The association between genetic variants at 3'-UTR and 5'-URR of HLA-G gene and the clinical outcomes of patients with leukemia receiving hematopoietic stem cell transplantation.

In addition to the classical human leukocyte antigen (HLA) genes, the outcomes of post-hematopoietic stem cell transplantation (HSCT) are associated with human leukocyte antigen (HLA)-related genes and non-HLA genes involved in immune regulation. HLA-G gene plays an important role in immune tolerance, assisting immune escape of tumor cells, and decrease of transplant rejection. In this study, we explored the association of genetic variants at the 3'-untranslated region (3'-UTR) and 5'-upstream regulatory region (5'-URR) of HLA-G gene with the adverse outcomes of patients with leukemia receiving HSCT. The genomic DNAs of 164 patients who had acute leukemia and received HSCT were collected for analysis. Nine single nucleotide polymorphisms (SNPs) and six haplotypes in the 3'-UTR and 27 SNPs and 6 haplotypes in the 5'-URR were selected to investigate their relationship with the development of adverse outcomes for patients receiving HSCT, including mortality, relapse, and graft-versus-host disease. Our results revealed that two SNPs (rs371194629 and rs9380142) and one haplotype (UTR-3) located in the 3'-UTR and two SNPs (rs3823321 and rs1736934) and one haplotype (G0104a) located in the 5'-URR of HLA-G were associated with the occurrence of chronic GVHD or development of any forms of GVHD. No SNP was found to associate with the occurrence of mortality and relapse for patients receiving HSCT. These SNPs and haplotypes may play important roles in regulating immune tolerance of allografts post-HSCT that can be used to predict the risk of poor outcomes after receiving HSCT and giving preventive treatment to patients on time.

Single-Nucleotide Polymorphisms Within Non-HLA Regions Are Associated With Engraftment Effectiveness for Patients With Unrelated Cord Blood Transplantation.

Clinically, stem cells with matched human leukocyte antigens (HLAs) must be selected for allogeneic transplantation to avoid graft rejection. However, adverse reactions still occur after cord blood transplantation (CBT). It was inferred that the HLA system is not the only regulatory factor that may influence CBT outcomes. Therefore, we plan to investigate whether the single-nucleotide polymorphisms (SNPs) located in non-HLA genes are associated with the effectiveness of CBT. In this study, the samples of 65 donors from CBT cases were collected for testing. DNA sequencing was focused on the SNPs of non-HLA genes, cytotoxic T-lymphocyte-associated protein 4 (CTLA4), CD28, tumor necrosis factor ligand superfamily 4 (TNFSF4), and programmed cell death protein 1 (PDCD1), which were selected in regard to the literatures published in 2017 and 2018, which indicated that they were related to stem cell transplantation. Then, in combination with the detailed follow-up transplantation tracking database, these SNPs were analyzed with the risk of mortality, relapse, cytomegalovirus (CMV) infection, and graft-versus-host disease (GVHD). We found that there were 2 SNPs of CTLA4, 1 SNP of TNFSF4, and 2 SNPs of PDCD1 associated with the effectiveness of unrelated CBT. These statistically significant SNPs and haplotypes would be used in clinical to choose the best donor for the patient receiving CBT. Moreover, the polygenic risk scores (PRSs) with these SNPs could be used to predict the risk of CBT adverse reactions with an area under the receiver operating characteristic curve (AUC) of 0.7692. Furthermore, these SNPs were associated with several immune-related diseases or cancer susceptibility, which implied that SNPs play an important role in immune regulation.

The adverse events of haematopoietic stem cell transplantation are associated with gene polymorphism within human leukocyte antigen region.

Adverse reactions may still occur in some patients after receiving haematopoietic stem cell transplantation (HSCT), even when choosing a human leukocyte antigen (HLA)-matched donor. The adverse reactions of transplantation include disease relapse, graft-versus-host disease (GVHD), mortality and CMV infection. However, only the relapse was discussed in our previous study. Therefore, in this study, we investigated the correlation between the gene polymorphisms within the HLA region and the adverse reactions of post-HSCT in patients with acute leukaemia (n = 176), where 72 patients were diagnosed with acute lymphocytic leukaemia (ALL) and 104 were acute myeloid leukaemia (AML). The candidate single nucleotide polymorphisms were divided into three models: donor, recipient, and donor-recipient pairs and the data of ALL and AML were analysed individually. Based on the results, we found 16 SNPs associated with the survival rates, the risk of CMV infection, or the grade of GVHD in either donor, recipient, or donor-recipient matching models. In the ALL group, the rs209132 of TRIM27 in the donor group was related to CMV infection (p = 0.021), the rs213210 of RING1 in the recipient group was associated with serious GVHD (p = 0.003), and the rs2227956 of HSPA1L in the recipient group correlated with CMV infection (p = 0.001). In the AML group, the rs3130048 of BAG6 in the donor-recipient pairs group was associated with serious GVHD (p = 0.048). Moreover, these SNPs were further associated with the duration time of survival after transplantation. These results could be applied to select the best donor in HSCT.

Genetic Diversity of Landraces and Improved Varieties of Rice (Oryza sativa L.) in Taiwan.

BACKGROUND: Rice, the most important crop in Asia, has been cultivated in Taiwan for more than 5000 years. The landraces preserved by indigenous peoples and brought by immigrants from China hundreds of years ago exhibit large variation in morphology, implying that they comprise rich genetic resources. Breeding goals according to the preferences of farmers, consumers and government policies also alter gene pools and genetic diversity of improved varieties. To unveil how genetic diversity is affected by natural, farmers', and breeders' selections is crucial for germplasm conservation and crop improvement. RESULTS: A diversity panel of 148 rice accessions, including 47 cultivars and 59 landraces from Taiwan and 42 accessions from other countries, were genotyped by using 75 molecular markers that revealed an average of 12.7 alleles per locus with mean polymorphism information content of 0.72. These accessions could be grouped into five subpopulations corresponding to wild rice, japonica landraces, indica landraces, indica cultivars, and japonica cultivars. The genetic diversity within subpopulations was: wild rices > landraces > cultivars; and indica rice > japonica rice. Despite having less variation among cultivars, japonica landraces had greater genetic variation than indica landraces because the majority of Taiwanese japonica landraces preserved by indigenous peoples were classified as tropical japonica. Two major clusters of indica landraces were formed by phylogenetic analysis, in accordance with immigration from two origins. Genetic erosion had occurred in later japonica varieties due to a narrow selection of germplasm being incorporated into breeding programs for premium grain quality. Genetic differentiation between early and late cultivars was significant in japonica (FST = 0.3751) but not in indica (FST = 0.0045), indicating effects of different breeding goals on modern germplasm. Indigenous landraces with unique intermediate and admixed genetic backgrounds were untapped, representing valuable resources for rice breeding. CONCLUSIONS: The genetic diversity of improved rice varieties has been substantially shaped by breeding goals, leading to differentiation between indica and japonica cultivars. Taiwanese landraces with different origins possess various and unique genetic backgrounds. Taiwanese rice germplasm provides diverse genetic variation for association mapping to unveil useful genes and is a precious genetic reservoir for rice improvement.

Investigation of the Correlation between Graves' Ophthalmopathy and CTLA4 Gene Polymorphism.

Graves' disease (GD) is an autoimmune inflammatory disease, and Graves' ophthalmopathy (GO) occurs in 25-50% of patients with GD. Several susceptible genes were identified to be associated with GO in some genetic analysis studies, including the immune regulatory gene CTLA4. We aimed to find out the correlation of CTLA4 gene polymorphism and GO. A total of 42 participants were enrolled in this study, consisting of 22 patients with GO and 20 healthy controls. Chi-square or Fisher's exact test were used to appraise the association between Graves' ophthalmopathy and CTLA4 single nucleotide polymorphisms (SNPs). All regions of CTLA4 including promoter, exon and 3'UTR were investigated. There was no nucleotide substitution in exon 2 and exon 3 of CTLA4 region, and the allele frequencies of CTLA4 polymorphisms had no significant difference between patients with GO and controls. However, the genotype frequency of "TT" genotype in rs733618 significantly differed between patients with GO and healthy controls (OR = 0.421, 95%CI: 0.290-0.611, p = 0.043), and the "CC" and "CT" genotype in rs16840252 were nearly significantly differed in genotype frequency (p = 0.052). Haplotype analysis showed that CTLA4 Crs733618Crs16840252 might increase the risk of GO (OR = 2.375, 95%CI: 1.636-3.448, p = 0.043). In conclusion, CTLA4 Crs733618Crs16840252 was found to be a potential marker for GO, and these haplotypes would be ethnicity-specific. Clinical application of CTLA4 Crs733618Crs16840252 in predicting GO in GD patients may be beneficial.

Comparative genomics revealed the gene evolution and functional divergence of magnesium transporter families in Saccharum.

BACKGROUND: Sugarcane served as the model plant for discovery of the C4 photosynthetic pathway. Magnesium is the central atom of chlorophyll, and thus is considered as a critical nutrient for plant development and photosynthesis. In plants, the magnesium transporter (MGT) family is composed of a number of membrane proteins, which play crucial roles in maintaining Mg homeostasis. However, to date there is no information available on the genomics of MGTs in sugarcane due to the complexity of the Saccharum genome. RESULTS: Here, we identified 10 MGTs from the Saccharum spontaneum genome. Phylogenetic analysis of MGTs suggested that the MGTs contained at least 5 last common ancestors before the origin of angiosperms. Gene structure analysis suggested that MGTs family of dicotyledon may be accompanied by intron loss and pseudoexon phenomena during evolution. The pairwise synonymous substitution rates corresponding to a divergence time ranged from 142.3 to 236.6 Mya, demonstrating that the MGTs are an ancient gene family in plants. Both the phylogeny and Ks analyses indicated that SsMGT1/SsMGT2 originated from the recent ρWGD, and SsMGT7/SsMGT8 originated from the recent σ WGD. These 4 recently duplicated genes were shown low expression levels and assumed to be functionally redundant. MGT6, MGT9 and MGT10 weredominant genes in the MGT family and werepredicted to be located inthe chloroplast. Of the 3 dominant MGTs, SsMGT6 expression levels were found to be induced in the light period, while SsMGT9 and SsMTG10 displayed high expression levels in the dark period. These results suggested that SsMGT6 may have a function complementary to SsMGT9 and SsMTG10 that follows thecircadian clock for MGT in the leaf tissues of S. spontaneum. MGT3, MGT7 and MGT10 had higher expression levels Insaccharum officinarum than in S. spontaneum, suggesting their functional divergence after the split of S. spontaneum and S. officinarum. CONCLUSIONS: This study of gene evolution and expression of MGTs in S. spontaneum provided basis for the comprehensive genomic study of the entire MGT genes family in Saccharum. The results are valuable for further functional analyses of MGT genes and utilization of the MGTs for Saccharum genetic improvement.

The Application and Future of Big Database Studies in Cardiology: A Single-Center Experience.

As medical research techniques and quality have improved, it is apparent that cardiovascular problems could be better resolved by more strict experiment design. In fact, substantial time and resources should be expended to fulfill the requirements of high quality studies. Many worthy ideas and hypotheses were unable to be verified or proven due to ethical or economic limitations. In recent years, new and various applications and uses of databases have received increasing attention. Important information regarding certain issues such as rare cardiovascular diseases, women's heart health, post-marketing analysis of different medications, or a combination of clinical and regional cardiac features could be obtained by the use of rigorous statistical methods. However, there are limitations that exist among all databases. One of the key essentials to creating and correctly addressing this research is through reliable processes of analyzing and interpreting these cardiologic databases.

Differences in the frequency of Alzheimer's disease-associated genomic variations in populations of different races.

AIM: The general genetic background is important when studying major common diseases, such as Alzheimer's disease (AD). Determining the underlying genetic factors in populations of different races might allow for the tailored management of such diseases. The aim of the present study was to identify potential single-nucleotide polymorphisms (SNP) and genes associated with racial differences. METHODS: We identified AD-associated SNP with different carrier frequencies among races through the National Human Genome Research Institute and 1000 Genome Project databases. We generated heatmaps and carried out principle component analysis and pathway analysis. A total of 99 AD-associated SNP from genome-wide association studies were found to have different frequencies among races. Principle component analysis showed that specific SNP had higher or lower frequencies in specific races, and that similar races were clustered together. RESULTS: Pathway analysis showed that a total of 15 pathways involving intracellular endocytosis, inflammation, immune response and lipid metabolism were significant, and that apolipoprotein E was involved in the most significant pathways. A literature review showed that 16 genes were involved in the pathogenesis of AD, and that the identified SNP could be used to cluster different races, suggesting that these SNP represented different genomic backgrounds of races. CONCLUSIONS: As disease-associated genes might have several functional variants across different populations, these genes could be candidates for further studies, such as target gene sequencing or functional follow up of putative loci regarding racial differences. Geriatr Gerontol Int 2017; 17: 2184-2193.

Microarray meta-analysis to explore abiotic stress-specific gene expression patterns in Arabidopsis.

BACKGROUND: Abiotic stresses are the major limiting factors that affect plant growth, development, yield and final quality. Deciphering the underlying mechanisms of plants' adaptations to stresses using few datasets might overlook the different aspects of stress tolerance in plants, which might be simultaneously and consequently operated in the system. Fortunately, the accumulated microarray expression data offer an opportunity to infer abiotic stress-specific gene expression patterns through meta-analysis. In this study, we propose to combine microarray gene expression data under control, cold, drought, heat, and salt conditions and determined modules (gene sets) of genes highly associated with each other according to the observed expression data. RESULTS: By analyzing the expression variations of the Eigen genes from different conditions, we had identified two, three, and five gene modules as cold-, heat-, and salt-specific modules, respectively. Most of the cold- or heat-specific modules were differentially expressed to a particular degree in shoot samples, while most of the salt-specific modules were differentially expressed to a particular degree in root samples. A gene ontology (GO) analysis on the stress-specific modules suggested that the gene modules exclusively enriched stress-related GO terms and that different genes under the same GO terms may be alternatively disturbed in different conditions. The gene regulatory events for two genes, DREB1A and DEAR1, in the cold-specific gene module had also been validated, as evidenced through the literature search. CONCLUSIONS: Our protocols study the specificity of the gene modules that were specifically activated under a particular type of abiotic stress. The biplot can also assist to visualize the stress-specific gene modules. In conclusion, our approach has the potential to further elucidate mechanisms in plants and beneficial for future experiments design under different abiotic stresses.

Genetic and Evolutionary Analysis of Purple Leaf Sheath in Rice.

BACKGROUND: Anthocyanin accumulates in many plant tissues or organs, in rice for example leading to red, purple red and purple phenotypes for protection from damage by biotic and abiotic stresses and for reproduction. Purple leaf, leaf sheath, stigma, pericarp, and apiculus are common in wild rice and landraces and occasionally found in modern cultivars. No gene directly conferring anthocyanin deposited in a purple leaf sheath has yet been isolated by using natural variants. An F2 population derived from ssp. japonica cv. Tainung 72 (TNG72) with purple leaf sheath (PSH) crossed with ssp. indica cv. Taichung Sen 17 (TCS17) with green leaf sheath (GSH) was utilized to isolate a gene conferring leaf sheath color. RESULTS: By positional cloning, 10-and 3-bp deletions in the R2R3 Myb domain of OsC1 were uncovered in GSH varieties TCS17 and Nipponbare, respectively. Allelic diversity, rather than gene expression levels of OsC1, might be responsible for anthocyanin accumulation. Parsimony-based analysis of genetic diversity in 50 accessions, including cultivars, landraces, and A-genome wild rice, suggests that independent mutation occurred in Asian, African, South American, and Australian species, while O. meridionalis had a divergent sequence. OsC1 was thought of as a domestication related gene, with up to 90 % reduction of genetic diversity in GSH; however, no values from three tests showed significant differences from neutral expectations, implying that OsC1 had not been subjected to recent selection. Haplotype network analysis revealed that species from different continents formed unique haplotypes with no gene flow. Two major groups of haplotypes corresponding to 10-bp deletion and other sequences were formed in Asian rice, including O. rufipogon, O. nivara and O. sativa. Introgressions of OsC1 between subspecies through natural and artificial hybridization were not rare. Because artificial and natural selection imposed admixture on rice germplasm in Taiwan, the genealogy of OsC1 might not be congruent with the current distribution of alleles through lineage diversification. CONCLUSION: OsC1 is responsible for purple leaf sheath, and much new information about OsC1 is provided e.g., new alleles, non-domestication syndrome, and incongruence of genealogy with geographic distribution.

Validation of a commercial insulated isothermal PCR-based POCKIT test for rapid and easy detection of white spot syndrome virus infection in Litopenaeus vannamei.

Timely pond-side detection of white spot syndrome virus (WSSV) plays a critical role in the implementation of bio-security measures to help minimize economic losses caused by white spot syndrome disease, an important threat to shrimp aquaculture industry worldwide. A portable device, namely POCKIT™, became available recently to complete fluorescent probe-based insulated isothermal PCR (iiPCR), and automatic data detection and interpretation within one hour. Taking advantage of this platform, the IQ Plus™ WSSV Kit with POCKIT system was established to allow simple and easy WSSV detection for on-site users. The assay was first evaluated for its analytical sensitivity and specificity performance. The 95% limit of detection (LOD) of the assay was 17 copies of WSSV genomic DNA per reaction (95% confidence interval [CI], 13 to 24 copies per reaction). The established assay has detection sensitivity similar to that of OIE-registered IQ2000™ WSSV Detection and Protection System with serial dilutions of WSSV-positive Litopenaeus vannamei DNA. No cross-reaction signals were generated from infectious hypodermal and haematopoietic necrosis virus (IHHNV), monodon baculovirus (MBV), and hepatopancreatic parvovirus (HPV) positive samples. Accuracy analysis using 700 L. vannamei of known WSSV infection status shows that the established assayhassensitivity93.5% (95% CI: 90.61-95.56%) and specificity 97% (95% CI: 94.31-98.50%). Furthermore, no discrepancy was found between the two assays when 100 random L. vannamei samples were tested in parallel. Finally, excellent correlation was observed among test results of three batches of reagents with 64 samples analyzed in three different laboratories. Working in a portable device, IQ Plus™ WSSV Kit with POCKIT system allows reliable, sensitive and specific on-site detection of WSSV in L. vannamei.

Characterization of tilapia (Oreochromis niloticus) viperin expression, and inhibition of bacterial growth and modulation of immune-related gene expression by electrotransfer of viperin DNA into zebrafish muscle.

Viperin is an anti-viral protein, induced by viral infection. In this study, we examined whether over-expression of viperin in fish muscle could inhibit bacterial growth. We first obtained the cDNA sequence of tilapia viperin, through RT-PCR-mediated cloning and sequencing. The cDNA sequence was similar to those of several fish viperins in GenBank, and it was predicted to encode the conserved domain of radical S-adenosylmethionine superfamily proteins. Phylogenetic analysis revealed that tilapia viperin was most closely related to viperin of Sciaenops ocellatus, Coreoperca kawamebari, and C. whiteheadi. Expression of tilapia viperin was significantly up-regulated in the kidney, liver, spleen, and gills upon challenge with lipopolysaccharide (LPS) and poly(I:C) in a time- and dose-dependent manner. Injection of Vibrio vulnificus (204) and Streptococcus agalactiae (SA47) bacteria into tilapia resulted in significant induction of viperin expression in the whole body, kidney, liver, and spleen. Electrotransfer of a viperin-expressing plasmid into zebrafish muscles decreased bacterial numbers and altered expression of immune-related genes. These data indicate that such altered expression may account for the improvement in bacterial clearance following electroporation of viperin, suggesting that fish viperin has antiviral and antibacterial activities.

Five different piscidins from Nile tilapia, Oreochromis niloticus: analysis of their expressions and biological functions.

Piscidins are antimicrobial peptides (AMPs) that play important roles in helping fish resist pathogenic infections. Through comparisons of tilapia EST clones, the coding sequences of five piscidin-like AMPs (named TP1∼5) of Nile tilapia, Oreochromis niloticus, were determined. The complete piscidin coding sequences of TP1, -2, -3, -4, and -5 were respectively composed of 207, 234, 231, 270, and 195 bases, and each contained a translated region of 68, 77, 76, 89, and 64 amino acids. The tissue-specific, Vibrio vulnificus stimulation-specific, and Streptococcus agalactiae stimulation-specific expressions of TP2, -3, and -4 mRNA were determined by a comparative RT-PCR. Results of the tissue distribution analysis revealed high expression levels of TP2 mRNA in the skin, head kidneys, liver, and spleen. To study bacterial stimulation, S. agalactiae (SA47) was injected, and the TP4 transcript was upregulated by >13-fold (compared to the wild-type (WT) control, without injection) and was 60-fold upregulated (compared to the WT control, without injection) 24 h after the S. agalactiae (SA47) injection in the spleen and gills. Synthesized TP3 and TP4 peptides showed antimicrobial activities against several bacteria in this study, while the synthesized TP1, -2, and -5 peptides did not. The synthesized TP2, -3, and -4 peptides showed hemolytic activities and synthesized TP3 and TP4 peptides inhibited tilapia ovary cell proliferation with a dose-dependent effect. In summary, the amphiphilic α-helical cationic peptides of TP3 and TP4 may represent novel and potential antimicrobial agents for further peptide drug development.

Identification of the nanogold particle-induced endoplasmic reticulum stress by omic techniques and systems biology analysis.

Growth inhibition and apoptotic/necrotic phenotype was observed in nanogold particle (AuNP)-treated human chronic myelogenous leukemia cells. To elucidate the underlying cellular mechanisms, proteomic techniques including two-dimensional electrophoresis/mass spectrometry and protein microarrays were utilized to study the differentially expressed proteome and phosphoproteome, respectively. Systems biology analysis of the proteomic data revealed that unfolded protein-associated endoplasmic reticulum (ER) stress response was the predominant event. Concomitant with transcriptomic analysis using mRNA expression, microarrays show ER stress response in the AuNP-treated cells. The ER stress protein markers' expression assay unveiled AuNPs as an efficient cellular ER stress elicitor. Upon ER stress, cellular responses, including reactive oxygen species increase, mitochondrial cytochrome c release, and mitochondria damage, chronologically occurred in the AuNP-treated cells. Conclusively, this study demonstrates that AuNPs cause cell death through induction of unmanageable ER stress.

Comparative analyses of linkage maps and segregation distortion of two F2 populations derived from japonica crossed with indica rice.

To facilitate genetic research, we constructed two linkage maps by employing two F2 populations derived from rice inter-subspecific crosses, japonica Tainung 67 (TNG67)/indica Taichung Sen 10 (TCS10) and japonica TNG67/indica Taichung Sen 17 (TCS17). We established linkage map lengths of 1481.6 cM and 1267.4 cM with average intervals of 13.8 cM and 14.4 cM by using 107 and 88 PCR markers for coverage of 88% of the rice genome in TNG67/TCS10 and TNG67/TCS17, respectively. The discrepancy in genetic maps in the two populations could be due to different cross combinations, crossing-over events, progeny numbers and/or markers. The most plausible explanation was segregation distortion; 18 markers (16.8%) distributed at nine regions of seven chromosomes and 10 markers (11.4%) at four regions of four chromosomes displayed severe segregation distortion (p < 0.01)in TNG67/TCS10 and TNG67/TCS17, respectively. All segregation-distorted markers in these two populations corresponded to reported reproductive barriers, either gametophytic or zygotic genes but not to hybrid breakdown genes. The observed recombination frequency, which was higher or lower than the intrinsic frequency, revealed the association of segregation distortion skewed to the same or different genotypes at the consecutive markers. The segregation distortion, possibly caused by reproductive barriers, affects the evaluation recombination frequencies and consequently the linkage analysis of QTLs and positional cloning.

A proteomics-based translational approach reveals an antifolate resistance inherent in human plasma derived from blood donation.

The inhibition of dihydrofolate reductase (DHFR) by antifolates is a common practice both in cell culture and in chemotherapy. Surprisingly, antifolate resistance was also observed in cultured murine myeloma cells (SP2/0) in the presence of human plasma (HP); thus, we used a proteomic approach to identify novel plasma biomarker(s) for this condition. In contrast to the in vitro antifolate response, metabolic enzymes and translation machinery proteins were found to be up-regulated in the presence of HP. The antifolate resistance inherent in HP may be explained by a simultaneous promotion of cell proliferation and the maintenance of DNA integrity. Furthermore, the factor(s) was found to be extrinsic, heat stable and very small in size. Adenine, a supplemented additive in erythrocyte preservation, was subsequently identified as the contributing factor and exogenous addition in cultures reversed the cytotoxicity induced by antifolates. Importantly, adenine-containing blood components, which may provide enhanced survival to otherwise sensitive antifolate-targeted cells, showed a dose-dependent adverse effect in transfusion recipients receiving antifolate (methotrexate) medications. These findings not only highlight a previously unnoticed role of adenine, but also emphasize a novel mechanistic link between transfusion and subsequently reduced survival in patients taking methotrexate.

A comprehensive evaluation of imidazole-zinc reverse stain for current proteomic researches.

In this paper, we comprehensively evaluated the capability of imidazole-zinc reverse stain (ZN) in comparative proteomics. Three commonly used protein gel staining methods, including silver (SN), SYPRO Ruby (SR), and CB stain were investigated alongside for comparison purpose. A transparency scanning procedure, which may deliver more even and contrasting gel images, was found best for documenting ZN stained gels. Our results showed that ZN was more sensitive than SN, SR, and CB. It may reveal as few as 1.8 ng of proteins in a gel. Moreover, ZN was found to provide a linear dynamic range of staining for revealing proteins up to 140 ng, and show an insignificant staining preference. To analyze a ZN stained 2-D gel image that generally comprises an apparent but even background, the Melanie 4 software was found more suitable than others. Furthermore, ZN demonstrated an equivalent or better MS compatibility than the other three staining methods. Intense and comprehensive MS profiles were frequently observed for ZN stained gel spots. Approximate two-third of ZN stained gel spots were successfully identified for protein identities. Taken together, our results suggest that the prompt, cost effective and versatile ZN is well suited for current proteomic researches.

Molecular cloning and functional analysis of zebrafish (Danio rerio) chemokine genes.

Chemokines control leukocyte trafficking which plays important roles in resistance to pathogenic infection. Five CXC chemokines have been reported in the zebrafish (Danio rerio) in GenBank, and herein we named them CXC-46, -56, -64, -66, and scyba. Through RT-PCR for cloning and sequencing these chemokines, the cDNA sequences of CXC-46, -56, -64, and -66 of zebrafish were determined, and it was found that the cDNA sequences were the same as those published in GenBank. Phylogenetic analysis revealed that zebrafish scyba is closest to the CXCL14 subgroup, CXC-46 is closest to the human CCL25 and catfish CXCL-2-like gene, and CXC-56, -64, and -66 are closest to the catfish CXCL10 subgroup. Further study of the tissue-specific, lipopolysaccharide (LPS) stimulation-specific, and polyinosinic-polycytidylic acid (poly I:C) stimulation-specific expressions of these five zebrafish CXC chemokine messenger (m)RNAs were determined by a comparative reverse-transcription polymerase chain reaction (RT-PCR). The RT-PCR revealed a high level of constitutive expression of CXC-56 in many tissues including the eyes, fins, heart, liver, muscles, and skin. Starvation had significant effects on the gene expressions of several zebrafish CXC chemokines including CXC-56, -64, -66, and scyba compared to the control group. Zebrafish CXC chemokines showed a concave pattern of expression after stimulation with LPS. Following poly I:C treatment of between 0.1 and 10 g/fish, dose-dependent effects were revealed. Temperature and acid-base conditions affected these zebrafish chemokines by increasing their induction compared to the control group, except for CXC-64 which exhibited no significant differences in either condition. Furthermore, these novel research results indicate that chemokines can be markers of different experimental conditions.

The sweet potato sporamin promoter confers high-level phytase expression and improves organic phosphorus acquisition and tuber yield of transgenic potato.

The sweet potato sporamin promoter was used to control the expression in transgenic potato of the E. coli appA gene, which encodes a bifunctional enzyme exhibiting both acid phosphatase and phytase activities. The sporamin promoter was highly active in leaves, stems and different size tubers of transgenic potato, with levels of phytase expression ranging from 3.8 to 7.4% of total soluble proteins. Phytase expression levels in transgenic potato tubers were stable over several cycles of propagation. Field tests showed that tuber size, number and yield increased in transgenic potato. Improved phosphorus (P) acquisition when phytate was provided as a sole P source and enhanced microtuber formation in cultured transgenic potato seedlings when phytate was provided as an additional P source were observed, which may account for the increase in leaf chloroplast accumulation (important for photosynthesis) and tuber yield of field-grown transgenic potato supplemented with organic fertilizers. Animal feeding tests indicated that the potato-produced phytase supplement was as effective as a commercially available microbial phytase in increasing the availability of phytate-P to weanling pigs. This study demonstrates that the sporamin promoter can effectively direct high-level recombinant protein expression in potato tubers. Moreover, overexpression of phytase in transgenic potato not only offers an ideal feed additive for improving phytate-P digestibility in monogastric animals but also improves tuber yield, enhances P acquisition from organic fertilizers, and has a potential for phytoremediation.

A rice phenomics study--phenotype scoring and seed propagation of a T-DNA insertion-induced rice mutant population.

With the completion of the rice genome sequencing project, the next major challenge is the large-scale determination of gene function. As an important crop and a model organism, rice provides major insights into gene functions important for crop growth or production. Phenomics with detailed information about tagged populations provides a good tool for functional genomics analysis. By a T-DNA insertional mutagenesis approach, we have generated a rice mutant population containing 55,000 promoter trap and gene activation or knockout lines. Approximately 20,000 of these lines have known integration sites. The T0 and T1 plants were grown in net "houses" for two cropping seasons each year since 2003, with the mutant phenotypes recorded. Detailed data describing growth and development of these plants, in 11 categories and 65 subcategories, over the entire four-month growing season are available in a searchable database, along with the genetic segregation information and flanking sequence data. With the detailed data from more than 20,000 T1 lines and 12 plants per line, we estimated the mutation rates of the T1 population, as well the frequency of the dominant T0 mutants. The correlations among different mutation phenotypes are also calculated. Together, the information about mutant lines, their integration sites, and the phenotypes make this collection, the Taiwan Rice Insertion Mutants (TRIM), a good resource for rice phenomics study. Ten T2 seeds per line can be distributed to researchers upon request.