Transcriptomic analysis reveals peripheral pathway in 3-phenoxybenzoic acid degradation by Aspergillus oryzae M-4.

As a major intermediate metabolite of synthetic pyrethroids, the occurrence of 3-phenoxybenzoic acid hinders the decomposition of the parent pesticide and poses uncertain risks to environmental ecology and living organisms. Strain Aspergillus oryzae M-4 was previously reported to degrade 3-PBA and several substances were identified as downstream transformation products (TPs). But the mechanism underlying the cleavage of ether bond remains largely unclear. Here, we attempted to address such concern through identifying the peripheral TPs and analyzing transcriptomics, coupled with serial batch degradation experiments. Analysis results of chromatographic/mass spectrometry suggested that 3-PBA underwent twice hydroxylation, to yield mono- and dihydroxylated 3-PBA successively. In parallel, a mutual transformation between 3-PBA and 3-phenoxybenzyl alcohol (3-PBOH) also existed. The proposal of peripheral pathway represents an important advance towards fully understanding the whole 3-PBA metabolism in M-4. A specific altered metabolization was found for the first time, that is, resting cells of M-4 skipped the reduction step and initiate hydroxylation directly, by comparison with growing cells. Transcriptome analysis indicated that 3-PBA induced the up-regulation of genes related to energy investment, oxidative stress response, membrane transport and DNA repair. In-depth functional interpretation of differential expression genes suggested that the generation 3-PBOH and hydroxylated 3-PBA may be due to the participation of flavin-dependent monooxygenases (FMOs) and cytochrome P450 (CYP450), respectively. This study provides new insight to reveal the biodegradation mechanism of 3-PBA by A. oryzae M-4.

Seasonal dynamics of microbiota and physicochemical indices in the industrial-scale fermentation of Sichuan Baoning vinegar.

We investigated the impact of seasons of the year on microbiota and physicochemical indices in industrial-scale fermentation of Sichuan Baoning vinegar. Illumina HiSeq sequencing results showed significant differences (P < 0.05) between the microbiomes of vinegar Pei in every-two seasons, except for bacterial communities between summer and autumn. Total acid, reducing sugar, starch, and alcohol contents of vinegar Pei from the same sampling day of each season were measurably different. Although total acid content in vinegar Pei was similar at the end of fermentation (P > 0.05), the increase in total acidity was highest in the autumn. Acetic acid content in raw vinegar was highest in the autumn (3472.42 mg/100 mL), and lowest in the summer (2304.01 mg/100 mL). This study provides a theoretical basis for the production of Sichuan bran vinegar with consistent quality and provides insights into the quality control of traditional fermented foods.

Structure, Antioxidant, and Hypoglycemic Activities of Arabinoxylans Extracted by Multiple Methods from Triticale.

Different methods of isolating arabinoxylans (AXs) from triticale were performed to investigate the extraction methods' effects on the physiological functions of the AXs. Structural, antioxidant, and hypoglycemic activities were determined. The molecular weights (MWs) of enzyme- or water-extracted AXs were lower than those of alkali-extracted AXs. Opposite trends were shown by the arabinose-xylose ratio. Enzyme-extracted AXs exhibited higher glucose adsorption capacity and hydroxyl radical-scavenging efficiency than alkali-extracted AXs. The α-amylase inhibition ability, DPPH radical-scavenging capacity, and metal-chelating activity of alkali-extracted AXs were higher than those of enzyme-extracted AXs. Water-extracted AXs had the highest glucose dialysis retardation index. In conclusion, extraction methods can influence the physiological function of AXs through their structural features. AXs with higher MWs and esterified ferulic acid (FA) levels had higher antioxidant ability, whereas AXs with higher solubility and free FA level exhibited higher hypoglycemic activity.

Microbial BOD sensors based on Zr (IV)-loaded collagen fiber.

Biochemical oxygen demand (BOD) sensors based on Zr (IV)-loaded collagen fiber (ZrCF), a novel material with great porous structure, were developed. This novel material shows adsorbability by microorganisms. Saccharomyces cerevisiae and Escherichia coli were used for the construction of BOD sensors. Factors affecting BOD sensor performance were examined. The ZrCF-based BOD sensor showed different sensitivities and linear response ranges with different biofilm densities. The amount of microorganisms strongly affected the performance of the BOD sensor. Poor permeability of previously reported immobilization carriers were greatly circumvented by ZrCF. The service life of the ZrCF-based BOD sensor was more than 42 days. The immobilized microorganisms can be stored for more than 6 months under 4°C in PB solution. There was good correlation between the results of the sensor method and the standard 5-day BOD method in the determination of pure organic substrates and real water samples.

Preparation and Application of Starch/Polyvinyl Alcohol/Citric Acid Ternary Blend Antimicrobial Functional Food Packaging Films.

Ternary blend films were prepared with different ratios of starch/polyvinyl alcohol (PVA)/citric acid. The films were characterized by field emission scanning electron microscopy (FE-SEM), thermogravimetric analysis, as well as Fourier transform infrared (FTIR) analysis. The influence of different ratios of starch/polyvinyl alcohol (PVA)/citric acid and different drying times on the performance properties, transparency, tensile strength (TS), water vapor permeability (WVP), water solubility (WS), color difference (ΔE), and antimicrobial activity of the ternary blends films were investigated. The starch/polyvinyl alcohol/citric acid (S/P/C1:1:0, S/P/C3:1:0.08, and S/P/C3:3:0.08) films were all highly transparent. The S/P/C3:3:0.08 had a 54.31 times water-holding capacity of its own weight and its mechanical tensile strength was 46.45 MPa. In addition, its surface had good uniformity and compactness. The S/P/C3:1:0.08 and S/P/C3:3:0.08 showed strong antimicrobial activity to Listeria monocytogenes and Escherichia coli, which were the food-borne pathogenic bacteria used. The freshness test results of fresh figs showed that all of the blends prevented the formation of condensed water on the surface of the film, and the S/P/C3:1:0.08 and S/P/C3:3:0.08 prevented the deterioration of figs during storage. The films can be used as an active food packaging system due to their strong antibacterial effect.

Wheat bran components modulate intestinal bacteria and gene expression of barrier function relevant proteins in a piglet model.

The objective of this study was to determine the impact of wheat bran and its main polysaccharides on intestinal bacteria and gene expression of intestinal barrier function relevant proteins. Thirty freshly weaned male piglets were assigned randomly to five dietary treatment groups with six piglets per group. Accordingly, five synthetic diets including a basal control diet without fiber components (CON), wheat bran diet (10% wheat bran, WB), arabinoxylan diet (AX), cellulose diet (CEL) and combined diet of arabinoxylan and cellulose (CB) were studied. The piglets were fed ad libitum for 30 d. Lower Escherichia coli (E. coli) populations in WB group and higher probiotic (Lactobacillus and Bifidobacterium) populations in groups fed diets containing arabinoxylan (WB, AX and CB) were observed and compared with CON group. Compared with CON group, the gene expressions of cystic fibrosis transmembrane conductance regulator (CFTR), calcium-activated chloride channel regulator 1 (CLCA1) and voltage-gated chloride channel 2 (CIC2) were suppressed in the WB group. And wheat bran down-regulated gene expression of pro-inflammation (TNF-α, IL-1ß, IL-6) and TLRs/MyD88/NF-κB pathway compared with CON group. In conclusion, wheat bran and its main polysaccharides could change intestinal microflora and down-regulate the gene expression of intestinal barrier function relevant proteins in the distal small intestinal mucosa.

Chryseobacterium chengduensis sp. nov. isolated from the air of captive giant panda enclosures in Chengdu, China.

A Gram-negative, aerobic, non-motile, rod-shaped bacterial strain, designated 25-1(T), was isolated from the air inside giant panda enclosures at the Chengdu Research Base of Giant Panda Breeding, China. Strain 25-1(T) grew optimally at pH 7.0-8.0, at 28-30 °C and in the presence of NaCl concentrations from 0.0% to 0.5 %. 16S rRNA gene sequence analysis indicated that strain 25-1(T) belongs to the genus Chryseobacterium within the family Flavobacteriaceae and is related most closely to C. carnis G81(T) (96.4% similarity), C. lathyri RBA2-6(T) (95.8% similarity), and C. zeae JM1085(T) (95.8% similarity). Its genomic DNA G+C molar composition was 36.2%. The major cellular fatty acids were iso-C15:0 (44.0%), iso-C17:0 3OH (19.8%) and C16:1 ω7c/16:1 ω6c (12.7%). The only isoprenoid quinone was menaquinone 6 (MK-6). The major polar lipids were phosphatidylethanolamine, two unidentified amino lipids and two unidentified lipids. The DNA-DNA relatedness between strain 25-1(T) and C. lathyri RBA2-6(T) was 38%. Phenotypic, genotypic, and phylogenetic characteristics indicated that strain 25-1(T) is a novel member of the genus Chryseobacterium, for which the name C. chengduensis sp. nov. is proposed. The type strain is 25-1(T) (CCTCC AB2015133(T)=DSM 100396(T)).

Nutritional composition of boletus mushrooms from Southwest China and their antihyperglycemic and antioxidant activities.

Thirteen samples representing five species were collected from different provinces of Southwest China, and their chemical composition, antihyperglycemic activity, and antioxidant activity were evaluated. These mushrooms had high crude protein (21.72-30.59g/100g dw) and total carbohydrate (49.18-62.58g/100g dw) contents, but low crude fat contents (1.96-7.87g/100g dw). They also accumulated notable quantities of potassium, zinc, sodium, magnesium and copper from the soil. The potassium content, in particular, was 18.75-39.21 times that found in the soil at the collection site. The natural habitat of these mushrooms, especially the mineral content of the soil, seems to have more influence on the mineral content of these mushrooms than their species. Most of the samples possessed antihyperglycemic and antioxidant activities. Suillellus luridus showed the highest antioxidant activity and antihyperglycemic activities, suggesting that S. luridus shows potential for development as a dietary nutritional supplement.

Lysobacter chengduensis sp. nov. Isolated from the Air of Captive Ailuropoda melanoleuca Enclosures in Chengdu, China.

A novel bacterial strain, designated as CF21(T), was isolated from the air of Ailuropoda melanoleuca enclosures in China. Cells were gram-negative, aerobic, non-motile, and rod shaped. Strain CF21(T) grew at 10-40 °C (optimum 28-30 °C) and pH 6.0-9.0 (optimum pH 7.0-8.0) and in the presence of NaCl concentrations ranging from 0.0% (w/v) to 2.0 % (optimum 0.0-1.0%). 16SrRNA gene sequence analysis indicated that strain CF21(T) belonged to genus Lysobacter within class Gammaproteobacteria and was most closely related to Luteimonas dalianensi OB44-3(T) (95.8% similarity), Lysobacter ruishenii CTN-1(T) (95.1%), Lysobacter spongiicola KMM329(T) (94.8 %), and Lysobacter daejeonensis GH1-9T (94.6%). The genomic G+C DNA content was 68.72 mol%. Major cellular fatty acids of CF21(T) were iso-C16:0 (30.22%), iso-C15:0 (25.70%), and the sum of 10-methyl C16 : 0 and/or iso-C17 : 1ω9c (21.94%). The prominent isoprenoid quinone was ubiquinone 8 (Q-8). Primary polar lipids included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and an unknown phospholipid. DNA sequence relatedness between strain CF21(T) and L. ruishenii CTN-1(T) was 56%, which was clearly below the 70% threshold for prokaryotic species delineation. These analyses indicated that CF21(T) is a novel member of genus Lysobacter, for which the name Lysobacter chengduensis sp. nov. is proposed. The type strain is CF21(T) (=CGMCC1.15145(T) = DSM 100306(T)).

Effect of 25-hydroxyvitamin D3 on rotavirus replication and gene expressions of RIG-I signalling molecule in porcine rotavirus-infected IPEC-J2 cells.

The study evaluated whether a 25-hydroxyvitamin D3 (25D3) supplementation decreases the replication of rotavirus by the retinoic acid-inducible gene I (RIG-I) signalling pathway in a porcine small intestinal epithelial cell line (IPEC-J2). The results show that IPEC-J2 cells express high baseline levels of 1α-hydroxylase (CYP27B1), which converts inactive 25D3 to the active 1,25-dihydroxyvitamin D3 (1,25D3). Porcine rotavirus (PRV) infection alone resulted in a significant increase in CYP27B1 mRNA, which augmented the production of active vitamin D. Physiological concentrations of 25D3 were found to decrease PRV replication in IPEC-J2 cells. RIG-I plays an important role in the recognition of double-stranded RNA virus by host cells. Upon recognition, RIG-I triggers a series of signalling molecules such as interferon-ß (IFN-ß) promoter stimulator 1 (IPS-1) leading to the expression of type I interferons (IFN-ß). Active 25D3 that was generated by PRV-infected IPEC-J2 cells led to an increased expression of toll-like receptors 3 (TLR3), RIG-I, IPS-1, IFN-ß and IFN-stimulated genes 15 (ISG15) with important innate immune functions. Inhibiting CYP27B1 also failed to increase RIG-I, IPS-1, IFN-ß and ISG15 mRNA expression. These observations suggest that 25D3 can directly inhibit PRV in IPEC-J2 cells, which requires this active form of vitamin D. The anti-rotavirus effect of 25D3 is mediated at least in part by RIG-I signalling pathways in IPEC-J2 cells.

Characterization of selenium-enriched mycelia of Catathelasma ventricosum and their antihyperglycemic and antioxidant properties.

This is the first report concerning the selenium enrichment of Catathelasma ventricosum mycelia. The selenium-containing proteins present in selenium-enriched mycelia (Se-MC) were identified using size-exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS). The selenium-containing amino acids liberated by hydrolysis of these proteins were identified using anion exchange-ICP-MS. Se-MC was found to contain selenoproteins with molecular weights ranging from 1.7 to 60.5 kDa. The main selenium-containing amino acids within them were selenomethionine and selenocysteine. Furthermore, Se-MC possessed excellent antihyperglycemic and antioxidant properties. Se-MC normalized biochemical parameters like insulin level, blood glucose level, body weight, and antioxidant enzyme activity in streptozocin-induced diabetic mice. It also inhibited the α-amylase and α-glucosidase activities present in in vitro gastric and intestinal models. In conclusion, Se-MC has the potential to serve as a dietary supplement of selenium, an antioxidant, or an ingredient for the formulation of nutraceuticals.

Chronic glucocorticoid exposure-induced epididymal adiposity is associated with mitochondrial dysfunction in white adipose tissue of male C57BL/6J mice.

Prolonged and excessive glucocorticoids (GC) exposure resulted from Cushing's syndrome or GC therapy develops central obesity. Moreover, mitochondria are crucial in adipose energy homeostasis. Thus, we tested the hypothesis that mitochondrial dysfunction may contribute to chronic GC exposure-induced epididymal adiposity in the present study. A total of thirty-six 5-week-old male C57BL/6J mice (∼20 g) were administrated with 100 µg/ml corticosterone (CORT) or vehicle through drinking water for 4 weeks. Chronic CORT exposure mildly decreased body weight without altering food and water intake in mice. The epididymal fat accumulation was increased, but adipocyte size was decreased by CORT. CORT also increased plasma CORT, insulin, leptin, and fibroblast growth factor 21 concentrations as measured by RIA or ELISA. Interestingly, CORT increased plasma levels of triacylglycerols and nonesterified fatty acids, and up-regulated the expression of both lipolytic and lipogenic genes as determined by real-time RT-PCR. Furthermore, CORT impaired mitochondrial biogenesis and oxidative function in epididymal WAT. The reactive oxygen species production was increased and the activities of anti-oxidative enzymes were reduced by CORT treatment as well. Taken together, these findings reveal that chronic CORT administration-induced epididymal adiposity is, at least in part, associated with mitochondrial dysfunction in mouse epididymal white adipose tissue.

Dietary vitamin D supplementation attenuates immune responses of pigs challenged with rotavirus potentially through the retinoic acid-inducible gene I signalling pathway.

In the present study, twenty-four Duroc × Landrance × Yorkshire (initial body weight (BW) of 21·82 (sem 2·06) kg) cross-bred pigs were used to determine whether dietary vitamin D supplementation could confer protection against viral infections through the retinoic acid-inducible gene I (RIG-I) signalling pathway in pigs. Experimental treatments were arranged in a 2 × 2 factorial manner with the main effects of immune challenge (control v. porcine rotavirus (PRV) challenge) and dietary concentrations of vitamin D (200 and 5000 IU; where 1 IU of vitamin D is defined as the biological activity of 0.025 mg of cholecalciferol). The pigs were fed a diet containing 200 or 5000 IU vitamin D in the first week of the study period. On day 8, the pigs were orally dosed with 4 ml of Dulbecco's modified Eagle's medium/Ham's F-12 medium containing PRV or essential medium (control). Serum samples were collected on day 8 (pre-challenge), and 6 d after the PRV challenge, the pigs were killed to evaluate intestinal morphology and tissue gene expression following the last blood collection. Pigs challenged with PRV had decreased BW gain (P< 0·01), feed intake (P< 0·01), villus height (P< 0·01), faecal consistency (P< 0·05), and serum 1,25-dihydroxyvitamin D concentration (P< 0·01) and increased (P< 0·01) serum IL-2, IL-6 and interferon (IFN)-ß concentrations. Vitamin D supplementation mitigated these effects. The mRNA expression of RIG-I (P< 0·01), IFN-ß promoter stimulator 1 (P< 0·01), IFN-ß (P< 0·01) and interferon-stimulated gene 15 (ISG 15 ) (P< 0·01) was up-regulated by the PRV challenge and vitamin D supplementation in the intestine. In conclusion, vitamin D supplementation could activate the RIG-I signalling pathway and thus alleviate the negative effects caused by PRV challenge.

Codon optimization significantly improves the expression level of a keratinase gene in Pichia pastoris.

The main keratinase (kerA) gene from the Bacillus licheniformis S90 was optimized by two codon optimization strategies and expressed in Pichia pastoris in order to improve the enzyme production compared to the preparations with the native kerA gene. The results showed that the corresponding mutations (synonymous codons) according to the codon bias in Pichia pastoris were successfully introduced into keratinase gene. The highest keratinase activity produced by P. pastoris pPICZαA-kerAwt, pPICZαA-kerAopti1 and pPICZαA-kerAopti2 was 195 U/ml, 324 U/ml and 293 U/ml respectively. In addition, there was no significant difference in biomass concentration, target gene copy numbers and relative mRNA expression levels of every positive strain. The molecular weight of keratinase secreted by recombinant P. pastori was approx. 39 kDa. It was optimally active at pH 7.5 and 50°C. The recombinant keratinase could efficiently degrade both α-keratin (keratin azure) and ß-keratin (chicken feather meal). These properties make the P. pastoris pPICZαA-kerAopti1 a suitable candidate for industrial production of keratinases.

Comparative analysis of mesenchymal stem cells from adult mouse adipose, muscle, and fetal muscle.

Recently, increasing evidence supports that adult stem cells are the part of a natural system for tissue growth and repair. This study focused on the differences of mesenchymal stem cells from adult adipose (ADSCs), skeletal muscle (MDSCs) and fetal muscle (FMSCs) in biological characteristics, which is the key to cell therapy success. Stem cell antigen 1 (Sca-1) expression of MDSCs and FMSCs at passage 3 was two times more than that at passage 1 (P < 0.0001). After 28-day myogenic induction, higher expression levels of skeletal muscle-specific genes were observed in MDSCs than FMSCs (P < 0.01), and the lowest expression levels were demonstrated in ADSCs among three cells (P < 0.01). Besides, M-Cad and MyHC expressions in ADSCs were not detected by immunofluorescence or real-time quantitative PCR. Furthermore, after 14 days adipogenic induction, PPARγ2, LPL and aP2 mRNA expressions were higher in ADSCs vs. MDSCs (P < 0.01). Besides, MSCs from adult or fetal muscle expressed higher OCN and OPN than ADSCs after 28 days osteogenic induction (P < 0.01). Taken together, our results suggested that cell source and developmental stage had great impacts on biological properties of mesenchymal stem cells, and proper consideration of all the issues is necessary.

Expression of a keratinase (kerA) gene from Bacillus licheniformis in Escherichia coli and characterization of the recombinant enzymes.

The gene kerA (1,047 bp) encoding the main keratinase from Bacillus licheniformis was cloned into two conventional vectors, pET30α and pET32α, and expressed in Escherichia coli. From SDS-PAGE analysis, the recombinant keratinases were 45 and 55 kDa. They had different optimal pH values (7.5 and 8.5) but the same optimum temperature of 50 °C. The recombinant keratinase produced in E. coli pET30α-kerA was more stable than that produced in E. coli pET32α-kerA, and retained approx. 70 % of its total enzyme activity after 30 min at 70 °C.

Molecular cloning and expression analysis of IFN-ß promoter stimulator 1 in Tibetan pigs.

IFN-ß promoter stimulator 1 (IPS-1) is an important adaptor protein linking RIG-I/MDA5 to the downstream signaling molecules and plays the pivotal role in type I interferons induction. In this study, we cloned and characterized Tibetan porcine IPS-1, investigated the tissue distribution, compared different messenger RNA expression for IPS-1 between Tibetan and Crossbred (Duroc × Yorkshire × Landrace) pigs (DLY). The Tibetan porcine IPS-1 gene was first cloned from spleen. The entire open reading frame (ORF) of the IPS-1 is 1,575 bp and encodes for 524 amino acid residues, has 1 putative transmembrane domains, with a higher degree of sequence similarity with common pig (99.37%) and cattle (81.23%) than with human (70.20%) or mouse (63.44%). Real-time quantitative PCR analysis indicated that Tibetan porcine IPS-1 mRNA was most abundant in the liver and kidney. The expression of IPS-1 of Tibetan pigs in most tissues was higher than DLY pigs.

Effects of different starch sources on Bacillus spp. in intestinal tract and expression of intestinal development related genes of weanling piglets.

The study was conducted to evaluate the effects of different starch sources on Bacillus spp. in intestinal tract and expression of intestinal development related genes of weanling piglets. Twenty-eight PIC male piglets were divided into four homogeneous groups according to initial body weight (similar birth and parity, weaned at 21 ± 1.5 days). Diets for the four treatments consisted of corn starch, wheat starch, tapioca starch and pea starch with the determined ratio for amylose to amylopectin of 0.21, 0.24, 0.12 and 0.52 respectively. Real-time quantitative polymerase chain reaction was applied to: (1) detect genomic DNA of Bacillus and to quantify the number of Bacillus in the intestinal tract chyme of piglets with the primers and probe which designed based on the 16S rRNA sequences of maximum species of Bacillus on GenBank; (2) measure the mRNA level of glucagon-like peptide 2 (GLP-2), insulin-like growth factors 1 (IGF-1) and epidermal growth factor (EGF) in duodenum, jejunum and ileum. Results showed that the number of Baciilus and the percentage based on all bacteria in the whole intestinal content of piglets fed pea starch was highest in all groups (P < 0.05). There was no significant differance on copy numbers for all bacteria and Bacillus in the whole intestinal tract of piglets between the corn starch group and wheat starch group (P > 0.05). In addition, the expression level of GLP-2, IGF-1 mRNA in jejunum and ileum of pea starch treatment (the high amylose/amylopectin ratio) were increased while the tapioca starch decreased their mRNA level significantly compared to other three treatments (P < 0.05). There was no significant difference for the mRNA level of EGF in each group. The present study revealed that high amylose/amylopectin ratio of starches significantly enhanced the numbers of Bacillus in all segments of intestine and the mRNA level of intestinal development related genes.

Inhibition of adipogenic differentiation by myostatin is alleviated by arginine supplementation in porcine-muscle-derived mesenchymal stem cells.

Porcine mesenchymal stem cells in postnatal muscle have been demonstrated to differentiate into adipocytes. This increases adipocyte number and lipid accumulation, and is thought to be the origin of intramuscular fat. In this study, the effects of myostatin and arginine on adipogenic differentiation in mesenchymal stem cells derived from porcine muscle (pMDSCs) were investigated in vitro. Intracellular triglyceride levels were reduced by exogenous myostatin and increased by arginine supplementation or myostatin antibody (P<0.01). The inhibition of lipid accumulation by myostatin in pMDSCs was alleviated by arginine supplementation (P<0.01). Expression patterns of adipogenic transcription factors showed that exogenous myostatin suppressed PPARγ2 and aP2 expression (P<0.01), while supplemental arginine or myostatin antibody promoted ADD1 expression (P<0.01). Furthermore, compared with the addition of either myostatin protein or antibody alone, ADD1 and PPARδ expression were promoted by the combination of arginine and myostatin (P<0.01), and arginine combined with myostatin antibody promoted the expression of ADD1, PPARδ, C/EBPα, PPARγ2 and LPL in pMDSCs (P<0.05). These results suggest that myostatin inhibits adipogenesis in pMDSCs, and that this can be alleviated by arginine supplementation, at least in part, through promoting ADD1 and PPARδ expression.

Molecular cloning, expression in Escherichia coli of Attacin A gene from Drosophila and detection of biological activity.

Attacin, a 20 kDa antibacterial peptide, plays an important role in immunity. To understand this gene better, gene cloning, expression and biological activity detection of Attacin A was carried out in present study. The full-length open reading frame (ORF) coding for Attacin A gene was generated using RT-PCR which takes total RNA extracted from Drosophila as the template. The gene was inserted directionally into the prokaryotic expression vector pET-32a (+). The resulting recombinant plasmid was transformed into E. coli Rosetta. SDS-PAGE was carried out to detect the expression product which was induced by IPTG. The antimicrobial activity and hemolysis activity were tested in vitro after purification. Agarose gel electrophoresis indicated that the complete ORF of Attacin A gene has been cloned successfully from Drosophila stimulated by E. coli which includes 666 bp and encodes 221 AA. The gene encoding mature Attacin A protein was amplified by PCR from the recombinant plasmid containing Attacin A, which includes 570 bp in all. SDS-PAGE analysis demonstrated that the fusion protein expressed was approximately 39.2 kDa. Biological activities detection showed that this peptide exhibited certain antibacterial activity to several G- bacteria, as well as minor hemolysis activity for porcine red blood cells. In conclusion, Attacin A gene was cloned and expressed successfully. It was the basis for further study of Attacin.