Objective To study the gene expression profiles of megakaryocytes(MKs) from human cord blood CD34+ cells in vitro expanded and to understand megakaryopoiesis at the molecular level. Methods CD34+ cells were isolated using density gradient centrifugation and magnetic activated cell sorting. The cells were cultured and stimulated with recombinant human TPO ( 100 ng/ml). After 12 days, the MKs fraction was separated using an anti-CD41 monoclonal antibody by immunomagnetic sorting. The gene expression profiles of MKs, non-MKs as well as meg-01 cells were studied by gene chip assay. THBSI, HOX A9,β-actin, lL-8,Annexin A6, FGF-8 were selected to validate the gene chip results by RT-PCR. Results A total of 116 genes between MKs and non-MKs cells were significantly different, 52 genes were up-regulated and 64 genes were down-regulated. In addition, 158 genes between MKs and meg-01 cells were significantly different, 71 genes were up-regulated and 87 genes were down-regulated. THBSI showed higher expression in MKs than in non-MKs. HOXA9 showed lower expression in MKs than in non-MKs. The expression of β-actin did not show any significant difference in MKs and non-MKs. IL-8 showed higher expression in MKs than in meg-01 cells, while ANXA6 showed lower expression in MKs than in meg-01 cells. The expression of FGF-8 did not show any significant difference between MKs and meg-01 cells. Conclusions MKs, non-MKs and meg-01 cells show different gene expression profiles. The regulatory genes include stress response genes,immune related genes, DNA synthesis and repair genes, metabolism genes, pro-onco genes and tumor suppressor genes.

Objective To screen the factors that can affect α-toxin expression of CA-MRSA except for quorum-sensing system and to investigate the regulative mechanism of the interesting genes. Methods S. aureus CA-MRSA transposon mutagenesis library was constructed by using mariner based transposon mutagenesis system. The clones with significantly changed level of hemolysis were selected, the location of erm insertion in a gene was confirmed by arbitrary primed (inverse) PCR and nucleotide sequence. Genetic complementation, mice bacteremia and skin abscess models and real time RT-PCR were used to study the function of the interesting gene. Results Twenty-five mutants with down-expression of α-toxin were selected by screening about 104 isolates of transposon mutagenesis library. The hemolytic diameter of CA-MRSA wild type was about 212 mm, no clear hemolysis was found in AraC-, The hemolytic diameter of AraC-pT181 araC was about 197 mm. Real time RT-PCR results showed that compared to the expression of the virulence factors in CA-MRSA wild type( PSMα 257. 30 ±37. 33 ;agr 115. 60 ±0. 81 and α-toxin 3.23 ±0. 21), in AraC-, α-toxin, PSMα and agr were significantly down regulated(α-toxin 1.09 ±0.01 :t = 10. 18, P <0.01 ;PSMα 34.85 ±2. 15:t=5.95,P<0.05;agr35. 19 ±1. 72:t =42. 33, P<0. 01). The result of mice bacteremia model showed that the virulence of wild type and AraC- ( (x) ± s ) were significantly different (x2 = 21. 34, P < 0.01). The expression of PSMα, agr and α-toxin in AraC-pT181araC ( PSMa 180.10 ± 15.29;agr 101. 50 ±8. 96;α-toxin 2.59 ±0.26) had no significant difference compared to the expression of the virulent factors in CA-MRSA wild type (PSMα: t =1.914, P>0.05;agr:t= 1.563, P>0.05;α-toxm: t = 1. 923, P > 0. 05 ). There were no significant difference of the expression of ClpP in AraC-(0. 21 ±0.01) and in AraC-pT181araC(0.17 ±0.03)compared to the expression of ClpP in CA-MRSA wild type (0. 20 ± 0.01) (t=0.555, P>0.05 and t=0. 851, P>0.05). The result of mice skin abscess model showed that the dermonecrosis area caused by CA-MRSA was (136. 5 ±21.45) mm2, the dermonecrosis area caused by AraC- was (55. 69 ± 13. 81) mm2, the different was significant (t = 3.169, P < 0. 05). Conclusion In CA-MRSA, AraC-type transcriptional regulator controlled the pathogenesis of CA-MRSA by regulating the expression of the most important virulence factors such as hla, PSMα and agr.

Nitrate is the main form of inorganic nitrogen that crop absorbs, and nitrate transporter 2 (NRT2) is a high affinity transporter using nitrate as a specific substrate. When the available nitrate is limited, the high affinity transport systems are activated and play an important role in the process of nitrate absorption and transport. Most NRT2 cannot transport nitrates alone and require the assistance of a helper protein belonging to nitrate assimilation related family (NAR2) to complete the absorption or transport of nitrates. Crop nitrogen utilization efficiency is affected by environmental conditions, and there are differences between varieties, so it is of great significance to develop varieties with high nitrogen utilization efficiency. Sorghum bicolor has high stress tolerance and is more efficient in soil nitrogen uptake and utilization. The S. bicolor genome database was scanned to systematically analyze the gene structure, chromosomal localization, physicochemical properties, secondary structure and transmembrane domain, signal peptide and subcellular localization, promoter region cis-acting elements, phylogenetic evolution, single nucleotide polymorphism (SNP) recognition and annotation, and selection pressure of the gene family members. Through bioinformatics analysis, 5 NRT2 gene members (designated as SbNRT2-1a, SbNRT2-1b, SbNRT2-2, SbNRT2-3, and SbNRT2-4) and 2 NAR2 gene members (designated as SbNRT3-1 and SbNRT3-2) were identified, the number of which was less than that of foxtail millet. SbNRT2/3 were distributed on 3 chromosomes, and could be divided into four subfamilies. The genetic structure of the same subfamilies was highly similar. The average value of SbNRT2/3 hydrophilicity was positive, indicating that they were all hydrophobic proteins, whereas α-helix and random coil accounted for more than 70% of the total secondary structure. Subcellular localization occurred on plasma membrane, where SbNRT2 proteins did not contain signal peptides, but SbNRT3 proteins contained signal peptides. Further analysis revealed that the number of transmembrane domains of the SbNRT2s family members was greater than 10, while that of the SbNRT3s were 2. There was a close collinearity between NRT2/3s of S. bicolor and Zea mays. Protein domains analysis showed the presence of MFS_1 and NAR2 protein domains, which supported executing high affinity nitrate transport. Phylogenetic tree analysis showed that SbNRT2/3 were more closely related to those of Z. mays and Setaria italic. Analysis of gene promoter cis-acting elements indicated that the promoter region of SbNRT2/3 had several plant hormones and stress response elements, which might respond to growth and environmental cues. Gene expression heat map showed that SbNRT2-3 and SbNRT3-1 were induced by nitrate in the root and stem, respectively, and SbNRT2-4 and SbNRT2-3 were induced by low nitrogen in the root and stem. Non-synonymous SNP variants were found in SbNRT2-4 and SbNRT2-1a. Selection pressure analysis showed that the SbNRT2/3 were subject to purification and selection during evolution. The expression of SbNRT2/3 gene and the effect of aphid infection were consistent with the expression analysis results of genes in different tissues, and SbNRT2-1b and SbNRT3-1 were significantly expressed in the roots of aphid lines 5-27sug, and the expression levels of SbNRT2-3, SbNRT2-4 and SbNRT3-2 were significantly reduced in sorghum aphid infested leaves. Overall, genome-wide identification, expression and DNA variation analysis of NRT2/3 gene family of Sorghum bicolor provided a basis for elucidating the high efficiency of sorghum in nitrogen utilization.
Nitrate Transporters ; Nitrates/metabolism* ; Sorghum/metabolism* ; Anion Transport Proteins/metabolism* ; Phylogeny ; Protein Sorting Signals/genetics* ; Nitrogen/metabolism* ; DNA ; Gene Expression Regulation, Plant ; Plant Proteins/metabolism*