Multi-genome evolutionary study of the ABC1 gene family and identification of the pleiotropic effects of OsABC1-13 in rice development

In four rice genomes,85 ABC1-family genes were identified by comparative genomics,evolution,genetics,and physiology.One,OsABC1-13,was shown by knockdown and knockout experiments to affect plant height,grain size,and photosynthetic capability.

FIT interacts with AtbHLH38 and AtbHLH39 in regulating iron uptake gene expression for iron homeostasis in Arabidopsis

Iron is an essential element for plant growth and development. Iron homeostasis in plants is tightly regulated at both transcriptional and posttranscriptional level. Several bHLH transcription factors involved in iron homeostasis have been identified recently. However, their regulatory mechanisms remain unknown. In this work, we demonstrate that the transcription factor FIT interacted with AtbHLH38 and AtbHLH39 and directly conferred the expression regulation of iron uptake genes for iron homeostasis in Arabidopsis. Yeast two-hybrid analysis and transient expression in Arabidopsis protoplasts showed that AtbHLH38 or AtbHLH39 interacted with FIT, a central transcription factor involved in iron homeostasis in Arabidopsis. Expression of FIT/AtbHLH38 or FIT/AtbHLH39 in yeast cells activated GUS expression driven by ferric chelate reductase （FRO2） and ferrous transporter （IRT1） promoters. Overexpression of FITwith either AtbHLH38 or AtbHLH39 in plants converted the expression of the iron uptake genes FRO2 and IRT1 from induced to constitutive. Further analysis revealed that FRO2 and IRT1 were not regulated at the posttranscriptional level in these plants because IRT1 protein accumulation and high ferric chelate reductase activity were detected in the overexpression plants under both iron deficiency and iron sufficiency. The double overexpression plants accumulated more iron in their shoots than wild type or the plants overexpressing either AtbHLH38, AtbHLH39 or FIT. Our data support that ferric-chelate reductase FRO2 and ferrous-transporter IRT1 are the targets of the three transcription factors and the transcription of FRO2 and IRT1 is directly regulated by a complex of FIT/AtbHLH38 or FIT/AtbHLH39.

Expression of SNC73, a transcript of the immunoglobulin α-1 gene, in human epithelial carcinomas

AIM： To investigate the expression of SNC73, a transcript of the immunoglobulin α-1 gene （IgA1-H chain）, in human epitheliα-derived tumor cells. METHODS： Total RNAs and cell lysates were prepared from five different human epithelial cell lines derived from lung, stomach, liver, skin, and breast, respectively. RT-PCR and immunoblot analysis of these five cell lines were done. Both RT-PCR and immunochemistry were used to detect the expression of SNC73 in these cell lines. We also examined the expression of SNC73 in normal epithelial cells of colon mucosa by in situ hybridization. RT-PCR and immunoblot analysis were used to determine whether the recombination activating gene1/2 （RAG1 and RAG2） is present. The expression of three immunoglobulin transcription factors, EBF, E2A and Pax5, and the heavy chain of IgA1 and two types of light chains of immunoglobulin （κ and λ） in the aforementioned cell lines were analyzed by RT-PCR and immunochemistry, respectively. All the RT-PCR products were analyzed by sequencing. RESULTS： The results of RT-PCR and immunochemistry showed that both mRNA and protein of SNC73 were expressed in five human epitheliα-derived cancer cell lines. These data were further confirmed in the normal epithelial cells of colon mucosa by in situ hybridization. Also, the heavy chain of IgA1 and κ light chain were detected in these cells, but no λ light chain was observed. Both RAG1 and RAG2 were expressed in these human epitheliα-derived cancer cell lines and the sequence was identical to that expressed in pre-B and pre-T cells. In addition to RAG1 and RAG2, the mRNA in one of the immunoglobulin transcription factors, EBF, was also detected in these cell lines, and Pax5 was only expressed in SW480 cells, but no expression of E2A was observed in all the five cell lines. CONCLUSION： Immunoglobulin A1 is originally expressed and V（D）J recombination machine is also present in non-lymphoid cells, suggesting that V（D）J recombination machine mediates the assembly of immunoglobulin A1 in non-lymphoid cells as in prelymphocytes.

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.

Assessing effect of interaction between the FTO A/T polymorphism(rs9939609)and physical activity on obesity-related traits

Background： The first described obesity-susceptibility gene was the fat mass and obesity-associated （FTO） gene. However, knowledge about FTO＇s potential modifying effect on changes in body weight achieved through a training program is still limited. We decided to study the association between the FTO A/T polymorphism （rs9939609） and obesity-related traits. Additionally, we investigated whether body mass and body composition, as well as metabolic variables observed in physically active participants, are modulated by the FTO polymorphism. Methods： A group of 201 young Polish women were recruited for the study. The genotype distribution was examined in participants measured for selected changes before and after the completion of a 12-week training program. Results： Our results confirm the association between the common FTO A/T polymorphism and increased body mass index （BMI）. Subjects with AA and AT genotypes had higher BMI during the entire study period compared with the TT genotype. Although parameters such as BMI, basal metabolism rate, tissue independence, fat mass percentage, fat mass, fat-free mass, total body water, high-density lipoprotein, and glucose changed significantly during the training program, none of the examined parameters changed significantly across the FTO genotypes （genotype × training interaction）. Conclusion： We confirm an association between the FTO A/T polymorphism and increased BMI; this polymorphism is therefore a candidate for influencing obesity and other disease-related phenotypes. Although the gene x physical activity interaction was not shown, we want to point out that promoting physical activity is an important approach to controlling the increasing obesity epidemic.2018 Published by Elsevier B.V. on behalf of Shanghai University of Sport. This is an open access article under the CC BY-NC-ND license. （http：//creativecommons.org/licenses/by-nc-nd/4.0/）.

Systematic Identification of Rice ABC1 Gene Family and Its Response to Abiotic Stress

Members of the activity of bc1 complex (ABC1) family are protein kinases that are widely found in prokaryotes and eukaryotes. Previous studies showed that several plant ABC1 genes participated in the abiotic stress response. Here, we present the systematic identification of rice and Arabidopsis ABC1 genes and the expression analysis of rice ABC1 genes. A total of 15 and 17 ABC1 genes from the rice and Arabidopsis genomes, respectively, were identified using a bioinformatics approach. Phylogenetic analyses of these proteins suggested that the divergence of this family had occurred and their main characteristics were established before the monocot-dicot split. Indeed, species-specific expansion contributed to the evolution of this family in rice and Arabidopsis after the monocot-dicot split. Intron/exon structure analysis indicated that most of the orthologous genes had similar exon sizes, but diverse intron sizes, and the rice genes contained larger introns, moreover, intron gain was an important event accompanying the recent evolution of the rice ABC1 family. Multiple sequence alignment revealed one conserved amino acid segment and four conserved amino acids in the ABC1 domain. Online subcellular localization predicted that nine rice ABC1 proteins were localized in chloroplasts. Real-time RT-PCR established that the rice ABC1 genes were primarily expressed in leaves and the expression could be modulated by a broad range of abiotic factors such as H2O2, abscisic acid, low temperature, drought, darkness and high salinity. These results reveal that the rice ABC1 gene family plays roles in the environmental stress response and specific biological processes of rice.

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.

Telomerase activity and homozygous deletions of the p16 gene in liver metastases of colorectal carcinoma

Objective To study the interaction between telomerase activity and abnormalities of the p16 gene in liver metastases of colorectal carcinoma.Methods Telomerase activity was detected by a non-isotopic PCR-based telomeric repeat amplification protocol (TRAP) assay, and homozygous deletions of the p16 gene were detected by a semiquantitative multiplex polymerase chain reaction in tissue samples from 24 liver metastases of colorectal carcinoma and 5 primary colorectal carcinomas.Results Telomerase activity was observed in 19 (79.2%) of 24 liver metastases of colorectal carcinoma.Telomerase activity was also observed in all 5 primary colorectal carcinomas and in 3 of their liver metastatic samples. The incidence of telomerase activity in liver metastases of colorectal carcinoma was not significantly correlated to tumor diameter, number of tumors, cirrhosis, and HBsAg. Homozygous deletions of the p16 gene were found in 9 of 24 (37.5%) liver metastases of colorectal carcinoma. Homozygous deletions of the p16 gene were observed in 2 of the 5 primary colorectal carcinomas and in 1 of the matching liver metastatic cancers. There was a correlation between telomerase activity and homozygous deletions of the p16 gene.Conclusions There is a correlation between telomerase activity and homozygous deletions of the p16gene in liver metastases of colorectal carcinoma, suggesting its crucial role in liver metastases. However,telomerase activation and homozygous deletions of the p16 gene might not be the initiating event in liver metastases of colorectal carcinoma.

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.

Interleukin-1 gene polymorphism disease activity and bone mineral metabolism in rheumatoid arthritis

Objective To determine whether interleukin-1α and 1β gene polymorphism is associated with rheumatoid arthritis disease activity and bone mineral metabolism, and whether there is any relationship between IL-1β and rheumatoid arthritis (RA) motif gene. Methods IL-1 gene polymorphisms were analyzed in 65 RA patients who met American College of Radiology (ACR) criteria and 60 controls. From genomic DNA, 2 polymorphisms in each gene for IL1α-889 and IL-1β+3953 were typed by PCR-RFLP and HLA-DRB1 allele typing was also undertaken by PCR-SSOP. Some clinical and laboratory parameters were collected. The allelic frequencies and carriage rates were compared between RA patients and controls and between patients with active and quiescent disease. Comparison was also made between IL-1 polymorphism and parameters of bone mineral metabolism and between patients with the HLA-DRB1 RA motif plus IL-1β 2 and patients without the two alleles. Fisher test and the analysis of variance was used to analyze the data.Results There was no significant difference in the frequency and carriage rate of IL-1α polymorphisms between RA patients and the controls. The β2/2 genotype of IL-1β was more common in female RA patients compared with controls (P=0.001). A lower carriage rate of IL-1β 2 occurred in male RA patients (P=0.001). A higher carriage rate of IL-1α2 is associated with a higher ESR (P=0.008), HAQ score (P=0.03), and vit-D 3 (P【0.001), but conversely a lower SJC (p=0.002), a lower RF (P=0.002) and a lower BMD at the lumbar spine (P=0.001). A higher frequency of IL-1α1 is associated with a lower CRP value (P=0.009). An increased IL-1β2 carriage is associated with active rheumatoid disease as indicated by a higher CRP (P【0.001), ESR (P【0.001) and pain score (P=0.001) and a higher BMD at the lumbar spine (P=0.007), lower vit-D 3 and. Udpd/Crea level The presence of the HLA DRB1 RA motif and IL-1β allele 2 at same time did not contribute to disease activity.Conclution Polymorphisms of the IL-β gene may affect the RA occurrence. Carriage of IL-1β2 polymorphisms is associated with more active disease in RA and the presence of both the IL-1α2 and the IL-1β1 allele in RA influences bone resorption.

Relationship between gene polymorphism of the PAI-1 promoter and myocardial infarction

abstractObjective To investigate the association between gene polymorphism of the plasminogen activator inhibitor 1 (PAI 1) and myocardial infarction (MI) in Chinese Methods PAI 1 genotyping with polymerase chain reaction restriction fragment length polymorphism (PCR RFLP) and allele specific polymerase chain reaction (ASPCR) was performed in 87 myocardial infarction patients and 92 unrelated healthy controls All subjects'clinical features and PAI 1 activity were tested Results There were two polymorphisms within the promoter, a G/A single base substitution polymorphism upstream at -844?bp, and a single guanosine deletion/insertion 4G/5G polymorphism -675?bp upstream from the start of transcription Significant differences between the patients and the controls were observed neither for the frequencies of the GG, GA and AA genotypes nor for the PAI 1 activities of these three types But for the 4G/5G polymorphism, there were significant differences between patients and controls for the frequencies of the 4G/4G, 4G/5G and 5G/5G genotypes ( P <0 05) In the MI group, the PAI 1 activity of the 4G/4G type was significantly higher than that of the 5G/5G type ( P <0 05) Further more, a positive correlation between the glucose level and PAI 1 activity was found ( r =0 34, P =0 02) Conclusion This study indicates that the 4G/5G gene polymorphism of PAI 1 is associated with myocardial infarction, that 4G/4G type is probably an important hereditary risk factor, and that glucose has functional importance in regulating PAI 1 activity

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.

Stochastic equations for a self-regulating gene

Expression of cellular genes is regulated by binding of transcription factors to their promoter, either activating or inhibiting transcription of a gene. Particularly interesting is the case when the expressed protein regulates its own transcription. In this paper, the features of this self-regulating process are investigated. In the presented model here, the gene can be in two states. Either a protein is bound to its promoter or not. The steady state distributions of protein during and just before switching from one state to the next state are analyzed. Moreover, a powerful numerical method based on the corresponding master equation to compute the protein distribution in the steady state is presented and compared to an already-existing method. Additionally the special case of self-regulation, in which protein can only be produced, if one of these proteins is bound to the promoter region, is analyzed. Furthermore, a self-regulating gene is compared to a similar gene, which also has two states and produces the same amount of proteins but is not regulated by its protein-product.

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.