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MicroRNAs (miRNAs) are endogenous single-stranded non-coding RNAs of ~22 nucleotides in length and they act as post-transcriptional regulators in bacteria, animals and plants. Almost all current methods for computational prediction of miRNAs use hairpin structure and minimum of free energy as characteristics to identify putative pre-miRNAs from a pool of candidates. We discovered a new effective feature named “basic-n-units” (BNU) to distinguish pre-miRNAs from pseudo ones. This feature describes pairing continuity of RNA secondary structure. Simulation results show that a classification method, called Triplet-SVM-classifier, achieved an accuracy of 97.24% when this BNU feature was used. This is a 3% increase caused solely by adding this new feature. We anticipate that this BNU feature may increase the accuracy for most classification methods.

Background Identifying genes with essential roles in resisting environmental stress rates high in agronomic importance. Although massive DNA microarray gene expression data have been generated for plants, current computational approaches underutilize these data for studying genotype-trait relationships. Some advanced gene identification methods have been explored for human diseases, but typically these methods have not been converted into publicly available software tools and cannot be applied to plants for identifying genes with agronomic traits. Methodology In this study, we used 22 sets of Arabidopsis thaliana gene expression data from GEO to predict the key genes involved in water tolerance. We applied an SVM-RFE (Support Vector Machine-Recursive Feature Elimination) feature selection method for the prediction. To address small sample sizes, we developed a modified approach for SVM-RFE by using bootstrapping and leave-one-out cross-validation. We also expanded our study to predict genes involved in water susceptibility. Conclusions We analyzed the top 10 genes predicted to be involved in water tolerance. Seven of them are connected to known biological processes in drought resistance. We also analyzed the top 100 genes in terms of their biological functions. Our study shows that the SVM-RFE method is a highly promising method in analyzing plant microarray data for studying genotype-phenotype relationships. The software is freely available with source code at http://ccst.jlu.edu.cn/JCSB/RFET/.

Soybean (Glycine max L.) is a major crop providing an important source of protein and oil, which can also be converted into biodiesel. A major milestone in soybean research was the recent sequencing of its genome. The sequence predicts 69,145 putative soybean genes, with 46,430 predicted with high confidence. In order to examine the expression of these genes, we utilized the Illumina Solexa platform to sequence cDNA derived from 14 conditions (tissues). The result is a searchable soybean gene expression atlas accessible through a browser (http://digbio.missouri.edu/soybean_atlas). The data provide experimental support for the transcription of 55,616 annotated genes and also demonstrate that 13,529 annotated soybean genes are putative pseudogenes, and 1736 currently unannotated sequences are transcribed. An analysis of this atlas reveals strong differences in gene expression patterns between different tissues, especially between root and aerial organs, but also reveals similarities between gene expression in other tissues, such as flower and leaf organs. In order to demonstrate the full utility of the atlas, we investigated the expression patterns of genes implicated in nodulation, and also transcription factors, using both the Solexa sequence data and large-scale qRT-PCR. The availability of the soybean gene expression atlas allowed a comparison with gene expression documented in the two model legume species, Medicago truncatula and Lotus japonicus, as well as data available for Arabidopsis thaliana, facilitating both basic and applied aspects of soybean research.

*SUMMARY Soybean (Glycine max L.) is a major crop providing an important source of protein and oil, which can also be converted into biodiesel. A major milestone in soybean research was the recent sequencing of its genome. The sequence predicts 69 145 putative soybean genes, with 46 430 predicted with high confidence. In order to examine the expression of these genes, we utilized the Illumina Solexa platform to sequence cDNA derived from 14 conditions (tissues). The result is a searchable soybean gene expression atlas accessible through a browser (http://digbio.missouri.edu/soybean_atlas). The data provide experimental support for the transcription of 55 616 annotated genes and also demonstrate that 13 529 annotated soybean genes are putative pseudogenes, and 1736 currently unannotated sequences are transcribed. An analysis of this atlas reveals strong differences in gene expression patterns between different tissues, especially between root and aerial organs, but also reveals similarities between gene expression in other tissues, such as flower and leaf organs. In order to demonstrate the full utility of the atlas, we investigated the expression patterns of genes implicated in nodulation, and also transcription factors, using both the Solexa sequence data and large-scale qRT-PCR. The availability of the soybean gene expression atlas allowed a comparison with gene expression documented in the two model legume species, Medicago truncatula and Lotus japonicus, as well as data available for Arabidopsis thaliana, facilitating both basic and applied aspects of soybean research.

Background Transcription factors play the crucial rule of regulating gene expression and influence almost all biological processes. Systematically identifying and annotating transcription factors can greatly aid further understanding their functions and mechanisms. In this article, we present SoyDB, a user friendly database containing comprehensive knowledge of soybean transcription factors. Description The soybean genome was recently sequenced by the Department of Energy-Joint Genome Institute (DOE-JGI) and is publicly available. Mining of this sequence identified 5,671 soybean genes as putative transcription factors. These genes were comprehensively annotated as an aid to the soybean research community. We developed SoyDB - a knowledge database for all the transcription factors in the soybean genome. The database contains protein sequences, predicted tertiary structures, putative DNA binding sites, domains, homologous templates in the Protein Data Bank (PDB), protein family classifications, multiple sequence alignments, consensus protein sequence motifs, web logo of each family, and web links to the soybean transcription factor database PlantTFDB, known EST sequences, and other general protein databases including Swiss-Prot, Gene Ontology, KEGG, EMBL, TAIR, InterPro, SMART, PROSITE, NCBI, and Pfam. The database can be accessed via an interactive and convenient web server, which supports full-text search, PSI-BLAST sequence search, database browsing by protein family, and automatic classification of a new protein sequence into one of 64 annotated transcription factor families by hidden Markov models. Conclusions A comprehensive soybean transcription factor database was constructed and made publicly accessible at http://casp.rnet.missouri.edu/soydb/.

There exist many heterogeneous data sources that are closely related to gene regulatory networks. These data sources provide rich information for depicting complex biological processes at different levels and from different aspects. Here, we introduce a linear programming framework to infer the gene regulatory networks. Within this framework, we extensively integrate the available information derived from multiple time-course expression datasets, ChIP-chip data, regulatory motif-binding patterns, protein-protein interaction data, protein-small molecule interaction data, and documented regulatory relationships in literature and databases. Results on synthetic and real experimental data both demonstrate that the linear programming framework allows us to recover gene regulations in a more robust and reliable manner.

Soybean (Glycine max) is one of the most important crop plants for seed protein and oil content, and for its capacity to fix atmospheric nitrogen through symbioses with soil-borne microorganisms. We sequenced the 1.1-gigabase genome by a whole-genome shotgun approach and integrated it with physical and high-density genetic maps to create achromosome-scale draft sequence assembly. We predict 46,430 protein-coding genes, 70percent more than Arabidopsis and similar to the poplar genome which, like soybean, is an ancient polyploid (palaeopolyploid). About 78percent of the predicted genes occur in chromosome ends, which comprise less than one-half of the genome but account for nearly all of the genetic recombination. Genome duplications occurred at approximately 59 and 13 million years ago, resulting in a highly duplicated genome with nearly 75percent of the genes present in multiple copies. The two duplication events were followed by genediversification and loss, and numerous chromosome rearrangements. An accurate soybean genome sequence will facilitate the identification of the genetic basis of many soybean traits, and accelerate the creation of improved soybean varieties.

Experimental visceral leishmaniasis (VL) represents an exquisite model to study CD8+ T cell responses in a context of chronic inflammation and antigen persistence, since it is characterized by chronic infection in the spleen and CD8+ T cells are required for the development of protective immunity. However, antigen-specific CD8+ T cell responses in VL have so far not been studied, due to the absence of any defined Leishmania-specific CD8+ T cell epitopes. In this study, transgenic Leishmania donovani parasites expressing ovalbumin were used to characterize the development, function, and fate of Leishmania-specific CD8+ T cell responses. Here we show that L. donovani parasites evade CD8+ T cell responses by limiting their expansion and inducing functional exhaustion and cell death. Dysfunctional CD8+ T cells could be partially rescued by in vivo B7-H1 blockade, which increased CD8+ T cell survival but failed to restore cytokine production. Nevertheless, B7-H1 blockade significantly reduced the splenic parasite burden. These findings could be exploited for the design of new strategies for immunotherapeutic interventions against VL., Author Summary The protozoan parasite Leishmania donovani is the cause of visceral leishmaniasis, a chronic disease that currently affects 12 million people worldwide. We are interested in understanding the immune mechanisms that can control infection. Preliminary studies suggested that CD8+ T cells can kill parasites and limit disease; however, studying these important killer cells has been hindered, because we do not know what parasite molecules they recognize. To overcome this, we engineered parasites to express ovalbumin. Since many tools exist to track and measure immune cells targeted at ovalbumin, we can now track the specific CD8+ T cell responses that develop upon infection with Leishmania. We found that Leishmania initially induced CD8+ T cells to divide and produce molecules such as IFN-gamma that may help them to kill parasites. However, the CD8+ T cells rapidly lost their effector function and died off as infection progressed. More encouragingly, though, we were able to recover some CD8+ T cell function by blocking immune inhibitory molecules that are induced by parasite infection. The recovered T cells killed parasites and controlled infection. These results are important as they could be exploited for the design of new therapeutic vaccine strategies aimed at inducing protective CD8+ T cells.

Antibody-based therapies, such as rituximab and alemtuzumab, have contributed significantly to the treatment of Chronic Lymphocytic leukaemia (CLL). The CD40 antigen is expressed predominantly on B-cells and represents a potential target for immune-based therapies. SGN-40 is a humanized IgG1 monoclonal antibody currently in Phase I/II clinical trials for indolent lymphomas, diffuse large B cell lymphomas and Multiple Myeloma. Its biological effect on CLL cells has not been studied. The present study demonstrated that SGN-40 mediated modest apoptosis in a subset of patients with secondary cross-linking but did not mediate complement-dependent cytotoxicity. SGN-40 also mediated antibody-dependent cellular cytotoxicity (ADCC) predominantly through natural killer (NK) cells. Previous studies by our group and others have demonstrated that lenalidomide upregulates CD40 expression on primary B CLL cells and activates NK-cells. We therefore examined for the combinatorial effect of lenalidomide and SGN-40 and demonstrated that both enhanced direct apoptosis and ADCC against primary CLL B cells. These data together provide justification for clinical trials of SGN-40 and lenalidomide in combination for CLL therapy.

Root hairs are single tubular cells formed from the differentiation of epidermal cells on roots. They are involved in water and nutrient uptake and represent the infection site on leguminous roots by rhizobia, soil bacteria that establish a nitrogen-fixing symbiosis. Root hairs develop by polar cell expansion or tip growth, a unique mode of plant growth shared only with pollen tubes. A more complete characterization of root hair cell biology will lead to a better understanding of tip growth, the rhizobial infection process, and also lead to improvements in plant water and nutrient uptake. We analyzed the proteome of isolated soybean (Glycine max) root hair cells using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and shotgun proteomics (1D-PAGE-liquid chromatography and multidimensional protein identification technology) approaches. Soybean was selected for this study due to its agronomic importance and its root size. The resulting soybean root hair proteome reference map identified 1,492 different proteins. 2D-PAGE followed by mass spectrometry identified 527 proteins from total cell contents. A complementary shotgun analysis identified 1,134 total proteins, including 443 proteins that were specific to the microsomal fraction. Only 169 proteins were identified by the 2D-PAGE and shotgun methods, which highlights the advantage of using both methods. The proteins identified are involved not only in basic cell metabolism but also in functions more specific to the single root hair cell, including water and nutrient uptake, vesicle trafficking, and hormone and secondary metabolism. The data presented provide useful insight into the metabolic activities of a single, differentiated plant cell type.

Characterizing gene function is one of the major challenging tasks in the postgenomic era. To address this challenge, we developed GeneFAS (gene function annotation system), a computer system with a graphical user interface for cellular function prediction by integrating information from protein-protein interactions, protein complexes, microarray gene expression profiles, and annotations of known proteins. GeneFAS can provide biologists a workspace for their organism of interest, to integrate different types of experimental data and annotation information, and facilitate biological discovery and hypothesis generation using all the information. It also provides testing and training capabilities for users to utilize and integrate their data more efficiently. GeneFAS is freely available for download at http://digbio.missouri.edu/genefas .

Characterizing gene function is one of the major challenging tasks in the postgenomic era. To address this challenge, we developed GeneFAS (gene function annotation system), a computer system with a graphical user interface for cellular function prediction by integrating information from protein-protein interactions, protein complexes, microarray gene expression profiles, and annotations of known proteins. GeneFAS can provide biologists a workspace for their organism of interest, to integrate different types of experimental data and annotation information, and facilitate biological discovery and hypothesis generation using all the information. It also provides testing and training capabilities for users to utilize and integrate their data more efficiently. GeneFAS is freely available for download at http://digbio.missouri.edu/genefas .

Background: Several years after sequencing the human genome and the mouse genome, much remains to be discovered about the functions of most human and mouse genes. Computational prediction of gene function promises to help focus limited experimental resources on the most likely hypotheses. Several algorithms using diverse genomic data have been applied to this task in model organisms; however, the performance of such approaches in mammals has not yet been evaluated. Results: In this study, a standardized collection of mouse functional genomic data was assembled; nine bioinformatics teams used this data set to independently train classifiers and generate predictions of function, as defined by Gene Ontology (GO) terms, for 21,603 mouse genes; and the best performing submissions were combined in a single set of predictions. We identified strengths and weaknesses of current functional genomic data sets and compared the performance of function prediction algorithms. This analysis inferred functions for 76% of mouse genes, including 5,000 currently uncharacterized genes. At a recall rate of 20%, a unified set of predictions averaged 41% precision, with 26% of GO terms achieving a precision better than 90%. Conclusion: We performed a systematic evaluation of diverse, independently developed computational approaches for predicting gene function from heterogeneous data sources in mammals. The results show that currently available data for mammals allows predictions with both breadth and accuracy. Importantly, many highly novel predictions emerge for the 38% of mouse genes that remain uncharacterized.

The growth and persistence of rhizobia and bradyrhizobia in soils are negatively impacted by drought conditions. In this study, we used genome-wide transcriptional analyses to obtain a comprehensive understanding of the response of Bradyrhizobium japonicum to drought. Desiccation of cells resulted in the differential expression of 15 to 20% of the 8,480 B. japonicum open reading frames, with considerable differentiation between early (after 4 h) and late (after 24 and 72 h) expressed genes. While 225 genes were universally up-regulated at all three incubation times in response to desiccation, an additional 43 and 403 up-regulated genes were common to the 4/24- and 24/72-h incubation times, respectively. Desiccating conditions resulted in the significant induction (>2.0-fold) of the trehalose-6-phosphate synthetase ( otsA ), trehalose-6-phosphate phosphatase ( otsB ), and trehalose synthase ( treS ) genes, which encode two of the three trehalose synthesis pathways found in B. japonicum . Gene induction was correlated with an elevated intracellular concentration of trehalose and increased activity of trehalose-6-phosphate synthetase, collectively supporting the hypothesis that this disaccharide plays a prominent and important role in promoting desiccation tolerance in B. japonicum . Microarray data also indicated that σ 54 - and σ 24 -associated transcriptional regulators and genes encoding isocitrate lyase, oxidative stress responses, the synthesis and transport of exopolysaccharides, heat shock response proteins, enzymes for the modification and repair of nucleic acids, and the synthesis of pili and flagella are also involved in the response of B. japonicum to desiccation. Polyethylene glycol-generated osmotic stress induced significantly fewer genes than those transcriptionally activated by desiccation. However, 67 genes were commonly induced under both conditions. Taken together, these results suggest that B. japonicum directly responds to desiccation by adapting to changes imparted by reduced water activity, such as the synthesis of trehalose and polysaccharides and, secondarily, by the induction of a wide variety of proteins involved in protection of the cell membrane, repair of DNA damage, stability and integrity of proteins, and oxidative stress responses.

Determination of protein function and biological pathway is one of the most challenging problems in the post-genomic era. To address this challenge, we have developed a new integrated probabilistic method for cellular function prediction using microarray gene expression profiles, in conjunction with predicted protein-protein interactions and annotations of known proteins. Our approach is based on a novel assessment for the relationship between correlation of two genes' expression profiles and their functional relationship in terms of the Gene Ontology (GO) hierarchy. We applied the method for function prediction of hypothetical genes in Arabidopsis. We have also extended our method using Dijkstra's algorithm to identify the components and topology of signaling pathway of phosphatidic acid as a second messenger in Arabidopsis.

Purpose: Natural killer (NK) cells express an activating Fc receptor (FcγRIIIa) that mediates antibody-dependent cellular cytotoxicity (ADCC) and production of immune modulatory cytokines in response to antibody-coated targets. Cetuximab is a therapeutic monoclonal antibody directed against the HER1 antigen. We hypothesized that the NK cell response to cetuximab-coated tumor cells could be enhanced by the administration of NK cell–stimulatory cytokines. Experimental Design: Human NK cells stimulated with cetuximab-coated tumor cells and interleukin-2 (IL-2), IL-12, or IL-21 were assessed for ADCC and secretion of IFN-γ and T cell–recruiting chemokines. IL-21 and cetuximab were given to nude mice bearing HER1-positive xenografts. Results: Stimulation of human NK cells with cetuximab-coated tumor cells and IL-2, IL-12, or IL-21 resulted in 3-fold to 10-fold higher IFN-γ production than was observed with either agent alone. NK cell–derived IFN-γ significantly enhanced monocyte ADCC against cetuximab-coated tumor cells. Costimulated NK cells also secreted elevated levels of chemokines (IL-8, macrophage inflammatory protein-1α, and RANTES) that could direct the migration of naive and activated T cells. IL-2, IL-12, and IL-21 enhanced NK cell ADCC against tumor cells treated with cetuximab. The combination of cetuximab, trastuzumab (an anti-HER2 monoclonal antibody), and IL-21 mediated greater NK cell cytokine secretion and ADCC than any agent alone. Furthermore, administration of IL-21 enhanced the effects of cetuximab in a murine tumor model. Conclusions: These results show that cetuximab-mediated NK cell activity can be significantly enhanced in the presence of NK cell–stimulatory cytokines. These factors, therefore, may be effective adjuvants to administer, in combination with cetuximab, to patients with HER1-positive malignancies.

A DNA microarray, comprising 70-mer oligonucleotides, representing 8,453 open reading frames (ORFs), was constructed based on the Bradyrhizobium japonicum strain USDA110 genomic sequence. New annotation predicted 199 additional genes, which were added to the microarray and were shown to be transcribed. These arrays were used to profile transcription in cells under a variety of conditions, including growth in minimal versus rich medium, osmotic stress, and free-living cells versus bacteroids. Increased expression was seen for genes involved in translation, motility, and cell envelope synthesis in rich medium whereas expression increased in minimal medium for genes involved in vitamin biosynthesis and stress responses. Treatment with 50 mM NaCl activated stress-inducible genes but repressed genes involved in chemotaxis and motility. Strikingly, no known transport systems for accumulation of compatible solutes or osmoprotectants were induced in response to osmotic stress. A number of nif, fix, and hup genes, but not all, were upregulated in bacteroids. The B. japonicum type III secretion system, known to be important in early nodulation, was downregulated in bacteroids. The availability of a reliable, low-cost B. japonicum microarray provides a useful tool for functional genomic studies of one of the most agriculturally important bacteria.

Background Comparative sequence analysis is considered as the first step towards annotating new proteins in genome annotation. However, sequence comparison may lead to creation and propagation of function assignment errors. Thus, it is important to perform a thorough analysis for the quality of sequence-based function assignment using large-scale data in a systematic way. Results We present an analysis of the relationship between sequence similarity and function similarity for the proteins in four model organisms, i.e., Arabidopsis thaliana, Saccharomyces cerevisiae, Caenorrhabditis elegans, and Drosophila melanogaster. Using a measure of functional similarity based on the three categories of Gene Ontology (GO) classifications (biological process, molecular function, and cellular component), we quantified the correlation between functional similarity and sequence similarity measured by sequence identity or statistical significance of the alignment and compared such a correlation against randomly chosen protein pairs. Conclusion Various sequence-function relationships were identified from BLAST versus PSI-BLAST, sequence identity versus Expectation Value, GO indices versus semantic similarity approaches, and within genome versus between genome comparisons, for the three GO categories. Our study provides a benchmark to estimate the confidence in assignment of functions purely based on sequence similarity.

CD37 is a lineage-specific B-cell antigen that to date has been neglected as an attractive therapeutic target. To exploit this, novel CD37-specific small modular immunopharmaceuticals (CD37-SMIP) that include variable regions linked to modified human IgG1 hinge, CH2, and CH3 domains were designed. The lead CD37-SMIP molecule induces potent apoptosis in the presence of a cross-linker, and antibody-dependent cellular cytotoxicity against B-cell leukemia/lymphoma cell lines and primary chronic lymphocytic leukemia (CLL) cells superior to therapeutic antibodies used in these diseases. The CD37-SMIP–dependent ADCC function in vitro was mediated by natural killer (NK) cells but not naive or activated monocytes. Significant in vivo therapeutic efficacy was demonstrated in a SCID mouse xenograft leukemia/lymphoma model. Depletion of NK cells in this mouse model resulted in diminished efficacy further supported the in vivo importance of NK cells in SMIP therapy. These findings provide strong justification for CD37 as a therapeutic target and introduce small modular immunopharmaceuticals as a novel class of targeted therapies for B-cell malignancies.

Fcgamma receptors are among the best-studied phagocytic receptors. The key features of Fcgamma receptor-mediated phagocytosis include phagocytic cup formation by extensive actin cytoskeletal rearrangements, particle engulfment, and the release of proinflammatory mediators such as cytokines and reactive oxygen species. These events are elegantly regulated by the simultaneous engagement of activating and inhibitory Fcgamma receptors and by intracellular signaling molecules. Extensive studies in the past several years have defined the molecular mechanisms of the phagocytic process. The purpose of this review is to revisit some of the well-established signaling pathways as well as to summarize the new findings in this field.

The inhibitory receptor FcgammaRIIb becomes tyrosine phosphorylated and associates with the inositol phosphatase SHIP to downregulate phagocytosis. The two splice variants of FcgammaRIIb, b1 and b2, are differentially expressed in hematopoetic cells. Both isoforms of FcgammaRIIb are expressed in human myeloid cells although FcgammaRIIb2 predominates. In murine B cells FcgammaRIIb2 associates with clathrin-coated pits and undergoes endocytosis, whereas FcgammaRIIbl is excluded from the coated pits, indicating that the two isoforms serve partially differing functions. In humans, there are conflicting reports with regard to the ability of FcgammaRIIb2 to become tyrosine phosphorylated, and the functional capacities of the two isoforms are poorly understood. We, and others, have previously reported that the expression of FcgammaRIIb is upregulated in human monocytes by the anti-inflammatory cytokine IL-4. Here, we extend these findings to demonstrate that the IL-4-induced upregulation of FcgammaRIIb is synergistically enhanced by the addition of IL-10, both at the protein and the mRNA level. The upregulated receptors are functional as assessed by their ability to become tyrosine phosphorylated and to downregulate phagocytosis. Interestingly, both b1 and b2 isoforms are upregulated by anti-inflammatory cytokines. Transfection experiments expressing human FcgammaRIIbl or b2 in Raw 264.7 murine macrophage cells revealed that both isoforms are tyrosine phosphorylated and promote SHIP phosphorylation. Finally, both b1 and b2 isoforms of FcgammaRIIb downregulate phagocytosis to a similar extent. Thus we conclude that FcgammaRIIbl and b2 are both functional inhibitory receptors in the phagocytic process.

Background: There is enormous burden of thyroid diseases in the general population. Among all the endocrine disorders, thyroid disorder are the most common in India, though most of them are benign. Multinodular goitre is the commonest cause of thyroid enlargement followed by thyroid tumors. Aims & Objectives: The aim of this study is to describe the clinicopathological findings of thyroidectomy specimens. Materials and Methods: It was a 24 month (May 2012 to May 2014) non-interventional retrospective study, including all cases of thyroidectomy specimens in Azeezia Medical College, Kollam, Kerala. All histology reports, clinical information and stained slides were reviewed. Thyroid diseases were grouped into different categories according to gender and age distribution. Results: A total of 179 lesions were reviewed, 167 were from females and 12 from males. 164 cases were found non-neoplastic and 15 were neoplastic lesions. Multinodular goitre was found to be the commonest - 116 (64.8%) non-neoplastic lesion, followed by Hashimoto’s thyroiditis 21(11.73%). Among 179 specimens, 15 (8.37%) were of thyroid malignancy, and papillary carcinoma was found to be the commonest malignant thyroid lesion, observed in (10 /15) 66.66% of all thyroid malignant lesions. This was followed by follicular carcinoma (3/15) 20% and while medullary and poorly differentiated carcinoma were noted in one patients each. Conclusion: The commonest cause of goitre was multinodular goitre. Papillary carcinoma was the commonest malignant lesion.

Abstract Sodium butyrate (SB), a naturally occurring short-chain fatty acid, was investigated for its therapeutic value as an antiproliferative agent for vascular smooth muscle cells (SMCs). At 5-mmol/L concentration, SB had no significant effect on rat SMC proliferation. However, at the same concentration, SB inhibited platelet-derived growth factor (PDGF)-AA–, -AB–, and -BB–induced proliferation of SMCs. Exposure of SMCs to PDGF-BB resulted in activation of receptor intrinsic tyrosine kinase activity and autophosphorylation of β-PDGF–receptor (β-PDGFR). The activated β-PDGFR physically associated and phosphorylated signaling molecules such as ras -GTPase activating protein (GAP) and phospholipase Cγ (PLCγ). SB, in the absence of PDGF-BB, caused neither β-PDGFR tyrosine phosphorylation nor phosphorylation and association of GAP and PLCγ with β-PDGFR. PDGF-BB–enhanced activation of receptor intrinsic tyrosine kinase activity and autophosphorylation of tyrosine residues of β-PDGFR were unaffected by SB irrespective of whether SMCs were preincubated with SB before exposure to PDGF-BB plus SB or incubated concomitantly with PDGF-BB plus SB. Likewise, phosphorylation and association of GAP and PLCγ with PDGF-BB–activated β-PDGFR were unaffected. In addition, SB did not block PDGF-BB–stimulated, PLCγ-mediated production of inositol triphosphate. Similarly, PDGF-BB–induced β-PDGFR degradation was unaffected when SMCs were exposed to PDGF-BB plus SB, and SB by itself had no influence on β-PDGFR degradation. Unlike β-PDGFR kinase activity, mitogen-activated protein kinase (MAP-kinase) activity was stimulated by SB by about 2.7-fold. Exposure of SMCs to PDGF-BB caused an ≈11.4-fold increase in MAP-kinase activity and this increase in activity was not significantly affected when cells were coincubated with PDGF-BB and SB (10.3-fold). However, pretreatment of SMCs with SB for 30 minutes and subsequent incubation in PDGF-BB plus SB abolished most of the PDGF-BB–induced MAP-kinase activity (4.6-fold). Transcription of growth response genes such as c- fos , c- jun , and c- myc were induced by PDGF-BB, and their induction was suppressed, particularly c- myc , by incubating SMCs with PDGF-BB plus SB. Similarly, preincubation of cells with SB for 30 minutes and subsequent incubation in PDGF-BB plus SB diminished PDGF-BB–induced transcription of c- fos , c- jun , and c- myc . However, SB by itself had no significant effect on c- fos , c- jun , and c- myc transcription. Our data suggest that the inhibition of PDGF-BB–induced proliferation of SMCs by SB involves MAP-kinase–regulated events as well as transcription of growth-response genes.

We applied a solution hybrid selection approach to the enrichment of CpG islands (CGIs) and promoter sequences from the human genome for targeted high-throughput bisulfite sequencing. A single lane of Illumina sequences allowed accurate and quantitative analysis of ~1 million CpGs in more than 21,408 CGIs and more than 15,946 transcriptional regulatory regions. Of the CpGs analyzed, 77-84% fell on or near capture probe sequences; 69-75% fell within CGIs. More than 85% of capture probes successfully yielded quantitative DNA methylation information of targeted regions. Differentially methylated regions (DMRs) were identified in the 5'-end regulatory regions, as well as the intra- and intergenic regions, particularly in the X-chromosome among the three breast cancer cell lines analyzed. We chose 46 candidate loci (762 CpGs) for confirmation with PCR-based bisulfite sequencing and demonstrated excellent correlation between two data sets. Targeted bisulfite sequencing of three DNA methyltransferase (DNMT) knockout cell lines and the wild-type HCT116 colon cancer cell line revealed a significant decrease in CpG methylation for the DNMT1 knockout and DNMT1, 3B double knockout cell lines, but not in DNMT3B knockout cell line. We demonstrated the targeted bisulfite sequencing approach to be a powerful method to uncover novel aberrant methylation in the cancer epigenome. Since all targets were captured and sequenced as a pool through a series of single-tube reactions, this method can be easily scaled up to deal with a large number of samples.

Background With the advance of new massively parallel genotyping technologies, quantitative trait loci (QTL) fine mapping and map-based cloning become more achievable in identifying genes for important and complex traits. Development of high-density genetic markers in the QTL regions of specific mapping populations is essential for fine-mapping and map-based cloning of economically important genes. Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation existing between any diverse genotypes that are usually used for QTL mapping studies. The massively parallel sequencing technologies (Roche GS/454, Illumina GA/Solexa, and ABI/SOLiD), have been widely applied to identify genome-wide sequence variations. However, it is still remains unclear whether sequence data at a low sequencing depth are enough to detect the variations existing in any QTL regions of interest in a crop genome, and how to prepare sequencing samples for a complex genome such as soybean. Therefore, with the aims of identifying SNP markers in a cost effective way for fine-mapping several QTL regions, and testing the validation rate of the putative SNPs predicted with Solexa short sequence reads at a low sequencing depth, we evaluated a pooled DNA fragment reduced representation library and SNP detection methods applied to short read sequences generated by Solexa high-throughput sequencing technology. Results A total of 39,022 putative SNPs were identified by the Illumina/Solexa sequencing system using a reduced representation DNA library of two parental lines of a mapping population. The validation rates of these putative SNPs predicted with low and high stringency were 72% and 85%, respectively. One hundred sixty four SNP markers resulted from the validation of putative SNPs and have been selectively chosen to target a known QTL, thereby increasing the marker density of the targeted region to one marker per 42 K bp. Conclusions We have demonstrated how to quickly identify large numbers of SNPs for fine mapping of QTL regions by applying massively parallel sequencing combined with genome complexity reduction techniques. This SNP discovery approach is more efficient for targeting multiple QTL regions in a same genetic population, which can be applied to other crops.

Nature 463, 178–183 (2010) During resubmission of this work, a paper was published1 that used a comparative genomics approach between soybean and maize to show that a single-base mutation in chromosome 19 accounts for the duplicate recessive epistasis needed to greatly reduce phytate production in soybean seed.

The transcription factor Interferon Regulatory Factor 5 (IRF5) has been shown to be involved in the induction of proinflammatory cytokines in response to viral infections and TLR activation and to play an essential role in the innate inflammatory response. In this study, the experimental model of visceral leishmaniasis was used to investigate the role of IRF5 in the generation of Th1 responses and subsequent formation of Th1-type liver granulomas in Leishmania donovani infected mice. Here we show that IRF5 deficiency severely impairs the development of Th1 responses to L.donovani leading to the formation of Th2-type-like granulomas. These granulomas are characterized by a strong IL-4 response, upregulation of arginase 1 and Fizz1 mRNA and concomitant low iNOS expression. This study identifies IRF5 as a critical molecular switch for the development of Th1 immune responses following L.donovani infections and as an important molecule in the regulation of arginase 1 and iNOS expression.

IL-10 is an immunoregulatory cytokine produced by many cell types. Studies in mice suggest the cellular source of IL-10 plays a critical role in determining disease outcome. Yet the effects of tissue specific human IL-10 (hIL-10) expression are not well understood. To address this issue, we generated a hIL-10 transgenic mouse using a bacterial artificial chromosome (hIL10BAC). The hIL10BAC contains most if not all the regulatory sequences that govern hIL-10 production thus allowing tissue specific expression. As hIL-10 is biologically active in the mouse, reconstituting Il10-/- mice with the hIL10BAC allowed us to evaluate the effects of hIL-10 on known IL-10 dependant phenotypes. Expression of IL-10 from myeloid sources in WT mice protects against lethal endotoxic shock. Faithful expression of the hIL10BAC in myeloid cells rescued Il10-/- mice from this fate. In contrast, T-cell-derived IL-10 leads to high parasite burdens in the livers and spleens of WT mice infected with Leishmania Donovani. However both Il10-/- and Il10-/-/hIL10BAC mice cleared the parasites. The failure to reproduce the susceptibility phenotype is attributed to weak hIL-10 expression in CD4+ T cells. Similarly, inflammatory bowel disease (IBD) is also IL-10-dependant and resistance is thought to involve T-cell-derived IL-10. Surprisingly, we found that the hIL10BAC rescues Il10-/- mice from IBD, suggesting differential regulation of hIL-10 in CD4+ T cells found in the gut versus the spleen.

In order to expand our knowledge of the soybean genome and to create a useful DNA repeat sequence database, over 24 000 DNA fragments from a soybean [Glycine max (L.) Merr.] cv. Williams 82 genomic shotgun library were sequenced. Additional sequences came from over 29 000 bacterial artificial chromosome (BAC) end sequences derived from a BstI library of the cv. Williams 82 genome. Analysis of these sequences identified 348 different DNA repeats, many of which appear to be novel. To extend the utility of the work, a pilot study was also conducted using methylation filtration to estimate the hypomethylated, soybean gene space. A comparison between 8366 sequences obtained from a filtered library and 23 788 from an unfiltered library indicate a gene-enrichment of ~3.2-fold in the hypomethylated sequences. Given the 1.1-Gb soybean genome, our analysis predicts a ~343-Mb hypomethylated, gene-rich space.

Key message We performed QTL analysis for SCN resistance in PI 437655 in two mapping populations, characterized CNV of Rhg1 through whole-genome resequencing and evaluated the effects of QTL pyramiding to enhance resistance. Abstract Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is one of the most serious pests of soybean worldwide. PI 437655 has broader resistance to SCN HG types than PI 88788. The objectives of this study were to identify quantitative trait loci (QTL) underlying SCN resistance in PI 437655, and to evaluate the QTL for their contribution to SCN resistance. Two F6:7 recombinant inbred line populations, derived from cv. Williams 82 × PI 437655 and cv. Hutcheson × PI 437655 crosses, were evaluated for resistance to SCN HG types 1.2.5.7 (PA2), 0 (PA3), 1.3.5.6.7 (PA14), and 1.2.3.4.5.6.7 (LY2). The 1,536 SNP array was used to genotype the mapping populations and construct genetic linkage maps. Two significant QTL were consistently mapped on chromosomes (Chr.) 18 and 20 in these two populations. One QTL on Chr. 18, which corresponds to the known Rhg1 locus, contributed resistance to SCN HG types 1.2.5.7, 0, 1.3.5.6.7, and 1.2.3.4.5.6.7 (PA2, PA3, PA14, and LY2, respectively). Copy number variation (CNV) analysis by whole-genome resequencing showed that PI 437655 and PI 88788 had similar CNV at the Rhg1 locus. The QTL on Chr. 20 contributed resistance to SCN HG types 1.3.5.6.7 (PA14) and 1.2.3.4.5.6.7 (LY2). Evaluation of both QTL showed that pyramiding of Rhg1 and the QTL on Chr. 20 significantly improved the resistance to SCN HG types 1.3.5.6.7 (PA14) and 1.2.3.4.5.6.7 (LY2) in both populations. Our studies provided useful information for deploying PI 437655 as a donor for SCN resistance in soybean breeding through marker-assisted selection. Electronic supplementary material The online version of this article (doi:10.1007/s00122-014-2409-5) contains supplementary material, which is available to authorized users.

Soybean cyst nematode (SCN, Heterodera glycine Ichinohe), southern root-knot nematode [SRKN, Meloidogyne incognita (Kofoid and White) Chitwood] and reniform nematode (RN, Rotylenchulus reniformis Linford and Oliveira) are three important plant–parasitic pests in soybean. Previous study showed that plant introduction (PI) 567516C harbored novel quantitative trait loci (QTL) conferring SCN resistance to soybean. However, QTL underlying resistance to SRKN and RN in PI 567516C remain unknown. The objectives of this study were to identify QTL for resistance to SRKN and RN in PI 567516C. Two hundred and forty-seven F6:9 recombinant inbred lines, derived from a cross between cultivar Magellan and PI 567516C, were evaluated for resistance to SRKN and RN. Two hundred and thirty-eight simple sequence repeats and 687 single nucleotide polymorphism markers were used to construct a genetic linkage map. Three significant QTL associated with resistance to SRKN were mapped on chromosomes (Chrs.) 10, 13 and 17. Two significant QTL associated with resistance to RN were detected on Chrs. 11 and 18. Whole-genome resequencing revealed that there might be Peking-type Rhg1 in PI 567516C. Our study provides useful information to employ PI 567516C in soybean breeding in order to develop new cultivars with resistance to multiple nematodes. Electronic supplementary material The online version of this article (doi:10.1007/s11032-015-0330-5) contains supplementary material, which is available to authorized users.

Realization of precision medicine ideas requires significant research effort to be able to spot subtle differences in complex diseases at the molecular level to develop personalized therapies. It is especially important in many cases of highly heterogeneous cancers. Precision diagnostics and therapeutics of such diseases demands interrogation of vast amounts of biological knowledge coupled with novel analytic methodologies. For instance, pathway-based approaches can shed light on the way tumorigenesis takes place in individual patient cases and pinpoint to novel drug targets. However, comprehensive analysis of hundreds of pathways and thousands of genes creates a combinatorial explosion, that is challenging for medical practitioners to handle at the point of care. Here we extend our previous work on mapping clinical omics data to curated Resource Description Framework (RDF) knowledge bases to derive influence diagrams of interrelationships of biomarker proteins, diseases and signal transduction pathways for personalized theranostics. We present RDF Sketch Maps - a computational method to reduce knowledge complexity for precision medicine analytics. The method of RDF Sketch Maps is inspired by the way a sketch artist conveys only important visual information and discards other unnecessary details. In our case, we compute and retain only so-called RDF Edges - places with highly important diagnostic and therapeutic information. To do this we utilize 35 maps of human signal transduction pathways by transforming 300 KEGG maps into highly processable RDF knowledge base. We have demonstrated potential clinical utility of RDF Sketch Maps in hematopoietic cancers, including analysis of pathways associated with Hairy Cell Leukemia (HCL) and Chronic Myeloid Leukemia (CML) where we achieved up to 20-fold reduction in the number of biological entities to be analyzed, while retaining most likely important entities. In experiments with pathways associated with HCL a generated RDF Sketch Map of the top 30% paths retained important information about signaling cascades leading to activation of proto-oncogene BRAF, which is usually associated with a different cancer, melanoma. Recent reports of successful treatments of HCL patients by the BRAF-targeted drug vemurafenib support the validity of the RDF Sketch Maps findings. We therefore believe that RDF Sketch Maps will be invaluable for hypothesis generation for precision diagnostics and therapeutics as well as drug repurposing studies.

Background Small non-coding RNAs (21 to 24 nucleotides) regulate a number of developmental processes in plants and animals by silencing genes using multiple mechanisms. Among these, the most conserved classes are microRNAs (miRNAs) and small interfering RNAs (siRNAs), both of which are produced by RNase III-like enzymes called Dicers. Many plant miRNAs play critical roles in nutrient homeostasis, developmental processes, abiotic stress and pathogen responses. Currently, only 70 miRNA have been identified in soybean. Methods We utilized Illumina's SBS sequencing technology to generate high-quality small RNA (sRNA) data from four soybean (Glycine max) tissues, including root, seed, flower, and nodules, to expand the collection of currently known soybean miRNAs. We developed a bioinformatics pipeline using in-house scripts and publicly available structure prediction tools to differentiate the authentic mature miRNA sequences from other sRNAs and short RNA fragments represented in the public sequencing data. Results The combined sequencing and bioinformatics analyses identified 129 miRNAs based on hairpin secondary structure features in the predicted precursors. Out of these, 42 miRNAs matched known miRNAs in soybean or other species, while 87 novel miRNAs were identified. We also predicted the putative target genes of all identified miRNAs with computational methods and verified the predicted cleavage sites in vivo for a subset of these targets using the 5' RACE method. Finally, we also studied the relationship between the abundance of miRNA and that of the respective target genes by comparison to Solexa cDNA sequencing data. Conclusion Our study significantly increased the number of miRNAs known to be expressed in soybean. The bioinformatics analysis provided insight on regulation patterns between the miRNAs and their predicted target genes expression. We also deposited the data in a soybean genome browser based on the UCSC Genome Browser architecture. Using the browser, we annotated the soybean data with miRNA sequences from four tissues and cDNA sequencing data. Overlaying these two datasets in the browser allows researchers to analyze the miRNA expression levels relative to that of the associated target genes. The browser can be accessed at http://digbio.missouri.edu/soybean_mirna/.

Characterizing gene function is one of the major challenging tasks in the post-genomic era. To address this challenge, we have developed GeneFAS (Gene Function Annotation System), a new integrated probabilistic method for cellular function prediction by combining information from protein–protein interactions, protein complexes, microarray gene expression profiles, and annotations of known proteins through an integrative statistical model. Our approach is based on a novel assessment for the relationship between (1) the interaction/correlation of two proteins' high-throughput data and (2) their functional relationship in terms of their Gene Ontology (GO) hierarchy. We have developed a Web server for the predictions. We have applied our method to yeast Saccharomyces cerevisiae and predicted functions for 1548 out of 2472 unannotated proteins.

Background Phaseolus vulgaris (common bean) is the second most important legume crop in the world after soybean. Consequently, yield losses due to fungal infection, like Uromyces appendiculatus (bean rust), have strong consequences. Several resistant genes were identified that confer resistance to bean rust infection. However, the downstream genes and mechanisms involved in bean resistance to infection are poorly characterized. Results A subtractive bean cDNA library composed of 10,581 unisequences was constructed and enriched in sequences regulated by either bean rust race 41, a virulent strain, or race 49, an avirulent strain on cultivar Early Gallatin carrying the resistance gene Ur-4. The construction of this library allowed the identification of 6,202 new bean ESTs, significantly adding to the available sequences for this plant. Regulation of selected bean genes in response to bean rust infection was confirmed by qRT-PCR. Plant gene expression was similar for both race 41 and 49 during the first 48 hours of the infection process but varied significantly at the later time points (72–96 hours after inoculation) mainly due to the presence of the Avr4 gene in the race 49 leading to a hypersensitive response in the bean plants. A biphasic pattern of gene expression was observed for several genes regulated in response to fungal infection. Conclusion The enrichment of the public database with over 6,000 bean ESTs significantly adds to the genomic resources available for this important crop plant. The analysis of these genes in response to bean rust infection provides a foundation for further studies of the mechanism of fungal disease resistance. The expression pattern of 90 bean genes upon rust infection shares several features with other legumes infected by biotrophic fungi. This finding suggests that the P. vulgaris-U. appendiculatus pathosystem could serve as a model to explore legume-rust interaction.