Rapid gene expression change in a novel synthesized allopolyploid population of cultivated peanut×Arachis doigoi cross by cDNA-SCoT and HFO-TAG technique

AIIopolyploidy has played an important role in plant evolution and heterosis. Recent studies indicate that the process of wide hybridization and （or） polyploidization may induce rapid and extensive genetic and epigenetic changes in some plant species. To better understand the allopolyploidy evolutionism and the genetic mechanism of Arachis interspecific hybridization, this study was conducted to monitor the gene expression variation by cDNA start codon targeted polymorphism （cDNA-SCoT） and cDNA high-frequency oligonucleotide-targeting active gene （cDNA-HFO-TAG） techniques, from the hybrids （F1） and newly synthesized allopolyploid generations （S0-$3） between tetraploid cultivated peanut Zhongkaihua 4 with diploid wild one Arachis doigoi. Rapid and considerable gene expression variations began as early as in the FI hybrid or immediately after chromosome doubling. Three types of gene expression changes were observed, including complete silence （gene from progenitors was not expressed in all progenies）, incomplete silence （gene expressed only in some progenies） and new genes activation. Those silent genes mainly involved in RNA transcription, metabolism, disease resistance, signal transduction and unknown functions. The activated genes with known function were almost retroelements by cDNA-SCoT technique and all metabolisms by cDNA-HFO-TAG. These findings indicated that interspecific hybridization and ploidy change affected gene expression via genetic and epigenetic alterations immediately upon allopolyploid formation, and some obtained transcripts derived fragments （TDFs） probably could be used in the research of molecular mechanism of Arachis allopolyploidization which contribute to thwe genetic diploidization of newly formed allopolyploids. Our research is valuable for understanding of peanut evolution and improving the utilization of putative and beneficial genes from the wild peanut.

Down-Regulated Expression of RACK1 Gene by RNA Interference Enhances Drought Tolerance in Rice

The receptor for activated C-kinase 1 （RACK1） is a highly conserved scaffold protein with versatile functions, and plays important roles in the regulation of plant growth and development. Transgenic rice plants, in which the expression of RACK1 gene was inhibited by RNA interference （RNAi）, were studied to elucidate the possible functions of RACK1 in responses to drought stress in rice. Real-time PCR analysis showed that the expression of RACK1 in transgenic rice plants was inhibited by more than 50%. The tolerance to drought stress of the transgenic rice plants was higher as compared with the non-transgenic rice plants. The peroxidation of membrane and the production of malondialdehyde were significantly lower and the superoxide dismutase activity in transgenic rice plants was significantly higher than those in non-trangenic rice plants It is suggested that RACK1 negatively regulated the redox system-related tolerance to drought stress of rice plants.

Application of the back-error propagation artificial neural network(BPANN) on genetic variants in the PPAR-γ and RXR-α gene and risk of metabolic syndrome in a Chinese Han population

This study was aimed to explore the associations between the combined effects of several polymorphisms in the PPAR-γ and RXR-α gene and environmental factors with the risk of metabolic syndrome by back-error propaga- tion artificial neural network （BPANN）. We established the model based on data gathered from metabolic syndrome patients （n = 1012） and normal controls （n = 1069） by BPANN. Mean impact value （MIV） for each input variable was calculated and the sequence of factors was sorted according to their absolute MIVs. Generalized multifactor dimensionality reduction （GMDR） confirmed a joint effect of PPAR-9＂ and RXR-a based on the results from BPANN. By BPANN analysis, the sequences according to the importance of metabolic syndrome risk fac- tors were in the order of body mass index （BMI）, serum adiponectin, rs4240711, gender, rs4842194, family history of type 2 diabetes, rs2920502, physical activity, alcohol drinking, rs3856806, family history of hypertension, rs1045570, rs6537944, age, rs17817276, family history of hyperlipidemia, smoking, rs1801282 and rs3132291. However, no polymorphism was statistically significant in multiple logistic regression analysis. After controlling for environmental factors, A1, A2, B1 and B2 （rs4240711, rs4842194, rs2920502 and rs3856806） models were the best models （cross-validation consistency 10/10, P = 0.0107） with the GMDR method. In conclusion, the interaction of the PPAR-γ and RXR-α gene could play a role in susceptibility to metabolic syndrome. A more realistic model is obtained by using BPANN to screen out determinants of diseases of multiple etiologies like metabolic syndrome.

Cloning, expression profiling and promoter functional analysis of bone morphogenetic protein 2 in the tongue sole(Cynoglossus semilaevis)

BMP2 plays crucial roles in vertebrate developmental process and acts as a bone inducer during osteogenesis. We present here the molecular cloning of bmp2 cDNA from the marine flatfish Cynoglossus semilaevis, and the analysis of bmp2 expression profiling and promoter function. The full length of bmp2 cDNA sequence is 2 048 bp,which encodes a protein of 422 amino acids. Tissue expression distribution of bmp2 was examined in 14 tissues of mature individuals by quantitative real time PCR（qRT-PCR）. The results revealed that bmp2 was expressed ubiquitously, and the highest expression level was detected in the spinal cord. Moreover, bmp2 expression levels were detected at 15 sampling time points of early developmental stages（egg, larva, juvenile and fingerling stages）.The highest expression level of bmp2 was observed at the gastrula stage, which was about ten times higher than those at the other three embryo stages. Whole-mount in situ hybridization showed that the bmp2 signal was strongly detected at the location of the crown-like larval fin, heart and liver, and slightly expressed in the notochord at one day post hatch（dph）; then the expression of bmp2 started to be concentrated in notochord at three dph. Subsequently, we characterized the 5′-flanking region of bmp2 by testing the promoter activity by Luciferase reporter assays. Positive regulatory region was detected at the location of –179 to ＋109. The predicted transcription factor binding sites（E-box binding factors, zinc finger transcription factor, etc.） in this region might participate in the transcriptional regulation of the bmp2 gene.

THE DNase-1 SENSITIVE REGIONS IN GENOMES OF BURKITT' S LYMPHOMA CELLS

This article reported the distribution of DNase-1 sensitive regions in genomes of three Burkitt's lymphoma cell lines, P3HR-1, Raji and Ramos cell lines using a new method of in situ nick translation of chromosomes substituted completely by BrdU. The results showed that the Blym locus on chromosomes In three cell lines and the c-myc locus on chromosomes in P3HR-1 were the DNase-1 sensitive regions and found that the rearrangemental sites of chromosomes present in three Burkltt' s lymphoma cell lines were sensitive to DNase-1 digestion, Indicating that c-myc, bcl-1 genes located at the rearrangemental sites and the Blym gene in Burkltt' s lymphoma are the active genes having the capability of expression.

Puromycin A, B and C, cryptic nucleosides identified from Streptomyces alboniger NRRL B-1832 by PPtase-based activation

Natural product discovery is pivot for drug development,however,this endeavor is often challenged by the wide inactivation or silence of natural products biosynthetic pathways.We recently developed a highly efficient approach to activate cryptic/silenced biosynthetic pathways through augmentation of the phosphopantetheinylation of carrier proteins.By applying this approach in the Streptomyces alboniger NRRL B-1832,we herein identified three cryptic nucleosides products,including one known puromycin A and two new derivatives(puromycin B and C).The biosynthesis of these products doesn't require the involvement of carrier protein,indicating the phosphopantetheinyl transferase(PPtase)indeed plays a fundamental regulatory role in metabolites biosynthesis.These results demonstrate that the PPtasebased approach have a much broader effective scope than the previously assumed carrier proteininvolving pathways,which will benefit future natural products discovery and biosynthetic studies.

Osteogenic potential of the human bone morphogenetic protein 2 gene activated nanobone putty

Background Nanobone putty is an injectable and bioresorbable bone substitute. The neutral-pH putty resembles hard bone tissue, does not contain polymers or plasticizers, and is self-setting and nearly isothermic, properties which are helpful for the adhesion, proliferation, and function of bone cells. The aim of this study was to investigate the osteogenic potential of human bone morphogenetic protein 2 （hBMP2） gene activated nanobone putty in inducing ectopic bone formation, and the effects of the hBMP2 gene activated nanobone putty on repairing bone defects. Methods Twenty four Kunming mice were randomly divided into two groups. The nanobone putty ＋ hBMP2 plasmid was injected into the right thigh muscle pouches of the mice （experiment side）. The nanobone putty ＋ blank plasmid or nanobone putty was injected into the left thigh muscle pouches of the group 1 （control side 1） or group 2 （control side 2）, respectively. The effects of ectopic bone formation were evaluated by radiography, histology, and molecular biology analysis at 2 and 4 weeks after operation. Bilateral 15 mm radial defects were made in forty-eight rabbits. These rabbits were randomly divided into three groups： Group A, nanobone putty ＋ hBMP2 plasmid; Group B, putty ＋ blank plasmid; Group C, nanobone putty only. Six rabbits with left radial defects served as blank controls. The effect of bone repairing was evaluated by radiography, histology, molecular biology, and biomechanical analysis at 4, 8, and 12 weeks after operation. Results The tissue from the experimental side of the mice expressed hBMP2. Obvious cartilage and island-distributed immature bone formation in implants of the experiment side were observed at 2 weeks after operation, and massive mature bone observed at 4 weeks. No bone formation was observed in the control side of the mice. The ALP activity in the experiment side of the mice was higher than that in the control side. The tissue of Group A rabbits expressed hBMP2 protein and higher ALP level. The new bone formation rate and antibending strength of group A was significantly higher than those of group B and C. The defects in blank control were not healed. Conclusions The hBMP2 gene activated nanobone putty exhibited osteoinductive ability, and had a better bone defect repair capabilitv than that of nanobone putty only.

N-carboxymethyl chitosan/sodium alginate composite hydrogel loading plasmid DNA as a promising gene activated matrix for in-situ burn wound treatment

Improving the degree of vascularization through the regulation of wound microenvironment is crucial for wound repair.Gene activated matrix(GAM)technology provides a new approach for skin regeneration.It is a local gene delivery system that can not only maintain a moist environment,but also increase the concentration of local active factors.For this purpose,we fabricated the mVEGF165/TGF-β1 gene-loaded N-carboxymethyl chitosan/sodium alginate hydrogel and studied its effect on promoting deep second degree burn wound repair.The average diameter of the hydrogel pores was 100μm and the porosity was calculated as 50.9%.SEM and CLSM images showed that the hydrogel was suitable for cell adhesion and growth.The NS-GAM could maintain continuous expression for at least 9 days in vitro,showing long-term gene release and expression effect.Deep second-degree burn wound model was made on the backs of Wistar rats to evaluate the healing effect.The wounds were healed by day 22 in NS-GAM group with the prolonged high expression of VEGF and TGF-β1 protein.A high degree of neovascularization and high expression level of CD34 were observed in NS-GAM group in 21 days.The histological results showed that NS-GAM had good tissue safety and could effectively promote epithelialization and collagen regeneration.These results indicated that the NS-GAM could be applied as a promising local gene delivery system for the repair of deep second-degree burn wounds.

Evaluation of bacterial pathogen diversity,abundance and health risks in urban recreational water by amplicon next-generation sequencing and quantitative PCR

The microbial quality of urban recreational water is of great concern to public health.The monitoring of indicator organisms and several pathogens alone is not sufficient to accurately and comprehensively identify microbial risks.To assess the levels of bacterial pathogens and health risks in urban recreational water,we analyzed pathogen diversity and quantified four pathogens in 46 water samples collected from waterbodies in Beijing Olympic Forest Park in one year.The pathogen diversity revealed by 16 S r RNA gene targeted next-generation sequencing（NGS） showed that 16 of 40 genera and 13 of 76 reference species were present.The most abundant species were Acinetobacter johnsonii,Mycobacterium avium and Aeromonas spp.Quantitative polymerase chain reaction（q PCR） of Escherichia coli（uid A）,Aeromonas（aer A）,M.avium（16S r RNA）,Pseudomonas aeruginosa（oaa） and Salmonella（inv A） showed that the aer A genes were the most abundant,occurring in all samples with concentrations of 10^（4–6） genome copies/100 m L,followed by oaa,inv A and M.avium.In total,34.8% of the samples harbored all genes,indicating the prevalence of these pathogens in this recreational waterbody.Based on the q PCR results,a quantitative microbial risk assessment（QMRA） showed that the annual infection risks of Salmonella,M.avium and P.aeruginosa in five activities were mostly greater than the U.S.EPA risk limit for recreational contacts,and children playing with water may be exposed to the greatest infection risk.Our findings provide a comprehensive understanding of bacterial pathogen diversity and pathogen abundance in urban recreational water by applying both NGS and q PCR.

Tissue engineering in mandibular reconstruction: osteogenesis-inducing scaffolds

Currently, the gold standard for aesthetic and functional reconstruction of critical mandibular defects is an autologous fibular flap;however, this carries risk of donor site morbidity, and is not a promising option in patients with depleted donor sites due to previous surgeries. Tissue engineering presents a potential solution in the design of a biomimetic scaffold that must be osteoconductive, osteoinductive, and support osseointegration. These osteogenesis-inducing scaffolds are most successful when they mimic and interact with the surrounding native macro- and micro-environment of the mandible. This is accomplished via the regeneration triad: (1) a biomimetic, bioactive osteointegrative scaffold, most likely a resorbable composite of collagen or a synthetic polymer with collagen-like properties combined with beta-tri calcium phosphate that is 3D printed according to defect morphology;(2) growth factor, most frequently bone morphogenic protein 2 (BMP-2);and (3) stem cells, most commonly bone marrow mesenchymal stem cells. Novel techniques for scaffold modification include the use of nano-hydroxyapatite, or combining a vector with a biomaterial to create a gene activated matrix that produces proteins of interest (typically BMP-2) to support osteogenesis. Here, we review the current literature in tissue engineering in order to discuss the success of varying use and combinations of scaffolding materials (i.e., ceramics, biological polymers, and synthetic polymers) with stem cells and growth factors, and will examine their success in vitro and in vivo to induce and guide osteogenesis in mandibular defects.

An Experimental Study on the Flexibility of Prevention against Thrombosis Following Mechanical Valve Replacement by tPA Gene Transduction

Objectives Use a gene suture immersed recombinant tissue-type plasminogen activator （r-tPA）expression plasmid to transduce myocardia to prevent the thrombosis after mechanical tricuspid valve replacement in pigs. Methods A r-tPA gene plasmid was constructed and conjugated to a novel cationic phosphonolipid and a r-tPA gene suture was made. Eighteen pigs were selected and divided into two groups at randomization. There were 9 pigs in the experimental group and 9 in the control group, all the 18 pigs＇ tricuspids were replaced with mechanical valves. The gene threads were sutured into the right ventricular walls near mechanical valves and an ultrasound was used on the surfaces of the right ventricular walls for the gene transfer in the experimental group. Coagulative function, D-dimer level of the blood and the thrombosis on the surfaces of the valves were observed. Results r-tPA gene plasmid was successfully constructed and r-tPA protein was expressed in the ventricular cells around the gene sutures. D-dimer reached its peak level （ 1.67 ±0. 79） μg · mL^-1 in 1 week after operation in two groups, but it decreased to preoperation level thereafter in control group and kept on the high level and reincreased to a new high level （ 1.89 ± 0.79 ） μg · mL^-1 until the end of the experiment in experimental group. The thromboses around the valves were found in all the control group （100%） but only 1 （ 11.11% ） case in experimental group. There were no changes in prothrombin time pre and post operation in two groups. Conclusions Using gene suture immersed r-tPA expression plasmid to transduce myocardia might be a best substitution for life long anti-coagulation therapy for the patients, who underwent operation.

Quantitative characterization of filamentous fungal promoters on a single-cell resolution to discover cryptic natural products

Characterization of filamentous fungal regulatory elements remains challenging because of time-consuming transformation technologies and limited quantitative methods.Here we established a method for quantitative assessment of filamentous fungal promoters based on flow cytometry detection of the superfolder green fluorescent protein at single-cell resolution.Using this quantitative method,we acquired a library of 93 native promoter elements from Aspergillus nidulans in a high-throughput format.The strengths of identified promoters covered a 37-fold range by flow cytometry.P_(zipA) and P_(sltA)were identified as the strongest promoters,which were 2.9-and 1.5-fold higher than that of the commonly used constitutive promoter P_(gpdA).Thus,we applied P_(zipA)and P_(sltA)to activate the silent nonribosomal peptide synthetase gene Afpes1 from Aspergillus fumigatus in its native host and the heterologous host A.nidulans.The metabolic products of Afpes1 were identified as new cyclic tetrapeptide derivatives,namely,fumiganins A and B.Our method provides an innovative strategy for natural product discovery in fungi.

Transcription-Coupled Replacement of Histones:Degradation or Recycling?

Histone modifications are proposed to constitute a ＂histone code＂ for epigenetic regulation of gene expression. However, recent studies demonstrate that histones have to be disassembled from chromatin during transcription. Recent evidence, though not conclusive, suggests that histories might be degradable after being removed from chromatin during transcription. Degradation of overexpressed excessive histones, instead of native histones, has been shown to be dependent on proteasomes and ubiquitination. Since the 26S proteasome usually recognizes polyubiquitinated substrates, it is critical to demonstrate whether degradation of histones is mediated by polyubiquitination. Unexpectedly, there is almost no evidence that any ubiquitin ligase can promote polyubiquitination-dependent degradation of constitutive histones. Meanwhile, acetylation and phosphorylation are also associated with histone degradation. This review attempts to summarize the current knowledge on the transcription-coupled degradation of histones and its regulation by posttranslational protein modifications.

Expression and significance of urokinase-type plasminogen activator in human gliomas

To investigate the expression and clinical significance of urokinase type plasminogen activator (uPA) in human gliomas Methods mRNA and protein expressions of uPA were examined by Northern blot hybridization and immunohistochemical method in 43 cases of gliomas and 5 cases of normal brain tissues and their relationship to clinical indexes was comprehensively analyzed Results All tissues expressed the 2 5*!kb transcript of uPA mRNA The uPA mRNA level in high-grade gliomas was considerably higher than that in low-grade gliomas and normal brain tissues ( P <0 01) Levels of uPA mRNA expression in tumor tissues with recurrence during 18 postoperative months and a survival period less than 3 years, were significantly higher than counterparts ( P <0 01) uPA mRNA expression was strongly correlated with the microvessel quantity (MVQ) in gliomas ( r =0 56, P <0 01) uPA protein was mainly distributed in tumor cells and endothelial cells of glioblastomas and anaplastic astrocytomas Conclusion Expression of uPA is associated with the malignant progression, invasion and angiogenesis of gliomas, and it may play a critical role in the recurrence and prognosis of gliomas