Identification of microRNAs in Wool Follicles during Anagen,Catagen, and Telogen Phases in Tibetan Sheep

Background

Wool quality is one of the most important economic traits in sheep. The wool fiber is derived from specialized skin cells that are referred to as wool follicles. To understand the roles of microRNAs (miRNAs) in wool fiber growth, we detected the expression patterns of miRNAs in wool follicles at the anagen, catagen, and telogen stages from Tibetan sheep through Solexa sequencing.

Results

A total of 244 mature miRNAs were identified. Of these, only five miRNAs are listed in the database of sheep miRNAs (miRBase Database V19), and the other 239 miRNAs have not been previously described in this species. Further analyses indicated that 204 miRNAs are evolutionarily conserved among mammal species, whereas 35 of the identified miRNAs were first found specifically in sheep. The expression pattern analyses showed that the expression levels of 39, 34, and 20 of the miRNAs significantly change between anagen and catagen, between anagen and telogen, and between catagen and telogen, respectively. The results of the bioinformatics analysis show that these differentially expressed miRNAs might regulate wool follicle development by targeting genes in many different pathways, such as the MAPK and Wnt pathways, as well as the pathways that regulate the actin cytoskeleton, focal adhesion, and tight junctions. Furthermore, we identified six differentially expressed miRNAs (oar-miR-103-3P, oar-miR-148b-3P, oar-miR-320-3P, oar-miR-31-5P, oar-novel-1-5P, and oar-novel-2-3P) that might target the key genes of the Wnt pathway. It has been reported that the Wnt pathway is critical for wool follicle development. Therefore, these miRNAs may regulate wool development through the Wnt pathway.

Conclusions

Our results provide new information on the identification and expression pattern of miRNAs in wool follicles. Our data might therefore aid in the understanding of the mechanisms of wool follicle development in sheep.     

Comparative proteome analysis of Aspergillus oryzae 3.042 and A. oryzae 100-8 strains: Towards the production of different soy sauce flavors

Aspergillus oryzae plays a central role in soybean fermentation, particularly in its contribution to the flavor of soy sauce. We present a comparative assessment of the intracellular differences between wild-type strain 3.042 and mutant strain A100-8, at the proteome level. 522 different protein spots were identified by MALDI-TOF MS, with 134 spots being confirmed by MALDI-TOF MS/MS. Of these, 451 were differentially expressed proteins (DEPs). There was at least a two-fold increase for 288 spots, and at least a two-fold decrease for 163 spots, in strain A100-8 when compared to 3.042. Further analysis showed that 63 of the more abundant proteins were involved in glycolysis and the citrate cycle; 43 more abundant proteins and 10 less abundant proteins were related to amino acid biosynthesis and metabolism; two of the more abundant proteins were involved in vitamin biosynthesis; and five of the more abundant proteins and four of the less abundant proteins were related to secondary metabolites. Moreover, quantitative real time PCR showed that the mRNA expression levels of six typical genes we selected were consistent with changes in protein expression. We postulate that there may be a relationship between DEPs and the flavor formation mechanism in A. oryzae.     

A novel carbonyl reductase from Pichia stipitis for the production of ethyl (S)-4-chloro-3-hydroxybutanoate

An NADPH-dependent carbonyl reductase (PsCR) gene from Pichia stipitis was cloned. It contains an open reading frame of 849 bp encoding 283 amino acids whose sequence had less than 60% identity to known reductases that produce ethyl (S)-4-chloro-3-hydroxybutanoates (S-CHBE). When expressed in Escherichia coli, the recombinant PsCR exhibited an activity of 27 U/mg using ethyl 4-chloro-3-oxobutanoate (COBE) as a substrate. Reduction of COBE to (S)-CHBE by transformants in an aqueous mono-phase system for 18 h, gave a molar yield of 94% and an optical purity of the (S)-isomer of more than 99% enantiomeric excess.     

Comparison of three ELISA kits for the differentiation of foot-and-mouth disease virus-infected from vaccinated animals

A study was performed to validate 3 FMDV 3ABC-I-ELISA kits developed in China for the differentiation of FMDV infected and vaccinated animals. Sets of sera from naive and vaccinated cattle as well as from cattle that had been infected were tested for antibodies against nonstructural proteins (NSPs) of FMDV by commercial diagnosis kits, Ceditest® FMDV-NS (Ceditest® kit), UBI® FMDV NONSTRUCTURAL PROTEIN ELISA DIRECTION INSERT (UBI® kit) and a FMDV 3ABC-I-ELISA kit developed at the Lanzhou Veterinary Research Institute. The test parameters (sensitivity and specificity) of the three kits were determined, and the result obtained from FMD 3ABC-I-ELISA kit was compared with that obtained from two foreign kits. The results indicated that the coincidence rate between the FMDV 3ABC-I-ELISA and Ceditest® kits was 98.05%, and the coincidence rate between the FMDV 3ABC-I-ELISA and UBI® kits was 94.4%; the sensitivity of both Ceditest® and FMDV 3ABC-I-ELISA kit was 100%. However, the sensitivity of the UBI® kit was only 81.8%. With sera from naive or vaccinated non-infected animals, the specificity of all tests exceeded 90%.     

Activation of STAT3 stimulates AHSP expression in K562 cells

Studies on the chaperone proteinα-hemoglobin stabilizing protein(AHSP)reveal that abundant AHSP in erythroid cells enhance the cells’tolerance to oxidative stress imposed by excessα-hemoglobin in pathological conditions.However,the potential intracellular modulation of AHSP expression itself in response to oxidative stress is still unknown.The present study examined the effect and molecular mechanism of STAT3,an oxidative regulator,on the expression of AHSP.AHSP expression increased in K562 cells upon cytokine IL-6-induced STAT3 activation and decreased in STAT3 knock-down K562 cells.Regulation of AHSP in oxidative circumstance was then examined inα-globin-overloaded K562 cells,and real-time PCR showed strengthened expression of both AHSP and STAT3.ChIP analysis showed binding of STAT3 to AHSP promoter and binding was significantly augmented with IL6 stimulation and uponα-globin overexpression.Dual luciferase reporter assays of the wildtype and mutated SB3 element,an IL-6RE site,in the AHSP promoter in K562 cells highlighted the direct regulatory effect of STAT3 on AHSP gene.Finally,direct binding of STAT3 to SB3 site of AHSP promoter was confirmed with EMSA assays.Our work reveals an adaptive AHSP regulation mediated by the redox-sensitive STAT3 signaling pathway,and provides clues to the therapeutic strategy for AHSP enhancement.     

A novel and highly efficient production system for recombinant adeno-associated virus vector

Recombinant adeno-associated virus(rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1(rHSV-1) designated HSV1-rc/△UL2, which expressed adeno-associated virus type2(AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein(GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/△UL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit(TU) or 4.28×104 particles per cell. Therefore, compared with the conventional transfection method, the yield of rAAV using this "one proviral cell line, one helper virus" strategy was increased by two orders of magnitude. Large-scale production of rAAV can be easily achieved using this strategy and might meet the demands for clinical trials of rAAV-mediated gene therapy.     

一株红壤溶磷菌的分离、鉴定及溶磷特性

【目的】为了提高红壤磷素利用率,探讨溶磷菌溶磷机理。【方法】利用难溶性无机盐培养基从花生根际土壤样品中分离到一株溶磷菌C5-A,结合菌落形态特征、生理生化和16S rRNA序列确定该菌株的系统发育地位;通过菌株C5-A在NBRIP液体培养基培养过程中培养液pH变化确定其溶磷能力;利用液体发酵实验测定不同的碳源、氮源对菌株C5-A溶磷的影响;通过高效液相色谱检测C5-A在不同氮源培养液中有机酸的种类和浓度。【结果】菌株C5-A鉴定为洋葱伯克霍尔德氏菌(Burkholderia cepacia),遗传稳定性较好。在FePO4和AlPO4培养液中,菌株C5-A的溶磷量和pH变化呈显著负相关;菌株C5-A对磷酸三钙、磷酸铝、磷酸铁、磷矿粉均有较强的溶解能力,最高溶磷量分别为125.79、227.34、60.02和321.15 mg/L;菌株C5-A对不同浓度的两种磷矿粉有较强的溶解能力;分别以麦芽糖和草酸铵为碳源和氮源时溶磷量最高。高效液相色谱检测出10种有机酸,分别为草酸(葡萄糖酸)、乙酸、苹果酸、琥珀酸和5种未知有机酸,然而,乙酸而非草酸似乎是影响C5-A溶磷的重要有机酸。【结论】从红壤花生根际土壤中筛选到一株对难溶性无机盐具有较强溶解能力溶的菌株C5-A,有望为开发高效红壤微生物磷肥提供种质资源。     

Functions of poly-gamma-glutamic acid (γ-PGA) degradation genes in γ-PGA synthesis and cell morphology maintenance

Poly-γ-glutamic acid (γ-PGA) is an important biopolymer with greatly potential in industrial and medical applications. In the present study, we constructed a metabolically engineered glutamate-independent Bacillus amyloliquefaciens LL3 strain with considerable γ-PGA production, which was carried out by single, double, and triple markerless deletions of three degradation genes pgdS, ggt, and cwlO. The highest γ-PGA production (7.12 g/L) was obtained from the pgdS and cwlO double-deletion strain NK-pc, which was 93 % higher than that of wild-type LL3 strain (3.69 g/L). The triple-gene-deletion strain NK-pgc showed a 28 % decrease in γ-PGA production, leading to a yield of 2.69 g/L. Furthermore, the cell morphologies of the mutant strains were also characterized. The cell length of cwlO deletion strains NK-c and NK-pc was shorter than that of the wild-type strain, while the ggt deletion strains NK-g, NK-pg, NK-gc, and NK-pgc showed longer cell lengths. This is the first report concerning the markerless deletion of γ-PGA degradation genes to improve γ-PGA production in a glutamate-independent strain and the first observation that γ-glutamyltranspeptidase (encoded by ggt) could be involved in the inhibition of cell elongation.