Proteomic Analysis of Bacillus thuringiensis Strain 4.0718 at Different Growth Phases

The growth process of Bacillus thuringiensis Bt4.0718 strain was studied using proteomic technologies. The proteins of Bt whole cells at three phases—middle vegetative, early sporulation, and late sporulation—were extracted with lysis buffer, followed with separation by 2-DE and identified by MALDI-TOF/TOF MS. Bioactive factors such as insecticidal crystal proteins (ICPs) including Cry1Ac(3), Cry2Aa, and BTRX28, immune inhibitor (InhA), and InhA precursor were identified. InhA started to express at the middle vegetative phase, suggesting its contribution to the survival of Bt in the host body. At the early sporulation phase, ICPs started their expression. CotJC, OppA, ORF1, and SpoIVA related to the formation of crystals and spores were identified, the expression characteristics of which ensured the stable formation of crystals and spores. This study provides an important foundation for further exploration of the stable expression of ICPs, the smooth formation of crystals, and the construction of recombinant strains

Comparative Proteomic Analysis of saccharopolyspora spinosa SP06081 and PR2 strains reveals the differentially expressed proteins correlated with the increase of spinosad yield

<p>Abstract</p> <p>Background</p> <p><it>Saccharopolyspora spinosa </it>produces the environment-friendly biopesticide spinosad, a mixture of two polyketide-derived macrolide active ingredients called spinosyns A and D. Therefore considerable interest is in the improvement of spinosad production because of its low yield in wild-type <it>S. spinosa</it>. Recently, a spinosad-hyperproducing PR2 strain with stable heredity was obtained from protoplast regeneration of the wild-type <it>S. spinosa </it>SP06081 strain. A comparative proteomic analysis was performed on the two strains during the first rapid growth phase (RG1) in seed medium (SM) by using label-free quantitative proteomics to investigate the underlying mechanism leading to the enhancement of spinosad yield.</p> <p>Results</p> <p>In total, 224 proteins from the SP06081 strain and 204 proteins from the PR2 strain were unambiguously identified by liquid chromatography-tandem mass spectrometry analysis, sharing 140 proteins. A total of 12 proteins directly related to spinosad biosynthesis were identified from the two strains in RG1. Comparative analysis of the shared proteins revealed that approximately 31% of them changed their abundance significantly and fell in all of the functional groups, such as tricarboxylic acid cycles, glycolysis, biosynthetic processes, catabolic processes, transcription, translation, oxidation and reduction. Several key enzymes involved in the synthesis of primary metabolic intermediates used as precursors for spinosad production, energy supply, polyketide chain assembly, deoxysugar methylation, and antioxidative stress were differentially expressed in the same pattern of facilitating spinosad production by the PR2 strain. Real-time reverse transcriptase polymerase chain reaction analysis revealed that four of five selected genes showed a positive correlation between changes at the translational and transcriptional expression level, which further confirmed the proteomic analysis.</p> <p>Conclusions</p> <p>The present study is the first comprehensive and comparative proteome analysis of <it>S. spinosa </it>strains. Our results highlight the differentially expressed proteins between the two <it>S. spinosa </it>strains and provide some clues to understand the molecular and metabolic mechanisms that could lead to the increased spinosad production yield.</p

Evaluating the Insecticidal Genes and Their Expressed Products in Bacillus thuringiensis Strains by Combining PCR with Mass Spectrometry▿ †

By a combination of PCR and mass spectrometry, a total of five cry genes (cry1Aa, cry1Ac, cry2Aa, cry2Ab, and cry1Ia) were detected in genomic DNA from the wild-type Bacillus thuringiensis strain 4.0718, and three protoxins (Cry1Aa, Cry1Ac, and Cry2Aa) were identified in the strain's parasporal crystals. These results indicated that this complementary method may be useful in evaluating B. thuringiensis strains at both the gene and protein levels

Shared and divergent contribution of vitamin A and oxytocin to the aetiology of autism spectrum disorder

Rare genetic variations contribute to the heterogeneity of autism spectrum disorder (ASD) and the responses to various interventions for ASD probands. However, the associated molecular underpinnings remain unclear. Herein, we estimated the association between rare genetic variations in 410 vitamin A (VA)-related genes (VARGs) and ASD aetiology using publicly available de novo mutations (DNMs), rare inherited variants, and copy number variations (CNVs) from about 50,000 ASD probands and 20,000 normal controls (discovery and validation cohorts). Additionally, given the functional relevance of VA and oxytocin, we systematically compared the similarities and differences between VA and oxytocin with respect to ASD aetiology and evaluated their potential for clinical applications. Functional DNMs and pathogenic CNVs in VARGs contributed to ASD pathogenesis in the discovery and validation cohorts. Additionally, 324 potential VA-related biomarkers were identified, 243 of which were shared with previously identified oxytocin-related biomarkers, while 81 were unique VA biomarkers. Moreover, multivariable logistic regression analysis revealed that both VA- and oxytocin-related biomarkers were able to predict ASD aetiology for individuals carrying functional DNM in corresponding biomarkers with an average precision of 0.94. As well as, convergent and divergent functions were also identified between VA- and oxytocin-related biomarkers. The findings of this study provide a basis for future studies aimed at understanding the pathophysiological mechanisms underlying ASD while also defining a set of potential molecular biomarkers for adjuvant diagnosis and intervention in ASD

Achieving Good Protection on Ultra-High Molecular Weight Polythene by In Situ Growth of Amorphous Carbon Film

Ultra-high molecular weight polythene (UHMWPE), with outstanding characteristics, is widely applied in modern industry, while it is also severely limited by its inherent shortcomings, which include low hardness, poor wear resistance, and easy wear. Implementation of feasible protection on ultra-high molecular weight polythene to overcome its shortcomings would be of significance. In the present study, amorphous carbon (a-C) film was fabricated on ultra-high molecular weight polythene (UHMWPE) to provide good protection, and the relevant growth mechanism of a-C film was revealed by controlling carbon plasma currents. The results showed the in situ transition layer, in the form of chemical bonds, was formed between the UHMWPE substrate and the a-C film with the introduction of carbon plasma, which provided strong adhesion, and then the a-C film continued epitaxial growth on the in situ transition layer with the treatment of carbon plasma. This in situ growth of a-C film, including the in situ transition layer and the epitaxial growth layer, significantly improved the wetting properties, mechanical properties, and tribological properties of UHMWPE. In particular, good protection by in situ growth a-C film on UHMWPE was achieved during sliding wear

Isovitexin Suppresses Stemness of Lung Cancer Stem-Like Cells through Blockage of MnSOD/CaMKII/AMPK Signaling and Glycolysis Inhibition

Background. Manganese superoxide dismutase (MnSOD) has been reported to promote stemness of lung cancer stem-like cells (LCSLCs) which had higher glycolytic rates compared with non-CSLCs. Isovitexin exhibited an inhibitory effect on the stemness of hepatocellular carcinoma cells. However, whether isovitexin could inhibit the promotion of stemness of LCSLCs mediated by MnSOD through glycolysis remains unclear. Objective. Our study was aimed at investigating whether isovitexin inhibits lung cancer stem-like cells (LCSLCs) through MnSOD signaling blockage and glycolysis suppression. Methods. Sphere formation and soft agar assays were conducted to determine self-renewal ability. The migration and invasion of LCSLCs were determined by wound healing and transwell assay. The glycolytic activity was assessed by determination of L-lactate metabolism rate. The influences of isovitexin on MnSOD, CaMKII, and AMPK activations as well as the metabolic shift to glycolysis were determined by manipulating MnSOD expression. Results. It was found that MnSOD and glycolysis enhanced simultaneously in LCSLCs compared with parental H460 cells. Overexpression of MnSOD activated CaMKII/AMPK signaling and glycolysis in LCSLCs with increased self-renewal, migration, invasion, and expression of stemness-associated markers in vitro and elevated carcinogenicity in vivo. Knockdown of MnSOD induced an inverse effect in LCSLCs. Isovitexin blocked MnSOD/CaMKII/AMPK signaling axis and suppressed glycolysis in LCSLCs, resulting in inhibition of stemness features in LCSLCs. The knockdown of MnSOD significantly augmented isovitexin-associated inhibition of CaMKII/AMPK signaling, glycolysis, and stemness in LCSLCs. However, the overexpression of MnSOD could attenuate the inhibition of isovitexin on LCSLCs. Importantly, isovitexin notably suppressed tumor growth in nude mice bearing LCSLCs by downregulation of MnSOD expression. Conclusion. MnSOD promotion of stemness of LCSLCs derived from H460 cell line is involved in the activation of the CaMKII/AMPK pathway and induction of glycolysis. Isovitexin-associated inhibition of stemness in LCSLCs is partly dependent on blockage of the MnSOD/CaMKII/AMPK signaling axis and glycolysis suppression

A Proteomic Analysis Provides Novel Insights into the Stress Responses of Caenorhabditis elegans towards Nematicidal Cry6A Toxin from Bacillus thuringiensis

Abstract Cry6A represents a novel family of nematicidal crystal proteins from Bacillus thuringiensis. It has distinctive architecture as well as mechanism of action from Cry5B, a highly focused family of nematicidal crystal proteins, and even from other insecticidal crystal proteins containing the conserved three-domain. However, how nematode defends against Cry6A toxin remains obscure. In this study, the global defense pattern of Caenorhabditis elegans against Cry6Aa2 toxin was investigated by proteomic analysis. In response to Cry6Aa2, 12 proteins with significantly altered abundances were observed from worms, participating in innate immune defense, insulin-like receptor (ILR) signaling pathway, energy metabolism, and muscle assembly. The differentially expressed proteins (DEPs) functioning in diverse biological processes suggest that a variety of defense responses participate in the stress responses of C. elegans to Cry6Aa2. The functional verifications of DEPs suggest that ILR signaling pathway, DIM-1, galectin LEC-6 all are the factors of defense responses to Cry6Aa2. Moreover, Cry6Aa2 also involves in accelerating the metabolic energy production which fulfills the energy demand for the immune responses. In brief, our findings illustrate the global pattern of defense responses of nematode against Cry6A for the first time, and provide a novel insight into the mechanism through which worms respond to Cry6A

Heterologous expression and antitumor activity analysis of syringolin from Pseudomonas syringae pv. syringae B728a

Abstract Background Syringolin, synthesized by a mixed non-ribosomal peptide synthetase/polyketide synthetase in Pseudomonas syringae pv. syringae (Pss) B728a, is a novel eukaryotic proteasome inhibitor. Meanwhile, directly modifying large fragments in the PKS/NRPS gene cluster through traditional DNA engineering techniques is very difficult. In this study, we directly cloned the syl gene cluster from Pss B301D-R via Red/ET recombineering to effectively express syringolin in heterologous hosts. Results A 22 kb genomic fragment containing the sylA–sylE gene cluster was cloned into the pASK vector, and the obtained recombinant plasmid was transferred into Streptomyces coelicolor and Streptomyces lividans for the heterologous expression of syringolin. Transcriptional levels of recombinant syl gene in S. coelicolor M145 and S. lividans TK24 were evaluated via RT-PCR and the production of syringolin compounds was detected via LC–MS analysis. The extracts of the engineered bacteria showed cytotoxic activity to B16, 4T1, Meth-A, and HeLa tumor cells. It is noteworthy that the syringolin displayed anticancer activity against C57BL/6 mice with B16 murine melanoma tumor cells. Together, our results herein demonstrate the potential of syrinolin as effective antitumor agent that can treat various cancers without apparent adverse effects. Conclusions This present study is the first to report the heterologous expression of the entire syl gene cluster in Streptomyces strains and the successful expression of syringolin in both S. coelicolor M145 and S. lividans TK24. Syringolin derivatives demonstrated high cytotoxicity in vitro and in vivo. Hence, this paper provided an important foundation for the discovery and production of new antitumor compounds

Dietary SWF® enhanced growth performance and disease resistance in hybrid sturgeon (Acipenser baerii x Acipenser schrenckii) mediated by the gut microbiota

The presence of healthy gut microbiota in the gastrointestinal tract of fish is important for the optimal function of gut, which plays a significant role in the host growth and health. The aim of the present study was to investigate the effect of dietary stress worry free (SWF®) on growth, feed utilization and disease resistance of hybrid sturgeon (Acipenser baerii x Acipenser schrenckii). Sturgeon were fed for three weeks with SWF® supplemented or basal diet. The weight gain and FCR of sturgeon fed on the diet supplemented with SWF® were significantly improved (P<0.05). SWF® supplemented diet provoked an increase in the resistance of sturgeon against A. veronii Hm091 (P=0.09). In terms of gut microbiota, the number of total bacteria, Fusobacteria, Firmicutes, and Proteobacteria were increased significantly in the SWF® group (P<0.05), whereas significant reduction of Actinobacteria was observed in the gut of the SWF® group compared with the control group (P<0.01). Moreover, at the end of the experiment the gut microbiota of sturgeon, were colonized to germ-free (GF) zebrafish for three days. Results indicated that, the expression of growth promoter genes mTOR, MyoD and Myogenin was significantly higher in GF zebrafish colonized with gut microbiota of SWF® group of sturgeon. Furthermore, TGF-β was increased significantly in GF zebrafish colonized with gut microbiota from SWF® group (P<0.01), whereas the expression of TNF-α was significantly decreased (P<0.05). The expression of non-specific immune related genes DEFBL-1, C3a and Lysozyme was significantly increased in GF zebrafish colonized with gut microbiota of sturgeon fed on SWF® (P<0.05). Group of GF zebrafish colonized with gut microbiota of sturgeon fed on SWF® had significantly higher survival rate against A. veronii Hm091 (P<0.05). Our study suggests that, the gut microbiota induced by SWF® played a great role in growth and disease resistance of sturgeon using GF zebrafish model