[Show abstract][Hide abstract] ABSTRACT: Ergothioneine is a histidine thio-derivative isolated in 1909. In ergothioneine biosynthesis, the combination of a mononuclear non-heme iron enzyme catalyzed oxidative C-S bond formation reaction and a PLP-mediated C-S lyase (EgtE) reaction results in a net sulfur transfer from cysteine to histidine side-chain. This demonstrates a new sulfur transfer strategy in the biosynthesis of sulfur-containing natural products. Due to difficulties associated with the overexpression of Mycobacterium smegmatis EgtE protein, the proposed EgtE functionality remained to be verified biochemically. In this study, we have successfully overexpressed and purified M. smegmatis EgtE enzyme and evaluated its activities under different in vitro conditions: C-S lyase reaction using either thioether or sulfoxide as a substrate in the presence or absence of reductants. Results from our biochemical characterizations support the assignment of sulfoxide 4 as the native EgtE substrate and the involvement of a sulfenic acid intermediate in the ergothioneine C-S lyase reaction.
[Show abstract][Hide abstract] ABSTRACT: Profiling immune repertoires by high throughput sequencing enhances our understanding of immune system complexity and immune-related diseases in humans. Previously, cloning and Sanger sequencing identified limited numbers of T cell receptor (TCR) nucleotide sequences in rhesus monkeys, thus their full immune repertoire is unknown. We applied multiplex PCR and Illumina high throughput sequencing to study the TCRβ of rhesus monkeys. We identified 1.26 million TCRβ sequences corresponding to 643,570 unique TCRβ sequences and 270,557 unique complementarity-determining region 3 (CDR3) gene sequences. Precise measurements of CDR3 length distribution, CDR3 amino acid distribution, length distribution of N nucleotide of junctional region, and TCRV and TCRJ gene usage preferences were performed. A comprehensive profile of rhesus monkey immune repertoire might aid human infectious disease studies using rhesus monkeys.
[Show abstract][Hide abstract] ABSTRACT: Single-cell genomic analysis has grown rapidly in recent years and finds widespread applications in various fields of biology, including cancer biology, development, immunology, preimplantation genetic diagnosis, and neurobiology. To date, the amplification bias, amplification uniformity and reproducibility of the three major single cell whole genome amplification methods (GenomePlex WGA4, MDA and MALBAC) have not been systematically investigated using mammalian cells. In this study, we amplified genomic DNA from individual hippocampal neurons using three single-cell DNA amplification methods, and sequenced them at shallow depth. We then systematically evaluated the GC-bias, reproducibility, and copy number variations among individual neurons. Our results showed that single-cell genome sequencing results obtained from the MALBAC and WGA4 methods are highly reproducible and have a high success rate. The MALBAC displays significant biases towards high GC content. We then attempted to correct the GC bias issue by developing a bioinformatics pipeline, which allows us to call CNVs in single cell sequencing data, and chromosome level and sub-chromosomal level CNVs among individual neurons can be detected. We also proposed a metric to determine the CNV detection limits. Overall, MALBAC and WGA4 have better performance than MDA in detecting CNVs.
[Show abstract][Hide abstract] ABSTRACT: Human induced pluripotent stem cells (hiPSCs) have potential applications in cell replacement therapy and regenerative medicine. However, limited information is available regarding the immunologic features of iPSCs. In this study, expression of MHC and T cell co-stimulatory molecules in hiPSCs, and the effects on activation, proliferation and cytokine production in allogeneic human peripheral blood mononuclear cells were examined. We found that no-integrate hiPSCs had no MHC-II and T cell co-stimulatory molecules expressions but had moderate level of MHC-I and HLA-G expressions. In contrast to human skin fibroblasts (HSFs) which significantly induced allogeneic T cell activation and proliferation, hiPSCs failed to induce allogeneic CD45+ lymphocyte and CD8+ T cell activation and proliferation but could induce a low level of allogeneic CD4+ T cell proliferation. Unlike HSFs which induced allogeneic lymphocytes to produce high levels of IFN-γ, TNF-α and IL-17, hiPSCs only induced allogeneic lymphocytes to produce IL-2 and IL-10, and promote IL-10-secreting regulatory T cell (Treg) generation. Our study suggests that the integration-free hiPSCs had low or negligible immunogenicity, which may result from their induction of IL-10-secreting Treg.
PLoS ONE 12/2014; 9(12):e114949. DOI:10.1371/journal.pone.0114949 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Previous studies indicated that Plasmodium infection activates the immune system, including memory CD4+ T cells, which constitute the reservoir of human immunodeficiency virus type-1 (HIV-1). Therefore, we postulated that co-infection with malaria might activate the reservoir of HIV-1. To test this hypothesis, we used a rhesus macaque model of co-infection with malaria and simian immunodeficiency virus (SIV), along with antiretroviral therapy (ART).ResultsOur results showed that Plasmodium infection reduced both the replication-competent virus pool in resting CD4+ T cells and the integrated virus DNA (iDNA) load in peripheral blood mononuclear cells in the monkeys. This reduction might be attributable to malaria-mediated activation and apoptotic induction of memory CD4+ T cells. Further studies indicated that histone acetylation and NF-kappaB (NF-¿B) activation in resting CD4+ T cells may also play an important role in this reduction.Conclusions
The findings of this work expand our knowledge of the interaction between these two diseases. As more HIV-1-infected individuals in malaria-endemic areas receive ART, we should explore whether any of the patients co-infected with Plasmodium experience virologic benefits.
[Show abstract][Hide abstract] ABSTRACT: Ergothioneine is a histidine thiol derivative. Its mycobacterial biosynthetic pathway has five steps (EgtA-E catalysis) with two novel reactions: a mononuclear nonheme iron enzyme (EgtB) catalyzed oxidative C-S bond formation and a PLP-mediated C-S lyase (EgtE) reaction. Our bioinformatic and biochemical analyses indicate that the fungus Neurospora crassa has a more concise ergothioneine biosynthetic pathway because its nonheme iron enzyme, Egt1, makes use of cysteine instead of γ-Glu-Cys as the substrate. Such a change of substrate preference eliminates the competition between ergothioneine and glutathione biosyntheses. In addition, we have identified the N. crassa C-S lyase (NCU11365) and reconstituted its activity in vitro, which makes the future ergothioneine production through metabolic engineering feasible.
[Show abstract][Hide abstract] ABSTRACT: The peanut is a special plant for its aerial flowering but subterranean fructification. The failure of peg penetration into the soil leads to form aerial pod and finally seed abortion. However, the mechanism of seed abortion during aerial pod development remains obscure. Here, a comparative transcriptome analysis between aerial and subterranean pods at different developmental stages was produced using a customized NimbleGen microarray representing 36,158 unigenes. By comparing 4 consecutive time-points, totally 6,203 differentially expressed genes, 4,732 stage-specific expressed genes and 2,401 specific expressed genes only in aerial or subterranean pods were identified in this study. Functional annotation showed their mainly involvement in biosynthesis, metabolism, transcription regulation, transporting, stress response, photosynthesis, signal transduction, cell division, apoptosis, embryonic development, hormone response and light signaling, etc. Emphasis was focused on hormone response, cell apoptosis, embryonic development and light signaling relative genes. These genes might function as potential candidates to provide insights into seed abortion during aerial pod development. Ten candidate genes were validated by Real-time RT-PCR. Additionally, consistent with up-regulation of auxin response relative genes in aerial pods, endogenous IAA content was also significantly increased by HPLC analysis. This study will further provide new molecular insight that auxin and auxin response genes potentially contribute to peanut seed and pod development.
Electronic supplementary material
The online version of this article (doi:10.1007/s11103-014-0193-x) contains supplementary material, which is available to authorized users.
[Show abstract][Hide abstract] ABSTRACT: Given the threat of drug resistance, there is an acute need for new classes of antimalarial agents that act via a unique mechanism of action relative to currently used drugs. We have identified a set of druglike compounds within the Tres Cantos Anti-Malarial Set (TCAMS) which likely act via inhibition of a Plasmodium aspartic protease. Structure-activity relationship analysis and optimization of these aminohydantoins demonstrate that these compounds are potent nanomolar inhibitors of the Plasmodium aspartic proteases PM-II and PM-IV and likely one or more other Plasmodium aspartic proteases. Incorporation of a bulky group, such as a cyclohexyl group, on the aminohydantion N-3 position gives enhanced antimalarial potency while reducing inhibition of human aspartic proteases such as BACE. We have identified compound 8p (CWHM-117) as a promising lead for optimization as an antimalarial drug with a low molecular weight, modest lipophilicity, oral bioavailability, and in vivo antimalarial activity in mice.
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
The peanut plant produces flowers aerially, while develops the fruits and seeds underground. Pod swelling is a vital process of peanut pod and seed development only occurring after the gynophore carrying the ovule into the soil. The failure of gynophore penetration into the soil leads to suppression of pod swelling initiation. However, the molecular mechanism underlying the process remains unknown. A comparative proteome analysis between developing aerial and subterranean pods at various developmental stages was performed using 2-DE approach. 47 significantly differentially expressed spots were selected to further identification by MALDI-TOF-TOF MS. They were corresponded to 31 distinct proteins, suggesting that many identified spots were modified in post-translation. Functional annotation revealed their involvement in twelve important biological processes, such as photosynthesis, oxidative stress response, lignin synthesis, fatty acid biosynthesis, glycolysis, protein catabolic process, cellular metabolic process, regulation process, etc. Furthermore, 10 identified proteins were validated by real-time RT-PCR analysis. Several photosynthesis and oxidative stress proteins displayed elevated expression levels in aerial pods. Otherwise, enzymes in lignin synthesis and ubiquitin proteasome system were down-accumulation in subterranean pods. These enzymes might function as potential candidate proteins and play critical roles to regulate pods swelling and development.
Pod swelling plays a crucial role in peanut fruit and seed development. However, a large number of aerial pods can't form normal pods due to suppression of swelling initiation by the failure of penetration into the soil, thereby causing to seed yield loss. Limited knowledge is available underlying molecular mechanism regulating initiation of swelling in peg tips and pod development. The results generated in this study may provide evidence for some functional proteins as potential candidates to pod swelling and new molecular insights to improve our understanding of pod development under light and darkness conditions, which may contribute valuable information to high yield breeding in future.
Journal of proteomics 07/2013; 91. DOI:10.1016/j.jprot.2013.07.002 · 3.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been successfully used to knock out endogenous genes in stem cell research. However, the deficiencies of current gene-based delivery systems may hamper the clinical application of these nucleases. A new delivery method that can improve the utility of these nucleases is needed.
In this study, we utilized a cell-penetrating peptide-based system for ZFN and TALEN delivery. Functional TAT-ZFN and TAT-TALEN proteins were generated by fusing the cell-penetrating TAT peptide to ZFN and TALEN, respectively. However, TAT-ZFN was difficult to purify in quantities sufficient for analysis in cell culture. Purified TAT-TALEN was able to penetrate cells and disrupt the gene encoding endogenous human chemokine (C-C motif) receptor 5 (CCR5, a co-receptor for HIV-1 entry into cells). Hypothermic treatment greatly enhanced the TAT-TALEN-mediated gene disruption efficiency. A 5% modification rate was observed in human induced pluripotent stem cells (hiPSCs) treated with TAT-TALEN as measured by the Surveyor assay.
TAT-TALEN protein-mediated gene disruption was applicable in hiPSCs and represents a promising technique for gene knockout in stem cells. This new technique may advance the clinical application of TALEN technology.
[Show abstract][Hide abstract] ABSTRACT: Germin-like superfamily members are ubiquitously expressed in various plant species and play important roles in plant development and defense. Although several GLPs have been identified in peanut (Arachis hypogaea L.), their roles in development and defense remain unknown. In this research, we study the spatiotemporal expression of AhGLPs in peanut and their functions in plant defense.
We have identified three new AhGLP members (AhGLP3b, AhGLP5b and AhGLP7b) that have distinct but very closely related DNA sequences. The spatial and temporal expression profiles revealed that each peanut GLP gene has its distinct expression pattern in various tissues and developmental stages. This suggests that these genes all have their distinct roles in peanut development. Subcellular location analysis demonstrated that AhGLP2 and 5 undergo a protein transport process after synthesis. The expression of all AhGLPs increased in responding to Aspergillus flavus infection, suggesting AhGLPs' ubiquitous roles in defense to A. flavus. Each AhGLP gene had its unique response to various abiotic stresses (including salt, H2O2 stress and wound), biotic stresses (including leaf spot, mosaic and rust) and plant hormone stimulations (including SA and ABA treatments). These results indicate that AhGLPs have their distinct roles in plant defense. Moreover, in vivo study of AhGLP transgenic Arabidopsis showed that both AhGLP2 and 3 had salt tolerance, which made transgenic Arabidopsis grow well under 100 mM NaCl stress.
For the first time, our study analyzes the AhGLP gene expression profiles in peanut and reveals their roles under various stresses. These results provide an insight into the developmental and defensive roles of GLP gene family in peanut.
PLoS ONE 04/2013; 8(4):e61722. DOI:10.1371/journal.pone.0061722 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Peanut (Arachis hypogaea L.) is one of the most susceptible host crops to Aspergillus flavus invasion and subsequent aflatoxin contamination. In this report, a new member of PR10 family putative resistant gene (designated as ARAhPR10, No. EU661964.1) encoding a PR10 protein was isolated and characterized. Analysis of qRT-PCR showed that the ex- pression of ARAhPR10 was induced by pre-harvested A. flavus infection, but no significant difference was observed between resistant genotype “GT-C20” and susceptible genotype “Yueyou 7”. Seven transgenic peanut lines expressing the ARAhPR10 gene under the control of 35S promoter were obtained using the Agrobacterium tumefaciens-mediated method. Real time RT-PCR results showed that the expression level of the ARAhPR10 was significantly higher and the A. flavus infection and aflatoxin content were significantly lower in seeds of transgenic lines than that of the wild type. A significant negative correlation between ARAhPR10 expression at transcript level and seeds aflatoxin production was observed. Combining the previous results, it is suggested that ARAhPR10 expression play an important role in peanut host resistance to A. flavus infection and aflatoxin producing.
American Journal of Plant Sciences 02/2013; DOI:10.4236/ajps.2013.43079
[Show abstract][Hide abstract] ABSTRACT: Low genetic diversity makes peanut (Arachis hypogaea L.) very vulnerable to plant pathogens, causing severe yield loss and reduced seed quality. Several hundred partial genomic DNA sequences as nucleotide-binding-site leucine-rich repeat (NBS-LRR) resistance genes (R) have been identified, but a small portion with expressed transcripts has been found. We aimed to identify resistance gene analogs (RGAs) from peanut expressed sequence tags (ESTs) and to develop polymorphic markers. The protein sequences of 54 known R genes were used to identify homologs from peanut ESTs from public databases. A total of 1,053 ESTs corresponding to six different classes of known R genes were recovered, and assembled 156 contigs and 229 singletons as peanut-expressed RGAs. There were 69 that encoded for NBS-LRR proteins, 191 that encoded for protein kinases, 82 that encoded for LRR-PK/transmembrane proteins, 28 that encoded for Toxin reductases, 11 that encoded for LRR-domain containing proteins and 4 that encoded for TM-domain containing proteins. Twenty-eight simple sequence repeats (SSRs) were identified from 25 peanut expressed RGAs. One SSR polymorphic marker (RGA121) was identified. Two PCR-based markers (Ahsw-1 and Ahsw-2) developed from RGA013 were homologous to the Tomato Spotted Wilt Virus (TSWV) resistance gene. All three markers were mapped on the same linkage group AhIV. These expressed RGAs are the source for RGA-tagged marker development and identification of peanut resistance genes.
[Show abstract][Hide abstract] ABSTRACT: Legumes are important food crops worldwide, contributing to more than 33% of human dietary protein. The production of crop legumes is frequently impacted by abiotic and biotic stresses. It is therefore important to identify genes conferring resistance to biotic stresses and tolerance to abiotic stresses that can be used to both understand molecular
mechanisms of plant response to the environment and to accelerate crop improvement. Recent advances in genomics offer a range of approaches such as the sequencing of genomes and transcriptomes, gene expression microarray as well as RNA-seq
based gene expression profiling, and map-based cloning for the identification and isolation of biotic and abiotic stress responsive genes in several crop legumes. These candidate stress associated genes should provide insights into the molecular
mechanisms of stress tolerance and ultimately help to develop legume varieties with improved stress tolerance and productivity under adverse conditions. This review provides an overview on recent advances in the functional genomics of crop legumes that includes the discovery as well as validation of candidate genes.
[Show abstract][Hide abstract] ABSTRACT: The failure of peg penetration into the soil leads to seed abortion in peanut. Knowledge of genes involved in these processes is comparatively deficient. Here, we used RNA-seq to gain insights into transcriptomes of aerial and subterranean pods. More than 2 million transcript reads with an average length of 396 bp were generated from one aerial (AP) and two subterranean (SP1 and SP2) pod libraries using pyrosequencing technology. After assembly, sets of 49 632, 49 952 and 50 494 from a total of 74 974 transcript assembly contigs (TACs) were identified in AP, SP1 and SP2, respectively. A clear linear relationship in the gene expression level was observed between these data sets. In brief, 2194 differentially expressed TACs with a 99.0% true-positive rate were identified, among which 859 and 1068 TACs were up-regulated in aerial and subterranean pods, respectively. Functional analysis showed that putative function based on similarity with proteins catalogued in UniProt and gene ontology term classification could be determined for 59 342 (79.2%) and 42 955 (57.3%) TACs, respectively. A total of 2968 TACs were mapped to 174 KEGG pathways, of which 168 were shared by aerial and subterranean transcriptomes. TACs involved in photosynthesis were significantly up-regulated and enriched in the aerial pod. In addition, two senescence-associated genes were identified as significantly up-regulated in the aerial pod, which potentially contribute to embryo abortion in aerial pods, and in turn, to cessation of swelling. The data set generated in this study provides evidence for some functional genes as robust candidates underlying aerial and subterranean pod development and contributes to an elucidation of the evolutionary implications resulting from fruit development under light and dark conditions.
[Show abstract][Hide abstract] ABSTRACT: With 9 figures and 1 table AbstractOver the past five decades, cultivated peanut in China has been subjected to strong artificial selection in breeding programmes. To investigate the impact of artificial selection on expression diversity, we compared gene expression profiles in pod and leaf of five widespread cultivars in Southern China. In terms of tissues, hierarchical clustering analysis revealed that expression data of pod and leaf generated different dendrograms owing to artificial selection. K‐means analysis also showed that there were 16 gene expression patterns in leaf, while only eight in pod. In considering cultivars, a cultivar specificity index (τ) was employed to characterize expression patterns, which suggested that genes having 0.15 80% of all expression patterns. Additionally, the diversity of gene expression in pod among cultivars of the ‘YY7’ pedigree decreased from 23.8% to 3.9%. Taken together, nucleotide polymorphisms in regulatory elements owing to artificial selection led to low‐expression polymorphisms in both tissues and cultivars, contributed to the narrow genetic diversity and might be a driving force behind the breeding of cultivated peanut.
[Show abstract][Hide abstract] ABSTRACT: Only a few genetic maps based on recombinant inbred line (RIL) and backcross (BC) populations have been developed for tetraploid groundnut. The marker density, however, is not very satisfactory especially in the context of large genome size (2800 Mb/1C) and 20 linkage groups (LGs). Therefore, using marker segregation data for 10 RILs and one BC population from the international groundnut community, with the help of common markers across different populations, a reference consensus genetic map has been developed. This map is comprised of 897 marker loci including 895 simple sequence repeat (SSR) and 2 cleaved amplified polymorphic sequence (CAPS) loci distributed on 20 LGs (a01-a10 and b01-b10) spanning a map distance of 3, 863.6 cM with an average map density of 4.4 cM. The highest numbers of markers (70) were integrated on a01 and the least number of markers (21) on b09. The marker density, however, was lowest (6.4 cM) on a08 and highest (2.5 cM) on a01. The reference consensus map has been divided into 20 cM long 203 BINs. These BINs carry 1 (a10_02, a10_08 and a10_09) to 20 (a10_04) loci with an average of 4 marker loci per BIN. Although the polymorphism information content (PIC) value was available for 526 markers in 190 BINs, 36 and 111 BINs have at least one marker with >0.70 and >0.50 PIC values, respectively. This information will be useful for selecting highly informative and uniformly distributed markers for developing new genetic maps, background selection and diversity analysis. Most importantly, this reference consensus map will serve as a reliable reference for aligning new genetic and physical maps, performing QTL analysis in a multi-populations design, evaluating the genetic background effect on QTL expression, and serving other genetic and molecular breeding activities in groundnut.
PLoS ONE 07/2012; 7(7):e41213. DOI:10.1371/journal.pone.0041213 · 3.23 Impact Factor