Postoperative ileus appears to be underestimated after cardiac surgery. We conducted this study to analyse the incidence, risk factors and outcomes of postoperative ileus.P < 0.1 for inputs P < 0.05 for outputs.In this single-centre observational study we prospectively enrolled all patients undergoing elective cardiac surgery. The primary output was the time to faeces (TTFE) as representing the postoperative ileus. Secondary outputs were the occurrence of ischaemic colitis and pneumonia. Quantitative variables were compared by ANOVA or Wilcoxon tests, qualitative variables by chi-square tests. Multivariate analyses were performed by logistic regression, P < 0.01), had shorter extracorporeal circulation duration, 67 ± 27 versus 75 ± 24 minutes (P < 0.01), and received less blood products (P < 0.0001). Bypasses had lower postoperative levels of troponin  and LDH (330 ± 162 vs. 420 ± 175 pg/ ml). In contrast, the intra-abdominal pressure (IAP) was higher and related to the number of grafts at day 0 , and to the number of mammal grafts at day 0 and day 1 (P = 0.01 and 0.04 respectively). The TTFE was longer but did not reach significance (P = 0.13) as well as the occurrence of abdominal ischaemia (P = 0.22). The occurrence of pneumonia was higher (P = 0.01). In multivariate analysis, the IAP at day 0 and day 1 was related to propofol quantities only. The predictors of pneumonia were: duration of mechanical ventilation, peak lactate in the postoperative 24 hours, and coronary bypass: OR = 163, 2.6, and 4.2 respectively.We included 349 patients: age 67.5 ± 10.8 years, M/F sex ratio 252/97, preoperative left ventricle ejection fraction 58.8 ± 10.6%, bypass/valve ratio 234/154, number of grafts 2.7 ± 0.9, mammal arteries 1.8 ± 0.5. In univariate analyses, bypasses received more anaesthetic drugs (The number of coronary grafts and of mammal artery used in cardiac surgery is associated with higher IAP and higher risk of pneumonia. However, whether this is due to direct bowel ischaemia or longer anaesthesia remains to be studied in larger trials.


Paenibacillus polymyxa strain CCI-25, which displays strong antifungal and antibacterial activities in vitro. The genome encompasses nonribosomal peptide synthetases predicted to encode a tridecaptin, polymyxin, fusaricidin, an iturin-like synthetase, a lantibiotic similar to paenicidin A, as well as a type 1 polyketide synthase.We report here the draft genome sequence of  Paenibacillus polymyxa strains are well noted for their production of a wide range of secondary metabolites , and Rhizoctonia solani CBS101769, on plate assays.Plant-associated abolites –3, predobolites ––6. Here,extracts , 8 exhibN50 size of 95,765 bp. The draft genome size is 5.61 Mb, with a G+C content of 44.95%. The identification of 40 highly conserved single-copy marker genes in the assembly by PhyloSift version 1.0.1 (P. polymyxa CR1. The NCBI Prokaryotic Genome Annotation Pipeline (PGAP) identified 5,146 genes, 4,953 coding sequences (CDSs), 15 complete 5S rRNAs, 30 partial 16S rRNAs, 37 partial 23S rRNAs , 107 tRNAs, 4 noncoding RNAs (ncRNAs), and 241 pseudogenes. The rRNAs were further confirmed by RNAmmer 1.2 . Library sequencing was performed using an Illumina MiSeq platform (MiSeq reagent kit version 3). Sequencing generated 2,213,773 paired-end reads with 124 ± 53-fold coverage after PhiX sequence removal by Bowtie2 . Adapteron 1.0.1  indicatemmer 1.2 .Paenibacillus terrae NRRL B-30644 and fusaricidin C, and a polymyxin with leucine instead of phenylalanine at the 6th position compared to P. polymyxa M1. In addition, an iturin- and paenilarvin-like compound with altered monomer composition  compared to the published metabolites from Bacillus amyloliquefaciens FZB42 (<58% identity) and Paenibacillus larvae DSM 25430 (<40% identity)   has beensynthase . Given tLTYJ00000000. The version described in this paper is the first version.The nucleotide sequences have been deposited at the DDBJ/EMBL/GenBank under the accession no.


We report the complete genome sequence of a European genotype porcine reproductive and respiratory syndrome virus isolated from swine in northern China in 2012. Genome alignment revealed that the virus (LNEU12) strain shares 90.1% nucleotide identity with the European prototype Lelystad virus. Here, we announce the complete genome sequence of LNEU12. Arteriviridae family, that is a significant cause of swine morbidity and mortality worldwide (The porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped RNA virus, belonging to the orldwide , 2. PRRSorldwide . Currentorldwide . Since Porldwide , 6, 7, 8orldwide , 10. To The LNEU12 PRRSV was isolated from serum samples of piglets suspected to be infected in northern China in 2012. The complete genome sequence was generated with PCR using 15 pairs of oligonucleotide primers to amplify different regions of the PRRSV. The PCR products were purified and cloned into pMD18-T vector (TaKaRa), sequenced with an ABI3730xl genome sequencer, and assembled using DNAStar version 7.1 to obtain the complete genome sequence. The whole genome of LNEU12 is 15,083 bp, not including poly(A) sequences. Genetic analyses demonstrated that LNEU12 exhibits 90.1%, 93.1%, and 90.8% sequence identities with PRRSV strains Lelystad virus (LV), NVDC-NM1-2011, and BJEU06-1, respectively, and 57.5% sequence identity with the North American prototype VR2332, indicating that it belongs to EU-PRRSV.However, LNEU12 has 18-nt discontinuous deletions in comparison with the EU-PRRSV prototypic of strain Lelystad virus by DNAStar version 7.1, which are distributed in different regions as follows: 12 nt(ATTAACCTGGTA) deletions at positions 2,448 to 2,459, 0.3 nt(GTG) deletions at positions 2,608 to 2,610, and 3 nt(CCT) deletions at positions 13,142 to 13,144. Moreover, LNEU12 has similar characteristics to BJEU06-1, NMEU09-1, and NVDC-NM1-2011 (KM196101.The complete genome sequence of LNEU12 is available in GenBank under the accession number


These endophytic extracts were previously found to exhibit potent neuroprotective effect against LPS-challenged microglial cells.Excessive production of inflammatory mediators such as nitric oxide (NO) and proinflammatory cytokines like tumour necrosis factor-alpha (TNF-α) from activated microglia contributes to uncontrolled inflammation in neurodegenerative diseases. This study investigated the protective role of five endophytic extracts  against LPS-induced inflammatory events The effects of these fungal endophytic extracts against nitric oxide (NO), CD40 phenotype and, pro- and anti-inflammatory cytokine production in lipopolysaccharide (LPS)-stimulated BV2 microglia cells were examined using commercially available assay kits, immunophenotyping and flow cytometry, respectively.P < 0.05) inhibited by these endophytic extracts.Microglia pre-treated with the five endophytic extracts (0.1 mg/mL) reduced NO production without compromising cell viability. Whilst CD40 expression in LPS-stimulated microglia was not significantly different with or without the influence of endophytic extracts, expression of the proinflammatory cytokines, IL-6 and TNF-α in LPS-stimulated microglia was significantly  contains supplementary material, which is available to authorized users. Inflammation in the brain was previously observed only as a reactive response to neuronal damage . There iMicroglia account for approximately 12 % of cell population  in the cin vitro inflammatory responses of LPS-activated BV2 microglia cells.Medicinal plants, plant extracts and isolated secondary metabolites have traditionally been used to treat a variety of diseases including those associated with acute and chronic inflammation . EndophyCinnamomum porrectum . Cells were plated in 96-well microtiter plates at a density of 1 × 105 cells per well and incubated for 24 h. Cells were then treated with medium supplemented with endophytic extracts (0.1–1000 μg/mL) and after 24 h, the media was changed to 250 μL DMEM (without phenol red) per well and the standard iNOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME) (250 μM)  added to designated wells. The cells were then stimulated with 1 μg/mL lipopolysaccharide (LPS). Supernatants were collected at 18, 24 and 48 h for nitrite (NO2−) production assay. An equal volume of Griess reagent was mixed with cell culture supernatants and color development assessed at absorbance 530 nm with a microplate reader . The amount of NO2− in the samples was calculated from a standard curve (0–100 μM) of freshly prepared sodium nitrite. Cell viability was determined using the MTT reduction assay . Then, 50 μL of the mouse inflammation PE detection reagents were added to the entire test assay tubes and incubated for 2 h at room temperature in darkness. Wash buffer (1 mL) was added to each assay tube and centrifuged at 200 × g for 5 min. The bead pellet was resuspended in 300 μL wash buffer and analysed using FACSCalibur cytometer . The BD FACSCalibur flow cytometer is a four-color, dual laser and bench top flow cytometry system that provides both cell analysis and sorting. For intra-assay precision, ten replicates of each of the three different levels of IL-6, IL10, MCP-1, IFN-γ, TNF-α and IL-12p70 were tested and for inter-assay precision, three different levels of IL-6, IL10, MCP-1, IFN-γ, TNF-α and IL-12p70  were tested in four experiments. The detection limit of IL-6, IL10, MCP-1, IFN-γ, TNF-α and IL-12p70 is 5, 17.5, 52.7, 2.5, 7.3 and 10.7 pg/mL, respectively.BV2 microglia cells were seeded onto a 6-well plate at a density of 2 × 10P < 0.05.Differences in the anti-inflammatory effects of the extracts after treatment were evaluated using the One-Way ANOVA procedure of the SPSS version 17.0. When there was a difference, the LSD’s post hoc test was used to identify pairs that differed significantly. Differences were considered as significant at P < 0.05). For pre-treatment at 0.1 mg/mL, NO was significantly reduced by approximately 57.5–66.1 %, 64.2–74.7 % and 61.6–74.4 % following exposure to LPS for 18, 24 and 48 h, respectively (P < 0.05). Pre-treatment with extracts at lower concentrations (0.01–0.0001 mg/mL), however, reduced NO production by only 6.6–21.8 %, 8.0–19.7 % and 4.8–18.8 % following exposure to LPS for 18 h, 24 h and 48 h, respectively. For pre-treatment at 1.0 mg/mL NO production was significantly (P < 0.05) reduced by approximately 61.5–65.9 %, 68.9–76.0 % and 85.0–86.4 % following exposure to LPS for 18 h, 24 h and 48 h, respectively. The iNOS inhibitor, L-NAME (250 μM) that was added to BV2 simultaneously with LPS, significantly (P < 0.05) reduced NO production by 46.0 %, 50.7 % and 53.4 % at 18 h, 24 h and 48 h, respectively. The present study found the NO lowering effect of pre-treatment with endophytic extracts (0.1–1 mg/mL) to be either comparable to (at 0.1 mg/mL) or otherwise better (at 1.0 mg/mL) than that of standard nitric oxide synthase (NOS) inhibitor, L-NAME. It is noteworthy that the protective effects of pre-treatment with endophytic extracts as observed had no effect on basal NO release by microglia  was present in unstimulated BV2 microglia cells (resting cells) at all time points Fig. . The basP < 0.05), pre-treatment with all five extracts did not significantly affect the cell viability of BV2 microglia cells between concentrations of 0.0001–0.1 mg/mL, with or without LPS  increased CD40 expression on unstimulated BV2 cells [Fig. P < 0.05) suppressed CD40 expression on unstimulated BV2 cells by 53.4 %  were examined for their suppressive effects against CD40 expression on LPS-stimulated BV2 microglia cells. BV2 cells expressed CD40 at resting state and the expression of this antigen was found to increase following stimulation with LPS  suppressed IL-6 production (47.5–87.3 %) with extract HAB16R12 showing the greatest effect  (Table P < 0.05). Similar trend was also observed with TNF-α production. All five extracts reduced TNF-α (50.7–89.7 %) with extract HAB16R14 yielding the greatest suppression (89.7 % and 81.3 %) at 24 h and 48 h (Table P < 0.05). The extracts, however, elicited no significant effect against IL-10, IL-12p70 and IFN-γ in BV2 microglia cells (data not shown). BV2 microglia cells exhibited very low expression of these cytokines (<45 pg/mL) be it in the presence (stimulated by) or absence (unstimulated by) of LPS. For MCP-1, the levels produced by the microglia cells were beyond the detectable range of this assay.In the CNS, proinflammatory cytokines are produced primarily from activated microglia and are involved in the pathogenesis of brain inflammation . In ordein vitro and in vivo model. Phomol, 4-arylcoumarins and ergoflavin produced by endophytes showed anti-inflammatory activity using reporter gene assays and in ear oedema model in mice [in vitro model of microglia cells.Efforts in the past decade have been made to develop anti-inflammatory agents that are capable of inhibiting microglia activation and preventing neuronal cell death. The long-term use of the anti-inflammatory drugs is limited by of their side effects. As such, there have been many attempts uncover alternative anti-inflammatory agents from natural sources. There were already reports of anti-inflammatory activity by metabolites of several fungal and actinomycetous endophytes using  in mice , murine  in mice  and huma in mice , respectCytospora sp. has been previously reported to produce antibiotics: cytosporacin [50 of 20 μM [50 > 5 μg/ml) in vitro [sporacin , grahamisporacin , cytoskysporacin , cytosposporacin  and cytosporacin . Apart fof 20 μM . The purin vitro . Grahamiin vitro . To dateInflammation is closely related to pathogenesis of neurodegenerative diseases like Alzheimer’s disease , Parkinsp < 0.05) lowered NO production in LPS-activated BV2 cells with no significant effect on basal NO release by the cells. Extracts at 1.0 mg/mL, however, was toxic to BV2 cells and the reduced NO levels for cells pre-treated at this dosage was likely attributed to the pronounced cell loss. Interestingly, pre-treatment with extracts at 0.1 mg/ml was generally more effective than L-NAME, an iNOS inhibitor, in lowering NO production in activated microglia. The mechanism underlying the NO lowering effects by endophytic extracts remains poorly understood. There is, however, a large number of studies have considered that the antagonistic effects of natural compounds on NO production are due to suppression of NFĸB [In this study, the anti-inflammatory effect of endophytic extracts was investigated against LPS stimulated microglia. These extracts were previously found to exhibit excellent BACE1 inhibitory activity  and here of NFĸB .Microglial cells express the costimulatory molecule CD40 upon activation –32. CD40Besides NO, excessive production of inflammatory mediators such as proinflammatory cytokines including interleukin-1 beta (IL-1β) and tumor necrosis factor (TNF) from activated microglia contributes to uncontrolled inflammation in neurodegenerative diseases . Here, tA potential anti-inflammatory role of these endophytic fungal extracts on microglia is indicated. Although this study was performed on LPS-stimulated BV2 microglia cells, this paradigm partially reflects the pathological condition where activated microglia influences neuron viability . FurtherIn conclusion, the results demonstrated that these endophytic extracts exerted inhibitory effect against NO and proinflammatory cytokines (IL-6 and TNF-α) produced by activated BV2 microglia. It is likely that they would have a neuroprotective effect against inflammation resulting from the presence of inflammatory mediators such as NO, IL-6 and TNF-α in activated BV2 microglia. To our knowledge, this is the first report of the effect of a fungal endophytic extract in controlling inflammation in BV2 microglia cells.


Drug product purity and potency are of most significance in the regulatory market as we notice many recalled batches worldwide, particularly in the US and Japan. Olmesartan Medoxomil is an anti-hypertensive drug. The present invention relates to a process for the preparation of Olmesartan Medoxomil with 99.9% purity in an overall 62% yield. The synthesis includes three isolations and one purification with easy plant operations. This process describes the formation and control of each individual impurity in all stages. This process for Olmesartan Medoxomil and its intermediates is competent for industrial production in very short reaction time intervals with an appreciable yield and high purity. Ethyl acetate and water were added to the reaction mixture and the product was extracted into ethyl acetate. The purification of the product was achieved by flash column chromatography  and an additional crystallization from diisopropyl ether, hexane, ethyl acetate, or in mixtures of them.Yanagisawa escribed  the procet al. disclosed benzyl bromide (3) in the presence of anhydrous K2CO3 in N,N-Dimethylacetamide (DMA) afforded Trityl Olmesartan Ethyl Ester (4).The present study was initiated with kylation  of the iN-alkylation were low yield and isolation of compound 4. The lower yield in the reaction was due to the formation of impurities 5, 8, 9, 10, 11, & 12. The impurities 5, 8, & 9 were formed by ester hydrolysis of compounds 2 & 4 .The specified impurities mentioned should be controlled at this stage or else they may carry forward to the final stage, leading to a lower yield and purity of Olmesartan Medoxomil  and dibromomethylbiphenyl trityl tetrazole . The crystallized process of compound 4 was successfully executed even after selecting compound 3 with a total raw material impurity of ~9.6%. These impurities were found undetected after the purification process  from 100 mesh (90%).This first stage was taken in plant-scale and it was observed that the reaction time intervals were enhanced to 48 h against 24 h as obtained in lab-scale. To overcome the issue, an additional mole ratio of KThe potassium carbonate particle size impact on reaction time is summarized in 4 was studied at room temperature up to 48 hrs and was found to be stable.The stability of 4 which afforded compound 5. The reaction under the specified conditions has  +.To a solution of 4  in a mixture of tetrahydrofuran (1200 L), ethanol (200 L) was added to a pre-cooled aqueous sodium hydroxide  solution in DM water (50 L) at 10–15°C and stirred for 5 h. Thereafter, we concentrated the reaction mass to below 20°C under reduced pressure to afford Trityl Olmesartan sodium salt (5) as a thick oily mass. To the solution of 5 in DMA (600 L) was added 4-(chloromethyl)-5-methyl-1,3-dioxol-2-one (6)  and sodium iodide , DM water (2000 L), followed by sodium metabisulphite (2 kg) at 30–35°C and stirred for 15 min. The layers were separated and the organic layer was washed with 20% w/w aqueous sodium chloride solution (2×1000 L) at 30-35°C. The organic layer was concentrated at 30–40°C under reduced pressure till the distillate volume was 1150 L, and then diisopropylether (1000 L) was added at 30–40°C. The slurry was cooled to 0–5°C and stirred for 30 min. The product was filtered and dried under reduced pressure to obtain 7 as a white powder  with 99.47% purity by HPLC. Mp 104–106°C. IR (KBr): 3398, 3059, 3027, 2873, 1819, 1805, 1737, 1707, 1527, 1492, 1465, 1393, 1357, 1256, 1170, 1094, 1004, 726, 678 cm-1. 1H-NMR : δ = 7.78–7.29 , 7.02 , 6.87 , 6.77 , 5.35 , 5.25 , 5.00 , 2.43 , 2.02 , 1.54 , 1.49 , 0.75 . MS: m/z = 801.4 [M+H]+.To a pre-cooled solution of 7 (175 kg) in 75% v/v aqueous acetic acid (875 L) was stirred at 25–30°C for 10 h. The byproduct, trityl alcohol, was filtered through hyflo and washed with 75% v/v aqueous acetic acid (200 L). Methylene chloride (1225 L) was added to the filtrate followed by DM water (875 L), which was added at 20–30°C and stirred for 15 min. The layers were separated and the aqueous layer was extracted with methylene chloride (525 L) at 20–30°C. The combined organic extract was washed with DM water (2×1750 L) at 20-30°C. Water was added at 20–30°C to the organic layer and its pH was adjusted to 7.30–7.50 with 5% w/w aqueous sodium bicarbonate (4.3 kg in 81 L of Dm water) at 20–30°C. The organic layer was separated and concentrated at 30-40 °C under reduced pressure. Acetone (700 L) was added to the concentrated mass and distillation was continued at 50–55°C at ambient pressure, till the distillate volume was 600 L. The resulting slurry was cooled to 0–5°C and stirring was continued for 1 h. The product was filtered and dried to afford 1  with 99.7% purity by HPLC. Mp 182–184°C. IR (KBr): 3290, 3039, 3003, 2930, 1922, 1832, 1707, 1551, 1474, 1389, 1301, 1258, 1225, 1168, 1135, 1088, 1002, 953, 816, 782, 760 cm-1. 1H-NMR : δ = 7.67 , 7.56 , 7.04 , 6.86 , 5.42 , 5.32  5.24 , 2.62 , 2.08 , 1.59 , 1.47 , 0.88 . MS: m/z = 559.3 [M + H]+.The suspension of Olmesartan Medoxomil crude (94 kg), carbon  was added to acetone (1600 L) and we heated the contents to reflux temperature, and stirring was continued for 30 min. The contents were cooled to 45–50°C and the carbon was filtered through hyflo, washed with hot acetone . Ethyl acetate (94 L) was added to the filtrate and concentrated to below 55–60°C at ambient pressure to collect 1560 L of the distillate. Thereafter, the resulting slurry was cooled to 0–5°C and stirred for 30 min. The product was filtered and dried to afford pure Olmesartan Medoxomil  as white crystalline powder having 99.9% of HPLC purity.1  having residual acetone less than 1000 ppm.The obtained Olmesartan Medoxomil (80 kg) was suspended in DM water (1600 L) at 25–30°C and stirring was continued for 1 h at this temperature. The product was filtered and dried under reduced pressure to afford pure


Line 13–19 on p. 5, right column, should read as follows:2 receptor binding relative to baseline amounted to 21 ± 35% after 5 mg/kg L-DOPA/benserazide and to 19 ± 15% after 10 mg/kg L-DOPA/benserazide. Multiplication of percentual decreases by Kexo/Kendo yielded mean increases of synaptic DA by 213 ± 360% after 5 mg/kg L-DOPA/benserazide and by 199 ± 160% after 10 mg/kg L-DOPA/benserazide.”“DLikewise, l. 9–14 on p. 13, right column, should read as follows:exo/Kendo), resulting in mean increases of DA by 213 ± 360% after 5 mg/kg and to 199 ± 160% after 10 mg/kg L-DOPA/benserazide.”“Changes of synaptic DA concentrations were estimated by multiplying the percentual alteration of exogenous ligand binding with the ratio of affinities (KThis error does not change the scientific conclusions of the article in any way.Writing of the manuscript: SN. Editing of the manuscript: MB, MASS, JPH, HH, CM, CA, and HWM.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Gordonia sihwensis strain 9 is a Gram-positive bacterium capable of efficient aerobic degradation of branched and normal alkanes. The draft genome of G. sihwensis S9 is 4.16 Mb in size, with 3,686 coding sequences and 68.1% G+C content. Alkane monooxygenase and P-450 cytochrome genes required for alkane degradation are predicted in G. sihwensis S9. Gordonia sihwensis strain 9, a Gram-positive actinomycete bacterium, was isolated from raw sludge from a wastewater treatment facility in Dayton, OH, USA after enrichment with hydrocarbons. This strain was shown to metabolize a gamut of normal and branched alkanes. The type strain of G. sihwensis was originally isolated from a wastewater-treatment bioreactor in South Korea (http://blast.ncbi.nlm.nih.gov/Blast.cgi) of the 16S rRNA gene sequence, G. sihwensis S9 is 100% similar to G. sihwensis NBRC 108236, DSM 44576, and SPR2, 99% similar to G. neofelifaecis NRRL B-59395, and 98% similar to G. alkanivorans NBRC 16433. The gyrase subunit A gene (gyrA) of strain S9 is 100% similar to G. sihwensis NBRC 108236 but only 92% and 84% similar to G. neofelifaecis and G. alkanivorans, respectively. Gordonia species, including G. alkanivorans have been shown to actively degrade branched and aromatic hydrocarbons and contain desulfurization pathways  approach, producing 360,174 reads. The Roche de novo assembly software aligned reads into 348 large (>500 bp) contigs with an average depth of 40 reads and N50 of 25,140 bp. The largest contig extended 183,625 bp. The draft genome sequence was 4,155,610 bases in length with a G+C content of 68.1%. The NCBI Prokaryotic Genome Annotation Pipeline (http://www.ncbi.nlm.nih.gov/genome/annotation_prok/) predicted 4,115 genes, including 3,686 coding sequences (CDSs), 51 tRNAs, and 372 pseudogenes. BLAST analysis indicated three copies of the 16S rRNA gene. Rapid genome annotations using the RAST server (n = 403 CDSs); carbohydrates (n = 308); cofactors, vitamins, prosthetic groups, and pigments (n = 243); protein metabolism (n = 231); fatty acids, lipids, and isoprenoids (n = 229); RNA metabolism (n = 108); nucleosides and nucleotides (n = 88); and virulence, disease, and defense (n = 80) were most abundant.alkB1). Immediately downstream of alkB1 in contig 00033, 2 rubredoxin genes and a rubredoxin reductase gene were identified. Similar three-part genetic system for the degradation of normal and branched alkanes has been observed in P. aeruginosa and Rhodococcus sp., respectively (Alcanivorax borkumensis P-450 alkane hydroxylase cytochromes were predicted. A. borkumensis cytochromes are involved in the degradation of branched alkanes (Gordonia species adapt to and degrade hydrocarbons.The NCBI Prokaryotic Genome Annotation Pipeline predicted 3 alkane 1-monooxygenase genes (ectively , 7. EighJZDP00000000.This project has been deposited at DDBJ/EMBL/GenBank under the accession no.


Physalis peruviana L.) juice was determined by GC-MS. Quercetin and kaempferol were active components in the juice. In this study we have evaluated its potential protective effect on hepatic injury and fibrosis induced by carbon tetrachloride (CCl4). Twenty-eight rats divided into 4 groups: Group I served as control group, and Group II received weekly i.p. injection of 2 mL CCl4/kg bwt for 12 weeks. Group III were supplemented with Physalis juice via the drinking water. The animals of Group IV received Physalis juice as Group III and also were intraperitoneally injected weekly with 2 mL CCl4/kg bwt for 12 weeks. Hepatoprotective effect was evaluated by improvement in liver enzymes serum levels, reduction in collagen areas, downregulation in expression of the fibrotic marker MMP-9, reduction in the peroxidative marker malonaldehyde and the inflammatory marker nitric oxide, and restoration of the activity of antioxidant enzymatic and nonenzymatic systems, namely, glutathione content, superoxide dismutase, catalase, glutathione-S-transferase, glutathione peroxidase, and glutathione reductase activities. The results show that the potential hepatoprotective effects of Physalis peruviana may be due to physalis acts by promotion of processes that restore hepatolobular architecture and through the inhibition of oxidative stress pathway.The active constituent profile in Cape gooseberry ( Liver diseases are amongst the most serious health problems in the world today and their prevention and treatment options still remain limited despite tremendous advances in modern medicine. The pathogenesis of hepatic diseases as well as the role of oxidative stress and inflammation therein is well established and, accordingly, blocking or retarding the chain reactions of oxidation and inflammation process could be promising therapeutic strategies for prevention and treatment of liver injury .4. Documented evidences suggested that CCl4 has been commonly used as hepatotoxins in experimental hepatopathy  − 100, where A is the absorbance.The free radical scavenging capacity was evaluated by the 2, 2-diphenyl -1-picrylhydrazyl (DPPH) assay. In its radical form, DPPH is monitored at 517 nm but, upon reduction by an antioxidant or a radical species, the absorption decreases. Briefly, 1 mL of 0.25 mM solution of DPPH in methanol was added to 50, 100, 150, and 200 uL of sample in 950, 900, 850, and 800 μL methanol, respectively, and 1 mL of 1 mM PMS were added. After incubation at 25°C for 5 min, the absorbance was measured at 560 nm against a blank. Vit. C was used as a positive control.The superoxide anion scavenging activity was determined by the method of Nishikimi et al. . SuperoxμL methanol, respectively, and incubated at room temperature for 150 min followed by addition of 0.5 mL of Griess reagent ethylenediamine dihydrochloride). The absorbance of the chromophore formed was read at 546 nm.The nitric oxide radical inhibition activity was measured using Griess reagent. Briefly, sodium nitroprusside (10 mM) in phosphate buffered saline was mixed with 50, 100, 150, and 200 uL of sample in 950, 900, 850, and 800 μL of 15 mM FeSO4 solution to 3 mL of liver homogenate . After 30 min, 100 μL of this reaction mixture was taken in a tube containing 1.5 mL of 0.67% thiobarbituric acid (TBA) in 50% acetic acid. Samples were incubated at 37°C for 1 hr, and then lipid peroxidation was measured using the reaction with TBA. The absorbance of the organic layer was measured at 532 nm. All reactions were carried out in triplicate. Vitamin C (Vit. C) was used as the positive control. The percentage of inhibition of lipid peroxidation was calculated, by the formula Inhibition (%) = (Acontrol − Asample) × 100/Acontrol.The total antioxidant potential was measured by the ability of sample to scavenge thiobarbituric acid reactive substances (TBARS) . BrieflyμL injected using the splitless injection technique; fused capillary silica column TG-5MS (30 m × 0.25 mm × 0.25 μm). Temperatures: injector: 260°C, detector: 300°C, column: 70°C, 10°C min−1, and 260°C (10 min). The total GC running time is at 60 min. The MS was taken at 70 eV. The MS scan parameters included a mass range of m/z 40–1000, a scan interval of 0.5 s, a scan speed of 2000 amu s−1, and a detector voltage of 1.0 kV. Identification of compounds was conducted using the database of Wiley9 combined with NIST 11 mass spectral database. The name, molecular weight, molecular formula, and area under peak of the components of the test materials were ascertained.The GC-MS analysis of physalis juice was performed with Thermo Scientific, Trace GC Ultra & ISQ Single Quadruple MS. The inert gas helium (99.9995%) was used as carrier gas, at flow rate of 1.5 mL/min; split ratio 10 : 1; sample size, 1 μL of saline by intraperitoneal (i.p.) injection route each week. Group II (CCl4) received weekly i.p. injection of 2 mL CCl4/kg bwt for 12 weeks as described by Sohn et al. , diacetate (1.45%), Pregna-4,6-diene-21-carboxylic acid, 17-hydroxy-3-oxo-, γ-lactone (0.75%), Hexadecatrienoic acid, methyl ester (0.64%), beta-k-strophanthin (2.48%), 2,2,4,9,11,11-Hexamethyl dodecane (0.66%), Cholest-5-en-3-one (0.63%), 9,12,15-Octadecatrienoic acid methyl ester (0.89%), 9-cis-Hexadecenoic acid (0.93%), 3,7,11-Trihydroxypregnan-20-one (0.99%), Ceanothine C (1.26%), Methyl-9,9,10,10-D4-octadecanoate (4.01%), Lucenin-2 (1.50%), Betulin (0.62%), (5á)Pregnane-3,20á-diol (0.97%), Anodendroside G, monoacetate (0.64%) and 7,8-Epoxylanostan-11-ol, 3-acetoxy- (0.61%).GC-MS chromatogram of the physalis showed 29 peaks indicating the presence of 29 phytochemical constituents. On comparison of the mass spectra of the constituents with Wiley9 combined with NIST 11 libraries, the 29 phytoconstituents were characterized and identified as it shown in Table S1. The identified phytochemical constituent's mass spectra are Oleic acid, eicosyl ester (5.04%), Colchifoleine (1.48 %), 3Click here for additional data file.


Here, caged RET inhibitors were obtained from 3-substituted pyrazolopyrimidine-based compounds by attaching photolabile groups to the exocyclic amino function. The most promising compound displayed excellent inhibitory effect in cell-free, as well as live-cell assays upon decaging. Furthermore, inhibition could be efficiently activated with light in vivo in zebrafish embryos and was shown to effect motoneuron development.Proto-oncogene tyrosine-protein kinase receptor RET is implicated in the development and maintenance of neurons of the central and peripheral nervous systems. Attaching activity-compromising photocleavable groups ( It follows that caged effectors would represent a powerful technique for manipulating biological processes234in situ release of for example ATP78912Receptor tyrosine kinases and their related signalling pathways are essential for cell developmente.g., the development of the central and peripheral nervous systems. Dysregulation of RET has been found in thyroid cancers, including papillary thyroid carcinomas and multiple endocrine neoplasia type 214151  phosphorylation, all downstream of RET. It was also found to inhibit proliferation to a similar degree as tamoxifen. No cytotoxicity was observed in any of the cell lines used in these experiments.Ret, also referred to as RET , is a receptor tyrosine kinase involved in several processes of biological importance, Here, we report the design, synthesis, and biological evaluation of a caged small-molecule inhibitor of RET in cell-free and live-cell assays, as well as in zebrafish.1. NVOC can be removed at wavelengths longer than 350 nm, i.e. wavelengths sufficiently low in energy to avoid extensive cell damage. Moreover, NVOC-caged retinoic acid has been used to study the effect of retinoic acid on the development of zebrafish embryos and it was reported that no effects of the nitrosoaldehyde byproduct formed in the deprotection were observedA wide range of photolabile caging groups is described in the literature1 on the exocylic amino functionality that interacts with the backbone of RET in the ATP-binding site through a hydrogen bond to the amide oxygen of Glu805  gave 2  gave 2  in 42% ytert-butyldimethylsilyl)oxy)ethyl)-3-(phenylethynyl)-1H-pyrazolopyrimidin-4-amine (see SI for details) with 6-nitroveratryloxycarbonyltetrazolide as described for 1 followed by removal of the silyl protecting group on the hydroxyl function by tetrabutyl ammonium fluoride (TBAF) resulted in 3. Again, the solubility in aqueous media was too low to be practical for biological assays. The high lipophilicity of the protected compounds necessitated a new approach in which we instead modified the protecting group to increase the hydrophilicity.Reacting 1-. Using a protocol developed for tBoc-protection of primary anilines1 was heated at 105 °C with carbonyldiimidazole (CDI) in DMF followed by addition of 4 which resulted in 5 .Carboxylic acid analogs of nitrobenzyl protecting groups have been reported216 was readily achieved with 365 nm light, as monitored by HPLC. The decaging followed first order kinetics  up to 100 μM. Photoinduced cleavage of  mW/cm2,  with resin 37 °C , indicatin vitro assay with purified RET kinase was used to measure ADP production during substrate phosphorylation. Compound 6 was added to two parallel preparations comprising RET kinase and substrate. One preparation was exposed to light , while the other was kept in the dark.With the caged compound in hand, the ability to photocontrol the inhibition of RET kinase was assessed. For this purpose, an 6 increased 12-fold upon light exposure (IC50: 6.8 μM → 590 nM), clearly illustrating light-activation of the RET kinase inhibitor , while the other was kept in the dark. Again, 15 min is clearly not sufficient to complete the decaging (see above). Addition of the growth factor neurturin (required to activate the RET kinase) and incubation for 3 h at 22 °C allowed for activity-correlating signal induction using the supplemented detection reagents. In accordance with the cell-free assay, a clear difference in kinase activity was observed after incubation with irradiated (365 nm for 15 min) vs. non-irradiated 6. Irradiated 6 showed an IC50 of 8.7 μM  at concentrations higher than 6 μM. However, no IC50 value could be obtained for non-irradiated 6. As expected from the decaging experiments and in vitro assay, incubations with free 1 resulted in a lower IC50 (470 nM), than for irradiated 6. In the live-cell assay, it is seen that the activity of RET can be suppressed to lower values than the positive control (in which no growth factor was added), leading to negative activity readings in 6 in zebrafish embryos.The photoactivation of f 8.7 μM  while noDanio rerio) is a popular model organism for studying developmental biology. Reasons for this include its fast early development, embryonic transparency, and mapped genome. Similar to humans29Ret mutant mice show aberrant motoneuron innervationThe zebrafish (6 in the transgenic zebrafish line tg(olig2:dsRed)346 at 3 hours post fertilization (hpf) for 11 h. We opted for decaging at 14 hpf since primary motoneurons start to sprout growth cones at 18 hpf6 and irradiated for 15 min at 14 hpf displayed motoneurons with shortened and malformed axons  during development or with 1 (50 μM) for 90 min at 6 hpf  or irradiation (15 min at 365 nm) alone does not affect motoneuron development of embryos. Furthermore, the concentration of 6 is clearly influential for the development of the irradiated embryos, as 10 μM had virtually no effect when contrasted to 50 μM. We also irradiated embryos at 24 hpf after 10 h of incubation with 6 and subsequent wash  was purchased from Sigma Aldrich. Reactions were monitored by TLC (Merck silica gel 60 F254) and analyzed under UV (254 nm). Microwave reactions were performed in a Biotage Initiator reactor with fixed hold time. Column chromatography was performed by manual flash chromatography  or by automated column chromatography on a Biotage SP-4 instrument using pre-packed silica columns. Analytical high-performance liquid chromatography (HPLC) analysis was carried out on a Waters separation module 2690 connected to a Waters photodiode array detector 996 using an Atlantis® 5 μm C18 AQ (250*4.6 mm) column eluting with a gradient of 20–100% acetonitrile in water using 0.1% TFA as buffer. 1H- and 13C-NMR spectra were obtained at 400 and 100 MHz respectively, using a Varian 400/54 spectrometer. For compound 6, additional 13C-NMR experiments were acquired on a Bruker Advance III HD 800 MHz spectrometer. All reactions where photolabile protecting groups were involved were carried out avoiding direct light, i.e. covering reaction vessels and columns with aluminum foil and working with the fume hood lamp turned off (ceiling lamp was left on).All commercial chemicals were used without prior purification. CH1  was dissolved in DMF (1.5 ml) in a MW-vial (0.5–2 ml) and CDI  was added. The vial was capped, flushed with nitrogen and heated at 105 °C. After 2 h, the vial was removed from the heat source and a solution of 4  in DMF (1 ml) was added after 5 min. The reaction mixture was stirred at room temperature for 19 h at which point full consumption of 1 was confirmed by TLC (5% methanol in CHCl3). The reaction mixture was poured in to ice cold water (40 ml) which resulted in precipitation, ethyl acetate (100 ml) was added, the precipitate was dissolved and the phases were separated. The aqueous phase was extracted with ethyl acetate (50 ml) and the organic phases were pooled and concentrated. The resulting residue was dissolved in CH2Cl2 and passed through a silica column (10 g) eluting with 2% methanol in CH2Cl2. After evaporation of the solvents, the resulting yellow residue was washed with ice cold acetonitrile to provide 5 as a white solid . 1H NMR : 8.77 , 8.50 , 7.69 , 7.62 , 7.46 – 7.34 , 7.27 , 5.74 , 5.22 , 4.75 , 4.28 , 3.89 , 1.61 , 1.31 ; 13C NMR : 167.9, 156.2, 154.1, 152.9, 152.5, 150.3, 146.9, 140.1, 131.8, 130.0, 129.0, 127.2, 125.1, 121.0, 112.9, 110.9, 104.0, 96.2, 80.5, 66.3, 65.2, 61.9, 56.7, 50.2, 22.1, 14.3; HRMS (m/z): [M + H]+ calculated for C29H28N6O8, 589.2047; found, 589.2052.Following a procedure for carbamate formation5  in THF (1 ml) and water (1 ml) was added LiOH monohydrate . The reaction mixture was stirred at room temperature for 20 min. Full consumption of 5 was confirmed by TLC (5% methanol in CHCl3). The solvents were removed, the residue was taken up in water (10 ml) and the pH was set to 3–5 with HCl . The aqueous phase was extracted with ethyl acetate (3 × 15 ml) and the organic phases were pooled and concentrated. The white residue was dissolved in CHCl3 (60 ml) and MQ-water (10 ml), the aqueous phase was washed with ethyl acetate (100 ml), the organic phases were pooled and the solvents were removed to provide 6 as a white solid  after drying under vacuum overnight. 1H NMR : 13.21 , 10.98 , 8.78 , 7.64 , 7.56–7.49 , 7.45–7.39 , 7.38–7.30 , 7.22 , 5.45 , 5.19 , 4.84 , 3.84 , 1.53 ; 13C NMR : 169.6, 154.9 (broad), 153.5, 153.1 , 152.8 (broad), 146.2, 138.8, 131.2, 129.4, 128.7, 126.9, 126.8, 121.2, 110.8, 109.6, 107.1, 93.0, 81.9, 65.4, 63.9, 56.2, 49.4, 21.7; HRMS (m/z): [M + H]+ calculated for C27H24N6O8, 561.1734; found, 561.1710.To a suspension of 6, see 2 flux ca. 1 cm from the lamp. Irradiation in the assays was accomplished by placing the lamp directly on top of the sample plates/dishes. It should be noted that the exact light conditions in the assays may differ slightly depending on the type of plates/dishes used (see Methods sections below for plate specifications).Steady state absorption measurements , substrate (IGFlRtide), and ATP were purchased in an assay ready kit from Promega2 in a white flat-bottom 96-well plate. Incubation volume was 25 μL. Brief procedure; Kinase (0.8 μg/mL), substrate (40 μg/mL), ATP (10 μM) and inhibitor were combined and incubated for 30 min at room temperature in the dark, with shaking. After the kinase reaction, two detection reagents were added in a stepwise manner. The first reagent depleted the remaining (unreacted) ATP in the reaction mix. The second reagent converted the ADP produced in the kinase reaction to ATP and generated a luminescence signal using a luciferase/luciferin reaction. The light generated is proportional to the amount ADP present and, consequently, to the kinase activity. Luminescence was recorded on a BMG Labtech Fluostar Omega luminometer. All incubations were done in duplicates.Human recombinant RET expressed in Sf9 insect cells  and incubated 3 h at 22 °C in the dark. An activity-correlating luminescence signal was induced using the detection reagent provided in the kit according to the recommended protocol. Luminescence was recorded on a BMG Labtech Fluostar Omega luminometer. All incubations were done in duplicates.PathHunter express c-RET-GFRα2 functional assay and recombinant human neurturin were purchased from DiscoveRx  and used according to instructions. For a thorough description of the key concepts of the assay, see Danio rerio) embryos of strain tg(olig2:dsRed)2·2H2O, 0.33 mM MgCl2·6H2O), in darkness, in an incubator at 28.5 °C. Eggs were incubated in either 6 or 1% DMSO from 3 hours post-fertilization (hpf) and then dechorinated at 9 hpf to allow for better compound uptake. After 11  hours of incubation, 14 hpf embryos were washed three times with EM and moved to clean petri dishes containing 1-phenyl 2-thiourea  and diluted in EM to prevent pigmentation. Embryos were irradiated for 15 min (365 nm) and then kept in the incubator until time of confocal imaging. A second set of animals were incubated in 6 from 14 hpf to 24 hpf, after which animals were washed and illuminated in the same way as the first group. Prior to imaging at 2 days post-fertilization (dpf), the embryos were anesthetized using 0.02% tricaine methanesulfonate (MS-222) diluted in EM and embedded in low-melting agarose. The imaging was performed on a ZEISS LSM710 confocal microscope at the Centre for Cellular Imaging at the Sahlgrenska Academy. Stacks of labeled spinal cord neurons were collected using a 561 nm laser. Maximal intensity pictures of spinal cord hemisegments and axonal phenotypes were analyzed using ImageJ  according to Abramsson et al. 2013All experimental procedures were carried out in compliance with relevant guidelines and regulations of the Swedish National Board for Laboratory Animals. The protocols were also approved by the medical ethics committee of University of Gothenburg. Zebrafish .


We aimed to evaluate the effect of oral l-glutamine, compared with whole protein low in glutamine, on insulin response in well-controlled T2D patients. In a randomized study with a crossover design, T2D patients  aged 65.1 ± 5.8, with glycosylated hemoglobin (HbA1c) 6.6% ± 0.7% (48 ± 8 mmol/mol), received oral l-glutamine (25 g), protein (25 g) or water, followed by an intravenous glucose bolus (0.3 g/kg) and hyperglycemic glucose clamp for 2 h. Blood was frequently collected for analyses of glucose, serum insulin and plasma total and active GLP-1 and area under the curve of glucose, insulin, total and active GLP-1 excursions calculated. Treatments were tested 1–2 weeks apart. Both l-glutamine and protein increased first-phase insulin response (p ≤ 0.02). Protein (p = 0.05), but not l-glutamine (p = 0.2), increased second-phase insulin response. Total GLP-1 was increased by both l-glutamine and protein (p ≤ 0.02). We conclude that oral l-glutamine and whole protein are similarly effective in restoring first-phase insulin response in T2D patients. Larger studies are required to further investigate the utility of similar approaches in improving insulin response in diabetes. Numerouex vivo . In typeng GLP-1 ,8,9, delng GLP-1 .The aim of the present study was to compare the effects of oral glutamine with whole protein low in glutamine on: (i) first- and second- phase insulin response using the gold standard hyperglycemic glucose clamp; and (ii) total and active GLP-1 in well-controlled type 2 diabetes patients. We hypothesized that glutamine would be more efficacious than whole protein in restoring insulin response due to greater increases in GLP-1.1c) 6.5%–9% (48–75 mmol/mol), body mass index (BMI) 40 kg/m2 or less and stable body weight in the preceding 6 months (±2 kg). Exclusion criteria were treatment with oral hypoglycemic agents other than metformin, ethanol intake >20 or 40 g/day for women and men, respectively, liver or kidney disease or abnormal full blood count, renal or liver function tests, use of weight loss medications, previous bowel surgery or documented malabsorption. The study was approved by the Human Research and Ethics Committee at St Vincent’s Hospital and participants gave written informed consent prior to commencement of the study. The study was registered at ClinicalTrials.gov (NCT-00673894).Type 2 diabetes patients were recruited through advertisements at the St Vincent’s Hospital precinct, Sydney and in local newspapers. Inclusion criteria were age 40–70 years, diabetes duration of 5 years or less, treatment with diet or metformin in a stable dose , glycosylated hemoglobin  supplemented with 16 g Philadelphia cheese ; or (2) 200 g low fat cottage cheese  25 g  2 g Gln , 5 g fatTwo intravenous cannulas were inserted for glucose infusion and blood withdrawal. Glutamine was mixed in cold water (250 mL) and the same water volume was given in all studies. Blood was drawn fasting twice 10 min apart , then treatment ingested within 8 min and blood collected at t = 15 and 30 min. At t = 30, glucose  was injected intravenously over 1 min followed by frequent blood sampling for 10 min (t = 30–40). Hyperglycemia was then maintained (target 10.8 mmol/L) for additional 110 min (t = 40–150 min) by adjusting glucose infusion rate (GIR) according to 10 min blood glucose readings.g, snap frozen, and stored at −80 °C until analysis. Total GLP-1 was measured by radioimmunoassay after extraction of plasma with 70% ethanol. Carboxy-terminal GLP-1 immunoreactivity was determined using antiserum 89390. Active GLP-1 was analyzed using an ELISA, as previously described [Blood glucose  and serum insulin  were measured at t = 0, 15 and 30 min post Gln/protein/water treatment, at t = 1, 2, 3, 4, 5, 6, 8 and 10 min post intravenous glucose bolus (t = 30–40 min) and at t = 60, 90, 120, 130, 140 and 150 min during the hyperglycemic glucose clamp. Blood samples for total and active GLP-1 were collected into EDTA-coated tubes, with dipeptidyl peptidase (DPP)4 inhibitor and trasylol in the active GLP-1 testing tube, and immediately centrifuged for 7 min at 4100 p > 0.1). AUCs were calculated for the 30 min after ingestion of Gln/protein/water (AUC0–30 min), 10 min post IV glucose injection  and for the hyperglycemic glucose clamp . Data are presented as means ± SD, unless stated otherwise. Insulin data were log10-transformed prior to statistical analysis. One-way ANOVA with Tukey posthoc comparisons was used to test for differences in AUCs of endpoints between l-glutamine, whole protein and water. Data were analyzed using SPSS version 21 .Area under the curves (AUCs) of glucose, insulin and total and active GLP-1 excursions were calculated  participated in the study. Participants were 65.1 ± 5.8 years old with diabetes duration of 3.5 ± 1.5 years, BMI 27.1 ± 2.4 kg/m2 and HbA1c 6.6% ± 0.7% (48 ± 8 mmol/mol).Well-controlled type 2 diabetes patients (p = 0.7). Blood glucose peaked 2 min after the IV glucose bolus (17.3 ± 2.7 mmol/L), without significant differences between treatments (p = 0.2). Blood glucose was clamped at an average of 10.8 ± 0.4 mmol/L during clamp steady state (t = 120–150 min), without a significant difference between treatments (p = 0.3). Glucose AUCs were not significantly different between the treatments , consistent with similar acute effects of the treatments on insulin sensitivity.The treatments were well tolerated. Baseline blood glucose was 6.6 ± 1.3 mmol/L, without significant differences between treatments  was blunted after water and augmented by both Gln (p = 0.02) and protein  was significantly augmented by protein (p = 0.05), but not Gln (p = 0.2) compared with water , but not protein (p = 0.8) compared with water. During the hyperglycemic glucose clamp, total GLP-1 remained significantly increased after both Gln (p = 0.02) and protein (p = 0.02) compared with water  and tended to increase after Gln (p = 0.08) compared with water  could have been enhanced by ingestion of whole protein . While pversus glucose administered IV. Furthermore, increases of approximately 1.5-fold in circulating GLP-1 concentrations in the present study were related to first-phase insulin restoration. In support, similar scale increases in circulating GLP-1 in response to OGTT were associated with restoration of first-phase insulin response to IVGTT in severely obese type 2 diabetes patients undergoing bariatric surgery [Previously, we have reported that a similar dose of glutamine ingested with a meal increased the early active GLP-1 response, but not the early insulin response to the meal . However surgery .The main strengths of this study are the use of the hyperglycemic glucose clamp, enabling the investigation of the treatments on first- and second- phase insulin response. Furthermore, the test treatments were energy and fat matched to negate possible effects of energy and fat on gastric emptying, insulin and incretin response. Furthermore, the randomized crossover design increased the power to detect differences in response in a relatively small cohort. The two main limitations were the lack of a healthy control group and the relatively small cohort studied.l-glutamine and whole protein restored first-phase insulin response in type 2 diabetes patients. Larger studies are required to further investigate the utility of similar approaches in improving insulin secretory capacity in type 2 diabetes.Both


The objective of this study was to study how changing the ratio of Lys to Thr, Lys to His, and Lys to Val affects the expression of lipogenic genes and microRNA (miRNA) in bovine mammary epithelial cells.ACSS2, FABP3, ACACA, FASN, SCD, LPIN1, INSIG1, SREBF1, PPARD, and NR1H3 (commonly known as LXR-α). Furthermore, LV1.6 up-regulated expression of ACSL1, DGAT1, and RXRA and down-regulated PPARG expression. Although no effect of OPAA on expression of PPARG was observed, compared with the control, OPAA up-regulated expression of the PPAR targets ACSS2, FABP3, ACACA, FASN, SCD, LPIN1, INSIG1, and SREBF1. Compared with the control, the expression of the anti-lipogenic MIR27AB was down-regulated by OPAA, LT2.1, LT1.3 and LH3.0. In contrast, compared with the control, the expression of the pro-lipogenic MIR21 was up-regulated by LT2.1, LT1.3, LH3.0, and LV1.6.Triplicate cultures with the respective “optimal” amino acid (AA) ratio  plus rapamycin , OPAA, Lys:Thr 2.1:1 (LT2.1), Lys:Thr 1.3:1 (LT1.3), Lys:His 3.05:1 (LH3.0), or Lys:Val 1.62:1 (LV1.6) were incubated in lactogenic medium for 12 h. The expression of 15 lipogenic genes and 7 miRNA were evaluated. Responses to LT2.1, LT1.3, LH3.0, and LV1.6 relative to the control (OPAARMC) included up-regulated expression of The observed up-regulation of lipogenic gene networks and the changes in expression of key miRNA involved in the control of lipogenic balance are indicative of a potentially important role of EAA ratios and mTOR signaling in the regulation of milk fat synthesis.The online version of this article (doi:10.1186/s40104-016-0104-x) contains supplementary material, which is available to authorized users. Previous research has underscored that provision of adequate levels of essential AA (EAA) is critical for improving N utilization efficiency as well as maximizing bovine milk protein synthesis . RulquinFASN) [ACACA) [Both Lys and Met, at supraphysiological concentrations, are known to stimulate β-casein synthesis in dairy cow mammary epithelial cells (MEC) , 6. It wFASN) . Further [ACACA)  in rat hPPARG) and/or sterol-responsive element binding factor 1 (SREBF1) expression and/or signaling. Both of these transcription regulators are up-regulated  in bovine mammary gland at the onset and throughout lactation [PPARG via microRNA (miRNA) was reported recently in studies where up-regulation of MIR27AB led to downregulation of PPARG expression and lipogenesis in adipocytes [PPARG itself alters transcription of MIR103 and MIR378, hence, these miRNA could elicit a pro-lipogenic effect through targets such as FASN [A “missing link” between EAA availability and alterations in lipogenic gene expression in the studies reported to date could be alterations in peroxisome proliferator-activated receptor-γ  and SREBF1 [The general hypothesis was that the EAA profile could affect mRNA expression of lipogenic gene networks and selected lipogenic miRNA in bovine mammary cells. Because of the newly-demonstrated linkage between mTOR signaling and activation of lipogenesis via the Liver-X-receptor α  in Minimum Essential Medium/Earle’s Balanced Salts HyQ . Cultures were maintained in a water-jacketed incubator  with 5 % in air at 37 °C. Culture medium was changed every 24 h and cells were subcultured to 80 to 90 % confluence by rinsing once with (10 mL) of PBS buffer without Ca and Mg , 3 mL of 0.25 % trypsin  plus 3 mL of Cellstripper , and incubated at 37 °C for 5 to 10 min . Trypsin activity was inhibited by addition of 6 mL of fresh culture media at 37 °C.Bovine mammary epithelial cells (Mac-T) were allowed to grow in canted neck, vented, 75 cmApproximately 48 h prior to the last subculture before initializing the experiment, cells were allowed to grow in a basal medium composed of Minimum Essential Medium/Earle’s Balanced Salts HyQ , insulin , hydrocortisone , transferrin , ascorbic acid , sodium acetate , and penicillin/streptomycin . The basal medium was supplemented with fetal bovine serum  and growth-promoting hormones . When the cells reached ~90 % confluence they were cultured overnight with 2.5 mL of a lactogenic medium in 6-well plates as reported by Kadegowda et al. . The lacTriplicate cultures were incubated for 12 h with the following treatments . For RNA extraction, cells were cultured in duplicate to generate three specific treatments: an essential AA-free treatment , OPAARMC and OPAA. These treatments were performed for 0, 1, 6, 12 h. Cells were harvested in 1 mL of QIAzol reagent (Invitrogen) and stored at −80 °C until RNA extraction.The Mac-T cells from several 75 cmThe preliminary study was conducted to determine a suitable incubation time based on peak mRNA expression of the genes eukaryotic translation initiation factor 4E, eukaryotic translation initiation factor 4E-binding protein 1, mechanistic target of rapamycin (Ser/Thr kinase), ras homolog enriched in brain, ribosomal protein S6 kinase beta-1, and tuberous sclerosis 1 (data not shown). The final data for statistical analysis were calculated as the percentage change relative to time 0 for all treatments. Statistical analysis was performed using the MIXED procedure of SAS  to evaluate the effects of treatment, time, and treatment × time on normalized mRNA expression of genes. The model included the fixed effect of time , treatment , and the random effect of replicate nested within treatment and time. Results (not shown) indicated that a 12-h incubation was suitable to obtain maximal responses in mRNA expression. Clearly, this represented a compromise because it is unlikely that all genes would have maximal expression at the same time point.2 flasks with lactogenic medium. In doing this we attempted to obtain the same initial number of cells in each flask and, thus, ensure consistency for the initial conditions among all samples. Before initiating the experiment, cells remained in the basal medium (changed every 24 h) for approximately 2 d, when they reached ≥ 90 % confluence. Subsequently, cells were cultured in lactogenic medium overnight before the treatments were applied. The OPAA, OPAARMC, LT2.1, LT1.3, LH3.0, and LV1.6 treatments in lactogenic medium  before the cells were harvested after 12 h of incubation.To obtain a large number of cells to initiate the main experiment, several subcultures were performed. When the numbers of cells were deemed adequate to initiate the experiment, all the cells were pooled in a 50-mL sterile tube with fresh medium at 37 °C and mixed thoroughly before plating in 75 cmTotal RNA was extracted from the cells using the RNeasy Mini Kit columns . Concentrations of RNA were measured with a NanoDrop ND-1000 spectrophotometer . RNA integrity (RIN) was assessed using Aglient 2100 Bioanalyzer . The RIN factor was above 7.5. A portion of the RNA was diluted to 100 ng/μL using RNase free water before reverse transcriptase. Adequate cDNA was prepared to run all selected genes. Each cDNA was synthesized using 100 ng RNA, 1 μL Random Primers , and 9 μL RNase free water. The mixture was incubated at 65 °C for 5 min in an Eppendorf Mastercycler Gradient and keep on ice for 3 min. A total of 9 μL of Master Mix, composed of 4 μL 5 × First-Strand Buffer , 1 μL Oligo dT18, 2 μL 10 mmol/L deoxynucleotide 5′-triphosphate mix (Invitrogen), 0.25 μL Reverse aid RT (Fermentas), 0.125 μL RNase Inhibitor (Fermentas), and 1.625 μL RNase free water was added. The reaction was performed using the following temperature program: 25 °C for 5 min, 42 °C for 60 min, and 70 °C for 5 min. Complementary DNA was then diluted 1:4 with RNase free water.The qPCR was performed in a MicroAmp Optical 384-well reaction plate  using 4 μL diluted cDNA combined with 6 μL of a mixture composed by 5 μL 1 × SYBR Green Master Mix , 0.4 μL each of 10 μmol/L forward and reverse primers, and 0.2 μL RNase free water. Each sample was run in triplicate and a 6-point relative standard curve plus the nontemplate control were used. The reactions were performed in an ABI Prism 7900 HT SDS instrument (Applied Biosystems) under the following conditions: 5 min at 95 °C, 40 cycles of 1 s at 95 °C, and 30 s at 60 °C. The presence of a single PCR product was verified by the dissociation protocol using incremental temperatures to 95 °C for 15 s, 65 °C for 15 s, and 95 °C for 15 s. Data were analyzed using with the 7900 HT Sequence Detection Systems Software . Methods for primer design and validation were reported previously . AdditioGADPH, UXT, and RPS9) [n = 3 cultures/treatment). The Kenward-Roger statement was used for computing the denominator degrees of freedom. Treatment means were compared using the PDIFF statement in SAS after correction using Tukey’s. Significance was declared at a PDIFF Tukey-adjusted P < 0.05.The relative percentage of mRNA abundance among genes was analyzed as reported previously . The qPCnd RPS9) . The MIXACSL1) was greatest with LV1.6 (P < 0.01) compared with other treatments. Compared with the control (OPAARMC), the expression of acyl-CoA synthetase short-chain family member 2 (ACSS2) and fatty acid-binding protein 3, muscle and heart (FABP3) was greater with OPAA, LT2.1, LT1.3, LH3.0 and LV1.6. Incubation with LT1.3 up-regulated the expression of ACSS2 compared with LH3.0 (P < 0.01) and LV1.6 (P < 0.01), while LV1.6 down-regulated ACSS2 compared with LH3.0 (P < 0.01). Treatment with OPAA led to greater expression of FABP3 compared with LH3.0 (P = 0.04) and LV1.6 (P < 0.01), while incubation with LV1.6 resulted in lower expression compared with LT2.1 (P < 0.01) and LT1.3 (P < 0.01).The responses of genes associated to FA metabolism are reported in Fig. ACACA), fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD) had greater expression in response to OPAA, LT2.1, LT1.3, LH3.0 and LV1.6 compared with the control. The LH3.0 resulted in lower expression of FASN compared with the other treatments (P < 0.01), while the expression of SCD was up-regulated in OPAA compared with LH3.0 (P < 0.01).Among the genes associated with de novo FA synthesis and desaturation, acetyl-coenzyme A carboxylase alpha (AGPAT6) was up-regulated with LT1.3 (P < 0.01) and LH3.0 (P < 0.01) compared with the control. The mRNA expression of diacylglycerol acyltransferase 1 (DGAT1) was down-regulated with OPAA compared with the control (P < 0.01), but was upregulated by LV1.6 compared with the other treatments (P = 0.01). The mRNA abundance of lipin 1 (LPIN1) was greater (P = 0.04) with OPAA, LT2.1, LT1.3, LH3.0 and LV1.6 compared with the control, while LH3.0 had greater (P < 0.01) expression compared with OPAA and LV1.6.Expression of 1-acylglycerol-3-phosphate O-acyltransferase 6 (INSIG1) and sterol regulatory element-binding transcription factor 1 (SREBF1) was up-regulated (P < 0.05) with OPAA, LT2.1, LT1.3, LH3.0 and LV1.6 compared with the control. Furthermore, incubation with LT1.3 up-regulated the expression of INSIG1 compared with LH3.0 (P = 0.02) and LV1.6 (P < 0.01), while LV1.6 resulted in lower expression compared with LH3.0 (P < 0.01). For SREBF1, treatment with LV1.6 resulted in greater expression compared with the other treatments (P < 0.01). The mRNA expression of peroxisome proliferator-activated receptor gamma (PPARG) was lowest with LV1.6 compared with other treatments (P < 0.01). Compared with the control, expression of peroxisome proliferator-activated receptor beta (PPARD) was up-regulated with OPAA, LT2.1, LT1.3, LH3.0 and LV1.6 (P < 0.01). In contrast, OPAA resulted in lower expression of LT2.1, LT1.3, LH3.0 and LV1.6 (P < 0.01). The expression of retinoid X receptor alpha (RXRA) was down-regulated with OPAA (P = 0.01) and up-regulated with LV1.6 (P < 0.01) compared with the control. The expression of liver X receptor α (NR1H3) was up-regulated (P < 0.01) with LT2.1, LT1.3, LH3.0 and LV1.6 compared with the control, while LV1.6 resulted in greater (P < 0.01) expression than LT2.1, LT1.3 and LH3.0.The responses of genes associated with regulation of transcription are reported in Fig. P < 0.01) in response to LT2.1, LT1.3, LH3.0 and LV1.6. Treatment with LV1.6 resulted in lower (P < 0.01) expression of MIR21 compared with LT2.1, LT1.3 and LH3.0. Compared with the control, the expression of MIR27AB was lower (P < 0.01) in response to OPAA, LT2.1, LT1.3 and LH3.0, while LV1.6 led to similar expression than the control.MicroRNA expression responses to treatments are reported in Fig. Signaling through mTOR plays a crucial role in mammary protein synthesis, and some amino acids, e.g. Leu, Ile and Val, are known to be potent stimulators of mTOR signaling and protein synthesis in mouse and bovine –7. Otherde novo FA synthesis  and desaturation (SCD), triacylglycerol (TAG) synthesis (LPIN1), and transcription regulation of lipid synthesis , relative to OPAARMC the present data demonstrated that mTOR is, in fact, a regulator of lipid metabolism in mammary cells. The role of mTOR in lipogenesis has been previously demonstrated in murine hepatic cells [SREBF1 in an S6K-independent manner [SREBF1 expression when OPAA was added to the medium without rapamycin, relative to OPAARMC, is further suggestive of this mechanism.Because the OPAA treatment stimulated gene expression of enzymes related to ic cells , 23. In SREBF1 plays an important role regulating lipid synthesis in bovine mammary epithelial cells [ACSS2, FABP3, SCD, and LPIN1 decreased by blocking transcription of SREBF1 with small interfering RNA technology. The response was indicative of a positive correlation among these genes. Therefore, the up-regulation of SREBF1 with OPAA may have contributed to the greater expression of genes related to de novo FA and TAG synthesis.Evidence indicates that al cells . Ma and al cells  reportedSREBF1, Peterson et al. [LPIN1 gene expression measured in this experiment is more likely reflective of its enzymatic role, which is in agreement with the upregulation of the FA synthesis enzymes. Because we did not attempt to determine intracellular location of lipin protein, we cannot infer directly about its function as transcriptional co-activator.Regarding the pathway by which mTOR stimulates n et al.  demonstrn et al. . TherefoNR1H3), a transcriptional regulator that senses oxysterols, dimerizes with retinoid-X-receptor-α (RXRA) and activates transcription of SREBF1, FASN, SCD and ACACA [NR1H3) is a potentially-important transcription regulator of milk fat synthesis that enhances de novo FA synthesis in bovine MEC treated with the NR1H3 agonist T0901317 [RXRA expression with OPAA relative to OPAARMC is a stronger indication that NR1H3 was not involved in the observed lipogenic up-regulation, because NR1H3 needs to form heterodimers with RXRA to regulate the transcription of lipogenic enzymes [Another pathway that links mTOR with lipogenic regulation is through S6K-mediated phosphorylation of liver-X-receptor-α  and desaturation (SCD) might have been controlled by other transcriptional regulators such as SREBF1 and INSIG1. This idea is supported by data from Oppi-Williams et al. [SCD mRNA expression in SREBF1-knockdown Mac-T cells [INSIG1, if extended to the protein level, might have resulted in a lower activity of SREBF1, which would have been retained in the ER via SCAP-INSIG1 binding [In the absence of up-regulation of s et al. , who rep-T cells . However binding , despitePPARG in bovine mammary tissue during lactation indicated that milk fat synthesis might be under partial control by PPARγ [ACACA, FASN), TAG synthesis , and transcriptional regulation of lipid synthesis  were upregulated in Mac-T cells by treatment with a PPARG agonist (rosiglitazone). Despite the lack of effect of mTOR on PPARG, the up-regulation of lipogenic genes in OPAA compared with OPAARMC indicates that EAA could indirectly alter PPARG, hence, play a role in de novo FA and TAG synthesis regulation [This would suggest the involvement of other key transcription regulators in lipid synthesis, as observed for non-ruminants . For insby PPARγ . The PPAby PPARγ . Kadegowby PPARγ  demonstrgulation .PPARG mRNA expression. The authors suggested that the regulatory mechanisms might involve covalent and allosteric modulation of PPARG and/or its co-regulators [PPARGC1A and LPIN1 are substantially up-regulated during lactation in bovine mammary cells, indicating an important role in milk fat synthesis [LPIN1 in the rapamycin-free medium is noteworthy.In a similar manner, Blanchard et al.  working gulators . In factynthesis . In thatPPARD was associated with greater expression of genes related to de novo FA synthesis , desaturation (SCD), and intracellular FA transport (FABP3), all of which agrees with a positive correlation reported among these genes in a previous Mac-T cell study [PPARD in de novo FA synthesis will have to be performed because correlations were weaker compared with that for SREBF1 [The up-regulation of ll study . Furtherr SREBF1 .ACACA, FASN, SCD) in response to OPAA provides some indication of EAA as regulators of transcription in bovine mammary cells. The insulin effect alone or in combination with a mixture of EAA on lipogenic gene expression  in cells such as hepatocytes is clear [FASN and SREBF1 appears to require mTOR because incubating EAA plus insulin plus RMC completely prevented the up-regulation of lipogenic genes [FASN and other lipogenic genes [Although we did not include a treatment devoid of EAA and insulin, the observed expression level of the lipogenic genes  . BesidesThe observed up-regulation of lipogenic gene networks and the changes in expression of key miRNA involved in the control of lipogenic balance are indicative of a potentially important role of EAA ratios and mTOR signaling in the regulation of milk fat synthesis.AA, amino acid; ACACA, Acetyl-coenzyme A carboxylase alpha; ACSL1, Acyl-CoA synthetase long-chain family member 1; ACSS2, Acyl-CoA synthetase short-chain family member 2; AGPAT6, 1-acylglycerol-3-phosphate O-acyltransferase 6; DGAT1, diacylglycerol acyltransferase 1; EAA, essential amino acids; EIF4E, eukaryotic translation initiation factor 4E; EIF4EBP1, eukaryotic translation initiation factor 4E-binding protein 1; FA, fatty acid; FABP3, fatty acid-binding protein, heart; FASN, fatty acid synthase; GADPH, glyceraldehyde 3-phosphate dehydrogenase; INSIG1, insulin induced gene 1; LCFA, long-chain fatty acids; LPIN1, lipin 1; Mac-T, bovine mammary epithelial cells; MEC, mammary epithelial cells; MFD, milk fat-depressing diet; MIR103, microRNA 103; MIR130a, microRNA 130a; MIR21, microRNA 21; MIR27ab, microRNA 27ab; MIR34a, microRNA 34a; MIR378, microRNA 378; MIR448, microRNA 448; miRNA, microRNA; MTOR, mechanistic target of rapamycin (Ser/Thr kinase); mTORC1, mTOR complex 1; NR1H3, liver X receptor α; PPARD, peroxisome proliferator-activated receptor beta; PPARG, peroxisome proliferator-activated receptor gamma; RHEB, ras homolog enriched in brain; RIN, RNA integrity; RMC, rapamycin; RPS6KB1, ribosomal protein S6 kinase beta-1; RPS9, ribosomal protein S9; RXRA, retinoid X receptor, alpha; SCD, stearoyl-CoA desaturase; SREBF1, sterol regulatory element-binding transcription factor 1; TAG, triacylglycerol; TSC1, tuberous sclerosis 1; UXT, ubiquitously expressed prefoldin like chaperone


In the crystal lattice, the cations are arranged in layers in the ab plane divided by the nitrate anions and solvent molecules. All of the amine H atoms are involved in hydrogen bonding to nitrate, DMF or ligand O atoms or to one of the Br atoms, forming two-dimensional networks.In [Co(C 8H8BrN4O)2]NO3·C3H7NO, is formed of discrete [CoL2]+ cations, nitrate anions and dimethylformamide (DMF) molecules of crystallization. The cation has no crystallographically imposed symmetry. The ligand molecules are deprotonated at the phenol O atom and octahedrally coordinate the CoIII atoms through the azomethine N and phenolate O atoms in a mer configuration. The deprotonated ligand molecules adopt an almost planar conformation. In the crystal lattice, the cations are arranged in layers in the ab plane divided by the nitrate anions and solvent molecules. No π–π stacking is observed. All of the amine H atoms are involved in hydrogen bonding to nitrate, DMF or ligand O atoms or to one of the Br atoms, forming two-dimensional networks parallel to (100).The title compound, [Co(C The isolated dark-red microcrystalline product was identified crystallographically to be the mononuclear CoIII Schiff base complex CoL2NO3·DMF (I)The title compound was isolated in an attempt to prepare a heterometallic Co/Mn compound with the ligand, HO3 Fig. 1 that was8H8BrN4O)2]NO3·C3H7NO, (I)L2]+ cations, nitrate anions and DMF molecules of crystallization. The cation has no crystallographically imposed symmetry  to 177.14 (8)°, the cis angles lie in the range 82.62 (9) to 94.35 (8)°. The deprotonated ligand molecules adopt an almost planar conformation.The title compound [Comethyl]phenolato-κ3N,N′,O]}cobalt(III) chloride hemihydrate –176.71 (4)°, the cis angles are equal to 82.33 (4)–94.86 (4)°. Obviously, the use of the 5-bromo-derivative of salicylaldehyde in the present study does not change the coordination properties of the resulting Schiff base ligand compared to that of parent salicylaldehyde-aminoguanidine Schiff base.The coordination geometry around the metal atom has a close resemblance to that found in Coab plane divided by the nitrate anions and DMF molecules  and CuCl42– salts (four) with protonated Schiff base ligands as cations. Other mononuclear complexes and hybrid metal salts of AG-based Schiff base ligands comprise V, Co, and Ni, Cd structures, respectively. The Schiff base ligands derived from AG do not show any coordination variability in their metal complexes - the ligand tends to coordinate through two azomethine N atoms and phenoxy O atom from the ring if such one is present.Of 18 crystal structures of Schiff base metal complexes derived from AG, six are Fe, Cu and Zn compounds that contain a pyridoxal-aminoguanidine ligand. The latter has been of much interest due to its suggested superiority to AG in the treatment of diabetic complications. The remaining 12 compounds are mostly mononuclear Cu3 aminoguanidine HNOL·3) ligand:HNO 5-Bromosalicylaldehyde  in ethanol (10 ml) was poured into an aqueous solution (10 ml) of AG·HNO3  and 5 drops of concentrated nitric acid were added to the resulting clear solution. It was heated to 353 K under stirring for 20 min and then cooled in air. A white crystalline precipitate of HL·HNO3 deposited shortly. It was filtered off, washed with distilled water and dried out in air (yield: 82%).1H NMR ; 10.20, s ; 8.34, s ; 8.13, s ; 7.52, br ; 7.27, d ; 6.82, d . FT–IR (solid) ν (cm−1): 3500w, 3446m, 3418m, 3322m, 3208s, 3124m, 2922m, 2892m, 2854m, 2816m, 1692s, 1632vs, 1476s, 1420s, 1384vs, 1346s, 1336s, 1256s, 1190m, 1048m, 956w, 904w, 836w, 820w, 654w, 622m, 538w, 480w.Synthesis of 1: Mn powder , Co(NO3)2·6H2O  and HL·HNO3  were added to methanol (20 ml) and the mixture was heated to 323 K under stirring until total dissolution of the manganese powder was observed (1 h). The resulting red solution was filtered and allowed to stand at room temperature. Dark-red microcrystals of the title compound were formed over several days. They were collected by filter-suction, washed with dry PriOH and finally dried in vacuo (yield: 39%). FT–IR (solid) ν (cm−1): 3476m, 3406m, 3358m, 3226s, 3180s, 3092m, 3054m, 2998m, 2940m, 2900m, 2800m, 1660sh, 1650vs, 1596s, 1556s, 1522m, 1454s, 1384s, 1354m, 1334s, 1290s, 1250m, 1182m, 1134m, 1102m, 1046w, 926m, 822m, 969m, 656m, 620m, 574m, 526m, 468w.Uiso(H) = 1.2UeqC for CH, C—H = 0.98 Å, Uiso(H) = 1.5UeqC for CH3). NH hydrogen atoms were refined with bond lengths restrained to ideal values (N—H = 0.88 Å). Anisotropic displacement parameters were employed for the non-hydrogen atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989016008690/hg5475sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989016008690/hg5475Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016008690/hg5475sup3.tifIR spectrum of the ligand. DOI: Click here for additional data file.10.1107/S2056989016008690/hg5475sup4.tifIR spectrum of the complex. DOI: 1482509CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Anoxybacillus suryakundensis strain JS1T, a facultative anaerobic, moderately thermophilic, alkalitolerant bacterium, was isolated from a hot spring. The estimated genome is 2.6 Mb and encodes 2,668 proteins. Anoxybacillus suryakundensis strain JS1T is a Gram-positive, nonmotile, facultative anaerobe. A moderately thermophilic and alkalitolerant bacterium, it was isolated from sediment samples from a hot spring in Suryakund, Jharkhand, India. Heterotrophic growth occurred at 40 to 60°C and pH 5.5 to 11.5 . Illumina reads were assembled with simulated read pairs using Allpaths-LG version r46652 .The genome was annotated using the JGI Microbial Genome Annotation Pipeline . Genes wN50 contig size of 148.6 kb. The largest contig was 245.9 kb with a G+C content of 42.0 mol%. The draft genome sequence has 2,668 CDSs, 61 tRNAs, 16 rRNAs, and two clustered regularly interspaced short palindromic repeats.The ﬁnal draft assembly contained 44 contigs in 41 scaffolds, totaling 2.6 Mb with an LIOK00000000. The version described in this paper is version LIOK01000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number


GJB2 gene) and 97 subjects with GJB2 genotype wt/wt in the Republic of Sakha/Yakutia . This subarctic territory was found as the region with the most extensive accumulation of mutation IVS1+1G>A in the world as a result of founder effect in the unique Yakut population isolate. The GJB2 gene resequencing and detailed audiological analysis in the frequency range 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 kHz were performed in all examined subjects that allowed to investigate genotype-phenotype correlations between the presence of single mutation IVS1+1G>A and hearing of subjects from examined groups. We revealed the linear correlation between increase of average hearing thresholds at speech frequencies  and age of individuals with GJB2 genotype IVS1+1G>A/wt . Moreover, the average hearing thresholds on high frequency (8.0 kHz) in individuals with genotype IVS1+1G>A/wt (both sexes) were significantly worse than in individuals with genotype wt/wt (p<0.05). Age of hearing loss manifestation in individuals with genotype IVS1+1G>A/wt was estimated to be ∼40 years . These findings demonstrate that the single IVS1+1G>A mutation (GJB2) is associated with age-related hearing impairment (ARHI) of the IVS1+1G>A carriers in the Yakuts.Age-Related Hearing Impairment (ARHI) is one of the frequent sensory disorders registered in 50% of individuals over 80 years. ARHI is a multifactorial disorder due to environmental and poor-known genetic components. In this study, we present the data on age-related hearing impairment of 48 heterozygous carriers of mutation IVS1+1G>A ( Age-related hearing impairment (ARHI), or presbyacusis, is a common sensory disorder characterized by bilateral sensorineural hearing loss more evident at high frequencies GJB2 gene coding protein connexin 26 (Cx26) in homozygous and compound-heterozygous states are responsible for a significant part of inherited autosomal recessive forms of hearing loss in different populations GJB2 gene (in heterozygous state) have influence on hearing function GJB2 gene – c.35delG, p.Met34Thr, c.167delT (most frequently registered in Europe), and c.235delC single mutation (the study was carried out in China) – had controversial results Genes already known to be associated with hearing impairment are not investigated enough for the presence of genetic associations with ARHI. Mutations in GJB2 gene is relatively rare, and audiological analysis of hearing threshold in carriers of this single mutation has not been performed previously. High rates of IVS1+1G>A mutation (2–11.7%) were found among some indigenous populations of Eastern Siberia, Russia , with the highest carrier frequency (11.7%) in isolated Yakut population GJB2 gene mutation.Splice site mutation IVS1+1G>A in GJB2 mutation IVS1+1G>A carriers versus individuals with normal GJB2 genotype in Yakut population.The purpose of this study is to analyze age-related hearing alteration in GJB2 gene mutations ; had no recent history of fever, otitis media, tinnitus; and had no obvious contacts with occupational noise. From those 145 individuals based on genotyping data we formed two groups: Cx26-H – the group included the carriers of single mutation IVS1+1G>A ; and Cx26-Wt – the group of non-carriers of the IVS1+1G>A mutation . Subsequently, we stratified these two groups according to age and sex for the purpose of correct comparison  and informed the potential participants about the aims of upcoming study. We also invited the parents and siblings of deaf children homozygous for the IVS1+1G>A mutation, as they could be the carriers of this mutation. All the volunteers  passed otologic-audiological evaluation and genotyping for the mparison .0.5,1.0,2.0,4.0 kHz separately.Audiological examination was conducted on all 214 individuals (n = 428 ears). Otologic examination was performed with the «Basic-Diagnostik-Set» , tuning fork standard set. Acoustic impedance was measured with impedance meter . Air conduction thresholds were measured on frequencies 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 kHz and bone conduction thresholds – on frequencies 0.25, 0.5, 1.0, 2.0, 4.0 kHz with audiometer «MAICO ST20»  in the same conditions for all participants. The results were assessed separately for each ear on air conduction thresholds as on all measured frequencies, and speech range of frequencies – PTAGJB2 gene mutation analysis. Amplification of the coding (exon 2) and noncoding (exon 1) and flanking intronic regions of GJB2 gene was conducted with PCR on thermocycler «MJ Mini» (Bio-Rad) using appropriate primers GJB2 gene are shown in the A total of 214 genomic DNA samples, which were extracted from leukocytes of peripheral blood, were used for GJB2 gene were summarized in the unified database. Regression and correlation analysis of «age-hearing threshold» association were assessed with Spearman's rank correlation coefficient sr. Significance of hearing thresholds on measured frequencies was statistically evaluated with the Mann-Whitney U test using software STATISTICA version 8.0.  and Biostatd . Differences were statistically significant when p<0.05.The results of audiological examination and mutation analysis of Written informed consent was obtained from all individuals. This study was approved by the local Committee on Biomedical Ethics of Yakut Scientific Center of Complex Medical Problems, Siberian Branch of the Russian Academy of Medical Sciences, Yakutsk, Russia .0.5,1.0,2.0,4.0 kHz» in IVS1+1G>A single mutation carriers (group Cx26-H) and non-carriers (group Cx26-Wt), for females and males separately. Statistically significant (p<0.05) correlation of increase in hearing threshold according to age was detected in IVS1+1G>A carriers  and not observed in non-carriers    A, B.0.5,1.0,2.0,4.0 kHz» was performed in these age cohorts (p<0.05) correlation was observed in hearing thresholds of individuals from group Cx26-H at ages 40–49 years , and 50–59 years   . Thus, h0.5,1.0,2.0,4.0 kHz) did not identify differences between groups Cx26-Wt and Cx26-H at age 20–39 years . However, statistically significant differences in hearing thresholds were observed at age 40–59 years in both groups Cx26-Wt and Cx26-H   .Following analysis at all measured frequencies  revealed that in group Cx26-Wt averaged audiological profile of females and males aged 20–39 and 40–59 years had a shape of flat curve. Averaged audiological profile of females and males aged 20–39 years in group Cx26-H had a shape of sloping curve, aged 40–59 years – steep-sloping curve in high-frequent range (4.0–8.0 kHz) .p<0.05) differences were observed at frequency 8.0 kHz in both sexes and at frequency 4.0 kHz – in females  . However females .p<0.05) in hearing thresholds at frequencies 4.0–8.0 kHz between groups Cx26-Wt and Cx26-H were observed in females and males aged 40–59 years . Moreover, median value of hearing threshold was ≥20.0 dB in single mutation IVS1+1G>A carriers at 4.0–8.0 kHz for females, and at 8.0 kHz – for males  and individuals with normal GJB2 genotype (n = 29) in Italy, however, this study failed to find a correlation of hearing threshold increase with age GJB2 c.35delG heterozygous females in comparison with controls GJB2 single mutations, c.35delG and p.Met34Thr (c.101T>C), conducted in a UK population from 1991 to 2011 with three-step audiological test of children aged 3, 7 and 11 years (9202 children) showed that hearing thresholds on extra-high frequency (16.0 kHz) in c.35delG and p.Met34Thr (c.101T>C) carriers was significantly worse than in children without GJB2 gene mutation and tend to increase with age Moreover, there is a study that detected statistically significant differences in mean hearing thresholds on high frequencies (6.0 and 8.0 kHz) between mutation c.35delG carriers (n = 31) and individuals with normal A recently published study provides the data comparable to our results. The study carried out in China (2011) showed that in c.235delC heterozygous carriers hearing for high-frequency (4.0 and 8.0 kHz) increases at the age of 30–59 years and hearing thresholds in the intermediate frequencies may deteriorate over the age of 40 0.5,1.0,2.0,4.0 kHz) linearly increase (p<0.05) with age in the heterozygous carriers of single site splicing mutation IVS1+1G>A (GJB2 gene) of both sexes. This correlation is more expressed in males, which probably associated with physiological features and environmental factors. Moreover, audiometric analysis showed that hearing thresholds at high frequency (8.0 kHz) in heterozygous for IVS1+1G>A females and males aged 20–39 years were definitely worse (p<0.05) than in non-carriers. We also found that hearing on lower frequencies (1.0–2.0 kHz) was affected in both heterozygous for IVS1+1G>A females and males aged 40–59 years . Thus, we identified that hearing acuity of single mutation IVS1+1G>A carriers on particular frequencies was decreased compared to non-carriers, regardless of sex and age. Splice site mutation IVS1+1G>A, probably leads to a deficiency of normal Cx26 molecules that form intercellular gap junction channels, and this results in impaired recycling of potassium ions in the cochlea with a local organ of Corti intoxication Our study provides the first evidence that in a relatively isolated Yakut population the average hearing thresholds at the speech frequencies (PTA59 years . At the GJB2) is associated with age-related hearing impairment (ARHI) of the IVS1+1G>A carriers among the Yakuts.Despite the controversial results of previous audiological analysis of the c.35delG mutation in heterozygous carriers from Caucasian populations