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1.  Over-Expression of a Rice Tau Class Glutathione S-Transferase Gene Improves Tolerance to Salinity and Oxidative Stresses in Arabidopsis 
PLoS ONE  2014;9(3):e92900.
Glutathione S-transferases (GSTs) are multifunctional proteins encoded by large gene family in plants, which play important role in cellular detoxification of several endobiotic and xenobiotic compounds. Previously, we suggested the diverse roles of rice GST gene family members in plant development and various stress responses based on their differential expression. In this study, we report the functional characterization of a rice tau class GST gene, OsGSTU4. OsGSTU4 fusion protein was found to be localized in nucleus and cytoplasm. The over-expression of OsGSTU4 in E. coli resulted in better growth and higher GST activity under various stress conditions. Further, we raised over-expression transgenic Arabidopsis plants to reveal its in planta function. These transgenic lines showed reduced sensitivity towards plant hormones, auxin and abscisic acid. Various analyses revealed improved tolerance in transgenic Arabidopsis plants towards salinity and oxidative stresses, which may be attributed to the lower accumulation of reactive oxygen species and enhanced GST activity. In addition, microarray analysis revealed up-regulation of several genes involved in stress responses and cellular detoxification processes in the transgenic plants as compared to wild-type. These results suggest that OsGSTU4 can be used as a good candidate for the generation of stress-tolerant crop plants.
PMCID: PMC3963979  PMID: 24663444
2.  Endothelial KLF4: Crippling Vascular Injury? 
PMCID: PMC3959667  PMID: 24572258
Editorials; endothelium; Krüppel‐like transcription factor; vascular injury
3.  Myeloid KLF4 deficiency augments atherogenesis in ApoE-/- mice 
To investigate the role of Krüppel-like factor 4 (KLF4), an essential transcriptional regulator of macrophage polarization (M1/M2), in the pathogenesis of atherosclerosis.
Methods and Results
Despite the acknowledged importance of macrophages in atherosclerosis, the role of M1 (classically activated or pro-inflammatory) versus M2 (alternatively activated or anti-inflammatory) macrophages in this process remains incompletely understood. We recently identified KLF4 as a regulator of macrophage subset specification i.e. KLF4 promotes the M2 and inhibits the M1 phenotype. Here, we provide evidence that KLF4 deficient macrophages exhibit enhanced pro-inflammatory activation and foam cell formation in response to oxidized lipids. In vivo, myeloid KLF4 deficient mice (ApoE-/- background) develop significantly more vascular inflammation and atherosclerotic lesion formation.
Our findings identify myeloid KLF4 as an essential regulator of vascular inflammation and experimental atherogenesis.
PMCID: PMC3574634  PMID: 23065827
atherosclerosis; inflammation; KLF4; macrophage
4.  The Glucocorticoid Receptor and KLF15 Regulate Gene Expression Dynamics and Integrate Signals through Feed-Forward Circuitry 
Molecular and Cellular Biology  2013;33(11):2104-2115.
The glucocorticoid receptor (GR) regulates adaptive transcriptional programs that alter metabolism in response to stress. Network properties that allow GR to tune gene expression to match specific physiologic demands are poorly understood. We analyzed the transcriptional consequences of GR activation in murine lungs deficient for KLF15, a transcriptional regulator of amino acid metabolism that is induced by glucocorticoids and fasting. Approximately 7% of glucocorticoid-regulated genes had altered expression in Klf15-knockdown (Klf15−/−) mice. KLF15 formed coherent and incoherent feed-forward circuits with GR that correlated with the expression dynamics of the glucocorticoid response. Coherent feed-forward gene regulation by GR and KLF15 was characterized by combinatorial activation of linked GR-KLF15 regulatory elements by both factors and increased GR occupancy, while expression of KLF15 reduced GR occupancy at the incoherent target, MT2A. Serum deprivation, which increased KLF15 expression in a GR-independent manner in vitro, enhanced glucocorticoid-mediated induction of feed-forward targets of GR and KLF15, such as the loci for the amino acid-metabolizing enzymes proline dehydrogenase and alpha-aminoadipic semialdehyde synthase. Our results establish feed-forward architecture as an organizational principle for the GR network and provide a novel mechanism through which GR integrates signals and regulates expression dynamics.
PMCID: PMC3648070  PMID: 23508109
5.  Apelin/APJ Signaling is a Critical Regulator of Statin Effects in Vascular Endothelial Cells 
The endothelial response elicited by the G protein coupled receptor (GPCR) pathway involving apelin and APJ predict an overall vasoprotective effect. As the downstream endothelial targets of apelin/APJ signaling are also known to be targeted by statins (HMG-CoA reductase inhibitors) as potential mediators of their known pleiotropic effects, we evaluated for the involvement of apelin/APJ signaling in statin endothelial effects.
Methods and Results
We found that disruption of apelin/APJ signaling in endothelial cells leads to significantly decreased expression of Krűppel-Like-Factor 2 (KLF2), endothelial nitric oxide synthase (eNOS) and thrombomodulin (THBD). We found that statin-mediated induction of KLF2, eNOS and THBD expression, as well as inhibition of monocyte-endothelial adhesion, was abrogated by concurrent apelin knockdown. Moreover, we found that statins can transcriptionally regulate APJ in a KLF2-dependent manner, demonstrating the presence of a positive feedback loop.
Our findings provide a novel mechanism by which the apelin/APJ pathway serves as a critical intermediary that links statin to its pleiotropic effects in regulating endothelial gene targets and function.
PMCID: PMC3495062  PMID: 22995518
6.  KLF15 Is a Molecular Link between Endoplasmic Reticulum Stress and Insulin Resistance 
PLoS ONE  2013;8(10):e77851.
Obesity places major demands on the protein folding capacity of the endoplasmic reticulum (ER), resulting in ER stress, a condition that promotes hepatic insulin resistance and steatosis. Here we identify the transcription factor, Kruppel-like factor 15 (KLF15), as an essential mediator of ER stress-induced insulin resistance in the liver. Mice with a targeted deletion of KLF15 exhibit increased hepatic ER stress, inflammation, and JNK activation compared to WT mice; however, KLF15-/- mice are protected against hepatic insulin resistance and fatty liver under high-fat feeding conditions and in response to pharmacological induction of ER stress. The mammalian target of rapamycin complex 1 (mTORC1), a key regulator of cellular energy homeostasis, has been shown to cooperate with ER stress signaling pathways to promote hepatic insulin resistance and lipid accumulation. We find that the uncoupling of ER stress and insulin resistance in KLF15-/- liver is associated with the maintenance of a low energy state characterized by decreased mTORC1 activity, increased AMPK phosphorylation and PGC-1α expression and activation of autophagy, an intracellular degradation process that enhances hepatic insulin sensitivity. Furthermore, in primary hepatocytes, KLF15 deficiency markedly inhibits activation of mTORC1 by amino acids and insulin, suggesting a mechanism by which KLF15 controls mTORC1-mediated insulin resistance. This study establishes KLF15 as an important molecular link between ER stress and insulin action.
PMCID: PMC3805598  PMID: 24167585
7.  Deep Transcriptome Sequencing of Wild Halophyte Rice, Porteresia coarctata, Provides Novel Insights into the Salinity and Submergence Tolerance Factors 
Porteresia coarctata is a wild relative of rice with capability of high salinity and submergence tolerance. The transcriptome analyses of Porteresia can lead to the identification of candidate genes involved in salinity and submergence tolerance. We sequenced the transcriptome of Porteresia under different conditions using Illumina platform and generated about 375 million high-quality reads. After optimized assembly, a total of 152 367 unique transcript sequences with average length of 794 bp were obtained. Many of these sequences might represent fragmented transcripts. Functional annotation revealed the presence of genes involved in diverse cellular processes and 2749 transcription factor (TF)-encoding genes in Porteresia. The differential gene expression analyses identified a total of 15 158 genes involved in salinity and/or submergence response(s). The stress-responsive members of different TF families, including MYB, bHLH, AP2-EREBP, WRKY, bZIP and NAC, were identified. We also revealed key metabolic pathways, including amino acid biosynthesis, hormone biosynthesis, secondary metabolite biosynthesis, carbohydrate metabolism and cell wall structures, involved in stress tolerance in Porteresia. The transcriptome analyses of Porteresia are expected to highlight genes/pathways involved in salinity and submergence tolerance of this halophyte species. The data can serve as a resource for unravelling the underlying mechanism and devising strategies to engineer salinity and submergence tolerance in rice.
PMCID: PMC3925395  PMID: 24104396
metabolic pathways; Porteresia; salinity tolerance; submergence; transcriptome analysis
8.  Kruppel-like factor 15 is critical for vascular inflammation 
The Journal of Clinical Investigation  2013;123(10):4232-4241.
Activation of cells intrinsic to the vessel wall is central to the initiation and progression of vascular inflammation. As the dominant cellular constituent of the vessel wall, vascular smooth muscle cells (VSMCs) and their functions are critical determinants of vascular disease. While factors that regulate VSMC proliferation and migration have been identified, the endogenous regulators of VSMC proinflammatory activation remain incompletely defined. The Kruppel-like family of transcription factors (KLFs) are important regulators of inflammation. In this study, we identified Kruppel-like factor 15 (KLF15) as an essential regulator of VSMC proinflammatory activation. KLF15 levels were markedly reduced in human atherosclerotic tissues. Mice with systemic and smooth muscle–specific deficiency of KLF15 exhibited an aggressive inflammatory vasculopathy in two distinct models of vascular disease: orthotopic carotid artery transplantation and diet-induced atherosclerosis. We demonstrated that KLF15 alters the acetylation status and activity of the proinflammatory factor NF-κB through direct interaction with the histone acetyltransferase p300. These studies identify a previously unrecognized KLF15-dependent pathway that regulates VSMC proinflammatory activation.
PMCID: PMC3785338  PMID: 23999430
9.  Inhibition of ARNT severely compromises endothelial cell viability and function in response to moderate hypoxia 
Angiogenesis  2012;15(3):409-420.
Hypoxia Inducible Factor (HIF) is a master heterodimeric transcriptional regulator of oxygen (O2) homeostasis critical to proper angiogenic responses. Due to the distinctive coexpression of HIF-1α and HIF-2α subunits in endothelial cells, our goal was to examine the genetic elimination of HIF transcriptional activity in response to physiological hypoxic conditions by using a genetic model in which the required HIF-β subunit (ARNT, Aryl hydrocarbon Receptor Nuclear Translocator) to HIF transcriptional responses was depleted. Endothelial cells (ECs) and aortic explants were isolated from ArntloxP/loxP mice and infected with Adenovirus -Cre/GFP or control -GFP. We observed that moderate levels of 2.5% O2 promoted vessel sprouting, growth, and branching in control aortic ring assays while growth from Adenovirus -Cre infected explants was compromised. Primary Adenovirus -Cre infected EC cultures featured adverse migration and tube formation phenotypes. Primary pulmonary or cardiac ARNT-deleted ECs also failed to proliferate and survive in response to 8 or 2.5% O2 and hydrogen peroxide treatment. Our data demonstrates that ARNT promotes EC migration and vessel outgrowth and indispensible for the proliferation and preservation of ECs in response to the physiological environmental cue of hypoxia. Thus, these results demonstrate that ARNT plays a critical intrinsic role in ECs and support a critical role for the collaboration of HIF-1 and HIF-2 transcriptional activity in these cells.
PMCID: PMC3661274  PMID: 22484908
Angiogenesis; ARNT; HIF; physiological hypoxia; endothelium
10.  Correction: Optimization of De Novo Short Read Assembly of Seabuckthorn (Hippophae rhamnoides L.) Transcriptome 
PLoS ONE  2013;8(8):10.1371/annotation/8af6a452-11e9-45c1-995d-7fee9b0456eb.
PMCID: PMC3796628  PMID: 24205393
11.  Optimization of De Novo Short Read Assembly of Seabuckthorn (Hippophae rhamnoides L.) Transcriptome 
PLoS ONE  2013;8(8):e72516.
Seabuckthorn (Hippophaerhamnoides L.) is known for its medicinal, nutritional and environmental importance since ancient times. However, very limited efforts have been made to characterize the genome and transcriptome of this wonder plant. Here, we report the use of next generation massive parallel sequencing technology (Illumina platform) and de novo assembly to gain a comprehensive view of the seabuckthorn transcriptome. We assembled 86,253,874 high quality short reads using six assembly tools. At our hand, assembly of non-redundant short reads following a two-step procedure was found to be the best considering various assembly quality parameters. Initially, ABySS tool was used following an additive k-mer approach. The assembled transcripts were subsequently subjected to TGICL suite. Finally, de novo short read assembly yielded 88,297 transcripts (> 100 bp), representing about 53 Mb of seabuckthorn transcriptome. The average length of transcripts was 610 bp, N50 length 1198 BP and 91% of the short reads uniquely mapped back to seabuckthorn transcriptome. A total of 41,340 (46.8%) transcripts showed significant similarity with sequences present in nr protein databases of NCBI (E-value < 1E-06). We also screened the assembled transcripts for the presence of transcription factors and simple sequence repeats. Our strategy involving the use of short read assembler (ABySS) followed by TGICL will be useful for the researchers working with a non-model organism’s transcriptome in terms of saving time and reducing complexity in data management. The seabuckthorn transcriptome data generated here provide a valuable resource for gene discovery and development of functional molecular markers.
PMCID: PMC3749127  PMID: 23991119
12.  RiceSRTFDB: A database of rice transcription factors containing comprehensive expression, cis-regulatory element and mutant information to facilitate gene function analysis 
Rice is one of the most important crop plants, representing the staple food for more than half the world’s population. However, its productivity is challenged by various stresses, including drought and salinity. Transcription factors (TFs) represent a regulatory component of the genome and are the most important targets for engineering stress tolerance. Here, we constructed a database, RiceSRTFDB, which provides comprehensive expression information for rice TFs during drought and salinity stress conditions and various stages of development. This information will be useful to identify the target TF(s) involved in stress response at a particular stage of development. The curated information for cis-regulatory elements present in their promoters has also been provided, which will be important to study the binding proteins. In addition, we have provided the available mutants and their phenotype information for rice TFs. All these information have been integrated in the database to facilitate the selection of target TFs of interest for functional analysis. This database aims to accelerate functional genomics research of rice TFs and understand the regulatory mechanisms underlying abiotic stress responses.
Database URL:
PMCID: PMC3649641  PMID: 23660286
13.  Functionally Relevant Microsatellite Markers From Chickpea Transcription Factor Genes for Efficient Genotyping Applications and Trait Association Mapping 
We developed 1108 transcription factor gene-derived microsatellite (TFGMS) and 161 transcription factor functional domain-associated microsatellite (TFFDMS) markers from 707 TFs of chickpea. The robust amplification efficiency (96.5%) and high intra-specific polymorphic potential (34%) detected by markers suggest their immense utilities in efficient large-scale genotyping applications, including construction of both physical and functional transcript maps and understanding population structure. Candidate gene-based association analysis revealed strong genetic association of TFFDMS markers with three major seed and pod traits. Further, TFGMS markers in the 5′ untranslated regions of TF genes showing differential expression during seed development had higher trait association potential. The significance of TFFDMS markers was demonstrated by correlating their allelic variation with amino acid sequence expansion/contraction in the functional domain and alteration of secondary protein structure encoded by genes. The seed weight-associated markers were validated through traditional bi-parental genetic mapping. The determination of gene-specific linkage disequilibrium (LD) patterns in desi and kabuli based on single nucleotide polymorphism-microsatellite marker haplotypes revealed extended LD decay, enhanced LD resolution and trait association potential of genes. The evolutionary history of a strong seed-size/weight-associated TF based on natural variation and haplotype sharing among desi, kabuli and wild unravelled useful information having implication for seed-size trait evolution during chickpea domestication.
PMCID: PMC3738162  PMID: 23633531
association mapping; chickpea; microsatellite; SNP; transcription factor
14.  Klf15 orchestrates circadian nitrogen homeostasis 
Cell Metabolism  2012;15(3):311-323.
Diurnal variation in nitrogen homeostasis is observed across phylogeny. But whether these are endogenous rhythms, and if so, molecular mechanisms that link nitrogen homeostasis to the circadian clock remain unknown. Here, we provide evidence that a clock-dependent peripheral oscillator, Krüppel-like factor15 transcriptionally coordinates rhythmic expression of multiple enzymes involved in mammalian nitrogen homeostasis. In particular, Krüppel-like factor15-deficient mice exhibit no discernable amino acid rhythm, and the rhythmicity of ammonia to urea detoxification is impaired. Of the external cues, feeding plays a dominant role in modulating Krüppel-like factor15 rhythm and nitrogen homeostasis. Further, when all behavioral, environmental and dietary cues were controlled in humans, nitrogen homeostasis still expressed endogenous circadian rhythmicity. Thus, in mammals, nitrogen homeostasis exhibits circadian rhythmicity, and is orchestrated by Krüppel-like factor15.
PMCID: PMC3299986  PMID: 22405069
15.  Microarray analysis reveals overlapping and specific transcriptional responses to different plant hormones in rice 
Plant Signaling & Behavior  2012;7(8):951-956.
Hormones exert pleiotropic effects on plant growth and development throughout the life cycle. Many of these effects are mediated at molecular level via altering gene expression. In this study, we investigated the exogenous effect of plant hormones, including auxin, cytokinin, abscisic acid, ethylene, salicylic acid and jasmonic acid, on the transcription of rice genes at whole genome level using microarray. Our analysis identified a total of 4171 genes involved in several biological processes, whose expression was altered significantly in the presence of different hormones. Further, 28% of these genes exhibited overlapping transcriptional responses in the presence of any two hormones, indicating crosstalk among plant hormones. In addition, we identified genes showing only a particular hormone-specific response, which can be used as hormone-specific markers. The results of this study will facilitate further studies in hormone biology in rice.
PMCID: PMC3474693  PMID: 22827941
gene expression; hormones; microarray; rice (Oryza sativa); transcriptional response
16.  Comparative Analysis of Kabuli Chickpea Transcriptome with Desi and Wild Chickpea Provides a Rich Resource for Development of Functional Markers 
PLoS ONE  2012;7(12):e52443.
Chickpea (Cicer arietinum L.) is an important crop legume plant with high nutritional value. The transcriptomes of desi and wild chickpea have already been sequenced. In this study, we sequenced the transcriptome of kabuli chickpea, C. arietinum (genotype ICCV2), having higher commercial value, using GS-FLX Roche 454 and Illumina technologies. The assemblies of both Roche 454 and Illumina datasets were optimized using various assembly programs and parameters. The final optimized hybrid assembly generated 43,389 transcripts with an average length of 1065 bp and N50 length of 1653 bp representing 46.2 Mb of kabuli chickpea transcriptome. We identified a total of 5409 simple sequence repeats (SSRs) in these transcript sequences. Among these, at least 130 and 493 SSRs were polymorphic with desi (ICC4958) and wild (PI489777) chickpea, respectively. In addition, a total of 1986 and 37,954 single nucleotide polymorphisms (SNPs) were predicted in kabuli/desi and kabuli/wild genotypes, respectively. The SNP frequency was 0.043 SNP per kb for kabuli/desi and 0.821 SNP per kb for kabuli/wild, reflecting very low genetic diversity in chickpea. Further, SSRs and SNPs present in tissue-specific and transcription factor encoding transcripts have been identified. The experimental validation of a selected set of polymorphic SSRs and SNPs exhibited high intra-specific polymorphism potential between desi and kabuli chickpea, suggesting their utility in large-scale genotyping applications. The kabuli chickpea gene index assembled, and SSRs and SNPs identified in this study will serve as useful genomic resource for genetic improvement of chickpea.
PMCID: PMC3531472  PMID: 23300670
17.  Endothelial Kruppel-like factor 4 protects against atherothrombosis in mice 
The Journal of Clinical Investigation  2012;122(12):4727-4731.
The endothelium regulates vascular homeostasis, and endothelial dysfunction is a proximate event in the pathogenesis of atherothrombosis. Stimulation of the endothelium with proinflammatory cytokines or exposure to hemodynamic-induced disturbed flow leads to a proadhesive and prothrombotic phenotype that promotes atherothrombosis. In contrast, exposure to arterial laminar flow induces a gene program that confers a largely antiadhesive, antithrombotic effect. The molecular basis for this differential effect on endothelial function remains poorly understood. While recent insights implicate Kruppel-like factors (KLFs) as important regulators of vascular homeostasis, the in vivo role of these factors in endothelial biology remains unproven. Here, we show that endothelial KLF4 is an essential determinant of atherogenesis and thrombosis. Using in vivo EC-specific KLF4 overexpression and knockdown murine models, we found that KLF4 induced an antiadhesive, antithrombotic state. Mechanistically, we demonstrated that KLF4 differentially regulated pertinent endothelial targets via competition for the coactivator p300. These observations provide cogent evidence implicating endothelial KLFs as essential in vivo regulators of vascular function in the adult animal.
PMCID: PMC3533563  PMID: 23160196
18.  “Go With the Flow”: How Krüppel-Like Factor 2 Regulates the Vasoprotective Effects of Shear Stress 
Antioxidants & Redox Signaling  2011;15(5):1449-1461.
Laminar shear stress is known to confer potent anti-inflammatory, antithrombotic, and antiadhesive effects by differentially regulating endothelial gene expression. The identification of Krüppel-like factor 2 as a flow-responsive molecule has greatly advanced our understanding of molecular mechanisms governing vascular homeostasis. This review summarizes the current understanding of Krüppel-like factor 2 action in endothelial gene expression and function. Antioxid. Redox Signal. 15, 1449–1461.
PMCID: PMC3144441  PMID: 20919941
19.  Expression Profiling Identifies Klf15 as a Glucocorticoid Target That Regulates Airway Hyperresponsiveness 
Glucocorticoids (GCs), which activate GC receptor (GR) signaling and thus modulate gene expression, are widely used to treat asthma. GCs exert their therapeutic effects in part through modulating airway smooth muscle (ASM) structure and function. However, the effects of genes that are regulated by GCs on airway function are not fully understood. We therefore used transcription profiling to study the effects of a potent GC, dexamethasone, on human ASM (HASM) gene expression at 4 and 24 hours. After 24 hours of dexamethasone treatment, nearly 7,500 genes had statistically distinguishable changes in expression; quantitative PCR validation of a 40-gene subset of putative GR-regulated genes in 6 HASM cell lines suggested that the early transcriptional targets of GR signaling are similar in independent HASM lines. Gene ontology analysis implicated GR targets in controlling multiple aspects of ASM function. One GR-regulated gene, the transcription factor, Kruppel-like factor 15 (Klf15), was already known to modulate vascular smooth and cardiac muscle function, but had no known role in the lung. We therefore analyzed the pulmonary phenotype of Klf15−/− mice after ovalbumin sensitization and challenge. We found diminished airway responses to acetylcholine in ovalbumin-challenged Klf15−/− mice without a significant change in the induction of asthmatic inflammation. In cultured cells, overexpression of Klf15 reduced proliferation of HASM cells, whereas apoptosis in Klf15−/− murine ASM cells was increased. Together, these results further characterize the GR-regulated gene network in ASM and establish a novel role for the GR target, Klf15, in modulating airway function.
PMCID: PMC3175579  PMID: 21257922
asthma; glucocorticoid; Klf15
20.  Circadian rhythms govern cardiac repolarization and arrhythmogenesis 
Nature  2012;483(7387):96-99.
Sudden cardiac death exhibits diurnal variation in both acquired and hereditary forms of heart disease 1, 2, but the molecular basis is unknown. A common mechanism that underlies susceptibility to ventricular arrhythmias is abnormalities in the duration (e.g. short or long QT syndromes, heart failure) 3-5 or pattern (e.g. Brugada syndrome) 6 of myocardial repolarization. Here we provide the first molecular evidence that links circadian rhythms to vulnerability in ventricular arrhythmias in mice. Specifically, we show that cardiac ion channel expression and QT interval duration (an index of myocardial repolarization) exhibit endogenous circadian rhythmicity under the control of a novel clock-dependent oscillator, Krüppel-like factor 15 (Klf15). Klf15 transcriptionally controls rhythmic expression of KChIP2, a critical subunit required for generating the transient outward potassium current (Ito). 7 Deficiency or excess of Klf15 causes loss of rhythmic QT variation, abnormal repolarization and enhanced susceptibility to ventricular arrhythmias. In sum, these findings identify circadian transcription of ion channels as a novel mechanism for cardiac arrhythmogenesis.
PMCID: PMC3297978  PMID: 22367544
21.  High-Throughput SNP Discovery and Genotyping for Constructing a Saturated Linkage Map of Chickpea (Cicer arietinum L.) 
The present study reports the large-scale discovery of genome-wide single-nucleotide polymorphisms (SNPs) in chickpea, identified mainly through the next generation sequencing of two genotypes, i.e. Cicer arietinum ICC4958 and its wild progenitor C. reticulatum PI489777, parents of an inter-specific reference mapping population of chickpea. Development and validation of a high-throughput SNP genotyping assay based on Illumina's GoldenGate Genotyping Technology and its application in building a high-resolution genetic linkage map of chickpea is described for the first time. In this study, 1022 SNPs were identified, of which 768 high-confidence SNPs were selected for designing the custom Oligo Pool All (CpOPA-I) for genotyping. Of these, 697 SNPs could be successfully used for genotyping, demonstrating a high success rate of 90.75%. Genotyping data of the 697 SNPs were compiled along with those of 368 co-dominant markers mapped in an earlier study, and a saturated genetic linkage map of chickpea was constructed. One thousand and sixty-three markers were mapped onto eight linkage groups spanning 1808.7 cM (centiMorgans) with an average inter-marker distance of 1.70 cM, thereby representing one of the most advanced maps of chickpea. The map was used for the synteny analysis of chickpea, which revealed a higher degree of synteny with the phylogenetically close Medicago than with soybean. The first set of validated SNPs and map resources developed in this study will not only facilitate QTL mapping, genome-wide association analysis and comparative mapping in legumes but also help anchor scaffolds arising out of the whole-genome sequencing of chickpea.
PMCID: PMC3473369  PMID: 22864163
chickpea; SNP; linkage map; genotyping; NGS
A complex and diverse vascular system is requisite for the survival of higher organisms. The process of vascular development is highly regulated involving the de novo formation of vessels (vasculogenesis), followed by expansion and remodeling of the primitive vasculature (angiogenesis), culminating in differentiation of endothelial phenotypes as found in the mature vascular system. Over the last decade significant advances have been accomplished in understanding the molecular regulation of endothelial cell development and differentiation. Endothelial development, in particular the mechanisms in play during vasculogenesis and angiogenesis, is discussed in a sister review to this article. This review highlights the key pathways governing in endothelial differentiation with a focus on the major molecular mechanisms of endothelial specification and heterogeneity.
PMCID: PMC3134408  PMID: 21677290
Vascular biology; endothelium; angiogenesis; developmental biology; endothelial differentiation
23.  The Myeloid Transcription Factor KLF2 Regulates the Host Response to Polymicrobial Infection and Endotoxic Shock 
Immunity  2011;34(5):715-728.
Precise control of myeloid cell activation is required for optimal host defense. However, this activation process must be under exquisite control to prevent uncontrolled inflammation. Herein, we identify the Kruppel-like transcription factor 2 (KLF2) as a potent regulator of myeloid cell activation in vivo. Exposure of myeloid cells to hypoxia and/or bacterial products reduced KLF2 expression while inducing hypoxia indusable factor-1α (HIF-1α), findings that were recapitulated in human septic patients. Myeloid KLF2 was found to be a potent inhibitor of nuclear factor-kappaB (NFκB)-dependent HIF-1α transcription and, consequently, a critical determinant of outcome in models of polymicrobial infection and endotoxemia. Collectively, these observations identify KLF2 as a tonic repressor of myeloid cell activation in vivo and an essential regulator of the innate immune system.
PMCID: PMC3103656  PMID: 21565532
24.  Spatial and temporal profiles of cytokinin biosynthesis and accumulation in developing caryopses of maize 
Annals of Botany  2010;107(7):1235-1245.
Background and Aims
Cytokinins are a major group of plant hormones and are associated with various developmental processes. Developing caryopses of maize have high levels of cytokinins, but little is known about their spatial and temporal distribution. The localization and quantification of cytokinins was investigated in maize (Zea mays) caryopsis from 0 to 28 d after pollination together with the expression and localization of isopentenyltransferase ZmIPT1 involved in cytokinin biosynthesis and ZmCNGT, the gene putatively involved in N9-glucosylation.
Biochemical, cellular and molecular approaches resolved the overall cytokinin profiles, and several gene expression assays were used for two critical genes to assess cytokinin cell-specific biosynthesis and conversion to the biologically inactive form. Cytokinins were immunolocalized for the first time in maize caryopses.
Key Results
During the period 0–28 d after pollination (DAP): (1) large quantities of cytokinins were detected in the maternal pedicel region relative to the filial tissues during the early stages after fertilization; (2) unpollinated ovules did not accumulate cytokinins; (3) the maternal nucellar region showed little or no cytokinin signal; (4) the highest cytokinin concentrations in filial endosperm and embryo were detected at 12 DAP, predominantly zeatin riboside and zeatin-9-glucoside, respectively; and (5) a strong cytokinin immuno-signal was detected in specific cell types in the pedicel, endosperm and embryo.
The cytokinins of developing maize caryopsis may originate from both local syntheses as well as by transport. High levels of fertilization-dependent cytokinins in the pedicel suggest filial control on metabolism in the maternal tissue; they may also trigger developmental programmed cell death in the pedicel.
PMCID: PMC3091798  PMID: 21169292
Caryopsis; cytokinins; immunolocalization; maize; N9-glucosylation; programmed cell death; Zea mays
25.  Endothelial Gab1 is crucial for postnatal angiogenesis 
Grb2-associated binder 1 (Gab1), a scaffolding adaptor protein, plays an important role in transmitting key signals that control cell growth, differentiation and function from multiple tyrosine kinase receptors. The study was designed to investigate the role of endothelial Gab1 in angiogenesis and underlying molecular mechanisms.
Methods and Results
Using cre-loxp technology, we generated endothelial-specific Gab1 knockout (Gab1-ecKO) mice. Gab1-ecKO mice are viable and showed no obvious developmental defects in the vascular system. To analyze the role of Gab1 in postnatal angiogenesis, we used hindlimb ischemia and Matrigel plug models. We found that loss of endothelial Gab1 in mice dramatically impaired postnatal angiogenesis. Gab1-ecKO mice had impaired ischemia-initiated blood flow recovery, exhibited reduced angiogenesis and were associated with marked limb necrosis. We further observed significant EC death in the ischemic hindlimb of Gab1-ecKO mice. Matrigel plug assay showed that hepatocyte growth factor (HGF)-mediated angiogenesis was inhibited in Gab1-ecKO mice. In vitro studies showed that Gab1 was required for HGF-induced EC migration, tube formation and microvessel sprouting. Mechanistically, HGF stimulated Gab1 tyrosine phosphorylation in ECs, leading to activation of ERK1/2 and Akt, which are angiogenic and survival signaling.
Gab1 is essential for postnatal angiogenesis through mediating angiogenic and survival signaling.
PMCID: PMC3094153  PMID: 21372298
Gab1; angiogenesis; hindlimb ischemia; hepatocyte growth factor; endothelial cells

Results 1-25 (54)