A 14-amino acid cationic peptide Bolespleenin334-347 from the marine fish mudskipper Boleophthalmus pectinirostris exhibiting potent antimicrobial activity and therapeutic potential.

Antimicrobial peptides (AMPs) are an important component of innate immunity in both vertebrates and invertebrates, and some of the unique characteristics of AMPs are usually associated with their living environment. The marine fish, mudskipper Boleophthalmus pectinirostris, usually live amphibiously in intertidal environments that are quite different from other fish species, which would be an exceptional source of new AMPs. In the study, an AMP named Bolespleenin334-347 was identified, which was a truncated peptide derived from a new functional gene found in B. pectinirostris, that was up-regulated in response to bacterial challenge. Bolespleenin334-347 had only 14 amino acid residues, including five consecutive arginine residues. It was found that the peptide had broad-spectrum antibacterial activity, good thermal stability and sodium ion tolerance. Bolespleenin334-347 killed Acinetobacter baumannii and Staphylococcus aureus by disrupting the structural integrity of the bacterial membrane, leading to leakage of the cellular contents, and inducing accumulation of bacterial endogenous reactive oxygen species (ROS). In addition, Bolespleenin334-347 effectively inhibited biofilm formation of A. baumannii and S. aureus and long-term treatment did not lead to the development of resistance. Importantly, Bolespleenin334-347 maintained stable activity against clinically multi-drug resistant bacterial strains. In addition, it was noteworthy that Bolespleenin334-347 showed superior efficacy to LL-37 and vancomycin in a constructed mouse model of MRSA-induced superficial skin infections, as evidenced by a significant reduction in bacterial load and more favorable wound healing. This study provides an effective antimicrobial agent for topical skin infections with potential therapeutic efficacy for infections with drug-resistant bacteria, including MRSA.

A flexible 'plug and play' bicistronic construct and its application in the screening of protein expression system in Escherichia coli.

The bicistronic expression system that utilizes fluorescent reporters has been demonstrated to be a straightforward method for detecting recombinant protein expression levels, particularly when compared to polyacrylamide gel electrophoresis and immunoblot analysis, which are tedious and labor-intensive. However, existing bicistronic reporter systems are less capable of quantitative measurement due to the lag in reporter expression and its negative impact on target protein. In this work, a plug and play bicistronic construct using mCherry as reporter was applied in the screening of optimal replicon and promoter for Sortase expression in Escherichia coli (E. coli). The bicistronic construct allowed the reporter gene and target open reading frame (ORF) to be co-transcribed under the same promoter, resulting in a highly positive quantitative correlation between the expression titer of Sortase and the fluorescent intensity (R2 > 0.97). With the correlation model, the titer of target protein can be quantified by noninvasively measuring the fluorescent intensity. On top of this, the expression of reporter has no significant effect on the yield of target protein, thus favoring a plug and play design for removing reporter gene to generate a plain plasmid for industrial use.

SYNJ2BP Improves the Production of Lentiviral Envelope Protein by Facilitating the Formation of Mitochondrion-Associated Endoplasmic Reticulum Membrane.

Equine infectious anemia virus (EIAV) and HIV are both members of the Lentivirus genus and are similar in major virological characters. EIAV endangers the horse industry. In addition, EIAV can also be used as a model for HIV research. The maturation of the lentiviral Env protein, which is necessary for viral entry, requires Env to be folded in the endoplasmic reticulum (ER). It is currently unclear how this process is regulated. Mitochondrion-associated endoplasmic reticulum membrane (MAM) is a specialized part of the close connection between the ER and mitochondria, and one of the main functions of MAM is to promote oxidative protein production in the ER. SYNJ2BP is one of the key proteins that make up the MAM, and we found that SYNJ2BP is essential for EIAV replication. We therefore constructed a SYNJ2BP knockout HEK293T cell line in which the number of MAMs is significantly reduced. Moreover, overexpression of SYNJ2BP could increase the number of MAMs. Our study demonstrates that SYNJ2BP can improve the infectivity of the EIAV virus with elevated production of the viral Env protein through increased MAM formation. Interestingly, SYNJ2BP was able to improve the production of not only EIAV Env but also HIV. Further investigation showed that MAMs can provide more ATP and calcium ions, which are essential factors for Env production, to the ER and can also reduce ER stress induced by HIV or EIAV Envs to increase the Env production level in cells. These results may help us to understand the key production mechanisms of lentiviral Env. IMPORTANCE Lentiviral Env proteins, which are rich in disulfide bonds, need to be fully folded in the ER; otherwise, misfolded Env proteins will induce ER stress and be degraded by ER-associated protein degradation (ERAD). To date, it is still unclear about Env production mechanism in the ER. MAM is the structure of closely connection between the ER and mitochondria. MAMs play important roles in the calcium steady state and oxidative stress, especially in the production of oxidative protein. For the first time, we found that SYNJ2BP can promote the production of lentiviral Env proteins by providing the ATP and calcium ions required for oxidative protein production in the ER and by reducing ER stress through facilitating formation of MAMs. These studies shed light on how MAMs improve lentiviral Env production, which will lay the foundation for the study of replication mechanisms in other lentiviruses from the perspective of the cellular organelle microenvironment.

Attenuation of Equine Lentivirus Alters Mitochondrial Protein Expression Profile from Inflammation to Apoptosis.

Equine infectious anemia virus (EIAV) is an equine lentivirus similar to HIV-1, targets host immune cells, and causes a life-long infection in horses. The Chinese live EIAV vaccine is attenuated from long-term passaging of a highly virulent strain in vitro The parent pathogenic strain (EIAVDLV34) induces a host inflammatory storm to cause severe pathological injury of animals. However, the vaccine strain (EIAVDLV121) induces a high level of apoptosis to eliminate infected cells. To investigate how these processes are regulated, we performed a comparative proteomics analysis and functional study in equine monocyte-derived macrophages (eMDMs) and found that the divergent mitochondrial protein expression profiles caused by EIAV strains with different virulence led to disparate mitochondrial function, morphology, and metabolism. This in turn promoted the distinct transformation of macrophage inflammatory polarization and intrinsic apoptosis. In EIAVDLV34-infected cells, a high level of glycolysis and increased mitochondrial fragmentation were induced, resulting in the M1-polarized proinflammatory-type transformation of macrophages and the subsequent production of a strong inflammatory response. Following infection with EIAVDLV121, the infected cells were transformed into M2-polarized anti-inflammatory macrophages by inhibition of glycolysis. In this case, a decrease in the mitochondrial membrane potential and impairment of the electron transport chain led to increased levels of apoptosis and reactive oxygen species. These results correlated with viral pathogenicity loss and may help provide an understanding of the key mechanism of lentiviral attenuation.IMPORTANCE Following viral infection, the working pattern and function of the cell can be transformed through the impact on mitochondria. It still unknown how the mitochondrial response changes in cells infected with viruses in the process of virulence attenuation. EIAVDLV121 is the only effective lentiviral vaccine for large-scale use in the world. EIAVDLV34 is the parent pathogenic strain. Unlike EIAVDLV34-induced inflammation storms, EIAVDLV121 can induce high levels of apoptosis. For the first time, we found that, after the mitochondrial protein expression profile is altered, EIAVDLV34-infected cells are transformed into M1-polarized-type macrophages and cause inflammatory injury and that the intrinsic apoptosis pathway is activated in EIAVDLV121-infected cells. These studies shed light on how the mitochondrial protein expression profile changes between cells infected by pathogenic lentivirus strains and cells infected by attenuated lentivirus strains to drive different cellular responses, especially from inflammation to apoptosis.

NRPS Protein MarQ Catalyzes Flexible Adenylation and Specific S-Methylation.

Maremycins are a group of structurally diverse 2,5-diketopiperazine natural products featuring a rare amino acid building block, S-methyl-l-cysteine (Me-Cys). Three freestanding nonribosomal peptide synthetase (NRPS) proteins from the maremycins biosynthetic pathway were proposed for the formation of the 2,5-diketopiperazine scaffold: MarQ, MarM, and MarJ. MarQ displays flexible adenylation activity toward Cys, Me-Cys, Ser, and ( S)-2,3-diaminopropanoic acid (DAP) and transfers these substrates to MarJ, which is the discrete peptidyl carrier protein (PCP). MarQ could also activate several other amino acids. The embedded methyltransferase (MT) domain in MarQ specifically catalyzes the thiol methylation of MarJ-tethered Cys. The in vitro reconstitution of MarQ and MarJ further provides clear evidence for the reaction sequence of methylation step on Cys. Our study on MarJ/Q tridomain cassette gains valuable insights into maremycins structure diversity and will be exploited to incorporate Me-Cys into natural products by combinatorial biosynthesis.

Divergent biosynthesis of indole alkaloids FR900452 and spiro-maremycins.

FR900452 and spirocyclic maremycins, including F and G components, are structurally related indole alkaloids, previously identified from different Streptomyces species. These alkaloids feature an indole diketopiperazine motif linked with a cyclopentenone moiety, but the linkage differs in FR900452 and the spirocyclic maremycins. Here, FR900452 and its two new analogues were identified from the fermentation broth of Streptomyces sp. B9173, the producer of maremycins. Gene inactivation and heterologous expression of the mar gene cluster confirmed that production of FR900452 shares the same biosynthetic machinery that produces maremycins. FR900452 was identified as the precursor of maremycin A/B by feeding studies. MarP, a SnoaL-like protein, was demonstrated to differentiate the biosynthesis of FR900452 from that of spiro-form maremycin G.

Influence of heart failure on the prognosis of patients with acute myocardial infarction in southwestern China.

The impact of heart failure (HF) on acute myocardial infarction (AMI) in patients from southwestern China remains unclear. The present study aimed to compare in-hospital cardiovascular events, mortality and clinical therapies in AMI patients with or without HF in southwestern China. In total, 591 patients with AMI hospitalized between February 2009 and December 2012 were examined; those with a history of HF were excluded. The patients were divided into four groups according to AMI type (ST-elevated or non-ST-elevated AMI) and the presence of HF during hospitalization. Clinical characteristics, in-hospital cardiovascular events, mortality, coronary angiography and treatment were compared. Clinical therapies, specifically evidence-based drug use were analyzed in patients with HF during hospitalization, including angiotensin converting enzyme inhibitors (ACEIs) and ß-blockers (BBs). AMI patients with HF had a higher frequency of co-morbidities, lower left ventricular ejection fraction, longer length of hospital stay and a greater risk of in-hospital mortality compared with AMI patients without HF. AMI patients with HF were less likely to be examined by cardiac angiography or treated with reperfusion therapy or recommended medications. AMI patients with HF co-treated with ACEIs and BBs had a significantly higher survival rate (94.4 vs. 67.5%; P<0.001) compared with untreated patients or patients treated with either ACEIs or BBs alone. Logistic regression analysis revealed that HF and cardiogenic shock in patients with AMI were the strongest predictors of in-hospital mortality. AMI patients with HF were at a higher risk of adverse outcomes. Cardiac angiography and timely standard recommended medications were associated with improved clinical outcomes.

Screening of Ganoderma strains with high polysaccharides and ganoderic acid contents and optimization of the fermentation medium by statistical methods.

Polysaccharides and ganoderic acids (GAs) are the major bioactive constituents of Ganoderma species. However, the commercialization of their production was limited by low yield in the submerged culture of Ganoderma despite improvement made in recent years. In this work, twelve Ganoderma strains were screened to efficiently produce polysaccharides and GAs, and Ganoderma lucidum 5.26 (GL 5.26) that had been never reported in fermentation process was found to be most efficient among the tested stains. Then, the fermentation medium was optimized for GL 5.26 by statistical method. Firstly, glucose and yeast extract were found to be the optimum carbon source and nitrogen source according to the single-factor tests. Ferric sulfate was found to have significant effect on GL 5.26 biomass production according to the results of Plackett-Burman design. The concentrations of glucose, yeast extract and ferric sulfate were further optimized by response surface methodology. The optimum medium composition was 55 g/L of glucose, 14 g/L of yeast extract, 0.3 g/L of ferric acid, with other medium components unchanged. The optimized medium was testified in the 10-L bioreactor, and the production of biomass, IPS, total GAs and GA-T enhanced by 85, 27, 49 and 93 %, respectively, compared to the initial medium. The fermentation process was scaled up to 300-L bioreactor; it showed good IPS (3.6 g/L) and GAs (670 mg/L) production. The biomass was 23.9 g/L in 300-L bioreactor, which was the highest biomass production in pilot scale. According to this study, the strain GL 5.26 showed good fermentation property by optimizing the medium. It might be a candidate industrial strain by further process optimization and scale-up study.