Enzyme discovery is a promising approach to aid in the deconstruction of recalcitrant plant biomass in an industrial process. Novel enzymes can be readily discovered by applying metagenomics on whole microbiomes. Our goal was to select, examine, and characterize eight novel glycoside hydrolases that were previously detected in metagenomic libraries, to serve biotechnological applications with high performance.Escherichia coli. Four of the eight enzymes had significant activities on either pNP-β-d-galactopyranoside, pNP-β-d-xylopyranoside, pNP-α-l-arabinopyranoside or pNP-α-d-glucopyranoside. These proteins, denoted as proteins 1, 2, 5 and 6, were his-tag purified and their nature and activities further characterized using molecular and activity screens with the pNP-labeled substrates. Proteins 1 and 2 showed high homologies with (1) a β-galactosidase (74%) and (2) a β-xylosidase (84%), whereas the remaining two (5 and 6) were homologous with proteins reported as a diguanylate cyclase and an aquaporin, respectively. The β-galactosidase- and β-xylosidase-like proteins 1 and 2 were confirmed as being responsible for previously found thermo-alkaliphilic glycosidase activities of extracts of E. coli carrying the respective source fosmids. Remarkably, the β-xylosidase-like protein 2 showed activities with both pNP-Xyl and pNP-Ara in the temperature range 40–50 °C and pH range 8.0–10.0. Moreover, proteins 5 and 6 showed thermotolerant α-glucosidase activity at pH 10.0. In silico structure prediction of protein 5 revealed the presence of a potential “GGDEF” catalytic site, encoding α-glucosidase activity, whereas that of protein 6 showed a “GDSL” site, encoding a ‘new family’ α-glucosidase activity.Here, eight glycosyl hydrolase family candidate genes were selected from metagenomes of wheat straw-degrading microbial consortia using molecular cloning and subsequent gene expression studies in Using a rational screening approach, we identified and characterized four thermo-alkaliphilic glycosyl hydrolases that have the potential to serve as constituents of enzyme cocktails that produce sugars from lignocellulosic plant remains.The online version of this article (doi:10.1186/s13068-017-0808-y) contains supplementary material, which is available to authorized users. Thus, in the light of the currently still imperfect substrate unlocking approaches  and Escherichia coli [PDB: 2O9D] and a transport protein, also from E. coli [PDB: 3NKA]. Further manipulations, structural alignments and comparisons between the 3D models of the proteins were completed using PyMOL (http://www.pymol.org).We used Phyre2 to reconstruct the tertiary structures of proteins 5 and 6 . Next, wIn our previous study, we successfully screened two metagenomic libraries generated from wheat straw-degrading microbial consortia produced by the dilution-to-stimulation approach for genes encoding (hemi)cellulose-degrading enzymes . In totaEnterobacter hormaechei by Blast-P. Genes 2 (fosmid T5-5) and 3 (fosmid T4-1) were both predicted to encode a family GH3 β-xylosidase of ~86 kDa . These two genes revealed 86% identity between them. The remaining five genes, i.e., genes 4 and 5 (source fosmid 10BT) and 6, 7 and 8 (source fosmid NT18-17), were predicted to encode enzymes belonging to CAZy families GH39, GH53, GH27, GH20 and GH58, respectively. Blast-P comparisons showed homologies of the predicted gene products with a suite of different proteins, i.e. a transcriptional regulator of the AraC family , an inner membrane protein , an aquaporin Z , a beta-hexosaminidase , and a hypothetical protein , respectively  was annotated as a gene for a family GH2 β-galactosidase (EC. 3.2.1.23), with a molecular size of ~116 kDa. Its predicted amino acid sequence revealed 74% identity with a β-galactosidase of E. coli strains BL21(DE3) and Origami2 (DE3) pLysS. To confirm the fidelity of the cloning, the pET28b(+) plasmids with inserts were extracted from selected clones, per gene, of each of the two strains and the presence of the cloned fragments detected by restriction with specific restriction enzymes followed by gel electrophoresis. The observed sizes of the restriction fragments (data not shown) were consistent with the fragment sizes as predicted from the fosmid-derived gene sequences  and Origami2 (DE3) pLysS) in 2x-PY medium, following induction by IPTG. Both the cell and the soluble fractions (culture supernatants) were used in the tests. We investigated the effects of IPTG concentration , temperature , and glucose concentration  on the gene expression levels. In strain BL21(DE3), the target proteins were, unfortunately, mostly found in the cell fractions, except the product of gene 2, where ~50% of protein occurred in the soluble fraction (data not shown). In contrast, the use of E. coli Origami2 (DE3) pLysS resulted in seven of the eight proteins being present in the soluble fractions, albeit at different levels. Unfortunately, the single remaining protein (product of gene 4) was only detectable in the cell fraction. The collective data revealed that maximal protein production took place, in the selected strain E. coli Origami2 (DE3) pLysS, with 0.5 mM IPTG at 18 °C, in the absence of glucose. This was true for all genes (data not shown).Expression of the eight cloned genes was then investigated using extracts of grown cultures of both E. coli Origami2 (DE3) pLysS for analysis of the selected seven gene products, i.e., proteins 1, 2, 3, 5, 6, 7 and 8, on a series of pNP-labeled substrates. Indeed, protein 1 revealed high activity on pNP-β-d-galactopyranoside, indicating beta-galactosidase-like activity. This was consistent with the activity that had previously been detected in its source fosmid NT2-2. Interestingly, the cultures of E. coli with cloned genes 2 and 3 both yielded soluble fractions that revealed dual activities, i.e. transformation of pNP-β-d-xylopyranoside (β-xylosidase) and pNP-α-l-arabinopyranoside (α-arabinosidase). These genes had been selected from fosmid clones T5-5 and T4-1, respectively, which had previously shown high β-xylosidase activities, but had not shown α-arabinosidase activity. Expectedly, the cultures with cloned gene 4, with predicted β-xylosidase activity, did not yield lysates with any activity on the substrates used. Clearly, the expressed protein of gene 4 was in inclusion bodies, and unfortunately we were unsuccessful in several attempts to recover native forms of it by refolding. In fact, its source fosmid clone 10BT had previously been found to have the gene 4 encoded GH39 family protein linked to β-xylosidase activity [pNP-α-d-glucopyranoside, and there was no detectable enzymatic activity with any of the other pNP substrates (such as pNP-β-d-xylopyranoside and pNP-α-l-arabinopyranoside). Gene 5 had also been selected from fosmid clone 10BT, which had shown consistent enzyme activity on pNP-β-d-xylopyranoside, but not on α-d-glucopyranoside [N-acetylneuraminidase) did not reveal activities with any of the substrates.We thus used the total protein-containing lysates of the cultures of activity . Finallyactivity ) both reranoside . Gene 6,For all further work, we selected the four genes 1, 2, 5 and 6 that had yielded soluble proteins with key promising activities, i.e. β-galactosidase, β-xylosidase and α-glucosidase. Specifically, gene 2 was selected instead of the similar gene 3, because our previous study showed that its mother fosmid clone, T5-5, had yielded extracts in which proteins with thermo-alkaliphilic β-xylosidase activity were present, whereas fosmid clone T4-1 (source for gene 3) showed only slight β-xylosidase activity .E. coli Origami2 (DE3) pLysS cultures containing copies of intact genes 1, 2, 5 and 6 in the pET28b(+) expression vector , revealing both activities. The purified enzyme was found to be active on pNP-Xyl in the temperature range 40–60 °C  between 20 and 70 °C, with maximal activity at 40 °C  at pH 7.5 and at temperatures ranging from 10 to 70 °C. An optimal temperature of 50 °C for protein 5 activity was found  was maximal at pH 10.0  were 0.1 mM and 58.8 U/mg and for protein 2 (pNP-β-d-xylopyranoside and pNP-α-l-arabinopyranoside) 1.0 and 0.3 mM, and 666.7 and 102 U/mg, respectively. The Km values of proteins 5 and 6, with pNP-α-d-glucopyranoside, were 7.4 and 21.4 mM, and the Vmax values were 196 and 588.2 U/mg, respectively.The kinetic parameters of all enzymes were calculated from Lineweaver–Burk plots of specific activities at various substrate concentrations (0–10 mM). The pNP-Gal), 5 (pNP-Glu) and 6 (pNP-Glu) products retained 95% of activity after over 120 min of incubation at 50 °C, after which the activities decreased—at 180 min—to about 50% of the initial levels. At 50 °C, the gene 2 product showed 100% activity with pNP-Xyl for 15 min, and then enzyme activity even increased, to over 180% of the control, maintaining the raised activity level until 180 min. On pNP-Ara, the gene 2 encoded protein retained 100% activity for 30 min, showing a 20% increased activity in the period between 120 and 180 min  , 5-HMF inhibited all enzymes by 10–40%   and furfural were tested for their effects on the activity proteins 1, 2, 5 and 6 using selected concentrations of The efficiency of degradation of raw wheat straw by the four enzymes was determined by measuring the total sugars released. The data suggested that the 24 h treatment showed higher hydrolysis effect for all enzymes  when compared with the 12 h treatment . Protein 2, on pNP-Ara, showed significantly increased (P < 0.05) activity (50%) in the presence of 5 mM of Mg2+, as compared to the controls without ions, and almost 80% elevated activity with 200 mM of NaCl. Finally, protein 6 showed increased α-glucosidase activity (50%) in the presence of 200 mM of NaCl when compared to the controls without NaCl. In fact, all additives, except 5 mM of Mg2+, significantly increased this enzymatic activity. Strikingly, the protein 5 α-glucosidase activity decreased in the presence of all additives; the activity was significantly lowered upon addition of both 5 mM of Mg2+ and 2000 mM NaCl (p<0.05).To examine the effects of ions (including NaCl) on the four selected enzymes Table , activitKluyvera cryocrescens and Enterobacter cloacae ; the presence of such a GDSL motif may indicate the potential for multiple functional properties, such as broad substrate specificity, given its active site flexibility. Due to this, multiple activities, such as lipase and esterase activities, could be present, which are of use in hydrolysis processes of biological interest . FurtherMartelella endophytica (80%) and Devosia sp. root635 (97%), which are both Alphaproteobacteria from the order Rhizobiales  signature motif located at the segment interface between helices M3 and M7 in residues 103–105  and Origami2 (DE3) pLysS, and gene expression was examined. Strain BL21(DE3) is useful for gene expression studies from a T7-type promoter on the vector. In our study, BL21(DE3) cell growth was arrested, possibly as a result of the emergence of inhibitory compounds, i.e., the intended proteins, in the culture. In the case of E. coli Origami2 (DE3) pLysS, the T7 lysozyme produced suppresses the basal expression of T7 RNA polymerase prior to induction. Thus, we found E. coli Origami2 (DE3) pLysS to produce more proteins in the soluble fractions than BL21(DE3), giving rise to five  measurable enzyme activities. In our previous work [Metagenomics constitutes a true ‘breakthrough tool’ that allows to examine natural and manipulated microbiomes for the presence of genes for novel enzymes that may fuel biomedical and industrial applications . In a prous work , fosmid pNP-Xyl, whereas the gene 3 mother fosmid clone T4-1 had shown only slight activity against pNP-Xyl [Based on the initial screening, we thus considered the selected five genes for further study of their products. Among these, proteins 2 and 3 were similar in activity and size. With reference to our previous study, here we selected gene 2 instead of gene 3 (from fosmid clone T5-5) based on the high thermo-alkaliphilic activity with  pNP-Xyl .Arthrobacter psychrolactophilus F2 showed maximal enzymatic activity  at pH 8.0/10 °C (33.3 U/mg). Such β-galactosidases are widely used in the food industry given their capabilities to hydrolyze lactose at extreme (low or high) temperatures, and to produce glucose and galactose. Remarkably, our gene 1 encoded protein showed increased enzymatic activity at raised temperature and similar pH when compared to the BglA enzyme, with increased p-nitrophenol release , several new β-galactosidase family -GH2 enzymes have recently been discovered and characterized –29. Spec0 enzyme , 29. ThepNP-Xyl and pNP-Ara, with optimum pH values of 9.0 (and 6.0) at temperatures of 50 and 40 °C. This was superior as compared to the previously reported enzymes Rubgx1, GH43  and XynB5. Specifically, the Rubgx1 (GH3 family) enzyme  [Vmax (666.6 and 102 U/mg), than a previously-characterized bifunctional (β-xylosidase/α-arabinosidase) enzyme [Vmax values of 285.71/78.12 U/mg, respectively (unpublished data). Also, protein 2 progressively showed higher enzyme activity (with the corresponding substrates) with increasing pH. Such changes in pH will alter the attractions between groups in the side chains of the protein, potentially modifying the protein domain shape. Moreover, the binding of substrate to the active site may be modulated and/or it cannot undergo catalysis.Gene 2 was annotated as a gene encoding a protein with β-xylosidase/α-arabinosidase activity belonging to CAZy family GH3 (EC. 3.2.1.37). As shown in Table d 50 °C) , the GH4d 50 °C)  and, find 50 °C) . In addi) enzyme . In anotCaulobacter crescentus) [pNP-Xyl and pNP-Ara dual activity may have been due to two distinct catalytic properties in the same polypeptide chain, usually catalyzing two consecutive reactions [As indicated above, protein 2 was similar to the GH3 family proteins Rubgx1 (81.4 kDa) and XynB5  , 32. Itseactions . Dual- oeactions .pNP-Xyl. Thus, expression of the α-glucosidase encoding gene may have been repressed at the genomic level, becoming expressed from the inducible promoter of the expression system of the used vector. Protein 5 clearly belongs to the “GGDEF” domain protein family [C. crescentus [Salmonella enterica enzymes involved in cellulose biosynthesis and biofilm formation [− oversupply may have caused a change in the shape and/or charge of the enzyme’s active site, spurring activity. Further detailed structural studies could reveal the dynamics of such glycoside hydrolase activities at varying pH values in the lignocellulose degradation processes. Also, protein 5 was found to have a different motif, of the “GDSL” class by CDART prediction.Interestingly, protein 5 showed α-glucosidase activity, whereas no such activity was detected from its mother fosmid clone 10BT . Fosmid n family , 36. Thiescentus . It was ormation . For morormation . The forormation . Howeverormation , allowinormation . Surprisaxe2 product (acetyl xylan esterase), which has a GDSL motif, removes acetyl groups from the hemicellulose polymer xylan [Remarkably, protein 6 was found to possess two different conserved motifs, NPA and GDSL. The NPA motif is a key structural feature of proteins that plays crucial roles as water channels across membranes, supporting membrane localization of the protein. Surprisingly, protein 6 also belongs to the GDSL hydrolase family (encompassing esterases/lipases), not sharing sequence homology with any of the CAZy database glycosyl hydrolases. GDSL motif enzymes constitute a rather new group of proteins, with characteristics that have not yet been precisely described . Furtherer xylan . Such acer xylan . Indeed,Trichoderma reesei, Thermobifida fusca and Clostridium thermocellum. The degradation of lignocellulose requires the intensive action of multiple enzymes. This is due to the structural aspects that are typical for the substrate, i.e., its high molecular weight and the association of hemicellulose with cellulose and lignin. The required hemicellulases need to hydrolyze glycosidic bonds. In many habitats, the degradation process is rather slow because of the poor substrate accessibility, inhibitors , and untimely presence of efficient enzymes. However, in industrial processes, such enzyme availabilities can be steered [In recent years, several enzyme cocktails have been proposed that can enhance plant biomass degradation rates . Such co steered , 49. The steered , 51. How steered . InteresThe four (thermo-stable) enzymes described here may pave the way towards improved (hemi) cellulolytic action of existing enzyme cocktails. Using the his-tags, the enzymes, provided they are sufficiently stable, can be easily recovered from the treatment process and re-used in continuous applications, allowing alkaline and enhanced temperature conditions . The herAdditional file 1: Figure S1. Protein 5 Blast-P multiple sequence alignment.Additional file 2: Figure S2. Protein 6 Blast-P multiple sequence alignment.


ABCB1 and CYP4F2 genes were associated with stable warfarin dose requirements in Brazilian patients. This retrospective study included samples from 309 individuals. Genotyping of ABCB1 c.3435C>T and CYP4F2 c.1297G>A were performed by polymerase chain reaction followed by melting curve analysis (HRM-PCR) and TaqMan® genotyping assay, respectively. Stable doses were adjusted in a linear multiple regression model for age, gender, body mass index, self-reported race, use of amiodarone, CYP2C9 (*2 and *3), VKORC1 c.1639G>A, and ABCB1 c.3435C>T or CYP4F2 c.1297G>A. By performing a univariate analysis of variance, we found that the warfarin patients who carry ABCB1 c.3435T variant alleles (CT and TT genotypes) need fewer warfarin stable doses in comparison with the individuals that are CC wild-type: 2.5 (p = 0.003) and 4.3 (p < 0.001) mg/week less, respectively, for the overall group of patients on stable anticoagulation therapeutics (n = 309); and 5.5 (p = 0.006) and 10.2 (p < 0.001) mg/week less, respectively, for the self-declared non-white stable subgroup (n = 76). No statistically significant differences in dose requirements were observed according to CYP4F2 genotypes. In conclusion, our results suggest ABCB1 c.3435C>T variant may influence warfarin dose requirements in Brazilian patients, when associated with other genotypic, demographic and clinical factors.The ideal dose of the oral anticoagulant warfarin varies widely among patients, mainly due to genetic factors. Genetic variations that impact warfarin pharmacokinetics and the vitamin K cycle are plausible candidates for being associated with warfarin dose requirements. Therefore, the aim of this study was to assess whether polymorphisms in the  In the last 65 years, warfarin has been the most widely prescribed oral anticoagulant for treating and preventing thromboembolic events  in warfarin sensitivity , which is responsible for the primary metabolization of vitamin K in the liver. Warfarin mechanism of action consists of blocking the vitamin K redox cycle in order to diminish the concentration of the vitamin K reduced form, which is a coactivator of several coagulation factors   codes for the well-known multidrug efflux pump P-glycoprotein (P-gp), responsible for restricting the absorption of many xenobiotics, as well as excreting them and their metabolites through the kidneys and liver. The referred ABCB1 polymorphism has been extensively studied and associated with the variability dosage of many drugs, including warfarin, which is one of the many substrates of P-gp , University of São Paulo Medical School (FMUSP), São Paulo, Brazil , CYP2C9*2  and CYP2C9*3  genotypes were obtained from the previous study of our group  and CYP4F2 p.Val433Met (rs2108622) were performed by polymerase chain reaction followed by melting curve analysis (HRM-PCR) and TaqMan® genotyping assay , respectively, in a Rotor-Gene Q® (Qiagen)  presented stable warfarin maintenance doses. Positive and negative controls were added in all experiments. Furthermore, as genotyping quality control, 6% of the samples were reanalyzed and gave 100% data consistency.Genomic DNA was extracted from peripheral blood leukocytes by the standard salting-out method. Data of ABCB1 c.3435C>T  and CYP4F2 c.1297G>A  genotypes.For descriptive analysis purposes of the cohort characteristics, categorical variables are presented as percentages, and continuous variables are presented as mean ± standard deviation (SD) or standard error (SE) of the mean. Chi-square and Fisher's exact tests were performed for comparative analysis of categorical variables  according to genotypes. One-way ANOVA  test was used for analysis of warfarin stable maintenance dose according to CYP2C9 phenotypes , and VKORC1 c.-1639G>A genotypes . Tukey's HSD (honest significant difference) post hoc tests were performed to identify the different groups. The level of significance was set at p ≤ 0.05. All statistical analyses were carried out using SPSS statistical package (20.0 version) and R (3.4.4 version).Mean warfarin stable doses were adjusted through a linear multiple regression model for age (years), gender , Body Mass Index (BMI) (kg/m2), self-declared race , amiodarone use , predicted metabolic n = 309). Of the 309 patients (mean age 64 ± 14), 154 (49.8%) were female, most of them (75.4%) declared their race/color as white, 4.6% were smokers and 10.7% used the warfarin concurrent medication amiodarone. The observed genotypic distributions for VKORC1 and frequencies of CYP2C9 predicted metabolic phenotypes are shown in Table Table ABCB1 and CYP4F2 genotypes, respectively , the frequencies of the T variant allele and of the homozygous genotype for the ABCB1 c.3435C>T were 44 and 18.8%, respectively. The frequencies of the A variant allele and of the homozygous genotype for the CYP4F2 c.1297G>A were 32 and 8.7%, respectively. ABCB1 c.3435C>T and CYP4F2 c.1297G>A genotypic distributions are in accordance with Hardy-Weinberg equilibrium (Table Table n = 309), we found different mean warfarin doses according to ABCB1 c.3435C>T genotypes p-value (ANOVA) < 0.001; CC: 30.5 ± 0.6 (n = 98), CT: 28.0 ± 0.5 (n = 153), TT: 26.2 ± 0.6 (n = 58) (mean stable dose ± SE in mg/week). A Tukey's HSD post hoc test revealed that patients with ABCB1 3435CC genotype presented higher doses than CT or TT carriers . The data were adjusted for age, gender, BMI, self-reported race/color, use of amiodarone, CYP2C9 (*2 and *3), and VKORC1 c.-1639G>A  for the overall group of patients on stable warfarin treatment n = 09, we fon = 309) into two distinct subgroups according to self-declared race/color: white (n = 233) and non-white (n = 76). For this analysis, we adjusted the dose for all covariates cited above, except self-reported race. Table ABCB1 and CYP4F2 genotypes of the patients with stable warfarin therapy according to self-declared racial subgroups  < 0.001; CC = 35.3 ± 1.3 (n = 24), CT = 29.8 ± 1.0 (n = 38); TT = 25.1 ± 2.0 (n = 14) (mean stable dose ± SE in mg/week). Carriers of ABCB1 3435CC genotype presented higher doses than CT or TT, according to Tukey's HSD test   = 0.084; CC = 30.0 ± 0.7 (n = 74), CT = 27.3 ± 0.6 (n = 115); TT = 26.8 ± 0.7 (n = 44) (mean stable dose ± SE in mg/week)  = 0.062; GG = 27.7 ± 0.4 (n = 156), GA = 29.3 ± 0.5 (n = 126), AA = 28.9 ± 0.9 (n = 27). P-values according to Tukey's HSD test were: GG vs. AA: p = 0.567; GG vs. GA: p = 0.053; GA vs. AA: p = 0.946. No significant differences were found when the white subgroup was assessed p-value (ANOVA) = 0.257; GG = 27.1 ± 0.6 (n = 109), GA = 28.3 ± 0.6 (n = 99), AA = 28.5 ± 1.1 (n = 25)  = 0.048; GG = 29.2 ± 1.1 (n = 47), GA = 33.1 ± 1.0 (n = 27), AA = 33.0 ± 1.9 (n = 2). P values according to Tukey's HSD test were: GG vs. AA: p = 0.705; GG vs. GA: p = 0.042; GA vs. AA: p = 1.000. However, only two patients were CYP4F2 1297AA carriers and self-declared their race/color as non-white  need, on average, fewer warfarin stable maintenance doses in comparison to the individuals that are wild-type CC carriers. Similar data were obtained in the study of Wadelius et al. They found that an ABCB1 haplotype containing only the 3435T allele as variant  was over-represented [p(χ2) = 0.0242] among the low warfarin dose patients  subgroup in comparison to the medium dose  and the high dose  subgroups (p = 0.0094). On average, the patients that carried one 3435T variant allele for the D haplotype (n = 20) needed 24% lower warfarin dose and dose/body weight (BW) than the other patients (n = 181) who did not carry this haplotype : CC: 59.5 (n = 70); CT: 56.9 (n = 90); TT: 55.6 (n = 36). Nevertheless, no statistical differences between the genotypic subgroups were found. However, according to race, it is possible to verify that the African, Hispanic, and Asian Americans (the non-white subgroup in Kim's study), when analyzed together presented a decreasing pattern of mean warfarin doses according to the addition of the 3435T allele   are quite controversial  and the synonymous c.1236T>C (p.Gly412=) variants, some authors hypothesize that the c.3435C>T itself does not directly influence the P-gp activity, but this common haplotype does  criteria. Self-declared race/color does not correlate completely with genetic ancestry in Brazilians  and genetic factors (VKORC1 and CYP2C9 genotypes), it did not contribute significantly to the model, even when self-declared race/color subgroups were assessed. Nevertheless, we verified a descriptive bias of augmented dose in individuals that carry the polymorphic allele A, but with no statistical significance (corresponding to GA and AA genotypes): GG = 27.7 ± 0.4 (n = 156), GA = 2 9.3 ± 0.5 (n = 126), AA = 28.9 ± 0.9 (n = 27).Regarding the lack of association of the CYP4F2 c.1297G>A polymorphism as a minor significant influencer of warfarin dose variability. The authors found that in three independent cohorts of white patients who had a stable warfarin dose , the CYP4F2 c.1297G>A variant contributed to approximately 1 mg/day more in homozygous AA individuals, when compared to wild-type , Caldwell et al. implicated the ABCB1 c.3435C>T polymorphism may marginally influence warfarin dose requirements in Brazilian patients, when associated with other genotypic, demographic and clinical factors.In conclusion, our results suggest This study was approved by the ethics committee and all included subjects provided written informed consent .All authors have contributed substantially to the conception and design of this paper. LT performed the experiments, collected and analyzed the data, and wrote the paper. LM recruited the patients, collected data and critically revised the manuscript. RS recruited the patients. JK and AP provided the facilities. PS analyzed the data and critically revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The effect of diabetes on vascular mechano-transductive response is of great concern. Given the higher rate of vein graft failures associated with diabetes, understanding the multiple cellular and molecular events associated with vascular remodeling is of vital importance. This article represents data related to a study published in Cardiovascular Diabetology  Specifications TableValue of the data•The data presented in this Brief is vital to understanding the effect of diabetes on tissue•This data gives insight into the how diabetes alters tissue response to stimuli•The data can provide comprehensive analysis of the effect of diabetes on vascular signaling in vein transplant surgery•These data provides a more thorough understanding of the SHP-2 involvement in pressure mediated signaling in both diabetic and lean IVC11.1p<0.05). Pressurization resulted in a significant increase in SHP-2 in the LNZ IVC (108 ± 10.2% p<0.05) and OSXZ IVC (52 ± 6.6% p<0.05) (p<0.05) (p<0.05) and the OSXZ IVC (74 ± 8.9% p<0.05) (p<0.05) compared to LNZ controls. Pressurization decrease the LNZ (26 ± 6.6% p<0.05) ratio of p-SHP-2 to SHP-2 but failed to change the OSXZ ratio of p-SHP-2 to SHP-2, this absence of a pressure induced decrease in the OSXZ IVC was significantly different compared to lean (To determine the effect of pressurization of inferior vena cava (IVC) from diabetic male obese syndrome-X Zucker (OSXZ) diabetic and nondiabetic male normal lean Zucker (LNZ) animals we evaluated the expression of Tyrosine-protein phosphatase non-receptor type 11 (PTPN11) also known as Src homology region 2 domain-containing phosphatase-2 (SHP-2)  p<0.05) -B. Howev(p<0.05) -B. Compa p<0.05) -A. The r to lean -C.Fig. 11.2p<0.05) but no change in OSXZ animals, this absence of a pressure induced increase in the OSXZ IVC was significantly different compared to lean  and OSXZ (72 ± 12.7% p<0.05). However the pressure induced elevation in the OSXZ IVC was significantly less than that of the lean (34 ± 21.0% p<0.05)  -B.1.4The small heat shock protein HSP 27 has been shown to be involved in a number of cellular processes. Recently research has indicated that it is required for proper heart tube formation 1.5p<0.05). Pressurization resulted in a significant increase in HSP 70 in the LNZ IVC (54 ± 2.9% p<0.05) and OSXZ IVC (91 ± 3.0% p<0.05) (p<0.05) (The 70 kDa heat shock proteins (HSP 70) is a ubiquitously expressed chaperone protein activated by various cellular stresses including oxygen-free radicals, inflammation, heat shock, oncogenes, and proto-oncogenes  p<0.05) -B. Howev(p<0.05) -B.1.6p<0.05) and OSXZ IVC (21 ± 4.5% p<0.05). However the pressure induced elevation in the OSXZ IVC was significantly less than that of the lean (21 ± 8.6% p<0.05)  is a family of enzymes involved in proliferation, motility, cell growth, differentiation and intracellular trafficking. PI3K is composed of a heterodimer between a p85 regulatory subunit and a p110 catalytic subunit  p<0.05) .Fig. 4Di22.1n=12) male normal lean Zucker (non-diabetic) (LNZ) and young  male obese syndrome-X Zucker (diabetic) (OSXZ) rats were obtained from the Charles River Laboratories and barrier housed one per cage in an AAALAC approved vivarium. All procedures were conducted in strict accordance with the Public Health Service policy on animal welfare. Housing conditions consisted of a 12 H: 12 H dark-light cycle and temperature was maintained at 22±2 °C. Animals were provided food and water ad libitum. Rats were allowed to recover from shipment stress for at least two weeks before the commencement of experimentation during which time the animals were carefully observed and weighed weekly. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals as approved by the Council of the American Physiological Society and the Animal Use Review Board of Marshall University. Young  [cat #3751], and Bcl-XL [cat #2762], p70 S6 Kinase Control Cell Extracts [cat #9203], mouse IgG and rabbit IgG were purchased from Cell Signaling Technology . Purchased antibodies against Bcl-3 (H-146) [sc-13038], from Santa Cruz Biotechnology . SDS-PAGE precast gels were purchased from Lonza . Enhanced chemiluminescence (ECL) western blotting detection reagent was purchased from Amersham Biosciences . All other chemicals were from Sigma .2.3Inferior venae cavae and experimental design were performed as described by Rice et. al 2.4Immunoblotting was performed according to the protocol outlined by Rice et al. 3a priori was ≤ 0.05.Data were analyzed using the Sigma Stat 3.0 and results were presented as mean ± SEM. A one-way analysis of variance (ANOVA) was performed for overall comparisons followed by the Student-Newman-Keuls post hoc test to determine differences between groups. The level of significance accepted


Pseudomonas putida Y-9 was investigated and exhibited excellent capability for nitrogen removal at 15°C. The strain capable of heterotrophic nitrification and aerobic denitrification could efficiently remove ammonium, nitrate, and nitrite at an average removal rate of 2.85 mg, 1.60 mg, and 1.83 mg NL−1 h−1, respectively. Strain Y-9 performed nitrification in preference to denitrification when ammonium and nitrate or ammonium and nitrite coexisted in the solution. Meantime, the presence of nitrate had no effect on the ammonium removal rate of strain Y-9, and yet the presence of high concentration of nitrite would inhibit the cell growth and decrease the nitrification rate. The experimental results indicate that P. putida Y-9 has potential application for the treatment of wastewater containing high concentrations of ammonium along with its oxidation products at low temperature.The cold-adapted bacterium Providencia rettgeri YL [ Bacillus methylotrophicus strain L7 [ Alcaligenes faecalis strain number 4 [ V. diabolicus SF16 [Excess nitrogen concentration, often in the form of nitrate and ammonium, would lead to eutrophication of water. And this would affect the balance of natural water ecosystems, even human health . Therefotgeri YL , Bacillutrain L7 , Alcalignumber 4 , and V. Nevertheless, the growth and bioactivity of microorganisms are affected significantly by temperature. Previous research has shown that both nitrification and denitrification would be inhibited strongly at temperatures below 20°C , 9. Most Pseudomonas putida. And P. Putida Y-9 exhibited an excellent performance of denitrification with a low concentration of nitrite at low temperature. But its ability of heterotrophic nitrification and aerobic denitrification with other nitrogen compounds was unclear. In the study, the nitrogen removal performance of strain Y-9 with high concentrations of ammonium, nitrate, nitrite, and organic nitrogen was investigated at 15°C, and the characteristics of nitrification and denitrification were assessed by analyzing the intermediate products during the nitrification and denitrification process. The results indicated that strain Y-9 is the heterotrophic nitrifying-aerobic denitrifying psychrophile bacteria and could quickly remove high concentration ammonium in wastewater. Although several strains belong to P. putida are reported as heterotrophic nitrifier [ P. putida capable of heterotrophic nitrification and aerobic denitrification at low temperature. All attempts above would be applied to elucidate the nitrogen removal characteristics of strain Y-9. And strain Y-9 might enhance aerobic denitrification activity of indigenous bacteria by quickly decreasing ammonium concentration in wastewater, especially cold wastewater.In our previous study , a novelitrifier  or aerob Pseudomonas putida and used for the investigation on nitrogen removal performance.Strain Y-9 was isolated from the long-term flooded paddy soil and successfully identified as2HPO4, 3.0 g KH2PO4, 0.1 g MgSO4·7H2O, 1.0 g (NH4)2SO4, 0.05 g FeSO4·7H2O, and 10 g CH3COONa, pH 7.2. HNM was used to measure the nitrification ability of strain Y-9. Aerobic denitrification medium (ADM) contained (per liter) 7.0 g K2HPO4, 3.0 g KH2PO4, 0.1 g MgSO4·7H2O, 1.45 g KNO3 (ADM-1) or 0.986 g NaNO2 (ADM-2), 0.05 g FeSO4·7H2O, and 10 g CH3COONa, pH 7.2. ADM was used to test the aerobic denitrification ability of strain Y-9. Tryptone (1.575 g/L) was used instead of inorganic nitrogen in the ADM to detect the aerobic denitrification ability of strain Y-9 with organic nitrogen. Simultaneous nitrification and denitrification medium (SNDM) consisted of the following components (per liter): 7.0 g K2HPO4, 3.0 g KH2PO4, 0.1 g MgSO4·7H2O, 1.0 g (NH4)2SO4, 1.45 g KNO3 (SNDM-1) or 0.986 g NaNO2 (SNDM-2), 0.05 g FeSO4·7H2O, and 10 g CH3COONa, pH 7.2. SNDM-1 and SNDM-2 were used to analyze simultaneous heterotrophic nitrification and aerobic denitrification capability of strain Y-9. Luria-Bertani (LB) medium contained (per liter) 10 g tryptone, 5 g yeast extract, and 10 g NaCl, pH 7.5.Heterotrophic nitrification medium (HNM)  containeAll chemicals were of analytical grade. And conical flasks (250 ml capacity) containing 100 ml medium was autoclaved for 30 min at 121°C.4+-N, NO2−-N, NO3-N, TN, and optical density (OD600) at 24 intervals. All experiments were conducted in triplicate.Single colony of Y-9 was cultivated for 36 h in 100 ml sterile LB broth medium at 15°C and 150 rpm. After 36 h cultivation, the strain cell in 8 ml cultivation medium was harvested by centrifuging at 4000 rpm for 8 min and washed once with sterile water. The pellets were inoculated into 100 ml HNM, ADM-1, ADM-2, organic medium, SNDM-1, or SNDM-2, respectively. The cultures were incubated at 15°C with 150 rpm shaking speed for 4 days. And the medium without inoculation was used as control. During incubation, the different cultures were sampled to determine the concentration of NH600 by using a spectrophotometer . Total nitrogen was calculated by the absorbance value at 220 nm subtracting the two times background absorbance value at 275 nm after using alkaline potassium persulfate digestion. Ammonium, nitrate, and nitrite were detected using the supernatant after samples centrifuged at 8000 rpm for 5 min. Ammonium nitrogen was analyzed by indophenols blue method. Nitrate was calculated by the absorbance value at 220 nm subtracting the two times background absorbance value at 275 nm. The concentration of NO2−-N was determined by N--diaminoethane spectrophotometry method according to the State Environmental Protection Administration of China [Cell density was tested ODof China .Statistical analysis and graphical work were carried out by using Excel, SPSS Statistics, and Origin 8.6. The results were presented as means ± SD (standard deviation of means).600 reached 1.644 at 2 d, which was higher than that of Pseudomonas fluorescens wsw-1001 at 48 h with an OD600 of 0.554 under the same conditions [4+-N/L/h, which was observably higher than that of heterotrophic nitrification-aerobic denitrification bacterium Aeromonas sp. HN-02 possessed an average rate of 2.32 mg NH4+-N/L/h at the same temperature [To assess heterotrophic nitrification with ammonium of strain Y-9 at low temperature, strain Y-9 was cultivated in the HNM at 15°C. The growth curve and ammonium removal capability of strain Y-9 were shown in perature . P. alcaligenes AS-1 (1.15 mg NH4+-N/L/h) [ Diaphorobacter sp. PD-7 (1.61 mg NH4+-N/L/h) [ Marinobacter strain NNA5 (1.23 mg NH4+-N/L/h) [2−-N concentration of 0.31 mg/L was detected and then declined, and nitrate was undetectable during the whole process. This would be a significant advantage in practical application of ammonium removal to avoid high accumulation of nitrification products under low temperature. Additionally, the production of gaseous nitrogen by strain Y-9 might be via the intermediate nitrite when ammonium was used as the sole nitrogen source. Similar result had been reported on Vibrio diabolicus SF16 [ Zobellella taiwanensis DN-7 [ Acinetobacter sp. Y16 [ Rhodococcus sp. CPZ24 [ Marinobacter sp. F6 [ Chryseobacterium sp. R31 [Furthermore, it was attractive that ammonium removal performance of strain Y-9 at low temperature was even better than most bacterial at moderate temperature (around 30°C), such as+-N/L/h) , Diaphor+-N/L/h) , and Mar+-N/L/h) . The consis DN-7  in which sp. Y16 . In addi3 was used as the sole nitrogen source in the ADM-1 at 15°C. 600 increased from 0.235 to 1.748, and cell growth reached the stationary phase after 3 d. By 4 d of cultivation, approximately 74.7% of nitrate nitrogen  and 18.3% of total nitrogen  were removed. The average denitrification rate of strain Y-9 was 1.60 mg NO3−-N/L/h, which was similar to that of P. migulae AN-1 which possessed an average rate of 1.57 mg NO3−-N/L/h at 10°C [To demonstrate the denitrification by strain Y-9 at low temperature, KNO at 10°C . Meanwhi at 10°C . This ma P. alcaliphila strain MBR could conduct denitrification and DNRA simultaneously with an electrode as the sole electron donor in bioelectrochemical systems (BESs), but not in an open culture. The experimental results indicated that strain Y-9 could perform denitrification with nitrate at low temperature.In addition, ammonium nitrogen gradually increased from 1 d to 4 d, and the final concentration was approximately 8.04 mg/L. This phenomenon was different from the report that the production of ammonium might be the decomposition of cell, and part of nitrogen containing in the cell was released into the medium in senescent phases . Therefo2) as the sole nitrogen source was evaluated. 2−-N by strain Y-9 was observed. The growth of strain Y-9 slowly increased at the initial 2 d. Subsequently, the NO2−-N was rapidly reduced, and OD600 reached 1.775 at 4 d. By 4 d of cultivation, the concentration of total nitrogen decreased dramatically from 214.12 mg/L to 97.45 mg/L, and approximately 54.5% of total nitrogen was removed. Meantime, 85.6% of nitrite  was removed. The average denitrification rate of strain Y-9 was approximately 1.83 mg NO2−-N/L/h, which was higher than that of Pseudomonas sp. yy7 which possessed an average rate of 0.76 mg NO2−-N/L/h [Denitrification performance of strain Y-9 with nitrite 2SO4 and KNO3 as the nitrogen sources at 15°C. 4+-N) was completely removed after 3 d of cultivation, and the average nitrification rate of strain Y-9 was 2.84 mg NH4+-N/L/h, which was almost equivalent to that of ammonium as the sole nitrogen source. Thus, the presence of nitrate had no material impact on the nitrification rate of strain Y-9, which was obviously different from previous report that the presence of nitrate could decrease the nitrification rate [Biological simultaneous nitrification and denitrification is a more efficient and economic method for nitrogen removal from wastewater . Strain ion rate . Rhodococcus sp. HY-1 utilized ammonium preferentially when ammonium and nitrate coexisted in the medium, and nitrate could be utilized when ammonium was completely exhausted under aerobic conditions. However, this result was different from the report that Klebsiella pneumoniae CF-S9 had the ability to achieve simultaneous removal of ammonium and nitrate, and nitrate removal rate was higher than that of ammonium when ammonium and nitrate coexisted in the medium [Nevertheless, the removal rate of nitrate was far lower than that of nitrate as the single nitrogen source, and removal efficiency of nitrate was only 6.4% after 4 d of cultivation. This phenomenon was consistent with the report thate medium . There w4)2SO4 and NaNO2 as the nitrogen sources at 15°C was plotted in 600 only slightly increased to 0.232 at 4 d, which correlated well with the low nitrogen removal efficiency. Hence, the concentration of ammonium nitrogen decreased inconspicuously from 203.43 mg/L to 188.13 mg/L, and only 7.5% of ammonium was removed after 4 d of cultivation. The average nitrification rate of strain Y-9 was 0.16 mg NH4+-N/L/h, which was much less than that of ammonium or nitrite as the nitrogen source. Moreover, nitrate was undetectable during the whole process, and 203.58 mg/L initial NO2−-N was almost unchanged, which was inconsistent with the report that the presence of ammonium together with nitrite could promote the nitrite removal efficiency by Marichromatium gracile YL28 [ Pseudomonas sp. qy37 [ A. Faecalis TUD would be inhibited from the presence of nitrite [ P. versutus LYM, which could synchronize ammonium removal with nitrite removal and could not reduce nitrite when nitrite was used as the sole nitrogen source even in presence of enough carbon source [The capability of simultaneous nitrification and denitrification by strain Y-9 with (NHile YL28  or promosp. qy37 . This phn source .Strain Y-9 could simultaneously reduce ammonium and its nitrification product (nitrite) when ammonium was used as the sole nitrogen source. But ammonium and adscititious nitrite could not be removed synchronously by strain Y-9. This might be explained in that enzyme activity (such as ammonia monooxygenase) related to the nitrification could be inhibited from the presence of high concentration of nitrite, thereby affecting the ammonium nitrogen removal. The results indicated that the presence of high concentration of nitrite would restrain the cell growth and decrease the ammonium removal. Pseudomonas putida Y-9 could perform heterotrophic nitrification with ammonium and aerobic denitrification with nitrate or nitrite at 15°C. The strain could efficiently remove ammonium, nitrate, and nitrite at average removal rate of 2.85 mg, 1.60 mg, and 1.83 mg NL−1 h−1, respectively. Strain Y-9 performed nitrification in preference to denitrification when ammonium and nitrate or ammonium and nitrite coexisted in the solution. Meantime, the presence of nitrate had no impact on the nitrification rate of strain Y-9. However, the presence of high concentration of nitrite could suppress cell growth and lower the nitrification rate. In addition, the process of dissimilatory nitrate or nitrite reduction to ammonium (DNRA) might present in strain Y-9. The results indicated that P. putida Y-9 has potential application for wastewater treatment of nitrogen pollution under cold temperature conditions. What is more, the nitrogen transformation mechanism of strain Y-9 needs further research.In this study,


Clostridium botulinum produce botulinum toxin complex (TC), which is comprised of botulinum neurotoxin, nontoxic nonhemagglutinin, and hemagglutinins (HAs). The TC is capable of aggregating equine erythrocytes via interaction between one of the HAs, namely HA-33, and sugar chains on the cell surface. This hemagglutination is inhibited by specific sugars. In this data article, we used four TCs from serotype C and D strains. The hemagglutination-inhibiting effects of 18 sugars and 8 glycoproteins were studied. The purified TC was mixed with the sugar to enable binding of the sugar to the TC; then, the erythrocytes were added to the mixture. Specific binding between the sugar and TC resulted in inhibition of cell aggregation. Here, data illustrating the inhibitory effects of various sugars and glycoproteins against hemagglutination induced by TC of C. botulinum serotypes C and D are presented.Serotype C and D of  Specifications TableValue of the data1.C. botulinum.The data presented illustrate the specific inhibitory effect exerted by 18 sugars and 8 glycoproteins on hemagglutination by the toxin complex (TC) of serotypes C and D of 2.C. botulinum serotypes C and D strains may be distinguished via the inhibitory profiles exerted by the present sugars and glycoproteins.The results in this article indicate that two modes of hemagglutination mediated by the TCs of 3.C. botulinum serotypes C and D.The data in this article should be useful for elucidation of the mechanism underlying the recognition of sugars by the TCs of 1C. botulinum serotype C strain Stockholm (C-St), Yoichi (C-Yoichi), D strain CB-16 (D-CB16), and 1873 (D-1873).Here, we present data illustrating the inhibitory effects of 18 sugars and 8 glycoproteins  or galactose moieties (of C-Yoichi and D-1873) at their termini L-TC was purified from the culture of 33.1SDS-PAGE was performed as described by Laemmli 3.2g, 5 min, 4 °C)  and rinsed with 0.15 M phosphate buffer, pH 7.0. Suspension of the erythrocytes (1%) was prepared with the same buffer. Erythrocytes were treated with Clostridium perfringens neuraminidase (Sigma-Aldrich). Neuraminidase (0.5 U) was added to a 10% equine erythrocyte suspension, and the mix was incubated at 37 °C for 1 h. Treated erythrocytes were collected, rinsed, and suspended in 0.15 M phosphate buffer (pH 7.0) to create a 1% suspension.Erythrocytes in defibrinated equine blood  were collected by centrifugation . Samples (35 µl) of each preparation (100 µg/ml) were diluted in serial two-fold steps with 0.15 M phosphate buffer (pH 7.0) and mixed with an equal volume of a 1% equine erythrocyte suspension. After incubation at 20–25 °C for 2 h, the reciprocal hemagglutination titer was determined as 2d-glucose, d-mannose, galactose, N-acetyl-d-glucosamine, methyl-α-d-mannoside, l-fucose, xylose, fructose, rhamnose, N-acetyl-d-galactosamine, sucrose, N-acetylneuraminic acid, maltose, cellobiose, lactose, chitin oligomers, N-acetyllactosamine, and chitobiose) and glycoproteins  were used in this study. The L-TC solution  was incubated with an equal volume of sugar or glycoprotein solution , at room temperature, for 1 h. Thereafter, the solution was incubated with 20 µl of equine erythrocyte suspension (1%) at 37 °C for 1 h.For the inhibitory test, sugars (


Streptomyces viridochromogenes DSM 40736 was identified. The discovery of UMP C5-methylase opens the way to rational pathway engineering for polyoxin component optimization, and will also enrich the toolbox for natural nucleotide chemistry.Polyoxin is a group of structurally-related peptidyl nucleoside antibiotics bearing C-5 modifications on the nucleoside skeleton. Although the structural diversity and bioactivity preference of polyoxin are, to some extent, affected by such modifications, the biosynthetic logic for their occurence remains obscure. Here we report the identification of PolB in polyoxin pathway as an unusual UMP C-5 methylase with thymidylate synthase activity which is responsible for the C-5 methylation of the nucleoside skeleton. To probe its molecular mechanism, we determined the crystal structures of PolB alone and in complexes with 5-Br UMP and 5-Br dUMP at 2.15 Å, 1.76 Å and 2.28 Å resolutions, respectively. Loop 1 (residues 117–131), Loop 2 (residues 192–201) and the substrate recognition peptide (residues 94–102) of PolB exhibit considerable conformational flexibility and adopt distinct structures upon binding to different substrate analogs. Consistent with the structural findings, a PolB homolog that harbors an identical function from The online version of this article (doi:10.1007/s13238-016-0289-y) contains supplementary material, which is available to authorized users. Sreptomyces cacaoi var. asoensis (S. cacaoi hereafter) and Streptomyces aureochromogenes . To determine its involvement and the corresponding function, we first performed a gene knockout of polB in S. cacaoi. This was achieved via conjugation of a polB disruption vector pJTU2183 (Table S1) and a double-crossover replacement of the corresponding region in the chromosome of S. cacaoi. The sample of the resulting mutant CY5 was found to display higher bioactivity against the indicator strain Trichosporon cutaneum , implicating that the metabolites might be different. LC-MS analysis showed that CY5 was unable to produce polyoxin A (5-hydoxymethyl), polyoxin F (5-carboxyl) and polyoxin H (5-methyl), but instead accumulated polyoxin K (without C5 modification)  , suggesting that PolB may carry ThyX function. In bacteria, ThyXs catalyze the biosynthesis of dTMP by C5-methylation of dUMP, as dTMP is the key building block for DNA synthesis, deletion of thyX usually causes a lethal effect on cell growth in minimal medium. To evaluate the function of polB, we performed targeted gene knockout of thyX in the chromosome of S. cacaoi. Interestingly, CY3, the thyX mutant of S. cacaoi, survived and exhibited sporadic growth phenotype , implicating that PolB might complement the dTMP biosynthesis of ThyX. Indeed, when thyX and polB of S. cacaoi were both deleted, the resulting mutant CY6 could not grow at all in minimal medium /pLysE . By incubating PolB with dUMP, NADPH and CH2H4folate in vitro, we observed the target product of dTMP using LC-MS analysis  than UMP (Km = 19.48 ± 4.15 μmol/L), the kcat of UMP (kcat = 3.09 ± 0.17 min−1) is 78% higher than that of dUMP (kcat = 1.74 ± 0.05 min−1).Next, we measured the kinetic parameters of PolB to substrates UMP and dUMP . Although dUMP exhibits higher affinity   using molecular replacement . Because the C5-position is substituted by the Br atom, these two structures should mimic the state of substrate binding. Both structures can be superimposed with the apo-PolB structure within rmsd of 1.26 Å over all the Cα atoms . In apo-PolB, the electron densities of SRP were weak or difficult to observe. However, upon substrate binding, Glu94, Ser96, Ala97 and Arg98 in SRP are able to form hydrogen bonds to the substrate, which renders SRP ordered . We also discerned that the side chain of Arg98 in SRP forms hydrogen bond to Q206 in Loop 2. Notably, the side chain of Tyr99 in SRP adopts different rotamer structures in the two complex structures . The Loop 1 and Loop 2 regions in SVIThyX2 were different from those of ThyX proteins but highly conserved with the corresponding parts of PolB  in in vivo and in vitro assays, was able to catalyze the C5-methylation of both UMP and dUMP while the classic ThyXs only possess the dUMP methylase activity . We proposed that Loop 1, Loop 2 and the substrate recognition peptide constituted the gate-keeper for substrate entrance and cooperatively regulated the catalysis of PolB on UMP/dUMP methylation. Our hypothesis that Loop 1 and Loop 2 regulated the substrate specificity of PolB was further validated by identification of SVIThyX2 as the second thymidylate synthase with UMP methylase activity.S. lividians TK24, only the polyoxin H components were detected  from S. avermitilis  via a potential pyrimidine salvage pathway. This hypothesis is supported by our previous report that when the biosynthetic gene cluster of polyoxin was heterologously expressed in In summary, we have reported the identification and structural basis of an unprecedented C5 methylase that employs FAD-dependent reductive mechanism for the methylation of UMP/dUMP. We also revealed that Loop 1, Loop 2 and substrate recognition peptide of the protein collectively constitute a gate-keeper for substrate selective-entrance and preferred-catalysis. The present data will provide insights for ThyXs evolution and enrich the chemical diversity of natural nucleotides.2H4folate was a gift from Merck. Materials and primers were individually listed in Table S1 and Table S2, and general methods and procedures were described by Kieser et al.  unless otherwise indicated. 5-Br UMP, 5-Br dUMP and 5-methyl UMP were purchased from Hongene Biotechnology Ltd. . CHr et al.  and Sambr et al. .S. cacaoi and SVIThyX2 from S. viridochromogens DSM40736  as N-terminally His6-tagged proteins. The soluble fraction of the cell lysate was first purified using nickel affinity column  and further purified by gel-filtration chromatography .All constructs and point mutations were generated using a standard PCR-based cloning strategy and verified through DNA sequencing. The recombinant PolB from 2H4folate, 0.2 mmol/L UMP (or dUMP), 50 mmol/L Tris-HCl (pH 8.0), and 10 μg of protein . The reaction was terminated by adding TCA with final concentration 10% (v/v) and further analyzed by LC-MS using a ZORBA SB-C18 Column . The LC conditions were as follows. The elution buffer for LC was 10% methanol (v/v) contained 0.1% aqueous trifluoroacetic acid (v/v). The flow rate was 0.3 mL/min and the eluted fraction was monitored at 260 nm with a DAD detector. The parameters for MS analysis are 10 l/mL of drying gas flow, 30 psi of nebulizer pressure, and 325°C of drying gas temperature.All tests were performed in triplicates in 2 mL centrifuge tubes. A typical methylase activity assay (200 µL) contained 2.0 mmol/L NADPH, 0.2 mmol/L CHw/v) PEG4000, 0.2 mol/L Li2SO4 and 0.1 mol/L Tris-HCl, pH 8.5. Crystals of the PolB/5-BrdUMP complex were obtained by mixing PolB (11.4 mg/mL) with 4 mmol/L of 5-Br dUMP and growing under the conditions of 1.4 mol/L sodium acetate and 0.1 mol/L sodium cacodylate, pH 6.5. Crystals of the PolB/5-Br UMP complex were obtained by mixing PolB (11.4 mg/mL) with 4 mmol/L of 5-Br UMP and growing under the conditions of 10% (w/v) PEG4000, 5% isopropanol and 0.1 mol/L HEPES, pH 7.5. Prior to data collection, all crystals were flash-cooled in liquid nitrogen using Paratone-N  as cryo-protectants. Diffraction data were collected on a Mar225 detector at 100 K on the beamline BL17U1 at Shanghai Synchrotron Radiation Facility . The data sets were integrated and scaled with HKL2000.All crystallization experiments were performed at 20°C using the sitting-drop vapor-diffusion method. Protein samples  stored in 25 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl and 5 mmol/L β-mercaptoethanol were mixed with well solution (1 µL) and equilibrated against the well solution (75 µL) in 96-well plates . The crystal of apo-PolB was grown under the condition of 17% (The structure of the PolB/5-Br dUMP complex was solved by molecular replacement using Phaser and the structure of TMAThyX (PDB code: 1O2A) as the search molecule. The structure of the PolB/5-Br UMP complex was solved by molecular replacement using Phaser and the PolB/5-Br dUMP complex as the search molecule. The structure of the PolB was solved by molecular replacement using Phaser and the PolB/5-Br UMP complex as the search molecule. Manual model building was performed with COOT  , the National Grand Project for Medicine Innovation (2012ZX10002006 to J.Z.), the National Natural Science Foundation of Chin (Grant No. 31270100 to W.C.), Wuhan Youth Chenguang Program of Science and Technology (201507040401018 to W.C.).SMD, steered molecular dynamics; SRP, substrate recognition peptide; ThyX, thymidylate synthase.The authors declare that they have no conflicts of interest pertaining to the contents of this article. This article does not contain any studies with human subjects performed by any of the authors.W.C., Y.L., J.Z. and Z.D. conceived the project. W.C. performed genetics experiments. W.C., Y.L. and S.L. performed biochemical experiments. Y.L. L.W. and J.L carried out crystallographic studies, Y.L. and R.X. calculated the molecular modeling data. W.C., Y.L., J.Z. and Z.D. analyzed data and wrote the manuscript.Supplementary material 1 (PDF 2243 kb)Below is the link to the electronic supplementary material.


Hypocitraturia has been associated with metabolic acidosis and mineral disorders. The aim of this study was to investigate the occurrence of urinary acidification defects underlying hypocitraturia.3- (<22 mmol/L) were studied by bicarbonate loading or furosemide/fludrocortisone tests. Patients with normal baseline plasma HCO3- had an ammonium-chloride challenge test. A normal response was a decrease in urinary pH <5.3 and an increase in urinary NH4+ ≥33 μmol/min and defined idiopathic hypocitraturia.This retrospective observational study included 67 patients (32 men), aged 40.7±15.1 years with hypocitraturia (<1.67 mmol/24-h) and nephrolithiasis, nephrocalcinosis, and/or bone demineralization, referred to our center from 2000 to 2015. We aimed to assess renal distal acidification capacity, prevalence and mechanisms of urinary acidification defects. Patients with low baseline plasma HCO3- and overt distal acidification defect. Three had a mutation in the gene encoding AE1, 4 had Gougerot-Sjögren syndrome and no cause was found in the remaining 4 cases. Fifty-six patients (83.6%) had normal HCO3-; of those, 33 (58.9%) had idiopathic hypocitraturia. Among the 23 (41%) remaining patients, 12 were unable to increase urinary NH4+ excretion  whereas 11 were able to increase urinary NH4+ excretion but unable to decrease urinary pH. These 11 patients had higher fasting urinary calcium, reflecting bone resorption, than the other 12 patients: median 0.41 [0.24–0.47] vs. 0.22 [0.08–0.37] mmol/mmol creatinine (P = 0.04).Eleven patients (16.4%) had low HCO3- frequently show a latent acidification defect that can be further dissected into one of several subtypes based on urinary pH and NH4+ response to the acid load. Those patients with impaired urine acidification capacity but preserved NH4+ excretion exhibit particularly high calciuria and should be identified to optimize nephrolithiasis prevention.Patients with hypocitraturia and normal plasma HCO SLC4A1 gene encoding the chloride bicarbonate exchanger AE1 are the main causes of autosomal dominant dRTA . Ca2+: calcium; P: plasma. * p < 0.05 compared to the overt metabolic acidosis group. Table B. Biological characteristics of patients with a normal acid-base status undergoing the acute acid load test. Values are expressed as median [interquartile range]. Ca2+: calcium; NH4+: ammonium; P: plasma; NH4Cl: ammonium chloride. Subgroups classification: Idiopathic Hypocitraturia: urine pH min < 5.3, NH4+ max ≥ 33 μEq/min.; high U.pH, high U.NH4: urine pH min < 5.3, NH4+ max ≥ 33 μEq/min.; high U.pH, low U.NH4: urine pH min ≥5.3, NH4+ max ≥ 33 μEq/min.; low U.pH, low U.NH4: urine pH min < 5.3, NH4+ max < 33 μEq/min.(DOCX)Click here for additional data file.


Interestingly, no antiviral effects of arbidol are observed in virus infected porcine stable kidney cells (PS), human neuroblastoma cells (UKF-NB-4), and human hepatoma cells (Huh-7 cells) indicating that the antiviral effect of arbidol is strongly cell-type dependent. Arbidol shows increasing cytotoxicity when tested in various cell lines, in the order: Huh-7 < HBCA < PS < UKF-NB-4 < Vero with CC50 values ranging from 18.69 ± 0.1 to 89.72 ± 0.19 µM. Antiviral activities and acceptable cytotoxicity profiles suggest that arbidol could be a promising candidate for further investigation as a potential therapeutic agent in selective treatment of flaviviral infections.Arthropod-borne flaviviruses are human pathogens of global medical importance, against which no effective small molecule-based antiviral therapy has currently been reported. Arbidol (umifenovir) is a broad-spectrum antiviral compound approved in Russia and China for prophylaxis and treatment of influenza. This compound shows activities against numerous DNA and RNA viruses. The mode of action is based predominantly on impairment of critical steps in virus-cell interactions. Here we demonstrate that arbidol possesses micromolar-level anti-viral effects (EC Flavivirus, family Flaviviridae) include human pathogens of global medical importance such as dengue virus (DENV), Yellow fever virus (YFV), West Nile virus (WNV), Japanese encephalitis virus (JEV), Zika virus (ZIKV), Kyasanur Forest disease virus (KFDV), Omsk hemorrhagic fever virus (OHFV) and tick-borne encephalitis virus (TBEV). These viruses are causative of many serious diseases with a broad spectrum of clinical symptoms ranging from near asymptomatic or mild flu-like infections through neurological diseases  to viscerotropic (DENV), hemorrhagic  or even terratogenic manifestations (ZIKV) -5-hydroxy-1-methyl-2-[(phenylthio)methyl]-indole-3-carboxylate hydrochloride monohydrate) A was obt50), which represents the concentration of compound that reduced cell viability by 50%. A viral titer reduction assay was performed to determine the flavivirus sensitivity to arbidol in cell culture. Host cells were seeded in 96-well plates (approximately 2 × 104 cells per well) and incubated for 24 h at 37 °C to form a confluent monolayer. The medium was then aspirated from the wells and replaced with 200 µL of fresh medium containing 0–12.5 µM (for Huh-7), 0–25 µM (for HBCA and PS), 0–30 µM (for UKF-NB-4), or 0–50 µM (for Vero) of arbidol and incubated for 24 h . The medium was then removed from wells and replaced with fresh medium containing arbidol and appropriate virus strain at a multiplicity of infection (MOI) of 0.1. After 2 h incubation, the medium was replaced with fresh medium containing arbidol and incubated for 48 h at 37 °C. Then, the supernatant medium was collected and viral titers were determined by plaque assays, expressed as PFU/mL [50). For construction of growth curves, Vero cells were infected as described above and treated with arbidol at a concentration of 50 µM. Supernatant media were collected every six hours until 48 h p.i. Viral titers in supernatants were determined using plaque assay. In all experiments, DMSO was added to virus-infected cells as a negative control at a concentration corresponding to a dilution of the initial arbidol-DMSO stock. A cell-based flavivirus immunostaining assay was performed to measure the arbidol-induced inhibition of viral surface antigen (E protein) expression, as previously described [The compound cytotoxicity was determined in terms of cell viability using the Cell Counting Kit-8  according to the manufacturer’s instructions and expressed as the 50% cytotoxic concentration , version 7.04. Differences with p < 0.05 were considered to be statistically significant.Differences in viral titer between groups were evaluated by unpaired parametric two-tailed 50 = 89.72 ± 0.19 µM). This level of cytotoxicity was about 5 times lower than observed with Huh-7 cells (CC50 = 18.69 ± 0.1 µM) that were the most susceptible cells studied. Experiments with other cell types, namely PS, UKF-NB-4, and HBCA gave rise to CC50 values ranging from 24.78 ± 0.01 to 46.99 ± 0.1 µM  were evident in both Vero and HBCA cells 48 h after infection. Whereas in HBCA the highest arbidol concentration tested (25 µM) caused a reduction in ZIKV titer of about 10ependent C. This pemployed ,23. For employed .50 values for ZIKV infections in Vero cells were found to be 12.09 ± 0.77 and 10.57 ± 0.74 µM respectively for MR766 and Paraiba_01 strains  in comparison to values found with ZIKV . As with ZIKV, the anti-WNV effects of arbidol were most obvious when incubated at 25 and 50 µM with WNV-infected HBCA and Vero cells for 48 h. By contrast, there were little or no antiviral effects observed in UKF-NB-4, PS, and Huh-7 virus infected cells C. EC50 vith ZIKV . This obith ZIKV .50 values of 18.67 ± 0.15 µM  infections in HBCA and Vero cells C, also p 0.15 µM . Howevered cells . 6.5 to 108 PFU/mL  until the end of the experiment. In untreated cells, there was a peak in virus production between 12 and 30 h p.i. reaching a maximum of 108 PFU/mL .50 values ranging from 3 to 9 µg/mL [50 values < 10 µg/mL [50 value of 2.8 µg/mL [3-fold [Our data agree with numerous reports of in vitro arbidol-mediated inhibition of other viruses of medical interest. Arbidol inhibits replication of various subtypes of human influenza A and B viruses with EC 9 µg/mL . Arbidol10 µg/mL , plus in.8 µg/mL . Further [3-fold . Overall [3-fold ,32,33. B50 values ranging from 10.57 ± 0.74 to 19.16 ± 0.29 µM. The observed antiviral effects were strongly cell-type dependent being substantial only in HBCA and Vero cells. Arguably, such cell line variability results from differences in arbitol up-take or metabolic processing by the different individual cell lines concerned. Arbidol-mediated cytotoxicities are also cell line dependent, with Huh-7 cells being the most susceptible and Vero cells the least. Our data broaden the possibility for future in vivo testing of arbidol in animal models of flavivirus infection.In conclusion, we have demonstrated that arbidol has substantial antiviral activities against ZIKV, WNV and TBEV in vitro with EC


Lipodystrophic syndromes are uncommon medical conditions which are normally associated with metabolic disorders, such as diabetes mellitus, dyslipidemia, and fatty liver disease. These complications are generally difficult to treat, particularly diabetes, due to severe insulin resistance. We present two case reports of successful treatment of diabetes with glucagon-like peptide-1 analogues in patients with clinical features of lipodystrophic syndromes.Two white women aged 49 and 60 years manifested marked central body fat deposition with severe lipoatrophy of their limbs and buttocks and pronounced acanthosis nigricans. They had hypertension, dyslipidemia, fatty liver disease, and poorly controlled diabetes  despite the use of three classes of oral antidiabetic drugs taken in combination in the first case, and high doses of insulin in the second case. Four months after the addition of glucagon-like peptide-1 analogue to their previous treatment they both showed a pronounced improvement in metabolic control . In the first case, a weight loss of nearly 30 kg was recorded.We intend to demonstrate that glucagon-like peptide-1 analogues could be a valuable tool for patients with lipodystrophic disorders, probably by improving body fat distribution, with favorable results in insulin-sensitivity and glycemic control. Lipodystrophic syndromes are rare disorders characterized by selective loss of adipose tissue, mainly from subcutaneous compartments. Lipoatrophy may vary from being partial, co-existing with adipose tissue depots in ectopic sites, to generalized . These s2, and presented an evident abdominal fat deposition (waist circumference 123 cm), with markedly reduced adipose tissue in her limbs and buttocks. She had a round face with cervical acanthosis nigricans, and an accumulation of fat in the chin and in the cervicodorsal and supraclavicular regions. Laboratory tests revealed glycated hemoglobin (A1c) 8.3%; total cholesterol 273 mg/dL (<200), high-density lipoprotein (HDL) cholesterol 49 mg/dL (>60), low-density lipoprotein (LDL) cholesterol 196 mg/dL (<130), triglycerides 350 mg/dL (<150), under statin; and uric acid 6.9 mg/dL (2.3 to 6.1). She presented normal 24-hour urinary free cortisol of 39 mcg/24 hours (36 to 137) and an overnight dexamethasone suppression test recorded a serum cortisol of 0.4 mcg/dL (<1.8mcg/dL). She started taking exenatide 2 mg once a week, with good tolerance. Sitagliptin was suspended, while maintaining the other antidiabetic drugs. After 4 months of therapy, she lost 29.2 kg  and her A1c was 5.6%. The results of genetic tests for mutations in LMNA gene and PPAR-gamma gene were negative.A 49-year-old white woman was referred to our Endocrinology department for obesity. She presented diabetes treated with metformin 2500 mg/day, sitagliptin 100 mg/day, and gliclazide 120 mg/day. She had hypertension, dyslipidemia, hyperuricemia, fatty liver disease, and obstructive sleep apnea. Furthermore, she was being treated for a major depression, partly motivated by body image problems. She weighed 89.5 kg, had a body mass index (BMI) of 33.7 kg/m2), with central body fat deposition (waist circumference 105 cm) and prominent lipoatrophy of her limbs and buttocks. She presented pronounced cervical and axillary acanthosis nigricans, and hirsutism. Laboratory tests showed an A1c of 10.2% and dyslipidemia: total cholesterol 226 mg/dL (<200), HDL cholesterol 51 mg/dL (>60), LDL cholesterol 131 mg/dL (<130), triglycerides 221 mg/dL (<150), despite being medicated with statin. She had mildly elevated liver enzymes levels: aspartate aminotransferase 67 U/L (10 to 31), alanine aminotransferase 76 U/L (10 to 31), gamma glutamyl transferase 321 U/L (7 to 32), and alkaline phosphatase 175 U/L (30 to 120). Her renal function was normal. Cushing syndrome had been excluded: 24-hour urinary free cortisol 80.1 mcg/24 hours (36 to 137). An overnight dexamethasone suppression test registered serum cortisol of 0.8 mcg/dL (<1.8 mcg/dL). Treatment with liraglutide was initiated, in addition to insulin therapy, at 0.6 mg every day for a week, titrated to 1.2 mg for the next week, and then to 1.8 mg daily. An insulin dose reduction of 20 units was recommended at the time that treatment with liraglutide was initiated. She reported no side effects, such as nausea, vomiting, diarrhea, or hypoglycemia. After 3 months of therapy, a total daily insulin dose of 200 units, metformin 2000 mg/day, and liraglutide 1.8 mg daily resulted in a much improved glycemic control (A1c 6.2%). She lost 1.8 kg. Similarly, the results of genetic tests for mutations in LMNA gene and PPAR-gamma gene were negative.A 60-year-old white woman was observed in our Endocrinology department for diabetes. She presented hypertension, dyslipidemia, fatty liver disease, severe obstructive sleep apnea, depressive disorder, and a history of a previous ischemic stroke. She had poor glycemic control, with A1c values between 9 and 11% over recent years, requiring several medication adjustments. She was treated with metformin 2000 mg/day, and a total daily dose of insulin of nearly 240 units in a basal-bolus regimen. Her regimen was: neutral protamine Hagedorn (NPH) insulin three times daily ; insulin aspart at mealtimes ; and a correctional insulin dose was added, based on her pre-prandial glucose value, resembling a sliding scale. Her insulin sensitivity factor was 10. Her physical examination revealed that she was overweight -associated, with severe insulin resistance, with GLP-1 analogues. We intend to demonstrate that GLP-1 analogues could be used as new tools for the treatment of these metabolic and adipose tissue disorders, reducing fat mass and insulin resistance, decreasing insulin requirements, and improving A1c.