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Abstract Background Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiomyopathy characterized with progressive cardiac fibrosis and heart failure. However, the exact mechanism driving the progression of cardiac fibrosis and heart failure in ACM remains elusive. This study aims to investigate the underlying mechanisms of progressive cardiac fibrosis in ACM caused by newly identified Desmoglein-2 (DSG2) variation. Methods We identified homozygous DSG2 F531C variant in a family with 8 ACM patients using whole-exome sequencing and generated Dsg2 F536C knock-in mice. Neonatal and adult mouse ventricular myocytes isolated from Dsg2 F536C knock-in mice were used. We performed functional, transcriptomic and mass spectrometry analyses to evaluate the mechanisms of ACM caused by DSG2 F531C variant. Results All eight patients with ACM were homozygous for DSG2 F531C variant. Dsg2 F536C/F536C mice displayed cardiac enlargement, dysfunction, and progressive cardiac fibrosis in both ventricles. Mechanistic investigations revealed that the variant DSG2-F536C protein underwent misfolding, leading to its recognition by BiP within the endoplasmic reticulum, which triggered endoplasmic reticulum stress, activated the PERK-ATF4 signaling pathway and increased ATF4 levels in cardiomyocytes. Increased ATF4 facilitated the expression of TGF-β1 in cardiomyocytes, thereby activating cardiac fibroblasts through paracrine signaling and ultimately promoting cardiac fibrosis in Dsg2 F536C/F536C mice. Notably, inhibition of the PERK-ATF4 signaling attenuated progressive cardiac fibrosis and cardiac systolic dysfunction in Dsg2 F536C/F536C mice. Conclusions Hyperactivation of the ATF4/TGF-β1 signaling in cardiomyocytes emerges as a novel mechanism underlying progressive cardiac fibrosis in ACM. Targeting the ATF4/TGF-β1 signaling may be a novel therapeutic target for managing ACM.

Abstract Background We have previously discovered clusters of sequentially negative and positive modulators of acute inflammation during cytokine stimulation in epithelial cells and identified potential targets for therapy within these clusters. MAP3K8 is a druggable kinase that we found to be a hub of a principal interaction network. We describe here the results of MAP3K8 knockdown in the A549 lung cancer cell line, the BEAS-2B epithelial cell line and normal human bronchial epithelial (NHBE) cells following IL-1β stimulation. We analysed signalling transduction and global gene expression after IL-1β stimulation with and without MAP3K8 knockdown, quantifying levels of the inflammatory cytokines IL-6, IL-8 and RANTES levels by qPCRs and/or by ELISAs. We also examined potential small molecule inhibitors for MAP3K8 in the same models. Results IL-1β significantly and consistently increased MAP3K8 expression after 2 h in A549, BEAS-2B and NHBE cells. Phosphorylation of MAP3K8 occurred at 20 min after IL-1β stimulation and MAP3K8 protein was degraded at 30 min. MAP3K8 knockdown significantly reduced IL-6, IL-8 levels after IL-1β stimulation and yielded a 10-fold enhancement of the anti-inflammatory effects of dexamethasone. Phosphorylation of ERK1/2 (P-ERK1/2) and phosphorylation of SAPK/JNK (P-SAPK/JNK) decreased at 30 min after IL-1β stimulation with MAP3K8 knockdown. The combination of dexamethasone and MAP3K8 knockdown resulted in greater inhibition of phosphorylated ERK1/2 and SAPK/JNK. Nineteen genes including MMP1, MMP3, MMP10, ITGB8, LAMC2 and PLAT (P corrected

Abstract Background Biological nitrogen fixation converting atmospheric dinitrogen to ammonia is an important way to provide nitrogen for plants. Pseudomonas stutzeri DSM4166 is a diazotrophic Gram-negative bacterium isolated from the rhizosphere of cereal Sorghum nutans. Endogenous constitutive promoters are important for engineering of the nitrogen fixation pathway, however, they have not been systematically characterized in DSM4166. Results Twenty-six candidate promoters were identified from DSM4166 by RNA-seq analysis. These 26 promoters were cloned and characterized using the firefly luciferase gene. The strengths of nineteen promoters varied from 100 to 959% of the strength of the gentamicin resistance gene promoter. The strongest P12445 promoter was used to overexpress the biological nitrogen fixation pathway-specific positive regulator gene nifA. The transcription level of nitrogen fixation genes in DSM4166 were significantly increased and the nitrogenase activity was enhanced by 4.1 folds determined by the acetylene reduction method. The nifA overexpressed strain produced 359.1 µM of extracellular ammonium which was 25.6 times higher than that produced by the wild-type strain. Conclusions The endogenous strong constitutive promoters identified in this study will facilitate development of DSM4166 as a microbial cell factory for nitrogen fixation and production of other useful compounds.

Abstract Background Epithelial barrier is important for asthma development by shaping immune responses. Airway expressing-IL-1 receptor-associated kinase (IRAK)-M of Toll-like receptor pathway was involved in immunoregulation of airway inflammation through influencing activities of macrophages and dendritic cells or T cell differentiation. Whether IRAK-M has effect on cellular immunity in airway epithelial cells upon stimulation remains unclear. Methods We modeled cellular inflammation induced by IL-1β, TNF-α, IL-33, and house dust mite (HDM) in BEAS-2B and A549 cells. Cytokine production and pathway activation were used to reflect the effects of IRAK-M siRNA knockdown on epithelial immunity. Genotyping an asthma-susceptible IRAK-M SNP rs1624395 and measurement of serum CXCL10 levels were performed in asthma patients. Results IRAK-M expression was significantly induced in BEAS-2B and A549 cells after inflammatory stimulation. IRAK-M knockdown increased the lung epithelial production of cytokines and chemokines, including IL-6, IL-8, CXCL10, and CXCL11, at both mRNA and protein levels. Upon stimulation, IRAK-M silencing led to overactivation of JNK and p38 MAPK in lung epithelial cells. While antagonizing JNK or p38 MAPK inhibited increased secretion of CXCL10 in IRAK-M silenced-lung epithelium. Asthma patients carrying G/G genotypes had significantly higher levels of serum CXCL10 than those carrying homozygote A/A. Conclusion Our findings suggested that IRAK-M has effect on lung epithelial inflammation with an influence on epithelial secretion of CXCL10 partly mediated through JNK and p38 MAPK pathways. IRAK-M modulation might indicate a new insight into asthma pathogenesis from disease origin.

Abstract Background Spinosad is a macrolide insecticide with the tetracyclic lactone backbone to which forosamine and tri-o-methylrhamnose are attached. Both the sugar moieties are essential for its insecticidal activity. In biosynthesis of spinosad, the amino group of forosamine is dimethylated by SpnS and then transferred onto the lactone backbone by SpnP. Because the spinosad native producer is difficult to genetically manipulate, we previously changed promoters, ribosome binding sites and start codons of 23 spinosad biosynthetic genes to construct an artificial gene cluster which resulted in a 328-fold yield improvement in the heterologous host Streptomyces albus J1074 compared with the native gene cluster. However, in fermentation of J1074 with the artificial gene cluster, the N-monodesmethyl spinosad with lower insecticidal activity was always produced with the same titer as spinosad. Results By tuning expression of SpnS with an inducible promotor, we found that the undesired less active byproduct N-monodesmethyl spinosad was produced when SpnS was expressed at low level. Although N-monodesmethyl spinosad can be almost fully eliminated with high SpnS expression level, the titer of desired product spinosad was only increased by less than 38%. When the forosaminyl transferase SpnP was further overexpressed together with SpnS, the titer of spinosad was improved by 5.3 folds and the content of N-desmethyl derivatives was decreased by ~ 90%. Conclusion N-monodesmethyl spinosad was produced due to unbalanced expression of spnS and upstream biosynthetic genes in the refactored artificial gene cluster. The accumulated N-desmethyl forosamine was transferred onto the lactone backbone by SpnP. This study suggested that balanced expression of biosynthetic genes should be considered in the refactoring strategy to avoid accumulation of undesired intermediates or analogues which may affect optimal production of desired compounds.

Abstract Recent years, Burkholderia species have emerged as a new source of natural products (NPs) with increasing attractions. Genome mining suggests the Burkholderia genomes include many natural product biosynthetic gene clusters (BGCs) which are new targets for drug discovery. In order to collect more Burkholderia, here, a strain S-53 was isolated from the soil samples on a mountain area in Changde, P.R. China and verified by comparative genetic analysis to belong to Burkholderia. The complete genome of Burkholderia strain S-53 is 8.2 Mbps in size with an average G + C content of 66.35%. Its taxonomy was both characterized by 16S rRNA- and whole genome-based phylogenetic trees. Bioinformatic prediction in silico revealed it has a total of 15 NP BGCs, some of which may encode unknown products. It is expectable that availability of these BGCs will speed up the identification of new secondary metabolites from Burkholderia and help us understand how sophisticated BGC regulation works.

Abstract Backgroud Myocardial infarction (MI) is one of the leading causes of global death. Dendritic cell-derived exosomes (DEXs) provide us with the possibility of improving cardiac function after MI but are limited by low retention times and short-lived therapeutic effects. In this study, we developed a novel drug delivery system incorporating alginate hydrogel that continuously releases DEXs and investigated the mechanisms underlying the action of DEXs in the improvement of cardiac function after MI. Results We incorporated DEXs with alginate hydrogel (DEXs-Gel) and investigated controlled released ability and rheology, and found that DEXs-Gel release DEXs in a sustainable mammer and prolonged the retention time of DEXs but had no detrimental effects on the migration in vivo. Then DEXs-Gel was applicated in the MI model mice, we found that DEXs-Gel siginificantly enhanced the therapeutic effects of DEXs with regards to improving cardiac function after MI. Flow cytometry and immunofluorescence staining revealed that DEXs significantly upregulated the infiltration of Treg cells and M2 macrophages into the border zoom after MI, and DEXs activated regulatory T (Treg) cells and shifted macrophages to reparative M2 macrophages, both in vitro and in vivo. Conclusion Our novel delivery method provides an innovative tool for enhancing the therapeutic effects of DEXs after MI. Further analysis revealed that DEXs exert effect by activating Treg cells and by modifying the polarization of macrophages. Graphic Abstract

Abstract Background Oroscomucoid 3 (ORMDL3) has been linked to susceptibility of childhood asthma and respiratory viral infection. Polyinosinic-polycytidylic acid (poly I:C) is a synthetic analog of viral double-stranded RNA, a toll-like receptor 3 (TLR3) ligand and mimic of viral infection. Methods To investigate the functional role of ORMDL3 in the poly I:C-induced inflammatory response in airway epithelial cells, ORMDL3 knockdown and over-expression models were established in human A549 epithelial cells and primary normal human bronchial epithelial (NHBE) cells. The cells were stimulated with poly I:C or the Th17 cytokine IL-17A. IL-6 and IL-8 levels in supernatants, mRNA levels of genes in the TLR3 pathway and inflammatory response from cell pellets were measured. ORMDL3 knockdown models in A549 and BEAS-2B epithelial cells were then infected with live human rhinovirus (HRV16) followed by IL-6 and IL-8 measurement. Results ORMDL3 knockdown and over-expression had little influence on the transcript levels of TLR3 in airway epithelial cells. Time course studies showed that ORMDL3-deficient A549 and NHBE cells had an attenuated IL-6 and IL-8 response to poly I:C stimulation. A549 and NHBE cells over-expressing ORMDL3 released relatively more IL-6 and IL-8 following poly I:C stimulation. IL-17A exhibited a similar inflammatory response in ORMDL3 knockdown and over-expressing cells, but co-stimulation of poly I:C and IL-17A did not significantly enhance the IL-6 and IL-8 response. Transcript abundance of IFNB following poly I:C stimulation was not significantly altered by ORMDL3 knockdown or over-expression. Dampening of the IL-6 response by ORMDL3 knockdown was confirmed in HRV16 infected BEAS-2B and A549 cells. Conclusions ORMDL3 regulates the viral inflammatory response in airway epithelial cells via mechanisms independent of the TLR3 pathway.

Abstract Background Polyketide synthases (PKSs) include ketone synthase (KS), acyltransferase (AT) and acyl carrier protein (ACP) domains to catalyse the elongation of polyketide chains. Some PKSs also contain ketoreductase (KR), dehydratase (DH) and enoylreductase (ER) domains as modification domains. Insertion, deletion or substitution of the catalytic domains may lead to the production of novel polyketide derivatives or to the accumulation of desired products. Epothilones are 16-membered macrolides that have been used as anticancer drugs. The substrate promiscuity of the module 4 AT domain of the epothilone PKS (EPOAT4) results in production of epothilone mixtures; substitution of this domain may change the ratios of epothilones. In addition, there are two dormant domains in module 9 of the epothilone PKS. Removing these redundant domains to generate a simpler and more efficient assembly line is a desirable goal. Results The substitution of module 4 drastically diminished the activity of epothilone PKS. However, with careful design of the KS-AT linker and the post-AT linker, replacing EPOAT4 with EPOAT2, EPOAT6, EPOAT7 or EPOAT8 (specifically incorporating methylmalonyl-CoA (MMCoA)) significantly increased the ratio of epothilone D (4) to epothilone C (3) (the highest ratio of 4:3 = 4.6:1), whereas the ratio of 4:3 in the parental strain Schlegelella brevitalea 104-1 was 1.4:1. We also obtained three strains by swapping EPOAT4 with EPOAT3, EPOAT5, or EPOAT9, which specifically incorporate malonyl-CoA (MCoA). These strains produced only epothilone C, and the yield was increased by a factor of 1.8 compared to that of parental strain 104-1. Furthermore, mutations of five residues in the AT domain identified Ser310 as the critical factor for MMCoA recognition in EPOAT4. Then, the mutation of His308 to valine or tyrosine combined with the mutation of Phe310 to serine further altered the product ratios. At the same time, we successfully deleted the inactive module 9 DH and ER domains and fused the ΨKR domain with the KR domain through an ~ 25-residue linker to generate a productive and simplified epothilone PKS. Conclusions These results suggested that the substitution and deletion of catalytic domains effectively produces desirable compounds and that selection of the linkers between domains is crucial for maintaining intact PKS catalytic activity.

Abstract Background Epothilone B is a natural product that stabilizes microtubules, similar to paclitaxel (Taxol); therefore, epothilone B and several derivatives have shown obvious antitumour activities. Some of these products are in clinical trials, and one (ixabepilone, BMS) is already on the market, having been approved by the FDA in 2007. The terminal step in epothilone B biosynthesis is catalysed by the cytochrome P450 enzyme EpoK (CYP167A1), which catalyses the epoxidation of the C12–C13 double bond (in epothilone C and D) to form epothilone A and B, respectively. Although redox partners from different sources support the catalytic activity of EpoK in vitro, the conversion rates are low, and these redox partners are not applied to produce epothilone B in heterologous hosts. Results Schlegelella brevitalea DSM 7029 contains electron transport partners that efficiently support the catalytic activity of EpoK. We screened and identified one ferredoxin, Fdx_0135, by overexpressing putative ferredoxin genes in vivo and identified two ferredoxin reductases, FdR_0130 and FdR_7100, by whole-cell biotransformation of epothilone C to effectively support the catalytic activity of EpoK. In addition, we obtained strain H7029-3, with a high epothilone B yield and found that the proportion of epothilone A + B produced by this strain was 90.93%. Moreover, the whole-cell bioconversion strain 7029-10 was obtained; this strain exhibited an epothilone C conversion rate of 100% in 12 h. Further RT-qPCR experiments were performed to analyse the overexpression levels of the target genes. Gene knock-out experiments showed that the selected ferredoxin (Fdx_0135) and its reductases (FdR_0130 and FdR_7100) might participate in critical physiological processes in DSM 7029. Conclusion Gene overexpression and whole-cell biotransformation were effective methods for identifying the electron transport partners of the P450 enzyme EpoK. In addition, we obtained an epothilone B high-yield strain and developed a robust whole-cell biotransformation system. This strain and system hold promise for the industrial production of epothilone B and its derivatives.

Abstract Background Glycosylated hemoglobin (HbA1c) has a detrimental impact on the myocardium with left ventricular (LV) diastolic dysfunction. Obesity is a risk factor of type 2 diabetes. To understand the relationships between HbA1c, body mass index (BMI) and LV diastolic dysfunction, we performed this interaction analysis in patients with type 2 diabetes. Methods Total 925 type 2 diabetes patients were selected from the patients who were diagnosed and treated at the First Affiliated Hospital of Shenzhen University. Patients’ BMI levels were defined as normal (BMI

Abstract Traditional cancer therapies, such as surgery treatment, radiotherapy, and chemotherapy, often fail to completely eliminate tumor cells in an anaerobic microenvironment of tumor regions. In contrast to these traditional cancer therapies, the use of targeted delivery vectors to deliver anticancer genes or antitumor drugs to hypoxic areas in tumors is the most clinically promising cancer treatment with rapid development in recent years. In this study, E.coli Nissle 1917 (EcN), an intestinal probiotic, was utilized as a targeted transport vector to deliver p53 and Tum-5 protein to tumor hypoxic regions. The tumor-targeting characteristics of EcN were investigated using luciferase LuxCDABE operon, and the results demonstrated that EcN could specifically accumulate in the solid tumor areas of SMMC-7721 tumor-bearing BALB/c nude mice. The Tum 5-p53 bifunctional proteins were initially constructed and then delivered to solid tumor regions by using the targeted transporter EcN for cancer therapy. The antitumor effect and safety of three engineered bacteria, namely, EcN (Tum-5), EcN (p53), and EcN (Tum 5-p53), were also examined. The calculated tumor volume and tumor weight indicated that these three engineered bacteria could inhibit the growth of human hepatoma SMMC-7721 cells, and the antitumor effect of EcN (Tum 5-p53) expressing the Tum 5-p53 fusion protein was significantly better than those of EcN (Tum-5) and EcN (p53) alone. Immunofluorescence demonstrated that the expression of Ki-67, a nuclear proliferation-related protein, was inhibited in the tumor areas of the groups treated with the engineered bacteria, whereas the expression of caspase-3 was upregulated. The expression trends of Ki-67 and caspase-3 were consistent with the different antitumor efficacies of these three engineered bacteria. EcN did not elicit obvious side effects on mice. This research not only provids a foundation for tumor-targeted therapy but also contributes greatly to the development of antitumor agents and anticancer proteins.

Abstract Background Injury may induce a sequential activation of intrinsic reparative activity that supports the maintenance of tissue homeostasis. Method In the present experiments, we investigated whether myocardial infarction (MI) was able to reinstate the expression of Wilms’ tumor factor 1 (WT1) as a key hallmark of fetal reprograming in the pericardial adipose-derived stem cells (pADSC). We characterized the immunophenotypical markers, cardiac potential, and reparative activity of WT1-expressing pADSC (WT1pos) isolated MI Wistar rats with an intact pericardial sac in which cardiac transudate was accumulated, sampled, and analyzed. Results The WT1pos cells formed colony-like aggregates in culture that subsequently generated phase-bright cells that homogenously constituted WT1 expression (> 98%). The WT1pos cells shared identical surface markers with canonical pADSC, but enhanced transcripts for cardiogenesis (isl-1, gata-4, Sox2 and Tbx18) as well as cardiac commitment (endothelial: 28%; cardiomyogenic: 12.3%) in defined conditions. Remarkably, cardiac transplantation of WT1pos cells promoted regional angiogenesis and myogenesis which led to significant functional amelioration of the infarcted hearts. Furthermore, we demonstrated that WT1pos cells uniquely secreted hepatocyte growth factor (HGF) as a key antiapoptotic factor that promotes cardiac repair. Conclusion Injury-associated fetal reprogramming in pADSC facilitates cardiac differentiation and promotes the reparative activity by enhancing HGF production. As such, injury-“conditioned” pADSC may represent a useful autologous cell donor from infarcted patients for cell-based therapy.

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.