In vivo investigation to the macrolide-glycosylating enzyme pair DesVII/DesVIII in Saccharopolyspora erythraea.

Glycosyltransferase DesVII and its auxiliary partner DesVIII from Streptomyces venezulae, homologs of EryCIII and EryCII in Saccharopolyspora erythraea, have previously been demonstrated to be flexible on their substrates in vitro. Herein, we investigated their in vivo function by interspecies complementation in the mutant strains of Sac. erythraea A226. As desVII and desVIII were concomitantly expressed in the ΔeryCIII mutant, the erythromycin A (Er-A) production was restored. Interestingly, co-expression of desVII and desVIII in the ΔeryBV mutant exhibited an increased Er-A yield by 15 % in comparison to A226. Hence, DesVII/DesVIII not only replaced EryCIII to upload D-desosamine to C5 position of 3-O-mycarosyl erythronolide B (MEB) but also in vivo attached L-mycarose, not D-desosamine to C3 position of erythronolide B (EB) with a higher activity than EryBV. Furthermore, expression of desVII in ΔeryCIII and ΔeryBV-CIII partially restored the Er-A production; however, no Er-A was detected while desVII was expressed in ΔeryBV. It was implicated that DesVII coupled with EryCII to form the DesVII/EryCII complex for attaching above two deoxysugars in the absence of EryCIII in Sac. erythraea. In addition, when desVII and desVIII were co-expressed in ΔeryBV-CII, Er-A was recovered with a lower yield than ΔeryBV-CIII. Our study presents an opportunity with Sac. erythraea as a cell factory for macrolide glycodiversification.

Capturing the target genes of BldD in Saccharopolyspora erythraea using improved genomic SELEX method.

BldD (SACE_2077), a key developmental regulator in actinomycetes, is the first identified transcriptional factor in Saccharopolyspora erythraea positively regulating erythromycin production and morphological differentiation. Although the BldD of S. erythraea binds to the promoters of erythromycin biosynthetic genes, the interaction affinities are relatively low, implying the existence of its other target genes in S. erythraea. Through the genomic systematic evolution of ligands by exponential enrichment (SELEX) method that we herein improved, four DNA sequences of S. erythraea A226, corresponding to the promoter regions of SACE_0306 (beta-galactosidase), SACE_0811 (50S ribosomal protein L25), SACE_3410 (fumarylacetoacetate hydrolase), and SACE_6014 (aldehyde dehydrogenase), were captured with all three BldD concentrations of 0.5, 1, and 2 µM, while the previously identified intergenic regions of eryBIV-eryAI and ermE-eryCI plus the promoter region of SACE_7115, the amfC homolog for aerial mycelium formation, could be captured only when the BldD's concentration reached 2 µM. Electrophoretic mobility shift assay (EMSA) analysis indicated that BldD specifically bound to above seven DNA sequences, and quantitative real-time PCR (qRT-PCR) assay showed that the transcriptional levels of the abovementioned target genes decreased when bldD was disrupted in A226. Furthermore, SACE_7115 and SACE_0306 in A226 were individually inactivated, showing that SACE_7115 was predominantly involved in aerial mycelium formation, while SACE_0306 mainly controlled erythromycin production. This study provides valuable information for better understanding of the pleiotropic regulator BldD in S. erythraea, and the improved method may be useful for uncovering regulatory networks of other transcriptional factors.

Transcriptome Analysis of Glycerin Regulating Reuterin Production of Lactobacillus reuteri.

Reuterin can be produced from glycerol dehydration catalyzed by glycerol dehydratase (GDHt) in Lactobacillus reuteri and has broad application prospects in industry, agriculture, food, and other fields as it is active against prokaryotic and eukaryotic organisms and is resistant to proteases and lipases. However, high concentrations of glycerin inhibit reuterin production, and the mechanism behind this phenomenon is not clear. To elucidate the inhibitory mechanism of glycerol on reuterin synthesis in L. reuteri and provide reference data for constructing an L. reuteri culture system for highly effective 3-hydroxypropionaldehyde synthesis, we used transcriptome-sequencing technology to compare the morphologies and transcriptomes of L. reuteri cultured in a medium with or without 600 mM of glycerol. Our results showed that after the addition of 600 mM of glycerol to the culture medium and incubation for 10 h at 37 °C, the culture medium of L. reuteri LR301 exhibited the best bacteriostatic effect, and the morphology of L. reuteri cells had significantly changed. The addition of 600 mM of glycerol to the culture medium significantly altered the transcriptome and significantly downregulated the transcription of genes involved in glycol metabolism, such as gldA, dhaT, glpK, plsX, and plsY, but significantly upregulated the transcription of genes related to D-glucose synthesis.

Standard pancreatoduodenectomy versus extended pancreatoduodenectomy with modified retroperitoneal nerve resection in patients with pancreatic head cancer: a multicenter randomized controlled trial.

BACKGROUND: The extent of pancreatoduodenectomy for pancreatic head cancer remains controversial, and more high-level clinical evidence is needed. This study aimed to evaluate the outcome of extended pancreatoduodenectomy (EPD) with retroperitoneal nerve resection in pancreatic head cancer. METHODS: This multicenter randomized trial was performed at 6 Chinese high-volume hospitals that enrolled patients between October 3, 2012, and September 21, 2017. Four hundred patients with stage I or II pancreatic head cancer and without specific pancreatic cancer treatments (preoperative chemotherapy or chemoradiation) within three months were randomly assigned to undergo standard pancreatoduodenectomy (SPD) or EPD, with the latter followed by dissection of additional lymph nodes (LNs), nerves and soft tissues 270° on the right side surrounding the superior mesenteric artery and celiac axis. The primary endpoint was overall survival (OS) by intention-to-treat (ITT). The secondary endpoints were disease-free survival (DFS), mortality, morbidity, and postoperative pain intensity. RESULTS: The R1 rate was slightly lower with EPD (8.46%) than with SPD (12.56%). The morbidity and mortality rates were similar between the two groups. The median OS was similar in the EPD and SPD groups by ITT in the whole study cohort (23.0 vs. 20.2 months, P = 0.100), while the median DFS was superior in the EPD group (16.1 vs. 13.2 months, P = 0.031). Patients with preoperative CA19-9 < 200.0 U/mL had significantly improved OS and DFS with EPD (EPD vs. SPD, 30.8 vs. 20.9 months, P = 0.009; 23.4 vs. 13.5 months, P < 0.001). The EPD group exhibited significantly lower locoregional (16.48% vs. 35.20%, P < 0.001) and mesenteric LN recurrence rates (3.98% vs. 10.06%, P = 0.022). The EPD group exhibited less back pain 6 months postoperation than the SPD group. CONCLUSIONS: EPD for pancreatic head cancer did not significantly improve OS, but patients with EPD treatment had significantly improved DFS. In the subgroup analysis, improvements in both OS and DFS in the EPD arm were observed in patients with preoperative CA19-9 < 200.0 U/mL. EPD could be used as an effective surgical procedure for patients with pancreatic head cancer, especially those with preoperative CA19-9 < 200.0 U/mL.

Genomewide comparative analysis of codon usage bias in three sequenced Jatropha curcas.

Jatropha curcas has recently emerged as an important bioenergy plant which is an ideal alternative for fossil fuels. It is particularly significant to analyse the codon usage bias (CUB) and further evaluate the intraspecific genetic divergence of three J. curcas in Asia, considering its potential economic benefits and various utilities. In the present study, the patterns of CUB were systematically compared, and the factors shaping CUB were identified in all three genomes of J. curcas. Our observations indicate that the preference for A/T nucleotides and A/T ending codons was present in all the three genomes. Moreover, 11 identical high-frequency codons as well as the optimal expression receptor Nicotiana tabacum were confirmed. Besides, it was observed that CUB resulted from the combined effects of natural selection and mutation pressure, while the natural selection was the determining factor. Eventually, similarity indices based on relative synonymous codon usage (RSCU) values implied low intraspecific genetic divergence in three Asian J. curcas. This study provides useful clues for improving the expression level of exogenous genes and optimizing breeding programmes by molecular-assisted breeding in J. curcas.

Genome-wide characterization of bZIP transcription factors and their expression patterns in response to drought and salinity stress in Jatropha curcas.

The basic leucine zipper (bZIP) family is one of the largest families of transcription factors (TFs) in plants and is responsible for various functions, including regulating development and responses to abiotic/biotic stresses. However, the roles of bZIPs in the regulation of responses to drought stress and salinity stress remain poorly understood in Jatropha curcas L., a biodiesel crop. In the present study, 50 JcbZIP genes were identified and classified into ten groups. Cis-element analysis indicated that JcbZIP genes are associated with abiotic stress. Gene expression patterns and quantitative real-time PCR (qRT-PCR) showed that four JcbZIP genes (JcbZIPs 34, 36, 49 and 50) are key resistance-related genes under both drought and salinity stress conditions. On the basis of the results of cis-element and phylogenetic analyses, JcbZIP49 and JcbZIP50 are likely involved in responses to drought and salinity stress; moreover, JcbZIP34 and JcbZIP36 might also play important roles in seed development and response to abiotic stress. These findings advance our understanding of the comprehensive characteristics of JcbZIP genes and provide new insights for functional validation in the further.

Comparative Genomics Uncovers the Genetic Diversity and Synthetic Biology of Secondary Metabolite Production of Trametes.

The carbohydrate-active enzyme (CAZyme) genes of Trametes contribute to polysaccharide degradation. However, the comprehensive analysis of the composition of CAZymes and the biosynthetic gene clusters (BGCs) of Trametes remain unclear. Here, we conducted comparative analysis, detected the CAZyme genes, and predicted the BGCs for nine Trametes strains. Among the 82,053 homologous clusters obtained for Trametes, we identified 8518 core genes, 60,441 accessory genes, and 13,094 specific genes. A large proportion of CAZyme genes were cataloged into glycoside hydrolases, glycosyltransferases, and carbohydrate esterases. The predicted BGCs of Trametes were divided into six strategies, and the nine Trametes strains harbored 47.78 BGCs on average. Our study revealed that Trametes exhibits an open pan-genome structure. These findings provide insights into the genetic diversity and explored the synthetic biology of secondary metabolite production for Trametes.

Comparative analysis of codon usage patterns in chloroplast genomes of six Euphorbiaceae species.

Euphorbiaceae plants are important as suppliers of biodiesel. In the current study, the codon usage patterns and sources of variance in chloroplast genome sequences of six different Euphorbiaceae plant species have been systematically analyzed. Our results revealed that the chloroplast genomes of six Euphorbiaceae plant species were biased towards A/T bases and A/T-ending codons, followed by detection of 17 identical high-frequency codons including GCT, TGT, GAT, GAA, TTT, GGA, CAT, AAA, TTA, AAT, CCT, CAA, AGA, TCT, ACT, TAT and TAA. It was found that mutation pressure was a minor factor affecting the variation of codon usage, however, natural selection played a significant role. Comparative analysis of codon usage frequencies of six Euphorbiaceae plant species with four model organisms reflected that Arabidopsis thaliana, Populus trichocarpa, and Saccharomyces cerevisiae should be considered as suitable exogenous expression receptor systems for chloroplast genes of six Euphorbiaceae plant species. Furthermore, it is optimal to choose Saccharomyces cerevisiae as the exogenous expression receptor. The outcome of the present study might provide important reference information for further understanding the codon usage patterns of chloroplast genomes in other plant species.

[Characteristic comparison of mouse primary macrophages cultured in L929 cell conditioned medium].

The purpose of this study is to provide a culture for mouse bone marrow-derived macrophages (BMDM) and peritoneal macrophages (PM) and to characterize their molecular and cellular biology. The cell number and purity from the primary culture were assessed by cell counter and flow cytometry, respectively. Morphological features were evaluated by inverted microscope. Phagocytosis by macrophages was detected by the neutral red dye uptake assay. Phenotypic markers were analyzed by real-time fluorescent quantitative PCR. Our results show that the cell number was much higher from culture of BMDM than PM, while there was no significant difference regarding the percentage of F4/80+CD11b+ cells (98.30%±0.53% vs. 94.83%±1.42%; P>0.05). The proliferation rate of BMDM was significantly higher than PM in the presence of L929 cell conditioned medium, by using CCK-8 assay. However, PM appeared to adhere to the flask wall and extend earlier than BMDM. The phagocytosis capability of un-stimulated BMDM was significantly higher than PM, as well as lipopolysaccharide (LPS)-stimulated BMDM, except the BMDM stimulated by low dose LPS (0.1 µg/mL). Furthermore, Tnfα expression was significantly higher in un-stimulated BMDM than PM, while Arg1 and Ym1 mRNA expression were significantly lower than PM. The expression difference was persistent if stimulated by LPS+IFN-γ or IL-4. Our data indicate that bone marrow can get larger amounts of macrophages than peritoneal cavity. However, it should be aware that the molecular and cellular characteristics were different between these two culture systems.

Pterostilbene Attenuates Experimental Atherosclerosis through Restoring Catalase-Mediated Redox Balance in Vascular Smooth Muscle Cells.

Atherosclerosis, the major risk of cardiovascular events, is a chronic vascular inflammatory disease. Pterostilbene is a naturally occurring dimethylated analogue of resveratrol and has recently been demonstrated to be beneficial against cardiovascular diseases. However, the underlying mechanisms of pterostilbene on atherosclerosis remain elusive. Experimental atherosclerosis was induced by a high-fat diet (HFD) in apolipoprotein E knockout (ApoE-/-) mice. Pterostilbene was administered intragastrically for 16 weeks. We found that pterostilbene significantly attenuated thoracic and abdominal atherosclerotic plaque formation in HFD-fed ApoE-/-mice, accompanied by modulated lipid profiles and reduced production of proinflammatory cytokines (including IL-6, IFN-γ, and TNF-α). In addition, pterostilbene restored vascular redox balance in thoracic and abdominal aorta, evidenced by enhanced catalase (CAT) expression and activities, and decreased malondialdehyde and H2O2 production. Notably, pterostilbene specifically induced CAT expression and activities in the vascular smooth muscle cells (VSMCs) of thoracic and abdominal aorta. In vitro, pterostilbene markedly promoted the expression and activity of CAT and decreased ox-low-density lipoprotein (LDL)-mediated VSMC proliferation and intracellular H2O2 production, which was abolished by CAT siRNA knockdown or inhibition. Pterostilbene-induced CAT expression was associated with inhibition of Akt, PRAS40, and GSK-3ß signaling activation and upregulation of PTEN. Our data clearly demonstrated that pterostilbene exerted an antiatherosclerotic effect by inducing CAT and modulating the VSMC function.

Corrigendum: (-)-Epigallocatechin-3-Gallate Ameliorates Atherosclerosis and Modulates Hepatic Lipid Metabolic Gene Expression in Apolipoprotein E Knockout Mice: Involvement of TTC39B.

[This corrects the article on p. 195 in vol. 9, PMID: 29593532.].

(-)-Epigallocatechin-3-Gallate Ameliorates Atherosclerosis and Modulates Hepatic Lipid Metabolic Gene Expression in Apolipoprotein E Knockout Mice: Involvement of TTC39B.

Background: Aberrant chronic inflammation and excess accumulation of lipids play a pivotal role in the occurrence and progression of atherosclerosis. (-)-Epigallocatechin-3-gallate (EGCG), the major catechins in green tea, displayed anti-atherosclerotic properties in vivo and in vitro. However, the effects and underlying mechanism of EGCG on atherosclerosis remain unclear. Methods: Male apolipoprotein E-knockout (ApoE-/-) mice (7 weeks old) fed with high-fat diet (HFD) were treated with normal saline or EGCG (40 mg/kg/d, i.g.) for 18 weeks. Atherosclerotic plaque and liver lipid accumulation were measured by Oil Red staining. Plasma lipids and cytokines were detected using commercial kits. The expression of protein and mRNA was analyzed by western blot and quantitative real-time reverse transcription-polymerase chain reaction, respectively. Results: EGCG administration markedly attenuated atherosclerotic plaque formation in HFD-fed ApoE-/- mice, which were accompanied by increased plasma interleukin-10 (IL-10) level and decreased plasma IL-6 and tumor necrosis factor-α (TNF-α) levels. In addition, EGCG modulated high-fat-induced dyslipidemia, evidencing by decreased total cholesterol (TC) and low-density lipoprotein levels and increased high-density lipoprotein level. Meanwhile, EGCG treatment alleviated high-fat-mediated liver lipid accumulation and decreased liver TC and triglyceride. Mechanistically, EGCG significantly modulated high-fat-induced hepatic tetratricopeptide repeat domain protein 39B (TTC39B) expression and its related genes (Lxrß, Abcg5, Abcg8, Abca1, Srebf1, Scd1, Scd2, Fas, Elovl5, Mylip) expression in liver from ApoE-/- mice. Notably, EGCG remarkably induced hepatic liver X receptor α (LXRα) and LXRß expression and inhibited both precursor and mature sterol regulatory element binding transcription factor-1 (SREBP-1) expression. Conclusion: Taken together, our data for the first time suggested that TTC39B was involved in EGCG-mediated anti-atherosclerotic effects through modulation of LXR/SREBP-1 pathway.

Crystallization and preliminary X-ray characterization of the dephospho-CoA kinase from Legionella pneumophila.

Dephospho-CoA kinases (DPCKs) are members of the kinase family that catalyze the final step in CoA biosynthesis. Their function is phosphorylation of the 3-hydroxyl group of the ribose using ATP as a phosphate donor. Structural changes induced by ATP binding play an important role during the DPCK catalytic cycle. In this work, DPCK from Legionella pneumophila was overexpressed in Escherichia coli. The purification, crystallization and preliminary X-ray analysis of crystals of this protein are described. The protein was crystallized in space group P21212, with unit-cell parameters a = 36.29, b = 82.20, c = 81.80 Å, using the hanging-drop vapour-diffusion method. Diffraction data were collected at 100 K and the phases were determined using the molecular-replacement method.

[Remifentanil preconditioning lowers cardiac troponin I levels in patients undergoing off-pump coronary artery bypass graft surgery].

OBJECTIVE: To investigate the value of cardiac troponin I (cTnI) levels in assessing myocardial protection by remifentanil precondition against myocardial injury induced by off-pump coronary artery bypass (OPCAB). METHODS: Twenty-four patients undergoing OPCAB were randomized into control and remifentanil preconditioning group (n=12). All the patients received pretreatment with oral diazepam (10 mg), intramuscular morphine (10 mg) and hyosine (0.3 mg). General anesthesia was induced with midazolam (0.08 mg/kg), etomidate (0.1-0.3 mg/kg), fentanyl (5-10 microg/kg), and rocuronium (1 mg/kg), and maintained with isoflurane inhalation and propofol infusion. Intermittent fentanyl and pipecuronium were given intravenously. In remifentanil preconditioning group, remifentanil (5 microg/kg in 50 ml normal saline) was infused in 10 min after anesthesia induction, and only NS was administered in the control group. Blood samples were obtained before and at 0, 2, 6, 24, and 48 h after the operation to determine serum cTnI levels. RESULTS: In both of the two groups, the cTnI levels increased significantly at the postoperative time points (0, 2, 6, 24, and 48 h) as compared with those before the operation (P<0.05). The cTnI levels of remifentanil preconditioning group were markedly decreased after the operation in comparison with those of the control group (P<0.05). CONCLUSION: Remifentanil preconditioning decreases the cTnI levels and reduces myocardial injury induced by OPCAB.

[Effect of levosimendan on B-type natriuretic peptide levels in patients with advanced heart failure: a meta-analysis].

OBJECTIVE: To conduct a meta-analysis of the effect of levosimendan on B-type natriuretic peptide (BNP) levels and evaluate the therapeutic effect of levosimendan on advanced heart failure. METHODS: A meta-analysis was performed on the selected data to analyze the effect of levosimendan on BNP levels. RESULTS: Levosimendan decreased BNP by a mean of 337.66 [95%CI (-296.30, -379.02)] pg/ml 24 h after the administration, and by 259.92 [95%CI (-195.76, -324.08)] pg/ml at 48 h, and by 123.09 [95%CI(-53.32,-195.86)] pg/ml at 1 week. Levosimendan resulted in improvements of the cardiac function by about 29%, 22%, and 10% at 24 h, 48 h and 1 week after the administration. CONCLUSION: Levosimendan produces favorable effects on the cardiac functions and BNP levels.