New Biotransformation Mode of Zearalenone Identified in Bacillus subtilis Y816 Revealing a Novel ZEN Conjugate.

An increasing number of Bacillus strains have been identified, and the removal capacity of zearalenone (ZEN) was determined; however, they failed to reveal the detoxification mechanism and transformation product. Here, Bacillus subtilis Y816, which could transform 40 mg/L of ZEN within 7 h of fermentation, was identified and studied. First, the biotransformation products of ZEN and 17-ß-estradiol (E2) were identified as ZEN-14-phosphate and E2-3-phosphate by HPLC-TOF-MS and NMR, respectively. An intracellular zearalenone phosphotransferase (ZPH) was found through transcriptome sequencing analysis of B. subtilis Y816. The phosphorylated reaction conditions of ZEN by ZPH were further revealed in this work. Furthermore, the phosphorylated conjugates showed reduced estrogenic toxicity compared with their original substances (ZEN and α/ß-zearalenol) using an engineered yeast biosensor system. The first report on the phosphorylated conjugated mode of ZEN in B. subtilis Y816 will inspire new perspectives on the biotransformation of ZEN in Bacillus strains.

The association between interleukin-19 concentration and diabetic nephropathy.

BACKGROUND: Interleukin-19 (IL-19) is a newly discovered cytokine belonging to the Interleukin-10(IL-10) family. IL-19 have indispensable functions in many inflammatory processes and also can induce the angiogenic potential of endothelial cells. The purpose of present study was to investigate the relation of serum interleukin-19 (IL-19) levels with diabetic nephropathy (DN). METHODS: Two hundred study groups of patients with type 2 diabetes mellitus (T2DM) (109 males and 91 females) were recruited, included normoalbuminuria(n = 102), microalbuminuria(n = 72) and macroalbuminuria(n = 26) . The 50 healthy blood donors were enrolled for the control group. All subjects were assessed for: IL-19, High-sensitivity C-reactive protein (Hs-CRP), Cystatin C, urinary albumin excretion rate (UAE) and glycosylated hemoglobin A1c(HbA1c). RESULTS: The serum IL-19 levels in DN patients were found to be significantly higher compared to controls. IL-19 levels were significantly positively correlated with Hs-CRP, Cystatin C, UAE and HbA1c(r = 0.623, 0.611,0.591 and 0.526 respectively, P < 0.01). Multivariable logistic regression analysis showed IL-19 levels (P = 0.01) were found to be independently associated with patients with DN. CONCLUSIONS: IL-19 is significantly positive correlated with UAE and Cystatin C. IL-19 may play an important role that contributes to the progression of diabetic nephropathy.

Generation of Induced Cardiospheres via Reprogramming of Skin Fibroblasts for Myocardial Regeneration.

Recent pre-clinical and clinical studies have suggested that endogenous cardiospheres (eCS) are potentially safe and effective for cardiac regeneration following myocardial infarction (MI). Nevertheless the preparation of autologous eCS requires invasive myocardial biopsy with limited yield. We describe a novel approach to generate induced cardiospheres (iCS) from adult skin fibroblasts via somatic reprogramming. After infection with Sox2, Klf4, and Oct4, iCS were generated from mouse adult skin fibroblasts treated with Gsk3ß inhibitor-(2'Z,3'E)- 6-Bromoindirubin-3'-oxime and Oncostatin M. They resembled eCS, but contained a higher percentage of cells expressing Mesp1, Isl1, and Nkx2.5. They were differentiated into functional cardiomyocytes in vitro with similar electrophysiological properties, calcium transient and contractile function to eCS and mouse embryonic stem cell-derived cardiomyocytes. Transplantation of iCS (1 × 106 cells) into mouse myocardium following MI had similar effects to transplantation of eCS but significantly better than saline or fibroblast in improving left ventricular ejection fraction, increasing anterior/septal ventricular wall thickness and capillary density in the infarcted region 4 weeks after transplantation. No tumor formation was observed. iCS generated from adult skin fibroblasts by somatic reprogramming and a cocktail of Gsk3ß inhibitor-6-Bromoindirubin-3'-oxime and Oncostatin M may represent a novel source for cell therapy in MI. Stem Cells 2016;34:2693-2706.

Interleukin-19 and angiopoietin-2 can enhance angiogenesis of diabetic complications.

This study was to investigate the role of interleukin-19 (IL-19) and angiopoietin-2 (Ang-2) in angiogenesis of type 2 diabetes (T2DM). In 240 patients with T2DM, IL-19 and Ang-2 levels were higher, IL-19 was positively correlated with Ang-2. IL-19 and Ang-2 might be involved in angiogenesis of T2DM complications.

Structure and sequence analysis-based engineering of pullulanase from Anoxybacillus sp. LM18-11 for improved thermostability.

Pullulanase (EC 3.2.1.41) is a well-known starch-debranching enzyme. Enhancing the thermostability of Pullulanase is required for industrial application. In this study, we used two methods to improve the thermostability of the pullulanase from Anoxybacillus sp. LM18-11; these methods were the modified amino acid consensus method combined with the analyses of the residue water-exposed surface (ACC) and the deletion of flexible domains. Four mutants (Y477A, Y175C, L215C and R473E) were obtained via the modified consensus method exhibited varying degrees of improvements in terms of thermostability. One deletion mutant termed D3 (residues(686-688)) was obtained and exhibited enhanced thermostability due to deletion of the flexible region at the C-terminus. The combination of the two strategies yielded the mutant M18 (Y477A/D3/Y175C/L215P/R473E). It retained 66% of its initial activity after incubation at 60 °C for 72 hrs, whereas that of the wild-type enzyme was only 35%. After incubation at 65 °C for 4 h, M18 retained 50.6% of its initial activity, whereas that of the wild-type was only 16.8%, respectively. Additionally, kinetic studies revealed that the Km of M17 (Y477A/D3/Y175C/L215P) was decreased by 33.9% and that the Kcat/Km value of M17 increased by 50%, while M18 exhibited Km and Kcat/Km values that were similar to those of the wild-type enzyme. The attractive improved thermostability and the high catalytic efficiency made M17 and M18 more suitable for industrial application.

Therapeutic application of endothelial progenitor cells for treatment of cardiovascular diseases.

Cardiovascular disease is the leading cause of death worldwide. Despite significant progress in understanding of the disease mechanisms, most therapies remain at best palliative. Few therapeutic approaches offer direct tissue repair and regeneration. Cell-based therapy offers a promising approach that involves transplantation of healthy and functional cells to replenish damaged cells and repair injured tissue. Endothelial dysfunction is one of the most important mechanisms of cardiovascular disease, thus endothelial progenitor cells (EPC) and their derivatives have been investigated as a potential source for cell therapy. In pre-clinical and pilot clinical studies, treatment with EPCs or their derivatives as well as their co-transplantation with other cell types has shown some initial promising results. In this review, we will first describe the importance of endothelial cells and EPC homeostasis in the pathophysiology of cardiovascular disease. The potential sources of EPCs, including their isolation and purification, differentiation from pluripotent stem cells and adult stem cells, and trans-differentiation from somatic cells will then be summarized. Lastly, the application of target genome editing tools, such as Zinc Finger Nuclease (ZFN), Transcription Activator Like Effector Nucleases (TALEN) and RNA Guided Endo Nuclease (RGEN) to modify EPCs and their derivatives will be described. These technologies promise to further improve the therapeutic potential of EPCs and their derivatives to treat cardiovascular disease.

Construction of two BAC libraries from half-smooth tongue sole Cynoglossus semilaevis and identification of clones containing candidate sex-determination genes.

Half-smooth tongue sole (Cynoglossus semilaevis) is an increasingly important aquaculture species in China. It is also a tractable model to study sex chromosome evolution and to further elucidate the mechanism of sex determination in teleosts. Two bacterial artificial chromosome (BAC) libraries for C. semilaevis, with large, high-quality inserts and deep coverage, were constructed in the BamHI and HindIII sites of the vector pECBAC1. The two libraries contain a total of 55,296 BAC clones arrayed in 144 384-well microtiter plates and correspond to 13.36 haploid genome equivalents. The combined libraries have a greater than 99% probability of containing any single-copy sequence. Screening high-density arrays of the libraries with probes for female-specific markers and sex-related genes generated between 4-46 primary positive clones per probe. Thus, the two BAC libraries of C. semilaevis provided a readily useable platform for genomics research, illustrated by the isolation of sex determination gene(s).

A new method for SNP discovery.

Single nucleotide polymorphisms (SNPs) are high-density natural sequence variations in genomes. They are considered to be the major genetic source of phenotypic variability within a given species and serve as excellent genetic markers. SNPs are useful in identifying candidate genes that contribute to disease and phenotypic traits. In non-model organisms, the application of SNPs has been limited, because of the expense and technical difficulties entailed in currently available SNP isolation techniques. In the present study, we have developed a rapid and effective method to isolate SNPs throughout the genome randomly. The DNA fragments containing SNPs could be isolated efficiently from background DNA. We analyzed ten isolated DNA fragments with this method in half-smooth tongue sole (Cynoglossus semilaevis)--a newly exploited and commercially important cultured marine flatfish in China--and found that nine of the fragments contained SNPs. The findings were confirmed successfully in different individuals. The method presented here is cost-effective and applicable to essentially any organism.

Artificial gynogenesis and sex determination in half-smooth tongue sole (Cynoglossus semilaevis).

Half-smooth tongue sole (Cynoglossus semilaevis) is an important cultured marine fish as well as a promising model fish for the study of sex determination mechanisms. In the present study, a protocol for artificial gynogenesis of half-smooth tongue sole was developed in order to identify the sex determination mechanism and to generate all-female stock. The optimal UV-irradiation dose for genetically inactivating sea perch spermatozoa was determined to be > or =30 mJ/cm(2). The optimal initiation time for cold shock of gynogenetic embryos was determined to be 5 min after fertilization, while the optimal temperature and treatment duration were determined to be 20-25 min at 5 degrees C. Chromosomes from common diploids, gynogenetic haploids, and diploids were analyzed. WW chromosomes were discovered in some of the gynogenetic diploids. The microsatellite marker was applied to analyze gynogenetic diploid fry. Among the 30 gynogenetic diploid fry, 11 fry contained only one allele, while 19 contained two alleles, which had the same genotype as their mother. The female-specific DNA marker was observed in four individuals out of ten gynogenetic diploid fry. Ploidy analysis of 20 putative gynogenetic fry showed them all to be diploid. Thus, a protocol for the induction of artificial gynogenesis has been developed for the first time in half smooth tongue sole, and the sex determination mechanism in the tongue sole was determined to be female heterogametic with the ZW chromosome.

Molecular cloning and characterization of the Myf5 gene in sea perch (Lateolabrax japonicus).

The cDNA of myogenic factor (Myf5) was isolated from sea perch (Lateolabrax japonicus) using Reverse-transcription Polymerase Chain Reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The 5' flanking sequence of the cDNA contains a TATA box, GC box, CAAT box, several E box sites and muscle-specific regulatory elements determined by genome walking. The Myf5 gene consists of 3 exons and 2 introns. The open reading frame was found to code a protein with 238 amino-acid residues, containing the conserved basic helix-loop-helix domain (bHLH). RT-PCR indicated the Myf5 was highly expressed in muscle, and weakly expressed in brain, eyes, spleen, gill, liver, kidney, intestine and heart. In early embryonic stages, Myf5 mRNA transcripts are highly detectable in the early gastrula stage while decreasing up to a low level at the late gastrula stage, subsequently greatly increased up to the highest level in the somites stage, then gradually decreases from the tail-bud stage to 15 d larvae after hatching, but they are still detectable. Further, Myf5 mRNA was expressed in several sea perch cell lines such as LJES1, LJHK, LJH-1, LJH-2, LJS, LJL, although its expression level varied greatly among different cell lines.

Isolation of female-specific AFLP markers and molecular identification of genetic sex in half-smooth tongue sole (Cynoglossus semilaevis).

The sex-specific molecular marker is a useful gene resource for studying sex- determining mechanisms and controlling fish sex. Artificially produced male and female half-smooth tongue sole (Cynoglossus semilaevis) were used to screen sex-specific amplified fragment length polymorphism (AFLPs) molecular markers. The phenotypic sex of 28 tongue soles was determined by histological sectioning of gonads. The AFLP analysis of 15 females and 13 males via 64 primer combinations produced a total of 4681 scorable bands, of which 42.11% and 43.39% of bands were polymorphic in females and males, respectively. Seven female-specific AFLP markers were identified and designated as CseF382, CseF575, CseF783, CseF464, CseF136, CseF618, and CseF305, respectively. One female-specific AFLP marker (CseF382) was amplified, recovered from the gels, cloned, and sequenced (accession no. DQ487760). This female-specific AFLP marker was converted into a single-locus polymerase-chain reaction (PCR) marker of a sequence-characterized amplified region (SCAR). A simple PCR method of using the specific primers was developed for identifying genetic sex of half-smooth tongue sole. PCR products demonstrated that the initial 15 females produced the female-specific band of about 350 bp, but the initial 13 male individuals failed to produce the band. We also investigated the applicability of the PCR primers in other tongue sole individuals. The same female-specific fragment of about 350 bp was found in the additional 59 female individuals, but not in the additional 58 male individuals. This AFLP-based molecular sexing technique may have great application potential in elucidation of sex determination mechanisms and sex control in half-smooth tongue sole.

Molecular cloning, characterization and expression analysis of natural resistance associated macrophage protein (Nramp) cDNA from turbot (Scophthalmus maximus).

Nramp (natural resistance associated macrophage protein) has been identified as one of the major candidate genes for controlling natural resistance and/or susceptibility to intracellular pathogens in vertebrates. However, few reports are available about the structure and function of Nramp in teleost. We have recently isolated the cDNA encoding Nramp from turbot (Scophthalmus maximus). The full-length cDNA of the Nramp is 2584 bp in length, including 69 bp 5' terminal UTR, 850 bp 3' terminal UTR and 1665 bp open reading frame for a protein with 554 amino acid residues (Genbank accession number: DQ263240). Comparison of amino acid sequence indicated that turbot Nramp consists of 12 transmembrane regions (TM) domains. A consensus transport motif (CTM) containing 20 residues was observed between transmembrane domains 8 and 9. The deduced amino acid sequence of turbot Nramp exhibited between 60 and 92% homology with 13 other vertebrate Nramp sequences. Nramp transcripts were found to be highly abundant in head kidney, kidney and spleen, abundant in intestine and gill, less abundant in liver, brain, heart and gonad, least in muscle and skin. The level of Nramp mRNA in embryos gradually increases during embryogenesis from blastula stage to fry stage. Challenge of turbot with pathogenic bacteria, Vibrio anguillarum, elevated Nramp mRNA levels in liver and spleen. The Nramp transcripts were detected in turbot embryonic cell line (TEC). Challenge of the TEC cell cultures with pathogenic bacteria, V. anguillarum, significantly elevated Nramp mRNA levels in TEC cell cultures.