and C17:0 cyclo as their major cellular fatty acids (> 10%). Based on the phenotypic characters and genomic data, strains 4D117T and ZD32-2T represent two novel species of genus Paraburkholderia, for which the names Paraburkholderia flagellata sp. nov. (type strain 4D117T = GDMCC 1.2617T = NBRC 115278T) and Paraburkholderia adhaesiva sp. nov. (type strain ZD32-2T = GDMCC 1.2622T = NBRC 115282T) are proposed.

Paracidobacterium acidisoli gen. nov., sp. nov. and Alloacidobacterium dinghuense gen. nov., sp. nov., two acidobacteria isolated from forest soil, and reclassification of Acidobacterium ailaaui and Acidipila dinghuensis as Pseudacidobacterium ailaaui gen. nov., comb. nov. and Silvibacterium dinghuense comb. nov.

Two aerobic and obligately acidophilic bacteria, designated 4G-K13T and 4Y35T, were isolated from the forest soil sampled at Dinghushan Biosphere Reserve, Guangdong Province, PR China. These two strains were Gram-stain-negative, non-motile and short rods that multiplied by binary division. Strains 4G-K13T and 4Y35T had the highest 16S rRNA gene sequence similarity of 97.0 and 97.2 % to Silvibacterium bohemicum DSM 103733T and Acidisarcina polymorpha SBC82T, respectively. Phylogenetic trees based on the 16S rRNA gene and whole genome sequences showed consistently that these two strains formed a major clade with members of the genera Acidipila, Acidisarcina, Silvibacterium and Acidobacterium in the family Acidobacteriaceae, but each occupied an unique position. In both the UBCG and the PhyloPhlAn phylogenomic trees, strains 4G-K13T and 4Y35T congruently formed a highly supported subclade with Acidobacterium capsulatum DSM 11244T and Acidobacterium ailaaui DSM 27394T, respectively. The major fatty acids (>5 %) of strain 4G-K13T were iso-C15 : 0, iso-C17 : 0, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and summed feature 9 (iso-C17 : 1 ω9c and/or C16 : 0 10-methyl), while that of strain 4Y35T were C16 : 0, C18 : 1 ω9c, iso-C15 : 0, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and summed feature 9 (iso-C17 : 1 ω9c and/or C16 : 0 10-methyl). Strain 4G-K13T contained phosphatidylethanolamine, four unidentified phospholipids, four glycolipids, two unidentified aminolipids and two unknown lipids, while strain 4Y35T had phosphatidylethanolamine, three unidentified phospholipids, two glycolipids, five unidentified aminolipids and one unknown polar lipid. The DNA G+C contents of 4G-K13T and 4Y35T were 60.5 and 55.8 mol%, respectively. Based on all these phylogenetic, physiological and chemotaxonomic data, we suggest that strains 4G-K13T and 4Y35T represent two novel species of two novel genera in the family Acidobacteriaceae, for which the names Paracidobacterium acidisoli gen. nov., sp. nov. (type strain: 4G-K13T=GDMCC 1.1195T=NBRC 113249T) and Alloacidobacterium dinghuense gen. nov., sp. nov. (type strain: 4Y35T=KACC 21728T=NBRC 114261T) are proposed. We also propose to reclassify Acidobacterium ailaaui and Acidipila dinghuensis as Pseudacidobacterium ailaaui gen. nov., comb. nov. and Silvibacterium dinghuense comb. nov., respectively, based mainly on the results of phylogenomic analysis.

Dyella telluris sp. nov. and Dyella acidiphila sp. nov., isolated from forest soil of Dinghushan Biosphere Reserve, China.

Cells of bacterial strains G9T and 7MK23T, isolated from forest soil samples collected from the Dinghushan Biosphere Reserve, Guangdong Province, PR China, were Gram-stain-negative, aerobic and rod-shaped. Strain G9T was motile with single polar flagellum and grew at 12-37 °C (optimum, 28 °C), pH 4.5-8.0 (optimum, pH 6.0-7.5) and in the presence of 0-3.5 % NaCl (optimum, 1.5%, w/v); while strain 7MK23T was non-motile and grew at 12-42 °C (optimum, 28-33 °C), pH 2.5-8.5 (optimum, pH 4.5-6.5) and NaCl levels of 0-1.0 % (optimum, 0-0.5 %, w/v). Phylogenetic analysis based on 16S rRNA gene sequences revealed that both isolates fell within the cluster of the genus Dyella. The closely related species (with a 16S rRNA gene sequence similarity >98.65%) of strain G9T were Dyella terrae JS14-6T (99.0 %), D. kyungheensis THG-B117T (98.8 %) and D. amyloliquefaciens DHC06T (98.7 %) while that of strain 7MK23T were D. mobilis DHON07T (99.2 %) and D. flava DHOC52T (99.1 %), but the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strains G9T, 7MK23T and the closely related Dyella species listed above were in the ranges of 77.5-83.8 % and 22.0-27.0 %, much lower than the species demarcation lines of 95.5 and 70 %, respectively. Phylogenomic analyses using UBCG and Phylophlan also supported that these two strains represent two novel species of Dyella. The major fatty acids of strain G9T were iso-C15 : 0, iso-C17 : 1 ω9c and iso-C17 : 0 while that of strain 7MK23T were iso-C15 : 0 and anteiso-C15 : 0. Ubiquinone-8 was the only respiratory quinone detected in both strains. The polar lipids of strain G9T consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, and several unknown phospholipids, aminophospholipids, aminolipids and lipid while strain 7MK23T contained phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine and several unknown phospholipids and aminophospholipids. The DNA G+C contents of strains G9T and 7MK23T were 64.7 and 63.4 mol%, respectively. On the basis of 16S rRNA gene sequence phylogenetic and phylogenomic analyses as well as phenotypic data obtained, we propose that strains G9T and 7MK23T represent two novel species of the genus Dyella, for which the names Dyella telluris sp. nov. (type strain G9T=KACC 21725T=GDMCC 1.2132T) and Dyella acidiphila sp. nov. (type strain 7MK23T=KCTC 62739T=GDMCC 1.1446T) are proposed.

Paraburkholderia acidiphila sp. nov., Paraburkholderia acidisoli sp. nov. and Burkholderia guangdongensis sp. nov., isolated from forest soil, and reclassification of Burkholderia ultramafica as Paraburkholderia ultramafica comb. nov.

Three Gram-stain-negative, aerobic, motile and rod-shaped bacterial strains, 7Q-K02T, DHF22T and DHOM02T, were isolated from forest soil sampled at Dinghushan Biosphere Reserve, Guangdong Province, China. Strains 7Q-K02T, DHF22T and DHOM02T grew at 4-37, 4-42 and 12-37 °C, pH 3.0-8.5, 3.5-8.5 and 5.0-8.0, and in the presence of 0-3.0, 0-3.5 and 0-2.5 % (w/v) NaCl; with optima at 28-33, 28 and 28-33 °C, pH 3.5-6.5, 4.0-5.5 and 6.5-7.0, and 0-1.5, 0-1.5 and 0.5-1.5 % (w/v) NaCl, respectively. Strains 7Q-K02T and DHF22T have the highest 16S rRNA gene sequence similarities of 99.0 and 98.0 % to Paraburkholderia sacchari LMG 19450T and 97.7 % between themselves, while strain DHOM02T shares the highest similarity of 98.4 % to 'Burkholderia rinojensis' A396T followed by 98.3 % to Burkholderia plantarii ATCC 43733T. In the 16S rRNA gene sequence phylogram, strain 7Q-K02T formed a sister branch with Paraburkholderia sacchari, Paraburkholderia oxyphila and Paraburkholderia paradisi, and strain DHF22T was separated from all other species within the genus Paraburkholderia, while strain DHOM02T formed a separated clade with members of the genus Burkholderia. The DNA G+C contents of strains 7Q-K02T, DHF22T and DHOM02T wwe 64.3, 65.4 and 66.6 %, respectively. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values of strains 7Q-K02T, DHF22T and closely related Paraburkholderia strains were in the ranges of 25.5-43.7 % and 81.5-91.3 %, respectively. While dDDH and ANI values between strain DHOM02T and Burkholderia strains with genome sequence data were in the ranges of 22.4-31.0 % and 78.2-86.1 %, respectively. These three strains have the same major respiratory quinone: ubiquinone-8. Strains 7Q-K02T, DHF22T and DHOM02T have C16 : 0, C17 : 0 cyclo, C19 : 0 cyclo ω8c and summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c) as their major fatty acid compositions. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. On the basis of phenotypic, phylogenetic, genomic analyses and chemotaxonomic data, strains 7Q-K02T and DHF22T represent two novel species of the genus Paraburkholderia, for which the names Paraburkholderia acidiphila sp. nov. (type strain 7Q-K02T=CGMCC 1.15433T=KCTC 62472T=LMG 29209T) and Paraburkholderia acidisoli sp. nov. (type strain DHF22T=GDMCC 1.1448T=LMG 30262T) are proposed, while strain DHOM02T represents a novel species in the genus Burkholderia, for which the name Burkholderia guangdongensis sp. nov. (type strain DHOM02T=KCTC 42625T=LMG 28843T) is proposed. We also propose to transfer Burkholderia ultramafica to the genus Paraburkholderia as Paraburkholderia ultramafica comb. nov. based mainly on the results of phylogenomic analysis.

Protective Effects of Reduced Beta 2 Glycoprotein I on Liver Injury in Streptozotocin (STZ)-Diabetic Rats by Activation of AMP-Activated Protein Kinase.

BACKGROUND Protective effects of reduced beta 2 glycoprotein I (Rb2GPI) against vascular injury of diabetes mellitus have been extensively investigated. However, the effects of Rb2GPI on liver injury in diabetic animals have not been reported. MATERIAL AND METHODS A diabetic rat model of was produced by systemic injection of streptozotocin (STZ). Rats were divided into a normal control group, a model group, and an Rb2GPI treatment group (N=6 in each group). After treatments, blood serum and liver tissue were collected to test the protection of Rb2GPI. AMP-activated protein kinase (AMPK) was detected by immunohistochemistry and Western blotting. RESULTS Our results revealed that Rß2GPI reduced blood glucose, serum creatinine, and urea nitrogen levels, as well as serum inflammation cytokines, including interleukin (IL)-6, tumor necrosis factor (TNF)-α and C-reactive protein in the diabetic rats. Importantly, Rß2GPI prevented liver injury in the diabetic rats as confirmed by hematoxylin-eosin (H&E) staining, alanine transaminase, aspartate transaminase, and gamma-glutamyl transferase. Reactive oxygen species (ROS) were promoted by diabetic modeling and were attenuated by Rß2GPI administration. Moreover, Rß2GPI significantly reduced liver catalase, malondialdehyde, and superoxide dismutase levels in the diabetic rats. Rß2GPI reduced liver glycolipid storage in STZ diabetic rats. Both immunohistochemistry and Western blotting demonstrated that Rß2GPI promoted AMPK phosphorylation in the diabetic rats. CONCLUSIONS Our data proved that Rß2GPI prevented liver injury in diabetic rats, likely through activating the AMPK signaling pathway.

Association of Thyroid-Stimulating Hormone Levels with Microvascular Complications in Type 2 Diabetes Patients.

BACKGROUND Subclinical hypothyroidism (SCH) is typically featured by elevated serum concentration of thyroid-stimulating hormone (TSH). This study aimed to determine the relationship between TSH levels and microvascular complications in type 2 diabetes patients. MATERIAL AND METHODS A total of 860 type 2 diabetes patients were enrolled in this cross-sectional study. Subjects were evaluated for anthropometric measurements, thyroid function, diabetic retinopathy, and diabetic kidney disease. TSH was divided into 3 levels: 0.27-2.49 mU/l, 2.5-4.2 mU/l, and >4.2 mU/l. RESULTS Among the participants, 76 subjects (8.8%) were diagnosed with subclinical hypothyroidism (SCH) (male: 6.6% and female: 11.8%). The prevalence of diabetic retinopathy did not differ among the groups (P=0.259). Of the 860 type 2 diabetic subjects, we further excluded invalid or missing data. Therefore, 800 and 860 subjects were included in our study of diabetic retinopathy (DR) and diabetic kidney disease (DKD), respectively. The frequencies of microalbuminuria and macroalbuminuria differed significantly among the different groups. The frequency of DKD was significantly different among the 3 groups (P=0.001) and was higher in subjects with higher TSH levels. After an adjustment for confounding variables, TSH levels were significantly associated with DKD (P<0.001). When compared with subjects with TSH 0.27-2.49 mU/l, the frequency of DKD was higher in subjects with TSH >4.20 mU/l (OR 1.531, 95% CI 1.174-1.997) and with TSH 2.50-4.20 mU/l (OR 1.579, 95% CI 1.098-2.270). However, TSH levels was not significantly correlated with DR (P=0.126). CONCLUSIONS Type 2 diabetic patients with higher TSH values had a higher prevalence of DKD.

Changes in liraglutide-induced body composition are related to modifications in plasma cardiac natriuretic peptides levels in obese type 2 diabetic patients.

BACKGROUND AND AIMS: Liraglutide treatment can improve glycemic control with a concomitant weight loss, but the underlying mechanism on weight loss is not completely understood. Cardiac natriuretic peptides (NPs) can resist body fat accumulation through increasing adipocytes lypolysis. In this study, we tested the hypothesis that liraglutide-induced weight loss was associated with increased plasma NPs concentrations. METHODS: Thirty-one outpatients with type 2 diabetes (T2D) treated with metformin and other oral antidiabetic drugs except for thiazolidinediones (TZDs) were subcutaneously administered with liraglutide for 12 weeks. Body composition, abdominal visceral adipose tissue areas (VAT) and subcutaneous adipose tissue areas (SAT) were assessed at pre- and post-treatment by dual-energy X-ray absorptiometry (DXA) scanning and abdominal computerized tomography (CT). Plasma atrial natriuretic peptides (ANP) and B-type ventricular natriuretic peptides (BNP) concentrations were tested by commercial ELISA Kit quantitatively. RESULTS: Following 12-week liraglutide treatment, body weight, waist circumference, total fat and lean mass, fat percentage, SAT and VAT areas were significantly reduced from baseline. Concurrently, plasma ANP and BNP levels were significantly increased following 12-week liraglutide treatment. There were significant correlations between the reductions in body compositions and the increases in both plasma ANP and BNP levels. CONCLUSIONS: There were significant correlations between increases in both plasma ANP and BNP levels and changes in liraglutide-induced body composition. Our data implied that increases in plasma NPs may add a novel dimension to explain how liraglutide induces weight loss.

[Changes of mechanical pain threshold in rats with experimental autoimmune prostatitis].

OBJECTIVE: To observe the changes of the mechanical pain threshold in the rat model of autoimmune prostatitis, explore the mechanism of autoimmune prostatitis pain and offer some animal experimental evidence for the drug therapy of the condition. METHODS: Twenty male Wistar rats weighing 180 - 220 g were divided into a model and a control group. The autoimmune prostatitis model was established by subcutaneous injection of an extract of male rat prostate glands (RPG) at 60 mg/ml in Freund's complete adjuvant (FCA) and pertussis-diphtheria-tetanus vaccine at 0 and 30 days, respectively. Mechanical tactile hyperalgesia was measured once a week using Von Frey Filaments from the beginning of the study. At 8 weeks after modeling, the rats were sacrificed and the prostate tissues harvested for observation of histomorphological changes by HE staining. RESULTS: HE staining revealed different degrees of benign prostatitis in the model rats. Compared with the controls, the mechanical pain threshold in the model rats was significantly decreased with the increased time of modeling, from (65.52 +/- 6.27) g at 0 week to (23.67 +/- 4.09) g at 8 weeks (P < 0.01). Statistically significant differences were found in the variation trend at different time points between the two groups (P < 0.01). CONCLUSION: Autoimmune prostatitis models were successfully established in rats and hyperalgesia was induced after modeling.

[Study on Chemical Constituents of Oldenlandia diffusa].

OBJECTIVE: To study the chemical constituents of Oldenlandia diffusa. METHODS: The chemical constituents were isolated and purified by silica gel and Sephadex LH-20 column chromatography as well as recrystallization method, and their structures were elucidated on the basis of physical and spectral analyses. RESULTS: Nine compounds were isolated and their structures were identified as quercetin (1), kaempferol (2), scopoletin (3), 2-hydroxy-3-methylanthraquinone (4), 2-hydroxy-l-methoxyanthraquinone (5), α-linolenic acid (6), vanillic acid (7), p-hydroxyphenylethanol (8) and, ß-sitosterol (9). CONCLUSION: Compound 6 is obtained from this genus for the first time. Compounds 7 and 8 are obtained from this plant for the first time.

Ethanol-induced yeast flocculation directed by the promoter of TPS1 encoding trehalose-6-phosphate synthase 1 for efficient ethanol production.

Yeast flocculation is an important trait in the brewing industry as well as in ethanol production, through which biomass can be recovered by cost-effective sedimentation. However, mass transfer limitation may affect yeast growth and ethanol fermentation if the flocculation occurs earlier before fermentation is completed. In this article, a novel type of cell-cell flocculation induced by trehalose-6-phosphate synthase 1 (TPS1) promoter was presented. The linear cassette HO-P(TPS1)-FLO1(SPSC01)-KanMX4-HO was constructed to transform the non-flocculating industrial yeast S. cerevisiae 4126 by chromosome integration to obtain a new flocculating yeast strain, ZLH01, whose flocculation was induced by ethanol produced during fermentation. The experimental results illustrated that flocculation of ZLH01 was triggered by 3% (v/v) ethanol and enhanced as ethanol concentration increased till complete flocculation was achieved at ethanol concentration of 8% (v/v). Real time PCR analysis confirmed that the expression of FLO1(SPSC01) was dependent on ethanol concentration. The growth and ethanol fermentation of ZLH01 were improved significantly, compared with the constitutive flocculating yeast BHL01 engineered with the same FLO gene but directed by the constitutive 3-phosphoglycerate kinase promoter PGK1, particularly under high temperature conditions. These characteristics make the engineered yeast more suitable for ethanol production from industrial substrates under high gravity and temperature conditions. In addition, this strategy offers advantage in inducing differential expression of other genes for metabolic engineering applications of S. cerevisiae.

Efficacy and safety comparison between liraglutide as add-on therapy to insulin and insulin dose-increase in Chinese subjects with poorly controlled type 2 diabetes and abdominal obesity.

OBJECTIVE: To assess the efficacy and safety of adding liraglutide to established insulin therapy in poorly controlled Chinese subjects with type 2 diabetes and abdominal obesity compared with increasing insulin dose. METHODS: A 12-week, randomized, parallel-group study was carried out. A total of 84 patients completed the trial who had been randomly assigned to either the liraglutide-added group or the insulin-increasing group while continuing current insulin based treatment. Insulin dose was reduced by 0-30% upon the initiation of liraglutide. Insulin doses were subsequently adjusted to optimized glycemic control. Glycosylated hemoglobin (HbA1c) values, blood glucose, total daily insulin dose, body weight, waist circumference, and the number of hypoglycemic events and adverse events were evaluated. RESULTS: At the end of study, the mean reduction in HbA1c between the liraglutide-added group and the insulin-increasing group was not significantly different (1.9% vs. 1.77%, p>0.05). However, the percentage of subjects reaching the composite endpoint of HbA1c ≤ 7.0% with no weight gain and no hypoglycemia, was significantly higher in the liraglutide-added group than in the insulin-increasing group (67% vs. 19%, p<0.001). Add-on liraglutide treatment significantly reduced mean body weight (5.62 kg, p<0.01), waist circumference (5.70 cm, p<0.01), body mass index (BMI) (1.93 kg/m2, p<0.01) and daily total insulin dose (dropped by 66%) during 12-week treatment period, while all of these significantly increased with insulin increasing treatment. Add-on liraglutide treated patients had lower rate of hypoglycemic events and greater insulin and oral antidiabetic drugs discontinuation. Gastrointestinal disorders were the most common adverse events in the liraglutide added treatment, but were transient. CONCLUSIONS: Addition of liraglutide to abdominally obese, insulin-treated patients led to improvement in glycemic control similar to that achieved by increasing insulin dosage, but with a lower daily dose of insulin and fewer hypoglycemic events. Adding liraglutide to insulin also induced a significant reduction in body weight and waist circumference. Liraglutide combined with insulin may be the best treatment option for poorly controlled type 2 diabetes and abdominal obesity.

Human Nei-like protein NEIL3 has AP lyase activity specific for single-stranded DNA and confers oxidative stress resistance in Escherichia coli mutant.

Oxidative base damage leads to alteration of genomic information and is implicated as a cause of aging and carcinogenesis. To combat oxidative damage to DNA, cells contain several DNA glycosylases including OGG1, NTH1 and the Nei-like proteins, NEIL1 and NEIL2. A third Nei-like protein, NEIL3, is composed of an amino-terminal Nei-like domain and an unknown carboxy-terminal domain. In contrast to the other well-described DNA glycosylases, the DNA glycosylase activity and in vivo repair function of NEIL3 remains unclear. We show here that the structural modeling of the putative NEIL3 glycosylase domain (1-290) fits well to the known Escherichia coli Fpg crystal structure. In spite of the structural similarity, the recombinant NEIL3 and NEIL3(1-290) proteins do not cleave any of several test oligonucleotides containing a single modified base. Within the substrates, we detected AP lyase activity for single-stranded (ss) DNA but double-stranded (ds) DNA. The activity is abrogated completely in mutants with an amino-terminal deletion and at the zinc-finger motif. Surprisingly, NEIL3 partially rescues an E. coli nth nei mutant from hydrogen peroxide sensitivity. Taken together, repair of certain base damage including base loss in ssDNA may be mediated by NEIL3.

Cloning and characterization of uracil-DNA glycosylase and the biological consequences of the loss of its function in the nematode Caenorhabditis elegans.

Uracil arises in DNA from spontaneous deamination of cytosine and through incorporation of dUMP by DNA polymerase during DNA replication. Excision of uracil by the action of uracil-DNA glycosylase (Ung) initiates the base excision repair pathway to counter the promutagenic base modification. In this study, we cloned a cDNA-encoding Caenorhabditis elegans homologue (CeUng-1) of Escherichia coli Ung. There was 49% identity in amino acid sequence between E.coli Ung and CeUng-1. Purified CeUng-1 removed uracil from both U:G and U:A base pairs in DNA. It also removed uracil from single-stranded oligonucleotide substrate less efficiently than double-stranded oligonucleotide. The CeUng-1 activity was inhibited by Bacillus subtilis Ung inhibitor, indicating that CeUng-1 is a member of the family-1 Ung group. The mutation in the ung-1 gene did not affect development, fertility and lifespan in C.elegans, suggesting the existence of backup enzyme. However, we could not detect residual uracil excision activity in the extract derived from the ung-1 mutant. The present experiments also showed that the ung-1 mutant of C.elegans was more resistant to NaHSO(3)-inducing cytosine deamination than wild-type strain.

Serum adipocyte fatty acid-binding protein levels are associated with peripheral arterial disease in women, but not men, with type 2 diabetes mellitus.

BACKGROUND: Adipocyte fatty acid-binding protein (A-FABP) has been recognized as an important player in macrophage cholesterol trafficking and inflammation, and may promote the development of atherosclerosis. To further elucidate the role of A-FABP in atherosclerosis in diabetes, we investigated the relationship between serum A-FABP concentrations and peripheral arterial disease (PAD) in patients with type 2 diabetes mellitus (T2DM). METHODS: In all, 488 inpatients with T2DM were enrolled in the study (254 men, 234 women; mean (±SD) age 57.3 ± 13.0 years). The severity of peripheral arterial stenosis was assessed by ultrasound examination. Serum A-FABP concentrations were determined by ELISA. RESULTS: Serum A-FABP concentrations were significantly higher in patients with than without PAD (8.0 ± 3.3 vs 6.2 ± 1.6 ng/mL, respectively; P < 0.05). Interestingly, there was an obvious gender-related difference in PAD patients with T2DM, with the stenosis rate being higher for female than male T2DM patients in the third A-FABP tertile. Logistic regression analysis revealed that serum A-FABP concentrations were an independent risk factor for PAD in female T2DM patients (odds ratio 1.890, 95% confidence interval 1.041-3.432; P = 0.036), but not in male T2DM patients. Correlation analyses revealed that A-FABP concentrations were correlated with body mass index (BMI), diastolic blood pressure, urinary microalbumin, and serum creatinine in male patients, and with BMI, duration of T2DM, fasting blood glucose, and serum creatinine in female patients. CONCLUSIONS: Serum A-FABP concentrations are closely associated with PAD in Chinese women with T2DM. The study findings suggest that A-FABP may be a more specific marker of PAD in diabetic women than men.

Recombinant Schizosaccharomyces pombe Nth1 protein exhibits DNA glycosylase activities for 8-oxo-7,8-dihydroguanine and thymine residues oxidized in the methyl group.

Bacteria and eukaryotes possess redundant enzymes that recognize and remove oxidatively damaged bases from DNA through base excision repair. DNA glycosylases remove damaged bases to initiate the base excision repair. The exocyclic methyl group of thymine does not escape oxidative damage to produce 5-formyluracil (5-foU) and 5-hydroxymethyluracil (5-hmU). 5-foU is a potentially mutagenic lesion. A homolog of E. coli endonuclease III (SpNth1) had been identified and characterized in Schizosaccharomyces pombe. In this study, we found that SpNth1 recognizes and removes 5-foU and 5-hmU from DNA with similar efficiency. The specific activities for the removal of 5-foU and 5-hmU were comparable with that for thymine glycol. The expression of SpNth1 reduced the hydrogen peroxide toxicity and the frequency of spontaneous mutations in E. coli nth nei mutant. It was also revealed that SpNth1 had DNA glycosylase activity for removing 8-oxo-7,8-dihydroguanine (8-oxoG) from 8-oxoG/G and 8-oxoG/A mispairs. These results indicated that SpNth1 has a broad substrate specificity and is involved in the base excision repair of 8-oxoG and thymine residues oxidized in the methyl group in S. pombe.

Effects of reduced ß2 glycoprotein I on high glucose­induced cell death in HUVECs.

Reduced ß2 glycoprotein I (ß2GPI) has been demonstrated to exhibit a beneficial effect in diabetic atherosclerosis and retinal neovascularization. However, the effect of reduced ß2GPI on vascular disorders in diabetic mellitus (DM) remains to be elucidated. The present study established a high glucose­induced injury model using human umbilical cords veins (HUVECs) and evaluated the protective effects of reduced ß2GPI against the injury. The data demonstrated that a low concentration of reduced ß2GPI (0.5 µM) mitigated high glucose­induced cell loss, decreased nitric oxide (NO) production and resulted in calcium overloading. Mechanically, reduced ß2GPI additionally reversed high glucose­induced phosphatase and tensin homolog (PTEN) accumulation, decrease of protein kinase B phosphorylation and nitric oxide synthase activity, and increase of cyclooxygenase­2 activity. It was further confirmed that PTEN inhibitor­bpV (1 µM) exhibited similar effects to those resulting from reduced ß2GPI. Overall, the data revealed that reduced ß2GPI exerts protective effects from glucose­induced injury in HUVECs, potentially via decreasing PTEN levels. The present study suggests reduced ß2GPI may act as a novel therapeutic strategy for the treatment of vascular disorders in DM.

Reduced beta 2 glycoprotein I prevents high glucose-induced cell death in HUVECs through miR-21/PTEN.

High serum beta 2 glycoprotein I (ß2GPI) is associated with complications of type 2 diabetes mellitus (DM), and especially microvascular disorders. In contrast, reduced ß2GPI (Rß2GPI) can prevent diabetic vascular injury. This study aimed to investigate the protective function of Rß2GPI in DM vascular disorders, and to assess the under lying mechanisms. High glucose-induced injury in human umbilical vein endothelial cells (HUVECs) was used to model hyperglycemia. Alow concentration of Rß2GPI (0.5 µM), but not ß2GPI, mitigated high glucose-induced cell injury. High glucose decreased miR-21 expression and Akt phosphorylation at 6 h, but facilitated their expression at 48 h. Moreover, high glucose decreased phosphatase and tensin homolog deleted on chromosome ten(PTEN) expression at 6 h, but facilitatedits expression at 48 h. Importantly, by promoting miR-21 expression, Rß2GPI mitigated high glucose-induced PTEN expression, reduced Akt phosphorylation and nitric oxide synthase activity, and increased cyclooxygenase-2 activity and cell loss. Similar to Rß2GPI, an miR-21 mimic (1 pM) and PTEN inhibition (1 µM bpV, or PTEN silencing) exerted protective action, while an Akt signaling pathway inhibitor (LY294002, 1 µM) aborted the effect of Rß2GPI on high glucose-induced cell injury. Finally, Rß2GPI inhibited high glucose-induced apoptosis via a mitochondria-dependent pathway. These data reveal that Rß2GPI exerts protective action in high glucose-induced HUVEC injury. The mechanism is related to the miR-21-PTEN-Akt pathway and mitochondria-dependent apoptosis. This study provides in vitro data supporting the therapeutic effect of Rß2GPI in diabetic vascular injury.

DNA repair fidelity of base excision repair pathways in human cell extracts.

Base excision repair (BER), responsible for the removal of altered DNA bases, is accomplished via two pathways that involve different subsets of repair enzymes and result in removal and replacement of one (short-patch BER) or several (long-patch BER) nucleotides. In this study, we constructed single-lesion containing DNA substrates that are predominantly repaired via one of the two pathways and investigated the fidelity of pathway specific repair in human whole cell extracts. We find that a single nucleotide deletion generated during addition of the first nucleotide into the repair gap is the major mutation characteristic for both pathways. This data suggest that for both BER pathways, mutations generated during repair in human whole cell extracts are principally the result of a slippage of DNA polymerase during initiation of repair synthesis.