Transcriptome analysis of Spodoptera frugiperda Sf9 cells reveals putative apoptosis-related genes and a preliminary apoptosis mechanism induced by azadirachtin.

PubMed

Shu, Benshui; Zhang, Jingjing; Sethuraman, Veeran; Cui, Gaofeng; Yi, Xin; Zhong, Guohua

2017-10-16

As an important botanical pesticide, azadirachtin demonstrates broad insecticidal activity against many agricultural pests. The results of a previous study indicated the toxicity and apoptosis induction of azadirachtin in Spodoptera frugiperda Sf9 cells. However, the lack of genomic data has hindered a deeper investigation of apoptosis in Sf9 cells at a molecular level. In the present study, the complete transcriptome data for Sf9 cell line was accomplished using Illumina sequencing technology, and 97 putative apoptosis-related genes were identified through BLAST and KEGG orthologue annotations. Fragments of potential candidate apoptosis-related genes were cloned, and the mRNA expression patterns of ten identified genes regulated by azadirachtin were examined using qRT-PCR. Furthermore, Western blot analysis showed that six putative apoptosis-related proteins were upregulated after being treated with azadirachtin while the protein Bcl-2 were downregulated. These data suggested that both intrinsic and extrinsic apoptotic signal pathways comprising the identified potential apoptosis-related genes were potentially active in S. frugiperda. In addition, the preliminary results revealed that caspase-dependent or caspase-independent apoptotic pathways could function in azadirachtin-induced apoptosis in Sf9 cells.

Molecular biomarkers of resistance to anti-EGFR treatment in metastatic colorectal cancer, from classical to innovation.

PubMed

Giampieri, Riccardo; Scartozzi, Mario; Del Prete, Michela; Maccaroni, Elena; Bittoni, Alessandro; Faloppi, Luca; Bianconi, Maristella; Cecchini, Luca; Cascinu, Stefano

2013-11-01

Systematic dissection of the EGFR pathway was considered as the best way to identify putative markers of resistance to anti-EGFR therapies. This kind of approach leaves other, less known but by no means less important, putative mechanisms of resistance. We tried to shed some light on these mechanisms of resistance. We performed a research through Pubmed database of all published articles highlighting mechanisms of resistance to Cetuximab and Panitumumab based therapies, published in 2000-2012 period. We reviewed the "classical" molecular factors, extensively analyzed as predictive factors for efficacy to anti-EGFR therapy, such as K-ras, B-raf, and PI3K-mTOR-Akt, focusing on their predictive or prognostic value and on the controversial aspects of the biomarker analysis for clinical practice. On the second part we will then move on to other less known molecular markers, for the future understanding of biological mechanisms underlying anti-EGFR therapy resistance, such as non-canonical heterodimer candidates, microRNA, IGF1-IGF1R, HGF-cMET and secondary mutations of EGFR. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Comprehensive proteomic analysis of bovine spermatozoa of varying fertility rates and identification of biomarkers associated with fertility.

PubMed

Peddinti, Divyaswetha; Nanduri, Bindu; Kaya, Abdullah; Feugang, Jean M; Burgess, Shane C; Memili, Erdogan

2008-02-22

Male infertility is a major problem for mammalian reproduction. However, molecular details including the underlying mechanisms of male fertility are still not known. A thorough understanding of these mechanisms is essential for obtaining consistently high reproductive efficiency and to ensure lower cost and time-loss by breeder. Using high and low fertility bull spermatozoa, here we employed differential detergent fractionation multidimensional protein identification technology (DDF-Mud PIT) and identified 125 putative biomarkers of fertility. We next used quantitative Systems Biology modeling and canonical protein interaction pathways and networks to show that high fertility spermatozoa differ from low fertility spermatozoa in four main ways. Compared to sperm from low fertility bulls, sperm from high fertility bulls have higher expression of proteins involved in: energy metabolism, cell communication, spermatogenesis, and cell motility. Our data also suggests a hypothesis that low fertility sperm DNA integrity may be compromised because cell cycle: G2/M DNA damage checkpoint regulation was most significant signaling pathway identified in low fertility spermatozoa. This is the first comprehensive description of the bovine spermatozoa proteome. Comparative proteomic analysis of high fertility and low fertility bulls, in the context of protein interaction networks identified putative molecular markers associated with high fertility phenotype.

Comprehensive proteomic analysis of bovine spermatozoa of varying fertility rates and identification of biomarkers associated with fertility

PubMed Central

Peddinti, Divyaswetha; Nanduri, Bindu; Kaya, Abdullah; Feugang, Jean M; Burgess, Shane C; Memili, Erdogan

2008-01-01

Background Male infertility is a major problem for mammalian reproduction. However, molecular details including the underlying mechanisms of male fertility are still not known. A thorough understanding of these mechanisms is essential for obtaining consistently high reproductive efficiency and to ensure lower cost and time-loss by breeder. Results Using high and low fertility bull spermatozoa, here we employed differential detergent fractionation multidimensional protein identification technology (DDF-Mud PIT) and identified 125 putative biomarkers of fertility. We next used quantitative Systems Biology modeling and canonical protein interaction pathways and networks to show that high fertility spermatozoa differ from low fertility spermatozoa in four main ways. Compared to sperm from low fertility bulls, sperm from high fertility bulls have higher expression of proteins involved in: energy metabolism, cell communication, spermatogenesis, and cell motility. Our data also suggests a hypothesis that low fertility sperm DNA integrity may be compromised because cell cycle: G2/M DNA damage checkpoint regulation was most significant signaling pathway identified in low fertility spermatozoa. Conclusion This is the first comprehensive description of the bovine spermatozoa proteome. Comparative proteomic analysis of high fertility and low fertility bulls, in the context of protein interaction networks identified putative molecular markers associated with high fertility phenotype. PMID:18294385

De novo Transcriptome Analysis of Miscanthus lutarioriparius Identifies Candidate Genes in Rhizome Development

PubMed Central

Hu, Ruibo; Yu, Changjiang; Wang, Xiaoyu; Jia, Chunlin; Pei, Shengqiang; He, Kang; He, Guo; Kong, Yingzhen; Zhou, Gongke

2017-01-01

HIGHLIGHT De novo transcriptome profiling of five tissues reveals candidate genes putatively involved in rhizome development in M. lutarioriparius. Miscanthus lutarioriparius is a promising lignocellulosic feedstock for second-generation bioethanol production. However, the genomic resource for this species is relatively limited thus hampers our understanding of the molecular mechanisms underlying many important biological processes. In this study, we performed the first de novo transcriptome analysis of five tissues (leaf, stem, root, lateral bud and rhizome bud) of M. lutarioriparius with an emphasis to identify putative genes involved in rhizome development. Approximately 66 gigabase (GB) paired-end clean reads were obtained and assembled into 169,064 unigenes with an average length of 759 bp. Among these unigenes, 103,899 (61.5%) were annotated in seven public protein databases. Differential gene expression profiling analysis revealed that 4,609, 3,188, 1,679, 1,218, and 1,077 genes were predominantly expressed in root, leaf, stem, lateral bud, and rhizome bud, respectively. Their expression patterns were further classified into 12 distinct clusters. Pathway enrichment analysis revealed that genes predominantly expressed in rhizome bud were mainly involved in primary metabolism and hormone signaling and transduction pathways. Noteworthy, 19 transcription factors (TFs) and 16 hormone signaling pathway-related genes were identified to be predominantly expressed in rhizome bud compared with the other tissues, suggesting putative roles in rhizome formation and development. In addition, a predictive regulatory network was constructed between four TFs and six auxin and abscisic acid (ABA) -related genes. Furthermore, the expression of 24 rhizome-specific genes was further validated by quantitative real-time RT-PCR (qRT-PCR) analysis. Taken together, this study provide a global portrait of gene expression across five different tissues and reveal preliminary insights into rhizome growth and development. The data presented will contribute to our understanding of the molecular mechanisms underlying rhizome development in M. lutarioriparius and remarkably enrich the genomic resources of Miscanthus. PMID:28446913

Red blood cells open promising avenues for longitudinal studies of ageing in laboratory, non-model and wild animals.

PubMed

Stier, Antoine; Reichert, Sophie; Criscuolo, Francois; Bize, Pierre

2015-11-01

Ageing is characterized by a progressive deterioration of multiple physiological and molecular pathways, which impair organismal performance and increase risks of death with advancing age. Hence, ageing studies must identify physiological and molecular pathways that show signs of age-related deterioration, and test their association with the risk of death and longevity. This approach necessitates longitudinal sampling of the same individuals, and therefore requires a minimally invasive sampling technique that provides access to the larger spectrum of physiological and molecular pathways that are putatively associated with ageing. The present paper underlines the interest in using red blood cells (RBCs) as a promising target for longitudinal studies of ageing in vertebrates. RBCs provide valuable information on the following six pathways: cell maintenance and turnover (RBC number, size, and heterogeneity), glucose homeostasis (RBC glycated haemoglobin), oxidative stress parameters, membrane composition and integrity, mitochondrial functioning, and telomere dynamics. The last two pathways are specific to RBCs of non-mammalian species, which possess a nucleus and functional mitochondria. We present the current knowledge about RBCs and age-dependent changes in these pathways in non-model and wild species that are especially suitable to address questions related to ageing using longitudinal studies. We discuss how the different pathways relate with survival and lifespan and give information on their genetic and environmental determinants to appraise their evolutionary potential. Copyright © 2015 Elsevier Inc. All rights reserved.

Molecular Characterization of Macrophage-Biomaterial Interactions

PubMed Central

Moore, Laura Beth; Kyriakides, Themis R.

2015-01-01

Implantation of biomaterials in vascularized tissues elicits the sequential engagement of molecular and cellular elements that constitute the foreign body response. Initial events include the non-specific adsorption of proteins to the biomaterial surface that render it adhesive for cells such as neutrophils and macrophages. The latter undergo unique activation and in some cases undergo cell-cell fusion to form foreign body giant cells that contribute to implant damage and fibrotic encapsulation. In this review, we discuss the molecular events that contribute to macrophage activation and fusion with a focus on the role of the inflammasome, signaling pathways such as JAK/STAT and NF-κB, and the putative involvement of micro RNAs in the regulation of these processes. PMID:26306446

Molecular Characterization of Macrophage-Biomaterial Interactions.

PubMed

Moore, Laura Beth; Kyriakides, Themis R

2015-01-01

Implantation of biomaterials in vascularized tissues elicits the sequential engagement of molecular and cellular elements that constitute the foreign body response. Initial events include the non-specific adsorption of proteins to the biomaterial surface that render it adhesive for cells such as neutrophils and macrophages. The latter undergo unique activation and in some cases undergo cell-cell fusion to form foreign body giant cells that contribute to implant damage and fibrotic encapsulation. In this review, we discuss the molecular events that contribute to macrophage activation and fusion with a focus on the role of the inflammasome, signaling pathways such as JAK/STAT and NF-κB, and the putative involvement of micro RNAs in the regulation of these processes.

Heat Shock Proteins and Autophagy Pathways in Neuroprotection: From Molecular Bases to Pharmacological Interventions

PubMed Central

Penke, Botond; Bogár, Ferenc; Sántha, Miklós; Tóth, Melinda E.; Vígh, László

2018-01-01

Neurodegenerative diseases (NDDs) such as Alzheimer’s disease, Parkinson’s disease and Huntington’s disease (HD), amyotrophic lateral sclerosis, and prion diseases are all characterized by the accumulation of protein aggregates (amyloids) into inclusions and/or plaques. The ubiquitous presence of amyloids in NDDs suggests the involvement of disturbed protein homeostasis (proteostasis) in the underlying pathomechanisms. This review summarizes specific mechanisms that maintain proteostasis, including molecular chaperons, the ubiquitin-proteasome system (UPS), endoplasmic reticulum associated degradation (ERAD), and different autophagic pathways (chaperon mediated-, micro-, and macro-autophagy). The role of heat shock proteins (Hsps) in cellular quality control and degradation of pathogenic proteins is reviewed. Finally, putative therapeutic strategies for efficient removal of cytotoxic proteins from neurons and design of new therapeutic targets against the progression of NDDs are discussed. PMID:29361800

Simultaneous prediction of enzyme orthologs from chemical transformation patterns for de novo metabolic pathway reconstruction

PubMed Central

Tabei, Yasuo; Yamanishi, Yoshihiro; Kotera, Masaaki

2016-01-01

Motivation: Metabolic pathways are an important class of molecular networks consisting of compounds, enzymes and their interactions. The understanding of global metabolic pathways is extremely important for various applications in ecology and pharmacology. However, large parts of metabolic pathways remain unknown, and most organism-specific pathways contain many missing enzymes. Results: In this study we propose a novel method to predict the enzyme orthologs that catalyze the putative reactions to facilitate the de novo reconstruction of metabolic pathways from metabolome-scale compound sets. The algorithm detects the chemical transformation patterns of substrate–product pairs using chemical graph alignments, and constructs a set of enzyme-specific classifiers to simultaneously predict all the enzyme orthologs that could catalyze the putative reactions of the substrate–product pairs in the joint learning framework. The originality of the method lies in its ability to make predictions for thousands of enzyme orthologs simultaneously, as well as its extraction of enzyme-specific chemical transformation patterns of substrate–product pairs. We demonstrate the usefulness of the proposed method by applying it to some ten thousands of metabolic compounds, and analyze the extracted chemical transformation patterns that provide insights into the characteristics and specificities of enzymes. The proposed method will open the door to both primary (central) and secondary metabolism in genomics research, increasing research productivity to tackle a wide variety of environmental and public health matters. Availability and Implementation: Contact: maskot@bio.titech.ac.jp PMID:27307627

Genetics and the Placebo Effect: the Placebome

PubMed Central

Hall, Kathryn T.; Loscalzo, Joseph; Kaptchuk, Ted J.

2015-01-01

Placebos are indispensable controls in randomized clinical trials (RCTs), and placebo responses significantly contribute to routine clinical outcomes. Recent neurophysiological studies reveal neurotransmitter pathways that mediate placebo effects. Evidence that genetic variations in these pathways can modify placebo effects raises the possibility of using genetic screening to identify placebo responders and thereby increase RCT efficacy and improve therapeutic care. Furthermore, the possibility of interaction between placebo and drug molecular pathways warrants consideration in RCT design. The study of genomic effects on placebo response, “the placebome”, is in its infancy. Here, we review evidence from placebo studies and RCTs to identify putative genes in the placebome, examine evidence for placebo-drug interactions, and discuss implications for RCTs and clinical care. PMID:25883069

Proteome-wide prediction of targets for aspirin: new insight into the molecular mechanism of aspirin

PubMed Central

Dai, Shao-Xing; Li, Wen-Xing

2016-01-01

Besides its anti-inflammatory, analgesic and anti-pyretic properties, aspirin is used for the prevention of cardiovascular disease and various types of cancer. The multiple activities of aspirin likely involve several molecular targets and pathways rather than a single target. Therefore, systematic identification of these targets of aspirin can help us understand the underlying mechanisms of the activities. In this study, we identified 23 putative targets of aspirin in the human proteome by using binding pocket similarity detecting tool combination with molecular docking, free energy calculation and pathway analysis. These targets have diverse folds and are derived from different protein family. However, they have similar aspirin-binding pockets. The binding free energy with aspirin for newly identified targets is comparable to that for the primary targets. Pathway analysis revealed that the targets were enriched in several pathways such as vascular endothelial growth factor (VEGF) signaling, Fc epsilon RI signaling and arachidonic acid metabolism, which are strongly involved in inflammation, cardiovascular disease and cancer. Therefore, the predicted target profile of aspirin suggests a new explanation for the disease prevention ability of aspirin. Our findings provide a new insight of aspirin and its efficacy of disease prevention in a systematic and global view. PMID:26989626

Proteome-wide prediction of targets for aspirin: new insight into the molecular mechanism of aspirin.

PubMed

Dai, Shao-Xing; Li, Wen-Xing; Li, Gong-Hua; Huang, Jing-Fei

2016-01-01

Besides its anti-inflammatory, analgesic and anti-pyretic properties, aspirin is used for the prevention of cardiovascular disease and various types of cancer. The multiple activities of aspirin likely involve several molecular targets and pathways rather than a single target. Therefore, systematic identification of these targets of aspirin can help us understand the underlying mechanisms of the activities. In this study, we identified 23 putative targets of aspirin in the human proteome by using binding pocket similarity detecting tool combination with molecular docking, free energy calculation and pathway analysis. These targets have diverse folds and are derived from different protein family. However, they have similar aspirin-binding pockets. The binding free energy with aspirin for newly identified targets is comparable to that for the primary targets. Pathway analysis revealed that the targets were enriched in several pathways such as vascular endothelial growth factor (VEGF) signaling, Fc epsilon RI signaling and arachidonic acid metabolism, which are strongly involved in inflammation, cardiovascular disease and cancer. Therefore, the predicted target profile of aspirin suggests a new explanation for the disease prevention ability of aspirin. Our findings provide a new insight of aspirin and its efficacy of disease prevention in a systematic and global view.

Sugars and plant innate immunity.

PubMed

Bolouri Moghaddam, Mohammad Reza; Van den Ende, Wim

2012-06-01

Sugars are involved in many metabolic and signalling pathways in plants. Sugar signals may also contribute to immune responses against pathogens and probably function as priming molecules leading to pathogen-associated molecular patterns (PAMP)-triggered immunity and effector-triggered immunity in plants. These putative roles also depend greatly on coordinated relationships with hormones and the light status in an intricate network. Although evidence in favour of sugar-mediated plant immunity is accumulating, more in-depth fundamental research is required to unravel the sugar signalling pathways involved. This might pave the way for the use of biodegradable sugar-(like) compounds to counteract plant diseases as cheaper and safer alternatives for toxic agrochemicals.

De Novo Transcriptome Analysis of an Aerial Microalga Trentepohlia jolithus: Pathway Description and Gene Discovery for Carbon Fixation and Carotenoid Biosynthesis

PubMed Central

Li, Qianqian; Liu, Jianguo; Zhang, Litao; Liu, Qian

2014-01-01

Background Algae in the order Trentepohliales have a broad geographic distribution and are generally characterized by the presence of abundant β-carotene. The many monographs published to date have mainly focused on their morphology, taxonomy, phylogeny, distribution and reproduction; molecular studies of this order are still rare. High-throughput RNA sequencing (RNA-Seq) technology provides a powerful and efficient method for transcript analysis and gene discovery in Trentepohlia jolithus. Methods/Principal Findings Illumina HiSeq 2000 sequencing generated 55,007,830 Illumina PE raw reads, which were assembled into 41,328 assembled unigenes. Based on NR annotation, 53.28% of the unigenes (22,018) could be assigned to gene ontology classes with 54 subcategories and 161,451 functional terms. A total of 26,217 (63.44%) assembled unigenes were mapped to 128 KEGG pathways. Furthermore, a set of 5,798 SSRs in 5,206 unigenes and 131,478 putative SNPs were identified. Moreover, the fact that all of the C4 photosynthesis genes exist in T. jolithus suggests a complex carbon acquisition and fixation system. Similarities and differences between T. jolithus and other algae in carotenoid biosynthesis are also described in depth. Conclusions/Significance This is the first broad transcriptome survey for T. jolithus, increasing the amount of molecular data available for the class Ulvophyceae. As well as providing resources for functional genomics studies, the functional genes and putative pathways identified here will contribute to a better understanding of carbon fixation and fatty acid and carotenoid biosynthesis in T. jolithus. PMID:25254555

Multi-Targeted Molecular Effects of Hibiscus sabdariffa Polyphenols: An Opportunity for a Global Approach to Obesity

PubMed Central

Herranz-López, María; Olivares-Vicente, Mariló; Barrajón-Catalán, Enrique; Segura-Carretero, Antonio; Joven, Jorge; Micol, Vicente

2017-01-01

Improper diet can alter gene expression by breaking the energy balance equation and changing metabolic and oxidative stress biomarkers, which can result in the development of obesity-related metabolic disorders. The pleiotropic effects of dietary plant polyphenols are capable of counteracting by modulating different key molecular targets at the cell, as well as through epigenetic modifications. Hibiscus sabdariffa (HS)-derived polyphenols are known to ameliorate various obesity-related conditions. Recent evidence leads to propose the complex nature of the underlying mechanism of action. This multi-targeted mechanism includes the regulation of energy metabolism, oxidative stress and inflammatory pathways, transcription factors, hormones and peptides, digestive enzymes, as well as epigenetic modifications. This article reviews the accumulated evidence on the multiple anti-obesity effects of HS polyphenols in cell and animal models, as well as in humans, and its putative molecular targets. In silico studies reveal the capacity of several HS polyphenols to act as putative ligands for different digestive and metabolic enzymes, which may also deserve further attention. Therefore, a global approach including integrated and networked omics techniques, virtual screening and epigenetic analysis is necessary to fully understand the molecular mechanisms of HS polyphenols and metabolites involved, as well as their possible implications in the design of safe and effective polyphenolic formulations for obesity. PMID:28825642

Multi-Targeted Molecular Effects of Hibiscus sabdariffa Polyphenols: An Opportunity for a Global Approach to Obesity.

PubMed

Herranz-López, María; Olivares-Vicente, Mariló; Encinar, José Antonio; Barrajón-Catalán, Enrique; Segura-Carretero, Antonio; Joven, Jorge; Micol, Vicente

2017-08-20

Improper diet can alter gene expression by breaking the energy balance equation and changing metabolic and oxidative stress biomarkers, which can result in the development of obesity-related metabolic disorders. The pleiotropic effects of dietary plant polyphenols are capable of counteracting by modulating different key molecular targets at the cell, as well as through epigenetic modifications. Hibiscus sabdariffa (HS)-derived polyphenols are known to ameliorate various obesity-related conditions. Recent evidence leads to propose the complex nature of the underlying mechanism of action. This multi-targeted mechanism includes the regulation of energy metabolism, oxidative stress and inflammatory pathways, transcription factors, hormones and peptides, digestive enzymes, as well as epigenetic modifications. This article reviews the accumulated evidence on the multiple anti-obesity effects of HS polyphenols in cell and animal models, as well as in humans, and its putative molecular targets. In silico studies reveal the capacity of several HS polyphenols to act as putative ligands for different digestive and metabolic enzymes, which may also deserve further attention. Therefore, a global approach including integrated and networked omics techniques, virtual screening and epigenetic analysis is necessary to fully understand the molecular mechanisms of HS polyphenols and metabolites involved, as well as their possible implications in the design of safe and effective polyphenolic formulations for obesity.

Huntington disease iPSCs show early molecular changes in intracellular signaling, the expression of oxidative stress proteins and the p53 pathway

PubMed Central

Szlachcic, Wojciech J.; Switonski, Pawel M.; Krzyzosiak, Wlodzimierz J.; Figlerowicz, Marek; Figiel, Maciej

2015-01-01

ABSTRACT Huntington disease (HD) is a brain disorder characterized by the late onset of motor and cognitive symptoms, even though the neurons in the brain begin to suffer dysfunction and degeneration long before symptoms appear. There is currently no cure. Several molecular and developmental effects of HD have been identified using neural stem cells (NSCs) and differentiated cells, such as neurons and astrocytes. Still, little is known regarding the molecular pathogenesis of HD in pluripotent cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Therefore, we examined putative signaling pathways and processes involved in HD pathogenesis in pluripotent cells. We tested naïve mouse HD YAC128 iPSCs and two types of human HD iPSC that were generated from HD and juvenile-HD patients. Surprisingly, we found that a number of changes affecting cellular processes in HD were also present in undifferentiated pluripotent HD iPSCs, including the dysregulation of the MAPK and Wnt signaling pathways and the dysregulation of the expression of genes related to oxidative stress, such as Sod1. Interestingly, a common protein interactor of the huntingtin protein and the proteins in the above pathways is p53, and the expression of p53 was dysregulated in HD YAC128 iPSCs and human HD iPSCs. In summary, our findings demonstrate that multiple molecular pathways that are characteristically dysregulated in HD are already altered in undifferentiated pluripotent cells and that the pathogenesis of HD might begin during the early stages of life. PMID:26092128

The molecular basis of ethylene signalling in Arabidopsis

NASA Technical Reports Server (NTRS)

Woeste, K.; Kieber, J. J.; Evans, M. L. (Principal Investigator)

1998-01-01

The simple gas ethylene profoundly influences plants at nearly every stage of growth and development. In the past ten years, the use of a genetic approach, based on the triple response phenotype, has been a powerful tool for investigating the molecular events that underlie these effects. Several fundamental elements of the pathway have been described: a receptor with homology to bacterial two-component histidine kinases (ETR1), elements of a MAP kinase cascade (CTR1) and a putative transcription factor (EIN3). Taken together, these elements can be assembled into a simple, linear model for ethylene signalling that accounts for most of the well-characterized ethylene mediated responses.

Compatibility of SYTO 13 and Hoechst 33342 for Longitudinal Imaging of Neuron Viability and Cell Death

DTIC Science & Technology

2012-08-14

Defining the molecular and biochemical pathways re- sponsible for cell death phenotypes is essential for iden- tifying critical points that could be...clearly image nuclear structure resulted in PI-positive nuclei developing an orange hue. (B) Planimetric quantitation of nuclear size measured...metabolites on undifferentiated PC12 cells: a putative structure -toxicity relationship. Chem Res Toxicol 2006, 19(10):1294–1304. 10. McNutt P, Celver J

PUTATIVE ADVERSE OUTCOME PATHWAY FOR INHIBITON ...

EPA Pesticide Factsheets

The adverse outcome pathway (AOP) provides a framework for organizing knowledge to define links between a molecular initiating event (MIE) and an adverse outcome (AO) occurring at a higher level of biological organization, such as the individual or population. The AOP framework proceeds from a general (e.g., not chemical specific) molecular mode of action, designated as a MIE, through stepwise changes in biological status, defined as key events (KEs), to a final AO that can be used in risk assessment. Because aromatase-inhibiting pharmaceuticals are widely used to treat breast cancer patients, we explored the unintended consequences that might occur in fish exposed to these chemicals through wastewater discharge into the aquatic environment. Unlike mammals, fish have two isoforms of aromatase, one that predominates in the ovary (cyp19a1a) and a second (cyp19a1b) that prevails in the brain. Aromatase activity in fish brain can be 100 to 1000 times that in mammals and is associated with reproduction. We have developed a putative AOP for inhibition of brain aromatase in fish leading to reproductive dysfunction based on review of relevant literature and reproductive experiments with the marine fish cunner (Tautogolabrus adspersus) exposed to aromatase-inhibiting pharmaceuticals in the laboratory. The first KE in this AOP is a decrease in brain aromatase activity due to exposure to an aromatase inhibitor. KEs then progress through subsequent steps including decreas

De novo transcriptome analysis of rose-scented geranium provides insights into the metabolic specificity of terpene and tartaric acid biosynthesis.

PubMed

Narnoliya, Lokesh K; Kaushal, Girija; Singh, Sudhir P; Sangwan, Rajender S

2017-01-13

Rose-scented geranium (Pelargonium sp.) is a perennial herb that produces a high value essential oil of fragrant significance due to the characteristic compositional blend of rose-oxide and acyclic monoterpenoids in foliage. Recently, the plant has also been shown to produce tartaric acid in leaf tissues. Rose-scented geranium represents top-tier cash crop in terms of economic returns and significance of the plant and plant products. However, there has hardly been any study on its metabolism and functional genomics, nor any genomic expression dataset resource is available in public domain. Therefore, to begin the gains in molecular understanding of specialized metabolic pathways of the plant, de novo sequencing of rose-scented geranium leaf transcriptome, transcript assembly, annotation, expression profiling as well as their validation were carried out. De novo transcriptome analysis resulted a total of 78,943 unique contigs (average length: 623 bp, and N50 length: 752 bp) from 15.44 million high quality raw reads. In silico functional annotation led to the identification of several putative genes representing terpene, ascorbic acid and tartaric acid biosynthetic pathways, hormone metabolism, and transcription factors. Additionally, a total of 6,040 simple sequence repeat (SSR) motifs were identified in 6.8% of the expressed transcripts. The highest frequency of SSR was of tri-nucleotides (50%). Further, transcriptome assembly was validated for randomly selected putative genes by standard PCR-based approach. In silico expression profile of assembled contigs were validated by real-time PCR analysis of selected transcripts. Being the first report on transcriptome analysis of rose-scented geranium the data sets and the leads and directions reflected in this investigation will serve as a foundation for pursuing and understanding molecular aspects of its biology, and specialized metabolic pathways, metabolic engineering, genetic diversity as well as molecular breeding.

NiaoDuQing granules relieve chronic kidney disease symptoms by decreasing renal fibrosis and anemia

PubMed Central

Wang, Xu; Yu, Suyun; Jia, Qi; Chen, Lichuan; Zhong, Jinqiu; Pan, Yanhong; Shen, Peiliang; Shen, Yin; Wang, Siliang; Wei, Zhonghong; Cao, Yuzhu; Lu, Yin

2017-01-01

NiaoDuQing (NDQ) granules, a traditional Chinese medicine, has been clinically used in China for over fourteen years to treat chronic kidney disease (CKD). To elucidate the mechanisms underlying the therapeutic benefits of NDQ, we designed an approach incorporating chemoinformatics, bioinformatics, network biology methods, and cellular and molecular biology experiments. A total of 182 active compounds were identified in NDQ granules, and 397 putative targets associated with different diseases were derived through ADME modelling and target prediction tools. Protein-protein interaction networks of CKD-related and putative NDQ targets were constructed, and 219 candidate targets were identified based on topological features. Pathway enrichment analysis showed that the candidate targets were mostly related to the TGF-β, the p38MAPK, and the erythropoietin (EPO) receptor signaling pathways, which are known contributors to renal fibrosis and/or renal anemia. A rat model of CKD was established to validate the drug-target mechanisms predicted by the systems pharmacology analysis. Experimental results confirmed that NDQ granules exerted therapeutic effects on CKD and its comorbidities, including renal anemia, mainly by modulating the TGF-β and EPO signaling pathways. Thus, the pharmacological actions of NDQ on CKD symptoms correlated well with in silico predictions. PMID:28915563

Transcriptome Analysis and Discovery of Genes Involved in Immune Pathways from Coelomocytes of Sea Cucumber (Apostichopus japonicus) after Vibrio splendidus Challenge.

PubMed

Gao, Qiong; Liao, Meijie; Wang, Yingeng; Li, Bin; Zhang, Zheng; Rong, Xiaojun; Chen, Guiping; Wang, Lan

2015-07-17

Vibrio splendidus is identified as one of the major pathogenic factors for the skin ulceration syndrome in sea cucumber (Apostichopus japonicus), which has vastly limited the development of the sea cucumber culture industry. In order to screen the immune genes involving Vibrio splendidus challenge in sea cucumber and explore the molecular mechanism of this process, the related transcriptome and gene expression profiling of resistant and susceptible biotypes of sea cucumber with Vibrio splendidus challenge were collected for analysis. A total of 319,455,942 trimmed reads were obtained, which were assembled into 186,658 contigs. After that, 89,891 representative contigs (without isoform) were clustered. The analysis of the gene expression profiling identified 358 differentially expression genes (DEGs) in the bacterial-resistant group, and 102 DEGs in the bacterial-susceptible group, compared with that in control group. According to the reported references and annotation information from BLAST, GO and KEGG, 30 putative bacterial-resistant genes and 19 putative bacterial-susceptible genes were identified from DEGs. The qRT-PCR results were consistent with the RNA-Seq results. Furthermore, many DGEs were involved in immune signaling related pathways, such as Endocytosis, Lysosome, MAPK, Chemokine and the ERBB signaling pathway.

Transcriptome Analysis and Discovery of Genes Involved in Immune Pathways from Coelomocytes of Sea Cucumber (Apostichopus japonicus) after Vibrio splendidus Challenge

PubMed Central

Gao, Qiong; Liao, Meijie; Wang, Yingeng; Li, Bin; Zhang, Zheng; Rong, Xiaojun; Chen, Guiping; Wang, Lan

2015-01-01

Vibrio splendidus is identified as one of the major pathogenic factors for the skin ulceration syndrome in sea cucumber (Apostichopus japonicus), which has vastly limited the development of the sea cucumber culture industry. In order to screen the immune genes involving Vibrio splendidus challenge in sea cucumber and explore the molecular mechanism of this process, the related transcriptome and gene expression profiling of resistant and susceptible biotypes of sea cucumber with Vibrio splendidus challenge were collected for analysis. A total of 319,455,942 trimmed reads were obtained, which were assembled into 186,658 contigs. After that, 89,891 representative contigs (without isoform) were clustered. The analysis of the gene expression profiling identified 358 differentially expression genes (DEGs) in the bacterial-resistant group, and 102 DEGs in the bacterial-susceptible group, compared with that in control group. According to the reported references and annotation information from BLAST, GO and KEGG, 30 putative bacterial-resistant genes and 19 putative bacterial-susceptible genes were identified from DEGs. The qRT-PCR results were consistent with the RNA-Seq results. Furthermore, many DGEs were involved in immune signaling related pathways, such as Endocytosis, Lysosome, MAPK, Chemokine and the ERBB signaling pathway. PMID:26193268

G Protein-Coupled Receptors in Anopheles gambiae

NASA Astrophysics Data System (ADS)

Hill, Catherine A.; Fox, A. Nicole; Pitts, R. Jason; Kent, Lauren B.; Tan, Perciliz L.; Chrystal, Mathew A.; Cravchik, Anibal; Collins, Frank H.; Robertson, Hugh M.; Zwiebel, Laurence J.

2002-10-01

We used bioinformatic approaches to identify a total of 276 G protein-coupled receptors (GPCRs) from the Anopheles gambiae genome. These include GPCRs that are likely to play roles in pathways affecting almost every aspect of the mosquito's life cycle. Seventy-nine candidate odorant receptors were characterized for tissue expression and, along with 76 putative gustatory receptors, for their molecular evolution relative to Drosophila melanogaster. Examples of lineage-specific gene expansions were observed as well as a single instance of unusually high sequence conservation.

Molecular evolution and diversification of snake toxin genes, revealed by analysis of intron sequences.

PubMed

Fujimi, T J; Nakajyo, T; Nishimura, E; Ogura, E; Tsuchiya, T; Tamiya, T

2003-08-14

The genes encoding erabutoxin (short chain neurotoxin) isoforms (Ea, Eb, and Ec), LsIII (long chain neurotoxin) and a novel long chain neurotoxin pseudogene were cloned from a Laticauda semifasciata genomic library. Short and long chain neurotoxin genes were also cloned from the genome of Laticauda laticaudata, a closely related species of L. semifasciata, by PCR. A putative matrix attached region (MAR) sequence was found in the intron I of the LsIII gene. Comparative analysis of 11 structurally relevant snake toxin genes (three-finger-structure toxins) revealed the molecular evolution of these toxins. Three-finger-structure toxin genes diverged from a common ancestor through two types of evolutionary pathways (long and short types), early in the course of evolution. At a later stage of evolution in each gene, the accumulation of mutations in the exons, especially exon II, by accelerated evolution may have caused the increased diversification in their functions. It was also revealed that the putative MAR sequence found in the LsIII gene was integrated into the gene after the species-level divergence.

Identification of biosynthetic intermediates of teaghrelins and teaghrelin-like compounds in oolong teas, and their molecular docking to the ghrelin receptor.

PubMed

Hsieh, Sheng-Kuo; Lo, Yuan-Hao; Wu, Chia-Chang; Chung, Tse-Yu; Tzen, Jason T C

2015-12-01

Teaghrelins are unique acylated flavonoid tetraglycosides found in Chin-shin oolong tea, and have been demonstrated to be promising oral ghrelin analogues. The biosynthetic pathway of teaghrelins from quercetin-3-O-rutinoside (rutin) or kaempferol-3-O-rutinoside (nicotiflorin) was proposed to comprise three enzymatic steps according to the identification of putative intermediates in Chin-shin oolong tea. In addition to the two known teaghrelins in Chin-shin oolong tea, four teaghrelin-like compounds with different attachments of glycosides were identified in various oolong teas. Molecular modeling and docking were used to evaluate theoretically whether the putative biosynthetic intermediates of teaghrelins and the four teaghrelin-like compounds could be potential candidates of ghrelin analogues. The results showed that the attachment of a coumaroyl group was crucial for these tea compounds to bind to the ghrelin receptor. However, the additional attachment of a rhamnosyl glycoside to the flavonoid backbone of teaghrelin-like compounds at C-7 significantly reduced their binding affinity with the ghrelin receptor. Copyright © 2015. Published by Elsevier B.V.

Circadian signaling in Homarus americanus: Region-specific de novo assembled transcriptomes show that both the brain and eyestalk ganglia possess the molecular components of a putative clock system.

PubMed

Christie, Andrew E; Yu, Andy; Pascual, Micah G; Roncalli, Vittoria; Cieslak, Matthew C; Warner, Amanda N; Lameyer, Tess J; Stanhope, Meredith E; Dickinson, Patsy S; Joe Hull, J

2018-04-11

Essentially all organisms exhibit recurring patterns of physiology/behavior that oscillate with a period of ~24-h and are synchronized to the solar day. Crustaceans are no exception, with robust circadian rhythms having been documented in many members of this arthropod subphylum. However, little is known about the molecular underpinnings of their circadian rhythmicity. Moreover, the location of the crustacean central clock has not been firmly established, although both the brain and eyestalk ganglia have been hypothesized as loci. The American lobster, Homarus americanus, is known to exhibit multiple circadian rhythms, and immunodetection data suggest that its central clock is located within the eyestalk ganglia rather than in the brain. Here, brain- and eyestalk ganglia-specific transcriptomes were generated and used to assess the presence/absence of transcripts encoding the commonly recognized protein components of arthropod circadian signaling systems in these two regions of the lobster central nervous system. Transcripts encoding putative homologs of the core clock proteins clock, cryptochrome 2, cycle, period and timeless were found in both the brain and eyestalk ganglia assemblies, as were transcripts encoding similar complements of putative clock-associated, clock input pathway and clock output pathway proteins. The presence and identity of transcripts encoding core clock proteins in both regions were confirmed using PCR. These findings suggest that both the brain and eyestalk ganglia possess all of the molecular components needed for the establishment of a circadian signaling system. Whether the brain and eyestalk clocks are independent of one another or represent a single timekeeping system remains to be determined. Interestingly, while most of the proteins deduced from the identified transcripts are shared by both the brain and eyestalk ganglia, assembly-specific isoforms were also identified, e.g., several period variants, suggesting the possibility of region-specific variation in clock function, especially if the brain and eyestalk clocks represent independent oscillators. Copyright © 2018 Elsevier B.V. All rights reserved.

Radical Rearrangement Chemistry in Ultraviolet Photodissociation of Iodotyrosine Systems: Insights from Metastable Dissociation, Infrared Ion Spectroscopy, and Reaction Pathway Calculations.

PubMed

Ranka, Karnamohit; Zhao, Ning; Yu, Long; Stanton, John F; Polfer, Nicolas C

2018-05-29

We report on the ultraviolet photodissociation (UVPD) chemistry of protonated tyrosine, iodotyrosine, and diiodotyrosine. Distonic loss of the iodine creates a high-energy radical at the aromatic ring that engages in hydrogen/proton rearrangement chemistry. Based on UVPD kinetics measurements, the appearance of this radical is coincident with the UV irradiation pulse (8 ns). Conversely, sequential UVPD product ions exhibit metastable decay on ca. 100 ns timescales. Infrared ion spectroscopy is capable of confirming putative structures of the rearrangement products as proton transfers from the imine and β-carbon hydrogens. Potential energy surfaces for the various reaction pathways indicate that the rearrangement chemistry is highly complex, compatible with a cascade of rearrangements, and that there is no preferred rearrangement pathway even in small molecular systems like these. Graphical Abstract.

Developing and applying the adverse outcome pathway concept for understanding and predicting neurotoxicity

PubMed Central

Bal-Price, Anna; Lein, Pamela J.; Keil, Kimberly P.; Sethi, Sunjay; Shafer, Timothy; Barenys, Marta; Fritsche, Ellen; Sachana, Magdalini; Meek, M.E. (Bette)

2016-01-01

The Adverse Outcome Pathway (AOP) concept has recently been proposed to support a paradigm shift in regulatory toxicology testing and risk assessment. This concept is similar to the Mode of Action (MOA), in that it describes a sequence of measurable key events triggered by a molecular initiating event in which a stressor interacts with a biological target. The resulting cascade of key events includes molecular, cellular, structural and functional changes in biological systems, resulting in a measurable adverse outcome. Thereby, an AOP ideally provides information relevant to chemical structure-activity relationships as a basis for predicting effects of structurally similar compounds. AOPs could potentially also form the basis for qualitative and quantitative predictive modeling of the human adverse outcome resulting from molecular initiating or other key events for which higher-throughput testing methods are available or can be developed. A variety of cellular and molecular processes are known to be critical for normal function of the central (CNS) and peripheral nervous systems (PNS). Because of the biological and functional complexity of the CNS and PNS, it has been challenging to establish causative links and quantitative relationships between key events that comprise the pathways leading from chemical exposure to an adverse outcome in the nervous system. Following introduction of the principles of MOA and AOPs, examples of potential or putative adverse outcome pathways specific for developmental or adult neurotoxicity are summarized and aspects of their assessment considered. Their possible application in developing mechanistically informed Integrated Approaches to Testing and Assessment (IATA) is also discussed. PMID:27212452

Identification of Putative Precursor Genes for the Biosynthesis of Cannabinoid-Like Compound in Radula marginata

PubMed Central

Hussain, Tajammul; Plunkett, Blue; Ejaz, Mahwish; Espley, Richard V.; Kayser, Oliver

2018-01-01

The liverwort Radula marginata belongs to the bryophyte division of land plants and is a prospective alternate source of cannabinoid-like compounds. However, mechanistic insights into the molecular pathways directing the synthesis of these cannabinoid-like compounds have been hindered due to the lack of genetic information. This prompted us to do deep sequencing, de novo assembly and annotation of R. marginata transcriptome, which resulted in the identification and validation of the genes for cannabinoid biosynthetic pathway. In total, we have identified 11,421 putative genes encoding 1,554 enzymes from 145 biosynthetic pathways. Interestingly, we have identified all the upstream genes of the central precursor of cannabinoid biosynthesis, cannabigerolic acid (CBGA), including its two first intermediates, stilbene acid (SA) and geranyl diphosphate (GPP). Expression of all these genes was validated using quantitative real-time PCR. We have characterized the protein structure of stilbene synthase (STS), which is considered as a homolog of olivetolic acid in R. marginata. Moreover, the metabolomics approach enabled us to identify CBGA-analogous compounds using electrospray ionization mass spectrometry (ESI-MS/MS) and gas chromatography mass spectrometry (GC-MS). Transcriptomic analysis revealed 1085 transcription factors (TF) from 39 families. Comparative analysis showed that six TF families have been uniquely predicted in R. marginata. In addition, the bioinformatics analysis predicted a large number of simple sequence repeats (SSRs) and non-coding RNAs (ncRNAs). Our results collectively provide mechanistic insights into the putative precursor genes for the biosynthesis of cannabinoid-like compounds and a novel transcriptomic resource for R. marginata. The large-scale transcriptomic resource generated in this study would further serve as a reference transcriptome to explore the Radulaceae family.

De novo sequencing and analysis of the cranberry fruit transcriptome to identify putative genes involved in flavonoid biosynthesis, transport and regulation.

PubMed

Sun, Haiyue; Liu, Yushan; Gai, Yuzhuo; Geng, Jinman; Chen, Li; Liu, Hongdi; Kang, Limin; Tian, Youwen; Li, Yadong

2015-09-02

Cranberries (Vaccinium macrocarpon Ait.), renowned for their excellent health benefits, are an important berry crop. Here, we performed transcriptome sequencing of one cranberry cultivar, from fruits at two different developmental stages, on the Illumina HiSeq 2000 platform. Our main goals were to identify putative genes for major metabolic pathways of bioactive compounds and compare the expression patterns between white fruit (W) and red fruit (R) in cranberry. In this study, two cDNA libraries of W and R were constructed. Approximately 119 million raw sequencing reads were generated and assembled de novo, yielding 57,331 high quality unigenes with an average length of 739 bp. Using BLASTx, 38,460 unigenes were identified as putative homologs of annotated sequences in public protein databases, including NCBI NR, NT, Swiss-Prot, KEGG, COG and GO. Of these, 21,898 unigenes mapped to 128 KEGG pathways, with the metabolic pathways, secondary metabolites, glycerophospholipid metabolism, ether lipid metabolism, starch and sucrose metabolism, purine metabolism, and pyrimidine metabolism being well represented. Among them, many candidate genes were involved in flavonoid biosynthesis, transport and regulation. Furthermore, digital gene expression (DEG) analysis identified 3,257 unigenes that were differentially expressed between the two fruit developmental stages. In addition, 14,473 simple sequence repeats (SSRs) were detected. Our results present comprehensive gene expression information about the cranberry fruit transcriptome that could facilitate our understanding of the molecular mechanisms of fruit development in cranberries. Although it will be necessary to validate the functions carried out by these genes, these results could be used to improve the quality of breeding programs for the cranberry and related species.

Differentially expressed microRNAs associated with changes of transcript levels in detoxification pathways and DDT-resistance in the Drosophila melanogaster strain 91-R.

PubMed

Seong, Keon Mook; Coates, Brad S; Kim, Do-Hyup; Hansen, Allison K; Pittendrigh, Barry R

2018-01-01

Dichloro-diphenyl-trichloroethane (DDT) resistance among arthropod species is a model for understanding the molecular adaptations in response to insecticide exposures. Previous studies reported that DDT resistance may involve one or multiple detoxification genes, such as cytochrome P450 monooxygenases (P450s), glutathione S-transferases (GSTs), esterases, and ATP binding cassette (ABC) transporters, or changes in the voltage-sensitive sodium channel. However, the possible involvement of microRNAs (miRNAs) in the post-transcriptional regulation of genes associated with DDT resistance in the Drosophila melanogaster strain 91-R remains poorly understood. In this study, the majority of the resulting miRNAs discovered in small RNA libraries from 91-R and the susceptible control strain, 91-C, ranged from 16-25 nt, and contained 163 precursors and 256 mature forms of previously-known miRNAs along with 17 putative novel miRNAs. Quantitative analyses predicted the differential expression of ten miRNAs between 91-R and 91-C, and, based on Gene Ontology and pathway analysis, these ten miRNAs putatively target transcripts encoding proteins involved in detoxification mechanisms. RT-qPCR validated an inverse correlation between levels of differentially-expressed miRNAs and their putatively targeted transcripts, which implies a role of these miRNAs in the differential regulation of detoxification pathways in 91-R compared to 91-C. This study provides evidence associating the differential expression of miRNAs in response to multigenerational DDT selection in Drosophila melanogaster and provides important clues for understanding the possible roles of miRNAs in mediating insecticide resistance traits.

MinePath: Mining for Phenotype Differential Sub-paths in Molecular Pathways

PubMed Central

Koumakis, Lefteris; Kartsaki, Evgenia; Chatzimina, Maria; Zervakis, Michalis; Vassou, Despoina; Marias, Kostas; Moustakis, Vassilis; Potamias, George

2016-01-01

Pathway analysis methodologies couple traditional gene expression analysis with knowledge encoded in established molecular pathway networks, offering a promising approach towards the biological interpretation of phenotype differentiating genes. Early pathway analysis methodologies, named as gene set analysis (GSA), view pathways just as plain lists of genes without taking into account either the underlying pathway network topology or the involved gene regulatory relations. These approaches, even if they achieve computational efficiency and simplicity, consider pathways that involve the same genes as equivalent in terms of their gene enrichment characteristics. Most recent pathway analysis approaches take into account the underlying gene regulatory relations by examining their consistency with gene expression profiles and computing a score for each profile. Even with this approach, assessing and scoring single-relations limits the ability to reveal key gene regulation mechanisms hidden in longer pathway sub-paths. We introduce MinePath, a pathway analysis methodology that addresses and overcomes the aforementioned problems. MinePath facilitates the decomposition of pathways into their constituent sub-paths. Decomposition leads to the transformation of single-relations to complex regulation sub-paths. Regulation sub-paths are then matched with gene expression sample profiles in order to evaluate their functional status and to assess phenotype differential power. Assessment of differential power supports the identification of the most discriminant profiles. In addition, MinePath assess the significance of the pathways as a whole, ranking them by their p-values. Comparison results with state-of-the-art pathway analysis systems are indicative for the soundness and reliability of the MinePath approach. In contrast with many pathway analysis tools, MinePath is a web-based system (www.minepath.org) offering dynamic and rich pathway visualization functionality, with the unique characteristic to color regulatory relations between genes and reveal their phenotype inclination. This unique characteristic makes MinePath a valuable tool for in silico molecular biology experimentation as it serves the biomedical researchers’ exploratory needs to reveal and interpret the regulatory mechanisms that underlie and putatively govern the expression of target phenotypes. PMID:27832067

MinePath: Mining for Phenotype Differential Sub-paths in Molecular Pathways.

PubMed

Koumakis, Lefteris; Kanterakis, Alexandros; Kartsaki, Evgenia; Chatzimina, Maria; Zervakis, Michalis; Tsiknakis, Manolis; Vassou, Despoina; Kafetzopoulos, Dimitris; Marias, Kostas; Moustakis, Vassilis; Potamias, George

2016-11-01

Pathway analysis methodologies couple traditional gene expression analysis with knowledge encoded in established molecular pathway networks, offering a promising approach towards the biological interpretation of phenotype differentiating genes. Early pathway analysis methodologies, named as gene set analysis (GSA), view pathways just as plain lists of genes without taking into account either the underlying pathway network topology or the involved gene regulatory relations. These approaches, even if they achieve computational efficiency and simplicity, consider pathways that involve the same genes as equivalent in terms of their gene enrichment characteristics. Most recent pathway analysis approaches take into account the underlying gene regulatory relations by examining their consistency with gene expression profiles and computing a score for each profile. Even with this approach, assessing and scoring single-relations limits the ability to reveal key gene regulation mechanisms hidden in longer pathway sub-paths. We introduce MinePath, a pathway analysis methodology that addresses and overcomes the aforementioned problems. MinePath facilitates the decomposition of pathways into their constituent sub-paths. Decomposition leads to the transformation of single-relations to complex regulation sub-paths. Regulation sub-paths are then matched with gene expression sample profiles in order to evaluate their functional status and to assess phenotype differential power. Assessment of differential power supports the identification of the most discriminant profiles. In addition, MinePath assess the significance of the pathways as a whole, ranking them by their p-values. Comparison results with state-of-the-art pathway analysis systems are indicative for the soundness and reliability of the MinePath approach. In contrast with many pathway analysis tools, MinePath is a web-based system (www.minepath.org) offering dynamic and rich pathway visualization functionality, with the unique characteristic to color regulatory relations between genes and reveal their phenotype inclination. This unique characteristic makes MinePath a valuable tool for in silico molecular biology experimentation as it serves the biomedical researchers' exploratory needs to reveal and interpret the regulatory mechanisms that underlie and putatively govern the expression of target phenotypes.

The V-ATPase a2-subunit as a putative endosomal pH-sensor.

PubMed

Marshansky, V

2007-11-01

V-ATPase (vesicular H(+)-ATPase)-driven intravesicular acidification is crucial for vesicular trafficking. Defects in vesicular acidification and trafficking have recently been recognized as essential determinants of various human diseases. An important role of endosomal acidification in receptor-ligand dissociation and in activation of lysosomal hydrolytic enzymes is well established. However, the molecular mechanisms by which luminal pH information is transmitted to the cytosolic small GTPases that control trafficking events such as budding, coat formation and fusion are unknown. Here, we discuss our recent discovery that endosomal V-ATPase is a pH-sensor regulating the degradative pathway. According to our model, V-ATPase is responsible for: (i) the generation of a pH gradient between vesicular membranes; (ii) sensing of intravesicular pH; and (iii) transmitting this information to the cytosolic side of the membrane. We also propose the hypothetical molecular mechanism involved in function of the V-ATPase a2-subunit as a putative pH-sensor. Based on extensive experimental evidence on the crucial role of histidine residues in the function of PSPs (pH-sensing proteins) in eukaryotic cells, we hypothesize that pH-sensitive histidine residues within the intra-endosomal loops and/or C-terminal luminal tail of the a2-subunit could also be involved in the pH-sensing function of V-ATPase. However, in order to identify putative pH-sensitive histidine residues and to test this hypothesis, it is absolutely essential that we increase our understanding of the folding and transmembrane topology of the a-subunit isoforms of V-ATPase. Thus the crucial role of intra-endosomal histidine residues in pH-dependent conformational changes of the V-ATPase a2-isoform, its interaction with cytosolic small GTPases and ultimately in its acidification-dependent regulation of the endosomal/lysosomal protein degradative pathway remain to be determined.

Recent advances in prostate development and links to prostatic diseases

PubMed Central

Powers, Ginny L.

2013-01-01

The prostate is a branched ductal-acinar gland that is part of the male reproductive tract. Prostate development depends upon the integration of steroid hormone signals, paracrine interactions between the stromal and epithelial tissue layers, and the actions of cell autonomous factors. Several genes and signalling pathways are known to be required for one or more steps of prostate development including epithelial budding, duct elongation, branching morphogenesis, and/or cellular differentiation. Recent progress in the field of prostate development has included the application of genome-wide technologies including serial analysis of gene expression (SAGE), expression profiling microarrays, and other large scale approaches to identify new genes and pathways that are essential for prostate development. The aggregation of experimental results into online databases by organized multi-lab projects including the Genitourinary Developmental Molecular Atlas Project (GUDMAP) has also accelerated the understanding of molecular pathways that function during prostate development and identified links between prostate anatomy and molecular signaling. Rapid progress has also recently been made in understanding the nature and role of candidate stem cells in the developing and adult prostate. This has included the identification of putative prostate stem cell markers, lineage tracing, and organ reconstitution studies. However, several issues regarding their origin, precise nature, and possible role(s) in disease remain unresolved. Nevertheless, several links between prostatic developmental mechanisms and the pathogenesis of prostatic diseases including benign prostatic hyperplasia and prostate cancer have led to recent progress on targeting developmental pathways as therapeutic strategies for these diseases. PMID:23335485

Evolution of DMSP (dimethylsulfoniopropionate) biosynthesis pathway: Origin and phylogenetic distribution in polyploid Spartina (Poaceae, Chloridoideae).

PubMed

Rousseau, Hélène; Rousseau-Gueutin, Mathieu; Dauvergne, Xavier; Boutte, Julien; Simon, Gaëlle; Marnet, Nathalie; Bouchereau, Alain; Guiheneuf, Solène; Bazureau, Jean-Pierre; Morice, Jérôme; Ravanel, Stéphane; Cabello-Hurtado, Francisco; Ainouche, Abdelkader; Salmon, Armel; Wendel, Jonathan F; Ainouche, Malika L

2017-09-01

DMSP (dimethylsulfoniopropionate) is an ecologically important sulfur metabolite commonly produced by marine algae and by some higher plant lineages, including the polyploid salt marsh genus Spartina (Poaceae). The molecular mechanisms and genes involved in the DMSP biosynthesis pathways are still unknown. In this study, we performed comparative analyses of DMSP amounts and molecular phylogenetic analyses to decipher the origin of DMSP in Spartina that represents one of the major source of terrestrial DMSP in coastal marshes. DMSP content was explored in 14 Spartina species using 1 H Nuclear Magnetic Resonance (NMR) spectroscopy and Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS). Putative genes encoding the four enzymatic steps of the DMSP biosynthesis pathway in Spartina were examined and their evolutionary dynamics were studied. We found that the hexaploid lineage containing S. alterniflora, S. foliosa and S. maritima and their derived hybrids and allopolyploids are all able to produce DMSP, in contrast to species in the tetraploid clade. Thus, examination of DMSP synthesis in a phylogenetic context implicated a single origin of this physiological innovation, which occurred in the ancestor of the hexaploid Spartina lineage, 3-6MYA. Candidate genes specific to the Spartina DMSP biosynthesis pathway were also retrieved from Spartina transcriptomes, and provide a framework for future investigations to decipher the molecular mechanisms involved in this plant phenotypic novelty that has major ecological impacts in saltmarsh ecosystems. Copyright © 2017 Elsevier Inc. All rights reserved.

Low Temperature Extends the Lifespan of Bursaphelenchus xylophilus through the cGMP Pathway

PubMed Central

Wang, Bowen; Ma, Ling; Wang, Feng; Wang, Buyong; Hao, Xin; Xu, Jiayao; Ma, Yan

2017-01-01

The causal agent of pine wilt disease, pine wood nematode (PWN) (Bursaphelenchus xylophilus), revealed extended lifespan at low temperature. To discover the molecular mechanism of this phenomenon, we attempted to study the molecular characterization, transcript abundance, and functions of three genes of the cyclic guanosine monophosphate (cGMP) pathway from B. xylophilus. Three cGMP pathway genes were identified from B. xylophilus. Bioinformatic software was utilized to analyze the characteristics of the three putative proteins. Function of the three genes in cold tolerance was studied with RNA interference (RNAi). The results showed that the deduced protein of Bx-DAF-11 has an adenylate and guanylate cyclase catalytic domain, indicating an ability to bind to extracellular ligands and synthesizing cGMP. Both Bx-TAX-2 and Bx-TAX-4 have cyclic nucleotide-binding domains and ion transport protein domains, illustrating that they are cGMP-gated ion channels. The transcript level of Bx-daf-11, Bx-tax-2, and Bx-tax-4 increased at low temperature. The survival rates of three gene silenced B. xylophilus revealed a significant decrease at low temperature. This study illustrated that the cGMP pathway plays a key role in low-temperature-induced lifespan extension in B. xylophilus. PMID:29099744

MicroRNA Regulators of Anxiety and Metabolic Disorders.

PubMed

Meydan, Chanan; Shenhar-Tsarfaty, Shani; Soreq, Hermona

2016-09-01

Anxiety-related and metabolic disorders are under intense research focus. Anxiety-induced microRNAs (miRNAs) are emerging as regulators that are not only capable of suppressing inflammation but can also induce metabolic syndrome-related processes. We summarize here evidence linking miRNA pathways which share regulatory networks in metabolic and anxiety-related conditions. In particular, miRNAs involved in these disorders include regulators of acetylcholine signaling in the nervous system and their accompanying molecular machinery. These have been associated with anxiety-prone states in individuals, while also acting as inflammatory suppressors. In peripheral tissues, altered miRNA pathways can lead to dysregulated metabolism. Common pathways in metabolic and anxiety-related phenomena might offer an opportunity to reclassify 'healthy' and 'unhealthy', as well as metabolic and anxiety-prone biological states, and inform putative strategies to treat these disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

A proposed model for the flowering signaling pathway of sugarcane under photoperiodic control.

PubMed

Coelho, C P; Costa Netto, A P; Colasanti, J; Chalfun-Júnior, A

2013-04-25

Molecular analysis of floral induction in Arabidopsis has identified several flowering time genes related to 4 response networks defined by the autonomous, gibberellin, photoperiod, and vernalization pathways. Although grass flowering processes include ancestral functions shared by both mono- and dicots, they have developed their own mechanisms to transmit floral induction signals. Despite its high production capacity and its important role in biofuel production, almost no information is available about the flowering process in sugarcane. We searched the Sugarcane Expressed Sequence Tags database to look for elements of the flowering signaling pathway under photoperiodic control. Sequences showing significant similarity to flowering time genes of other species were clustered, annotated, and analyzed for conserved domains. Multiple alignments comparing the sequences found in the sugarcane database and those from other species were performed and their phylogenetic relationship assessed using the MEGA 4.0 software. Electronic Northerns were run with Cluster and TreeView programs, allowing us to identify putative members of the photoperiod-controlled flowering pathway of sugarcane.

Amplification of tumor inducing putative cancer stem cells (CSCs) by vitamin A/retinol from mammary tumors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharma, Rohit B.; Wang, Qingde; Khillan, Jaspal S., E-mail: khillan@pitt.edu

Highlights: •Vitamin A supports self renewal of putative CSCs from mammary tumors. •These cells exhibit impaired retinol metabolism into retinoic acid. •CSCs from mammary tumors differentiate into mammary specific cell lineages. •The cells express mammary stem cell specific CD29 and CD49f markers. •Putative CSCs form highly metastatic tumors in NOD SCID mouse. -- Abstract: Solid tumors contain a rare population of cancer stem cells (CSCs) that are responsible for relapse and metastasis. The existence of CSC however, remains highly controversial issue. Here we present the evidence for putative CSCs from mammary tumors amplified by vitamin A/retinol signaling. The cells exhibitmore » mammary stem cell specific CD29{sup hi}/CD49f{sup hi}/CD24{sup hi} markers, resistance to radiation and chemo therapeutic agents and form highly metastatic tumors in NOD/SCID mice. The cells exhibit indefinite self renewal as cell lines. Furthermore, the cells exhibit impaired retinol metabolism and do not express enzymes that metabolize retinol into retinoic acid. Vitamin A/retinol also amplified putative CSCs from breast cancer cell lines that form highly aggressive tumors in NOD SCID mice. The studies suggest that high purity putative CSCs can be isolated from solid tumors to establish patient specific cell lines for personalized therapeutics for pre-clinical translational applications. Characterization of CSCs will allow understanding of basic cellular and molecular pathways that are deregulated, mechanisms of tumor metastasis and evasion of therapies that has direct clinical relevance.« less

Application of Targeted Functional Assays to Assess a Putative Vascular Disruption Developmental Toxicity Pathway Informed By ToxCast High-Throughput Screening Data

EPA Science Inventory

Chemical perturbation of vascular development is a putative toxicity pathway which may result in developmental toxicity. EPA’s high-throughput screening (HTS) ToxCast program contains assays which measure cellular signals and biological processes critical for blood vessel develop...

An investigation of the molecular mechanisms engaged before and after the development of Alzheimer disease neuropathology in Down syndrome: a proteomics approach.

PubMed

Cenini, Giovanna; Fiorini, Ada; Sultana, Rukhsana; Perluigi, Marzia; Cai, Jian; Klein, Jon B; Head, Elizabeth; Butterfield, D Allan

2014-11-01

Down syndrome (DS) is one of the most common causes of intellectual disability, owing to trisomy of all or part of chromosome 21. DS is also associated with the development of Alzheimer disease (AD) neuropathology after the age of 40 years. To better clarify the cellular and metabolic pathways that could contribute to the differences in DS brain, in particular those involved in the onset of neurodegeneration, we analyzed the frontal cortex of DS subjects with or without significant AD pathology in comparison with age-matched controls, using a proteomics approach. Proteomics represents an advantageous tool to investigate the molecular mechanisms underlying the disease. From these analyses, we investigated the effects that age, DS, and AD neuropathology could have on protein expression levels. Our results show overlapping and independent molecular pathways (including energy metabolism, oxidative damage, protein synthesis, and autophagy) contributing to DS, to aging, and to the presence of AD pathology in DS. Investigation of pathomechanisms involved in DS with AD may provide putative targets for therapeutic approaches to slow the development of AD. Copyright © 2014 Elsevier Inc. All rights reserved.

Biochemistry of Mitochondrial Coenzyme Q Biosynthesis.

PubMed

Stefely, Jonathan A; Pagliarini, David J

2017-10-01

Coenzyme Q (CoQ, ubiquinone) is a redox-active lipid produced across all domains of life that functions in electron transport and oxidative phosphorylation and whose deficiency causes human diseases. Yet, CoQ biosynthesis has not been fully defined in any organism. Several proteins with unclear molecular functions facilitate CoQ biosynthesis through unknown means, and multiple steps in the pathway are catalyzed by currently unidentified enzymes. Here we highlight recent progress toward filling these knowledge gaps through both traditional biochemistry and cutting-edge 'omics' approaches. To help fill the remaining gaps, we present questions framed by the recently discovered CoQ biosynthetic complex and by putative biophysical barriers. Mapping CoQ biosynthesis, metabolism, and transport pathways has great potential to enhance treatment of numerous human diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cannabidiol Counteracts Amphetamine-Induced Neuronal and Behavioral Sensitization of the Mesolimbic Dopamine Pathway through a Novel mTOR/p70S6 Kinase Signaling Pathway

PubMed Central

Renard, Justine; Loureiro, Michael; Rosen, Laura G.; Zunder, Jordan; de Oliveira, Cleusa; Schmid, Susanne; Rushlow, Walter J.

2016-01-01

Schizophrenia-related psychosis is associated with disturbances in mesolimbic dopamine (DA) transmission, characterized by hyperdopaminergic activity in the mesolimbic pathway. Currently, the only clinically effective treatment for schizophrenia involves the use of antipsychotic medications that block DA receptor transmission. However, these medications produce serious side effects leading to poor compliance and treatment outcomes. Emerging evidence points to the involvement of a specific phytochemical component of marijuana called cannabidiol (CBD), which possesses promising therapeutic properties for the treatment of schizophrenia-related psychoses. However, the neuronal and molecular mechanisms through which CBD may exert these effects are entirely unknown. We used amphetamine (AMPH)-induced sensitization and sensorimotor gating in rats, two preclinical procedures relevant to schizophrenia-related psychopathology, combined with in vivo single-unit neuronal electrophysiology recordings in the ventral tegmental area, and molecular analyses to characterize the actions of CBD directly in the nucleus accumbens shell (NASh), a brain region that is the current target of most effective antipsychotics. We demonstrate that Intra-NASh CBD attenuates AMPH-induced sensitization, both in terms of DAergic neuronal activity measured in the ventral tegmental area and psychotomimetic behavioral analyses. We further report that CBD controls downstream phosphorylation of the mTOR/p70S6 kinase signaling pathways directly within the NASh. Our findings demonstrate a novel mechanism for the putative antipsychotic-like properties of CBD in the mesolimbic circuitry. We identify the molecular signaling pathways through which CBD may functionally reduce schizophrenia-like neuropsychopathology. SIGNIFICANCE STATEMENT The cannabis-derived phytochemical, cannabidiol (CBD), has been shown to have pharmacotherapeutic efficacy for the treatment of schizophrenia. However, the mechanisms by which CBD may produce antipsychotic effects are entirely unknown. Using preclinical behavioral procedures combined with molecular analyses and in vivo neuronal electrophysiology, our findings identify a functional role for the nucleus accumbens as a critical brain region whereby CBD can produce effects similar to antipsychotic medications by triggering molecular signaling pathways associated with the effects of classic antipsychotic medications. Specifically, we report that CBD can attenuate both behavioral and dopaminergic neuronal correlates of mesolimbic dopaminergic sensitization, via a direct interaction with mTOR/p70S6 kinase signaling within the mesolimbic pathway. PMID:27147666

Cannabidiol Counteracts Amphetamine-Induced Neuronal and Behavioral Sensitization of the Mesolimbic Dopamine Pathway through a Novel mTOR/p70S6 Kinase Signaling Pathway.

PubMed

Renard, Justine; Loureiro, Michael; Rosen, Laura G; Zunder, Jordan; de Oliveira, Cleusa; Schmid, Susanne; Rushlow, Walter J; Laviolette, Steven R

2016-05-04

Schizophrenia-related psychosis is associated with disturbances in mesolimbic dopamine (DA) transmission, characterized by hyperdopaminergic activity in the mesolimbic pathway. Currently, the only clinically effective treatment for schizophrenia involves the use of antipsychotic medications that block DA receptor transmission. However, these medications produce serious side effects leading to poor compliance and treatment outcomes. Emerging evidence points to the involvement of a specific phytochemical component of marijuana called cannabidiol (CBD), which possesses promising therapeutic properties for the treatment of schizophrenia-related psychoses. However, the neuronal and molecular mechanisms through which CBD may exert these effects are entirely unknown. We used amphetamine (AMPH)-induced sensitization and sensorimotor gating in rats, two preclinical procedures relevant to schizophrenia-related psychopathology, combined with in vivo single-unit neuronal electrophysiology recordings in the ventral tegmental area, and molecular analyses to characterize the actions of CBD directly in the nucleus accumbens shell (NASh), a brain region that is the current target of most effective antipsychotics. We demonstrate that Intra-NASh CBD attenuates AMPH-induced sensitization, both in terms of DAergic neuronal activity measured in the ventral tegmental area and psychotomimetic behavioral analyses. We further report that CBD controls downstream phosphorylation of the mTOR/p70S6 kinase signaling pathways directly within the NASh. Our findings demonstrate a novel mechanism for the putative antipsychotic-like properties of CBD in the mesolimbic circuitry. We identify the molecular signaling pathways through which CBD may functionally reduce schizophrenia-like neuropsychopathology. The cannabis-derived phytochemical, cannabidiol (CBD), has been shown to have pharmacotherapeutic efficacy for the treatment of schizophrenia. However, the mechanisms by which CBD may produce antipsychotic effects are entirely unknown. Using preclinical behavioral procedures combined with molecular analyses and in vivo neuronal electrophysiology, our findings identify a functional role for the nucleus accumbens as a critical brain region whereby CBD can produce effects similar to antipsychotic medications by triggering molecular signaling pathways associated with the effects of classic antipsychotic medications. Specifically, we report that CBD can attenuate both behavioral and dopaminergic neuronal correlates of mesolimbic dopaminergic sensitization, via a direct interaction with mTOR/p70S6 kinase signaling within the mesolimbic pathway. Copyright © 2016 the authors 0270-6474/16/365160-10$15.00/0.

New Insights on Drought Stress Response by Global Investigation of Gene Expression Changes in Sheepgrass (Leymus chinensis)

PubMed Central

Zhao, Pincang; Liu, Panpan; Yuan, Guangxiao; Jia, Junting; Li, Xiaoxia; Qi, Dongmei; Chen, Shuangyan; Ma, Tian; Liu, Gongshe; Cheng, Liqin

2016-01-01

Water is a critical environmental factor that restricts the geographic distribution of plants. Sheepgrass [Leymus chinensis, (Trin.) Tzvel] is an important forage grass in the Eurasia Steppe and a close germplasm for wheat and barley. This native grass adapts well to adverse environments such as cold, salinity, alkalinity and drought, and it can survive when the soil moisture may be less than 6% in dry seasons. However, little is known about how sheepgrass tolerates water stress at the molecular level. Here, drought stress experiment and RNA-sequencing (RNA-seq) was performed in three pools of RNA samples (control, drought stress, and rewatering). We found that sheepgrass seedlings could still survive when the soil water content (SWC) was reduced to 14.09%. Differentially expressed genes (DEGs) analysis showed that 7320 genes exhibited significant responses to drought stress. Of these DEGs, 2671 presented opposite expression trends before and after rewatering. Furthermore, ~680 putative sheepgrass-specific water responsive genes were revealed that can be studied deeply. Gene ontology (GO) annotation revealed that stress-associated genes were activated extensively by drought treatment. Interestingly, cold stress-related genes were up-regulated greatly after drought stress. The DEGs of MAPK and calcium signal pathways, plant hormone ABA, jasmonate, ethylene, brassinosteroid signal pathways, cold response CBF pathway participated coordinatively in sheepgrass drought stress response. In addition, we identified 288 putative transcription factors (TFs) involved in drought response, among them, the WRKY, NAC, AP2/ERF, bHLH, bZIP, and MYB families were enriched, and might play crucial and significant roles in drought stress response of sheepgrass. Our research provided new and valuable information for understanding the mechanism of drought tolerance in sheepgrass. Moreover, the identification of genes involved in drought response can facilitate the genetic improvement of crops by molecular breeding. PMID:27446180

Detecting recurrent gene mutation in interaction network context using multi-scale graph diffusion.

PubMed

Babaei, Sepideh; Hulsman, Marc; Reinders, Marcel; de Ridder, Jeroen

2013-01-23

Delineating the molecular drivers of cancer, i.e. determining cancer genes and the pathways which they deregulate, is an important challenge in cancer research. In this study, we aim to identify pathways of frequently mutated genes by exploiting their network neighborhood encoded in the protein-protein interaction network. To this end, we introduce a multi-scale diffusion kernel and apply it to a large collection of murine retroviral insertional mutagenesis data. The diffusion strength plays the role of scale parameter, determining the size of the network neighborhood that is taken into account. As a result, in addition to detecting genes with frequent mutations in their genomic vicinity, we find genes that harbor frequent mutations in their interaction network context. We identify densely connected components of known and putatively novel cancer genes and demonstrate that they are strongly enriched for cancer related pathways across the diffusion scales. Moreover, the mutations in the clusters exhibit a significant pattern of mutual exclusion, supporting the conjecture that such genes are functionally linked. Using multi-scale diffusion kernel, various infrequently mutated genes are found to harbor significant numbers of mutations in their interaction network neighborhood. Many of them are well-known cancer genes. The results demonstrate the importance of defining recurrent mutations while taking into account the interaction network context. Importantly, the putative cancer genes and networks detected in this study are found to be significant at different diffusion scales, confirming the necessity of a multi-scale analysis.

Pharmacophore based design of some multi-targeted compounds targeted against pathways of diabetic complications.

PubMed

Chadha, Navriti; Silakari, Om

2017-09-01

Diabetic complications is a complex metabolic disorder developed primarily due to prolonged hyperglycemia in the body. The complexity of the disease state as well as the unifying pathophysiology discussed in the literature reports exhibited that the use of multi-targeted agents with multiple complementary biological activities may offer promising therapy for the intervention of the disease over the single-target drugs. In the present study, novel thiazolidine-2,4-dione analogues were designed as multi-targeted agents implicated against the molecular pathways involved in diabetic complications using knowledge based as well as in-silico approaches such as pharmacophore mapping, molecular docking etc. The hit molecules were duly synthesized and biochemical estimation of these molecules against aldose reductase (ALR2), protein kinase Cβ (PKCβ) and poly (ADP-ribose) polymerase 1 (PARP-1) led to identification of compound 2 that showed good potency against PARP-1 and ALR2 enzymes. These positive results support the progress of a low cost multi-targeted agent with putative roles in diabetic complications. Copyright © 2017 Elsevier Inc. All rights reserved.

Genetic Characterization of the Carotenoid Biosynthetic Pathway in Methylobacterium extorquens AM1 and Isolation of a Colorless Mutant

PubMed Central

Van Dien, Stephen J.; Marx, Christopher J.; O'Brien, Brooke N.; Lidstrom, Mary E.

2003-01-01

Genomic searches were used to reconstruct the putative carotenoid biosynthesis pathway in the pink-pigmented facultative methylotroph Methylobacterium extorquens AM1. Four genes for putative phytoene desaturases were identified. A colorless mutant was obtained by transposon mutagenesis, and the insertion was shown to be in one of the putative phytoene desaturase genes. Mutations in the other three did not affect color. The tetracycline marker was removed from the original transposon mutant, resulting in a pigment-free strain with wild-type growth properties useful as a tool for future experiments. PMID:14660416

Genetic characterization of the carotenoid biosynthetic pathway in Methylobacterium extorquens AM1 and isolation of a colorless mutant.

PubMed

Van Dien, Stephen J; Marx, Christopher J; O'Brien, Brooke N; Lidstrom, Mary E

2003-12-01

Genomic searches were used to reconstruct the putative carotenoid biosynthesis pathway in the pink-pigmented facultative methylotroph Methylobacterium extorquens AM1. Four genes for putative phytoene desaturases were identified. A colorless mutant was obtained by transposon mutagenesis, and the insertion was shown to be in one of the putative phytoene desaturase genes. Mutations in the other three did not affect color. The tetracycline marker was removed from the original transposon mutant, resulting in a pigment-free strain with wild-type growth properties useful as a tool for future experiments.

Delineation of the Caffeine C-8 Oxidation Pathway in Pseudomonas sp. Strain CBB1 via Characterization of a New Trimethyluric Acid Monooxygenase and Genes Involved in Trimethyluric Acid Metabolism

PubMed Central

Mohanty, Sujit Kumar; Yu, Chi-Li; Das, Shuvendu; Louie, Tai Man; Gakhar, Lokesh

2012-01-01

The molecular basis of the ability of bacteria to live on caffeine via the C-8 oxidation pathway is unknown. The first step of this pathway, caffeine to trimethyluric acid (TMU), has been attributed to poorly characterized caffeine oxidases and a novel quinone-dependent caffeine dehydrogenase. Here, we report the detailed characterization of the second enzyme, a novel NADH-dependent trimethyluric acid monooxygenase (TmuM), a flavoprotein that catalyzes the conversion of TMU to 1,3,7-trimethyl-5-hydroxyisourate (TM-HIU). This product spontaneously decomposes to racemic 3,6,8-trimethylallantoin (TMA). TmuM prefers trimethyluric acids and, to a lesser extent, dimethyluric acids as substrates, but it exhibits no activity on uric acid. Homology models of TmuM against uric acid oxidase HpxO (which catalyzes uric acid to 5-hydroxyisourate) reveal a much bigger and hydrophobic cavity to accommodate the larger substrates. Genes involved in the caffeine C-8 oxidation pathway are located in a 25.2-kb genomic DNA fragment of CBB1, including cdhABC (coding for caffeine dehydrogenase) and tmuM (coding for TmuM). Comparison of this gene cluster to the uric acid-metabolizing gene cluster and pathway of Klebsiella pneumoniae revealed two major open reading frames coding for the conversion of TM-HIU to S-(+)-trimethylallantoin [S-(+)-TMA]. The first one, designated tmuH, codes for a putative TM-HIU hydrolase, which catalyzes the conversion of TM-HIU to 3,6,8-trimethyl-2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (TM-OHCU). The second one, designated tmuD, codes for a putative TM-OHCU decarboxylase which catalyzes the conversion of TM-OHCU to S-(+)-TMA. Based on a combination of enzymology and gene-analysis, a new degradative pathway for caffeine has been proposed via TMU, TM-HIU, TM-OHCU to S-(+)-TMA. PMID:22609920

Genome wide transcriptome profiling reveals differential gene expression in secondary metabolite pathway of Cymbopogon winterianus.

PubMed

Devi, Kamalakshi; Mishra, Surajit K; Sahu, Jagajjit; Panda, Debashis; Modi, Mahendra K; Sen, Priyabrata

2016-02-15

Advances in transcriptome sequencing provide fast, cost-effective and reliable approach to generate large expression datasets especially suitable for non-model species to identify putative genes, key pathway and regulatory mechanism. Citronella (Cymbopogon winterianus) is an aromatic medicinal grass used for anti-tumoral, antibacterial, anti-fungal, antiviral, detoxifying and natural insect repellent properties. Despite of having number of utilities, the genes involved in terpenes biosynthetic pathway is not yet clearly elucidated. The present study is a pioneering attempt to generate an exhaustive molecular information of secondary metabolite pathway and to increase genomic resources in Citronella. Using high-throughput RNA-Seq technology, root and leaf transcriptome was analysed at an unprecedented depth (11.7 Gb). Targeted searches identified majority of the genes associated with metabolic pathway and other natural product pathway viz. antibiotics synthesis along with many novel genes. Terpenoid biosynthesis genes comparative expression results were validated for 15 unigenes by RT-PCR and qRT-PCR. Thus the coverage of these transcriptome is comprehensive enough to discover all known genes of major metabolic pathways. This transcriptome dataset can serve as important public information for gene expression, genomics and function genomics studies in Citronella and shall act as a benchmark for future improvement of the crop.

Transcript Assembly and Quantification by RNA-Seq Reveals Differentially Expressed Genes between Soft-Endocarp and Hard-Endocarp Hawthorns

PubMed Central

Zhang, Feng; Liu, Zhongchi; Li, Xiaoming; Li, Wenran; Ma, Yue; Li, He; Liu, Yuexue; Zhang, Zhihong

2013-01-01

Hawthorn (Crataegus spp.) is an important pome with a long history as a fruit, an ornamental, and a source of medicine. Fruits of hawthorn are marked by hard stony endocarps, but a hawthorn germplasm with soft and thin endocarp was found in Liaoning province of China. To elucidate the molecular mechanism underlying the soft endocarp of hawthorn, we conducted a de novo assembly of the fruit transcriptome of Crataegus pinnatifida and compared gene expression profiles between the soft-endocarp and the hard-endocarp hawthorn varieties. De novo assembly yielded 52,673 putative unigenes, 20.4% of which are longer than 1,000 bp. Among the high-quality unique sequences, 35,979 (68.3%) had at least one significant match to an existing gene model. A total of 1,218 genes, represented 2.31% total putative unigenes, were differentially expressed between the soft-endocarp hawthorn and the hard-endocarp hawthorn. Among these differentially expressed genes, a number of lignin biosynthetic pathway genes were down-regulated while almost all the flavonoid biosynthetic pathway genes were strongly up-regulated, concomitant with the formation of soft endocarp. In addition, we have identified some MYB and NAC transcription factors that could potentially control lignin and flavonoid biosynthesis. The altered expression levels of the genes encoding lignin biosynthetic enzymes, MYB and NAC transcription factors were confirmed by quantitative RT-PCR. This is the first transcriptome analysis of Crataegus genus. The high quality ESTs generated in this study will aid future gene cloning from hawthorn. Our study provides important insights into the molecular mechanisms underlying soft endocarp formation in hawthorn. PMID:24039819

Exogenous γ-aminobutyric acid (GABA) affects pollen tube growth via modulating putative Ca2+-permeable membrane channels and is coupled to negative regulation on glutamate decarboxylase

PubMed Central

Yu, Guang-Hui; Zou, Jie; Feng, Jing; Peng, Xiong-Bo; Wu, Ju-You; Wu, Ying-Liang; Palanivelu, Ravishankar; Sun, Meng-Xiang

2014-01-01

γ-Aminobutyric acid (GABA) is implicated in pollen tube growth, but the molecular and cellular mechanisms that it mediates are largely unknown. Here, it is shown that exogenous GABA modulates putative Ca2+-permeable channels on the plasma membranes of tobacco pollen grains and pollen tubes. Whole-cell voltage-clamp experiments and non-invasive micromeasurement technology (NMT) revealed that the influx of Ca2+ increases in pollen tubes in response to exogenous GABA. It is also demonstrated that glutamate decarboxylase (GAD), the rate-limiting enzyme of GABA biosynthesis, is involved in feedback controls of Ca2+-permeable channels to fluctuate intracellular GABA levels and thus modulate pollen tube growth. The findings suggest that GAD activity linked with Ca2+-permeable channels relays an extracellular GABA signal and integrates multiple signal pathways to modulate tobacco pollen tube growth. Thus, the data explain how GABA mediates the communication between the style and the growing pollen tubes. PMID:24799560

Gramene 2013: comparative plant genomics resources.

PubMed

Monaco, Marcela K; Stein, Joshua; Naithani, Sushma; Wei, Sharon; Dharmawardhana, Palitha; Kumari, Sunita; Amarasinghe, Vindhya; Youens-Clark, Ken; Thomason, James; Preece, Justin; Pasternak, Shiran; Olson, Andrew; Jiao, Yinping; Lu, Zhenyuan; Bolser, Dan; Kerhornou, Arnaud; Staines, Dan; Walts, Brandon; Wu, Guanming; D'Eustachio, Peter; Haw, Robin; Croft, David; Kersey, Paul J; Stein, Lincoln; Jaiswal, Pankaj; Ware, Doreen

2014-01-01

Gramene (http://www.gramene.org) is a curated online resource for comparative functional genomics in crops and model plant species, currently hosting 27 fully and 10 partially sequenced reference genomes in its build number 38. Its strength derives from the application of a phylogenetic framework for genome comparison and the use of ontologies to integrate structural and functional annotation data. Whole-genome alignments complemented by phylogenetic gene family trees help infer syntenic and orthologous relationships. Genetic variation data, sequences and genome mappings available for 10 species, including Arabidopsis, rice and maize, help infer putative variant effects on genes and transcripts. The pathways section also hosts 10 species-specific metabolic pathways databases developed in-house or by our collaborators using Pathway Tools software, which facilitates searches for pathway, reaction and metabolite annotations, and allows analyses of user-defined expression datasets. Recently, we released a Plant Reactome portal featuring 133 curated rice pathways. This portal will be expanded for Arabidopsis, maize and other plant species. We continue to provide genetic and QTL maps and marker datasets developed by crop researchers. The project provides a unique community platform to support scientific research in plant genomics including studies in evolution, genetics, plant breeding, molecular biology, biochemistry and systems biology.

LncRNAs in Secondary Hair Follicle of Cashmere Goat: Identification, Expression, and Their Regulatory Network in Wnt Signaling Pathway.

PubMed

Bai, Wen L; Zhao, Su J; Wang, Ze Y; Zhu, Yu B; Dang, Yun L; Cong, Yu Y; Xue, Hui L; Wang, Wei; Deng, Liang; Guo, Dan; Wang, Shi Q; Zhu, Yan X; Yin, Rong H

2018-07-03

Long noncoding RNAs (lncRNAs) are a novel class of eukaryotic transcripts. They are thought to act as a critical regulator of protein-coding gene expression. Herein, we identified and characterized 13 putative lncRNAs from the expressed sequence tags from secondary hair follicle of Cashmere goat. Furthermore, we investigated their transcriptional pattern in secondary hair follicle of Liaoning Cashmere goat during telogen and anagen phases. Also, we generated intracellular regulatory networks of upregulated lncRNAs at anagen in Wnt signaling pathway based on bioinformatics analysis. The relative expression of six putative lncRNAs (lncRNA-599618, -599556, -599554, -599547, -599531, and -599509) at the anagen phase is significantly higher than that at telogen. Compared with anagen, the relative expression of four putative lncRNAs (lncRNA-599528, -599518, -599511, and -599497) was found to be significantly upregulated at telogen phase. The network generated showed that a rich and complex regulatory relationship of the putative lncRNAs and related miRNAs with their target genes in Wnt signaling pathway. Our results from the present study provided a foundation for further elucidating the functional and regulatory mechanisms of these putative lncRNAs in the development of secondary hair follicle and cashmere fiber growth of Cashmere goat.

Integrative Clinical Genomics of Metastatic Cancer

PubMed Central

Robinson, Dan R.; Wu, Yi-Mi; Lonigro, Robert J.; Vats, Pankaj; Cobain, Erin; Everett, Jessica; Cao, Xuhong; Rabban, Erica; Kumar-Sinha, Chandan; Raymond, Victoria; Schuetze, Scott; Alva, Ajjai; Siddiqui, Javed; Chugh, Rashmi; Worden, Francis; Zalupski, Mark M.; Innis, Jeffrey; Mody, Rajen J.; Tomlins, Scott A.; Lucas, David; Baker, Laurence H.; Ramnath, Nithya; Schott, Ann F.; Hayes, Daniel F.; Vijai, Joseph; Offit, Kenneth; Stoffel, Elena M.; Roberts, J. Scott; Smith, David C.; Kunju, Lakshmi P.; Talpaz, Moshe; Cieslik, Marcin; Chinnaiyan, Arul M.

2017-01-01

SUMMARY Metastasis is the primary cause of cancer-related deaths. While The Cancer Genome Atlas (TCGA) has sequenced primary tumor types obtained from surgical resections, much less comprehensive molecular analysis is available from clinically acquired metastatic cancers. Here, we perform whole exome and transcriptome sequencing of 500 adult patients with metastatic solid tumors of diverse lineage and biopsy site. The most prevalent genes somatically altered in metastatic cancer included TP53, CDKN2A, PTEN, PIK3CA, and RB1. Putative pathogenic germline variants were present in 12.2% of cases of which 75% were related to defects in DNA repair. RNA sequencing complemented DNA sequencing for the identification of gene fusions, pathway activation, and immune profiling. Integrative sequence analysis provides a clinically relevant, multi-dimensional view of the complex molecular landscape and microenvironment of metastatic cancers. PMID:28783718

Characterization of additional components of the environmental pH-sensing complex in the pathogenic fungus Cryptococcus neoformans.

PubMed

Pianalto, Kaila M; Ost, Kyla S; Brown, Hannah E; Alspaugh, J Andrew

2018-05-16

Pathogenic microorganisms must adapt to changes in their immediate surroundings, including alterations in pH, to survive the shift from the external environment to that of the infected host. In the basidiomycete fungal pathogen Cryptococcus neoformans , these pH changes are primarily sensed by the fungal-specific, alkaline pH-sensing Rim/Pal pathway. The C. neoformans Rim pathway has diverged significantly from that described in ascomycete fungi. We recently identified the C. neoformans putative pH sensor Rra1, which activates the Rim pathway in response to elevated pH. In this study, we probed the function of Rra1 by analyzing its cellular localization and performing protein co-immunoprecipitation to identify potential Rra1 interactors. We found that Rra1 does not strongly colocalize or interact with immediate downstream Rim pathway components. However, these experiments identified a novel Rra1 interactor, the previously uncharacterized C. neoformans nucleosome assembly protein 1 (Nap1), which was required for Rim pathway activation. We observed that Nap1 specifically binds to the C-terminal tail of the Rra1 sensor, likely promoting Rra1 protein stability. This function of Nap1 is conserved in fungi closely related to C. neoformans that contain Rra1 orthologs, but not in the more distantly-related ascomycete fungus Saccharomyces cerevisiae In conclusion, our findings have revealed the sophisticated, yet distinct, molecular mechanisms by which closely and distantly related microbial phyla rapidly adapt to environmental signals and changes such as alterations in pH. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Meta-Analysis of Global Transcriptomics Suggests that Conserved Genetic Pathways are Responsible for Quercetin and Tannic Acid Mediated Longevity in C. elegans

PubMed Central

Pietsch, Kerstin; Saul, Nadine; Swain, Suresh C.; Menzel, Ralph; Steinberg, Christian E. W.; Stürzenbaum, Stephen R.

2012-01-01

Recent research has highlighted that the polyphenols Quercetin and Tannic acid are capable of extending the lifespan of Caenorhabditis elegans. To gain a deep understanding of the underlying molecular genetics, we analyzed the global transcriptional patterns of nematodes exposed to three concentrations of Quercetin or Tannic acid, respectively. By means of an intricate meta-analysis it was possible to compare the transcriptomes of polyphenol exposure to recently published datasets derived from (i) longevity mutants or (ii) infection. This detailed comparative in silico analysis facilitated the identification of compound specific and overlapping transcriptional profiles and allowed the prediction of putative mechanistic models of Quercetin and Tannic acid mediated longevity. Lifespan extension due to Quercetin was predominantly driven by the metabolome, TGF-beta signaling, Insulin-like signaling, and the p38 MAPK pathway and Tannic acid’s impact involved, in part, the amino acid metabolism and was modulated by the TGF-beta and the p38 MAPK pathways. DAF-12, which integrates TGF-beta and Insulin-like downstream signaling, and genetic players of the p38 MAPK pathway therefore seem to be crucial regulators for both polyphenols. Taken together, this study underlines how meta-analyses can provide an insight of molecular events that go beyond the traditional categorization into gene ontology-terms and Kyoto encyclopedia of genes and genomes-pathways. It also supports the call to expand the generation of comparative and integrative databases, an effort that is currently still in its infancy. PMID:22493606

Molecular Pharmacology of Malignant Pleural Mesothelioma: Challenges and Perspectives From Preclinical and Clinical Studies.

PubMed

Thellung, Stefano; Favoni, Roberto E; Würth, Roberto; Nizzari, Mario; Pattarozzi, Alessandra; Daga, Antonio; Florio, Tullio; Barbieri, Federica

2016-01-01

Malignant pleural mesothelioma (MPM) is one of the deadliest and most heterogeneous tumors, highly refractory to multimodal therapeutic approach, including surgery, chemo- and radiotherapy. Preclinical and clinical studies exploring the efficacy of drugs targeting tyrosine kinases, angiogenesis and histone deacetylases, did not fulfil the expected clinical benefits. Thus, novel molecular targets should be identified from a definite knowledge of the unique biology and most relevant transduction pathways of MPM cells. Cancer stem cells (CSCs) are a subset of malignant precursors responsible for initiation, progression, resistance to cytotoxic drugs, recurrence and metastatic diffusion of tumor cells. CSCs are putative driving factors for MPM development and contribute to its clinical and biological heterogeneity; hence, targeted eradication of CSCs represents an ineludible goal to counteract MPM aggressiveness. In this context, innovative preclinical models could be exploited to identify novel intracellular pathway inhibitors able to target CSC viability. Novel drug targets have been identified among key factors responsible for the oncogenic transformation of mesothelial cells, often directly induced by asbestos. These include mitogenic and anti-apoptotic signaling that may also be activated by autocrine and paracrine cytokine pathways controlling cell plasticity. Both signaling pathways affecting proto-oncogene and transcription factor expression, or genetic and epigenetic alterations, such as mutations in cell cycle genes and silencing of tumor suppressor genes, represent promising disease-specific targets. In this review we describe current knowledge of MPM cell biology, focusing on potential targets to be tested in pharmacological studies, and highlighting results and challenges of clinical translation.

Predictive Genomic Analyses Inform the Basis for Vitamin Metabolism and Provisioning in Bacteria-Arthropod Endosymbioses

PubMed Central

Serbus, Laura R.; Rodriguez, Brian Garcia; Sharmin, Zinat; Momtaz, A. J. M. Zehadee; Christensen, Steen

2017-01-01

The requirement of vitamins for core metabolic processes creates a unique set of pressures for arthropods subsisting on nutrient-limited diets. While endosymbiotic bacteria carried by arthropods have been widely implicated in vitamin provisioning, the underlying molecular mechanisms are not well understood. To address this issue, standardized predictive assessment of vitamin metabolism was performed in 50 endosymbionts of insects and arachnids. The results predicted that arthropod endosymbionts overall have little capacity for complete de novo biosynthesis of conventional or active vitamin forms. Partial biosynthesis pathways were commonly predicted, suggesting a substantial role in vitamin provisioning. Neither taxonomic relationships between host and symbiont, nor the mode of host-symbiont interaction were clear predictors of endosymbiont vitamin pathway capacity. Endosymbiont genome size and the synthetic capacity of nonsymbiont taxonomic relatives were more reliable predictors. We developed a new software application that also predicted that last-step conversion of intermediates into active vitamin forms may contribute further to vitamin biosynthesis by endosymbionts. Most instances of predicted vitamin conversion were paralleled by predictions of vitamin use. This is consistent with achievement of provisioning in some cases through upregulation of pathways that were retained for endosymbiont benefit. The predicted absence of other enzyme classes further suggests a baseline of vitamin requirement by the majority of endosymbionts, as well as some instances of putative mutualism. Adaptation of this workflow to analysis of other organisms and metabolic pathways will provide new routes for considering the molecular basis for symbiosis on a comprehensive scale. PMID:28455417

Putative adverse outcome pathways relevant to neurotoxicity

PubMed Central

Bal-Price, Anna; Crofton, Kevin M.; Sachana, Magdalini; Shafer, Timothy J.; Behl, Mamta; Forsby, Anna; Hargreaves, Alan; Landesmann, Brigitte; Lein, Pamela J.; Louisse, Jochem; Monnet-Tschudi, Florianne; Paini, Alicia; Rolaki, Alexandra; Schrattenholz, André; Suñol, Cristina; van Thriel, Christoph; Whelan, Maurice; Fritsche, Ellen

2016-01-01

The Adverse Outcome Pathway (AOP) framework provides a template that facilitates understanding of complex biological systems and the pathways of toxicity that result in adverse outcomes (AOs). The AOP starts with an molecular initiating event (MIE) in which a chemical interacts with a biological target(s), followed by a sequential series of KEs, which are cellular, anatomical, and/or functional changes in biological processes, that ultimately result in an AO manifest in individual organisms and populations. It has been developed as a tool for a knowledge-based safety assessment that relies on understanding mechanisms of toxicity, rather than simply observing its adverse outcome. A large number of cellular and molecular processes are known to be crucial to proper development and function of the central (CNS) and peripheral nervous systems (PNS). However, there are relatively few examples of well-documented pathways that include causally linked MIEs and KEs that result in adverse outcomes in the CNS or PNS. As a first step in applying the AOP framework to adverse health outcomes associated with exposure to exogenous neurotoxic substances, the EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) organized a workshop (March 2013, Ispra, Italy) to identify potential AOPs relevant to neurotoxic and developmental neurotoxic outcomes. Although the AOPs outlined during the workshop are not fully described, they could serve as a basis for further, more detailed AOP development and evaluation that could be useful to support human health risk assessment in a variety of ways. PMID:25605028

Partial Activation of SA- and JA-Defensive Pathways in Strawberry upon Colletotrichum acutatum Interaction.

PubMed

Amil-Ruiz, Francisco; Garrido-Gala, José; Gadea, José; Blanco-Portales, Rosario; Muñoz-Mérida, Antonio; Trelles, Oswaldo; de Los Santos, Berta; Arroyo, Francisco T; Aguado-Puig, Ana; Romero, Fernando; Mercado, José-Ángel; Pliego-Alfaro, Fernando; Muñoz-Blanco, Juan; Caballero, José L

2016-01-01

Understanding the nature of pathogen host interaction may help improve strawberry (Fragaria × ananassa) cultivars. Plant resistance to pathogenic agents usually operates through a complex network of defense mechanisms mediated by a diverse array of signaling molecules. In strawberry, resistance to a variety of pathogens has been reported to be mostly polygenic and quantitatively inherited, making it difficult to associate molecular markers with disease resistance genes. Colletotrichum acutatum spp. is a major strawberry pathogen, and completely resistant cultivars have not been reported. Moreover, strawberry defense network components and mechanisms remain largely unknown and poorly understood. Assessment of the strawberry response to C. acutatum included a global transcript analysis, and acidic hormones SA and JA measurements were analyzed after challenge with the pathogen. Induction of transcripts corresponding to the SA and JA signaling pathways and key genes controlling major steps within these defense pathways was detected. Accordingly, SA and JA accumulated in strawberry after infection. Contrastingly, induction of several important SA, JA, and oxidative stress-responsive defense genes, including FaPR1-1, FaLOX2, FaJAR1, FaPDF1, and FaGST1, was not detected, which suggests that specific branches in these defense pathways (those leading to FaPR1-2, FaPR2-1, FaPR2-2, FaAOS, FaPR5, and FaPR10) were activated. Our results reveal that specific aspects in SA and JA dependent signaling pathways are activated in strawberry upon interaction with C. acutatum. Certain described defense-associated transcripts related to these two known signaling pathways do not increase in abundance following infection. This finding suggests new insight into a specific putative molecular strategy for defense against this pathogen.

Partial Activation of SA- and JA-Defensive Pathways in Strawberry upon Colletotrichum acutatum Interaction

PubMed Central

Amil-Ruiz, Francisco; Garrido-Gala, José; Gadea, José; Blanco-Portales, Rosario; Muñoz-Mérida, Antonio; Trelles, Oswaldo; de los Santos, Berta; Arroyo, Francisco T.; Aguado-Puig, Ana; Romero, Fernando; Mercado, José-Ángel; Pliego-Alfaro, Fernando; Muñoz-Blanco, Juan; Caballero, José L.

2016-01-01

Understanding the nature of pathogen host interaction may help improve strawberry (Fragaria × ananassa) cultivars. Plant resistance to pathogenic agents usually operates through a complex network of defense mechanisms mediated by a diverse array of signaling molecules. In strawberry, resistance to a variety of pathogens has been reported to be mostly polygenic and quantitatively inherited, making it difficult to associate molecular markers with disease resistance genes. Colletotrichum acutatum spp. is a major strawberry pathogen, and completely resistant cultivars have not been reported. Moreover, strawberry defense network components and mechanisms remain largely unknown and poorly understood. Assessment of the strawberry response to C. acutatum included a global transcript analysis, and acidic hormones SA and JA measurements were analyzed after challenge with the pathogen. Induction of transcripts corresponding to the SA and JA signaling pathways and key genes controlling major steps within these defense pathways was detected. Accordingly, SA and JA accumulated in strawberry after infection. Contrastingly, induction of several important SA, JA, and oxidative stress-responsive defense genes, including FaPR1-1, FaLOX2, FaJAR1, FaPDF1, and FaGST1, was not detected, which suggests that specific branches in these defense pathways (those leading to FaPR1-2, FaPR2-1, FaPR2-2, FaAOS, FaPR5, and FaPR10) were activated. Our results reveal that specific aspects in SA and JA dependent signaling pathways are activated in strawberry upon interaction with C. acutatum. Certain described defense-associated transcripts related to these two known signaling pathways do not increase in abundance following infection. This finding suggests new insight into a specific putative molecular strategy for defense against this pathogen. PMID:27471515

Molecular Diagnosis of Putative Stargardt Disease by Capture Next Generation Sequencing

PubMed Central

Shi, Wei; Huang, Ping; Min, Qingjie; Li, Minghan; Yu, Xinping; Wu, Yaming; Zhao, Guangyu; Tong, Yi; Jin, Zi-Bing; Qu, Jia; Gu, Feng

2014-01-01

Stargardt Disease (STGD) is the commonest genetic form of juvenile or early adult onset macular degeneration, which is a genetically heterogeneous disease. Molecular diagnosis of STGD remains a challenge in a significant proportion of cases. To address this, seven patients from five putative STGD families were recruited. We performed capture next generation sequencing (CNGS) of the probands and searched for potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes. Seven disease-causing mutations in ABCA4 and two in PROM1 were identified by CNGS, which provides a confident genetic diagnosis in these five families. We also provided a genetic basis to explain the differences among putative STGD due to various mutations in different genes. Meanwhile, we show for the first time that compound heterozygous mutations in PROM1 gene could cause cone-rod dystrophy. Our findings support the enormous potential of CNGS in putative STGD molecular diagnosis. PMID:24763286

Comparative genomics reveals candidate carotenoid pathway regulators of ripening watermelon fruit.

PubMed

Grassi, Stefania; Piro, Gabriella; Lee, Je Min; Zheng, Yi; Fei, Zhangjun; Dalessandro, Giuseppe; Giovannoni, James J; Lenucci, Marcello S

2013-11-12

Many fruits, including watermelon, are proficient in carotenoid accumulation during ripening. While most genes encoding steps in the carotenoid biosynthetic pathway have been cloned, few transcriptional regulators of these genes have been defined to date. Here we describe the identification of a set of putative carotenoid-related transcription factors resulting from fresh watermelon carotenoid and transcriptome analysis during fruit development and ripening. Our goal is to both clarify the expression profiles of carotenoid pathway genes and to identify candidate regulators and molecular targets for crop improvement. Total carotenoids progressively increased during fruit ripening up to ~55 μg g(-1) fw in red-ripe fruits. Trans-lycopene was the carotenoid that contributed most to this increase. Many of the genes related to carotenoid metabolism displayed changing expression levels during fruit ripening generating a metabolic flux toward carotenoid synthesis. Constitutive low expression of lycopene cyclase genes resulted in lycopene accumulation. RNA-seq expression profiling of watermelon fruit development yielded a set of transcription factors whose expression was correlated with ripening and carotenoid accumulation. Nineteen putative transcription factor genes from watermelon and homologous to tomato carotenoid-associated genes were identified. Among these, six were differentially expressed in the flesh of both species during fruit development and ripening. Taken together the data suggest that, while the regulation of a common set of metabolic genes likely influences carotenoid synthesis and accumulation in watermelon and tomato fruits during development and ripening, specific and limiting regulators may differ between climacteric and non-climacteric fruits, possibly related to their differential susceptibility to and use of ethylene during ripening.

Comparative genomics reveals candidate carotenoid pathway regulators of ripening watermelon fruit

PubMed Central

2013-01-01

Background Many fruits, including watermelon, are proficient in carotenoid accumulation during ripening. While most genes encoding steps in the carotenoid biosynthetic pathway have been cloned, few transcriptional regulators of these genes have been defined to date. Here we describe the identification of a set of putative carotenoid-related transcription factors resulting from fresh watermelon carotenoid and transcriptome analysis during fruit development and ripening. Our goal is to both clarify the expression profiles of carotenoid pathway genes and to identify candidate regulators and molecular targets for crop improvement. Results Total carotenoids progressively increased during fruit ripening up to ~55 μg g-1 fw in red-ripe fruits. Trans-lycopene was the carotenoid that contributed most to this increase. Many of the genes related to carotenoid metabolism displayed changing expression levels during fruit ripening generating a metabolic flux toward carotenoid synthesis. Constitutive low expression of lycopene cyclase genes resulted in lycopene accumulation. RNA-seq expression profiling of watermelon fruit development yielded a set of transcription factors whose expression was correlated with ripening and carotenoid accumulation. Nineteen putative transcription factor genes from watermelon and homologous to tomato carotenoid-associated genes were identified. Among these, six were differentially expressed in the flesh of both species during fruit development and ripening. Conclusions Taken together the data suggest that, while the regulation of a common set of metabolic genes likely influences carotenoid synthesis and accumulation in watermelon and tomato fruits during development and ripening, specific and limiting regulators may differ between climacteric and non-climacteric fruits, possibly related to their differential susceptibility to and use of ethylene during ripening. PMID:24219562

Structural Conversion of Aβ17–42 Peptides from Disordered Oligomers to U-Shape Protofilaments via Multiple Kinetic Pathways

PubMed Central

Cheon, Mookyung; Hall, Carol K.; Chang, Iksoo

2015-01-01

Discovering the mechanisms by which proteins aggregate into fibrils is an essential first step in understanding the molecular level processes underlying neurodegenerative diseases such as Alzheimer’s and Parkinson's. The goal of this work is to provide insights into the structural changes that characterize the kinetic pathways by which amyloid-β peptides convert from monomers to oligomers to fibrils. By applying discontinuous molecular dynamics simulations to PRIME20, a force field designed to capture the chemical and physical aspects of protein aggregation, we have been able to trace out the entire aggregation process for a system containing 8 Aβ17–42 peptides. We uncovered two fibrillization mechanisms that govern the structural conversion of Aβ17–42 peptides from disordered oligomers into protofilaments. The first mechanism is monomeric conversion templated by a U-shape oligomeric nucleus into U-shape protofilament. The second mechanism involves a long-lived and on-pathway metastable oligomer with S-shape chains, having a C-terminal turn, en route to the final U-shape protofilament. Oligomers with this C-terminal turn have been regarded in recent experiments as a major contributing element to cell toxicity in Alzheimer’s disease. The internal structures of the U-shape protofilaments from our PRIME20/DMD simulation agree well with those from solid state NMR experiments. The approach presented here offers a simple molecular-level framework to describe protein aggregation in general and to visualize the kinetic evolution of a putative toxic element in Alzheimer’s disease in particular. PMID:25955249

A fucose containing polymer-rich fraction from the brown alga Ascophyllum nodosum mediates lifespan increase and thermal-tolerance in Caenorhabditis elegans, by differential effects on gene and protein expression.

PubMed

Kandasamy, Saveetha; Khan, Wajahatullah; Evans, Franklin D; Critchley, Alan T; Zhang, Junzeng; Fitton, J H; Stringer, Damien N; Gardiner, Vicki-Anne; Prithiviraj, Balakrishnan

2014-02-01

The extracts of the brown alga, Ascophyllum nodosum, which contains several bioactive compounds, have been shown to impart biotic and abiotic stress tolerance properties when consumed by animals. However, the physiological, biochemical and molecular mechanism underlying such effects remain elusive. We investigated the effect of A. nodosum fucose-containing polymer (FCP) on tolerance to thermally induced stress using the invertebrate animal model, Caenorhabditis elegans. FCP at a concentration of 150 μg mL(-1) significantly improved the life span and tolerance against thermally induced stress in C. elegans. The treatment increased the C. elegans survival by approximately 24%, when the animals were under severe thermally induced stress (i.e. 35 °C) and 27% under mild stress (i.e. 30 °C) conditions. The FCP induced differential expression of genes and proteins is associated with stress response pathways. Under thermal stress, FCP treatment significantly altered the expression of 65 proteins (54 up-regulated & 11 down-regulated). Putative functional analysis of FCP-induced differential proteins signified an association of altered proteins in stress-related molecular and biochemical pathways of the model worm.

A Metabolic Probe-Enabled Strategy Reveals Uptake and Protein Targets of Polyunsaturated Aldehydes in the Diatom Phaeodactylum tricornutum

PubMed Central

Wolfram, Stefanie; Wielsch, Natalie; Hupfer, Yvonne; Mönch, Bettina; Lu-Walther, Hui-Wen; Heintzmann, Rainer; Werz, Oliver; Svatoš, Aleš; Pohnert, Georg

2015-01-01

Diatoms are unicellular algae of crucial importance as they belong to the main primary producers in aquatic ecosystems. Several diatom species produce polyunsaturated aldehydes (PUAs) that have been made responsible for chemically mediated interactions in the plankton. PUA-effects include chemical defense by reducing the reproductive success of grazing copepods, allelochemical activity by interfering with the growth of competing phytoplankton and cell to cell signaling. We applied a PUA-derived molecular probe, based on the biologically highly active 2,4-decadienal, with the aim to reveal protein targets of PUAs and affected metabolic pathways. By using fluorescence microscopy, we observed a substantial uptake of the PUA probe into cells of the diatom Phaeodactylum tricornutum in comparison to the uptake of a structurally closely related control probe based on a saturated aldehyde. The specific uptake motivated a chemoproteomic approach to generate a qualitative inventory of proteins covalently targeted by the α,β,γ,δ-unsaturated aldehyde structure element. Activity-based protein profiling revealed selective covalent modification of target proteins by the PUA probe. Analysis of the labeled proteins gave insights into putative affected molecular functions and biological processes such as photosynthesis including ATP generation and catalytic activity in the Calvin cycle or the pentose phosphate pathway. The mechanism of action of PUAs involves covalent reactions with proteins that may result in protein dysfunction and interference of involved pathways. PMID:26496085

Molecular Drivers of Pancreatic Cancer Pathogenesis: Looking Inward to Move Forward

PubMed Central

Khan, Mohammad Aslam; Azim, Shafquat; Zubair, Haseeb; Bhardwaj, Arun; Patel, Girijesh Kumar; Khushman, Moh’d; Singh, Seema; Singh, Ajay Pratap

2017-01-01

Pancreatic cancer (PC) continues to rank among the most lethal cancers. The consistent increase in incidence and mortality has made it the seventh leading cause of cancer-associated deaths globally and the third in the United States. The biggest challenge in combating PC is our insufficient understanding of the molecular mechanism(s) underlying its complex biology. Studies during the last several years have helped identify several putative factors and events, both genetic and epigenetic, as well as some deregulated signaling pathways, with implications in PC onset and progression. In this review article, we make an effort to summarize our current understanding of molecular and cellular events involved in the pathogenesis of pancreatic malignancy. Specifically, we provide up-to-date information on the genetic and epigenetic changes that occur during the initiation and progression of PC and their functional involvement in the pathogenic processes. We also discuss the impact of the tumor microenvironment on the molecular landscape of PC and its role in aggressive disease progression. It is envisioned that a better understanding of these molecular factors and the mechanisms of their actions can help unravel novel diagnostic and prognostic biomarkers and can also be exploited for future targeted therapies. PMID:28383487

Lipid nanocarriers and molecular targets for malaria chemotherapy.

PubMed

Jain, Kunal; Sood, Sumeet; Gowthamarajan, Kuppusamy

2014-03-01

Malaria is the most serious tropical disease of humankind and a cause of much debilitation and morbidity throughout the world especially in endemic areas like India and Africa. The development of drug resistance may be due to insufficient drug concentration in presence of high parasite load. In addition, the present pharmaceutical dosage forms are ineffective thereby necessitating the development of novel dosage forms which are effective, safe and affordable to underprivileged population of the developing world. The rapid advancement of nanotechnology has raised the possibility of using lipid nanocarriers that interact within biological environment for treatment of infectious diseases. Thus, lipid based nano-delivery systems offer a platform to formulate old and toxic antimalarial drugs thereby modifying their pharmacokinetic profile, biodistribution and targetability. Further, there is a need to develop new chemotherapy based approaches for inhibiting the parasite-specific metabolic pathways. The present review highlights the advances in lipid nanocarriers and putative molecular targets for antimalarial chemotherapy.

Ligand Entry and Exit Pathways in the β2-adrenergic Receptor

PubMed Central

Wang, Ting; Duan, Yong

2009-01-01

The recently determined crystal structure of the human β2-adrenergic (β2AR) G-protein coupled receptor provides an excellent structural basis for exploring β2AR -ligand binding and dissociation process. Based on this crystal structure, we simulated ligand exit from the β2AR receptor by applying the random acceleration molecular dynamics (RAMD) simulation method. The simulation results showed that the extracellular opening on the receptor surface was the most frequently observed egress point (referred to as pathway A) and a few other pathways through inter-helical clefts were also observed with significantly lower frequencies. In the egress trajectories along pathway A, the D192-K305 salt bridge between the extracellular loop 2 (ECL2) and the apex of the transmembrane helix 7 (TM7) was exclusively broken. The spatial occupancy maps of the ligand computed from the 100 RAMD simulation trajectories indicated that the receptor-ligand interactions that restrained the ligand in the binding pocket were the major resistance encountered by the ligand during exit and no second barrier was notable. We next performed RAMD simulations by using a putative ligand-free conformation of the receptor as input structure. This conformation was obtained in a standard MD simulation in the absence of the ligand and it differed from the ligand-bound conformation in a hydrophobic patch bridging ECL2 and TM7 due to the rotation of F193 of ECL2. Results from the RAMD simulations with this putative ligand-free conformation suggest that the cleft formed by the hydrophobic bridge, TM2, TM3 and TM7 on the extracellular surface likely serves as a more specific ligand-entry site and the ECL2-TM7 hydrophobic junction can be partially interrupted upon the entry of ligand that pushes F193 to rotate, resulting in a conformation as observed in the ligand-bound crystal structure. These results may help design β2AR-targeting drugs with improved efficacy as well as understand the receptor subtype-selectivity of ligand binding in the β family of the adrenergic receptors that share almost identical ligand-binding pockets but show notable amino acid sequence divergence in the putative ligand-entry site, including ECL2 and the extracellular end of TM7. PMID:19665031

Role of Aquaporins in a Composite Model of Water Transport in the Leaf.

PubMed

Yaaran, Adi; Moshelion, Menachem

2016-06-30

Water-transport pathways through the leaf are complex and include several checkpoints. Some of these checkpoints exhibit dynamic behavior that may be regulated by aquaporins (AQPs). To date, neither the relative weight of the different water pathways nor their molecular mechanisms are well understood. Here, we have collected evidence to support a putative composite model of water pathways in the leaf and the distribution of water across those pathways. We describe how water moves along a single transcellular path through the parenchyma and continues toward the mesophyll and stomata along transcellular, symplastic and apoplastic paths. We present evidence that points to a role for AQPs in regulating the relative weight of each path in the overall leaf water-transport system and the movement of water between these paths as a result of the integration of multiple signals, including transpiration demand, water potential and turgor. We also present a new theory, the hydraulic fuse theory, to explain effects of the leaf turgor-loss-point on water paths alternation and the subsequent reduction in leaf hydraulic conductivity. An improved understating of leaf water-balance management may lead to the development of crops that use water more efficiently, and responds better to environmental changes.

Safety and feasibility of targeted agent combinations in solid tumours.

PubMed

Park, Sook Ryun; Davis, Myrtle; Doroshow, James H; Kummar, Shivaani

2013-03-01

The plethora of novel molecular-targeted agents (MTAs) has provided an opportunity to selectively target pathways involved in carcinogenesis and tumour progression. Combination strategies of MTAs are being used to inhibit multiple aberrant pathways in the hope of optimizing antitumour efficacy and to prevent development of resistance. While the selection of specific agents in a given combination has been based on biological considerations (including the role of the putative targets in cancer) and the interactions of the agents used in combination, there has been little exploration of the possible enhanced toxicity of combinations resulting from alterations in multiple signalling pathways in normal cell biology. Owing to the complex networks and crosstalk that govern normal and tumour cell proliferation, inhibiting multiple pathways with MTA combinations can result in unpredictable disturbances in normal physiology. This Review focuses on the main toxicities and the lack of tolerability of some common MTA combinations, particularly where evidence of enhanced toxicity compared to either agent alone is documented or there is development of unexpected toxicity. Toxicities caused by MTA combinations highlight the need to introduce new preclinical testing paradigms early in the drug development process for the assessment of chronic toxicities resulting from such combinations.

Heterologous pathway assembly reveals molecular steps of fungal terreic acid biosynthesis.

PubMed

Kong, Chuixing; Huang, Hezhou; Xue, Ying; Liu, Yiqi; Peng, Qiangqiang; Liu, Qi; Xu, Qin; Zhu, Qiaoyun; Yin, Ying; Zhou, Xiangshan; Zhang, Yuanxing; Cai, Menghao

2018-02-01

Terreic acid is a potential anticancer drug as it inhibits Bruton's tyrosine kinase; however, its biosynthetic molecular steps remain unclear. In this work, the individual reactions of terreic acid biosynthesis were determined by stepwise pathway assembly in a heterologous host, Pichia pastoris, on the basis of previous knockout studies in a native host, Aspergillus terreus. Polyketide synthase AtX was found to catalyze the formation of partially reduced polyketide 6-methylsalicylic acid, followed by 3-methylcatechol synthesis by salicylate 1-monooxygenase AtA-mediated decarboxylative hydroxylation of 6-methylsalicylic acid. Our results show that cytochrome P450 monooxygenase AtE hydroxylates 3-methylcatechol, thus producing the next product, 3-methyl-1,2,4-benzenetriol. A smaller putative cytochrome P450 monooxygenase, AtG, assists with this step. Then, AtD causes epoxidation and hydroxyl oxidation of 3-methyl-1,2,4-benzenetriol and produces a compound terremutin, via which the previously unknown function of AtD was identified as cyclooxygenation. The final step involves an oxidation reaction of a hydroxyl group by a glucose-methanol-choline oxidoreductase, AtC, which leads to the final product: terreic acid. Functions of AtD and AtG were determined for the first time. All the genes were reanalyzed and all intermediates and final products were isolated and identified. Our model fully defines the molecular steps and corrects previous results from the literature.

Isolation and Identification of Putative Protein Substrates of the AAA+ Molecular Chaperone ClpB from the Pathogenic Spirochaete Leptospira interrogans.

PubMed

Krajewska, Joanna; Arent, Zbigniew; Zolkiewski, Michal; Kędzierska-Mieszkowska, Sabina

2018-04-18

Bacterial ClpB is an ATP-dependent Hsp100 chaperone that reactivates aggregated proteins in cooperation with the DnaK chaperone system and promotes survival of bacteria under stress conditions. A large number of publications also indicate that ClpB supports the virulence of bacteria, including a pathogenic spirochaete Leptospira interrogans responsible for leptospirosis in both animals and humans. However, the exact role of ClpB in bacterial pathogenicity remains poorly characterized. It can be assumed that ClpB, due to its role as the molecular chaperone, mediates refolding of essential bacterial proteins, including the known virulence factors, which may become prone to aggregation under infection-induced stresses. In this study, we identified putative substrates of ClpB from L. interrogans (ClpB Li ). For this purpose, we used a proteomic approach combining the ClpB-Trap affinity pull-down assays, Liquid chromatography-tandem mass spectrometry (LC-MS-MS/MS), and bioinformatics analyses. Most of the identified proteins were enzymes predominantly associated with major metabolic pathways like the tricarboxylic acid (TCA) cycle, glycolysis–gluconeogenesis and amino acid and fatty acid metabolism. Based on our proteomic study, we suggest that ClpB can support the virulence of L. interrogans by protecting the conformational integrity and catalytic activity of multiple metabolic enzymes, thus maintaining energy homeostasis in pathogen cells.

The CASTOR proteins are arginine sensors for the mTORC1 pathway

PubMed Central

Chantranupong, Lynne; Scaria, Sonia M.; Saxton, Robert A.; Gygi, Melanie P.; Shen, Kuang; Wyant, Gregory A.; Wang, Tim; Harper, J. Wade; Gygi, Steven P.; Sabatini, David M.

2016-01-01

Amino acids signal to the mTOR complex I (mTORC1) growth pathway through the Rag GTPases. Multiple distinct complexes regulate the Rags, including GATOR1, a GTPase activating protein (GAP), and GATOR2, a positive regulator of unknown molecular function. Arginine stimulation of cells activates mTORC1, but how it is sensed is not well understood. Recently, SLC38A9 was identified as a putative lysosomal arginine sensor required for arginine to activate mTORC1 but how arginine deprivation represses mTORC1 is unknown. Here, we show that CASTOR1, a previously uncharacterized protein, interacts with GATOR2 and is required for arginine deprivation to inhibit mTORC1. CASTOR1 homodimerizes and can also heterodimerize with the related protein, CASTOR2. Arginine disrupts the CASTOR1-GATOR2 complex by binding to CASTOR1 with a dissociation constant of ~30 μM, and its arginine-binding capacity is required for arginine to activate mTORC1 in cells. Collectively, these results establish CASTOR1 as an arginine sensor for the mTORC1 pathway. PMID:26972053

Predictive Genomic Analyses Inform the Basis for Vitamin Metabolism and Provisioning in Bacteria-Arthropod Endosymbioses.

PubMed

Serbus, Laura R; Rodriguez, Brian Garcia; Sharmin, Zinat; Momtaz, A J M Zehadee; Christensen, Steen

2017-06-07

The requirement of vitamins for core metabolic processes creates a unique set of pressures for arthropods subsisting on nutrient-limited diets. While endosymbiotic bacteria carried by arthropods have been widely implicated in vitamin provisioning, the underlying molecular mechanisms are not well understood. To address this issue, standardized predictive assessment of vitamin metabolism was performed in 50 endosymbionts of insects and arachnids. The results predicted that arthropod endosymbionts overall have little capacity for complete de novo biosynthesis of conventional or active vitamin forms. Partial biosynthesis pathways were commonly predicted, suggesting a substantial role in vitamin provisioning. Neither taxonomic relationships between host and symbiont, nor the mode of host-symbiont interaction were clear predictors of endosymbiont vitamin pathway capacity. Endosymbiont genome size and the synthetic capacity of nonsymbiont taxonomic relatives were more reliable predictors. We developed a new software application that also predicted that last-step conversion of intermediates into active vitamin forms may contribute further to vitamin biosynthesis by endosymbionts. Most instances of predicted vitamin conversion were paralleled by predictions of vitamin use. This is consistent with achievement of provisioning in some cases through upregulation of pathways that were retained for endosymbiont benefit. The predicted absence of other enzyme classes further suggests a baseline of vitamin requirement by the majority of endosymbionts, as well as some instances of putative mutualism. Adaptation of this workflow to analysis of other organisms and metabolic pathways will provide new routes for considering the molecular basis for symbiosis on a comprehensive scale. Copyright © 2017 Serbus et al.

Mechanisms of JAK/STAT pathway negative regulation by the short coreceptor Eye Transformer/Latran.

PubMed

Fisher, Katherine H; Stec, Wojciech; Brown, Stephen; Zeidler, Martin P

2016-02-01

Transmembrane receptors interact with extracellular ligands to transduce intracellular signaling cascades, modulate target gene expression, and regulate processes such as proliferation, apoptosis, differentiation, and homeostasis. As a consequence, aberrant signaling events often underlie human disease. Whereas the vertebrate JAK/STAT signaling cascade is transduced via multiple receptor combinations, the Drosophila pathway has only one full-length signaling receptor, Domeless (Dome), and a single negatively acting receptor, Eye Transformer/Latran (Et/Lat). Here we investigate the molecular mechanisms underlying Et/Lat activity. We demonstrate that Et/Lat negatively regulates the JAK/STAT pathway activity and can bind to Dome, thus reducing Dome:Dome homodimerization by creating signaling-incompetent Dome:Et/Lat heterodimers. Surprisingly, we find that Et/Lat is able to bind to both JAK and STAT92E but, despite the presence of putative cytokine-binding motifs, does not detectably interact with pathway ligands. We find that Et/Lat is trafficked through the endocytic machinery for lysosomal degradation but at a much slower rate than Dome, a difference that may enhance its ability to sequester Dome into signaling-incompetent complexes. Our data offer new insights into the molecular mechanism and regulation of Et/Lat in Drosophila that may inform our understanding of how short receptors function in other organisms. © 2016 Fisher et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Molecular characterization of Quercus suber MYB1, a transcription factor up-regulated in cork tissues.

PubMed

Almeida, Tânia; Menéndez, Esther; Capote, Tiago; Ribeiro, Teresa; Santos, Conceição; Gonçalves, Sónia

2013-01-15

The molecular processes associated with cork development in Quercus suber L. are poorly understood. A previous molecular approach identified a list of genes potentially important for cork formation and differentiation, providing a new basis for further molecular studies. This report is the first molecular characterization of one of these candidate genes, QsMYB1, coding for an R2R3-MYB transcription factor. The R2R3-MYB gene sub-family has been described as being involved in the phenylpropanoid and lignin pathways, both involved in cork biosynthesis. The results showed that the expression of QsMYB1 is putatively mediated by an alternative splicing (AS) mechanism that originates two different transcripts (QsMYB1.1 and QsMYB1.2), differing only in the 5'-untranslated region, due to retention of the first intron in one of the variants. Moreover, within the retained intron, a simple sequence repeat (SSR) was identified. The upstream regulatory region of QsMYB1 was extended by a genome walking approach, which allowed the identification of the putative gene promoter region. The relative expression pattern of QsMYB1 transcripts determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) revealed that both transcripts were up-regulated in cork tissues; the detected expression was several times higher in newly formed cork harvested from trees producing virgin, second or reproduction cork when compared with wood. Moreover, the expression analysis of QsMYB1 in several Q. suber organs showed very low expression in young branches and roots, whereas in leaves, immature acorns or male flowers, no expression was detected. These preliminary results suggest that QsMYB1 may be related to secondary growth and, in particular, with the cork biosynthesis process with a possible alternative splicing mechanism associated with its regulatory function. Copyright © 2012 Elsevier GmbH. All rights reserved.

The putative cellodextrin transporter-like protein CLP1 is involved in cellulase induction in Neurospora crassa.

PubMed

Cai, Pengli; Wang, Bang; Ji, Jingxiao; Jiang, Yongsheng; Wan, Li; Tian, Chaoguang; Ma, Yanhe

2015-01-09

Neurospora crassa recently has become a novel system to investigate cellulase induction. Here, we discovered a novel membrane protein, cellodextrin transporter-like protein 1 (CLP1; NCU05853), a putative cellodextrin transporter-like protein that is a critical component of the cellulase induction pathway in N. crassa. Although CLP1 protein cannot transport cellodextrin, the suppression of cellulase induction by this protein was discovered on both cellobiose and Avicel. The co-disruption of the cellodextrin transporters cdt2 and clp1 in strain Δ3βG formed strain CPL7. With induction by cellobiose, cellulase production was enhanced 6.9-fold in CPL7 compared with Δ3βG. We also showed that the suppression of cellulase expression by CLP1 occurred by repressing the expression of cellodextrin transporters, particularly cdt1 expression. Transcriptome analysis of the hypercellulase-producing strain CPL7 showed that the cellulase expression machinery was dramatically stimulated, as were the cellulase enzyme genes including the inducer transporters and the major transcriptional regulators. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Genome-wide identification and characterization of putative lncRNAs in the diamondback moth, Plutella xylostella (L.).

PubMed

Wang, Yue; Xu, Tingting; He, Weiyi; Shen, Xiujing; Zhao, Qian; Bai, Jianlin; You, Minsheng

2018-01-01

Long non-coding RNAs (lncRNAs) are of particular interest because of their contributions to many biological processes. Here, we present the genome-wide identification and characterization of putative lncRNAs in a global insect pest, Plutella xylostella. A total of 8096 lncRNAs were identified and classified into three groups. The average length of exons in lncRNAs was longer than that in coding genes and the GC content was lower than that in mRNAs. Most lncRNAs were flanked by canonical splice sites, similar to mRNAs. Expression profiling identified 114 differentially expressed lncRNAs during the DBM development and found that majority were temporally specific. While the biological functions of lncRNAs remain uncharacterized, many are microRNA precursors or competing endogenous RNAs involved in micro-RNA regulatory pathways. This work provides a valuable resource for further studies on molecular bases for development of DBM and lay the foundation for discovery of lncRNA functions in P. xylostella. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression analysis of Drosophila doublesex, transformer-2, intersex, fruitless-like, and vitellogenin homologs in the parahaploid predator Metaseiulus occidentalis (Chelicerata: Acari: Phytoseiidae).

PubMed

Pomerantz, Aaron F; Hoy, Marjorie A

2015-01-01

Characterization and expression analyses are essential to gain insight into sex-determination pathways in members of the Acari. Little is known about sex determination at the molecular level in the western orchard predatory mite Metaseiulus occidentalis (Arthropoda: Chelicerata: Arachnida: Acari: Phytoseiidae), a parahaploid species. In this study, eight genes previously identified as putative homologs to genes involved in the sex-determination pathway in Drosophila melanogaster were evaluated for sex-specific alternative splicing and sex-biased expression using reverse-transcriptase PCR and quantitative real-time PCR techniques, respectively. The homologs evaluated in M. occidentalis included two doublesex-like genes (Moccdsx1 and Moccdsx2), transformer-2 (Mocctra-2), intersex (Moccix), two fruitless-like genes (MoccBTB1 and MoccBTB2), as well as two vitellogenin-like genes (Moccvg1 and Moccvg2). Single transcripts of equal size were detected in males and females for Moccdsx1, Moccdsx2, Mocctra-2, Moccix, and MoccBTB2, suggesting that their pre-mRNAs do not undergo alternative splicing in a sex-specific manner. Three genes, Moccdsx1, Moccdsx2 and MoccBTB2, displayed male-biased expression relative to females. One gene, Moccix, displayed female-biased expression relative to males. Two genes, Mocctra-2 and MoccBTB1, did not display detectable differences in transcript abundance in males and females. Expression of Moccvg1 and Moccvg2 were detected in females only, and transcript levels were up-regulated in mated females relative to unmated females. To our knowledge, this represents the first attempt to elucidate expression patterns of putative sex-determination genes in an acarine. This study is an initial step towards understanding the sex-determination pathway in the parahaploid M. occidentalis.

Protein degradation pathways in Parkinson's disease: curse or blessing.

PubMed

Ebrahimi-Fakhari, Darius; Wahlster, Lara; McLean, Pamela J

2012-08-01

Protein misfolding, aggregation and deposition are common disease mechanisms in many neurodegenerative diseases including Parkinson's disease (PD). Accumulation of damaged or abnormally modified proteins may lead to perturbed cellular function and eventually to cell death. Thus, neurons rely on elaborated pathways of protein quality control and removal to maintain intracellular protein homeostasis. Molecular chaperones, the ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP) are critical pathways that mediate the refolding or removal of abnormal proteins. The successive failure of these protein degradation pathways, as a cause or consequence of early pathological alterations in vulnerable neurons at risk, may present a key step in the pathological cascade that leads to spreading neurodegeneration. A growing number of studies in disease models and patients have implicated dysfunction of the UPS and ALP in the pathogenesis of Parkinson's disease and related disorders. Deciphering the exact mechanism by which the different proteolytic systems contribute to the elimination of pathogenic proteins, like α-synuclein, is therefore of paramount importance. We herein review the role of protein degradation pathways in Parkinson's disease and elaborate on the different contributions of the UPS and the ALP to the clearance of altered proteins. We examine the interplay between different degradation pathways and provide a model for the role of the UPS and ALP in the evolution and progression of α-synuclein pathology. With regards to exciting recent studies we also discuss the putative potential of using protein degradation pathways as novel therapeutic targets in Parkinson's disease.

The role of the PI3K-Akt signaling pathway in the developmental competence of bovine oocytes.

PubMed

Andrade, Gabriella Mamede; da Silveira, Juliano Coelho; Perrini, Claudia; Del Collado, Maite; Gebremedhn, Samuel; Tesfaye, Dawit; Meirelles, Flávio Vieira; Perecin, Felipe

2017-01-01

The ovarian follicle encloses oocytes in a microenvironment throughout their growth and acquisition of competence. Evidence suggests a dynamic interplay among follicular cells and oocytes, since they are constantly exchanging "messages". We dissected bovine ovarian follicles and recovered follicular cells (FCs-granulosa and cumulus cells) and cumulus-oocyte complexes (COCs) to investigate whether the PI3K-Akt signaling pathway impacted oocyte quality. Following follicle rupture, COCs were individually selected for in vitro cultures to track the follicular cells based on oocyte competence to reach the blastocyst stage after parthenogenetic activation. Levels of PI3K-Akt signaling pathway components in FCs correlated with oocyte competence. This pathway is upregulated in FCs from follicles with high-quality oocytes that are able to reach the blastocyst stage, as indicated by decreased levels of PTEN and increased levels of the PTEN regulators bta-miR-494 and bta-miR-20a. Using PI3K-Akt responsive genes, we showed decreased FOXO3a levels and BAX levels in lower quality groups, indicating changes in cell cycle progression, oxidative response and apoptosis. Based on these results, the measurement of levels of PI3K-Akt pathway components in FCs from ovarian follicles carrying oocytes with distinct developmental competences is a useful tool to identify putative molecular pathways involved in the acquisition of oocyte competence.

Expression analysis in response to drought stress in soybean: Shedding light on the regulation of metabolic pathway genes.

PubMed

Guimarães-Dias, Fábia; Neves-Borges, Anna Cristina; Viana, Antonio Americo Barbosa; Mesquita, Rosilene Oliveira; Romano, Eduardo; de Fátima Grossi-de-Sá, Maria; Nepomuceno, Alexandre Lima; Loureiro, Marcelo Ehlers; Alves-Ferreira, Márcio

2012-06-01

Metabolomics analysis of wild type Arabidopsis thaliana plants, under control and drought stress conditions revealed several metabolic pathways that are induced under water deficit. The metabolic response to drought stress is also associated with ABA dependent and independent pathways, allowing a better understanding of the molecular mechanisms in this model plant. Through combining an in silico approach and gene expression analysis by quantitative real-time PCR, the present work aims at identifying genes of soybean metabolic pathways potentially associated with water deficit. Digital expression patterns of Arabidopsis genes, which were selected based on the basis of literature reports, were evaluated under drought stress condition by Genevestigator. Genes that showed strong induction under drought stress were selected and used as bait to identify orthologs in the soybean genome. This allowed us to select 354 genes of putative soybean orthologs of 79 Arabidopsis genes belonging to 38 distinct metabolic pathways. The expression pattern of the selected genes was verified in the subtractive libraries available in the GENOSOJA project. Subsequently, 13 genes from different metabolic pathways were selected for validation by qPCR experiments. The expression of six genes was validated in plants undergoing drought stress in both pot-based and hydroponic cultivation systems. The results suggest that the metabolic response to drought stress is conserved in Arabidopsis and soybean plants.

Transcriptome Analysis of Mango (Mangifera indica L.) Fruit Epidermal Peel to Identify Putative Cuticle-Associated Genes

PubMed Central

Tafolla-Arellano, Julio C.; Zheng, Yi; Sun, Honghe; Jiao, Chen; Ruiz-May, Eliel; Hernández-Oñate, Miguel A.; González-León, Alberto; Báez-Sañudo, Reginaldo; Fei, Zhangjun; Domozych, David; Rose, Jocelyn K. C.; Tiznado-Hernández, Martín E.

2017-01-01

Mango fruit (Mangifera indica L.) are highly perishable and have a limited shelf life, due to postharvest desiccation and senescence, which limits their global distribution. Recent studies of tomato fruit suggest that these traits are influenced by the expression of genes that are associated with cuticle metabolism. However, studies of these phenomena in mango fruit are limited by the lack of genome-scale data. In order to gain insight into the mango cuticle biogenesis and identify putative cuticle-associated genes, we analyzed the transcriptomes of peels from ripe and overripe mango fruit using RNA-Seq. Approximately 400 million reads were generated and de novo assembled into 107,744 unigenes, with a mean length of 1,717 bp and with this information an online Mango RNA-Seq Database (http://bioinfo.bti.cornell.edu/cgi-bin/mango/index.cgi) which is a valuable genomic resource for molecular research into the biology of mango fruit was created. RNA-Seq analysis suggested that the pathway leading to biosynthesis of the cuticle component, cutin, is up-regulated during overripening. This data was supported by analysis of the expression of several putative cuticle-associated genes and by gravimetric and microscopic studies of cuticle deposition, revealing a complex continuous pattern of cuticle deposition during fruit development and involving substantial accumulation during ripening/overripening. PMID:28425468

Transcriptome Analysis of Mango (Mangifera indica L.) Fruit Epidermal Peel to Identify Putative Cuticle-Associated Genes.

PubMed

Tafolla-Arellano, Julio C; Zheng, Yi; Sun, Honghe; Jiao, Chen; Ruiz-May, Eliel; Hernández-Oñate, Miguel A; González-León, Alberto; Báez-Sañudo, Reginaldo; Fei, Zhangjun; Domozych, David; Rose, Jocelyn K C; Tiznado-Hernández, Martín E

2017-04-20

Mango fruit (Mangifera indica L.) are highly perishable and have a limited shelf life, due to postharvest desiccation and senescence, which limits their global distribution. Recent studies of tomato fruit suggest that these traits are influenced by the expression of genes that are associated with cuticle metabolism. However, studies of these phenomena in mango fruit are limited by the lack of genome-scale data. In order to gain insight into the mango cuticle biogenesis and identify putative cuticle-associated genes, we analyzed the transcriptomes of peels from ripe and overripe mango fruit using RNA-Seq. Approximately 400 million reads were generated and de novo assembled into 107,744 unigenes, with a mean length of 1,717 bp and with this information an online Mango RNA-Seq Database (http://bioinfo.bti.cornell.edu/cgi-bin/mango/index.cgi) which is a valuable genomic resource for molecular research into the biology of mango fruit was created. RNA-Seq analysis suggested that the pathway leading to biosynthesis of the cuticle component, cutin, is up-regulated during overripening. This data was supported by analysis of the expression of several putative cuticle-associated genes and by gravimetric and microscopic studies of cuticle deposition, revealing a complex continuous pattern of cuticle deposition during fruit development and involving substantial accumulation during ripening/overripening.

Transcriptome Analysis of Mango (Mangifera indica L.) Fruit Epidermal Peel to Identify Putative Cuticle-Associated Genes

NASA Astrophysics Data System (ADS)

Tafolla-Arellano, Julio C.; Zheng, Yi; Sun, Honghe; Jiao, Chen; Ruiz-May, Eliel; Hernández-Oñate, Miguel A.; González-León, Alberto; Báez-Sañudo, Reginaldo; Fei, Zhangjun; Domozych, David; Rose, Jocelyn K. C.; Tiznado-Hernández, Martín E.

2017-04-01

Mango fruit (Mangifera indica L.) are highly perishable and have a limited shelf life, due to postharvest desiccation and senescence, which limits their global distribution. Recent studies of tomato fruit suggest that these traits are influenced by the expression of genes that are associated with cuticle metabolism. However, studies of these phenomena in mango fruit are limited by the lack of genome-scale data. In order to gain insight into the mango cuticle biogenesis and identify putative cuticle-associated genes, we analyzed the transcriptomes of peels from ripe and overripe mango fruit using RNA-Seq. Approximately 400 million reads were generated and de novo assembled into 107,744 unigenes, with a mean length of 1,717 bp and with this information an online Mango RNA-Seq Database (http://bioinfo.bti.cornell.edu/cgi-bin/mango/index.cgi) which is a valuable genomic resource for molecular research into the biology of mango fruit was created. RNA-Seq analysis suggested that the pathway leading to biosynthesis of the cuticle component, cutin, is up-regulated during overripening. This data was supported by analysis of the expression of several putative cuticle-associated genes and by gravimetric and microscopic studies of cuticle deposition, revealing a complex continuous pattern of cuticle deposition during fruit development and involving substantial accumulation during ripening/overripening.

Ups and downs of a transcriptional landscape shape iron deficiency associated chlorosis of the maize inbreds B73 and Mo17

PubMed Central

2013-01-01

Background Improving nutrient homeostasis is a major challenge of a sustainable maize cultivation, and cornerstone to ensure food supply for a growing world population. Although, iron constitutes an important nutrient, iron availability is limited. In this respect, iron deficiency associated chlorosis causes severe yield losses every year. Natural variation of the latter trait has yet not been addressed in maize and was therefore studied in the present analysis. Results In this study, we i) report about the contrasting chlorosis phenotypes of the inbreds B73 and Mo17 at 10 and 300 μM iron regime, ii) identified over 400 significantly regulated transcripts (FDR < 0.05) within both inbreds at these growth conditions by deep RNA-Sequencing, iii) linked the gained knowledge with QTL information about iron deficiency related traits within the maize intermated B73 by Mo17 (IBM) population, and iv) highlighted contributing molecular pathways. In this respect, several genes within methionine salvage pathway and phytosiderophore synthesis were found to present constitutively high expression in Mo17, even under sufficient iron supply. Moreover, the same expression pattern could be observed for two putative bHLH transcription factors. In addition, a number of differentially expressed genes showed a co-localisation with QTL confidence intervals for iron deficiency related traits within the IBM population. Conclusions Our study highlights differential iron deficiency associated chlorosis between B73 and Mo17 and represents a valuable resource for differentially expressed genes upon iron limitation and chlorosis response. Besides identifying two putative bHLH transcription factors, we propose that methionine salvage pathway and sterol metabolism amongst others; underlie the contrasting iron deficiency related chlorosis phenotype of both inbreds. Altogether, this study emphasizes a contribution of selected genes and pathways on natural trait variation within the IBM population. PMID:24330725

Pathways to Aggression in Urban Elementary School Youth

ERIC Educational Resources Information Center

Ozkol, Hivren; Zucker, Marla; Spinazzola, Joseph

2011-01-01

This study examined the pathways from violence exposure to aggressive behaviors in urban, elementary school youth. We utilized structural equation modeling to examine putative causal pathways between children's exposure to violence, development of posttraumatic stress symptoms, permissive attitudes towards violence, and engagement in aggressive…

Inhibin B and anti-Müllerian hormone/Müllerian-inhibiting substance may contribute to the male bias in autism.

PubMed

Pankhurst, M W; McLennan, I S

2012-08-14

The autistic spectrum disorders have a significant male bias in incidence, which is unexplained. The Sertoli cells of the immature testes secrete supra-adult levels of Müllerian-inhibiting substance/anti-Müllerian hormone (AMH) and inhibin B (InhB), with both hormones being putative regulators of brain development. We report here, that 82 boys with an autism spectrum disorder have normal levels of InhB and AMH. However, the boys' level of InhB correlated with their autism diagnostic interview-revised (ADI-R) scores for the social interaction (R=0.29, P=0.009, N=82) and communication domains (R=0.29, P=0.022, N=63), and with the number of autistic traits the boys exhibited (R=0.34 and 0.27, respectively). The strengths of the abovementioned correlates were stronger in the boys with milder autism (R=0.42 and 0.50, respectively), with AMH exhibiting a significant negative correlation to the ADI-R score in these boys (R=-0.44 and R=-0.39, respectively). Neither hormone correlated to the incidence of stereotyped and repetitive behaviours. This suggests that the male bias in the autistic spectrum has multiple determinants, which modulate the effects of an otherwise non-dimorphic pathology. Furthermore, AMH and InhB have opposing effects on the SMAD1/5/8 pathway, and opposing correlates to autistic traits, implicating the SMAD pathways as a putative point of molecular convergence for the autistic spectrum.

Mating type gene homologues and putative sex pheromone-sensing pathway in arbuscular mycorrhizal fungi, a presumably asexual plant root symbiont.

PubMed

Halary, Sébastien; Daubois, Laurence; Terrat, Yves; Ellenberger, Sabrina; Wöstemeyer, Johannes; Hijri, Mohamed

2013-01-01

The fungal kingdom displays a fascinating diversity of sex-determination systems. Recent advances in genomics provide insights into the molecular mechanisms of sex, mating type determination, and evolution of sexual reproduction in many fungal species in both ancient and modern phylogenetic lineages. All major fungal groups have evolved sexual differentiation and recombination pathways. However, sexuality is unknown in arbuscular mycorrhizal fungi (AMF) of the phylum Glomeromycota, an ecologically vital group of obligate plant root symbionts. AMF are commonly considered an ancient asexual lineage dating back to the Ordovician, approximately 460 M years ago. In this study, we used genomic and transcriptomic surveys of several AMF species to demonstrate the presence of conserved putative sex pheromone-sensing mitogen-activated protein (MAP) kinases, comparable to those described in Ascomycota and Basidiomycota. We also find genes for high mobility group (HMG) transcription factors, homologous to SexM and SexP genes in the Mucorales. The SexM genes show a remarkable sequence diversity among multiple copies in the genome, while only a single SexP sequence was detected in some isolates of Rhizophagus irregularis. In the Mucorales and Microsporidia, the sexM gene is flanked by genes for a triosephosphate transporter (TPT) and a RNA helicase, but we find no evidence for synteny in the vicinity of the Sex locus in AMF. Nonetheless, our results, together with previous observations on meiotic machinery, suggest that AMF could undergo a complete sexual reproduction cycle.

Transcriptome Analysis of Leaves, Flowers and Fruits Perisperm of Coffea arabica L. Reveals the Differential Expression of Genes Involved in Raffinose Biosynthesis

PubMed Central

dos Santos, Tiago Benedito; de Oliveira, Fernanda Freitas; Pot, David; Leroy, Thierry; Vieira, Luiz Gonzaga Esteves; Carazzolle, Marcelo Falsarella; Pereira, Gonçalo Amarante Guimarães

2017-01-01

Coffea arabica L. is an important crop in several developing countries. Despite its economic importance, minimal transcriptome data are available for fruit tissues, especially during fruit development where several compounds related to coffee quality are produced. To understand the molecular aspects related to coffee fruit and grain development, we report a large-scale transcriptome analysis of leaf, flower and perisperm fruit tissue development. Illumina sequencing yielded 41,881,572 high-quality filtered reads. De novo assembly generated 65,364 unigenes with an average length of 1,264 bp. A total of 24,548 unigenes were annotated as protein coding genes, including 12,560 full-length sequences. In the annotation process, we identified nine candidate genes related to the biosynthesis of raffinose family oligossacarides (RFOs). These sugars confer osmoprotection and are accumulated during initial fruit development. Four genes from this pathway had their transcriptional pattern validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, we identified ~24,000 putative target sites for microRNAs (miRNAs) and 134 putative transcriptionally active transposable elements (TE) sequences in our dataset. This C. arabica transcriptomic atlas provides an important step for identifying candidate genes related to several coffee metabolic pathways, especially those related to fruit chemical composition and therefore beverage quality. Our results are the starting point for enhancing our knowledge about the coffee genes that are transcribed during the flowering and initial fruit development stages. PMID:28068432

Transcriptome Analysis of Leaves, Flowers and Fruits Perisperm of Coffea arabica L. Reveals the Differential Expression of Genes Involved in Raffinose Biosynthesis.

PubMed

Ivamoto, Suzana Tiemi; Reis, Osvaldo; Domingues, Douglas Silva; Dos Santos, Tiago Benedito; de Oliveira, Fernanda Freitas; Pot, David; Leroy, Thierry; Vieira, Luiz Gonzaga Esteves; Carazzolle, Marcelo Falsarella; Pereira, Gonçalo Amarante Guimarães; Pereira, Luiz Filipe Protasio

2017-01-01

Coffea arabica L. is an important crop in several developing countries. Despite its economic importance, minimal transcriptome data are available for fruit tissues, especially during fruit development where several compounds related to coffee quality are produced. To understand the molecular aspects related to coffee fruit and grain development, we report a large-scale transcriptome analysis of leaf, flower and perisperm fruit tissue development. Illumina sequencing yielded 41,881,572 high-quality filtered reads. De novo assembly generated 65,364 unigenes with an average length of 1,264 bp. A total of 24,548 unigenes were annotated as protein coding genes, including 12,560 full-length sequences. In the annotation process, we identified nine candidate genes related to the biosynthesis of raffinose family oligossacarides (RFOs). These sugars confer osmoprotection and are accumulated during initial fruit development. Four genes from this pathway had their transcriptional pattern validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, we identified ~24,000 putative target sites for microRNAs (miRNAs) and 134 putative transcriptionally active transposable elements (TE) sequences in our dataset. This C. arabica transcriptomic atlas provides an important step for identifying candidate genes related to several coffee metabolic pathways, especially those related to fruit chemical composition and therefore beverage quality. Our results are the starting point for enhancing our knowledge about the coffee genes that are transcribed during the flowering and initial fruit development stages.

Genome-wide identification, classification, and functional analysis of the basic helix-loop-helix transcription factors in the cattle, Bos Taurus.

PubMed

Li, Fengmei; Liu, Wuyi

2017-06-01

The basic helix-loop-helix (bHLH) transcription factors (TFs) form a huge superfamily and play crucial roles in many essential developmental, genetic, and physiological-biochemical processes of eukaryotes. In total, 109 putative bHLH TFs were identified and categorized successfully in the genomic databases of cattle, Bos Taurus, after removing redundant sequences and merging genetic isoforms. Through phylogenetic analyses, 105 proteins among these bHLH TFs were classified into 44 families with 46, 25, 14, 3, 13, and 4 members in the high-order groups A, B, C, D, E, and F, respectively. The remaining 4 bHLH proteins were sorted out as 'orphans.' Next, these 109 putative bHLH proteins identified were further characterized as significantly enriched in 524 significant Gene Ontology (GO) annotations (corrected P value ≤ 0.05) and 21 significantly enriched pathways (corrected P value ≤ 0.05) that had been mapped by the web server KOBAS 2.0. Furthermore, 95 bHLH proteins were further screened and analyzed together with two uncharacterized proteins in the STRING online database to reconstruct the protein-protein interaction network of cattle bHLH TFs. Ultimately, 89 bHLH proteins were fully mapped in a network with 67 biological process, 13 molecular functions, 5 KEGG pathways, 12 PFAM protein domains, and 25 INTERPRO classified protein domains and features. These results provide much useful information and a good reference for further functional investigations and updated researches on cattle bHLH TFs.

Bifidobacterium breve C50 secretes lipoprotein with CHAP domain recognized in aggregated form by TLR2.

PubMed

Scuotto, Angelo; Djorie, Serge; Colavizza, Michel; Romond, Pierre-Charles; Romond, Marie-Bénédicte

2014-12-01

Extracellular components secreted by Bifidobacterium breve C50 can induce maturation, high IL-10 production and prolonged survival of dendritic cells via a TLR2 pathway. In this study, the components were isolated from the supernatant by gel filtration chromatography. Antibodies raised against the major compounds with molecular weight above 600 kDa (Bb C50BC) also recognized compounds of lower molecular weight (200–600 kDa). TLR2 and TLR6 bound to the components already recognized by the antibodies. Trypsin digestion of Bb C50BC released three major peptides whose sequences displayed close similarities to a putative secreted protein with a CHAP amidase domain from B. breve. The 1300-bp genomic region corresponding to the hypothetical protein was amplified by PCR. The deduced polypeptide started with an N-terminal signal sequence of 45 amino acids, containing the lipobox motif (LAAC) with the cysteine in position 25, and 2 positively charged residues within the first 14 residues of the signal sequence. Lipid detection in Bb C50BC by GC/MS further supported the implication of a lipoprotein. Sugars were also detected in Bb C50BC. Close similarity with the glucan-binding protein B from Bifidobacterium animalis of two released peptides from Bb C50BC protein suggested that glucose moieties, possibly in glucan form, could be bound to the lipoprotein. Finally, heating at 100 °C for 5 min led to the breakdown of Bb C50BC in compounds of molecular weight below 67 kDa, which suggested that Bb C50BC was an aggregate. One might assume that a basic unit was formed by the lipoprotein bound putatively to glucan. Besides the other sugars and hexosamines recognized by galectin 1 were localized at the surface of the Bb C50BC aggregate. In conclusion, the extracellular components secreted by B. breve C50 were constituted of a lipoprotein putatively associated with glucose moieties and acting in an aggregating form as an agonist of TLR2/TLR6.

In silico analysis of the fucosylation-associated genome of the human blood fluke Schistosoma mansoni: cloning and characterization of the enzymes involved in GDP-L-fucose synthesis and Golgi import

PubMed Central

2013-01-01

Background Carbohydrate structures of surface-expressed and secreted/excreted glycoconjugates of the human blood fluke Schistosoma mansoni are key determinants that mediate host-parasite interactions in both snail and mammalian hosts. Fucose is a major constituent of these immunologically important glycans, and recent studies have sought to characterize fucosylation-associated enzymes, including the Golgi-localized fucosyltransferases that catalyze the transfer of L-fucose from a GDP-L-fucose donor to an oligosaccharide acceptor. Importantly, GDP-L-fucose is the only nucleotide-sugar donor used by fucosyltransferases and its availability represents a bottleneck in fucosyl-glycotope expression. Methods A homology-based genome-wide bioinformatics approach was used to identify and molecularly characterize the enzymes that contribute to GDP-L-fucose synthesis and Golgi import in S. mansoni. Putative functions were further investigated through molecular phylogenetic and immunocytochemical analyses. Results We identified homologs of GDP-D-mannose-4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase (GMER), which constitute a de novo pathway for GDP-L-fucose synthesis, in addition to a GDP-L-fucose transporter (GFT) that putatively imports cytosolic GDP-L-fucose into the Golgi. In silico primary sequence analyses identified characteristic Rossman loop and short-chain dehydrogenase/reductase motifs in GMD and GMER as well as 10 transmembrane domains in GFT. All genes are alternatively spliced, generating variants of unknown function. Observed quantitative differences in steady-state transcript levels between miracidia and primary sporocysts may contribute to differential glycotope expression in early larval development. Additionally, analyses of protein expression suggest the occurrence of cytosolic GMD and GMER in the ciliated epidermal plates and tegument of miracidia and primary sporocysts, respectively, which is consistent with previous localization of highly fucosylated glycotopes. Conclusions This study is the first to identify and characterize three key genes that are putatively involved in the synthesis and Golgi import of GDP-L-fucose in S. mansoni and provides fundamental information regarding their genomic organization, genetic variation, molecular phylogenetics, and developmental expression in intramolluscan larval stages. PMID:23835114

In silico analysis of the fucosylation-associated genome of the human blood fluke Schistosoma mansoni: cloning and characterization of the enzymes involved in GDP-L-fucose synthesis and Golgi import.

PubMed

Peterson, Nathan A; Anderson, Tavis K; Wu, Xiao-Jun; Yoshino, Timothy P

2013-07-09

Carbohydrate structures of surface-expressed and secreted/excreted glycoconjugates of the human blood fluke Schistosoma mansoni are key determinants that mediate host-parasite interactions in both snail and mammalian hosts. Fucose is a major constituent of these immunologically important glycans, and recent studies have sought to characterize fucosylation-associated enzymes, including the Golgi-localized fucosyltransferases that catalyze the transfer of L-fucose from a GDP-L-fucose donor to an oligosaccharide acceptor. Importantly, GDP-L-fucose is the only nucleotide-sugar donor used by fucosyltransferases and its availability represents a bottleneck in fucosyl-glycotope expression. A homology-based genome-wide bioinformatics approach was used to identify and molecularly characterize the enzymes that contribute to GDP-L-fucose synthesis and Golgi import in S. mansoni. Putative functions were further investigated through molecular phylogenetic and immunocytochemical analyses. We identified homologs of GDP-D-mannose-4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase (GMER), which constitute a de novo pathway for GDP-L-fucose synthesis, in addition to a GDP-L-fucose transporter (GFT) that putatively imports cytosolic GDP-L-fucose into the Golgi. In silico primary sequence analyses identified characteristic Rossman loop and short-chain dehydrogenase/reductase motifs in GMD and GMER as well as 10 transmembrane domains in GFT. All genes are alternatively spliced, generating variants of unknown function. Observed quantitative differences in steady-state transcript levels between miracidia and primary sporocysts may contribute to differential glycotope expression in early larval development. Additionally, analyses of protein expression suggest the occurrence of cytosolic GMD and GMER in the ciliated epidermal plates and tegument of miracidia and primary sporocysts, respectively, which is consistent with previous localization of highly fucosylated glycotopes. This study is the first to identify and characterize three key genes that are putatively involved in the synthesis and Golgi import of GDP-L-fucose in S. mansoni and provides fundamental information regarding their genomic organization, genetic variation, molecular phylogenetics, and developmental expression in intramolluscan larval stages.

De novo Assembly of the Indo-Pacific Humpback Dolphin Leucocyte Transcriptome to Identify Putative Genes Involved in the Aquatic Adaptation and Immune Response

PubMed Central

Xia, Jia; Yang, Lili; Chen, Jialin; Wu, Yuping; Yi, Meisheng

2013-01-01

Background The Indo-Pacific humpback dolphin (Sousa chinensis), a marine mammal species inhabited in the waters of Southeast Asia, South Africa and Australia, has attracted much attention because of the dramatic decline in population size in the past decades, which raises the concern of extinction. So far, this species is poorly characterized at molecular level due to little sequence information available in public databases. Recent advances in large-scale RNA sequencing provide an efficient approach to generate abundant sequences for functional genomic analyses in the species with un-sequenced genomes. Principal Findings We performed a de novo assembly of the Indo-Pacific humpback dolphin leucocyte transcriptome by Illumina sequencing. 108,751 high quality sequences from 47,840,388 paired-end reads were generated, and 48,868 and 46,587 unigenes were functionally annotated by BLAST search against the NCBI non-redundant and Swiss-Prot protein databases (E-value<10−5), respectively. In total, 16,467 unigenes were clustered into 25 functional categories by searching against the COG database, and BLAST2GO search assigned 37,976 unigenes to 61 GO terms. In addition, 36,345 unigenes were grouped into 258 KEGG pathways. We also identified 9,906 simple sequence repeats and 3,681 putative single nucleotide polymorphisms as potential molecular markers in our assembled sequences. A large number of unigenes were predicted to be involved in immune response, and many genes were predicted to be relevant to adaptive evolution and cetacean-specific traits. Conclusion This study represented the first transcriptome analysis of the Indo-Pacific humpback dolphin, an endangered species. The de novo transcriptome analysis of the unique transcripts will provide valuable sequence information for discovery of new genes, characterization of gene expression, investigation of various pathways and adaptive evolution, as well as identification of genetic markers. PMID:24015242

De novo assembly of the Indo-Pacific humpback dolphin leucocyte transcriptome to identify putative genes involved in the aquatic adaptation and immune response.

PubMed

Gui, Duan; Jia, Kuntong; Xia, Jia; Yang, Lili; Chen, Jialin; Wu, Yuping; Yi, Meisheng

2013-01-01

The Indo-Pacific humpback dolphin (Sousa chinensis), a marine mammal species inhabited in the waters of Southeast Asia, South Africa and Australia, has attracted much attention because of the dramatic decline in population size in the past decades, which raises the concern of extinction. So far, this species is poorly characterized at molecular level due to little sequence information available in public databases. Recent advances in large-scale RNA sequencing provide an efficient approach to generate abundant sequences for functional genomic analyses in the species with un-sequenced genomes. We performed a de novo assembly of the Indo-Pacific humpback dolphin leucocyte transcriptome by Illumina sequencing. 108,751 high quality sequences from 47,840,388 paired-end reads were generated, and 48,868 and 46,587 unigenes were functionally annotated by BLAST search against the NCBI non-redundant and Swiss-Prot protein databases (E-value<10(-5)), respectively. In total, 16,467 unigenes were clustered into 25 functional categories by searching against the COG database, and BLAST2GO search assigned 37,976 unigenes to 61 GO terms. In addition, 36,345 unigenes were grouped into 258 KEGG pathways. We also identified 9,906 simple sequence repeats and 3,681 putative single nucleotide polymorphisms as potential molecular markers in our assembled sequences. A large number of unigenes were predicted to be involved in immune response, and many genes were predicted to be relevant to adaptive evolution and cetacean-specific traits. This study represented the first transcriptome analysis of the Indo-Pacific humpback dolphin, an endangered species. The de novo transcriptome analysis of the unique transcripts will provide valuable sequence information for discovery of new genes, characterization of gene expression, investigation of various pathways and adaptive evolution, as well as identification of genetic markers.

Fault tolerance in protein interaction networks: stable bipartite subgraphs and redundant pathways.

PubMed

Brady, Arthur; Maxwell, Kyle; Daniels, Noah; Cowen, Lenore J

2009-01-01

As increasing amounts of high-throughput data for the yeast interactome become available, more system-wide properties are uncovered. One interesting question concerns the fault tolerance of protein interaction networks: whether there exist alternative pathways that can perform some required function if a gene essential to the main mechanism is defective, absent or suppressed. A signature pattern for redundant pathways is the BPM (between-pathway model) motif, introduced by Kelley and Ideker. Past methods proposed to search the yeast interactome for BPM motifs have had several important limitations. First, they have been driven heuristically by local greedy searches, which can lead to the inclusion of extra genes that may not belong in the motif; second, they have been validated solely by functional coherence of the putative pathways using GO enrichment, making it difficult to evaluate putative BPMs in the absence of already known biological annotation. We introduce stable bipartite subgraphs, and show they form a clean and efficient way of generating meaningful BPMs which naturally discard extra genes included by local greedy methods. We show by GO enrichment measures that our BPM set outperforms previous work, covering more known complexes and functional pathways. Perhaps most importantly, since our BPMs are initially generated by examining the genetic-interaction network only, the location of edges in the protein-protein physical interaction network can then be used to statistically validate each candidate BPM, even with sparse GO annotation (or none at all). We uncover some interesting biological examples of previously unknown putative redundant pathways in such areas as vesicle-mediated transport and DNA repair.

Fault Tolerance in Protein Interaction Networks: Stable Bipartite Subgraphs and Redundant Pathways

PubMed Central

Brady, Arthur; Maxwell, Kyle; Daniels, Noah; Cowen, Lenore J.

2009-01-01

As increasing amounts of high-throughput data for the yeast interactome become available, more system-wide properties are uncovered. One interesting question concerns the fault tolerance of protein interaction networks: whether there exist alternative pathways that can perform some required function if a gene essential to the main mechanism is defective, absent or suppressed. A signature pattern for redundant pathways is the BPM (between-pathway model) motif, introduced by Kelley and Ideker. Past methods proposed to search the yeast interactome for BPM motifs have had several important limitations. First, they have been driven heuristically by local greedy searches, which can lead to the inclusion of extra genes that may not belong in the motif; second, they have been validated solely by functional coherence of the putative pathways using GO enrichment, making it difficult to evaluate putative BPMs in the absence of already known biological annotation. We introduce stable bipartite subgraphs, and show they form a clean and efficient way of generating meaningful BPMs which naturally discard extra genes included by local greedy methods. We show by GO enrichment measures that our BPM set outperforms previous work, covering more known complexes and functional pathways. Perhaps most importantly, since our BPMs are initially generated by examining the genetic-interaction network only, the location of edges in the protein-protein physical interaction network can then be used to statistically validate each candidate BPM, even with sparse GO annotation (or none at all). We uncover some interesting biological examples of previously unknown putative redundant pathways in such areas as vesicle-mediated transport and DNA repair. PMID:19399174

The CCDC55 couples cannabinoid receptor CNR1 to a putative DISC1 schizophrenia pathway.

PubMed

Xie, J; Gizatullin, R; Vukojevic, V; Leopardi, R

2015-12-03

Our previous study suggested that the coiled coil domain-containing 55 gene (CCDC55), also named as NSRP1 (nuclear speckle splicing regulatory protein 1 (NSRP1)), was encompassed in a haplotype block spanning over the serotonin transporter (5-HTT) gene in patients with schizophrenia (SCZ). However, the neurobiological function of CCDC55 gene remains unknown. This study aims to uncover the potential role of CCDC55 in SCZ-associated molecular pathways. Using molecular cloning, sequencing and immune blotting to identify basic properties, yeast two-hybrid screening and glutathione S-transferase (GST) pull-down assay to test protein-protein interaction, and confocal laser scanning microscopy (CSLM) to show intracellular interaction of proteins. (i) CCDC55 is expressed as a nuclear protein in human neuronal cells; (ii) Protein-protein interaction analyses showed CCDC55 physically interacted with Ran binding protein 9 (RanBP9) and disrupted in schizophrenia 1 (DISC1); (iii) CCDC55 and RanBP9 co-localized in the nucleus of human neuronal cells; (iv) CCDC55 also interacted with the cannabinoid receptor 1 (CNR1), and with the brain cannabinoid receptor-interacting protein 1a (CNRIP1a); (v) CNR1 activation in differentiated human neuronal cells resulted in an altered RanBP9 localization. CCDC55 may be involved in a functional bridging between the CNR1 activation and the DISC1/RanBP9-associated pathways. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Deep Sequencing-Based Analysis of the Cymbidium ensifolium Floral Transcriptome

PubMed Central

Li, Xiaobai; Luo, Jie; Yan, Tianlian; Xiang, Lin; Jin, Feng; Qin, Dehui; Sun, Chongbo; Xie, Ming

2013-01-01

Cymbidium ensifolium is a Chinese Cymbidium with an elegant shape, beautiful appearance, and a fragrant aroma. C. ensifolium has a long history of cultivation in China and it has excellent commercial value as a potted plant and cut flower. The development of C. ensifolium genomic resources has been delayed because of its large genome size. Taking advantage of technical and cost improvement of RNA-Seq, we extracted total mRNA from flower buds and mature flowers and obtained a total of 9.52 Gb of filtered nucleotides comprising 98,819,349 filtered reads. The filtered reads were assembled into 101,423 isotigs, representing 51,696 genes. Of the 101,423 isotigs, 41,873 were putative homologs of annotated sequences in the public databases, of which 158 were associated with floral development and 119 were associated with flowering. The isotigs were categorized according to their putative functions. In total, 10,212 of the isotigs were assigned into 25 eukaryotic orthologous groups (KOGs), 41,690 into 58 gene ontology (GO) terms, and 9,830 into 126 Arabidopsis Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and 9,539 isotigs into 123 rice pathways. Comparison of the isotigs with those of the two related orchid species P. equestris and C. sinense showed that 17,906 isotigs are unique to C. ensifolium. In addition, a total of 7,936 SSRs and 16,676 putative SNPs were identified. To our knowledge, this transcriptome database is the first major genomic resource for C. ensifolium and the most comprehensive transcriptomic resource for genus Cymbidium. These sequences provide valuable information for understanding the molecular mechanisms of floral development and flowering. Sequences predicted to be unique to C. ensifolium would provide more insights into C. ensifolium gene diversity. The numerous SNPs and SSRs identified in the present study will contribute to marker development for C. ensifolium. PMID:24392013

Serum miRNAs Signature Plays an Important Role in Keloid Disease.

PubMed

Luan, Y; Liu, Y; Liu, C; Lin, Q; He, F; Dong, X; Xiao, Z

2016-01-01

The molecular mechanism underlying the pathogenesis of keloid is largely unknown. MicroRNA (miRNA) is a class of small regulatory RNA that has emerged as a group of posttranscriptional gene repressors, participating in diverse pathophysiological processes of skin diseases. We investigated the expression profiles of miRNAs in the sera of patients to decipher the complicated factors involved in the development of keloid disease. MiRNA expression profiling in the sera from 9 keloid patients and 7 normal controls were characterized using a miRNA microarray containing established human mature and precursor miRNA sequences. Quantitative real-time PCR was performed to confirm the expression of miRNAs. The putative targets of differentially expressed miRNAs were functionally annotated by bioinformatics. MiRNA microarray analysis identified 37 differentially expressed miRNAs (17 upregulated and 20 downregulated) in keloid patients, compared to the healthy controls. Functional annotations revealed that the targets of those differentially expressed miRNAs were enriched in signaling pathways essential for scar formation and wound healing. The expression profiling of miRNAs is altered in the keloid, providing a clue for the molecular mechanisms underlying its initiation and progression. MiRNAs may partly contribute to the etiology of keloids by affecting the critical signaling pathways relevant to keloid pathogenesis.

An executioner caspase regulates autophagy.

PubMed

Hou, Y C Claire; Hannigan, Adrienne M; Gorski, Sharon M

2009-05-01

The relationships between autophagy and cell death are complex and still not well understood. To advance our understanding of the molecular connections between autophagy and apoptosis, we performed an RNAi-based screen of Drosophila melanogaster apoptosis-related genes for their ability to enhance or suppress starvation-induced autophagy. We discovered that six apoptosis-related genes, Dcp-1, hid, Bruce, buffy, debcl and p53 as well as Ras/Raf/MAPK signaling pathway components play a role in autophagy regulation in Drosophila cultured cells. Our study also provides the first in vivo evidence that the effector caspase Dcp-1 and IAP protein Bruce regulate both autophagy and starvation-induced cell death at two nutrient status checkpoints, germarium and mid-oogenesis, in the Drosophila ovary. Analysis of degenerating mid-stage egg chambers in DmAtg1 and DmAtg7 mutants reveal a reduction in TUNEL staining though DNA condensation appears unaffected. Based on these and previous findings, we propose here a putative molecular pathway that might regulate the sensitivity threshold of apoptotic and autophagic responses. We also discuss multiple interpretations of the Atg mutant egg chamber TUNEL phenotype that are consistent with a possible role for autophagy in either suppressing or enhancing the efficiency of cell degradation and/or promoting cell clearance associated with the death process.

High-throughput SNP discovery and transcriptome expression profiles from the salmon louse Caligus rogercresseyi (Copepoda: Caligidae).

PubMed

Nuñez-Acuña, Gustavo; Valenzuela-Muñoz, Valentina; Gallardo-Escárate, Cristian

2014-06-01

The salmon louse Caligus rogercresseyi is the dominant ectoparasite species affecting the salmon aquaculture industry in the Southern hemisphere, and it is currently the main cause for economic losses in Chilean aquaculture. However, despite the great concern over Caligus infestations, genomic information on this louse is still scarce, even while the need to develop high-resolution molecular markers is growing. This study provides the first deep transcriptome survey to identify thousands of SNP markers from C. rogercresseyi, with a total of 69,466 SNPs identified using the MiSeq platform (Illumina®), 30,605 (52%) of which were found in contigs successfully annotated against known protein databases. Furthermore, in silico gene expression profiles associated with SNP variants were evaluated, and the results evidenced a wide array of genes that were down- and upregulated throughout the developmental stages of C. rogercresseyi. Interestingly, putative KEGG pathways involved in resistance to antiparasitic agents were also identified, where ten pathways were associated with the nervous system and one was related to ABC transporters. Taken together, this information could be highly useful for investigating the molecular underpinnings involved in the susceptibility or resistance of salmon lice to chemical treatments. Copyright © 2014 Elsevier Inc. All rights reserved.

Analysis of Altered Micro RNA Expression Profiles in Focal Cortical Dysplasia IIB.

PubMed

Li, Lin; Liu, Chang-Qing; Li, Tian-Fu; Guan, Yu-Guang; Zhou, Jian; Qi, Xue-Ling; Yang, Yu-Tao; Deng, Jia-Hui; Xu, Zhi-Qing David; Luan, Guo-Ming

2016-04-01

Focal cortical dysplasia type IIB is a commonly encountered subtype of developmental malformation of the cerebral cortex and is often associated with pharmacoresistant epilepsy. In this study, to investigate the molecular etiology of focal cortical dysplasia type IIB, the authors performed micro ribonucleic acid (RNA) microarray on surgical specimens from 5 children (2 female and 3 male, mean age was 73.4 months, range 50-112 months) diagnosed of focal cortical dysplasia type IIB and matched normal tissue adjacent to the lesion. In all, 24 micro RNAs were differentially expressed in focal cortical dysplasia type IIB, and the microarray results were validated using quantitative real-time polymerase chain reaction (PCR). Then the putative target genes of the differentially expressed micro RNAs were identified by bioinformatics analysis. Moreover, biological significance of the target genes was evaluated by investigating the pathways in which the genes were enriched, and the Hippo signaling pathway was proposed to be highly related with the pathogenesis of focal cortical dysplasia type IIB. © The Author(s) 2015.

A catalog of putative adverse outcome pathways (AOPs) that ...

EPA Pesticide Factsheets

A number of putative AOPs for several distinct MIEs of thyroid disruption have been formulated for amphibian metamorphosis and fish swim bladder inflation. These have been entered into the AOP knowledgebase on the OECD WIKI. The EDSP has been actively advancing high-throughput screening for chemical activity toward estrogen, androgen and thyroid targets. However, it has been recently identified that coverage for thyroid-related targets is lagging behind estrogen and androgen assay coverage. As thyroid-related medium-high throughput assays are actively being developed for inclusion in the ToxCast chemical screening program, a parallel effort is underway to characterize putative adverse outcome pathways (AOPs) specific to these thyroid-related targets. This effort is intended to provide biological and ecological context that will enhance the utility of ToxCast high throughput screening data for hazard identification.

Informatics approaches in the Biological Characterization of ...

EPA Pesticide Factsheets

Adverse Outcome Pathways (AOPs) are a conceptual framework to characterize toxicity pathways by a series of mechanistic steps from a molecular initiating event to population outcomes. This framework helps to direct risk assessment research, for example by aiding in computational prioritization of chemicals, genes, and tissues relevant to an adverse health outcome. We have designed and implemented a computational workflow to access a wealth of public data relating genes, chemicals, diseases, pathways, and species, to provide a biological context for putative AOPs. We selected three AOP case studies: ER/Aromatase Antagonism Leading to Reproductive Dysfunction, AHR1 Activation Leading to Cardiotoxicity, and AChE Inhibition Leading to Acute Mortality, and deduced a taxonomic range of applicability for each AOP. We developed computational tools to automatically access and analyze the pathway activity of AOP-relevant protein orthologs, finding broad similarity among vertebrate species for the ER/Aromatase and AHR1 AOPs, and similarity extending to invertebrate animal species for AChE inhibition. Additionally, we used public gene expression data to find groups of highly co-expressed genes, and compared those groups across organisms. To interpret these findings at a higher level of biological organization, we created the AOPdb, a relational database that mines results from sources including NCBI, KEGG, Reactome, CTD, and OMIM. This multi-source database connects genes,

Integrating publicly-available data to generate computationally ...

EPA Pesticide Factsheets

The adverse outcome pathway (AOP) framework provides a way of organizing knowledge related to the key biological events that result in a particular health outcome. For the majority of environmental chemicals, the availability of curated pathways characterizing potential toxicity is limited. Methods are needed to assimilate large amounts of available molecular data and quickly generate putative AOPs for further testing and use in hazard assessment. A graph-based workflow was used to facilitate the integration of multiple data types to generate computationally-predicted (cp) AOPs. Edges between graph entities were identified through direct experimental or literature information or computationally inferred using frequent itemset mining. Data from the TG-GATEs and ToxCast programs were used to channel large-scale toxicogenomics information into a cpAOP network (cpAOPnet) of over 20,000 relationships describing connections between chemical treatments, phenotypes, and perturbed pathways measured by differential gene expression and high-throughput screening targets. Sub-networks of cpAOPs for a reference chemical (carbon tetrachloride, CCl4) and outcome (hepatic steatosis) were extracted using the network topology. Comparison of the cpAOP subnetworks to published mechanistic descriptions for both CCl4 toxicity and hepatic steatosis demonstrate that computational approaches can be used to replicate manually curated AOPs and identify pathway targets that lack genomic mar

Molecular cloning, structural analysis and expression of complement component Bf/C2 genes in the nurse shark, Ginglymostoma cirratum.

PubMed

Shin, Dong-Ho; Webb, Barbara; Nakao, Miki; Smith, Sylvia L

2007-01-01

Factor B and C2 are serine proteases that provide the catalytic subunits of C3 and C5 convertases of the alternative (AP) and classical (CP) complement pathways. Two Bf/C2 cDNAs, GcBf/C2-1 and -2 (previously referred to as nsBf/C2-A and nsBf/C2-B), were isolated from the nurse shark, Ginglymostoma cirratum. GcBf/C2-1 and -2 are 3364 and 3082bp in length and encode a leader peptide, three CCPs, one VWFA, the serine protease domain and have a putative factor D/C1s/MASP cleavage site. Southern blots show that there might be up to two Bf/C2-like genes for each of the two GcBf/C2 isoforms. GcBf/C2-1 and -2 are constitutively expressed, albeit at different levels, in all nine tissues examined. Expression in erythrocytes is a novel finding. Structural analysis has revealed that the localization of glycosylation sites in the SP domain of both putative proteins indicates that the molecular organization of the shark molecules is more like C2 than factor B. Phylogenetic analysis indicates that GcBf/C2-1 and -2 and TrscBf of Triakis scyllia (another shark species) originated from a common ancestor and share a remote ancestor with Bf and C2 of mammals and bony fish.

Identification of Putative Nuclear Receptors and Steroidogenic Enzymes in Murray-Darling Rainbowfish (Melanotaenia fluviatilis) Using RNA-Seq and De Novo Transcriptome Assembly.

PubMed

Bain, Peter A; Papanicolaou, Alexie; Kumar, Anupama

2015-01-01

Murray-Darling rainbowfish (Melanotaenia fluviatilis [Castelnau, 1878]; Atheriniformes: Melanotaeniidae) is a small-bodied teleost currently under development in Australasia as a test species for aquatic toxicological studies. To date, efforts towards the development of molecular biomarkers of contaminant exposure have been hindered by the lack of available sequence data. To address this, we sequenced messenger RNA from brain, liver and gonads of mature male and female fish and generated a high-quality draft transcriptome using a de novo assembly approach. 149,742 clusters of putative transcripts were obtained, encompassing 43,841 non-redundant protein-coding regions. Deduced amino acid sequences were annotated by functional inference based on similarity with sequences from manually curated protein sequence databases. The draft assembly contained protein-coding regions homologous to 95.7% of the complete cohort of predicted proteins from the taxonomically related species, Oryzias latipes (Japanese medaka). The mean length of rainbowfish protein-coding sequences relative to their medaka homologues was 92.1%, indicating that despite the limited number of tissues sampled a large proportion of the total expected number of protein-coding genes was captured in the study. Because of our interest in the effects of environmental contaminants on endocrine pathways, we manually curated subsets of coding regions for putative nuclear receptors and steroidogenic enzymes in the rainbowfish transcriptome, revealing 61 candidate nuclear receptors encompassing all known subfamilies, and 41 putative steroidogenic enzymes representing all major steroidogenic enzymes occurring in teleosts. The transcriptome presented here will be a valuable resource for researchers interested in biomarker development, protein structure and function, and contaminant-response genomics in Murray-Darling rainbowfish.

Aryl hydrocarbon receptor (AHR) is a potential tumour suppressor in pituitary adenomas.

PubMed

Formosa, R; Borg, J; Vassallo, J

2017-08-01

Pituitary adenomas (PA) represent the largest group of intracranial neoplasms and yet the molecular mechanisms driving this disease remain largely unknown. The aim of this study was to use a high-throughput screening method to identify molecular pathways that may be playing a significant and consistent role in PA. RNA profiling using microarrays on eight local PAs identified the aryl hydrocarbon receptor (AHR) signalling pathway as a key canonical pathway downregulated in all PA types. This was confirmed by real-time PCR in 31 tumours. The AHR has been shown to regulate cell cycle progression in various cell types; however, its role in pituitary tissue has never been investigated. In order to validate the role of AHR in PA behaviour, further functional studies were undertaken. Over-expression of AHR in GH3 cells revealed a tumour suppressor potential independent of exogenous ligand activation by benzo α-pyrene (BαP). Cell cycle analysis and quantitative PCR of cell cycle regulator genes revealed that both unstimulated and BαP-stimulated AHR reduced E2F-driven transcription and altered expression of cell cycle regulator genes, thus increasing the percentage of cells in G 0 /G 1 phase and slowing the proliferation rate of GH3 cells. Co-immunoprecipitation confirmed the interaction between AHR and retinoblastoma (Rb1) protein supporting this as a functional mechanism for the observed reduction. Endogenous Ahr reduction using silencing RNA confirmed the tumour suppressive function of the Ahr. These data support a mechanistic pathway for the putative tumour suppressive role of AHR specifically in PA, possibly through its role as a cell cycle co-regulator, even in the absence of exogenous ligands. © 2017 The authors.

Aryl hydrocarbon receptor (AHR) is a potential tumour suppressor in pituitary adenomas

PubMed Central

Formosa, R; Borg, J

2017-01-01

Pituitary adenomas (PA) represent the largest group of intracranial neoplasms and yet the molecular mechanisms driving this disease remain largely unknown. The aim of this study was to use a high-throughput screening method to identify molecular pathways that may be playing a significant and consistent role in PA. RNA profiling using microarrays on eight local PAs identified the aryl hydrocarbon receptor (AHR) signalling pathway as a key canonical pathway downregulated in all PA types. This was confirmed by real-time PCR in 31 tumours. The AHR has been shown to regulate cell cycle progression in various cell types; however, its role in pituitary tissue has never been investigated. In order to validate the role of AHR in PA behaviour, further functional studies were undertaken. Over-expression of AHR in GH3 cells revealed a tumour suppressor potential independent of exogenous ligand activation by benzo α-pyrene (BαP). Cell cycle analysis and quantitative PCR of cell cycle regulator genes revealed that both unstimulated and BαP-stimulated AHR reduced E2F-driven transcription and altered expression of cell cycle regulator genes, thus increasing the percentage of cells in G0/G1 phase and slowing the proliferation rate of GH3 cells. Co-immunoprecipitation confirmed the interaction between AHR and retinoblastoma (Rb1) protein supporting this as a functional mechanism for the observed reduction. Endogenous Ahr reduction using silencing RNA confirmed the tumour suppressive function of the Ahr. These data support a mechanistic pathway for the putative tumour suppressive role of AHR specifically in PA, possibly through its role as a cell cycle co-regulator, even in the absence of exogenous ligands. PMID:28649092

Transcriptome assembly and identification of genes and SNPs associated with growth traits in largemouth bass (Micropterus salmoides).

PubMed

Li, Shengjie; Liu, Hao; Bai, Junjie; Zhu, Xinping

2017-04-01

Growth is one of the most crucial economic traits of all aquaculture species, but the molecular mechanisms involved in growth of largemouth bass (Micropterus salmoides) are poorly understood. The objective of this study was to screen growth-related genes of M. salmoides by RNA sequencing and identify growth-related single-nucleotide polymorphism (SNP) markers through a growth association study. The muscle transcriptomes of fast- and slow-growing largemouth bass were obtained using the RNA-Seq technique. A total of 54,058,178 and 54,742,444 qualified Illumina read pairs were obtained for the fast-growing and slow-growing groups, respectively, giving rise to 4,865,236,020 and 4,926,819,960 total clean bases, respectively. Gene expression profiling showed that 3,530 unigenes were differentially expressed between the fast-growing and slow-growing phenotypes (false discovery rate ≤0.001, the absolute value of log 2 (fold change) ≥1), including 1,441 up-regulated and 2,889 down-regulated unigenes in the fast-growing largemouth bass. Analysis of these genes revealed that several signalling pathways, including the growth hormone-insulin-like growth factor 1 axis and signalling pathway, the glycolysis pathway, and the myostatin/transforming growth factor beta signalling pathway, as well as heat shock protein, cytoskeleton, and myofibril component genes might be associated with muscle growth. From these genes, 10 genes with putative SNPs were selected, and 17 SNPs were genotyped successfully. Marker-trait analysis in 340 individuals of Youlu No. 1 largemouth bass revealed three SNPs associated with growth in key genes (phosphoenolpyruvate carboxykinase 1, FOXO3b, and heat shock protein beta-1). This research provides information about key genes and SNPs related to growth, providing new clues to understanding the molecular basis of largemouth bass growth.

[Metabolic disturbance of tryptophan-nicotinamide conversion pathway by putative endocrine disruptors, bisphenol A and styrene monomer].

PubMed

Fukuwatari, Tsutomu; Toriochi, Mai; Ohta, Mari; Sasaki, Ryuzo; Shibata, Katsumi

2004-02-01

Bisphenol A, a monomer of polycarbonate plastics, disturbed the conversion pathway of the amino acid tryptophan to the vitamin nicotinamide. The conversion ratio of tryptophan to nicotinamide was reduced to 1/15 by feeding a diet containing 1% bisphenol A. A putative disturbing reaction is kynurenine-->3-hydroxykynurenine, which is catalyzed by kynurenine monohydroxylase. This is an FAD-enzyme and requires NADPH as a coenzyme. Styrene monomer (1% addition to a normal diet) did not affect the food intake or the body weight, but slightly reduced the conversion ratio of tryptophan-nicotinamide.

Evaluating between-pathway models with expression data.

PubMed

Hescott, B J; Leiserson, M D M; Cowen, L J; Slonim, D K

2010-03-01

Between-pathway models (BPMs) are network motifs consisting of pairs of putative redundant pathways. In this article, we show how adding another source of high-throughput data--microarray gene expression data from knockout experiments--allows us to identify a compensatory functional relationship between genes from the two BPM pathways. We evaluate the quality of the BPMs from four different studies, and we describe how our methods might be extended to refine pathways.

The prediction of a pathogenesis-related secretome of Puccinia helianthi through high-throughput transcriptome analysis.

PubMed

Jing, Lan; Guo, Dandan; Hu, Wenjie; Niu, Xiaofan

2017-03-11

Many plant pathogen secretory proteins are known to be elicitors or pathogenic factors,which play an important role in the host-pathogen interaction process. Bioinformatics approaches make possible the large scale prediction and analysis of secretory proteins from the Puccinia helianthi transcriptome. The internet-based software SignalP v4.1, TargetP v1.01, Big-PI predictor, TMHMM v2.0 and ProtComp v9.0 were utilized to predict the signal peptides and the signal peptide-dependent secreted proteins among the 35,286 ORFs of the P. helianthi transcriptome. 908 ORFs (accounting for 2.6% of the total proteins) were identified as putative secretory proteins containing signal peptides. The length of the majority of proteins ranged from 51 to 300 amino acids (aa), while the signal peptides were from 18 to 20 aa long. Signal peptidase I (SpI) cleavage sites were found in 463 of these putative secretory signal peptides. 55 proteins contained the lipoprotein signal peptide recognition site of signal peptidase II (SpII). Out of 908 secretory proteins, 581 (63.8%) have functions related to signal recognition and transduction, metabolism, transport and catabolism. Additionally, 143 putative secretory proteins were categorized into 27 functional groups based on Gene Ontology terms, including 14 groups in biological process, seven in cellular component, and six in molecular function. Gene ontology analysis of the secretory proteins revealed an enrichment of hydrolase activity. Pathway associations were established for 82 (9.0%) secretory proteins. A number of cell wall degrading enzymes and three homologous proteins specific to Phytophthora sojae effectors were also identified, which may be involved in the pathogenicity of the sunflower rust pathogen. This investigation proposes a new approach for identifying elicitors and pathogenic factors. The eventual identification and characterization of 908 extracellularly secreted proteins will advance our understanding of the molecular mechanisms of interactions between sunflower and rust pathogen and will enhance our ability to intervene in disease states.

Comparative transcriptome analysis of obligately asexual and cyclically sexual rotifers reveals genes with putative functions in sexual reproduction, dormancy, and asexual egg production.

PubMed

Hanson, Sara J; Stelzer, Claus-Peter; Welch, David B Mark; Logsdon, John M

2013-06-19

Sexual reproduction is a widely studied biological process because it is critically important to the genetics, evolution, and ecology of eukaryotes. Despite decades of study on this topic, no comprehensive explanation has been accepted that explains the evolutionary forces underlying its prevalence and persistence in nature. Monogonont rotifers offer a useful system for experimental studies relating to the evolution of sexual reproduction due to their rapid reproductive rate and close relationship to the putatively ancient asexual bdelloid rotifers. However, little is known about the molecular underpinnings of sex in any rotifer species. We generated mRNA-seq libraries for obligate parthenogenetic (OP) and cyclical parthenogenetic (CP) strains of the monogonont rotifer, Brachionus calyciflorus, to identify genes specific to both modes of reproduction. Our differential expression analysis identified receptors with putative roles in signaling pathways responsible for the transition from asexual to sexual reproduction. Differential expression of a specific copy of the duplicated cell cycle regulatory gene CDC20 and specific copies of histone H2A suggest that such duplications may underlie the phenotypic plasticity required for reproductive mode switch in monogononts. We further identified differential expression of genes involved in the formation of resting eggs, a process linked exclusively to sex in this species. Finally, we identified transcripts from the bdelloid rotifer Adineta ricciae that have significant sequence similarity to genes with higher expression in CP strains of B. calyciflorus. Our analysis of global gene expression differences between facultatively sexual and exclusively asexual populations of B. calyciflorus provides insights into the molecular nature of sexual reproduction in rotifers. Furthermore, our results offer insight into the evolution of obligate asexuality in bdelloid rotifers and provide indicators important for the use of monogononts as a model system for investigating the evolution of sexual reproduction.

The Diverse Forms of Lactose Intolerance and the Putative Linkage to Several Cancers

PubMed Central

Amiri, Mahdi; Diekmann, Lena; von Köckritz-Blickwede, Maren; Naim, Hassan Y.

2015-01-01

Lactase-phlorizin hydrolase (LPH) is a membrane glycoprotein and the only β-galactosidase of the brush border membrane of the intestinal epithelium. Besides active transcription, expression of the active LPH requires different maturation steps of the polypeptide through the secretory pathway, including N- and O-glycosylation, dimerization and proteolytic cleavage steps. The inability to digest lactose due to insufficient lactase activity results in gastrointestinal symptoms known as lactose intolerance. In this review, we will concentrate on the structural and functional features of LPH protein and summarize the cellular and molecular mechanism required for its maturation and trafficking. Then, different types of lactose intolerance are discussed, and the molecular aspects of lactase persistence/non-persistence phenotypes are investigated. Finally, we will review the literature focusing on the lactase persistence/non-persistence populations as a comparative model in order to determine the protective or adverse effects of milk and dairy foods on the incidence of colorectal, ovarian and prostate cancers. PMID:26343715

The Diverse Forms of Lactose Intolerance and the Putative Linkage to Several Cancers.

PubMed

Amiri, Mahdi; Diekmann, Lena; von Köckritz-Blickwede, Maren; Naim, Hassan Y

2015-08-28

Lactase-phlorizin hydrolase (LPH) is a membrane glycoprotein and the only β-galactosidase of the brush border membrane of the intestinal epithelium. Besides active transcription, expression of the active LPH requires different maturation steps of the polypeptide through the secretory pathway, including N- and O-glycosylation, dimerization and proteolytic cleavage steps. The inability to digest lactose due to insufficient lactase activity results in gastrointestinal symptoms known as lactose intolerance. In this review, we will concentrate on the structural and functional features of LPH protein and summarize the cellular and molecular mechanism required for its maturation and trafficking. Then, different types of lactose intolerance are discussed, and the molecular aspects of lactase persistence/non-persistence phenotypes are investigated. Finally, we will review the literature focusing on the lactase persistence/non-persistence populations as a comparative model in order to determine the protective or adverse effects of milk and dairy foods on the incidence of colorectal, ovarian and prostate cancers.

Aggressive behavior in transgenic animal models: A systematic review.

PubMed

Jager, Amanda; Maas, Dorien A; Fricke, Kim; de Vries, Rob B; Poelmans, Geert; Glennon, Jeffrey C

2018-08-01

Aggressive behavior is often core or comorbid to psychiatric and neurodegenerative disorders. Transgenic animal models are commonly used to study the neurobiological mechanisms underlying aggressive phenotypes and have led to new insights into aggression. This systematic review critically evaluates the available literature on transgenic animal models tested for aggression with the resident-intruder test. By combining the available literature on this topic, we sought to highlight effective methods for laboratory aggression testing and provide recommendations for study design as well as aggression induction and measurement in rodents that are translational to humans, taking into consideration possible confounding factors. In addition, we built a molecular landscape of interactions between the proteins encoded by the aggression-linked genes from our systematic search. Some molecular pathways within this landscape overlap with psychiatric and neurodegenerative disorders and the landscapes point towards a number of putative (drug) targets for aggression that need to be validated in future studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

Putative signaling action of amelogenin utilizes the Wnt/beta-catenin pathway.

PubMed

Matsuzawa, M; Sheu, T-J; Lee, Y-J; Chen, M; Li, T-F; Huang, C T; Holz, J D; Puzas, J E

2009-06-01

While it has long been known that amelogenin is essential for the proper development of enamel, its role has generally been seen as structural in nature. However, our new data implicate this protein in the regulation of cell signaling pathways in periodontal ligament cells and osteoblasts. In this article we report the successful purification of a recombinant mouse amelogenin protein and demonstrate that it has signaling activity in isolated mouse calvarial cells and human periodontal ligament cells. To determine the regulatory function of canonical Wnt signaling by amelogenin, we used TOPGAL transgenic mice. These mice express a beta-galactosidase transgene under the control of a LEF/TCF and beta-catenin-inducible promoter. To investigate in greater detail the molecular mechanisms involved in the beta-catenin signaling pathway, isolated osteoblasts and periodontal ligament cells were exposed to full-length recombinant mouse amelogenin and were evaluated for phenotypic changes and beta-catenin signaling using a TOPFLASH construct and the LacZ reporter gene. In these in vitro models, we showed that amelogenin can activate beta-catenin signaling. Using the TOPGAL transgenic mouse we showed that amelogenin expression in vivo is localized mainly around the root, the periodontal ligament and the alveolar bone.

Transcriptome analysis of Petunia axillaris flowers reveals genes involved in morphological differentiation and metabolite transport

PubMed Central

Amano, Ikuko; Kitajima, Sakihito; Suzuki, Hideyuki; Koeduka, Takao

2018-01-01

The biosynthesis of plant secondary metabolites is associated with morphological and metabolic differentiation. As a consequence, gene expression profiles can change drastically, and primary and secondary metabolites, including intermediate and end-products, move dynamically within and between cells. However, little is known about the molecular mechanisms underlying differentiation and transport mechanisms. In this study, we performed a transcriptome analysis of Petunia axillaris subsp. parodii, which produces various volatiles in its corolla limbs and emits metabolites to attract pollinators. RNA-sequencing from leaves, buds, and limbs identified 53,243 unigenes. Analysis of differentially expressed genes, combined with gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses, showed that many biological processes were highly enriched in limbs. These included catabolic processes and signaling pathways of hormones, such as gibberellins, and metabolic pathways, including phenylpropanoids and fatty acids. Moreover, we identified five transporter genes that showed high expression in limbs, and we performed spatiotemporal expression analyses and homology searches to infer their putative functions. Our systematic analysis provides comprehensive transcriptomic information regarding morphological differentiation and metabolite transport in the Petunia flower and lays the foundation for establishing the specific mechanisms that control secondary metabolite biosynthesis in plants. PMID:29902274

20170312 - Adverse Outcome Pathway (AOP) framework for ...

EPA Pesticide Factsheets

Vascular development commences with de novo assembly of a primary capillary plexus (vasculogenesis) followed by its expansion (angiogenesis) and maturation (angio-adaptation) into a hierarchical system of arteries and veins. These processes are tightly regulated by genetic signals and environmental factors linked to morphogenesis and microphysiology. Gestational exposure to some chemicals disrupts vascular development leading to adverse outcomes. To broadly assess consequences of gestational toxicant exposure on vascular development, an Adverse Outcome Pathway (AOP) framework was constructed that integrates data from ToxCast high-throughput screening (HTS) assays with pathway-level information from the literature and public databases. The AOP-based model resolved the ToxCast library (1065 compounds) into a matrix based on several dozen molecular functions critical for developmental angiogenesis. A sample of 38 ToxCast chemicals selected across the matrix tested model performance. Putative vascular disrupting chemical (pVDC) bioactivity was assessed by multiple laboratories utilizing diverse angiogenesis assays, including: transgenic zebrafish, complex human cell co-cultures, engineered microscale systems, and human-synthetic models. The ToxCast pVDC signature predicted vascular disruption in a manner that was chemical-specific and assay-dependent. An AOP for developmental vascular toxicity was constructed that focuses on inhibition of VEGF receptor (VEGFR2). Thi

Regulation of the protein kinase activity of Shaggy(Zeste-white3) by components of the wingless pathway in Drosophila cells and embryos.

PubMed

Ruel, L; Stambolic, V; Ali, A; Manoukian, A S; Woodgett, J R

1999-07-30

The protein-serine kinase Shaggy(Zeste-white3) (Sgg(Zw3)) is the Drosophila homolog of mammalian glycogen synthase kinase-3 and has been genetically implicated in signal transduction pathways necessary for the establishment of patterning. Sgg(Zw3) is a putative component of the Wingless (Wg) pathway, and epistasis analyses suggest that Sgg(Zw3) function is repressed by Wg signaling. Here, we have investigated the biochemical consequences of Wg signaling with respect to the Sgg(Zw3) protein kinase in two types of Drosophila cell lines and in embryos. Our results demonstrate that Sgg(Zw3) activity is inhibited following exposure of cells to Wg protein and by expression of downstream components of Wg signaling, Drosophila frizzled 2 and dishevelled. Wg-dependent inactivation of Sgg(Zw3) is accompanied by serine phosphorylation. We also show that the level of Sgg(Zw3) activity regulates the stability of Armadillo protein and modulates the level of phosphorylation of D-Axin and Armadillo. Together, these results provide direct biochemical evidence in support of the genetic model of Wg signaling and provide a model for dissecting the molecular interactions between the signaling proteins.

Adverse Outcome Pathway (AOP) framework for embryonic ...

EPA Pesticide Factsheets

Vascular development commences with de novo assembly of a primary capillary plexus (vasculogenesis) followed by its expansion (angiogenesis) and maturation (angio-adaptation) into a hierarchical system of arteries and veins. These processes are tightly regulated by genetic signals and environmental factors linked to morphogenesis and microphysiology. Gestational exposure to some chemicals disrupts vascular development leading to adverse outcomes. To broadly assess consequences of gestational toxicant exposure on vascular development, an Adverse Outcome Pathway (AOP) framework was constructed that integrates data from ToxCast high-throughput screening (HTS) assays with pathway-level information from the literature and public databases. The AOP-based model resolved the ToxCast library (1065 compounds) into a matrix based on several dozen molecular functions critical for developmental angiogenesis. A sample of 38 ToxCast chemicals selected across the matrix tested model performance. Putative vascular disrupting chemical (pVDC) bioactivity was assessed by multiple laboratories utilizing diverse angiogenesis assays, including: transgenic zebrafish, complex human cell co-cultures, engineered microscale systems, and human-synthetic models. The ToxCast pVDC signature predicted vascular disruption in a manner that was chemical-specific and assay-dependent. An AOP for developmental vascular toxicity was constructed that focuses on inhibition of VEGF receptor (VEGFR2). Thi

Serrated pathway colorectal cancer in the population: genetic consideration

PubMed Central

Young, Joanne; Jenkins, Mark; Parry, Susan; Young, Bruce; Nancarrow, Derek; English, Dallas; Giles, Graham; Jass, Jeremy

2007-01-01

A significant proportion of colorectal cancer (CRC) develops through the serrated neoplasia pathway. Such tumours show a distinctive molecular phenotype of somatic BRAF mutations and widespread concordant methylation events in CpG islands (CIMP). These features are also observed in the polyps developing in individuals with hyperplastic polyposis syndrome (HPS). In HPS, multiple serrated polyps develop in the colorectum, and approximately 50% of cases present with at least one CRC. Observations of rare affected sibships including identical twins, suggest a recessive or co‐dominant mode of inheritance. In addition, up to 50% of individuals with HPS report a family history of CRC. At a population level, recent work has demonstrated that patients with serrated pathway cancers characterised by BRAF mutation are four times more likely to have a family history of CRC than patients with common CRC. These findings suggest an increased genetic predisposition for serrated pathway CRC in the wider population. We propose that HPS results from the inheritance of two putative co‐dominant alleles in approximately 1 in 2000 individuals. Therefore carriers of one co‐dominant allele may number up to 1 in 25, and it is likely that these carriers are at increased risk of CRC, accounting for, at least in part, the burden of serrated pathway CRC in the population. This proposition may have important implications for screening and prevention of CRC in individuals who have an increased risk for development of serrated pathway cancers, namely those with multiple, proximal, large or advanced serrated polyps, and their relatives. PMID:17566021

PHTS, a novel putative tumor suppressor, is involved in the transformation reversion of HeLaHF cells independently of the p53 pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu Dehua; Fan, Wufang; Liu, Guohong

2006-04-01

HeLaHF is a non-transformed revertant of HeLa cells, likely resulting from the activation of a putative tumor suppressor(s). p53 protein was stabilized in this revertant and reactivated for certain transactivation functions. Although p53 stabilization has not conclusively been linked to the reversion, it is clear that the genes in p53 pathway are involved. The present study confirms the direct role of p53 in HeLaHF reversion by demonstrating that RNAi-mediated p53 silencing partially restores anchorage-independent growth potential of the revertant through the suppression of anoikis. In addition, we identified a novel gene, named PHTS, with putative tumor suppressor properties, and showedmore » that this gene is also involved in HeLaHF reversion independently of the p53 pathway. Expression profiling revealed that PHTS is one of the genes that is up-regulated in HeLaHF but not in HeLa. It encodes a putative protein with CD59-like domains. RNAi-mediated PHTS silencing resulted in the partial restoration of transformation (anchorage-independent growth) in HeLaHF cells, similar to that of p53 gene silencing, implying its tumor suppressor effect. However, the observed increased transformation potential by PHTS silencing appears to be due to an increased anchorage-independent proliferation rate rather than suppression of anoikis, unlike the effect of p53 silencing. p53 silencing did not affect PHTS gene expression, and vice versa, suggesting PHTS may function in a new and p53-independent tumor suppressor pathway. Furthermore, over-expression of PHTS in different cancer cell lines, in addition to HeLa, reduces cell growth likely via induced apoptosis, confirming the broad PHTS tumor suppressor properties.« less

Transcriptomic and Proteomic Responses of Sweetpotato Whitefly, Bemisia tabaci, to Thiamethoxam

PubMed Central

Yang, Nina; Xie, Wen; Yang, Xin; Wang, Shaoli; Wu, Qingjun; Li, Rumei; Pan, Huipeng; Liu, Baiming; Shi, Xiaobin; Fang, Yong; Xu, Baoyun; Zhou, Xuguo; Zhang, Youjun

2013-01-01

Background The sweetpotato whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae), is one of the most widely distributed agricultural pests. Although it has developed resistance to many registered insecticides including the neonicotinoid insecticide thiamethoxam, the mechanisms that regulate the resistance are poorly understood. To understand the molecular basis of thiamethoxam resistance, “omics” analyses were carried out to examine differences between resistant and susceptible B. tabaci at both transcriptional and translational levels. Results A total of 1,338 mRNAs and 52 proteins were differentially expressed between resistant and susceptible B. tabaci. Among them, 11 transcripts had concurrent transcription and translation profiles. KEGG analysis mapped 318 and 35 differentially expressed genes and proteins, respectively, to 160 and 59 pathways (p<0.05). Thiamethoxam treatment activated metabolic pathways (e.g., drug metabolism), in which 118 transcripts were putatively linked to insecticide resistance, including up-regulated glutathione-S-transferase, UDP glucuronosyltransferase, glucosyl/glucuronosyl transferase, and cytochrome P450. Gene Ontology analysis placed these genes and proteins into protein complex, metabolic process, cellular process, signaling, and response to stimulus categories. Quantitative real-time PCR analysis validated “omics” response, and suggested a highly overexpressed P450, CYP6CX1, as a candidate molecular basis for the mechanistic study of thiamethoxam resistance in whiteflies. Finally, enzymatic activity assays showed elevated detoxification activities in the resistant B. tabaci. Conclusions This study demonstrates the applicability of high-throughput omics tools for identifying molecular candidates related to thiamethoxam resistance in an agricultural important insect pest. In addition, transcriptomic and proteomic analyses provide a solid foundation for future functional investigations into the complex molecular mechanisms governing the neonicotinoid resistance in whiteflies. PMID:23671574

Increased PKA signaling disrupts recognition memory and spatial memory: role in Huntington's disease.

PubMed

Giralt, Albert; Saavedra, Ana; Carretón, Olga; Xifró, Xavier; Alberch, Jordi; Pérez-Navarro, Esther

2011-11-01

Huntington's disease (HD) patients and mouse models show learning and memory impairment even before the onset of motor symptoms. However, the molecular events involved in this cognitive decline are still poorly understood. Here, using three different paradigms, the novel object recognition test, the T-maze spontaneous alternation task and the Morris water maze, we detected severe cognitive deficits in the R6/1 mouse model of HD before the onset of motor symptoms. When we examined the putative molecular pathways involved in these alterations, we observed hippocampal cAMP-dependent protein kinase (PKA) hyper-activation in naïve R6/1 mice compared with wild-type (WT) mice, whereas extracellular signal-regulated kinase 1/2 and calcineurin activities were not modified. Increased PKA activity resulted in hyper-phosphorylation of its substrates N-methyl-D-aspartate receptor subunit 1, Ras-guanine nucleotide releasing factor-1 and striatal-enriched protein tyrosine phosphatase, but not cAMP-responsive element binding protein or the microtubule-associated protein tau. In correlation with the over-activation of the PKA pathway, we found a down-regulation of the protein levels of some phosphodiesterase (PDE) 4 family members. Similar molecular changes were found in the hippocampus of R6/2 mice and HD patients. Furthermore, chronic treatment of WT mice with the PDE4 inhibitor rolipram up-regulated PKA activity, and induced learning and memory deficits similar to those seen in R6 mice, but had no effect on R6/1 mice cognitive impairment. Importantly, hippocampal PKA inhibition by infusion of Rp-cAMPS restored long-term memory in R6/2 mice. Thus, our results suggest that occlusion of PKA-dependent processes is one of the molecular mechanisms underlying cognitive decline in R6 animals.

Microarray analysis of gene expression profiles in ripening pineapple fruits.

PubMed

Koia, Jonni H; Moyle, Richard L; Botella, Jose R

2012-12-18

Pineapple (Ananas comosus) is a tropical fruit crop of significant commercial importance. Although the physiological changes that occur during pineapple fruit development have been well characterized, little is known about the molecular events that occur during the fruit ripening process. Understanding the molecular basis of pineapple fruit ripening will aid the development of new varieties via molecular breeding or genetic modification. In this study we developed a 9277 element pineapple microarray and used it to profile gene expression changes that occur during pineapple fruit ripening. Microarray analyses identified 271 unique cDNAs differentially expressed at least 1.5-fold between the mature green and mature yellow stages of pineapple fruit ripening. Among these 271 sequences, 184 share significant homology with genes encoding proteins of known function, 53 share homology with genes encoding proteins of unknown function and 34 share no significant homology with any database accession. Of the 237 pineapple sequences with homologs, 160 were up-regulated and 77 were down-regulated during pineapple fruit ripening. DAVID Functional Annotation Cluster (FAC) analysis of all 237 sequences with homologs revealed confident enrichment scores for redox activity, organic acid metabolism, metalloenzyme activity, glycolysis, vitamin C biosynthesis, antioxidant activity and cysteine peptidase activity, indicating the functional significance and importance of these processes and pathways during pineapple fruit development. Quantitative real-time PCR analysis validated the microarray expression results for nine out of ten genes tested. This is the first report of a microarray based gene expression study undertaken in pineapple. Our bioinformatic analyses of the transcript profiles have identified a number of genes, processes and pathways with putative involvement in the pineapple fruit ripening process. This study extends our knowledge of the molecular basis of pineapple fruit ripening and non-climacteric fruit ripening in general.

Microarray analysis of gene expression profiles in ripening pineapple fruits

PubMed Central

2012-01-01

Background Pineapple (Ananas comosus) is a tropical fruit crop of significant commercial importance. Although the physiological changes that occur during pineapple fruit development have been well characterized, little is known about the molecular events that occur during the fruit ripening process. Understanding the molecular basis of pineapple fruit ripening will aid the development of new varieties via molecular breeding or genetic modification. In this study we developed a 9277 element pineapple microarray and used it to profile gene expression changes that occur during pineapple fruit ripening. Results Microarray analyses identified 271 unique cDNAs differentially expressed at least 1.5-fold between the mature green and mature yellow stages of pineapple fruit ripening. Among these 271 sequences, 184 share significant homology with genes encoding proteins of known function, 53 share homology with genes encoding proteins of unknown function and 34 share no significant homology with any database accession. Of the 237 pineapple sequences with homologs, 160 were up-regulated and 77 were down-regulated during pineapple fruit ripening. DAVID Functional Annotation Cluster (FAC) analysis of all 237 sequences with homologs revealed confident enrichment scores for redox activity, organic acid metabolism, metalloenzyme activity, glycolysis, vitamin C biosynthesis, antioxidant activity and cysteine peptidase activity, indicating the functional significance and importance of these processes and pathways during pineapple fruit development. Quantitative real-time PCR analysis validated the microarray expression results for nine out of ten genes tested. Conclusions This is the first report of a microarray based gene expression study undertaken in pineapple. Our bioinformatic analyses of the transcript profiles have identified a number of genes, processes and pathways with putative involvement in the pineapple fruit ripening process. This study extends our knowledge of the molecular basis of pineapple fruit ripening and non-climacteric fruit ripening in general. PMID:23245313

Annotation: a computational solution for streamlining metabolomics analysis

PubMed Central

Domingo-Almenara, Xavier; Montenegro-Burke, J. Rafael; Benton, H. Paul; Siuzdak, Gary

2017-01-01

Metabolite identification is still considered an imposing bottleneck in liquid chromatography mass spectrometry (LC/MS) untargeted metabolomics. The identification workflow usually begins with detecting relevant LC/MS peaks via peak-picking algorithms and retrieving putative identities based on accurate mass searching. However, accurate mass search alone provides poor evidence for metabolite identification. For this reason, computational annotation is used to reveal the underlying metabolites monoisotopic masses, improving putative identification in addition to confirmation with tandem mass spectrometry. This review examines LC/MS data from a computational and analytical perspective, focusing on the occurrence of neutral losses and in-source fragments, to understand the challenges in computational annotation methodologies. Herein, we examine the state-of-the-art strategies for computational annotation including: (i) peak grouping or full scan (MS1) pseudo-spectra extraction, i.e., clustering all mass spectral signals stemming from each metabolite; (ii) annotation using ion adduction and mass distance among ion peaks; (iii) incorporation of biological knowledge such as biotransformations or pathways; (iv) tandem MS data; and (v) metabolite retention time calibration, usually achieved by prediction from molecular descriptors. Advantages and pitfalls of each of these strategies are discussed, as well as expected future trends in computational annotation. PMID:29039932

Evaluating Between-Pathway Models with Expression Data

PubMed Central

Leiserson, M.D.M.; Cowen, L.J.; Slonim, D.K.

2010-01-01

Abstract Between-pathway models (BPMs) are network motifs consisting of pairs of putative redundant pathways. In this article, we show how adding another source of high-throughput data—microarray gene expression data from knockout experiments—allows us to identify a compensatory functional relationship between genes from the two BPM pathways. We evaluate the quality of the BPMs from four different studies, and we describe how our methods might be extended to refine pathways. PMID:20377458

Unexpected Diversity and High Abundance of Putative Nitric Oxide Dismutase (Nod) Genes in Contaminated Aquifers and Wastewater Treatment Systems

PubMed Central

Bradford, Lauren; Huang, Sichao; Szalay, Anna; Leix, Carmen; Weissbach, Max; Táncsics, András; Drewes, Jörg E.

2016-01-01

ABSTRACT It has recently been suggested that oxygenic dismutation of NO into N2 and O2 may occur in the anaerobic methanotrophic “Candidatus Methylomirabilis oxyfera” and the alkane-oxidizing gammaproteobacterium HdN1. It may represent a new pathway in microbial nitrogen cycling catalyzed by a putative NO dismutase (Nod). The formed O2 enables microbes to employ aerobic catabolic pathways in anoxic habitats, suggesting an ecophysiological niche space of substantial appeal for bioremediation and water treatment. However, it is still unknown whether this physiology is limited to “Ca. Methylomirabilis oxyfera” and HdN1 and whether it can be coupled to the oxidation of electron donors other than alkanes. Here, we report insights into an unexpected diversity and remarkable abundance of nod genes in natural and engineered water systems. Phylogenetically diverse nod genes were recovered from a range of contaminated aquifers and N-removing wastewater treatment systems. Together with nod genes from “Ca. Methylomirabilis oxyfera” and HdN1, the novel environmental nod sequences formed no fewer than 6 well-supported phylogenetic clusters, clearly distinct from canonical NO reductase (quinol-dependent NO reductase [qNor] and cytochrome c-dependent NO reductase [cNor]) genes. The abundance of nod genes in the investigated samples ranged from 1.6 × 107 to 5.2 × 1010 copies · g−1 (wet weight) of sediment or sludge biomass, accounting for up to 10% of total bacterial 16S rRNA gene counts. In essence, NO dismutation could be a much more widespread physiology than currently perceived. Understanding the controls of this emergent microbial capacity could offer new routes for nitrogen elimination or pollutant remediation in natural and engineered water systems. IMPORTANCE NO dismutation into N2 and O2 is a novel process catalyzed by putative NO dismutase (Nod). To date, only two bacteria, the anaerobic methane-oxidizing bacterium “Ca. Methylomirabilis oxyfera” and the alkane-oxidizing gammaproteobacterium HdN1, are known to harbor nod genes. In this study, we report efficient molecular tools that can detect and quantify a wide diversity of nod genes in environmental samples. A surprisingly high diversity and abundance of nod genes were found in contaminated aquifers as well as wastewater treatment systems. This evidence indicates that NO dismutation may be a much more widespread physiology in natural and man-made environments than currently perceived. The molecular tools presented here will facilitate further studies on these enigmatic microbes in the future. PMID:27986721

Unexpected Diversity and High Abundance of Putative Nitric Oxide Dismutase (Nod) Genes in Contaminated Aquifers and Wastewater Treatment Systems.

PubMed

Zhu, Baoli; Bradford, Lauren; Huang, Sichao; Szalay, Anna; Leix, Carmen; Weissbach, Max; Táncsics, András; Drewes, Jörg E; Lueders, Tillmann

2017-02-15

It has recently been suggested that oxygenic dismutation of NO into N 2 and O 2 may occur in the anaerobic methanotrophic "Candidatus Methylomirabilis oxyfera" and the alkane-oxidizing gammaproteobacterium HdN1. It may represent a new pathway in microbial nitrogen cycling catalyzed by a putative NO dismutase (Nod). The formed O 2 enables microbes to employ aerobic catabolic pathways in anoxic habitats, suggesting an ecophysiological niche space of substantial appeal for bioremediation and water treatment. However, it is still unknown whether this physiology is limited to "Ca Methylomirabilis oxyfera" and HdN1 and whether it can be coupled to the oxidation of electron donors other than alkanes. Here, we report insights into an unexpected diversity and remarkable abundance of nod genes in natural and engineered water systems. Phylogenetically diverse nod genes were recovered from a range of contaminated aquifers and N-removing wastewater treatment systems. Together with nod genes from "Ca Methylomirabilis oxyfera" and HdN1, the novel environmental nod sequences formed no fewer than 6 well-supported phylogenetic clusters, clearly distinct from canonical NO reductase (quinol-dependent NO reductase [qNor] and cytochrome c-dependent NO reductase [cNor]) genes. The abundance of nod genes in the investigated samples ranged from 1.6 × 10 7 to 5.2 × 10 10 copies · g -1 (wet weight) of sediment or sludge biomass, accounting for up to 10% of total bacterial 16S rRNA gene counts. In essence, NO dismutation could be a much more widespread physiology than currently perceived. Understanding the controls of this emergent microbial capacity could offer new routes for nitrogen elimination or pollutant remediation in natural and engineered water systems. NO dismutation into N 2 and O 2 is a novel process catalyzed by putative NO dismutase (Nod). To date, only two bacteria, the anaerobic methane-oxidizing bacterium "Ca Methylomirabilis oxyfera" and the alkane-oxidizing gammaproteobacterium HdN1, are known to harbor nod genes. In this study, we report efficient molecular tools that can detect and quantify a wide diversity of nod genes in environmental samples. A surprisingly high diversity and abundance of nod genes were found in contaminated aquifers as well as wastewater treatment systems. This evidence indicates that NO dismutation may be a much more widespread physiology in natural and man-made environments than currently perceived. The molecular tools presented here will facilitate further studies on these enigmatic microbes in the future. Copyright © 2017 American Society for Microbiology.

Molecular characterization and origin of novel bipartite cold-regulated ice recrystallization inhibition proteins from cereals.

PubMed

Tremblay, Karine; Ouellet, François; Fournier, Julie; Danyluk, Jean; Sarhan, Fathey

2005-06-01

To understand the molecular basis of freezing tolerance in plants, several low temperature-responsive genes have been identified from wheat. Among these are two genes named TaIRI-1 and TaIRI-2 (Triticum aestivum ice recrystallization inhibition) that are up-regulated during cold acclimation in freezing-tolerant species. Phytohormones involved in pathogen defense pathways (jasmonic acid and ethylene) induce the expression of one of the two genes. The encoded proteins are novel in that they have a bipartite structure that has never been reported for antifreeze proteins. Their N-terminal part shows similarity with the leucine-rich repeat-containing regions present in the receptor domain of receptor-like protein kinases, and their C-terminus is homologous to the ice-binding domain of some antifreeze proteins. The recombinant TaIRI-1 protein inhibits the growth of ice crystals, confirming its function as an ice recrystallization inhibition protein. The TaIRI genes were found only in the species belonging to the Pooideae subfamily of cereals. Comparative genomic analysis suggested that molecular evolutionary events took place in the genome of freezing-tolerant cereals to give rise to these genes with putative novel functions. These apparent adaptive DNA rearrangement events could be part of the molecular mechanisms that ensure the survival of hardy cereals in the harsh freezing environments.

Selective degeneration of a putative cholinergic pathway in the chinchilla cochlea following infusion with ethylcholine aziridinium ion.

PubMed

Morley, B J; Spangler, K M; Schneider, B L; Javel, E

1991-03-22

Ethylcholine aziridinium ion (AF64A) diluted in artificial perilymph, or artificial perilymph alone was infused into the cochlea of chinchillas. After a survival time of 7 days, the cochleas were fixed with aldehydes, post-fixed in osmium and embedded in epoxy resin for light and electron microscopy. The ultrastructure of the cochleas infused with artificial perilymph was normal. Infusion of 1 microM AF64A resulted in massive degeneration of the axons of the lateral efferent system, a putative cholinergic pathway that originates in the brainstem and terminates on dendrites of the spiral ganglion innervating cochlear inner hair cells. The axons and terminals of a second putative cholinergic pathway, the medial efferent system which terminates on the outer hair cells, were normal. Infusion of AF64A in a concentration of 10 microM resulted in significant pathology of cochlear and supporting cells as well as the loss of efferent terminals at both inner and outer hair cell regions. The results suggest that AF64A is a selective neurotoxin when used under low-dosage conditions, and that certain pathways may be more susceptible to the effects of AF64A than others. One interpretation of these findings is that lateral efferent axons may have a higher rate of high-affinity choline uptake than terminals of the medial efferent axons.

Use of Putative Adverse Outcome Pathways for Chemical Hazard Identification

EPA Science Inventory

The Adverse Outcome Pathway (AOP) framework provides a knowledge infrastructure for evaluating health effects of environmental chemicals. In this work we are examining proof-of-concept issues in the development and prospective application of AOPs in chemical safety. Key outputs i...

KCNE1 induces fenestration in the Kv7.1/KCNE1 channel complex that allows for highly specific pharmacological targeting

NASA Astrophysics Data System (ADS)

Wrobel, Eva; Rothenberg, Ina; Krisp, Christoph; Hundt, Franziska; Fraenzel, Benjamin; Eckey, Karina; Linders, Joannes T. M.; Gallacher, David J.; Towart, Rob; Pott, Lutz; Pusch, Michael; Yang, Tao; Roden, Dan M.; Kurata, Harley T.; Schulze-Bahr, Eric; Strutz-Seebohm, Nathalie; Wolters, Dirk; Seebohm, Guiscard

2016-10-01

Most small-molecule inhibitors of voltage-gated ion channels display poor subtype specificity because they bind to highly conserved residues located in the channel's central cavity. Using a combined approach of scanning mutagenesis, electrophysiology, chemical ligand modification, chemical cross-linking, MS/MS-analyses and molecular modelling, we provide evidence for the binding site for adamantane derivatives and their putative access pathway in Kv7.1/KCNE1 channels. The adamantane compounds, exemplified by JNJ303, are highly potent gating modifiers that bind to fenestrations that become available when KCNE1 accessory subunits are bound to Kv7.1 channels. This mode of regulation by auxiliary subunits may facilitate the future development of potent and highly subtype-specific Kv channel inhibitors.

FLOWERING LOCUS C Mediates Natural Variation in the High-Temperature Response of the Arabidopsis Circadian Clock[W

PubMed Central

Edwards, Kieron D.; Anderson, Paul E.; Hall, Anthony; Salathia, Neeraj S.; Locke, James C.W.; Lynn, James R.; Straume, Martin; Smith, James Q.; Millar, Andrew J.

2006-01-01

Temperature compensation contributes to the accuracy of biological timing by preventing circadian rhythms from running more quickly at high than at low temperatures. We previously identified quantitative trait loci (QTL) with temperature-specific effects on the circadian rhythm of leaf movement, including a QTL linked to the transcription factor FLOWERING LOCUS C (FLC). We have now analyzed FLC alleles in near-isogenic lines and induced mutants to eliminate other candidate genes. We showed that FLC lengthened the circadian period specifically at 27°C, contributing to temperature compensation of the circadian clock. Known upstream regulators of FLC expression in flowering time pathways similarly controlled its circadian effect. We sought to identify downstream targets of FLC regulation in the molecular mechanism of the circadian clock using genome-wide analysis to identify FLC-responsive genes and 3503 transcripts controlled by the circadian clock. A Bayesian clustering method based on Fourier coefficients allowed us to discriminate putative regulatory genes. Among rhythmic FLC-responsive genes, transcripts of the transcription factor LUX ARRHYTHMO (LUX) correlated in peak abundance with the circadian period in flc mutants. Mathematical modeling indicated that the modest change in peak LUX RNA abundance was sufficient to cause the period change due to FLC, providing a molecular target for the crosstalk between flowering time pathways and circadian regulation. PMID:16473970

Genome and Transcriptome Analysis of the Fungal Pathogen Fusarium oxysporum f. sp. cubense Causing Banana Vascular Wilt Disease

PubMed Central

Zeng, Huicai; Fan, Dingding; Zhu, Yabin; Feng, Yue; Wang, Guofen; Peng, Chunfang; Jiang, Xuanting; Zhou, Dajie; Ni, Peixiang; Liang, Changcong; Liu, Lei; Wang, Jun; Mao, Chao

2014-01-01

Background The asexual fungus Fusarium oxysporum f. sp. cubense (Foc) causing vascular wilt disease is one of the most devastating pathogens of banana (Musa spp.). To understand the molecular underpinning of pathogenicity in Foc, the genomes and transcriptomes of two Foc isolates were sequenced. Methodology/Principal Findings Genome analysis revealed that the genome structures of race 1 and race 4 isolates were highly syntenic with those of F. oxysporum f. sp. lycopersici strain Fol4287. A large number of putative virulence associated genes were identified in both Foc genomes, including genes putatively involved in root attachment, cell degradation, detoxification of toxin, transport, secondary metabolites biosynthesis and signal transductions. Importantly, relative to the Foc race 1 isolate (Foc1), the Foc race 4 isolate (Foc4) has evolved with some expanded gene families of transporters and transcription factors for transport of toxins and nutrients that may facilitate its ability to adapt to host environments and contribute to pathogenicity to banana. Transcriptome analysis disclosed a significant difference in transcriptional responses between Foc1 and Foc4 at 48 h post inoculation to the banana ‘Brazil’ in comparison with the vegetative growth stage. Of particular note, more virulence-associated genes were up regulated in Foc4 than in Foc1. Several signaling pathways like the mitogen-activated protein kinase Fmk1 mediated invasion growth pathway, the FGA1-mediated G protein signaling pathway and a pathogenicity associated two-component system were activated in Foc4 rather than in Foc1. Together, these differences in gene content and transcription response between Foc1 and Foc4 might account for variation in their virulence during infection of the banana variety ‘Brazil’. Conclusions/Significance Foc genome sequences will facilitate us to identify pathogenicity mechanism involved in the banana vascular wilt disease development. These will thus advance us develop effective methods for managing the banana vascular wilt disease, including improvement of disease resistance in banana. PMID:24743270

Role of IncP-1β Plasmids pWDL7::rfp and pNB8c in Chloroaniline Catabolism as Determined by Genomic and Functional Analyses

PubMed Central

Król, J. E.; Penrod, J. T.; McCaslin, H.; Rogers, L. M.; Yano, H.; Stancik, A. D.; Dejonghe, W.; Brown, C. J.; Parales, R. E.; Wuertz, S.

2012-01-01

Broad-host-range catabolic plasmids play an important role in bacterial degradation of man-made compounds. To gain insight into the role of these plasmids in chloroaniline degradation, we determined the first complete nucleotide sequences of an IncP-1 chloroaniline degradation plasmid, pWDL7::rfp and its close relative pNB8c, as well as the expression pattern, function, and bioaugmentation potential of the putative 3-chloroaniline (3-CA) oxidation genes. Based on phylogenetic analysis of backbone proteins, both plasmids are members of a distinct clade within the IncP-1β subgroup. The plasmids are almost identical, but whereas pWDL7::rfp carries a duplicate inverted catabolic transposon, Tn6063, containing a putative 3-CA oxidation gene cluster, dcaQTA1A2BR, pNB8c contains only a single copy of the transposon. No genes for an aromatic ring cleavage pathway were detected on either plasmid, suggesting that only the upper 3-CA degradation pathway was present. The dcaA1A2B gene products expressed from a high-copy-number vector were shown to convert 3-CA to 4-chlorocatechol in Escherichia coli. Slight differences in the dca promoter region between the plasmids and lack of induction of transcription of the pNB8c dca genes by 3-CA may explain previous findings that pNB8C does not confer 3-CA transformation. Bioaugmentation of activated sludge with pWDL7::rfp accelerated removal of 3-CA, but only in the presence of an additional carbon source. Successful bioaugmentation requires complementation of the upper pathway genes with chlorocatechol cleavage genes in indigenous bacteria. The genome sequences of these plasmids thus help explain the molecular basis of their catabolic activities. PMID:22101050

Physalis floridana Cell Number Regulator1 encodes a cell membrane-anchored modulator of cell cycle and negatively controls fruit size

PubMed Central

Li, Zhichao; He, Chaoying

2015-01-01

Physalis species show a significant variation in berry size; however, the underlying molecular basis is unknown. In this work, we showed that cell division difference in the ovaries might contribute to the ultimate berry size variation within Physalis species, and that mRNA abundance of Physalis floridana Cell Number Regulator1 (PfCNR1), the putative orthologue of the tomato fruit weight 2.2 (FW2.2), was negatively correlated with cell division in the ovaries. Moreover, heterochronic expression variation of the PfCNR1 genes in the ovaries concomitantly correlated with berry weight variation within Physalis species. In transgenic Physalis, multiple organ sizes could be negatively controlled by altering PfCNR1 levels, and cell division instead of cell expansion was primarily affected. PfCNR1 was shown to be anchored in the plasma membrane and to interact with PfAG2 (an AGAMOUS-like protein determining ovary identity). The expression of PfCYCD2;1, a putative orthologue of the mitosis-specific gene CyclinD2;1 in the cell cycle was negatively correlated with the PfCNR1 mRNA levels. PfAG2 was found to selectively bind to the CArG-box in the PfCYCD2;1 promoter and to repress PfCYCD2;1 expression, thus suggesting a PfAG2-mediated pathway for PfCNR1 to regulate cell division. The interaction of PfCNR1 with PfAG2 enhanced the repression of PfCYCD2;1 expression. The nuclear import of PfAG2 was essential in the proposed pathway. Our data provide new insights into the developmental pathways of a cell membrane-anchored protein that modulates cell division and governs organ size determination. This study also sheds light on the link between organ identity and organ growth in plants. PMID:25305759

Enzymes involved in the anaerobic oxidation of n-alkanes: from methane to long-chain paraffins

PubMed Central

Callaghan, Amy V.

2013-01-01

Anaerobic microorganisms play key roles in the biogeochemical cycling of methane and non-methane alkanes. To date, there appear to be at least three proposed mechanisms of anaerobic methane oxidation (AOM). The first pathway is mediated by consortia of archaeal anaerobic methane oxidizers and sulfate-reducing bacteria (SRB) via “reverse methanogenesis” and is catalyzed by a homolog of methyl-coenzyme M reductase. The second pathway is also mediated by anaerobic methane oxidizers and SRB, wherein the archaeal members catalyze both methane oxidation and sulfate reduction and zero-valent sulfur is a key intermediate. The third AOM mechanism is a nitrite-dependent, “intra-aerobic” pathway described for the denitrifying bacterium, ‘Candidatus Methylomirabilis oxyfera.’ It is hypothesized that AOM proceeds via reduction of nitrite to nitric oxide, followed by the conversion of two nitric oxide molecules to dinitrogen and molecular oxygen. The latter can be used to functionalize the methane via a particulate methane monooxygenase. With respect to non-methane alkanes, there also appear to be novel mechanisms of activation. The most well-described pathway is the addition of non-methane alkanes across the double bond of fumarate to form alkyl-substituted succinates via the putative glycyl radical enzyme, alkylsuccinate synthase (also known as methylalkylsuccinate synthase). Other proposed mechanisms include anaerobic hydroxylation via ethylbenzene dehydrogenase-like enzymes and an “intra-aerobic” denitrification pathway similar to that described for ‘Methylomirabilis oxyfera.’ PMID:23717304

YODA MAP3K kinase regulates plant immune responses conferring broad-spectrum disease resistance.

PubMed

Sopeña-Torres, Sara; Jordá, Lucía; Sánchez-Rodríguez, Clara; Miedes, Eva; Escudero, Viviana; Swami, Sanjay; López, Gemma; Piślewska-Bednarek, Mariola; Lassowskat, Ines; Lee, Justin; Gu, Yangnan; Haigis, Sabine; Alexander, Danny; Pattathil, Sivakumar; Muñoz-Barrios, Antonio; Bednarek, Pawel; Somerville, Shauna; Schulze-Lefert, Paul; Hahn, Michael G; Scheel, Dierk; Molina, Antonio

2018-04-01

Mitogen-activated protein kinases (MAPKs) cascades play essential roles in plants by transducing developmental cues and environmental signals into cellular responses. Among the latter are microbe-associated molecular patterns perceived by pattern recognition receptors (PRRs), which trigger immunity. We found that YODA (YDA) - a MAPK kinase kinase regulating several Arabidopsis developmental processes, like stomatal patterning - also modulates immune responses. Resistance to pathogens is compromised in yda alleles, whereas plants expressing the constitutively active YDA (CA-YDA) protein show broad-spectrum resistance to fungi, bacteria, and oomycetes with different colonization modes. YDA functions in the same pathway as ERECTA (ER) Receptor-Like Kinase, regulating both immunity and stomatal patterning. ER-YDA-mediated immune responses act in parallel to canonical disease resistance pathways regulated by phytohormones and PRRs. CA-YDA plants exhibit altered cell-wall integrity and constitutively express defense-associated genes, including some encoding putative small secreted peptides and PRRs whose impairment resulted in enhanced susceptibility phenotypes. CA-YDA plants show strong reprogramming of their phosphoproteome, which contains protein targets distinct from described MAPKs substrates. Our results suggest that, in addition to stomata development, the ER-YDA pathway regulates an immune surveillance system conferring broad-spectrum disease resistance that is distinct from the canonical pathways mediated by described PRRs and defense hormones. © 2018 Universidad Politécnica de Madrid (UPM) New Phytologist © 2018 New Phytologist Trust.

Carotenoid metabolic profiling and transcriptome-genome mining reveal functional equivalence among blue-pigmented copepods and appendicularia.

PubMed

Mojib, Nazia; Amad, Maan; Thimma, Manjula; Aldanondo, Naroa; Kumaran, Mande; Irigoien, Xabier

2014-06-01

The tropical oligotrophic oceanic areas are characterized by high water transparency and annual solar radiation. Under these conditions, a large number of phylogenetically diverse mesozooplankton species living in the surface waters (neuston) are found to be blue pigmented. In the present study, we focused on understanding the metabolic and genetic basis of the observed blue phenotype functional equivalence between the blue-pigmented organisms from the phylum Arthropoda, subclass Copepoda (Acartia fossae) and the phylum Chordata, class Appendicularia (Oikopleura dioica) in the Red Sea. Previous studies have shown that carotenoid-protein complexes are responsible for blue coloration in crustaceans. Therefore, we performed carotenoid metabolic profiling using both targeted and nontargeted (high-resolution mass spectrometry) approaches in four different blue-pigmented genera of copepods and one blue-pigmented species of appendicularia. Astaxanthin was found to be the principal carotenoid in all the species. The pathway analysis showed that all the species can synthesize astaxanthin from β-carotene, ingested from dietary sources, via 3-hydroxyechinenone, canthaxanthin, zeaxanthin, adonirubin or adonixanthin. Further, using de novo assembled transcriptome of blue A. fossae (subclass Copepoda), we identified highly expressed homologous β-carotene hydroxylase enzymes and putative carotenoid-binding proteins responsible for astaxanthin formation and the blue phenotype. In blue O. dioica (class Appendicularia), corresponding putative genes were identified from the reference genome. Collectively, our data provide molecular evidences for the bioconversion and accumulation of blue astaxanthin-protein complexes underpinning the observed ecological functional equivalence and adaptive convergence among neustonic mesozooplankton. © 2014 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.

Impact of Insulin Degrading Enzyme and Neprilysin in Alzheimer's Disease Biology: Characterization of Putative Cognates for Therapeutic Applications.

PubMed

Jha, Niraj Kumar; Jha, Saurabh Kumar; Kumar, Dhiraj; Kejriwal, Noopur; Sharma, Renu; Ambasta, Rashmi K; Kumar, Pravir

2015-01-01

Alzheimer's disease (AD) is a neurodegenerative process primarily characterized by amyloid-β (Aβ) agglomeration, neuroinflammation, and cognitive dysfunction. The prominent cause for dementia is the deposition of Aβ plaques and tau-neurofibrillary tangles that hamper the neuronal organization and function. Aβ pathology further affects numerous signaling cascades that disturb the neuronal homeostasis. For instance, Aβ deposition is responsible for altered expression of insulin encoding genes that lead to insulin resistance, and thereby affecting insulin signaling pathway and glucose metabolism in the brain. As a result, the common pathology of insulin resistance between Type-2 diabetes mellitus and AD has led AD to be proposed as a form of diabetes and termed 'Type-3 diabetes'. Since accumulation of Aβ is the prominent cause of neuronal toxicity in AD, its clearance is the prime requisite for therapeutic prospects. This purpose is expertly fulfilled by the potential role of Aβ degrading enzymes such as insulin degrading enzyme (IDE) and Neprilysin (NEP). Therefore, their molecular study is important to uncover the proteolytic and regulatory mechanism of Aβ degradation. Herein, (i) In silico sequential and structural analysis of IDE and NEP has been performed to identify the molecular entities for proteolytic degradation of Aβ in the AD brain, (ii) to analyze their catalytic site to demonstrate the enzymatic action played by IDE and NEP, (iii) to identify their structural homologues that could behave as putative partners of IDE and NEP with similar catalytic action and (iv) to illustrate various IDE- and NEP-mediated therapeutic approaches and factors for clearing Aβ in AD.

Neuroendocrine factors affecting the glycogen metabolism of purified Mytilus edulis glycogen cells: partial characterization of the putative glycogen mobilization hormone--demonstration of a factor that stimulates glycogen synthesis.

PubMed

Robbins, I; Lenoir, F; Mathieu, M

1991-04-01

A putative glycogen mobilizing hormone (GMH) from the marine mussel Mytilus edulis L. has been partially characterized. GMH activity is present in the cerebral ganglia and the hemolymph serum and promotes the mobilization of glycogen in isolated glycogen cells. The cerebral GMH is trypsin sensitive and partially heat labile and has an apparent molecular mass of greater than 20 kDa. Following fractionation of cerebral extracts by molecular mass, a second factor, with a molecular mass of ca. 1.5 kDa, was discovered. This factor stimulates post-incubation incorporation of 14C into glycogen in isolated glycogen cells.

MiR-155-regulated molecular network orchestrates cell fate in the innate and adaptive immune response to Mycobacterium tuberculosis.

PubMed

Rothchild, Alissa C; Sissons, James R; Shafiani, Shahin; Plaisier, Christopher; Min, Deborah; Mai, Dat; Gilchrist, Mark; Peschon, Jacques; Larson, Ryan P; Bergthaler, Andreas; Baliga, Nitin S; Urdahl, Kevin B; Aderem, Alan

2016-10-11

The regulation of host-pathogen interactions during Mycobacterium tuberculosis (Mtb) infection remains unresolved. MicroRNAs (miRNAs) are important regulators of the immune system, and so we used a systems biology approach to construct an miRNA regulatory network activated in macrophages during Mtb infection. Our network comprises 77 putative miRNAs that are associated with temporal gene expression signatures in macrophages early after Mtb infection. In this study, we demonstrate a dual role for one of these regulators, miR-155. On the one hand, miR-155 maintains the survival of Mtb-infected macrophages, thereby providing a niche favoring bacterial replication; on the other hand, miR-155 promotes the survival and function of Mtb-specific T cells, enabling an effective adaptive immune response. MiR-155-induced cell survival is mediated through the SH2 domain-containing inositol 5-phosphatase 1 (SHIP1)/protein kinase B (Akt) pathway. Thus, dual regulation of the same cell survival pathway in innate and adaptive immune cells leads to vastly different outcomes with respect to bacterial containment.

The study on serum and urine of renal interstitial fibrosis rats induced by unilateral ureteral obstruction based on metabonomics and network analysis methods.

PubMed

Xiang, Zheng; Sun, Hao; Cai, Xiaojun; Chen, Dahui

2016-04-01

Transmission of biological information is a biochemical process of multistep cascade from genes/proteins to metabolites. However, because most metabolites reflect the terminal information of the biochemical process, it is difficult to describe the transmission process of disease information in terms of the metabolomics strategy. In this paper, by incorporating network and metabolomics methods, an integrated approach was proposed to systematically investigate and explain the molecular mechanism of renal interstitial fibrosis. Through analysis of the network, the cascade transmission process of disease information starting from genes/proteins to metabolites was putatively identified and uncovered. The results indicated that renal fibrosis was involved in metabolic pathways of glycerophospholipid metabolism, biosynthesis of unsaturated fatty acids and arachidonic acid metabolism, riboflavin metabolism, tyrosine metabolism, and sphingolipid metabolism. These pathways involve kidney disease genes such as TGF-β1 and P2RX7. Our results showed that combining metabolomics and network analysis can provide new strategies and ideas for the interpretation of pathogenesis of disease with full consideration of "gene-protein-metabolite."

Functional expression of an ajmaline pathway-specific esterase from Rauvolfia in a novel plant-virus expression system.

PubMed

Ruppert, Martin; Woll, Jörn; Giritch, Anatoli; Genady, Ezzat; Ma, Xueyan; Stöckigt, Joachim

2005-11-01

Acetylajmalan esterase (AAE) plays an essential role in the late stage of ajmaline biosynthesis. Based on the partial peptide sequences of AAE isolated and purified from Rauvolfia cell suspensions, a full-length AAE cDNA clone was isolated. The amino acid sequence of AAE has the highest level of identity of 40% to putative lipases known from the Arabidopsis thaliana genome project. Based on the primary structure AAE is a new member of the GDSL lipase superfamily. The expression in Escherichia coli failed although a wide range of conditions were tested. With a novel virus-based plant expression system, it was possible to express AAE functionally in leaves of Nicotiana benthamiana Domin. An extraordinarily high enzyme activity was detected in the Nicotiana tissue, which exceeded that in Rauvolfia serpentina (L.) Benth. ex Kurz cell suspension cultures about 20-fold. This expression allowed molecular analysis of AAE for the first time and increased the number of functionally expressed alkaloid genes from Rauvolfia now to eight, and the number of ajmaline pathway-specific cDNAs to a total of six.

Optimal Management of Metastatic Melanoma: Current Strategies and Future Directions

PubMed Central

Batus, Marta; Waheed, Salman; Ruby, Carl; Petersen, Lindsay; Bines, Steven D.; Kaufman, Howard L.

2013-01-01

Melanoma is increasing in incidence and remains a major public health threat. Although the disease may be curable when identified early, advanced melanoma is characterized by widespread metastatic disease and a median survival of less than 10 months. In recent years, however, major advances in our understanding of the molecular nature of melanoma and the interaction of melanoma cells with the immune system have resulted in several new therapeutic strategies that are showing significant clinical benefit. Current therapeutic approaches include surgical resection of metastatic disease, chemotherapy, immunotherapy, and targeted therapy. Dacarbazine, interleukin-2, ipilimumab, and vemurafenib are now approved for the treatment of advanced melanoma. In addition, new combination chemotherapy regimens, monoclonal antibodies blocking the programmed death-1 (PD-1)/PD-ligand 1 pathway, and targeted therapy against CKIT, mitogen-activated protein/extracellular signal-regulated kinase (MEK), and other putative signaling pathways in melanoma are beginning to show promise in early-phase clinical trials. Further research on these modalities alone and in combination will likely be the focus of future clinical investigation and may impact the outcomes for patients with advanced melanoma. PMID:23677693

Systems Biology Approach Reveals a Calcium-Dependent Mechanism for Basal Toxicity in Daphnia magna.

PubMed

Antczak, Philipp; White, Thomas A; Giri, Anirudha; Michelangeli, Francesco; Viant, Mark R; Cronin, Mark T D; Vulpe, Chris; Falciani, Francesco

2015-09-15

The expanding diversity and ever increasing amounts of man-made chemicals discharged to the environment pose largely unknown hazards to ecosystem and human health. The concept of adverse outcome pathways (AOPs) emerged as a comprehensive framework for risk assessment. However, the limited mechanistic information available for most chemicals and a lack of biological pathway annotation in many species represent significant challenges to effective implementation of this approach. Here, a systems level, multistep modeling strategy demonstrates how to integrate information on chemical structure with mechanistic insight from genomic studies, and phenotypic effects to define a putative adverse outcome pathway. Results indicated that transcriptional changes indicative of intracellular calcium mobilization were significantly overrepresented in Daphnia magna (DM) exposed to sublethal doses of presumed narcotic chemicals with log Kow ≥ 1.8. Treatment of DM with a calcium ATPase pump inhibitor substantially recapitulated the common transcriptional changes. We hypothesize that calcium mobilization is a potential key molecular initiating event in DM basal (narcosis) toxicity. Heart beat rate analysis and metabolome analysis indicated sublethal effects consistent with perturbations of calcium preceding overt acute toxicity. Together, the results indicate that altered calcium homeostasis may be a key early event in basal toxicity or narcosis induced by lipophilic compounds.

De novo transcriptome analysis of immune response on cobia (Rachycentron canadum) infected with Photobacterium damselae subsp. piscicida revealed inhibition of complement components and involvement of MyD88-independent pathway.

PubMed

Tran, Hung Bao; Lee, Yen-Hung; Guo, Jiin-Ju; Cheng, Ta-Chih

2018-06-01

Cobia, Rachycentron canadum, one of the most important aquatic species in Taiwan, has suffered heavy losses from Photobacterium damselae subsp. piscicida, which is the causal agent of photobacteriosis. In this study, the transcriptomic profiles of livers and spleens from Pdp-infected and non-infected cobia were obtained for the first time by Illumina-based paired-end sequencing method with a focus on immune-related genes. In total, 164,882 high quality unigenes were obtained in four libraries. Following Pdp infection, 7302 differentially expressed unigenes from liver and 8600 differentially expressed unigenes from spleen were identified. Twenty-seven of the differently expressed genes were further validated by RT-qPCR (average correlation coefficient 0.839, p-value <0.01). Results indicated a negative regulation of complement components and increased expression of genes involved in MyD88-independent pathway. Moreover, a remarkable finding was the increased expression of IL-10, implying an inadequacy of immune responses. This study not only characterized several putative immune pathways, but also provided a better understanding of the molecular responses to photobacteriosis in cobia. Copyright © 2018 Elsevier Ltd. All rights reserved.

Transcriptome analysis of thermogenic Arum concinnatum reveals the molecular components of floral scent production

PubMed Central

Onda, Yoshihiko; Mochida, Keiichi; Yoshida, Takuhiro; Sakurai, Tetsuya; Seymour, Roger S.; Umekawa, Yui; Pirintsos, Stergios Arg; Shinozaki, Kazuo; Ito, Kikukatsu

2015-01-01

Several plant species can generate enough heat to increase their internal floral temperature above ambient temperature. Among thermogenic plants, Arum concinnatum shows the highest respiration activity during thermogenesis. However, an overall understanding of the genes related to plant thermogenesis has not yet been achieved. In this study, we performed de novo transcriptome analysis of flower organs in A. concinnatum. The de novo transcriptome assembly represented, in total, 158,490 non-redundant transcripts, and 53,315 of those showed significant homology with known genes. To explore genes associated with thermogenesis, we filtered 1266 transcripts that showed a significant correlation between expression pattern and the temperature trend of each sample. We confirmed five putative alternative oxidase transcripts were included in filtered transcripts as expected. An enrichment analysis of the Gene Ontology terms for the filtered transcripts suggested over-representation of genes involved in 1-deoxy-d-xylulose-5-phosphate synthase (DXS) activity. The expression profiles of DXS transcripts in the methyl-d-erythritol 4-phosphate (MEP) pathway were significantly correlated with thermogenic levels. Our results suggest that the MEP pathway is the main biosynthesis route for producing scent monoterpenes. To our knowledge, this is the first report describing the candidate pathway and the key enzyme for floral scent production in thermogenic plants. PMID:25736477

Zic2-associated holoprosencephaly is caused by a transient defect in the organizer region during gastrulation.

PubMed

Warr, Nicholas; Powles-Glover, Nicola; Chappell, Anna; Robson, Joan; Norris, Dominic; Arkell, Ruth M

2008-10-01

The putative transcription factor ZIC2 is associated with a defect of forebrain development, known as Holoprosencephaly (HPE), in humans and mouse, yet the mechanism by which aberrant ZIC2 function causes classical HPE is unexplained. The zinc finger domain of all mammalian Zic genes is highly homologous with that of the Gli genes, which are transcriptional mediators of Shh signalling. Mutations in Shh and many other Hh pathway members cause HPE and it has been proposed that Zic2 acts within the Shh pathway to cause HPE. We have investigated the embryological cause of Zic2-associated HPE and the relationship between Zic2 and the Shh pathway using mouse genetics. We show that Zic2 does not interact with Shh to produce HPE. Moreover, molecular defects that are able to account for the HPE phenotype are present in Zic2 mutants before the onset of Shh signalling. Mutation of Zic2 causes HPE via a transient defect in the function of the organizer region at mid-gastrulation which causes an arrest in the development of the prechordal plate (PCP), a structure required for forebrain midline morphogenesis. The analysis provides genetic evidence that Zic2 functions during organizer formation and that the PCP develops via a multi-step process.

Chemical methodology as a source of small-molecule checkpoint inhibitors and heat shock protein 70 (Hsp70) modulators.

PubMed

Huryn, Donna M; Brodsky, Jeffrey L; Brummond, Kay M; Chambers, Peter G; Eyer, Benjamin; Ireland, Alex W; Kawasumi, Masaoki; Laporte, Matthew G; Lloyd, Kayla; Manteau, Baptiste; Nghiem, Paul; Quade, Bettina; Seguin, Sandlin P; Wipf, Peter

2011-04-26

Unique chemical methodology enables the synthesis of innovative and diverse scaffolds and chemotypes and allows access to previously unexplored "chemical space." Compound collections based on such new synthetic methods can provide small-molecule probes of proteins and/or pathways whose functions are not fully understood. We describe the identification, characterization, and evolution of two such probes. In one example, a pathway-based screen for DNA damage checkpoint inhibitors identified a compound, MARPIN (ATM and ATR pathway inhibitor) that sensitizes p53-deficient cells to DNA-damaging agents. Modification of the small molecule and generation of an immobilized probe were used to selectively bind putative protein target(s) responsible for the observed activity. The second example describes a focused library approach that relied on tandem multicomponent reaction methodologies to afford a series of modulators of the heat shock protein 70 (Hsp70) molecular chaperone. The synthesis of libraries based on the structure of MAL3-101 generated a collection of chemotypes, each modulating Hsp70 function, but exhibiting divergent pharmacological activities. For example, probes that compromise the replication of a disease-associated polyomavirus were identified. These projects highlight the importance of chemical methodology development as a source of small-molecule probes and as a drug discovery starting point.

Chemical methodology as a source of small-molecule checkpoint inhibitors and heat shock protein 70 (Hsp70) modulators

PubMed Central

Huryn, Donna M.; Brodsky, Jeffrey L.; Brummond, Kay M.; Chambers, Peter G.; Eyer, Benjamin; Ireland, Alex W.; Kawasumi, Masaoki; LaPorte, Matthew G.; Lloyd, Kayla; Manteau, Baptiste; Nghiem, Paul; Quade, Bettina; Seguin, Sandlin P.; Wipf, Peter

2011-01-01

Unique chemical methodology enables the synthesis of innovative and diverse scaffolds and chemotypes and allows access to previously unexplored “chemical space.” Compound collections based on such new synthetic methods can provide small-molecule probes of proteins and/or pathways whose functions are not fully understood. We describe the identification, characterization, and evolution of two such probes. In one example, a pathway-based screen for DNA damage checkpoint inhibitors identified a compound, MARPIN (ATM and ATR pathway inhibitor) that sensitizes p53-deficient cells to DNA-damaging agents. Modification of the small molecule and generation of an immobilized probe were used to selectively bind putative protein target(s) responsible for the observed activity. The second example describes a focused library approach that relied on tandem multicomponent reaction methodologies to afford a series of modulators of the heat shock protein 70 (Hsp70) molecular chaperone. The synthesis of libraries based on the structure of MAL3-101 generated a collection of chemotypes, each modulating Hsp70 function, but exhibiting divergent pharmacological activities. For example, probes that compromise the replication of a disease-associated polyomavirus were identified. These projects highlight the importance of chemical methodology development as a source of small-molecule probes and as a drug discovery starting point. PMID:21502524

Patterns of population differentiation of candidate genes for cardiovascular disease.

PubMed

Kullo, Iftikhar J; Ding, Keyue

2007-07-12

The basis for ethnic differences in cardiovascular disease (CVD) susceptibility is not fully understood. We investigated patterns of population differentiation (FST) of a set of genes in etiologic pathways of CVD among 3 ethnic groups: Yoruba in Nigeria (YRI), Utah residents with European ancestry (CEU), and Han Chinese (CHB) + Japanese (JPT). We identified 37 pathways implicated in CVD based on the PANTHER classification and 416 genes in these pathways were further studied; these genes belonged to 6 biological processes (apoptosis, blood circulation and gas exchange, blood clotting, homeostasis, immune response, and lipoprotein metabolism). Genotype data were obtained from the HapMap database. We calculated FST for 15,559 common SNPs (minor allele frequency > or = 0.10 in at least one population) in genes that co-segregated among the populations, as well as an average-weighted FST for each gene. SNPs were classified as putatively functional (non-synonymous and untranslated regions) or non-functional (intronic and synonymous sites). Mean FST values for common putatively functional variants were significantly higher than FST values for nonfunctional variants. A significant variation in FST was also seen based on biological processes; the processes of 'apoptosis' and 'lipoprotein metabolism' showed an excess of genes with high FST. Thus, putative functional SNPs in genes in etiologic pathways for CVD show greater population differentiation than non-functional SNPs and a significant variance of FST values was noted among pairwise population comparisons for different biological processes. These results suggest a possible basis for varying susceptibility to CVD among ethnic groups.

The Putative Mevalonate Diphosphate Decarboxylase from Picrophilus torridus Is in Reality a Mevalonate-3-Kinase with High Potential for Bioproduction of Isobutene

PubMed Central

Hall, Stephen J.; Eastham, Graham; Licence, Peter; Stephens, Gill

2015-01-01

Mevalonate diphosphate decarboxylase (MVD) is an ATP-dependent enzyme that catalyzes the phosphorylation/decarboxylation of (R)-mevalonate-5-diphosphate to isopentenyl pyrophosphate in the mevalonate (MVA) pathway. MVD is a key enzyme in engineered metabolic pathways for bioproduction of isobutene, since it catalyzes the conversion of 3-hydroxyisovalerate (3-HIV) to isobutene, an important platform chemical. The putative homologue from Picrophilus torridus has been identified as a highly efficient variant in a number of patents, but its detailed characterization has not been reported. In this study, we have successfully purified and characterized the putative MVD from P. torridus. We discovered that it is not a decarboxylase per se but an ATP-dependent enzyme, mevalonate-3-kinase (M3K), which catalyzes the phosphorylation of MVA to mevalonate-3-phosphate. The enzyme's potential in isobutene formation is due to the conversion of 3-HIV to an unstable 3-phosphate intermediate that undergoes consequent spontaneous decarboxylation to form isobutene. Isobutene production rates were as high as 507 pmol min−1 g cells−1 using Escherichia coli cells expressing the enzyme and 2,880 pmol min−1 mg protein−1 with the purified histidine-tagged enzyme, significantly higher than reported previously. M3K is a key enzyme of the novel MVA pathway discovered very recently in Thermoplasma acidophilum. We suggest that P. torridus metabolizes MVA by the same pathway. PMID:25636853

Finding off-targets, biological pathways, and target diseases for chymase inhibitors via structure-based systems biology approach.

PubMed

Arooj, Mahreen; Sakkiah, Sugunadevi; Cao, Guang Ping; Kim, Songmi; Arulalapperumal, Venkatesh; Lee, Keun Woo

2015-07-01

Off-target binding connotes the binding of a small molecule of therapeutic significance to a protein target in addition to the primary target for which it was proposed. Progressively such off-targeting is emerging to be regular practice to reveal side effects. Chymase is an enzyme of hydrolase class that catalyzes hydrolysis of peptide bonds. A link between heart failure and chymase is ascribed, and a chymase inhibitor is in clinical phase II for treatment of heart failure. However, the underlying mechanisms of the off-target effects of human chymase inhibitors are still unclear. Here, we develop a robust computational strategy that is applicable to any enzyme system and that allows the prediction of drug effects on biological processes. Putative off-targets for chymase inhibitors were identified through various structural and functional similarity analyses along with molecular docking studies. Finally, literature survey was performed to incorporate these off-targets into biological pathways and to establish links between pathways and particular adverse effects. Off-targets of chymase inhibitors are linked to various biological pathways such as classical and lectin pathways of complement system, intrinsic and extrinsic pathways of coagulation cascade, and fibrinolytic system. Tissue kallikreins, granzyme M, neutrophil elastase, and mesotrypsin are also identified as off-targets. These off-targets and their associated pathways are elucidated for the effects of inflammation, cancer, hemorrhage, thrombosis, and central nervous system diseases (Alzheimer's disease). Prospectively, our approach is helpful not only to better understand the mechanisms of chymase inhibitors but also for drug repurposing exercises to find novel uses for these inhibitors. © 2014 Wiley Periodicals, Inc.

Molecular Basis for Mycophenolic Acid Biosynthesis in Penicillium brevicompactum▿†

PubMed Central

Regueira, Torsten Bak; Kildegaard, Kanchana Rueksomtawin; Hansen, Bjarne Gram; Mortensen, Uffe H.; Hertweck, Christian; Nielsen, Jens

2011-01-01

Mycophenolic acid (MPA) is the active ingredient in the increasingly important immunosuppressive pharmaceuticals CellCept (Roche) and Myfortic (Novartis). Despite the long history of MPA, the molecular basis for its biosynthesis has remained enigmatic. Here we report the discovery of a polyketide synthase (PKS), MpaC, which we successfully characterized and identified as responsible for MPA production in Penicillium brevicompactum. mpaC resides in what most likely is a 25-kb gene cluster in the genome of Penicillium brevicompactum. The gene cluster was successfully localized by targeting putative resistance genes, in this case an additional copy of the gene encoding IMP dehydrogenase (IMPDH). We report the cloning, sequencing, and the functional characterization of the MPA biosynthesis gene cluster by deletion of the polyketide synthase gene mpaC of P. brevicompactum and bioinformatic analyses. As expected, the gene deletion completely abolished MPA production as well as production of several other metabolites derived from the MPA biosynthesis pathway of P. brevicompactum. Our work sets the stage for engineering the production of MPA and analogues through metabolic engineering. PMID:21398490

Molecular Modeling, de novo Design and Synthesis of a Novel, Extracellular Binding Fibroblast Growth Factor Receptor 2 Inhibitor Alofanib (RPT835).

PubMed

Tsimafeyeu, Ilya; Daeyaert, Frits; Joos, Jean-Baptiste; Aken, Koen V; Ludes-Meyers, John; Byakhov, Mikhail; Tjulandin, Sergei

2016-01-01

Fibroblast growth factor (FGF) receptors (FGFRs) play a key role in tumor growth and angiogenesis. The present report describes our search for an extracellularly binding FGFR inhibitor using a combined molecular modeling and de novo design strategy. Based upon crystal structures of the receptor with its native ligand and knowledge of inhibiting peptides, we have developed a computational protocol that predicts the putative binding of a molecule to the extracellular domains of the receptor. This protocol, or scoring function, was used in combination with the de novo synthesis program 'SYNOPSIS' to generate high scoring and synthetically accessible compounds. Eight compounds belonging to 3 separate chemical classes were synthesized. One of these compounds, alofanib (RPT835), was found to be an effective inhibitor of the FGF/FGFR2 pathway. The preclinical in vitro data support an allosteric inhibition mechanism of RPT835. RPT835 potently inhibited growth of KATO III gastric cancer cells expressing FGFR2, with GI50 value of 10 nmol/L. These results provide strong rationale for the evaluation of compound in advanced cancers.

Mutations blocking side chain assembly, polymerization, or transport of a Wzy-dependent Streptococcus pneumoniae capsule are lethal in the absence of suppressor mutations and can affect polymer transfer to the cell wall.

PubMed

Xayarath, Bobbi; Yother, Janet

2007-05-01

Extracellular polysaccharides of many bacteria are synthesized by the Wzy polymerase-dependent mechanism, where long-chain polymers are assembled from undecaprenyl-phosphate-linked repeat units on the outer face of the cytoplasmic membrane. In gram-positive bacteria, Wzy-dependent capsules remain largely cell associated via membrane and peptidoglycan linkages. Like many Wzy-dependent capsules, the Streptococcus pneumoniae serotype 2 capsule is branched. In this study, we found that deletions of cps2K, cps2J, or cps2H, which encode a UDP-glucose dehydrogenase necessary for side chain synthesis, the putative Wzx transporter (flippase), and the putative Wzy polymerase, respectively, were obtained only in the presence of suppressor mutations. Most of the suppressor mutations were in cps2E, which encodes the initiating glycosyltransferase for capsule synthesis. The cps2K mutants containing the suppressor mutations produced low levels of high-molecular-weight polymer that was detected only in membrane fractions. cps2K-repaired mutants exhibited only modest increases in capsule production due to the effect of the secondary mutation, but capsule was detectable in both membrane and cell wall fractions. Lethality of the cps2K, cps2J, and cps2H mutations was likely due to sequestration of undecaprenyl-phosphate in the capsule pathway and either preclusion of its turnover for utilization in essential pathways or destabilization of the membrane due to an accumulation of lipid-linked intermediates. The results demonstrate that proper polymer assembly requires not only a functional transporter and polymerase but also complete repeat units. A central role for the initiating glycosyltransferase in controlling capsule synthesis is also suggested.

Enrichment of putative PAX8 target genes at serous epithelial ovarian cancer susceptibility loci.

PubMed

Kar, Siddhartha P; Adler, Emily; Tyrer, Jonathan; Hazelett, Dennis; Anton-Culver, Hoda; Bandera, Elisa V; Beckmann, Matthias W; Berchuck, Andrew; Bogdanova, Natalia; Brinton, Louise; Butzow, Ralf; Campbell, Ian; Carty, Karen; Chang-Claude, Jenny; Cook, Linda S; Cramer, Daniel W; Cunningham, Julie M; Dansonka-Mieszkowska, Agnieszka; Doherty, Jennifer Anne; Dörk, Thilo; Dürst, Matthias; Eccles, Diana; Fasching, Peter A; Flanagan, James; Gentry-Maharaj, Aleksandra; Glasspool, Rosalind; Goode, Ellen L; Goodman, Marc T; Gronwald, Jacek; Heitz, Florian; Hildebrandt, Michelle A T; Høgdall, Estrid; Høgdall, Claus K; Huntsman, David G; Jensen, Allan; Karlan, Beth Y; Kelemen, Linda E; Kiemeney, Lambertus A; Kjaer, Susanne K; Kupryjanczyk, Jolanta; Lambrechts, Diether; Levine, Douglas A; Li, Qiyuan; Lissowska, Jolanta; Lu, Karen H; Lubiński, Jan; Massuger, Leon F A G; McGuire, Valerie; McNeish, Iain; Menon, Usha; Modugno, Francesmary; Monteiro, Alvaro N; Moysich, Kirsten B; Ness, Roberta B; Nevanlinna, Heli; Paul, James; Pearce, Celeste L; Pejovic, Tanja; Permuth, Jennifer B; Phelan, Catherine; Pike, Malcolm C; Poole, Elizabeth M; Ramus, Susan J; Risch, Harvey A; Rossing, Mary Anne; Salvesen, Helga B; Schildkraut, Joellen M; Sellers, Thomas A; Sherman, Mark; Siddiqui, Nadeem; Sieh, Weiva; Song, Honglin; Southey, Melissa; Terry, Kathryn L; Tworoger, Shelley S; Walsh, Christine; Wentzensen, Nicolas; Whittemore, Alice S; Wu, Anna H; Yang, Hannah; Zheng, Wei; Ziogas, Argyrios; Freedman, Matthew L; Gayther, Simon A; Pharoah, Paul D P; Lawrenson, Kate

2017-02-14

Genome-wide association studies (GWAS) have identified 18 loci associated with serous ovarian cancer (SOC) susceptibility but the biological mechanisms driving these findings remain poorly characterised. Germline cancer risk loci may be enriched for target genes of transcription factors (TFs) critical to somatic tumorigenesis. All 615 TF-target sets from the Molecular Signatures Database were evaluated using gene set enrichment analysis (GSEA) and three GWAS for SOC risk: discovery (2196 cases/4396 controls), replication (7035 cases/21 693 controls; independent from discovery), and combined (9627 cases/30 845 controls; including additional individuals). The PAX8-target gene set was ranked 1/615 in the discovery (P GSEA <0.001; FDR=0.21), 7/615 in the replication (P GSEA =0.004; FDR=0.37), and 1/615 in the combined (P GSEA <0.001; FDR=0.21) studies. Adding other genes reported to interact with PAX8 in the literature to the PAX8-target set and applying an alternative to GSEA, interval enrichment, further confirmed this association (P=0.006). Fifteen of the 157 genes from this expanded PAX8 pathway were near eight loci associated with SOC risk at P<10 -5 (including six with P<5 × 10 -8 ). The pathway was also associated with differential gene expression after shRNA-mediated silencing of PAX8 in HeyA8 (P GSEA =0.025) and IGROV1 (P GSEA =0.004) SOC cells and several PAX8 targets near SOC risk loci demonstrated in vitro transcriptomic perturbation. Putative PAX8 target genes are enriched for common SOC risk variants. This finding from our agnostic evaluation is of particular interest given that PAX8 is well-established as a specific marker for the cell of origin of SOC.

A New Set of ESTs from Chickpea (Cicer arietinum L.) Embryo Reveals Two Novel F-Box Genes, CarF-box_PP2 and CarF-box_LysM, with Potential Roles in Seed Development

PubMed Central

Gupta, Shefali; Garg, Vanika; Bhatia, Sabhyata

2015-01-01

Considering the economic importance of chickpea (C. arietinum L.) seeds, it is important to understand the mechanisms underlying seed development for which a cDNA library was constructed from 6 day old chickpea embryos. A total of 8,186 ESTs were obtained from which 4,048 high quality ESTs were assembled into 1,480 unigenes that majorly encoded genes involved in various metabolic and regulatory pathways. Of these, 95 ESTs were found to be involved in ubiquitination related protein degradation pathways and 12 ESTs coded specifically for putative F-box proteins. Differential transcript accumulation of these putative F-box genes was observed in chickpea tissues as evidenced by quantitative real-time PCR. Further, to explore the role of F-box proteins in chickpea seed development, two F-box genes were selected for molecular characterization. These were named as CarF-box_PP2 and CarF-box_LysM depending on their C-terminal domains, PP2 and LysM, respectively. Their highly conserved structures led us to predict their target substrates. Subcellular localization experiment revealed that CarF-box_PP2 was localized in the cytoplasm and CarF-box_LysM was localized in the nucleus. We demonstrated their physical interactions with SKP1 protein, which validated that they function as F-box proteins in the formation of SCF complexes. Sequence analysis of their promoter regions revealed certain seed specific cis-acting elements that may be regulating their preferential transcript accumulation in the seed. Overall, the study helped in expanding the EST database of chickpea, which was further used to identify two novel F-box genes having a potential role in seed development. PMID:25803812

Differential Effects of the Putative GBF1 Inhibitors Golgicide A and AG1478 on Enterovirus Replication▿

PubMed Central

van der Linden, Lonneke; van der Schaar, Hilde M.; Lanke, Kjerstin H. W.; Neyts, Johan; van Kuppeveld, Frank J. M.

2010-01-01

The genus Enterovirus, belonging to the family Picornaviridae, includes well-known pathogens, such as poliovirus, coxsackievirus, and rhinovirus. Brefeldin A (BFA) impedes replication of several enteroviruses through inhibition of Golgi-specific BFA resistance factor 1 (GBF1), a regulator of secretory pathway integrity and transport. GBF1 mediates the GTP exchange of Arf1, which in activated form recruits coatomer protein complex I (COP-I) to Golgi vesicles, a process important in transport between the endoplasmic reticulum and Golgi vesicles. Recently, the drugs AG1478 and Golgicide A (GCA) were put forward as new inhibitors of GBF1. In this study, we investigated the effects of these putative GBF1 inhibitors on secretory pathway function and enterovirus replication. We show that both drugs induced fragmentation of the Golgi vesicles and caused dissociation of Arf1 and COP-I from Golgi membranes, yet they differed in their effect on GBF1 localization. The effects of AG1478, but not those of GCA, could be countered by overexpression of Arf1, indicating a difference in their molecular mechanism of action. Consistent with this idea, we observed that GCA drastically reduced replication of coxsackievirus B3 (CVB3) and other human enterovirus species, whereas AG1478 had no effect at all on enterovirus replication. Time-of-addition studies and analysis of RNA replication using a subgenomic replicon both showed that GCA suppresses RNA replication of CVB3, which could be countered by overexpression of GBF1. These results indicate that, in contrast to AG1478, GCA inhibits CVB3 RNA replication by targeting GBF1. AG1478 and GCA may be valuable tools to further dissect enterovirus replication. PMID:20504936

Synchronization of developmental processes and defense signaling by growth regulating transcription factors.

PubMed

Liu, Jinyi; Rice, J Hollis; Chen, Nana; Baum, Thomas J; Hewezi, Tarek

2014-01-01

Growth regulating factors (GRFs) are a conserved class of transcription factor in seed plants. GRFs are involved in various aspects of tissue differentiation and organ development. The implication of GRFs in biotic stress response has also been recently reported, suggesting a role of these transcription factors in coordinating the interaction between developmental processes and defense dynamics. However, the molecular mechanisms by which GRFs mediate the overlaps between defense signaling and developmental pathways are elusive. Here, we report large scale identification of putative target candidates of Arabidopsis GRF1 and GRF3 by comparing mRNA profiles of the grf1/grf2/grf3 triple mutant and those of the transgenic plants overexpressing miR396-resistant version of GRF1 or GRF3. We identified 1,098 and 600 genes as putative targets of GRF1 and GRF3, respectively. Functional classification of the potential target candidates revealed that GRF1 and GRF3 contribute to the regulation of various biological processes associated with defense response and disease resistance. GRF1 and GRF3 participate specifically in the regulation of defense-related transcription factors, cell-wall modifications, cytokinin biosynthesis and signaling, and secondary metabolites accumulation. GRF1 and GRF3 seem to fine-tune the crosstalk between miRNA signaling networks by regulating the expression of several miRNA target genes. In addition, our data suggest that GRF1 and GRF3 may function as negative regulators of gene expression through their association with other transcription factors. Collectively, our data provide new insights into how GRF1 and GRF3 might coordinate the interactions between defense signaling and plant growth and developmental pathways.

Enrichment of putative PAX8 target genes at serous epithelial ovarian cancer susceptibility loci

PubMed Central

Kar, Siddhartha P; Adler, Emily; Tyrer, Jonathan; Hazelett, Dennis; Anton-Culver, Hoda; Bandera, Elisa V; Beckmann, Matthias W; Berchuck, Andrew; Bogdanova, Natalia; Brinton, Louise; Butzow, Ralf; Campbell, Ian; Carty, Karen; Chang-Claude, Jenny; Cook, Linda S; Cramer, Daniel W; Cunningham, Julie M; Dansonka-Mieszkowska, Agnieszka; Doherty, Jennifer Anne; Dörk, Thilo; Dürst, Matthias; Eccles, Diana; Fasching, Peter A; Flanagan, James; Gentry-Maharaj, Aleksandra; Glasspool, Rosalind; Goode, Ellen L; Goodman, Marc T; Gronwald, Jacek; Heitz, Florian; Hildebrandt, Michelle A T; Høgdall, Estrid; Høgdall, Claus K; Huntsman, David G; Jensen, Allan; Karlan, Beth Y; Kelemen, Linda E; Kiemeney, Lambertus A; Kjaer, Susanne K; Kupryjanczyk, Jolanta; Lambrechts, Diether; Levine, Douglas A; Li, Qiyuan; Lissowska, Jolanta; Lu, Karen H; Lubiński, Jan; Massuger, Leon F A G; McGuire, Valerie; McNeish, Iain; Menon, Usha; Modugno, Francesmary; Monteiro, Alvaro N; Moysich, Kirsten B; Ness, Roberta B; Nevanlinna, Heli; Paul, James; Pearce, Celeste L; Pejovic, Tanja; Permuth, Jennifer B; Phelan, Catherine; Pike, Malcolm C; Poole, Elizabeth M; Ramus, Susan J; Risch, Harvey A; Rossing, Mary Anne; Salvesen, Helga B; Schildkraut, Joellen M; Sellers, Thomas A; Sherman, Mark; Siddiqui, Nadeem; Sieh, Weiva; Song, Honglin; Southey, Melissa; Terry, Kathryn L; Tworoger, Shelley S; Walsh, Christine; Wentzensen, Nicolas; Whittemore, Alice S; Wu, Anna H; Yang, Hannah; Zheng, Wei; Ziogas, Argyrios; Freedman, Matthew L; Gayther, Simon A; Pharoah, Paul D P; Lawrenson, Kate

2017-01-01

Background: Genome-wide association studies (GWAS) have identified 18 loci associated with serous ovarian cancer (SOC) susceptibility but the biological mechanisms driving these findings remain poorly characterised. Germline cancer risk loci may be enriched for target genes of transcription factors (TFs) critical to somatic tumorigenesis. Methods: All 615 TF-target sets from the Molecular Signatures Database were evaluated using gene set enrichment analysis (GSEA) and three GWAS for SOC risk: discovery (2196 cases/4396 controls), replication (7035 cases/21 693 controls; independent from discovery), and combined (9627 cases/30 845 controls; including additional individuals). Results: The PAX8-target gene set was ranked 1/615 in the discovery (PGSEA<0.001; FDR=0.21), 7/615 in the replication (PGSEA=0.004; FDR=0.37), and 1/615 in the combined (PGSEA<0.001; FDR=0.21) studies. Adding other genes reported to interact with PAX8 in the literature to the PAX8-target set and applying an alternative to GSEA, interval enrichment, further confirmed this association (P=0.006). Fifteen of the 157 genes from this expanded PAX8 pathway were near eight loci associated with SOC risk at P<10−5 (including six with P<5 × 10−8). The pathway was also associated with differential gene expression after shRNA-mediated silencing of PAX8 in HeyA8 (PGSEA=0.025) and IGROV1 (PGSEA=0.004) SOC cells and several PAX8 targets near SOC risk loci demonstrated in vitro transcriptomic perturbation. Conclusions: Putative PAX8 target genes are enriched for common SOC risk variants. This finding from our agnostic evaluation is of particular interest given that PAX8 is well-established as a specific marker for the cell of origin of SOC. PMID:28103614

Population Genetic Analysis Infers Migration Pathways of Phytophthora ramorum in US Nurseries

PubMed Central

Goss, Erica M.; Larsen, Meg; Chastagner, Gary A.; Givens, Donald R.; Grünwald, Niklaus J.

2009-01-01

Recently introduced, exotic plant pathogens may exhibit low genetic diversity and be limited to clonal reproduction. However, rapidly mutating molecular markers such as microsatellites can reveal genetic variation within these populations and be used to model putative migration patterns. Phytophthora ramorum is the exotic pathogen, discovered in the late 1990s, that is responsible for sudden oak death in California forests and ramorum blight of common ornamentals. The nursery trade has moved this pathogen from source populations on the West Coast to locations across the United States, thus risking introduction to other native forests. We examined the genetic diversity of P. ramorum in United States nurseries by microsatellite genotyping 279 isolates collected from 19 states between 2004 and 2007. Of the three known P. ramorum clonal lineages, the most common and genetically diverse lineage in the sample was NA1. Two eastward migration pathways were revealed in the clustering of NA1 isolates into two groups, one containing isolates from Connecticut, Oregon, and Washington and the other isolates from California and the remaining states. This finding is consistent with trace forward analyses conducted by the US Department of Agriculture's Animal and Plant Health Inspection Service. At the same time, genetic diversities in several states equaled those observed in California, Oregon, and Washington and two-thirds of multilocus genotypes exhibited limited geographic distributions, indicating that mutation was common during or subsequent to migration. Together, these data suggest that migration, rapid mutation, and genetic drift all play a role in structuring the genetic diversity of P. ramorum in US nurseries. This work demonstrates that fast-evolving genetic markers can be used to examine the evolutionary processes acting on recently introduced pathogens and to infer their putative migration patterns, thus showing promise for the application of forensics to plant pathogens. PMID:19774068

De novo sequencing analysis of the Rosa roxburghii fruit transcriptome reveals putative ascorbate biosynthetic genes and EST-SSR markers.

PubMed

Yan, Xiuqin; Zhang, Xue; Lu, Min; He, Yong; An, Huaming

2015-04-25

Rosa roxburghii Tratt. is a well-known ornamental rose species native to China. In addition, the fruits of this species are valued for their nutritional and medicinal characteristics, especially their high ascorbic acid (AsA) levels. Nevertheless, AsA biosynthesis in R. roxburghii fruit has not been explored in detail because of a lack of genomic resources for this species. High-throughput transcriptomic sequencing generating large volumes of transcript sequence data can aid in gene discovery and molecular marker development. In this study, we generated more than 53 million clean reads using Illumina paired-end sequencing technology. De novo assembly yielded 106,590 unigenes, with an average length of 343 bp. On the basis of sequence similarity to known proteins, 9301 and 2393 unigenes were classified into Gene Ontology and Clusters of Orthologous Group categories, respectively. There were 7480 unigenes assigned to 124 pathways in the Kyoto Encyclopedia of Gene and Genome pathway database. BLASTx searches identified 498 unique putative transcripts encoding various transcription factors, some known to regulate fruit development. qRT-PCR validated the expressions of most of the genes encoding the main enzymes involved in ascorbate biosynthesis. In addition, 9131 potential simple sequence repeat (SSR) loci were identified among the unigenes. One hundred and two primer pairs were synthesized and 71 pairs produced an amplification product during initial screening. Among the amplified products, 30 were polymorphic in the 16 R. roxburghii germplasms tested. Our study was the first to produce a large volume of transcriptome data from R. roxburghii. The resulting sequence collection is a valuable resource for gene discovery and marker-assisted selective breeding in this rose species. Copyright © 2015 Elsevier B.V. All rights reserved.

ESTs Analysis Reveals Putative Genes Involved in Symbiotic Seed Germination in Dendrobium officinale

PubMed Central

Zhao, Ming-Ming; Zhang, Gang; Zhang, Da-Wei; Hsiao, Yu-Yun; Guo, Shun-Xing

2013-01-01

Dendrobium officinale (Orchidaceae) is one of the world’s most endangered plants with great medicinal value. In nature, D . officinale seeds must establish symbiotic relationships with fungi to germinate. However, the molecular events involved in the interaction between fungus and plant during this process are poorly understood. To isolate the genes involved in symbiotic germination, a suppression subtractive hybridization (SSH) cDNA library of symbiotically germinated D . officinale seeds was constructed. From this library, 1437 expressed sequence tags (ESTs) were clustered to 1074 Unigenes (including 902 singletons and 172 contigs), which were searched against the NCBI non-redundant (NR) protein database (E-value cutoff, e-5). Based on sequence similarity with known proteins, 579 differentially expressed genes in D . officinale were identified and classified into different functional categories by Gene Ontology (GO), Clusters of orthologous Groups of proteins (COGs) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The expression levels of 15 selected genes emblematic of symbiotic germination were confirmed via real-time quantitative PCR. These genes were classified into various categories, including defense and stress response, metabolism, transcriptional regulation, transport process and signal transduction pathways. All transcripts were upregulated in the symbiotically germinated seeds (SGS). The functions of these genes in symbiotic germination were predicted. Furthermore, two fungus-induced calcium-dependent protein kinases (CDPKs), which were upregulated 6.76- and 26.69-fold in SGS compared with un-germinated seeds (UGS), were cloned from D . officinale and characterized for the first time. This study provides the first global overview of genes putatively involved in D . officinale symbiotic seed germination and provides a foundation for further functional research regarding symbiotic relationships in orchids. PMID:23967335

ESTs analysis reveals putative genes involved in symbiotic seed germination in Dendrobium officinale.

PubMed

Zhao, Ming-Ming; Zhang, Gang; Zhang, Da-Wei; Hsiao, Yu-Yun; Guo, Shun-Xing

2013-01-01

Dendrobiumofficinale (Orchidaceae) is one of the world's most endangered plants with great medicinal value. In nature, D. officinale seeds must establish symbiotic relationships with fungi to germinate. However, the molecular events involved in the interaction between fungus and plant during this process are poorly understood. To isolate the genes involved in symbiotic germination, a suppression subtractive hybridization (SSH) cDNA library of symbiotically germinated D. officinale seeds was constructed. From this library, 1437 expressed sequence tags (ESTs) were clustered to 1074 Unigenes (including 902 singletons and 172 contigs), which were searched against the NCBI non-redundant (NR) protein database (E-value cutoff, e(-5)). Based on sequence similarity with known proteins, 579 differentially expressed genes in D. officinale were identified and classified into different functional categories by Gene Ontology (GO), Clusters of orthologous Groups of proteins (COGs) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The expression levels of 15 selected genes emblematic of symbiotic germination were confirmed via real-time quantitative PCR. These genes were classified into various categories, including defense and stress response, metabolism, transcriptional regulation, transport process and signal transduction pathways. All transcripts were upregulated in the symbiotically germinated seeds (SGS). The functions of these genes in symbiotic germination were predicted. Furthermore, two fungus-induced calcium-dependent protein kinases (CDPKs), which were upregulated 6.76- and 26.69-fold in SGS compared with un-germinated seeds (UGS), were cloned from D. officinale and characterized for the first time. This study provides the first global overview of genes putatively involved in D. officinale symbiotic seed germination and provides a foundation for further functional research regarding symbiotic relationships in orchids.

Genomic analysis of the interaction between pesticide exposure and nutrition in honey bees (Apis mellifera).

PubMed

Schmehl, Daniel R; Teal, Peter E A; Frazier, James L; Grozinger, Christina M

2014-12-01

Populations of pollinators are in decline worldwide. These declines are best documented in honey bees and are due to a combination of stressors. In particular, pesticides have been linked to decreased longevity and performance in honey bees; however, the molecular and physiological pathways mediating sensitivity and resistance to pesticides are not well characterized. We explored the impact of coumaphos and fluvalinate, the two most abundant and frequently detected pesticides in the hive, on genome-wide gene expression patterns of honey bee workers. We found significant changes in 1118 transcripts, including genes involved in detoxification, behavioral maturation, immunity, and nutrition. Since behavioral maturation is regulated by juvenile hormone III (JH), we examined effects of these miticides on hormone titers; while JH titers were unaffected, titers of methyl farnesoate (MF), the precursor to JH, were decreased. We further explored the association between nutrition- and pesticide-regulated gene expression patterns and demonstrated that bees fed a pollen-based diet exhibit reduced sensitivity to a third pesticide, chlorpyrifos. Finally, we demonstrated that expression levels of several of the putative pesticide detoxification genes identified in our study and previous studies are also upregulated in response to pollen feeding, suggesting that these pesticides and components in pollen modulate similar molecular response pathways. Our results demonstrate that pesticide exposure can substantially impact expression of genes involved in several core physiological pathways in honey bee workers. Additionally, there is substantial overlap in responses to pesticides and pollen-containing diets at the transcriptional level, and subsequent analyses demonstrated that pollen-based diets reduce workers' pesticide sensitivity. Thus, providing honey bees and other pollinators with high quality nutrition may improve resistance to pesticides. Copyright © 2014 Elsevier Ltd. All rights reserved.

A membrane-embedded pathway delivers general anesthetics to two interacting binding sites in the Gloeobacter violaceus ion channel.

PubMed

Arcario, Mark J; Mayne, Christopher G; Tajkhorshid, Emad

2017-06-09

General anesthetics exert their effects on the central nervous system by acting on ion channels, most notably pentameric ligand-gated ion channels. Although numerous studies have focused on pentameric ligand-gated ion channels, the details of anesthetic binding and channel modulation are still debated. A better understanding of the anesthetic mechanism of action is necessary for the development of safer and more efficacious drugs. Herein, we present a computational study identifying two anesthetic binding sites in the transmembrane domain of the Gloeobacter violaceus ligand-gated ion channel (GLIC) channel, characterize the putative binding pathway, and observe structural changes associated with channel function. Molecular simulations of desflurane reveal a binding pathway to GLIC via a membrane-embedded tunnel using an intrasubunit protein lumen as the conduit, an observation that explains the Meyer-Overton hypothesis, or why the lipophilicity of an anesthetic and its potency are generally proportional. Moreover, employing high concentrations of ligand led to the identification of a second transmembrane site (TM2) that inhibits dissociation of anesthetic from the TM1 site and is consistent with the high concentrations of anesthetics required to achieve clinical effects. Finally, asymmetric binding patterns of anesthetic to the channel were found to promote an iris-like conformational change that constricts and dehydrates the ion pore, creating a 13.5 kcal/mol barrier to ion translocation. Together with previous studies, the simulations presented herein demonstrate a novel anesthetic binding site in GLIC that is accessed through a membrane-embedded tunnel and interacts with a previously known site, resulting in conformational changes that produce a non-conductive state of the channel. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Common developmental pathways link tooth shape to regeneration

PubMed Central

Fraser, Gareth J.; Bloomquist, Ryan F.; Streelman, J. Todd

2013-01-01

In many non-mammalian vertebrates, adult dentitions result from cyclical rounds of tooth regeneration wherein simple unicuspid teeth are replaced by more complex forms. Therefore and by contrast to mammalian models, the numerical majority of vertebrate teeth develop shape during the process of replacement. Here, we exploit the dental diversity of Lake Malawi cichlid fishes to ask how vertebrates generally replace their dentition and in turn how this process acts to influence resulting tooth morphologies. First, we used immunohistochemistry to chart organogenesis of continually replacing cichlid teeth and discovered an epithelial down-growth that initiates the replacement cycle via a labial proliferation bias. Next, we identified sets of co-expressed genes from common pathways active during de novo, lifelong tooth replacement and tooth morphogenesis. Of note, we found two distinct epithelial cell populations, expressing markers of dental competence and cell potency, which may be responsible for tooth regeneration. Related gene sets were simultaneously active in putative signaling centers associated with the differentiation of replacement teeth with complex shapes. Finally, we manipulated targeted pathways (BMP, FGF, Hh, Notch, Wnt/β-catenin) in vivo with small molecules and demonstrated dose-dependent effects on both tooth replacement and tooth shape. Our data suggest that the processes of tooth regeneration and tooth shape morphogenesis are integrated via a common set of molecular signals. This linkage has subsequently been lost or decoupled in mammalian dentitions where complex tooth shapes develop in first generation dentitions that lack the capacity for lifelong replacement. Our dissection of the molecular mechanics of vertebrate tooth replacement coupled to complex shape pinpoints aspects of odontogenesis that might be re-evolved in the lab to solve problems in regenerative dentistry. PMID:23422830

Breast cancer risk associated with genotypic polymorphism of the nonhomologous end-joining genes: a multigenic study on cancer susceptibility.

PubMed

Fu, Yi-Ping; Yu, Jyh-Cherng; Cheng, Ting-Chih; Lou, M Ann; Hsu, Giu-Cheng; Wu, Chia-Yun; Chen, Sou-Tong; Wu, Hurng-Sheng; Wu, Pei-Ei; Shen, Chen-Yang

2003-05-15

The role of the familial breast cancer susceptibility genes, BRCA1 and BRCA2, in the homologous recombination pathway for DNA double-strand break (DSB) repair suggests that the mechanisms involved in DNA DSB repair are of particular etiological importance during breast tumorigenesis. However, there is currently no evidence for an association between breast cancer and the other DSB repair pathway, the nonhomologous end-joining (NHEJ) pathway. It is possible that, because this DNA repair pathway is so crucial for mammalian cells to maintain genomic stability, any severe defects in it would result in serious outcomes, such as genomic instability and cell death, and block subsequent cell outgrowth and tumor formation. Thus, only subtle defects arising from low-penetrance alleles would escape lethality accumulating essential genetic changes and be associated with cancer formation, and the tumorigenic contribution of these alleles would become more obvious if individual putative high-risk genotypes of each NHEJ gene act jointly. Furthermore, this joint effect might be modified by specific environmental factors, and we hypothesized that estrogen exposure might be one such factor because estrogen is suggested to cause DNA DSBs, triggering breast tumorigenesis. Because single nucleotide polymorphisms (SNPs) are the most subtle genetic variation in the genome, to examine these hypotheses, we have genotyped 30 SNPs in all five NHEJ genes (Ku70, Ku80, DNA-PKcs, Ligase IV, and XRCC4) in 254 primary breast cancer patients and 379 healthy controls. Support for these hypotheses came from the observations that (a) two SNPs in Ku70 and XRCC4 were associated with breast cancer risk (P < 0.05); (b) a trend toward increased risk of developing breast cancer was found in women harboring a greater number of putative high-risk genotypes of NHEJ genes (an adjusted odds ratio of 1.46 for having one additional putative high-risk genotype; 95% confidence interval, 1.19-1.80); (c) this association between risk and the number of putative high-risk genotypes was stronger and more significant in women thought to be more susceptible to estrogen, i.e., those with no history of full-term pregnancy; and (d) the protective effect conferred by a history of full-term pregnancy was only significant in women with a lower number of putative high-risk genotypes of NHEJ genes. Based on comprehensive NHEJ gene profiles, this study provides new insights to suggest the role of the NHEJ pathway in breast cancer development and supports the possibility that breast cancer is initiated by estrogen exposure, which causes DNA DSBs.

Genome-wide digital transcript analysis of putative fruitlet abscission related genes regulated by ethephon in litchi

PubMed Central

Li, Caiqin; Wang, Yan; Ying, Peiyuan; Ma, Wuqiang; Li, Jianguo

2015-01-01

The high level of physiological fruitlet abscission in litchi (Litchi chinensis Sonn.) causes severe yield loss. Cell separation occurs at the fruit abscission zone (FAZ) and can be triggered by ethylene. However, a deep knowledge of the molecular events occurring in the FAZ is still unknown. Here, genome-wide digital transcript abundance (DTA) analysis of putative fruit abscission related genes regulated by ethephon in litchi were studied. More than 81 million high quality reads from seven ethephon treated and untreated control libraries were obtained by high-throughput sequencing. Through DTA profile analysis in combination with Gene Ontology and KEGG pathway enrichment analyses, a total of 2730 statistically significant candidate genes were involved in the ethephon-promoted litchi fruitlet abscission. Of these, there were 1867 early-responsive genes whose expressions were up- or down-regulated from 0 to 1 d after treatment. The most affected genes included those related to ethylene biosynthesis and signaling, auxin transport and signaling, transcription factors (TFs), protein ubiquitination, ROS response, calcium signal transduction, and cell wall modification. These genes could be clustered into four groups and 13 subgroups according to their similar expression patterns. qRT-PCR displayed the expression pattern of 41 selected candidate genes, which proved the accuracy of our DTA data. Ethephon treatment significantly increased fruit abscission and ethylene production of fruitlet. The possible molecular events to control the ethephon-promoted litchi fruitlet abscission were prompted out. The increased ethylene evolution in fruitlet would suppress the synthesis and polar transport of auxin and trigger abscission signaling. To the best of our knowledge, it is the first time to monitor the gene expression profile occurring in the FAZ-enriched pedicel during litchi fruit abscission induced by ethephon on the genome-wide level. This study will contribute to a better understanding for the molecular regulatory mechanism of fruit abscission in litchi. PMID:26217356

Tiered High-Throughput Screening Approach to Identify ...

EPA Pesticide Factsheets

High-throughput screening (HTS) for potential thyroid–disrupting chemicals requires a system of assays to capture multiple molecular-initiating events (MIEs) that converge on perturbed thyroid hormone (TH) homeostasis. Screening for MIEs specific to TH-disrupting pathways is limited in the US EPA ToxCast screening assay portfolio. To fill one critical screening gap, the Amplex UltraRed-thyroperoxidase (AUR-TPO) assay was developed to identify chemicals that inhibit TPO, as decreased TPO activity reduces TH synthesis. The ToxCast Phase I and II chemical libraries, comprised of 1,074 unique chemicals, were initially screened using a single, high concentration to identify potential TPO inhibitors. Chemicals positive in the single concentration screen were retested in concentration-response. Due to high false positive rates typically observed with loss-of-signal assays such as AUR-TPO, we also employed two additional assays in parallel to identify possible sources of nonspecific assay signal loss, enabling stratification of roughly 300 putative TPO inhibitors based upon selective AUR-TPO activity. A cell-free luciferase inhibition assay was used to identify nonspecific enzyme inhibition among the putative TPO inhibitors, and a cytotoxicity assay using a human cell line was used to estimate the cellular tolerance limit. Additionally, the TPO inhibition activities of 150 chemicals were compared between the AUR-TPO and an orthogonal peroxidase oxidation assay using

Chemogenomics profiling of drug targets of peptidoglycan biosynthesis pathway in Leptospira interrogans by virtual screening approaches.

PubMed

Bhattacharjee, Biplab; Simon, Rose Mary; Gangadharaiah, Chaithra; Karunakar, Prashantha

2013-06-28

Leptospirosis is a worldwide zoonosis of global concern caused by Leptospira interrogans. The availability of ligand libraries has facilitated the search for novel drug targets using chemogenomics approaches, compared with the traditional method of drug discovery, which is time consuming and yields few leads with little intracellular information for guiding target selection. Recent subtractive genomics studies have revealed the putative drug targets in peptidoglycan biosynthesis pathways in Leptospira interrogans. Aligand library for the murD ligase enzyme in the peptidoglycan pathway has also been identified. Our approach in this research involves screening of the pre-existing ligand library of murD with related protein family members in the putative drug target assembly in the peptidoglycan biosynthesis pathway. A chemogenomics approach has been implemented here, which involves screening of known ligands of a protein family having analogous domain architecture for identification of leads for existing druggable protein family members. By means of this approach, one murC and one murF inhibitor were identified, providing a platform for developing an antileptospirosis drug targeting the peptidoglycan biosynthesis pathway. Given that the peptidoglycan biosynthesis pathway is exclusive to bacteria, the in silico identified mur ligase inhibitors are expected to be broad-spectrum Gram-negative inhibitors if synthesized and tested in in vitro and in vivo assays.

Molecular characterization of SG33 and Borghi vaccines used against myxomatosis.

PubMed

Cavadini, Patrizia; Botti, Giuliana; Barbieri, Ilaria; Lavazza, Antonio; Capucci, Lorenzo

2010-07-26

Myxoma virus is a poxvirus responsible for myxomatosis in European Rabbits (Oryctolagus cuniculus). The entire genome of the myxoma virus has been sequenced, allowing a systemic survey of the functions of a large number of putative pathogenic factors that this virus expresses to subvert the immune and inflammatory pathways of infected rabbit hosts. In Italy, industrial rabbits are mostly vaccinated against myxomatosis using the attenuated myxoma virus strains Borghi or SG33. We have identified genetic markers specific for Borghi or SG33 vaccine strains and established a PCR-based assay that could be used to: (a) rapidly diagnose the presence of myxoma virus in infected organs; (b) discriminate between field strain-infected and vaccinated rabbits and (c) differentiate between Borghi or SG33 vaccine strain. Copyright 2010 Elsevier Ltd. All rights reserved.

The autophagy interaction network of the aging model Podospora anserina.

PubMed

Philipp, Oliver; Hamann, Andrea; Osiewacz, Heinz D; Koch, Ina

2017-03-27

Autophagy is a conserved molecular pathway involved in the degradation and recycling of cellular components. It is active either as response to starvation or molecular damage. Evidence is emerging that autophagy plays a key role in the degradation of damaged cellular components and thereby affects aging and lifespan control. In earlier studies, it was found that autophagy in the aging model Podospora anserina acts as a longevity assurance mechanism. However, only little is known about the individual components controlling autophagy in this aging model. Here, we report a biochemical and bioinformatics study to detect the protein-protein interaction (PPI) network of P. anserina combining experimental and theoretical methods. We constructed the PPI network of autophagy in P. anserina based on the corresponding networks of yeast and human. We integrated PaATG8 interaction partners identified in an own yeast two-hybrid analysis using ATG8 of P. anserina as bait. Additionally, we included age-dependent transcriptome data. The resulting network consists of 89 proteins involved in 186 interactions. We applied bioinformatics approaches to analyze the network topology and to prove that the network is not random, but exhibits biologically meaningful properties. We identified hub proteins which play an essential role in the network as well as seven putative sub-pathways, and interactions which are likely to be evolutionary conserved amongst species. We confirmed that autophagy-associated genes are significantly often up-regulated and co-expressed during aging of P. anserina. With the present study, we provide a comprehensive biological network of the autophagy pathway in P. anserina comprising PPI and gene expression data. It is based on computational prediction as well as experimental data. We identified sub-pathways, important hub proteins, and evolutionary conserved interactions. The network clearly illustrates the relation of autophagy to aging processes and enables further specific studies to understand autophagy and aging in P. anserina as well as in other systems.

MBD5 haploinsufficiency is associated with sleep disturbance and disrupts circadian pathways common to Smith-Magenis and fragile X syndromes.

PubMed

Mullegama, Sureni V; Pugliesi, Loren; Burns, Brooke; Shah, Zalak; Tahir, Raiha; Gu, Yanghong; Nelson, David L; Elsea, Sarah H

2015-06-01

Individuals with autism spectrum disorders (ASD) who have an identifiable single-gene neurodevelopmental disorder (NDD), such as fragile X syndrome (FXS, FMR1), Smith-Magenis syndrome (SMS, RAI1), or 2q23.1 deletion syndrome (del 2q23.1, MBD5) share phenotypic features, including a high prevalence of sleep disturbance. We describe the circadian deficits in del 2q23.1 through caregiver surveys in which we identify several frequent sleep anomalies, including night/early awakenings, coughing/snoring loudly, and difficulty falling asleep. We couple these findings with studies on the molecular analysis of the circadian deficits associated with haploinsufficiency of MBD5 in which circadian gene mRNA levels of NR1D2, PER1, PER2, and PER3 were altered in del 2q23.1 lymphoblastoid cell lines (LCLs), signifying that haploinsufficiency of MBD5 can result in dysregulation of circadian rhythm gene expression. These findings were further supported by expression microarrays of MBD5 siRNA knockdown cells that showed significantly altered expression of additional circadian rhythm signaling pathway genes. Based on the common sleep phenotypes observed in del 2q23.1, SMS, and FXS patients, we explored the possibility that MBD5, RAI1, and FMR1 function in overlapping circadian rhythm pathways. Bioinformatic analysis identified conserved putative E boxes in MBD5 and RAI1, and expression levels of NR1D2 and CRY2 were significantly reduced in patient LCLs. Circadian and mTOR signaling pathways, both associated with sleep disturbance, were altered in both MBD5 and RAI1 knockdown microarray data, overlapping with findings associated with FMR1. These data support phenotypic and molecular overlaps across these syndromes that may be exploited to provide therapeutic intervention for multiple disorders.

Comparative Analyses of the β-Tubulin Gene and Molecular Modeling Reveal Molecular Insight into the Colchicine Resistance in Kinetoplastids Organisms

PubMed Central

Luis, Luis; Serrano, María Luisa; Hidalgo, Mariana; Mendoza-León, Alexis

2013-01-01

Differential susceptibility to microtubule agents has been demonstrated between mammalian cells and kinetoplastid organisms such as Leishmania spp. and Trypanosoma spp. The aims of this study were to identify and characterize the architecture of the putative colchicine binding site of Leishmania spp. and investigate the molecular basis of colchicine resistance. We cloned and sequenced the β-tubulin gene of Leishmania (Viannia) guyanensis and established the theoretical 3D model of the protein, using the crystallographic structure of the bovine protein as template. We identified mutations on the Leishmania   β-tubulin gene sequences on regions related to the putative colchicine-binding pocket, which generate amino acid substitutions and changes in the topology of this region, blocking the access of colchicine. The same mutations were found in the β-tubulin sequence of kinetoplastid organisms such as Trypanosoma cruzi, T. brucei, and T. evansi. Using molecular modelling approaches, we demonstrated that conformational changes include an elongation and torsion of an α-helix structure and displacement to the inside of the pocket of one β-sheet that hinders access of colchicine. We propose that kinetoplastid organisms show resistance to colchicine due to amino acids substitutions that generate structural changes in the putative colchicine-binding domain, which prevent colchicine access. PMID:24083244

Contextualizing the Genes Altered in Bladder Neoplasms in Pediatric andTeen Patients Allows Identifying Two Main Classes of Biological ProcessesInvolved and New Potential Therapeutic Targets

PubMed Central

Porrello, A.; Piergentili, R. b

2016-01-01

Research on bladder neoplasms in pediatric and teen patients (BNPTP) has described 21 genes, which are variously involved in this disease and are mostly responsible for deregulated cell proliferation. However, due to the limited number of publications on this subject, it is still unclear what type of relationships there are among these genes and which are the chances that, while having different molecular functions, they i) act as downstream effector genes of well-known pro- or anti- proliferative stimuli and/or interplay with biochemical pathways having oncological relevance or ii) are specific and, possibly, early biomarkers of these pathologies. A Gene Ontology (GO)-based analysis showed that these 21 genes are involved in biological processes, which can be split into two main classes: cell regulation-based and differentiation/development-based. In order to understand the involvement/overlapping with main cancer-related pathways, we performed a meta-analysis dependent on the 189 oncogenic signatures of the Molecular Signatures Database (OSMSD) curated by the Broad Institute. We generated a binary matrix with 53 gene signatures having at least one hit; this analysis i) suggests that some genes of the original list show inconsistencies and might need to be experimentally re- assessed or evaluated as biomarkers (in particular, ACTA2) and ii) allows hypothesizing that important (proto)oncogenes (E2F3, ERBB2/HER2, CCND1, WNT1, and YAP1) and (putative) tumor suppressors (BRCA1, RBBP8/CTIP, and RB1-RBL2/p130) may participate in the onset of this disease or worsen the observed phenotype, thus expanding the list of possible molecular targets for the treatment of BNPTP. PMID:27013923

Pathology of Endometrial Carcinoma.

PubMed

Lax, Sigurd F

2017-01-01

On a clinicopathological and molecular level, two distinctive types of endometrial carcinoma, type I and type II, can be distinguished. Endometrioid carcinoma, the typical type I carcinoma, seems to develop through an estrogen-driven "adenoma carcinoma" pathway from atypical endometrial hyperplasia/endometrioid intraepithelial neoplasia (AEH/EIN). It is associated with elevated serum estrogen and high body mass index and expresses estrogen and progesterone receptors. They are mostly low grade and show a favorable prognosis. A subset progresses into high-grade carcinoma which is accompanied by loss of receptor expression and accumulation of TP53 mutations and behaves poorly. Other frequently altered genes in type I carcinomas are K-Ras, PTEN, and ß-catenin. Another frequent feature of type I carcinomas is microsatellite instability mainly caused by methylation of the MLH1 promoter. In contrast, the typical type II carcinoma, serous carcinoma, is not estrogen related since it usually occurs in a small uterus with atrophic endometrium. It is often associated with a flat putative precursor lesion called serous endometrial intraepithelial carcinoma (SEIC). The molecular pathogenesis of serous carcinoma seems to be driven by TP53 mutations, which are present in SEIC. Other molecular changes in serous carcinoma detectable by immunohistochemistry involve cyclin E and p16. Since many of the aforementioned molecular changes can be demonstrated by immunohistochemistry, they are useful ancillary diagnostic tools and may further contribute to a future molecular classification of endometrial carcinoma as recently suggested based on The Cancer Genome Atlas (TCGA) data.

Molecular detection of a putatively novel cyprinid herpesvirus in sichel (Pelecus cultratus) during a mass mortality event in Hungary.

PubMed

Doszpoly, Andor; Papp, Melitta; Deákné, Petra P; Glávits, Róbert; Ursu, Krisztina; Dán, Ádám

2015-05-01

In the early summer of 2014, mass mortality of sichel (Pelecus cultratus) was observed in Lake Balaton, Hungary. Histological examination revealed degenerative changes within the tubular epithelium, mainly in the distal tubules and collecting ducts in the kidneys and multifocal vacuolisation in the brain stem and cerebellum. Routine molecular investigations showed the presence of the DNA of an unknown alloherpesvirus in some specimens. Subsequently, three genes of the putative herpesviral genome (DNA polymerase, terminase, and helicase) were amplified and partially sequenced. A phylogenetic tree reconstruction based on the concatenated sequence of these three conserved genes implied that the virus belongs to the genus Cyprinivirus within the family Alloherpesviridae. The sequences of the sichel herpesvirus differ markedly from those of the cypriniviruses CyHV-1, CyHV-2 and CyHV-3, putatively representing a fifth species in the genus.

Characterisation of a Desmosterol Reductase Involved in Phytosterol Dealkylation in the Silkworm, Bombyx mori

PubMed Central

Ciufo, Leonora F.; Murray, Patricia A.; Thompson, Anu; Rigden, Daniel J.; Rees, Huw H.

2011-01-01

Most species of invertebrate animals cannot synthesise sterols de novo and many that feed on plants dealkylate phytosterols (mostly C29 and C28) yielding cholesterol (C27). The final step of this dealkylation pathway involves desmosterol reductase (DHCR24)-catalysed reduction of desmosterol to cholesterol. We now report the molecular characterisation in the silkworm, Bombyx mori, of such a desmosterol reductase involved in production of cholesterol from phytosterol, rather than in de novo synthesis of cholesterol. Phylogenomic analysis of putative desmosterol reductases revealed the occurrence of various clades that allowed for the identification of a strong reductase candidate gene in Bombyx mori (BGIBMGA 005735). Following PCR-based cloning of the cDNA (1.6 kb) and its heterologous expression in Saccharomyces cerevisae, the recombinant protein catalysed reduction of desmosterol to cholesterol in an NADH- and FAD- dependent reaction. Conceptual translation of the cDNA, that encodes a 58.9 kDa protein, and database searching, revealed that the enzyme belongs to an FAD-dependent oxidoreductase family. Western blotting revealed reductase protein expression exclusively in the microsomal subcellular fraction and primarily in the gut. The protein is peripherally associated with microsomal membranes. 2D-native gel and PAGE analysis revealed that the reductase is part of a large complex with molecular weight approximately 250kDa. The protein occurs in midgut microsomes at a fairly constant level throughout development in the last two instars, but is drastically reduced during the wandering stage in preparation for metamorphosis. Putative Broad Complex transcription factor-binding sites detectable upstream of the DHCR24 gene may play a role in this down-regulation. PMID:21738635

Chromosomal insertion and excision of a 30 kb unstable genetic element is responsible for phase variation of lipopolysaccharide and other virulence determinants in Legionella pneumophila.

PubMed

Lüneberg, E; Mayer, B; Daryab, N; Kooistra, O; Zähringer, U; Rohde, M; Swanson, J; Frosch, M

2001-03-01

We recently described the phase-variable expression of a virulence-associated lipopolysaccharide (LPS) epitope in Legionella pneumophila. In this study, the molecular mechanism for phase variation was investigated. We identified a 30 kb unstable genetic element as the molecular origin for LPS phase variation. Thirty putative genes were encoded on the 30 kb sequence, organized in two putative opposite transcription units. Some of the open reading frames (ORFs) shared homologies with bacteriophage genes, suggesting that the 30 kb element was of phage origin. In the virulent wild-type strain, the 30 kb element was located on the chromosome, whereas excision from the chromosome and replication as a high-copy plasmid resulted in the mutant phenotype, which is characterized by alteration of an LPS epitope and loss of virulence. Mapping and sequencing of the insertion site in the genome revealed that the chromosomal attachment site was located in an intergenic region flanked by genes of unknown function. As phage release could not be induced by mitomycin C, it is conceivable that the 30 kb element is a non-functional phage remnant. The protein encoded by ORF T on the 30 kb plasmid could be isolated by an outer membrane preparation, indicating that the genes encoded on the 30 kb element are expressed in the mutant phenotype. Therefore, it is conceivable that the phenotypic alterations seen in the mutant depend on high-copy replication of the 30 kb element and expression of the encoded genes. Excision of the 30 kb element from the chromosome was found to occur in a RecA-independent pathway, presumably by the involvement of RecE, RecT and RusA homologues that are encoded on the 30 kb element.

A proteomics survey on wheat susceptibility to Fusarium head blight during grain development

PubMed Central

Chetouhi, Cherif; Lecomte, Philippe; Cambon, Florence; Merlino, Marielle; Biron, David Georges

2014-01-01

The mycotoxigenic fungal species Fusarium graminearum is able to attack several important cereal crops, such as wheat and barley. By causing Fusarium Head Blight (FHB) disease, F. graminearum induces yield and quality losses and poses a public health concern due to in planta mycotoxin production. The molecular and physiological plant responses to FHB, and the cellular biochemical pathways used by F. graminearum to complete its infectious process remain still unknown. In this study, a proteomics approach, combining 2D-gel approach and mass spectrometry, has been used to determine the specific protein patterns associated with the development of the fungal infection during grain growth on susceptible wheat. Our results reveal that F. graminearum infection does not deeply alter the grain proteome and does not significantly disturb the first steps of grain ontogeny but impacts molecular changes during the grain filling stage (impact on starch synthesis and storage proteins). The differentially regulated proteins identified were mainly involved in stress and defence mechanisms, primary metabolism, and main cellular processes such as signalling and transport. Our survey suggests that F. graminearum could take advantage of putative susceptibility factors closely related to grain development processes and thus provide new insights into key molecular events controlling the susceptible response to FHB in wheat grains. PMID:25663750

Interactions of the SAP Domain of Human Ku70 with DNA Substrate: A Molecular Dynamics Study

NASA Technical Reports Server (NTRS)

Hu, Shaowen; Carra, Claudio; Huff, Janice; Pluth, Janice M.; Cucinotta, Francis A.

2007-01-01

NASA is developing a systems biology approach to improve the assessment of health risks associated with space radiation. The primary toxic and mutagenic lesion following radiation exposure is the DNA double strand break (DSB), thus a model incorporating proteins and pathways important in response and repair of this lesion is critical. One key protein heterodimer for systems models of radiation effects is the Ku70/80 complex. The Ku70/80 complex is important in the initial binding of DSB ends following DNA damage, and is a component of nonhomologous end joining repair, the primary pathway for DSB repair in mammalian cells. The SAP domain of Ku70 (residues 556-609), contains an a helix-extended strand-helix motif and similar motifs have been found in other nucleic acid-binding proteins critical for DNA repair. However, the exact mechanism of damage recognition and substrate specificity for the Ku heterodimer remains unclear in part due to the absence of a high-resolution structure of the SAP/DNA complex. We performed a series of molecular dynamics (MD) simulations on a system with the SAP domain of Ku70 and a 10 base pairs DNA duplex. Large-scale conformational changes were observed and some putative binding modes were suggested based on energetic analysis. These modes are consistent with previous experimental investigations. In addition, the results indicate that cooperation of SAP with other domains of Ku70/80 is necessary to explain the high affinity of binding as observed in experiments.

Roles for miR-375 in Neuroendocrine Differentiation and Tumor Suppression via Notch Pathway Suppression in Merkel Cell Carcinoma.

PubMed

Abraham, Karan J; Zhang, Xiao; Vidal, Ricardo; Paré, Geneviève C; Feilotter, Harriet E; Tron, Victor A

2016-04-01

Dysfunction of key miRNA pathways regulating basic cellular processes is a common driver of many cancers. However, the biological roles and/or clinical applications of such pathways in Merkel cell carcinoma (MCC), a rare but lethal cutaneous neuroendocrine (NE) malignancy, have yet to be determined. Previous work has established that miR-375 is highly expressed in MCC tumors, but its biological role in MCC remains unknown. Herein, we show that elevated miR-375 expression is a specific feature of well-differentiated MCC cell lines that express NE markers. In contrast, miR-375 is strikingly down-regulated in highly aggressive, undifferentiated MCC cell lines. Enforced miR-375 expression in these cells induced NE differentiation, and opposed cancer cell viability, migration, invasion, and survival, pointing to tumor-suppressive roles for miR-375. Mechanistically, miR-375-driven phenotypes were caused by the direct post-transcriptional repression of multiple Notch pathway proteins (Notch2 and RBPJ) linked to cancer and regulation of cell fate. Thus, we detail a novel molecular axis linking tumor-suppressive miR-375 and Notch with NE differentiation and cancer cell behavior in MCC. Our findings identify miR-375 as a putative regulator of NE differentiation, provide insight into the cell of origin of MCC, and suggest that miR-375 silencing may promote aggressive cancer cell behavior through Notch disinhibition. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Patterns of population differentiation of candidate genes for cardiovascular disease

PubMed Central

Kullo, Iftikhar J; Ding, Keyue

2007-01-01

Background The basis for ethnic differences in cardiovascular disease (CVD) susceptibility is not fully understood. We investigated patterns of population differentiation (FST) of a set of genes in etiologic pathways of CVD among 3 ethnic groups: Yoruba in Nigeria (YRI), Utah residents with European ancestry (CEU), and Han Chinese (CHB) + Japanese (JPT). We identified 37 pathways implicated in CVD based on the PANTHER classification and 416 genes in these pathways were further studied; these genes belonged to 6 biological processes (apoptosis, blood circulation and gas exchange, blood clotting, homeostasis, immune response, and lipoprotein metabolism). Genotype data were obtained from the HapMap database. Results We calculated FST for 15,559 common SNPs (minor allele frequency ≥ 0.10 in at least one population) in genes that co-segregated among the populations, as well as an average-weighted FST for each gene. SNPs were classified as putatively functional (non-synonymous and untranslated regions) or non-functional (intronic and synonymous sites). Mean FST values for common putatively functional variants were significantly higher than FST values for nonfunctional variants. A significant variation in FST was also seen based on biological processes; the processes of 'apoptosis' and 'lipoprotein metabolism' showed an excess of genes with high FST. Thus, putative functional SNPs in genes in etiologic pathways for CVD show greater population differentiation than non-functional SNPs and a significant variance of FST values was noted among pairwise population comparisons for different biological processes. Conclusion These results suggest a possible basis for varying susceptibility to CVD among ethnic groups. PMID:17626638

Efficient searching and annotation of metabolic networks using chemical similarity

PubMed Central

Pertusi, Dante A.; Stine, Andrew E.; Broadbelt, Linda J.; Tyo, Keith E.J.

2015-01-01

Motivation: The urgent need for efficient and sustainable biological production of fuels and high-value chemicals has elicited a wave of in silico techniques for identifying promising novel pathways to these compounds in large putative metabolic networks. To date, these approaches have primarily used general graph search algorithms, which are prohibitively slow as putative metabolic networks may exceed 1 million compounds. To alleviate this limitation, we report two methods—SimIndex (SI) and SimZyme—which use chemical similarity of 2D chemical fingerprints to efficiently navigate large metabolic networks and propose enzymatic connections between the constituent nodes. We also report a Byers–Waterman type pathway search algorithm for further paring down pertinent networks. Results: Benchmarking tests run with SI show it can reduce the number of nodes visited in searching a putative network by 100-fold with a computational time improvement of up to 105-fold. Subsequent Byers–Waterman search application further reduces the number of nodes searched by up to 100-fold, while SimZyme demonstrates ∼90% accuracy in matching query substrates with enzymes. Using these modules, we have designed and annotated an alternative to the methylerythritol phosphate pathway to produce isopentenyl pyrophosphate with more favorable thermodynamics than the native pathway. These algorithms will have a significant impact on our ability to use large metabolic networks that lack annotation of promiscuous reactions. Availability and implementation: Python files will be available for download at http://tyolab.northwestern.edu/tools/. Contact: k-tyo@northwestern.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25417203

Efficient searching and annotation of metabolic networks using chemical similarity.

PubMed

Pertusi, Dante A; Stine, Andrew E; Broadbelt, Linda J; Tyo, Keith E J

2015-04-01

The urgent need for efficient and sustainable biological production of fuels and high-value chemicals has elicited a wave of in silico techniques for identifying promising novel pathways to these compounds in large putative metabolic networks. To date, these approaches have primarily used general graph search algorithms, which are prohibitively slow as putative metabolic networks may exceed 1 million compounds. To alleviate this limitation, we report two methods--SimIndex (SI) and SimZyme--which use chemical similarity of 2D chemical fingerprints to efficiently navigate large metabolic networks and propose enzymatic connections between the constituent nodes. We also report a Byers-Waterman type pathway search algorithm for further paring down pertinent networks. Benchmarking tests run with SI show it can reduce the number of nodes visited in searching a putative network by 100-fold with a computational time improvement of up to 10(5)-fold. Subsequent Byers-Waterman search application further reduces the number of nodes searched by up to 100-fold, while SimZyme demonstrates ∼ 90% accuracy in matching query substrates with enzymes. Using these modules, we have designed and annotated an alternative to the methylerythritol phosphate pathway to produce isopentenyl pyrophosphate with more favorable thermodynamics than the native pathway. These algorithms will have a significant impact on our ability to use large metabolic networks that lack annotation of promiscuous reactions. Python files will be available for download at http://tyolab.northwestern.edu/tools/. Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Genome mining of the sordarin biosynthetic gene cluster from Sordaria araneosa Cain ATCC 36386: characterization of cycloaraneosene synthase and GDP-6-deoxyaltrose transferase.

PubMed

Kudo, Fumitaka; Matsuura, Yasunori; Hayashi, Takaaki; Fukushima, Masayuki; Eguchi, Tadashi

2016-07-01

Sordarin is a glycoside antibiotic with a unique tetracyclic diterpene aglycone structure called sordaricin. To understand its intriguing biosynthetic pathway that may include a Diels-Alder-type [4+2]cycloaddition, genome mining of the gene cluster from the draft genome sequence of the producer strain, Sordaria araneosa Cain ATCC 36386, was carried out. A contiguous 67 kb gene cluster consisting of 20 open reading frames encoding a putative diterpene cyclase, a glycosyltransferase, a type I polyketide synthase, and six cytochrome P450 monooxygenases were identified. In vitro enzymatic analysis of the putative diterpene cyclase SdnA showed that it catalyzes the transformation of geranylgeranyl diphosphate to cycloaraneosene, a known biosynthetic intermediate of sordarin. Furthermore, a putative glycosyltransferase SdnJ was found to catalyze the glycosylation of sordaricin in the presence of GDP-6-deoxy-d-altrose to give 4'-O-demethylsordarin. These results suggest that the identified sdn gene cluster is responsible for the biosynthesis of sordarin. Based on the isolated potential biosynthetic intermediates and bioinformatics analysis, a plausible biosynthetic pathway for sordarin is proposed.

High-Molecular-Mass Multi-c-Heme Cytochromes from Methylococcus capsulatus Bath†

PubMed Central

Bergmann, David J.; Zahn, James A.; DiSpirito, Alan A.

1999-01-01

The polypeptide and structural gene for a high-molecular-mass c-type cytochrome, cytochrome c553O, was isolated from the methanotroph Methylococcus capsulatus Bath. Cytochrome c553O is a homodimer with a subunit molecular mass of 124,350 Da and an isoelectric point of 6.0. The heme c concentration was estimated to be 8.2 ± 0.4 mol of heme c per subunit. The electron paramagnetic resonance spectrum showed the presence of multiple low spin, S = 1/2, hemes. A degenerate oligonucleotide probe synthesized based on the N-terminal amino acid sequence of cytochrome c553O was used to identify a DNA fragment from M. capsulatus Bath that contains occ, the gene encoding cytochrome c553O. occ is part of a gene cluster which contains three other open reading frames (ORFs). ORF1 encodes a putative periplasmic c-type cytochrome with a molecular mass of 118,620 Da that shows approximately 40% amino acid sequence identity with occ and contains nine c-heme-binding motifs. ORF3 encodes a putative periplasmic c-type cytochrome with a molecular mass of 94,000 Da and contains seven c-heme-binding motifs but shows no sequence homology to occ or ORF1. ORF4 encodes a putative 11,100-Da protein. The four ORFs have no apparent similarity to any proteins in the GenBank database. The subunit molecular masses, arrangement and number of hemes, and amino acid sequences demonstrate that cytochrome c553O and the gene products of ORF1 and ORF3 constitute a new class of c-type cytochrome. PMID:9922265

De Novo Assembly, Gene Annotation, and Marker Discovery in Stored-Product Pest Liposcelis entomophila (Enderlein) Using Transcriptome Sequences

PubMed Central

Wei, Dan-Dan; Chen, Er-Hu; Ding, Tian-Bo; Chen, Shi-Chun; Dou, Wei; Wang, Jin-Jun

2013-01-01

Background As a major stored-product pest insect, Liposcelis entomophila has developed high levels of resistance to various insecticides in grain storage systems. However, the molecular mechanisms underlying resistance and environmental stress have not been characterized. To date, there is a lack of genomic information for this species. Therefore, studies aimed at profiling the L. entomophila transcriptome would provide a better understanding of the biological functions at the molecular levels. Methodology/Principal Findings We applied Illumina sequencing technology to sequence the transcriptome of L. entomophila. A total of 54,406,328 clean reads were obtained and that de novo assembled into 54,220 unigenes, with an average length of 571 bp. Through a similarity search, 33,404 (61.61%) unigenes were matched to known proteins in the NCBI non-redundant (Nr) protein database. These unigenes were further functionally annotated with gene ontology (GO), cluster of orthologous groups of proteins (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. A large number of genes potentially involved in insecticide resistance were manually curated, including 68 putative cytochrome P450 genes, 37 putative glutathione S-transferase (GST) genes, 19 putative carboxyl/cholinesterase (CCE) genes, and other 126 transcripts to contain target site sequences or encoding detoxification genes representing eight types of resistance enzymes. Furthermore, to gain insight into the molecular basis of the L. entomophila toward thermal stresses, 25 heat shock protein (Hsp) genes were identified. In addition, 1,100 SSRs and 57,757 SNPs were detected and 231 pairs of SSR primes were designed for investigating the genetic diversity in future. Conclusions/Significance We developed a comprehensive transcriptomic database for L. entomophila. These sequences and putative molecular markers would further promote our understanding of the molecular mechanisms underlying insecticide resistance or environmental stress, and will facilitate studies on population genetics for psocids, as well as providing useful information for functional genomic research in the future. PMID:24244605

Identification of the Pr1 gene product completes the anthocyanin biosynthesis pathway of maize

USDA-ARS?s Scientific Manuscript database

In maize, mutations in the pr1 locus lead to the accumulation of pelargonidin (red) rather than cyanidin (purple) pigments in aleurone cells where the anthocyanin biosynthetic pathway is active. We characterized pr1 mutation and isolated a putative F3'H encoding gene (Zmf3'h1), and showed by segrega...

Evidence for asymmetrical hybridization despite pre- and post-pollination reproductive barriers between two Silene species

PubMed Central

Zhang, Jin-Ju; Montgomery, Benjamin R.; Huang, Shuang-Quan

2016-01-01

Interspecific hybridization is widespread among plants; nevertheless, pre- and post-zygotic isolating mechanisms may maintain species integrity for interfertile species in sympatry despite some gene flow. Interspecific hybridization and potential isolating barriers were evaluated between co-flowering Silene asclepiadea and Silene yunnanensis in an alpine community in southwest China. We investigated morphological and molecular (nuclear microsatellites and chloroplast gene sequence) variation in sympatric populations of S. asclepiadea and S. yunnanensis. Additionally, we analyzed pollinator behaviour and compared reproductive success between the putative hybrids and their parental species. Both the molecular and morphological data indicate that there were putative natural hybrids in the field, with S. asclepiadae the ovule parent and S. yunnanensis the pollen parent. Bumblebees were the primary visitors to S. asclepiadae and putative hybrids, while butterflies were the primary visitors to S. yunnanensis. Pollen production and viability were significantly lower in putative hybrids than the parental species. The direction of hybridization is quite asymmetric from S. yunnanensis to S. asclepiadea. Protandry combined with later peak flowering of S. yunnanensis, and pollinator preference may have contributed to the asymmetric pattern of hybridization, but putative hybrids were rare. Our results thus suggest that despite gene flow, S. asclepiadea and S. yunnanensis can maintain species boundaries, perhaps as a result of floral isolation and low fecundity of the hybrids. PMID:27178066

Evidence for asymmetrical hybridization despite pre- and post-pollination reproductive barriers between two Silene species.

PubMed

Zhang, Jin-Ju; Montgomery, Benjamin R; Huang, Shuang-Quan

2016-01-01

Interspecific hybridization is widespread among plants; nevertheless, pre- and post-zygotic isolating mechanisms may maintain species integrity for interfertile species in sympatry despite some gene flow. Interspecific hybridization and potential isolating barriers were evaluated between co-flowering Silene asclepiadea and Silene yunnanensis in an alpine community in southwest China. We investigated morphological and molecular (nuclear microsatellites and chloroplast gene sequence) variation in sympatric populations of S. asclepiadea and S. yunnanensis. Additionally, we analyzed pollinator behaviour and compared reproductive success between the putative hybrids and their parental species. Both the molecular and morphological data indicate that there were putative natural hybrids in the field, with S. asclepiadae the ovule parent and S. yunnanensis the pollen parent. Bumblebees were the primary visitors to S. asclepiadae and putative hybrids, while butterflies were the primary visitors to S. yunnanensis Pollen production and viability were significantly lower in putative hybrids than the parental species. The direction of hybridization is quite asymmetric from S. yunnanensis to S. asclepiadea Protandry combined with later peak flowering of S. yunnanensis, and pollinator preference may have contributed to the asymmetric pattern of hybridization, but putative hybrids were rare. Our results thus suggest that despite gene flow, S. asclepiadea and S. yunnanensis can maintain species boundaries, perhaps as a result of floral isolation and low fecundity of the hybrids. Published by Oxford University Press on behalf of the Annals of Botany Company.

Carbon and nitrogen nutrient balance signaling in plants.

PubMed

Zheng, Zhi-Liang

2009-07-01

Cellular carbon (C) and nitrogen (N) metabolism must be tightly coordinated to sustain optimal growth and development for plants and other cellular organisms. Furthermore, C/N balance is also critical for the ecosystem response to elevated atmospheric CO(2). Despite numerous physiological and molecular studies in C/N balance or ratio response, very few genes have been shown to play important roles in C/N balance signaling. During recent five years, exciting progress was made through genetic and genomic studies. Several DNA microarray studies have shown that more than half of the transcriptome is regulated by C, N and the C-N combination. Three genetic studies involving distinct bioassays have demonstrated that a putative nitrate transporter (NTR2.1), a putative glutamate receptor (GLR1.1) and a putative methyltransferase (OSU1) have important functions in the C/N balance response. OSU1 is identical to QUA2/TSD2 which has been implicated to act in cell wall biogenesis, indicating a link between cell wall property and the C/N balance signaling. Given that many investigations are only focused on C alone or N alone, the C/N balance bioassays and gene expression patterns are discussed to assist phenotypic characterization of C/N balance signaling. Further, re-examination of those previously reported sugar or nitrogen responsive genes in C/N balance response may be necessary to dissect the C/N signaling pathways. In addition, key components involved in C-N interactions in bacterial, yeast and animal systems and whether they are functionally conserved in plants are discussed. These rapid advances have provided the first important step towards the construction of the complex yet elegant C/N balance signaling networks in plants.

Tiered High-Throughput Screening Approach to Identify Thyroperoxidase Inhibitors Within the ToxCast Phase I and II Chemical Libraries

PubMed Central

Watt, Eric D.; Hornung, Michael W.; Hedge, Joan M.; Judson, Richard S.; Crofton, Kevin M.; Houck, Keith A.; Simmons, Steven O.

2016-01-01

High-throughput screening for potential thyroid-disrupting chemicals requires a system of assays to capture multiple molecular-initiating events (MIEs) that converge on perturbed thyroid hormone (TH) homeostasis. Screening for MIEs specific to TH-disrupting pathways is limited in the U.S. Environmental Protection Agency ToxCast screening assay portfolio. To fill 1 critical screening gap, the Amplex UltraRed-thyroperoxidase (AUR-TPO) assay was developed to identify chemicals that inhibit TPO, as decreased TPO activity reduces TH synthesis. The ToxCast phase I and II chemical libraries, comprised of 1074 unique chemicals, were initially screened using a single, high concentration to identify potential TPO inhibitors. Chemicals positive in the single-concentration screen were retested in concentration-response. Due to high false-positive rates typically observed with loss-of-signal assays such as AUR-TPO, we also employed 2 additional assays in parallel to identify possible sources of nonspecific assay signal loss, enabling stratification of roughly 300 putative TPO inhibitors based upon selective AUR-TPO activity. A cell-free luciferase inhibition assay was used to identify nonspecific enzyme inhibition among the putative TPO inhibitors, and a cytotoxicity assay using a human cell line was used to estimate the cellular tolerance limit. Additionally, the TPO inhibition activities of 150 chemicals were compared between the AUR-TPO and an orthogonal peroxidase oxidation assay using guaiacol as a substrate to confirm the activity profiles of putative TPO inhibitors. This effort represents the most extensive TPO inhibition screening campaign to date and illustrates a tiered screening approach that focuses resources, maximizes assay throughput, and reduces animal use. PMID:26884060

NG2/CSPG4-collagen type VI interplays putatively involved in the microenvironmental control of tumour engraftment and local expansion.

PubMed

Cattaruzza, Sabrina; Nicolosi, Pier Andrea; Braghetta, Paola; Pazzaglia, Laura; Benassi, Maria Serena; Picci, Piero; Lacrima, Katia; Zanocco, Daniela; Rizzo, Erika; Stallcup, William B; Colombatti, Alfonso; Perris, Roberto

2013-06-01

In soft-tissue sarcoma patients, enhanced expression of NG2/CSPG4 proteoglycan in pre-surgical primary tumours predicts post-surgical metastasis formation and thereby stratifies patients into disease-free survivors and patients destined to succumb to the disease. Both primary and secondary sarcoma lesions also up-regulate collagen type VI, a putative extracellular matrix ligand of NG2, and this matrix alteration potentiates the prognostic impact of NG2. Enhanced constitutive levels of the proteoglycan in isolated sarcoma cells closely correlate with a superior engraftment capability and local growth in xenogenic settings. This apparent NG2-associated malignancy was also corroborated by the diverse tumorigenic behaviour in vitro and in vivo of immunoselected NG2-expressing and NG2-deficient cell subsets, by RNAi-mediated knock down of endogenous NG2, and by ectopic transduction of full-length or deletion constructs of NG2. Cells with modified expression of NG2 diverged in their interaction with purified Col VI, matrices supplemented with Col VI, and cell-free matrices isolated from wild-type and Col VI null fibroblasts. The combined use of dominant-negative NG2 mutant cells and purified domain fragments of the collagen allowed us to pinpoint the reciprocal binding sites within the two molecules and to assert the importance of this molecular interaction in the control of sarcoma cell adhesion and motility. The NG2-mediated binding to Col VI triggered activation of convergent cell survival- and cell adhesion/migration-promoting signal transduction pathways, implicating PI-3K as a common denominator. Thus, the findings point to an NG2-Col VI interplay as putatively involved in the regulation of the cancer cell-host microenvironment interactions sustaining sarcoma progression.

Influence of Molecular Resolution on Sequence-Based Discovery of Ecological Diversity among Synechococcus Populations in an Alkaline Siliceous Hot Spring Microbial Mat ▿ †

PubMed Central

Melendrez, Melanie C.; Lange, Rachel K.; Cohan, Frederick M.; Ward, David M.

2011-01-01

Previous research has shown that sequences of 16S rRNA genes and 16S-23S rRNA internal transcribed spacer regions may not have enough genetic resolution to define all ecologically distinct Synechococcus populations (ecotypes) inhabiting alkaline, siliceous hot spring microbial mats. To achieve higher molecular resolution, we studied sequence variation in three protein-encoding loci sampled by PCR from 60°C and 65°C sites in the Mushroom Spring mat (Yellowstone National Park, WY). Sequences were analyzed using the ecotype simulation (ES) and AdaptML algorithms to identify putative ecotypes. Between 4 and 14 times more putative ecotypes were predicted from variation in protein-encoding locus sequences than from variation in 16S rRNA and 16S-23S rRNA internal transcribed spacer sequences. The number of putative ecotypes predicted depended on the number of sequences sampled and the molecular resolution of the locus. Chao estimates of diversity indicated that few rare ecotypes were missed. Many ecotypes hypothesized by sequence analyses were different in their habitat specificities, suggesting different adaptations to temperature or other parameters that vary along the flow channel. PMID:21169433

Galactose metabolism and toxicity in Ustilago maydis.

PubMed

Schuler, David; Höll, Christina; Grün, Nathalie; Ulrich, Jonas; Dillner, Bastian; Klebl, Franz; Ammon, Alexandra; Voll, Lars M; Kämper, Jörg

2018-05-01

In most organisms, galactose is metabolized via the Leloir pathway, which is conserved from bacteria to mammals. Utilization of galactose requires a close interplay of the metabolic enzymes, as misregulation or malfunction of individual components can lead to the accumulation of toxic intermediate compounds. For the phytopathogenic basidiomycete Ustilago maydis, galactose is toxic for wildtype strains, i.e. leads to growth repression despite the presence of favorable carbon sources as sucrose. The galactose sensitivity can be relieved by two independent modifications: (1) by disruption of Hxt1, which we identify as the major transporter for galactose, and (2) by a point mutation in the gene encoding the galactokinase Gal1, the first enzyme of the Leloir pathway. The mutation in gal1(Y67F) leads to reduced enzymatic activity of Gal1 and thus may limit the formation of putatively toxic galactose-1-phosphate. However, systematic deletions and double deletions of different genes involved in galactose metabolism point to a minor role of galactose-1-phosphate in galactose toxicity. Our results show that molecular triggers for galactose toxicity in U. maydis differ from yeast and mammals. Copyright © 2018 Elsevier Inc. All rights reserved.

Comparative proteomic analysis reveals the positive effect of exogenous spermidine on photosynthesis and salinity tolerance in cucumber seedlings.

PubMed

Sang, Ting; Shan, Xi; Li, Bin; Shu, Sheng; Sun, Jin; Guo, Shirong

2016-08-01

Our results based on proteomics data and physiological alterations proposed the putative mechanism of exogenous Spd enhanced salinity tolerance in cucumber seedlings. Current studies showed that exogenous spermidine (Spd) could alleviate harmful effects of salinity. It is important to increase our understanding of the beneficial physiological responses of exogenous Spd treatment, and to determine the molecular responses underlying these responses. Here, we combined a physiological analysis with iTRAQ-based comparative proteomics of cucumber (Cucumis sativus L.) leaves, treated with 0.1 mM exogenous Spd, 75 mM NaCl and/or exogenous Spd. A total of 221 differentially expressed proteins were found and involved in 30 metabolic pathways, such as photosynthesis, carbohydrate metabolism, amino acid metabolism, stress response, signal transduction and antioxidant. Based on functional classification of the differentially expressed proteins and the physiological responses, we found cucumber seedlings treated with Spd under salt stress had higher photosynthesis efficiency, upregulated tetrapyrrole synthesis, stronger ROS scavenging ability and more protein biosynthesis activity than NaCl treatment, suggesting that these pathways may promote salt tolerance under high salinity. This study provided insights into how exogenous Spd protects photosynthesis and enhances salt tolerance in cucumber seedlings.

Activation of DNA Damage Response Induced by the Kaposi’s Sarcoma-Associated Herpes Virus

PubMed Central

Di Domenico, Enea Gino; Toma, Luigi; Bordignon, Valentina; Trento, Elisabetta; D’Agosto, Giovanna; Cordiali-Fei, Paola; Ensoli, Fabrizio

2016-01-01

The human herpes virus 8 (HHV-8), also known as Kaposi sarcoma-associated herpes virus (KSHV), can infect endothelial cells often leading to cell transformation and to the development of tumors, namely Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and the plasmablastic variant of multicentric Castleman’s disease. KSHV is prevalent in areas such as sub-Saharan Africa and the Mediterranean region presenting distinct genotypes, which appear to be associated with differences in disease manifestation, according to geographical areas. In infected cells, KSHV persists in a latent episomal form. However, in a limited number of cells, it undergoes spontaneous lytic reactivation to ensure the production of new virions. During both the latent and the lytic cycle, KSHV is programmed to express genes which selectively modulate the DNA damage response (DDR) through the activation of the ataxia telangiectasia mutated (ATM) pathway and by phosphorylating factors associated with the DDR, including the major tumor suppressor protein p53 tumor suppressor p53. This review will focus on the interplay between the KSHV and the DDR response pathway throughout the viral lifecycle, exploring the putative molecular mechanism/s that may contribute to malignant transformation of host cells. PMID:27258263

Evidence for Apoplasmic Phloem Unloading in Developing Apple Fruit1

PubMed Central

Zhang, Ling-Yun; Peng, Yi-Ben; Pelleschi-Travier, Sandrine; Fan, Ying; Lu, Yan-Fen; Lu, Ying-Min; Gao, Xiu-Ping; Shen, Yuan-Yue; Delrot, Serge; Zhang, Da-Peng

2004-01-01

The phloem unloading pathway remains unclear in fleshy fruits accumulating a high level of soluble sugars. A structural investigation in apple fruit (Malus domestica Borkh. cv Golden Delicious) showed that the sieve element-companion cell complex of the sepal bundles feeding the fruit flesh is symplasmically isolated over fruit development. 14C-autoradiography indicated that the phloem of the sepal bundles was functional for unloading. Confocal laser scanning microscopy imaging of carboxyfluorescein unloading showed that the dye remained confined to the phloem strands of the sepal bundles from the basal to the apical region of the fruit. A 52-kD putative monosaccharide transporter was immunolocalized predominantly in the plasma membrane of both the sieve elements and parenchyma cells and its amount increased during fruit development. A 90-kD plasma membrane H+-ATPase was also localized in the plasma membrane of the sieve element-companion cell complex. Studies of [14C]sorbitol unloading suggested that an energy-driven monosaccharide transporter may be functional in phloem unloading. These data provide clear evidence for an apoplasmic phloem unloading pathway in apple fruit and give information on the structural and molecular features involved in this process. PMID:15122035

Fine Tuning of Antibiotic Activity by a Tailoring Hydroxylase in a Trans-AT Polyketide Synthase Pathway.

PubMed

Mohammad, Hadi H; Connolly, Jack A; Song, Zhongshu; Hothersall, Joanne; Race, Paul R; Willis, Christine L; Simpson, Thomas J; Winn, Peter J; Thomas, Christopher M

2018-04-16

The addition or removal of hydroxy groups modulates the activity of many pharmacologically active biomolecules. It can be integral to the basic biosynthetic factory or result from associated tailoring steps. For the anti-MRSA antibiotic mupirocin, removal of a C8-hydroxy group late in the biosynthetic pathway gives the active pseudomonic acid A. An extra hydroxylation, at C4, occurs in the related but more potent antibiotic thiomarinol A. We report here in vivo and in vitro studies that show that the putative non-haem-iron(II)/α-ketoglutaratedependent dioxygenase TmuB, from the thiomarinol cluster, 4-hydroxylates various pseudomonic acids whereas C8-OH, and other substituents around the tetrahydropyran ring, block enzyme action but not substrate binding. Molecular modelling suggested a basis for selectivity, but mutation studies had a limited ability to rationally modify TmuB substrate specificity. 4-Hydroxylation had opposite effects on the potency of mupirocin and thiomarinol. Thus, TmuB can be added to the toolbox of polyketide tailoring technologies for the in vivo generation of new antibiotics in the future. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

An in silico assessment of gene function and organization of the phenylpropanoid pathway metabolic networks in Arabidopsis thaliana and limitations thereof

NASA Technical Reports Server (NTRS)

Costa, Michael A.; Collins, R. Eric; Anterola, Aldwin M.; Cochrane, Fiona C.; Davin, Laurence B.; Lewis, Norman G.

2003-01-01

The Arabidopsis genome sequencing in 2000 gave to science the first blueprint of a vascular plant. Its successful completion also prompted the US National Science Foundation to launch the Arabidopsis 2010 initiative, the goal of which is to identify the function of each gene by 2010. In this study, an exhaustive analysis of The Institute for Genomic Research (TIGR) and The Arabidopsis Information Resource (TAIR) databases, together with all currently compiled EST sequence data, was carried out in order to determine to what extent the various metabolic networks from phenylalanine ammonia lyase (PAL) to the monolignols were organized and/or could be predicted. In these databases, there are some 65 genes which have been annotated as encoding putative enzymatic steps in monolignol biosynthesis, although many of them have only very low homology to monolignol pathway genes of known function in other plant systems. Our detailed analysis revealed that presently only 13 genes (two PALs, a cinnamate-4-hydroxylase, a p-coumarate-3-hydroxylase, a ferulate-5-hydroxylase, three 4-coumarate-CoA ligases, a cinnamic acid O-methyl transferase, two cinnamoyl-CoA reductases) and two cinnamyl alcohol dehydrogenases can be classified as having a bona fide (definitive) function; the remaining 52 genes currently have undetermined physiological roles. The EST database entries for this particular set of genes also provided little new insight into how the monolignol pathway was organized in the different tissues and organs, this being perhaps a consequence of both limitations in how tissue samples were collected and in the incomplete nature of the EST collections. This analysis thus underscores the fact that even with genomic sequencing, presumed to provide the entire suite of putative genes in the monolignol-forming pathway, a very large effort needs to be conducted to establish actual catalytic roles (including enzyme versatility), as well as the physiological function(s) for each member of the (multi)gene families present and the metabolic networks that are operative. Additionally, one key to identifying physiological functions for many of these (and other) unknown genes, and their corresponding metabolic networks, awaits the development of technologies to comprehensively study molecular processes at the single cell level in particular tissues and organs, in order to establish the actual metabolic context.

Identification of the Pr1 Gene Product Completes the Anthocyanin Biosynthesis Pathway of Maize

PubMed Central

Sharma, Mandeep; Cortes-Cruz, Moises; Ahern, Kevin R.; McMullen, Michael; Brutnell, Thomas P.; Chopra, Surinder

2011-01-01

In maize, mutations in the pr1 locus lead to the accumulation of pelargonidin (red) rather than cyanidin (purple) pigments in aleurone cells where the anthocyanin biosynthetic pathway is active. We characterized pr1 mutation and isolated a putative F3′H encoding gene (Zmf3′h1) and showed by segregation analysis that the red kernel phenotype is linked to this gene. Genetic mapping using SNP markers confirms its position on chromosome 5L. Furthermore, genetic complementation experiments using a CaMV 35S::ZmF3′H1 promoter–gene construct established that the encoded protein product was sufficient to perform a 3′-hydroxylation reaction. The Zmf3′h1-specific transcripts were detected in floral and vegetative tissues of Pr1 plants and were absent in pr1. Four pr1 alleles were characterized: two carry a 24 TA dinucleotide repeat insertion in the 5′-upstream promoter region, a third has a 17-bp deletion near the TATA box, and a fourth contains a Ds insertion in exon1. Genetic and transcription assays demonstrated that the pr1 gene is under the regulatory control of anthocyanin transcription factors red1 and colorless1. The cloning and characterization of pr1 completes the molecular identification of all genes encoding structural enzymes of the anthocyanin pathway of maize. PMID:21385724

Role of TGF Beta and PPAR Alpha Signaling Pathways in Radiation Response of Locally Exposed Heart: Integrated Global Transcriptomics and Proteomics Analysis.

PubMed

Subramanian, Vikram; Seemann, Ingar; Merl-Pham, Juliane; Hauck, Stefanie M; Stewart, Fiona A; Atkinson, Michael J; Tapio, Soile; Azimzadeh, Omid

2017-01-06

Epidemiological data from patients undergoing radiotherapy for thoracic tumors clearly show the damaging effect of ionizing radiation on cardiovascular system. The long-term impairment of heart function and structure after local high-dose irradiation is associated with systemic inflammatory response, contraction impairment, microvascular damage, and cardiac fibrosis. The goal of the present study was to investigate molecular mechanisms involved in this process. C57BL/6J mice received a single X-ray dose of 16 Gy given locally to the heart at the age of 8 weeks. Radiation-induced changes in the heart transcriptome and proteome were investigated 40 weeks after the exposure. The omics data were analyzed by bioinformatics tools and validated by immunoblotting. Integrated network analysis of transcriptomics and proteomics data elucidated the signaling pathways that were similarly affected at gene and protein level. Analysis showed induction of transforming growth factor (TGF) beta signaling but inactivation of peroxisome proliferator-activated receptor (PPAR) alpha signaling in irradiated heart. The putative mediator role of mitogen-activated protein kinase cascade linking PPAR alpha and TGF beta signaling was supported by data from immunoblotting and ELISA. This study indicates that both signaling pathways are involved in radiation-induced heart fibrosis, metabolic disordering, and impaired contractility, a pathophysiological condition that is often observed in patients that received high radiation doses in thorax.

The putative role of oxidative stress and inflammation in the pathophysiology of sleep dysfunction across neuropsychiatric disorders: Focus on chronic fatigue syndrome, bipolar disorder and multiple sclerosis.

PubMed

Morris, Gerwyn; Stubbs, Brendon; Köhler, Cristiano A; Walder, Ken; Slyepchenko, Anastasiya; Berk, Michael; Carvalho, André F

2018-04-04

Sleep and circadian abnormalities are prevalent and burdensome manifestations of diverse neuro-immune diseases, and may aggravate the course of several neuropsychiatric disorders. The underlying pathophysiology of sleep abnormalities across neuropsychiatric disorders remains unclear, and may involve the inter-play of several clinical variables and mechanistic pathways. In this review, we propose a heuristic framework in which reciprocal interactions of immune, oxidative and nitrosative stress, and mitochondrial pathways may drive sleep abnormalities across potentially neuroprogressive disorders. Specifically, it is proposed that systemic inflammation may activate microglial cells and astrocytes in brain regions involved in sleep and circadian regulation. Activated glial cells may secrete pro-inflammatory cytokines (for example, interleukin-1 beta and tumour necrosis factor alpha), nitric oxide and gliotransmitters, which may influence the expression of key circadian regulators (e.g., the Circadian Locomotor Output Cycles Kaput (CLOCK) gene). Furthermore, sleep disruption may further aggravate oxidative and nitrosative, peripheral immune activation, and (neuro) inflammation across these disorders in a vicious pathophysiological loop. This review will focus on chronic fatigue syndrome, bipolar disorder, and multiple sclerosis as exemplars of neuro-immune disorders. We conclude that novel therapeutic targets exploring immune and oxidative & nitrosative pathways (p.e. melatonin and molecular hydrogen) hold promise in alleviating sleep and circadian dysfunction in these disorders. Copyright © 2018 Elsevier Ltd. All rights reserved.

Methoxychlor affects multiple hormone signaling pathways in the largemouth bass (Micropterus salmoides) liver

PubMed Central

Martyniuk, Christopher J.; Spade, Daniel J.; Blum, Jason L.; Kroll, Kevin J.; Denslow, Nancy D.

2011-01-01

Methoxychlor (MXC) is an organochlorine pesticide that has been shown to have estrogenic activity by activating estrogen receptors and inducing vitellogenin production in male fish. Previous studies report that exposure to MXC induces changes in mRNA abundance of reproductive genes in the liver and testes of largemouth bass (Micropterus salmoides). The objective of the present study was to better characterize the mode of action of MXC by measuring the global transcriptomic response in the male largemouth liver using an oligonucleotide microarray. Microarray analysis identified highly significant changes in the expression of 37 transcripts (p<0.001) (20 induced and 17 decreased) in the liver after MXC injection and a total of 900 expression changes (p<0.05) in transcripts with high homology to known genes. Largemouth bass estrogen receptor alpha (esr1) and androgen receptor (ar) were among the transcripts that were increased in the liver after MXC treatment. Functional enrichment analysis identified the molecular functions of steroid binding and androgen receptor activity as well as steroid hormone receptor activity as being significantly over-represented gene ontology terms. Pathway analysis identified c-fos signaling as being putatively affected through both estrogen and androgen signaling. This study provides evidence that MXC elicits transcriptional effects through the estrogen receptor as well as androgen receptor-mediated pathways in the liver. PMID:21276474

Toxoplasma gondii acetyl-CoA synthetase is involved in fatty acid elongation (of long fatty acid chains) during tachyzoite life stages.

PubMed

Dubois, David; Fernandes, Stella; Amiar, Souad; Dass, Sheena; Katris, Nicholas J; Botté, Cyrille Y; Yamaryo-Botté, Yoshiki

2018-06-01

Apicomplexan parasites are pathogens responsible for major human diseases such as toxoplasmosis caused by Toxoplasma gondii and malaria caused by Plasmodium spp. Throughout their intracellular division cycle, the parasites require vast and specific amounts of lipids to divide and survive. This demand for lipids relies on a fine balance between de novo synthesized lipids and scavenged lipids from the host. Acetyl-CoA is a major and central precursor for many metabolic pathways, especially for lipid biosynthesis. T. gondii possesses a single cytosolic acetyl-CoA synthetase ( Tg ACS). Its role in the parasite lipid synthesis is unclear. Here, we generated an inducible Tg ACS KO parasite line and confirmed the cytosolic localization of the protein. We conducted 13 C-stable isotope labeling combined with mass spectrometry-based lipidomic analyses to unravel its putative role in the parasite lipid synthesis pathway. We show that its disruption has a minor effect on the global FA composition due to the metabolic changes induced to compensate for its loss. However, we could demonstrate that Tg ACS is involved in providing acetyl-CoA for the essential fatty elongation pathway to generate FAs used for membrane biogenesis. This work provides novel metabolic insight to decipher the complex lipid synthesis in T. gondii . Copyright © 2018 by the American Society for Biochemistry and Molecular Biology, Inc.

A systems biology-based investigation into the therapeutic effects of Gansui Banxia Tang on reversing the imbalanced network of hepatocellular carcinoma

NASA Astrophysics Data System (ADS)

Zhang, Yanqiong; Guo, Xiaodong; Wang, Danhua; Li, Ruisheng; Li, Xiaojuan; Xu, Ying; Liu, Zhenli; Song, Zhiqian; Lin, Ya; Li, Zhiyan; Lin, Na

2014-02-01

Several complex molecular events are involved in tumorigenesis of hepatocellular carcinoma (HCC). The interactions of these molecules may constitute the HCC imbalanced network. Gansui Banxia Tang (GSBXT), as a classic Chinese herbal formula, is a popular complementary and alternative medicine modality for treating HCC. In order to investigate the therapeutic effects and the pharmacological mechanisms of GSBXT on reversing HCC imbalanced network, we in the current study developed a comprehensive systems approach of integrating disease-specific and drug-specific networks, and successfully revealed the relationships of the ingredients in GSBXT with their putative targets, and with HCC significant molecules and HCC related pathway systems for the first time. Meanwhile, further experimental validation also demonstrated the preventive effects of GSBXT on tumor growth in mice and its regulatory effects on potential targets.

Past, Present and Future Therapeutics for Cerebellar Ataxias

PubMed Central

Marmolino, D; Manto, M

2010-01-01

Cerebellar ataxias are a group of disabling neurological disorders. Patients exhibit a cerebellar syndrome and can also present with extra-cerebellar deficits, namely pigmentary retinopathy, extrapyramidal movement disorders, pyramidal signs, cortical symptoms (seizures, cognitive impairment/behavioural symptoms), and peripheral neuropathy. Recently, deficits in cognitive operations have been unraveled. Cerebellar ataxias are heterogeneous both at the phenotypic and genotypic point of view. Therapeutical trials performed during these last 4 decades have failed in most cases, in particular because drugs were not targeting a deleterious pathway, but were given to counteract putative defects in neurotransmission. The identification of the causative mutations of many hereditary ataxias, the development of relevant animal models and the recent identifications of the molecular mechanisms underlying ataxias are impacting on the development of new drugs. We provide an overview of the pharmacological treatments currently used in the clinical practice and we discuss the drugs under development. PMID:20808545

Dissecting the Mutational Landscape of Cutaneous Melanoma: An Omic Analysis Based on Patients from Greece

PubMed Central

Piroti, Georgia; Papadodima, Olga

2018-01-01

Melanoma is a lethal type of skin cancer, unless it is diagnosed early. Formalin-fixed, paraffin-embedded (FFPE) tissue is a valuable source for molecular assays after diagnostic examination, but isolated nucleic acids often suffer from degradation. Here, for the first time, we examine primary melanomas from Greek patients, using whole exome sequencing, so as to derive their mutational profile. Application of a bioinformatic framework revealed a total of 10,030 somatic mutations. Regarding the genes containing putative protein-altering mutations, 73 were common in at least three patients. Sixty-five of these 73 top common genes have been previously identified in melanoma cases. Biological processes related to melanoma were affected by varied genes in each patient, suggesting differences in the components of a pathway possibly contributing to pathogenesis. We performed a multi-level analysis highlighting a short list of candidate genes with a probable causative role in melanoma. PMID:29596374

Investigating sesquiterpene biosynthesis in Ginkgo biloba: molecular cloning and functional characterization of (E,E)-farnesol and α-bisabolene synthases.

PubMed

Parveen, Iffat; Wang, Mei; Zhao, Jianping; Chittiboyina, Amar G; Tabanca, Nurhayat; Ali, Abbas; Baerson, Scott R; Techen, Natascha; Chappell, Joe; Khan, Ikhlas A; Pan, Zhiqiang

2015-11-01

Ginkgo biloba is one of the oldest living tree species and has been extensively investigated as a source of bioactive natural compounds, including bioactive flavonoids, diterpene lactones, terpenoids and polysaccharides which accumulate in foliar tissues. Despite this chemical diversity, relatively few enzymes associated with any biosynthetic pathway from ginkgo have been characterized to date. In the present work, predicted transcripts potentially encoding enzymes associated with the biosynthesis of diterpenoid and terpenoid compounds, including putative terpene synthases, were first identified by mining publicly-available G. biloba RNA-seq data sets. Recombinant enzyme studies with two of the TPS-like sequences led to the identification of GbTPS1 and GbTPS2, encoding farnesol and bisabolene synthases, respectively. Additionally, the phylogenetic analysis revealed the two terpene synthase genes as primitive genes that might have evolved from an ancestral diterpene synthase.

Challenges in carrier-mediated intracellular delivery: moving beyond endosomal barriers.

PubMed

Stewart, Martin P; Lorenz, Anna; Dahlman, James; Sahay, Gaurav

2016-05-01

The deployment of molecular to microscale carriers for intracellular delivery has tremendous potential for biology and medicine, especially for in vivo therapies. The field remains limited, however, by a poor understanding of how carriers gain access to the cell interior. In this review, we provide an overview of the different types of carriers, their speculated modes of entry, putative pathways of vesicular transport, and sites of endosomal escape. We compare this alongside pertinent examples from the cell biology of how viruses, bacteria, and their effectors enter cells and escape endosomal confinement. We anticipate insights into the mechanisms of cellular entry and endosomal escape will benefit future research efforts on effective carrier-mediated intracellular delivery. WIREs Nanomed Nanobiotechnol 2016, 8:465-478. doi: 10.1002/wnan.1377 For further resources related to this article, please visit the WIREs website. © 2015 Wiley Periodicals, Inc.

Characterization of the Polyoxin Biosynthetic Gene Cluster from Streptomyces cacaoi and Engineered Production of Polyoxin H*S⃞

PubMed Central

Chen, Wenqing; Huang, Tingting; He, Xinyi; Meng, Qingqing; You, Delin; Bai, Linquan; Li, Jialiang; Wu, Mingxuan; Li, Rui; Xie, Zhoujie; Zhou, Huchen; Zhou, Xiufen; Tan, Huarong; Deng, Zixin

2009-01-01

A gene cluster (pol) essential for the biosynthesis of polyoxin, a nucleoside antibiotic widely used for the control of phytopathogenic fungi, was cloned from Streptomyces cacaoi. A 46,066-bp region was sequenced, and 20 of 39 of the putative open reading frames were defined as necessary for polyoxin biosynthesis as evidenced by its production in a heterologous host, Streptomyces lividans TK24. The role of PolO and PolA in polyoxin synthesis was demonstrated by in vivo experiments, and their functions were unambiguously characterized as O-carbamoyltransferase and UMP-enolpyruvyltransferase, respectively, by in vitro experiments, which enabled the production of a modified compound differing slightly from that proposed earlier. These studies should provide a solid foundation for the elucidation of the molecular mechanisms for polyoxin biosynthesis, and set the stage for combinatorial biosynthesis using genes encoding different pathways for nucleoside antibiotics. PMID:19233844

Harnessing pain heterogeneity and RNA transcriptome to identify blood–based pain biomarkers: a novel correlational study design and bioinformatics approach in a graded chronic constriction injury model

PubMed Central

Grace, Peter M.; Hurley, Daniel; Barratt, Daniel T.; Tsykin, Anna; Watkins, Linda R.; Rolan, Paul E.; Hutchinson, Mark R.

2017-01-01

A quantitative, peripherally accessible biomarker for neuropathic pain has great potential to improve clinical outcomes. Based on the premise that peripheral and central immunity contribute to neuropathic pain mechanisms, we hypothesized that biomarkers could be identified from the whole blood of adult male rats, by integrating graded chronic constriction injury (CCI), ipsilateral lumbar dorsal quadrant (iLDQ) and whole blood transcriptomes, and pathway analysis with pain behavior. Correlational bioinformatics identified a range of putative biomarker genes for allodynia intensity, many encoding for proteins with a recognized role in immune/nociceptive mechanisms. A selection of these genes was validated in a separate replication study. Pathway analysis of the iLDQ transcriptome identified Fcγ and Fcε signaling pathways, among others. This study is the first to employ the whole blood transcriptome to identify pain biomarker panels. The novel correlational bioinformatics, developed here, selected such putative biomarkers based on a correlation with pain behavior and formation of signaling pathways with iLDQ genes. Future studies may demonstrate the predictive ability of these biomarker genes across other models and additional variables. PMID:22697386

Aging of human short-wave cone pathways

PubMed Central

Shinomori, Keizo; Werner, John S.

2012-01-01

The retinal image is sampled concurrently, and largely independently, by three physiologically and anatomically distinct pathways, each with separate ON and OFF subdivisions. The retinal circuitry giving rise to an ON pathway receiving input from the short-wave-sensitive (S) cones is well understood, but the S-cone OFF circuitry is more controversial. Here, we characterize the temporal properties of putative S-cone ON and OFF pathways in younger and older observers by measuring thresholds for stimuli that produce increases or decreases in S-cone stimulation, while the middle- and long-wave-sensitive cones are unmodulated. We characterize the data in terms of an impulse response function, the theoretical response to a flash of infinitely short duration, from which the response to any temporally varying stimulus may be predicted. Results show that the S-cone response to increments is faster than to decrements, but this difference is significantly greater for older individuals. The impulse response function amplitudes for increment and decrement responses are highly correlated across individuals, whereas the timing is not. This strongly suggests that the amplitude is controlled by neural circuitry that is common to S-cone ON and OFF responses (photoreceptors), whereas the timing is controlled by separate postreceptoral pathways. The slower response of the putative OFF pathway is ascribed to different retinal circuitry, possibly attributable to a sign-inverting amacrine cell not present in the ON pathway. It is significant that this pathway is affected selectively in the elderly by becoming slower, whereas the temporal properties of the S-cone ON response are stable across the life span of an individual. PMID:22847416

Dual targeting of the antagonistic pathways mediated by Sirt1 and TXNIP as a putative approach to enhance the efficacy of anti-aging interventions

PubMed Central

Mousa, Shaker A.; Gallati, Christine; Simone, Tessa; Dier, Emmy; Yalcin, Murat; Dyskin, Evgeny; Thangirala, Sudha; Hanko, Christine; Rebbaa, Abdelhadi

2009-01-01

The organism's ability to regulate oxidative stress and metabolism is well recognized as a major determinant of longevity. While much research interest in this area is directed towards the study of genes that inhibit oxidative stress and/or improve metabolism, contribution to the aging process of genes with antagonistic effects on these two pathways is still less understood. The present study investigated the respective roles of the histone deacetylase Sirt1 and the thioredoxin binding protein TXNIP, two genes with opposite effects on oxidative stress and metabolism, in mediating the action of putative anti-aging interventions. Experiments were carried out in vitro and in vivo to determine the effect of proven, limited calorie availability, and unproven, resveratrol and dehydroepiandrosterone (DHEA), on the expression of Sirt1 and TXNIP. The results indicated that limited calorie availability consistently inhibited TXNIP in cancer and in normal cells including stem cells, however, it only slightly induced Sirt1expression in cancer cells. In contrast, resveratrol had a biphasic effect, and DHEA inhibited the expression of these two genes in a tissue specific manner, both in vitro and in vivo. Whereas all the three approaches tested inhibited TXNIP through the glycolytic pathway, DHEA acted by inhibiting G6PD and resveratrol through the activation of AMPK. In light of previous reports that Sirt1 induces AMPK-mediated signaling pathway, our findings point to the possibility of a negative relationship between Sirt1 and TXNIP that, if validated, can be exploited to improve the efficacy of putative anti-aging interventions. PMID:20195491

From genes to brain oscillations: is the visual pathway the epigenetic clue to schizophrenia?

PubMed

González-Hernández, J A; Pita-Alcorta, C; Cedeño, I R

2006-01-01

Molecular data and gene expression data and recently mitochondrial genes and possible epigenetic regulation by non-coding genes is revolutionizing our views on schizophrenia. Genes and epigenetic mechanisms are triggered by cell-cell interaction and by external stimuli. A number of recent clinical and molecular observations indicate that epigenetic factors may be operational in the origin of the illness. Based on the molecular insights, gene expression profiles and epigenetic regulation of gene, we went back to the neurophysiology (brain oscillations) and found a putative role of the visual experiences (i.e. visual stimuli) as epigenetic factor. The functional evidences provided here, establish a direct link between the striate and extrastriate unimodal visual cortex and the neurobiology of the schizophrenia. This result support the hypothesis that 'visual experience' has a potential role as epigenetic factor and contribute to trigger and/or to maintain the progression of the schizophrenia. In this case, candidate genes sensible for the visual 'insult' may be located within the visual cortex including associative areas, while the integrity of the visual pathway before reaching the primary visual cortex is preserved. The same effect can be perceived if target genes are localised within the visual pathway, which actually, is more sensitive for 'insult' during the early life than the cortex per se. If this process affects gene expression at these sites a stably sensory specific 'insult', i.e. distorted visual information, is entering the visual system and expanded to fronto-temporo-parietal multimodal areas even from early maturation periods. The difference in the timing of postnatal neuroanatomical events between such areas and the primary visual cortex in humans (with the formers reaching the same development landmarks later in life than the latter) is 'optimal' to establish an abnormal 'cell- communication' mediated by the visual system that may further interfere with the local physiology. In this context the strategy to search target genes need to be rearrangement and redirected to visual-related genes. Otherwise, psychophysics studies combining functional neuroimage, and electrophysiology are strongly recommended, for the search of epigenetic clues that will allow to carrier gene association studies in schizophrenia.

De Novo Assembly of the Japanese Flounder (Paralichthys olivaceus) Spleen Transcriptome to Identify Putative Genes Involved in Immunity

PubMed Central

Huang, Lin; Li, Guiyang; Mo, Zhaolan; Xiao, Peng; Li, Jie; Huang, Jie

2015-01-01

Background Japanese flounder (Paralichthys olivaceus) is an economically important marine fish in Asia and has suffered from disease outbreaks caused by various pathogens, which requires more information for immune relevant genes on genome background. However, genomic and transcriptomic data for Japanese flounder remain scarce, which limits studies on the immune system of this species. In this study, we characterized the Japanese flounder spleen transcriptome using an Illumina paired-end sequencing platform to identify putative genes involved in immunity. Methodology/Principal Findings A cDNA library from the spleen of P. olivaceus was constructed and randomly sequenced using an Illumina technique. The removal of low quality reads generated 12,196,968 trimmed reads, which assembled into 96,627 unigenes. A total of 21,391 unigenes (22.14%) were annotated in the NCBI Nr database, and only 1.1% of the BLASTx top-hits matched P. olivaceus protein sequences. Approximately 12,503 (58.45%) unigenes were categorized into three Gene Ontology groups, 19,547 (91.38%) were classified into 26 Cluster of Orthologous Groups, and 10,649 (49.78%) were assigned to six Kyoto Encyclopedia of Genes and Genomes pathways. Furthermore, 40,928 putative simple sequence repeats and 47, 362 putative single nucleotide polymorphisms were identified. Importantly, we identified 1,563 putative immune-associated unigenes that mapped to 15 immune signaling pathways. Conclusions/Significance The P. olivaceus transciptome data provides a rich source to discover and identify new genes, and the immune-relevant sequences identified here will facilitate our understanding of the mechanisms involved in the immune response. Furthermore, the plentiful potential SSRs and SNPs found in this study are important resources with respect to future development of a linkage map or marker assisted breeding programs for the flounder. PMID:25723398

Comparative analysis of programmed cell death pathways in filamentous fungi.

PubMed

Fedorova, Natalie D; Badger, Jonathan H; Robson, Geoff D; Wortman, Jennifer R; Nierman, William C

2005-12-08

Fungi can undergo autophagic- or apoptotic-type programmed cell death (PCD) on exposure to antifungal agents, developmental signals, and stress factors. Filamentous fungi can also exhibit a form of cell death called heterokaryon incompatibility (HI) triggered by fusion between two genetically incompatible individuals. With the availability of recently sequenced genomes of Aspergillus fumigatus and several related species, we were able to define putative components of fungi-specific death pathways and the ancestral core apoptotic machinery shared by all fungi and metazoa. Phylogenetic profiling of HI-associated proteins from four Aspergilli and seven other fungal species revealed lineage-specific protein families, orphan genes, and core genes conserved across all fungi and metazoa. The Aspergilli-specific domain architectures include NACHT family NTPases, which may function as key integrators of stress and nutrient availability signals. They are often found fused to putative effector domains such as Pfs, SesB/LipA, and a newly identified domain, HET-s/LopB. Many putative HI inducers and mediators are specific to filamentous fungi and not found in unicellular yeasts. In addition to their role in HI, several of them appear to be involved in regulation of cell cycle, development and sexual differentiation. Finally, the Aspergilli possess many putative downstream components of the mammalian apoptotic machinery including several proteins not found in the model yeast, Saccharomyces cerevisiae. Our analysis identified more than 100 putative PCD associated genes in the Aspergilli, which may help expand the range of currently available treatments for aspergillosis and other invasive fungal diseases. The list includes species-specific protein families as well as conserved core components of the ancestral PCD machinery shared by fungi and metazoa.

Pro-apoptotic effect of a Mycoplasma hyopneumoniae putative type I signal peptidase on PK(15) swine cells.

PubMed

Paes, Jéssica A; Virginio, Veridiana G; Cancela, Martín; Leal, Fernanda M A; Borges, Thiago J; Jaeger, Natália; Bonorino, Cristina; Schrank, Irene S; Ferreira, Henrique B

2017-03-01

Mycoplasma hyopneumoniae is an economically significant swine pathogen that causes porcine enzootic pneumonia (PEP). Important processes for swine infection by M. hyopneumoniae depend on cell surface proteins, many of which are secreted by secretion pathways not completely elucidated so far. A putative type I signal peptidase (SPase I), a possible component of a putative Sec-dependent pathway, was annotated as a product of the sipS gene in the pathogenic M. hyopneumoniae 7448 genome. This M. hyopneumoniae putative SPase I (MhSPase I) displays only 14% and 23% of sequence identity/similarity to Escherichia coli bona fide SPase I, and, in complementation assays performed with a conditional E. coli SPase I mutant, only a partial restoration of growth was achieved with the heterologous expression of a recombinant MhSPase I (rMhSPase I). Considering the putative surface location of MhSPase I and its previously demonstrated capacity to induce a strong humoral response, we then assessed its potential to elicit a cellular and possible immunomodulatory response. In assays for immunogenicity assessment, rMhSPase I unexpectedly showed a cytotoxic effect on murine splenocytes. This cytotoxic effect was further confirmed using the swine epithelial PK(15) cell line in MTT and annexin V-flow cytometry assays, which showed that rMhSPase I induces apoptosis in a dose dependent-way. It was also demonstrated that this pro-apoptotic effect of rMhSPase I involves activation of a caspase-3 cascade. The potential relevance of the rMhSPase I pro-apoptotic effect for M. hyopneumoniae-host interactions in the context of PEP is discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterisation of the paralytic shellfish toxin biosynthesis gene clusters in Anabaena circinalis AWQC131C and Aphanizomenon sp. NH-5.

PubMed

Mihali, Troco K; Kellmann, Ralf; Neilan, Brett A

2009-03-30

Saxitoxin and its analogues collectively known as the paralytic shellfish toxins (PSTs) are neurotoxic alkaloids and are the cause of the syndrome named paralytic shellfish poisoning. PSTs are produced by a unique biosynthetic pathway, which involves reactions that are rare in microbial metabolic pathways. Nevertheless, distantly related organisms such as dinoflagellates and cyanobacteria appear to produce these toxins using the same pathway. Hypothesised explanations for such an unusual phylogenetic distribution of this shared uncommon metabolic pathway, include a polyphyletic origin, an involvement of symbiotic bacteria, and horizontal gene transfer. We describe the identification, annotation and bioinformatic characterisation of the putative paralytic shellfish toxin biosynthesis clusters in an Australian isolate of Anabaena circinalis and an American isolate of Aphanizomenon sp., both members of the Nostocales. These putative PST gene clusters span approximately 28 kb and contain genes coding for the biosynthesis and export of the toxin. A putative insertion/excision site in the Australian Anabaena circinalis AWQC131C was identified, and the organization and evolution of the gene clusters are discussed. A biosynthetic pathway leading to the formation of saxitoxin and its analogues in these organisms is proposed. The PST biosynthesis gene cluster presents a mosaic structure, whereby genes have apparently transposed in segments of varying size, resulting in different gene arrangements in all three sxt clusters sequenced so far. The gene cluster organizational structure and sequence similarity seems to reflect the phylogeny of the producer organisms, indicating that the gene clusters have an ancient origin, or that their lateral transfer was also an ancient event. The knowledge we gain from the characterisation of the PST biosynthesis gene clusters, including the identity and sequence of the genes involved in the biosynthesis, may also afford the identification of these gene clusters in dinoflagellates, the cause of human mortalities and significant financial loss to the tourism and shellfish industries.

Characterisation of the paralytic shellfish toxin biosynthesis gene clusters in Anabaena circinalis AWQC131C and Aphanizomenon sp. NH-5

PubMed Central

Mihali, Troco K; Kellmann, Ralf; Neilan, Brett A

2009-01-01

Background Saxitoxin and its analogues collectively known as the paralytic shellfish toxins (PSTs) are neurotoxic alkaloids and are the cause of the syndrome named paralytic shellfish poisoning. PSTs are produced by a unique biosynthetic pathway, which involves reactions that are rare in microbial metabolic pathways. Nevertheless, distantly related organisms such as dinoflagellates and cyanobacteria appear to produce these toxins using the same pathway. Hypothesised explanations for such an unusual phylogenetic distribution of this shared uncommon metabolic pathway, include a polyphyletic origin, an involvement of symbiotic bacteria, and horizontal gene transfer. Results We describe the identification, annotation and bioinformatic characterisation of the putative paralytic shellfish toxin biosynthesis clusters in an Australian isolate of Anabaena circinalis and an American isolate of Aphanizomenon sp., both members of the Nostocales. These putative PST gene clusters span approximately 28 kb and contain genes coding for the biosynthesis and export of the toxin. A putative insertion/excision site in the Australian Anabaena circinalis AWQC131C was identified, and the organization and evolution of the gene clusters are discussed. A biosynthetic pathway leading to the formation of saxitoxin and its analogues in these organisms is proposed. Conclusion The PST biosynthesis gene cluster presents a mosaic structure, whereby genes have apparently transposed in segments of varying size, resulting in different gene arrangements in all three sxt clusters sequenced so far. The gene cluster organizational structure and sequence similarity seems to reflect the phylogeny of the producer organisms, indicating that the gene clusters have an ancient origin, or that their lateral transfer was also an ancient event. The knowledge we gain from the characterisation of the PST biosynthesis gene clusters, including the identity and sequence of the genes involved in the biosynthesis, may also afford the identification of these gene clusters in dinoflagellates, the cause of human mortalities and significant financial loss to the tourism and shellfish industries. PMID:19331657

Analysis of the expression of putative heat-stress related genes in relation to thermotolerance of cork oak.

PubMed

Correia, Barbara; Rodriguez, José Luis; Valledor, Luis; Almeida, Tânia; Santos, Conceição; Cañal, Maria Jesús; Pinto, Glória

2014-03-15

Cork oak (Quercus suber L.) is a research priority in the Mediterranean area and because of cork oaks' distribution these stands are experiencing daily stress. Based on projections of intensifying climate change and considering the key role of exploring the recovery abilities, cork oak seedlings were subjected to a cumulative temperature increase from 25°C to 55°C and subsequent recovery. CO2 assimilation rate, chlorophyll fluorescence, anthocyanins, proline and lipid peroxidation were used to evaluate plant performance, while the relative abundance of seven genes encoding for proteins of cork oak with a putative role in thermal/stress regulation (POX1, POX2, HSP10.4, HSP17a.22, CHS, MTL and RBC) was analyzed by qPCR (quantitative Polymerase Chain Reaction). A temperature change to 35°C showed abundance alterations in the tested genes; at 45°C, the molecular changes were associated with an antioxidant response, possibly modulated by anthocyanins. At 55°C, HSP17a.22, MTL and proline accumulation were evident. After recovery, physiological balance was restored, whereas POX1, HSP10.4 and MTL abundances were suggested to be involved in increased thermotolerance. The data presented here are expected to pinpoint some pathways changes occurring during such stress and further recovery in this particular Mediterranean species. Copyright © 2013 Elsevier GmbH. All rights reserved.

Data from Tiered High-Throughput Screening Approach to Identify Thyroperoxidase Inhibitors within the ToxCast Phase I and II Chemical Libraries

EPA Pesticide Factsheets

High-throughput screening for potential thyroid-disrupting chemicals requires a system of assays to capture multiple molecular-initiating events (MIEs) that converge on perturbed thyroid hormone (TH) homeostasis. Screening for MIEs specific to TH-disrupting pathways is limited in the U.S. Environmental Protection Agency ToxCast screening assay portfolio. To fill 1 critical screening gap, the Amplex UltraRed-thyroperoxidase (AUR-TPO) assay was developed to identify chemicals that inhibit TPO, as decreased TPO activity reduces TH synthesis. The ToxCast phase I and II chemical libraries, comprised of 1074 unique chemicals, were initially screened using a single, high concentration to identify potential TPO inhibitors. Chemicals positive in the single-concentration screen were retested in concentration-response. Due to high false-positive rates typically observed with loss-of-signal assays such as AUR-TPO, we also employed 2 additional assays in parallel to identify possible sources of nonspecific assay signal loss, enabling stratification of roughly 300 putative TPO inhibitors based upon selective AUR-TPO activity. A cell-free luciferase inhibition assay was used to identify nonspecific enzyme inhibition among the putative TPO inhibitors, and a cytotoxicity assay using a human cell line was used to estimate the cellular tolerance limit. Additionally, the TPO inhibition activities of 150 chemicals were compared between the AUR-TPO and an orthogonal peroxidase oxidat

Evidence of Two Functionally Distinct Ornithine Decarboxylation Systems in Lactic Acid Bacteria

PubMed Central

Romano, Andrea; Trip, Hein; Lonvaud-Funel, Aline; Lolkema, Juke S.

2012-01-01

Biogenic amines are low-molecular-weight organic bases whose presence in food can result in health problems. The biosynthesis of biogenic amines in fermented foods mostly proceeds through amino acid decarboxylation carried out by lactic acid bacteria (LAB), but not all systems leading to biogenic amine production by LAB have been thoroughly characterized. Here, putative ornithine decarboxylation pathways consisting of a putative ornithine decarboxylase and an amino acid transporter were identified in LAB by strain collection screening and database searches. The decarboxylases were produced in heterologous hosts and purified and characterized in vitro, whereas transporters were heterologously expressed in Lactococcus lactis and functionally characterized in vivo. Amino acid decarboxylation by whole cells of the original hosts was determined as well. We concluded that two distinct types of ornithine decarboxylation systems exist in LAB. One is composed of an ornithine decarboxylase coupled to an ornithine/putrescine transmembrane exchanger. Their combined activities results in the extracellular release of putrescine. This typical amino acid decarboxylation system is present in only a few LAB strains and may contribute to metabolic energy production and/or pH homeostasis. The second system is widespread among LAB. It is composed of a decarboxylase active on ornithine and l-2,4-diaminobutyric acid (DABA) and a transporter that mediates unidirectional transport of ornithine into the cytoplasm. Diamines that result from this second system are retained within the cytosol. PMID:22247134

The Transcriptome Analysis of Strongyloides stercoralis L3i Larvae Reveals Targets for Intervention in a Neglected Disease

PubMed Central

Marcilla, Antonio; Garg, Gagan; Bernal, Dolores; Ranganathan, Shoba; Forment, Javier; Ortiz, Javier; Muñoz-Antolí, Carla; Dominguez, M. Victoria; Pedrola, Laia; Martinez-Blanch, Juan; Sotillo, Javier; Trelis, Maria; Toledo, Rafael; Esteban, J. Guillermo

2012-01-01

Background Strongyloidiasis is one of the most neglected diseases distributed worldwide with endemic areas in developed countries, where chronic infections are life threatening. Despite its impact, very little is known about the molecular biology of the parasite involved and its interplay with its hosts. Next generation sequencing technologies now provide unique opportunities to rapidly address these questions. Principal Findings Here we present the first transcriptome of the third larval stage of S. stercoralis using 454 sequencing coupled with semi-automated bioinformatic analyses. 253,266 raw sequence reads were assembled into 11,250 contiguous sequences, most of which were novel. 8037 putative proteins were characterized based on homology, gene ontology and/or biochemical pathways. Comparison of the transcriptome of S. strongyloides with those of other nematodes, including S. ratti, revealed similarities in transcription of molecules inferred to have key roles in parasite-host interactions. Enzymatic proteins, like kinases and proteases, were abundant. 1213 putative excretory/secretory proteins were compiled using a new pipeline which included non-classical secretory proteins. Potential drug targets were also identified. Conclusions Overall, the present dataset should provide a solid foundation for future fundamental genomic, proteomic and metabolomic explorations of S. stercoralis, as well as a basis for applied outcomes, such as the development of novel methods of intervention against this neglected parasite. PMID:22389732

Diguanylate cyclase null mutant reveals that C-Di-GMP pathway regulates the motility and adherence of the extremophile bacterium Acidithiobacillus caldus.

PubMed

Castro, Matías; Deane, Shelly M; Ruiz, Lina; Rawlings, Douglas E; Guiliani, Nicolas

2015-01-01

An understanding of biofilm formation is relevant to the design of biological strategies to improve the efficiency of the bioleaching process and to prevent environmental damages caused by acid mine/rock drainage. For this reason, our laboratory is focused on the characterization of the molecular mechanisms involved in biofilm formation in different biomining bacteria. In many bacteria, the intracellular levels of c-di-GMP molecules regulate the transition from the motile planktonic state to sessile community-based behaviors, such as biofilm development, through different kinds of effectors. Thus, we recently started a study of the c-di-GMP pathway in several biomining bacteria including Acidithiobacillus caldus. C-di-GMP molecules are synthesized by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs). We previously reported the existence of intermediates involved in c-di-GMP pathway from different Acidithiobacillus species. Here, we report our work related to At. caldus ATCC 51756. We identified several putative-ORFs encoding DGC and PDE and effector proteins. By using total RNA extracted from At. caldus cells and RT-PCR, we demonstrated that these genes are expressed. We also demonstrated the presence of c-di-GMP by mass spectrometry and showed that genes for several of the DGC enzymes were functional by heterologous genetic complementation in Salmonella enterica serovar Typhimurium mutants. Moreover, we developed a DGC defective mutant strain (Δc1319) that strongly indicated that the c-di-GMP pathway regulates the swarming motility and adherence to sulfur surfaces by At. caldus. Together, our results revealed that At. caldus possesses a functional c-di-GMP pathway which could be significant for ores colonization during the bioleaching process.

Diguanylate Cyclase Null Mutant Reveals That C-Di-GMP Pathway Regulates the Motility and Adherence of the Extremophile Bacterium Acidithiobacillus caldus

PubMed Central

Castro, Matías; Deane, Shelly M.; Ruiz, Lina; Rawlings, Douglas E.; Guiliani, Nicolas

2015-01-01

An understanding of biofilm formation is relevant to the design of biological strategies to improve the efficiency of the bioleaching process and to prevent environmental damages caused by acid mine/rock drainage. For this reason, our laboratory is focused on the characterization of the molecular mechanisms involved in biofilm formation in different biomining bacteria. In many bacteria, the intracellular levels of c-di-GMP molecules regulate the transition from the motile planktonic state to sessile community-based behaviors, such as biofilm development, through different kinds of effectors. Thus, we recently started a study of the c-di-GMP pathway in several biomining bacteria including Acidithiobacillus caldus. C-di-GMP molecules are synthesized by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs). We previously reported the existence of intermediates involved in c-di-GMP pathway from different Acidithiobacillus species. Here, we report our work related to At. caldus ATCC 51756. We identified several putative-ORFs encoding DGC and PDE and effector proteins. By using total RNA extracted from At. caldus cells and RT-PCR, we demonstrated that these genes are expressed. We also demonstrated the presence of c-di-GMP by mass spectrometry and showed that genes for several of the DGC enzymes were functional by heterologous genetic complementation in Salmonella enterica serovar Typhimurium mutants. Moreover, we developed a DGC defective mutant strain (Δc1319) that strongly indicated that the c-di-GMP pathway regulates the swarming motility and adherence to sulfur surfaces by At. caldus. Together, our results revealed that At. caldus possesses a functional c-di-GMP pathway which could be significant for ores colonization during the bioleaching process. PMID:25689133

Molecular and biological diagnostic tests for monitoring benzimidazole resistance in human soil-transmitted helminths.

PubMed

Diawara, Aïssatou; Schwenkenbecher, Jan M; Kaplan, Ray M; Prichard, Roger K

2013-06-01

In endemic countries with soil-transmitted helminths mass drug administration with albendazole or mebendazole are being implemented as a control strategy. However, it is well known in veterinary helminths that the use of the same benzimidazole drugs can place selection on the β-tubulin gene, leading to resistance. Given the concern that resistance could arise in human soil-transmitted helminths, there is an urgent need to develop accurate diagnostic tools for monitoring resistance. In this study, we developed molecular assays to detect putative resistance genetic changes in Ascaris lumbricoides, Trichuris trichiura, and hookworms, and we optimized an egg hatch assay for the canine hookworm Ancylostoma caninum and applied it to Necator americanus. Both assays were tested on field samples. The molecular assays demonstrated their reproducibility and capacity to detect the presence of worms carrying putative resistance-associated genetic changes. However, further investigations are needed to validate our molecular and biological tests on additional field isolates.

Physiological and Molecular Responses to Excess Boron in Citrus macrophylla W.

PubMed

Martínez-Cuenca, Mary-Rus; Martínez-Alcántara, Belén; Quiñones, Ana; Ruiz, Marta; Iglesias, Domingo J; Primo-Millo, Eduardo; Forner-Giner, M Ángeles

2015-01-01

This work provides insight into several mechanisms involved in boron (B) regulation pathway in response to high B conditions in Citrus. The study was carried out in Citrus macrophylla W. (Cm) seedlings cultured "in vitro" in media with 50 or 400 μM H3BO3 (control, Ct, and B-excess, +B, plants, respectively). Growth parameters, B concentration, leaf chlorophyll (Chl) concentration, the expression of the main putative genes involved in B transport and distribution, and leaf and root proline and malonaldehyde (MDA) concentrations, were assessed. Excess B led to high B concentration in +B plants (3.8- and 1.4-fold in leaves and roots, respectively) when compared with Ct ones. However, a minor effect was recorded in the plant (incipient visual symptoms, less than 27% reduction in root growth and 26% decrease in Chl b concentration). B toxicity down-regulated by half the expression level of putative B transporter genes NIP5 and PIP1. CmBOR1 gene was not repressed in +B plants and B accumulated in the shoots. High B level increased the transcripts of putative gene TIP5, involved in B transport across the tonoplast, by 3.3- and 2.4-fold in leaves and roots, respectively. The activity of V-PPiase proton pump, related with the electrochemical gradient in the vacuole, was also enhanced in +B organs. B toxicity up-regulated putative BOR4 gene (2.1- and 2.7-fold in roots and leaves, respectively), which codifies for an active efflux B transporter. Accordingly, B was located in +B plants preferently in an insoluble form on cell walls. Finally, excess B caused a significant rise in proline concentration (51% and 34% in roots and leaves, respectively), while the MDA level did not exceed 20%. In conclusion, Cm tolerance to a high B level is likely based on the synergism of several specific mechanisms against B toxicity, including: 1/ down-regulation of NIP5 and PIP1 boron transporters; 2/ activation of B efflux from cells due to the up-regulation of putative BOR4 gene; 3/ compartmentation of B in the vacuole through TIP5 transporter activation and the acidification of the organelle; 4/ insolubilisation of B and deposition in cell walls preventing from cytoplasm damage; and, 5/ induction of an efficient antioxidant system through proline accumulation.

Physiological and Molecular Responses to Excess Boron in Citrus macrophylla W

PubMed Central

Martínez-Cuenca, Mary-Rus; Martínez-Alcántara, Belén; Quiñones, Ana; Ruiz, Marta; Iglesias, Domingo J.; Primo-Millo, Eduardo; Forner-Giner, M. Ángeles

2015-01-01

This work provides insight into several mechanisms involved in boron (B) regulation pathway in response to high B conditions in Citrus. The study was carried out in Citrus macrophylla W. (Cm) seedlings cultured “in vitro” in media with 50 or 400 μM H3BO3 (control, Ct, and B-excess, +B, plants, respectively). Growth parameters, B concentration, leaf chlorophyll (Chl) concentration, the expression of the main putative genes involved in B transport and distribution, and leaf and root proline and malonaldehyde (MDA) concentrations, were assessed. Excess B led to high B concentration in +B plants (3.8- and 1.4-fold in leaves and roots, respectively) when compared with Ct ones. However, a minor effect was recorded in the plant (incipient visual symptoms, less than 27% reduction in root growth and 26% decrease in Chl b concentration). B toxicity down-regulated by half the expression level of putative B transporter genes NIP5 and PIP1. CmBOR1 gene was not repressed in +B plants and B accumulated in the shoots. High B level increased the transcripts of putative gene TIP5, involved in B transport across the tonoplast, by 3.3- and 2.4-fold in leaves and roots, respectively. The activity of V-PPiase proton pump, related with the electrochemical gradient in the vacuole, was also enhanced in +B organs. B toxicity up-regulated putative BOR4 gene (2.1- and 2.7-fold in roots and leaves, respectively), which codifies for an active efflux B transporter. Accordingly, B was located in +B plants preferently in an insoluble form on cell walls. Finally, excess B caused a significant rise in proline concentration (51% and 34% in roots and leaves, respectively), while the MDA level did not exceed 20%. In conclusion, Cm tolerance to a high B level is likely based on the synergism of several specific mechanisms against B toxicity, including: 1/ down-regulation of NIP5 and PIP1 boron transporters; 2/ activation of B efflux from cells due to the up-regulation of putative BOR4 gene; 3/ compartmentation of B in the vacuole through TIP5 transporter activation and the acidification of the organelle; 4/ insolubilisation of B and deposition in cell walls preventing from cytoplasm damage; and, 5/ induction of an efficient antioxidant system through proline accumulation. PMID:26225859

Transcriptome analysis of molecular mechanisms responsible for light-stress response in Mythimna separata (Walker)

PubMed Central

Duan, Yun; Gong, ZhongJun; Wu, RenHai; Miao, Jin; Jiang, YueLi; Li, Tong; Wu, XiaoBo; Wu, YuQing

2017-01-01

Light is an important environmental signal for most insects. The Oriental Armyworm, Mythimna separata, is a serious pest of cereal crops worldwide, and is highly sensitive to light signals during its developmental and reproductive stages. However, molecular biological studies of its response to light stress are scarce, and related genomic information is not available. In this study, we sequenced and de novo assembled the transcriptomes of M. separata exposed to four different light conditions: dark, white light (WL), UV light (UVL) and yellow light (YL). A total of 46,327 unigenes with an average size of 571 base pairs (bp) were obtained, among which 24,344 (52.55%) matched to public databases. The numbers of genes differentially expressed between dark vs WL, dark vs UVL, dark vs YL, and UVL vs YL were 12,012, 12,950, 14,855, and 13,504, respectively. These results suggest that light exposure altered gene expression patterns in M. separata. Putative genes involved in phototransduction-fly, phototransduction, circadian rhythm-fly, olfactory transduction, and taste transduction were identified. This study thus identified a series of candidate genes and pathways potentially related to light stress in M. separata. PMID:28345615

Regulatory function of Arabidopsis lipid transfer protein 1 (LTP1) in ethylene response and signaling.

PubMed

Wang, Honglin; Sun, Yue; Chang, Jianhong; Zheng, Fangfang; Pei, Haixia; Yi, Yanjun; Chang, Caren; Dong, Chun-Hai

2016-07-01

Ethylene as a gaseous plant hormone is directly involved in various processes during plant growth and development. Much is known regarding the ethylene receptors and regulatory factors in the ethylene signal transduction pathway. In Arabidopsis thaliana, REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1) can interact with and positively regulates the ethylene receptor ETHYLENE RESPONSE1 (ETR1). In this study we report the identification and characterization of an RTE1-interacting protein, a putative Arabidopsis lipid transfer protein 1 (LTP1) of unknown function. Through bimolecular fluorescence complementation, a direct molecular interaction between LTP1 and RTE1 was verified in planta. Analysis of an LTP1-GFP fusion in transgenic plants and plasmolysis experiments revealed that LTP1 is localized to the cytoplasm. Analysis of ethylene responses showed that the ltp1 knockout is hypersensitive to 1-aminocyclopropanecarboxylic acid (ACC), while LTP1 overexpression confers insensitivity. Analysis of double mutants etr1-2 ltp1 and rte1-3 ltp1 demonstrates a regulatory function of LTP1 in ethylene receptor signaling through the molecular association with RTE1. This study uncovers a novel function of Arabidopsis LTP1 in the regulation of ethylene response and signaling.

Insights into the TOR-S6K signaling pathway in maize (Zea mays L.). pathway activation by effector-receptor interaction.

PubMed

Garrocho-Villegas, Verónica; Aguilar C, Raúl; Sánchez de Jiménez, Estela

2013-12-23

The primordial TOR pathway, known to control growth and cell proliferation, has still not been fully described for plants. Nevertheless, in maize, an insulin-like growth factor (ZmIGF) peptide has been reported to stimulate this pathway. This research provides further insight into the TOR pathway in maize, using a biochemical approach in cultures of fast-growing (FG) and slow-growing (SG) calli, as a model system. Our results revealed that addition of either ZmIGF or insulin to SG calli stimulated DNA synthesis and increased the growth rate through cell proliferation and increased the rate of ribosomal protein (RP) synthesis by the selective mobilization of RP mRNAs into polysomes. Furthermore, analysis of the phosphorylation status of the main TOR and S6K kinases from the TOR pathway revealed stimulation by ZmIGF or insulin, whereas rapamycin inhibited its activation. Remarkably, a putative maize insulin-like receptor was recognized by a human insulin receptor antibody, as demonstrated by immunoprecipitation from membrane protein extracts of maize callus. Furthermore, competition experiments between ZmIGF and insulin for the receptor site on maize protoplasts suggested structural recognition of the putative receptor by either effector. These data were confirmed by confocal immunolocalization within the cell membrane of callus cells. Taken together, these data indicate that cell growth and cell proliferation in maize depend on the activation of the TOR-S6K pathway through the interaction of an insulin-like growth factor and its receptor. This evidence suggests that higher plants as well as metazoans have conserved this biochemical pathway to regulate their growth, supporting the conclusion that it is a highly evolved conserved pathway.

Comparative Transcriptome Analysis Identifies Putative Genes Involved in the Biosynthesis of Xanthanolides in Xanthium strumarium L.

PubMed

Li, Yuanjun; Gou, Junbo; Chen, Fangfang; Li, Changfu; Zhang, Yansheng

2016-01-01

Xanthium strumarium L. is a traditional Chinese herb belonging to the Asteraceae family. The major bioactive components of this plant are sesquiterpene lactones (STLs), which include the xanthanolides. To date, the biogenesis of xanthanolides, especially their downstream pathway, remains largely unknown. In X. strumarium, xanthanolides primarily accumulate in its glandular trichomes. To identify putative gene candidates involved in the biosynthesis of xanthanolides, three X. strumarium transcriptomes, which were derived from the young leaves of two different cultivars and the purified glandular trichomes from one of the cultivars, were constructed in this study. In total, 157 million clean reads were generated and assembled into 91,861 unigenes, of which 59,858 unigenes were successfully annotated. All the genes coding for known enzymes in the upstream pathway to the biosynthesis of xanthanolides were present in the X. strumarium transcriptomes. From a comparative analysis of the X. strumarium transcriptomes, this study identified a number of gene candidates that are putatively involved in the downstream pathway to the synthesis of xanthanolides, such as four unigenes encoding CYP71 P450s, 50 unigenes for dehydrogenases, and 27 genes for acetyltransferases. The possible functions of these four CYP71 candidates are extensively discussed. In addition, 116 transcription factors that are highly expressed in X. strumarium glandular trichomes were also identified. Their possible regulatory roles in the biosynthesis of STLs are discussed. The global transcriptomic data for X. strumarium should provide a valuable resource for further research into the biosynthesis of xanthanolides.

Transcriptome Analysis of Nine Tissues to Discover Genes Involved in the Biosynthesis of Active Ingredients in Sophora flavescens.

PubMed

Han, Rongchun; Takahashi, Hiroki; Nakamura, Michimi; Bunsupa, Somnuk; Yoshimoto, Naoko; Yamamoto, Hirobumi; Suzuki, Hideyuki; Shibata, Daisuke; Yamazaki, Mami; Saito, Kazuki

2015-01-01

Sophora flavescens AITON (kurara) has long been used to treat various diseases. Although several research findings revealed the biosynthetic pathways of its characteristic chemical components as represented by matrine, insufficient analysis of transcriptome data hampered in-depth analysis of the underlying putative genes responsible for the biosynthesis of pharmaceutical chemical components. In this study, more than 200 million fastq format reads were generated by Illumina's next-generation sequencing approach using nine types of tissue from S. flavescens, followed by CLC de novo assembly, ultimately yielding 83,325 contigs in total. By mapping the reads back to the contigs, reads per kilobase of the transcript per million mapped reads values were calculated to demonstrate gene expression levels, and overrepresented gene ontology terms were evaluated using Fisher's exact test. In search of the putative genes relevant to essential metabolic pathways, all 1350 unique enzyme commission numbers were used to map pathways against the Kyoto Encyclopedia of Genes and Genomes. By analyzing expression patterns, we proposed some candidate genes involved in the biosynthesis of isoflavonoids and quinolizidine alkaloids. Adopting RNA-Seq analysis, we obtained substantially credible contigs for downstream work. The preferential expression of the gene for putative lysine/ornithine decarboxylase committed in the initial step of matrine biosynthesis in leaves and stems was confirmed in semi-quantitative polymerase chain reaction (PCR) analysis. The findings in this report may serve as a stepping-stone for further research into this promising medicinal plant.

Network analysis of S. aureus response to ramoplanin reveals modules for virulence factors and resistance mechanisms and characteristic novel genes.

PubMed

Subramanian, Devika; Natarajan, Jeyakumar

2015-12-10

Staphylococcus aureus is a major human pathogen and ramoplanin is an antimicrobial attributed for effective treatment. The goal of this study was to examine the transcriptomic profiles of ramoplanin sensitive and resistant S. aureus to identify putative modules responsible for virulence and resistance-mechanisms and its characteristic novel genes. The dysregulated genes were used to reconstruct protein functional association networks for virulence-factors and resistance-mechanisms individually. Strong link between metabolic-pathways and development of virulence/resistance is suggested. We identified 15 putative modules of virulence factors. Six hypothetical genes were annotated with novel virulence activity among which SACOL0281 was discovered to be an essential virulence factor EsaD. The roles of MazEF toxin-antitoxin system, SACOL0202/SACOL0201 two-component system and that of amino-sugar and nucleotide-sugar metabolism in virulence are also suggested. In addition, 14 putative modules of resistance mechanisms including modules of ribosomal protein-coding genes and metabolic pathways such as biotin-synthesis, TCA-cycle, riboflavin-biosynthesis, peptidoglycan-biosynthesis etc. are also indicated. Copyright © 2015 Elsevier B.V. All rights reserved.

Metabolome searcher: a high throughput tool for metabolite identification and metabolic pathway mapping directly from mass spectrometry and using genome restriction.

PubMed

Dhanasekaran, A Ranjitha; Pearson, Jon L; Ganesan, Balasubramanian; Weimer, Bart C

2015-02-25

Mass spectrometric analysis of microbial metabolism provides a long list of possible compounds. Restricting the identification of the possible compounds to those produced by the specific organism would benefit the identification process. Currently, identification of mass spectrometry (MS) data is commonly done using empirically derived compound databases. Unfortunately, most databases contain relatively few compounds, leaving long lists of unidentified molecules. Incorporating genome-encoded metabolism enables MS output identification that may not be included in databases. Using an organism's genome as a database restricts metabolite identification to only those compounds that the organism can produce. To address the challenge of metabolomic analysis from MS data, a web-based application to directly search genome-constructed metabolic databases was developed. The user query returns a genome-restricted list of possible compound identifications along with the putative metabolic pathways based on the name, formula, SMILES structure, and the compound mass as defined by the user. Multiple queries can be done simultaneously by submitting a text file created by the user or obtained from the MS analysis software. The user can also provide parameters specific to the experiment's MS analysis conditions, such as mass deviation, adducts, and detection mode during the query so as to provide additional levels of evidence to produce the tentative identification. The query results are provided as an HTML page and downloadable text file of possible compounds that are restricted to a specific genome. Hyperlinks provided in the HTML file connect the user to the curated metabolic databases housed in ProCyc, a Pathway Tools platform, as well as the KEGG Pathway database for visualization and metabolic pathway analysis. Metabolome Searcher, a web-based tool, facilitates putative compound identification of MS output based on genome-restricted metabolic capability. This enables researchers to rapidly extend the possible identifications of large data sets for metabolites that are not in compound databases. Putative compound names with their associated metabolic pathways from metabolomics data sets are returned to the user for additional biological interpretation and visualization. This novel approach enables compound identification by restricting the possible masses to those encoded in the genome.

Thermal acclimation in Antarctic fish: transcriptomic profiling of metabolic pathways.

PubMed

Windisch, Heidrun Sigrid; Kathöver, Raphaela; Pörtner, Hans-Otto; Frickenhaus, Stephan; Lucassen, Magnus

2011-11-01

It is widely accepted that adaptation to the extreme cold has evolved at the expense of high thermal sensitivity. However, recent studies have demonstrated significant capacities for warm acclimation in Antarctic fishes. Here, we report on hepatic metabolic reorganization and its putative molecular background in the Antarctic eelpout (Pachycara brachycephalum) during warm acclimation to 5°C over 6 wk. Elevated capacities of cytochrome c oxidase suggest the use of warm acclimation pathways different from those in temperate fish. The capacity of this enzyme rose by 90%, while citrate synthase (CS) activity fell by 20% from the very beginning. The capacity of lipid oxidation by hydroxyacyl-CoA dehydrogenase remained constant, whereas phosphoenolpyruvate carboxykinase as a marker for gluconeogenesis displayed 40% higher activities. These capacities in relation to CS indicate a metabolic shift from lipid to carbohydrate metabolism. The finding was supported by large rearrangements of the related transcriptome, both functional genes and potential transcription factors. A multivariate analysis (canonical correspondence analyses) of various transcripts subdivided the incubated animals in three groups, one control group and two responding on short and long timescales, respectively. A strong dichotomy in the expression of peroxisome proliferator-activated receptors-1α and -β receptors was most striking and has not previously been reported. Altogether, we identified a molecular network, which responds sensitively to warming beyond the realized ecological niche. The shift from lipid to carbohydrate stores and usage may support warm hardiness, as the latter sustain anaerobic metabolism and may prepare for hypoxemic conditions that would develop upon warming beyond the present acclimation temperature.

Digital gene expression profiling of flax (Linum usitatissimum L.) stem peel identifies genes enriched in fiber-bearing phloem tissue.

PubMed

Guo, Yuan; Qiu, Caisheng; Long, Songhua; Chen, Ping; Hao, Dongmei; Preisner, Marta; Wang, Hui; Wang, Yufu

2017-08-30

To better understand the molecular mechanisms and gene expression characteristics associated with development of bast fiber cell within flax stem phloem, the gene expression profiling of flax stem peels and leaves were screened, using Illumina's Digital Gene Expression (DGE) analysis. Four DGE libraries (2 for stem peel and 2 for leaf), ranging from 6.7 to 9.2 million clean reads were obtained, which produced 7.0 million and 6.8 million mapped reads for flax stem peel and leave, respectively. By differential gene expression analysis, a total of 975 genes, of which 708 (73%) genes have protein-coding annotation, were identified as phloem enriched genes putatively involved in the processes of polysaccharide and cell wall metabolism. Differential expression genes (DEGs) was validated using quantitative RT-PCR, the expression pattern of all nine genes determined by qRT-PCR fitted in well with that obtained by sequencing analysis. Cluster and Gene Ontology (GO) analysis revealed that a large number of genes related to metabolic process, catalytic activity and binding category were expressed predominantly in the stem peels. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the phloem enriched genes suggested approximately 111 biological pathways. The large number of genes and pathways produced from DGE sequencing will expand our understanding of the complex molecular and cellular events in flax bast fiber development and provide a foundation for future studies on fiber development in other bast fiber crops. Copyright © 2017 Elsevier B.V. All rights reserved.

Integrated Enrichment Analysis of Variants and Pathways in Genome-Wide Association Studies Indicates Central Role for IL-2 Signaling Genes in Type 1 Diabetes, and Cytokine Signaling Genes in Crohn's Disease

PubMed Central

Carbonetto, Peter; Stephens, Matthew

2013-01-01

Pathway analyses of genome-wide association studies aggregate information over sets of related genes, such as genes in common pathways, to identify gene sets that are enriched for variants associated with disease. We develop a model-based approach to pathway analysis, and apply this approach to data from the Wellcome Trust Case Control Consortium (WTCCC) studies. Our method offers several benefits over existing approaches. First, our method not only interrogates pathways for enrichment of disease associations, but also estimates the level of enrichment, which yields a coherent way to promote variants in enriched pathways, enhancing discovery of genes underlying disease. Second, our approach allows for multiple enriched pathways, a feature that leads to novel findings in two diseases where the major histocompatibility complex (MHC) is a major determinant of disease susceptibility. Third, by modeling disease as the combined effect of multiple markers, our method automatically accounts for linkage disequilibrium among variants. Interrogation of pathways from eight pathway databases yields strong support for enriched pathways, indicating links between Crohn's disease (CD) and cytokine-driven networks that modulate immune responses; between rheumatoid arthritis (RA) and “Measles” pathway genes involved in immune responses triggered by measles infection; and between type 1 diabetes (T1D) and IL2-mediated signaling genes. Prioritizing variants in these enriched pathways yields many additional putative disease associations compared to analyses without enrichment. For CD and RA, 7 of 8 additional non-MHC associations are corroborated by other studies, providing validation for our approach. For T1D, prioritization of IL-2 signaling genes yields strong evidence for 7 additional non-MHC candidate disease loci, as well as suggestive evidence for several more. Of the 7 strongest associations, 4 are validated by other studies, and 3 (near IL-2 signaling genes RAF1, MAPK14, and FYN) constitute novel putative T1D loci for further study. PMID:24098138

Proteomic Characterization of Plasmid pLA1 for Biodegradation of Polycyclic Aromatic Hydrocarbons in the Marine Bacterium, Novosphingobium pentaromativorans US6-1

PubMed Central

Yun, Sung Ho; Choi, Chi-Won; Lee, Sang-Yeop; Lee, Yeol Gyun; Kwon, Joseph; Leem, Sun Hee; Chung, Young Ho; Kahng, Hyung-Yeel; Kim, Sang Jin; Kwon, Kae Kyoung; Kim, Seung Il

2014-01-01

Novosphingobium pentaromativorans US6-1 is a halophilic marine bacterium able to degrade polycyclic aromatic hydrocarbons (PAHs). Genome sequence analysis revealed that the large plasmid pLA1 present in N. pentaromativorans US6-1 consists of 199 ORFs and possess putative biodegradation genes that may be involved in PAH degradation. 1-DE/LC-MS/MS analysis of N. pentaromativorans US6-1 cultured in the presence of different PAHs and monocyclic aromatic hydrocarbons (MAHs) identified approximately 1,000 and 1,400 proteins, respectively. Up-regulated biodegradation enzymes, including those belonging to pLA1, were quantitatively compared. Among the PAHs, phenanthrene induced the strongest up-regulation of extradiol cleavage pathway enzymes such as ring-hydroxylating dioxygenase, putative biphenyl-2,3-diol 1,2-dioxygenase, and catechol 2,3-dioxygenase in pLA1. These enzymes lead the initial step of the lower catabolic pathway of aromatic hydrocarbons through the extradiol cleavage pathway and participate in the attack of PAH ring cleavage, respectively. However, N. pentaromativorans US6-1 cultured with p-hydroxybenzoate induced activation of another extradiol cleavage pathway, the protocatechuate 4,5-dioxygenase pathway, that originated from chromosomal genes. These results suggest that N. pentaromativorans US6-1 utilizes two different extradiol pathways and plasmid pLA1 might play a key role in the biodegradation of PAH in N. pentaromativorans US6-1. PMID:24608660

A de novo transcriptome and valid reference genes for quantitative real-time PCR in Colaphellus bowringi.

PubMed

Tan, Qian-Qian; Zhu, Li; Li, Yi; Liu, Wen; Ma, Wei-Hua; Lei, Chao-Liang; Wang, Xiao-Ping

2015-01-01

The cabbage beetle Colaphellus bowringi Baly is a serious insect pest of crucifers and undergoes reproductive diapause in soil. An understanding of the molecular mechanisms of diapause regulation, insecticide resistance, and other physiological processes is helpful for developing new management strategies for this beetle. However, the lack of genomic information and valid reference genes limits knowledge on the molecular bases of these physiological processes in this species. Using Illumina sequencing, we obtained more than 57 million sequence reads derived from C. bowringi, which were assembled into 39,390 unique sequences. A Clusters of Orthologous Groups classification was obtained for 9,048 of these sequences, covering 25 categories, and 16,951 were assigned to 255 Kyoto Encyclopedia of Genes and Genomes pathways. Eleven candidate reference gene sequences from the transcriptome were then identified through reverse transcriptase polymerase chain reaction. Among these candidate genes, EF1α, ACT1, and RPL19 proved to be the most stable reference genes for different reverse transcriptase quantitative polymerase chain reaction experiments in C. bowringi. Conversely, aTUB and GAPDH were the least stable reference genes. The abundant putative C. bowringi transcript sequences reported enrich the genomic resources of this beetle. Importantly, the larger number of gene sequences and valid reference genes provide a valuable platform for future gene expression studies, especially with regard to exploring the molecular mechanisms of different physiological processes in this species.

Identification and validation of FaP1D7, a putative marker associated with the biosynthesis of methyl butanoate in cultivated strawberry (Fragaria x ananassa).

PubMed

Gor, Mian Chee; Candappa, Chrishani; de Silva, Thishakya; Mantri, Nitin; Pang, Edwin

2017-12-12

Breeding strawberry (Fragaria x ananassa) with enhanced fruit flavour is one of the top breeding goals of many strawberry-producing countries. Although several genes involved in the biosynthetic pathways of key aroma compounds have been identified, the development and application of molecular markers associated with fruit flavour remain limited. This study aims to identify molecular markers closely linked to genes controlling strawberry aroma. A purpose-built Subtracted Diversity Array (SDA) known as Fragaria Discovery Panel (FDP) was used for marker screening. Polymorphic sequences associated with key aroma compounds were identified from two DNA bulks with extreme phenotypes, established using 50 F 1 progeny plants derived from Juliette X 07-102-41 cross, two strawberry genotypes differing in aroma profile. A total of 49 polymorphic markers for eight key aroma compounds were detected using genotypic data of the extreme DNA bulks and phenotypic data obtained from gas chromatography-mass spectrometry (GC-MS). A similarity search against the physical maps of Fragaria vesca revealed that FaP1D7 is linked to genes potentially involved in the synthesis of methyl butanoate. A C/T SNP was detected within the feature, which could possibly be converted to a molecular tool for rapid screening of the strawberry accessions for their methyl butanoate production capacity.

Transcriptomic analysis on the formation of the viable putative non-culturable state of beer-spoilage Lactobacillus acetotolerans

PubMed Central

Liu, Junyan; Deng, Yang; Peters, Brian M.; Li, Lin; Li, Bing; Chen, Lequn; Xu, Zhenbo; Shirtliff, Mark E.

2016-01-01

Lactic acid bacteria (LAB) are the most common beer-spoilage bacteria regardless of beer type, and thus pose significant problems for the brewery industry. The aim of this study was to investigate the genetic mechanisms involved in the ability of the hard-to-culture beer-spoilage bacterium Lactobacillus acetotolerans to enter into the viable putative non-culturable (VPNC) state. A genome-wide transcriptional analysis of beer-spoilage L. acetotolerans strains BM-LA14526, BM-LA14527, and BM-LA14528 under normal, mid-term and VPNC states were performed using RNA-sequencing (RNA-seq) and further bioinformatics analyses. GO function, COG category, and KEGG pathway enrichment analysis were conducted to investigate functional and related metabolic pathways of the differentially expressed genes. Functional and pathway enrichment analysis indicated that heightened stress response and reduction in genes associated with transport, metabolic process, and enzyme activity might play important roles in the formation of the VPNC state. This is the first transcriptomic analysis on the formation of the VPNC state of beer spoilage L. acetotolerans. PMID:27819317

Triterpenoid Saponin Biosynthetic Pathway Profiling and Candidate Gene Mining of the Ilex asprella Root Using RNA-Seq

PubMed Central

Zheng, Xiasheng; Xu, Hui; Ma, Xinye; Zhan, Ruoting; Chen, Weiwen

2014-01-01

Ilex asprella, which contains abundant α-amyrin type triterpenoid saponins, is an anti-influenza herbal drug widely used in south China. In this work, we first analysed the transcriptome of the I. asprella root using RNA-Seq, which provided a dataset for functional gene mining. mRNA was isolated from the total RNA of the I. asprella root and reverse-transcribed into cDNA. Then, the cDNA library was sequenced using an Illumina HiSeq™ 2000, which generated 55,028,452 clean reads. De novo assembly of these reads generated 51,865 unigenes, in which 39,269 unigenes were annotated (75.71% yield). According to the structures of the triterpenoid saponins of I. asprella, a putative biosynthetic pathway downstream of 2,3-oxidosqualene was proposed and candidate unigenes in the transcriptome data that were potentially involved in the pathway were screened using homology-based BLAST and phylogenetic analysis. Further amplification and functional analysis of these putative unigenes will provide insight into the biosynthesis of Ilex triterpenoid saponins. PMID:24722569

Transcriptomic analysis on the formation of the viable putative non-culturable state of beer-spoilage Lactobacillus acetotolerans.

PubMed

Liu, Junyan; Deng, Yang; Peters, Brian M; Li, Lin; Li, Bing; Chen, Lequn; Xu, Zhenbo; Shirtliff, Mark E

2016-11-07

Lactic acid bacteria (LAB) are the most common beer-spoilage bacteria regardless of beer type, and thus pose significant problems for the brewery industry. The aim of this study was to investigate the genetic mechanisms involved in the ability of the hard-to-culture beer-spoilage bacterium Lactobacillus acetotolerans to enter into the viable putative non-culturable (VPNC) state. A genome-wide transcriptional analysis of beer-spoilage L. acetotolerans strains BM-LA14526, BM-LA14527, and BM-LA14528 under normal, mid-term and VPNC states were performed using RNA-sequencing (RNA-seq) and further bioinformatics analyses. GO function, COG category, and KEGG pathway enrichment analysis were conducted to investigate functional and related metabolic pathways of the differentially expressed genes. Functional and pathway enrichment analysis indicated that heightened stress response and reduction in genes associated with transport, metabolic process, and enzyme activity might play important roles in the formation of the VPNC state. This is the first transcriptomic analysis on the formation of the VPNC state of beer spoilage L. acetotolerans.

Overlapping contributions of Msh1p and putative recombination proteins Cce1p, Din7p, and Mhr1p in large-scale recombination and genome sorting events in the mitochondrial genome of Saccharomyces cerevisiae.

PubMed

Mookerjee, Shona A; Sia, Elaine A

2006-03-20

The mechanisms that govern mutation avoidance in the mitochondrial genome, though believed to be numerous, are poorly understood. The identification of individual genes has implicated mismatch repair and several recombination pathways in maintaining the fidelity and structural stability of mitochondrial DNA. However, the majority of genes in these pathways have not been identified and the interactions between different pathways have not been extensively studied. Additionally, the multicopy presence of the mitochondrial genome affects the occurrence and persistence of mutant phenotypes, making mitochondrial DNA transmission and sorting important factors affecting mutation accumulation. We present new evidence that the putative recombination genes CCE1, DIN7, and MHR1 have overlapping function with the mismatch repair homolog MSH1 in point mutation avoidance and suppression of aberrant recombination events. In addition, we demonstrate a novel role for Msh1p in mtDNA transmission, a role not predicted by studies of its nuclear homologs.

Dynamic Redox Regulation of IL-4 Signaling.

PubMed

Dwivedi, Gaurav; Gran, Margaret A; Bagchi, Pritha; Kemp, Melissa L

2015-11-01

Quantifying the magnitude and dynamics of protein oxidation during cell signaling is technically challenging. Computational modeling provides tractable, quantitative methods to test hypotheses of redox mechanisms that may be simultaneously operative during signal transduction. The interleukin-4 (IL-4) pathway, which has previously been reported to induce reactive oxygen species and oxidation of PTP1B, may be controlled by several other putative mechanisms of redox regulation; widespread proteomic thiol oxidation observed via 2D redox differential gel electrophoresis upon IL-4 treatment suggests more than one redox-sensitive protein implicated in this pathway. Through computational modeling and a model selection strategy that relied on characteristic STAT6 phosphorylation dynamics of IL-4 signaling, we identified reversible protein tyrosine phosphatase (PTP) oxidation as the primary redox regulatory mechanism in the pathway. A systems-level model of IL-4 signaling was developed that integrates synchronous pan-PTP oxidation with ROS-independent mechanisms. The model quantitatively predicts the dynamics of IL-4 signaling over a broad range of new redox conditions, offers novel hypotheses about regulation of JAK/STAT signaling, and provides a framework for interrogating putative mechanisms involving receptor-initiated oxidation.

Dynamic Redox Regulation of IL-4 Signaling

PubMed Central

Dwivedi, Gaurav; Gran, Margaret A.; Bagchi, Pritha; Kemp, Melissa L.

2015-01-01

Quantifying the magnitude and dynamics of protein oxidation during cell signaling is technically challenging. Computational modeling provides tractable, quantitative methods to test hypotheses of redox mechanisms that may be simultaneously operative during signal transduction. The interleukin-4 (IL-4) pathway, which has previously been reported to induce reactive oxygen species and oxidation of PTP1B, may be controlled by several other putative mechanisms of redox regulation; widespread proteomic thiol oxidation observed via 2D redox differential gel electrophoresis upon IL-4 treatment suggests more than one redox-sensitive protein implicated in this pathway. Through computational modeling and a model selection strategy that relied on characteristic STAT6 phosphorylation dynamics of IL-4 signaling, we identified reversible protein tyrosine phosphatase (PTP) oxidation as the primary redox regulatory mechanism in the pathway. A systems-level model of IL-4 signaling was developed that integrates synchronous pan-PTP oxidation with ROS-independent mechanisms. The model quantitatively predicts the dynamics of IL-4 signaling over a broad range of new redox conditions, offers novel hypotheses about regulation of JAK/STAT signaling, and provides a framework for interrogating putative mechanisms involving receptor-initiated oxidation. PMID:26562652

Whole-Genome Analysis of the SHORT-ROOT Developmental Pathway in Arabidopsis

PubMed Central

Busch, Wolfgang; Cui, Hongchang; Wang, Jean Y; Blilou, Ikram; Hassan, Hala; Nakajima, Keiji; Matsumoto, Noritaka; Lohmann, Jan U; Scheres, Ben

2006-01-01

Stem cell function during organogenesis is a key issue in developmental biology. The transcription factor SHORT-ROOT (SHR) is a critical component in a developmental pathway regulating both the specification of the root stem cell niche and the differentiation potential of a subset of stem cells in the Arabidopsis root. To obtain a comprehensive view of the SHR pathway, we used a statistical method called meta-analysis to combine the results of several microarray experiments measuring the changes in global expression profiles after modulating SHR activity. Meta-analysis was first used to identify the direct targets of SHR by combining results from an inducible form of SHR driven by its endogenous promoter, ectopic expression, followed by cell sorting and comparisons of mutant to wild-type roots. Eight putative direct targets of SHR were identified, all with expression patterns encompassing subsets of the native SHR expression domain. Further evidence for direct regulation by SHR came from binding of SHR in vivo to the promoter regions of four of the eight putative targets. A new role for SHR in the vascular cylinder was predicted from the expression pattern of several direct targets and confirmed with independent markers. The meta-analysis approach was then used to perform a global survey of the SHR indirect targets. Our analysis suggests that the SHR pathway regulates root development not only through a large transcription regulatory network but also through hormonal pathways and signaling pathways using receptor-like kinases. Taken together, our results not only identify the first nodes in the SHR pathway and a new function for SHR in the development of the vascular tissue but also reveal the global architecture of this developmental pathway. PMID:16640459

In silico analysis of Mn transporters (NRAMP1) in various plant species.

PubMed

Vatansever, Recep; Filiz, Ertugrul; Ozyigit, Ibrahim Ilker

2016-03-01

Manganese (Mn) is an essential micronutrient in plant life cycle. It may be involved in photosynthesis, carbohydrate and lipid biosynthesis, and oxidative stress protection. Mn deficiency inhibits the plant growth and development, and causes the various plant symptoms such as interveinal chlorosis and tissue necrosis. Despite its importance in plant life cycle, we still have limited knowledge about Mn transporters in many plant species. Therefore, this study aimed to identify and characterize high affinity Arabidopsis Mn root transporter NRAMP1 orthologs in 17 different plant species. Various in silico methods and digital gene expression data were used in identification and characterization of NRAMP1 homologs; physico-chemical properties of sequences were calculated, putative transmembrane domains (TMDs) and conserved motif signatures were determined, phylogenetic tree was constructed, 3D models and interactome map were generated, and gene expression data was analyzed. 49 NRAMP1 homologs were identified from proteome datasets of 17 plant species using AtNRAMP1 as query. Identified sequences were characterized with a NRAMP domain structure, 10-12 putative TMDs with cytosolic N- and C-terminuses, and 10-14 exons encoding a protein of 500-588 amino acids and 53.8-64.3 kDa molecular weight with basic characteristics. Consensus transport residues, GQSSTITGTYAGQY(/F)V(/I)MQGFLD(/E/N) between TMD-8 and 9 were identified in all sequences but putative N-linked glycosylation sites were not highly conserved. In phylogeny, NRAMP1 sequences demonstrated divergence in lower and higher plants as well as in monocots and dicots. Despite divergence of lower plant Physcomitrella patens in phylogeny, it showed similarity in superposed 3D models. Phylogenetic distribution of AtNRAMP1 and 6 homologs inferred a functional relationship to NRAMP6 sequences in Mn transport, while distribution of OsNRAMP1 and 5 homologs implicated an involvement of NRAMP1 sequences in Mn transport or a cross-talk between in Fe-Mn homeostasis. Interactome analysis further confirmed this cross-talk between Mn and Fe pathways. Gene expression profile of AtNRAMP1 under Fe-, K-, P- and S-deficiencies, and cold, drought, heat and salt stresses revealed various proteins involving in transcription regulation, cofactor biosynthesis, diverse developmental roles, carbohydrate metabolism, oxidation-reduction reactions, cellular signaling and protein degradation pathways. Mn deficiency or toxicity could cause serious adverse effects in plants as well as in humans. To reduce these adversities mainly rely on understanding the molecular mechanisms underlying Mn uptake from the soil. However, we still have limited knowledge regarding the structural and functional roles of Mn transporters in many plant species. Therefore, identification and characterization of Mn root uptake transporter, NRAMP1 orthologs in various plant species will provide valuable theoretical knowledge to better understand Mn transporters as well as it may become an insight for future studies aiming to develop genetically engineered and biofortified plants.

Secondary water pore formation for proton transport in a ClC exchanger revealed by an atomistic molecular-dynamics simulation.

PubMed

Ko, Youn Jo; Jo, Won Ho

2010-05-19

Several prokaryotic ClC proteins have been demonstrated to function as exchangers that transport both chloride ions and protons simultaneously in opposite directions. However, the path of the proton through the ClC exchanger, and how the protein brings about the coupled movement of both ions are still unknown. In this work, we use an atomistic molecular dynamics (MD) simulation to demonstrate that a previously unknown secondary water pore is formed inside an Escherichia coli ClC exchanger. The secondary water pore is bifurcated from the chloride ion pathway at E148. From the systematic simulations, we determined that the glutamate residue exposed to the intracellular solution, E203, plays an important role as a trigger for the formation of the secondary water pore, and that the highly conserved tyrosine residue Y445 functions as a barrier that separates the proton from the chloride ion pathways. Based on our simulation results, we conclude that protons in the ClC exchanger are conducted via a water network through the secondary water pore, and we propose a new mechanism for the coupled transport of chloride ions and protons. It has been reported that several members of ClC proteins are not just channels that simply transport chloride ions across lipid bilayers; rather, they are exchangers that transport both the chloride ion and proton in opposite directions. However, the ion transit pathways and the mechanism of the coupled movement of these two ions have not yet been unveiled. In this article, we report a new finding (to our knowledge) of a water pore inside a prokaryotic ClC protein as revealed by computer simulation. This water pore is bifurcated from the putative chloride ion, and water molecules inside the new pore connect two glutamate residues that are known to be key residues for proton transport. On the basis of our simulation results, we conclude that the water wire that is formed inside the newly found pore acts as a proton pathway, which enables us to resolve many problems that could not be addressed by previous experimental studies. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Molecular characterization and mRNA expression of two key enzymes of hypoxia-sensing pathways in eastern oysters Crassostrea virginica (Gmelin): Hypoxia-inducible factor α (HIF-α) and HIF-prolyl hydroxylase (PHD)

PubMed Central

Piontkivska, Helen; Chung, J. Sook; Ivanina, Anna V.; Sokolov, Eugene P.; Techa, Sirinart; Sokolova, Inna M.

2010-01-01

Oxygen homeostasis is crucial for development, survival and normal function of all metazoans. A family of transcription factors called hypoxia-inducible factors (HIF) is critical in mediating the adaptive responses to reduced oxygen availability. The HIF transcription factor consists of a constitutively expressed β subunit and an oxygen-dependent α subunit; the abundance of the latter determines the activity of HIF and is regulated by a family of O2- and Fe2+-dependent enzymes prolyl hydroxylases (PHDs). Currently very little is known about the function of this important pathway and the molecular structure of its key players in hypoxia-tolerant intertidal mollusks including oysters, which are among the animal champions of anoxic and hypoxic tolerance and thus can serve as excellent models to study the role of HIF cascade in adaptations to oxygen deficiency. We have isolated transcripts of two key components of the oxygen sensing pathway - the oxygen-regulated HIF-α subunit and PHD - from an intertidal mollusk, the eastern oyster Crassostrea virginica, and determined the transcriptional responses of these two genes to anoxia, hypoxia and cadmium (Cd) stress. HIF-α and PHD homologs from eastern oysters C. virginica show significant sequence similarity and share key functional domains with the earlier described isoforms from vertebrates and invertebrates. Phylogenetic analysis shows that genetic diversification of HIF and PHD isoforms occurred within the vertebrate lineage indicating functional diversification and specialization of the oxygen-sensing pathways in this group, which parallels situation observed for many other important genes. HIF-α and PHD homologs are broadly expressed at the mRNA level in different oyster tissues and show transcriptional responses to prolonged hypoxia in the gills consistent with their putative role in oxygen sensing and the adaptive response to hypoxia. Similarity in amino acid sequence, domain structure and transcriptional responses between HIF-α and PHD homologs from oysters and other invertebrate and vertebrate species implies the highly conserved functions of these genes throughout the evolutionary history of animals, in accordance with their critical role in oxygen sensing and homeostasis. PMID:21106446

Next generation sequencing and de novo transcriptome analysis of Costus pictus D. Don, a non-model plant with potent anti-diabetic properties

PubMed Central

2012-01-01

Background Phyto-remedies for diabetic control are popular among patients with Type II Diabetes mellitus (DM), in addition to other diabetic control measures. A number of plant species are known to possess diabetic control properties. Costus pictus D. Don is popularly known as “Insulin Plant” in Southern India whose leaves have been reported to increase insulin pools in blood plasma. Next Generation Sequencing is employed as a powerful tool for identifying molecular signatures in the transcriptome related to physiological functions of plant tissues. We sequenced the leaf transcriptome of C. pictus using Illumina reversible dye terminator sequencing technology and used combination of bioinformatics tools for identifying transcripts related to anti-diabetic properties of C. pictus. Results A total of 55,006 transcripts were identified, of which 69.15% transcripts could be annotated. We identified transcripts related to pathways of bixin biosynthesis and geraniol and geranial biosynthesis as major transcripts from the class of isoprenoid secondary metabolites and validated the presence of putative norbixin methyltransferase, a precursor of Bixin. The transcripts encoding these terpenoids are known to be Peroxisome Proliferator-Activated Receptor (PPAR) agonists and anti-glycation agents. Sequential extraction and High Performance Liquid Chromatography (HPLC) confirmed the presence of bixin in C. pictus methanolic extracts. Another significant transcript identified in relation to anti-diabetic, anti-obesity and immuno-modulation is of Abscisic Acid biosynthetic pathway. We also report many other transcripts for the biosynthesis of antitumor, anti-oxidant and antimicrobial metabolites of C. pictus leaves. Conclusion Solid molecular signatures (transcripts related to bixin, abscisic acid, and geranial and geraniol biosynthesis) for the anti-diabetic properties of C. pictus leaves and vital clues related to the other phytochemical functions like antitumor, anti-oxidant, immuno-modulatory, anti-microbial and anti-malarial properties through the secondary metabolite pathway annotations are reported. The data provided will be of immense help to researchers working in the treatment of DM using herbal therapies. PMID:23176672

Harnessing pain heterogeneity and RNA transcriptome to identify blood-based pain biomarkers: a novel correlational study design and bioinformatics approach in a graded chronic constriction injury model.

PubMed

Grace, Peter M; Hurley, Daniel; Barratt, Daniel T; Tsykin, Anna; Watkins, Linda R; Rolan, Paul E; Hutchinson, Mark R

2012-09-01

A quantitative, peripherally accessible biomarker for neuropathic pain has great potential to improve clinical outcomes. Based on the premise that peripheral and central immunity contribute to neuropathic pain mechanisms, we hypothesized that biomarkers could be identified from the whole blood of adult male rats, by integrating graded chronic constriction injury (CCI), ipsilateral lumbar dorsal quadrant (iLDQ) and whole blood transcriptomes, and pathway analysis with pain behavior. Correlational bioinformatics identified a range of putative biomarker genes for allodynia intensity, many encoding for proteins with a recognized role in immune/nociceptive mechanisms. A selection of these genes was validated in a separate replication study. Pathway analysis of the iLDQ transcriptome identified Fcγ and Fcε signaling pathways, among others. This study is the first to employ the whole blood transcriptome to identify pain biomarker panels. The novel correlational bioinformatics, developed here, selected such putative biomarkers based on a correlation with pain behavior and formation of signaling pathways with iLDQ genes. Future studies may demonstrate the predictive ability of these biomarker genes across other models and additional variables. © 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.

ALMA 0.1-0.2 arcsec Resolution Imaging of the NGC 1068 Nucleus: Compact Dense Molecular Gas Emission at the Putative AGN Location

NASA Astrophysics Data System (ADS)

Imanishi, Masatoshi; Nakanishi, Kouichiro; Izumi, Takuma

2016-05-01

We present the results of our ALMA Cycle 2 high angular resolution (0.″1-0.″2) observations of the nuclear region of the nearby well-studied type-2 active galactic nucleus (AGN), NGC 1068, at HCN J = 3-2 and HCO+ J = 3-2 emission lines. For the first time, due to a higher angular resolution than previous studies, we clearly detected dense molecular gas emission at the putative AGN location, identified as a ˜1.1 mm (˜266 GHz) continuum emission peak, by separating this emission from brighter emission located at 0.″5-2.″0 on the eastern and western sides of the AGN. The estimated intrinsic molecular emission size and dense molecular mass, which are thought to be associated with the putative dusty molecular torus around an AGN, were ˜10 pc and ˜several × 105 M ⊙, respectively. HCN-to-HCO+ J = 3-2 flux ratios substantially higher than unity were found throughout the nuclear region of NGC 1068. The continuum emission displayed an elongated morphology along the direction of the radio jet located at the northern side of the AGN, as well as a weak spatially-resolved component at ˜2.″0 on the southwestern side of the AGN. The latter component most likely originated from star formation, with the estimated luminosity more than one order of magnitude lower than the luminosity of the central AGN. No vibrationally excited (v 2 = 1f) J = 3-2 emission lines were detected for HCN and HCO+ across the field of view.

Sugar uptake in the Aril of litchi fruit depends on the apoplasmic post-phloem transport and the activity of proton pumps and the putative transporter LcSUT4.

PubMed

Wang, Teng-Duan; Zhang, Hui-Fen; Wu, Zi-Chen; Li, Jian-Guo; Huang, Xu-Ming; Wang, Hui-Cong

2015-02-01

The post-phloem unloading pathway and the mechanism of sugar accumulation remain unclear in litchi fruit. A combination of electron microscopy, transport of phloem-mobile symplasmic tracer (carboxyfluorescein, CF) and biochemical and molecular assays was used to explore the post-phloem transport pathway and the mechanism of aril sugar accumulation in litchi. In the funicle, where the aril originates, abundant plasmodesmata were observed, and CF introduced from the peduncle diffused to the parenchyma cells. In addition, abundant starch and pentasaccharide were detected and the sugar concentration was positively correlated with activities of sucrose hydrolysis enzymes. These results clearly showed that the phloem unloading and post-phloem transport in the funicle were symplastic. On the other hand, imaging of CF showed that it remained confined to the parenchyma cells in funicle tissues connecting the aril. Infiltration of both an ATPase inhibitor [eosin B (EB)] and a sucrose transporter inhibitor [p-chloromercuribenzene sulfonate (PCMBS)] inhibited sugar accumulation in the aril. These results indicated an apoplasmic post-phloem sugar transport from the funicle to the aril. Although facilitated diffusion might help sucrose uptake from the cytosol to the vacuole in cultivars with high soluble invertase, membrane ATPases in the aril, especially tonoplast ATPase, are crucial for aril sugar accumulation. The expression of a putative aril vacuolar membrane sucrose transporter gene (LcSUT4) was highly correlated with the sugar accumulation in the aril of litchi. These data suggest that apoplasmic transport is critical for sugar accumulation in litchi aril and that LcSUT4 is involved in this step. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Composite fatty acid ether amides suppress growth of liver cancer cells in vitro and in an in vivo allograft mouse model.

PubMed

Cao, Mengde; Prima, Victor; Nelson, David; Svetlov, Stanislav

2013-06-01

The heterogeneity of liver cancer, in particular hepatocellular carcinoma (HCC), portrays the requirement of multiple targets for both its treatment and prevention. Multifaceted agents, minimally or non-toxic for normal hepatocytes, are required to address the molecular diversity of HCC, including the resistance of putative liver cancer stem cells to chemotherapy. We designed and synthesized two fatty acid ethers of isopropylamino propanol, C16:0-AIP-1 and C18:1-AIP-2 (jointly named AIPs), and evaluated their anti-proliferative effects on the human HCC cell line Huh7 and the murine hepatoma cell line BNL 1MEA.7R.1, both in vitro and in an in vivo allograft mouse model. We found that AIP-1 and AIP-2 inhibited proliferation and caused cell death in both Huh7 and BNL 1MEA.7R.1 cells. Importantly, AIP-1 and AIP-2 were found to block the activation of putative liver cancer stem cells as manifested by suppression of clonal 'carcinosphere' development in growth factor-free and anchorage-free medium. The AIPs exhibited a relatively low toxicity against normal human or rat hepatocytes in primary cultures. In addition, we found that the AIPs utilized multifaceted pathways that mediate both autophagy and apoptosis in HCC, including the inhibition of AKTs and CAMK-1. In immune-competent mice, the AIPs significantly reduced BNL 1MEA.7R.1 cell-driven tumor allograft development, with a higher efficiency than sorafenib. A combination of AIP-1 + AIP-2 was most effective in reducing the tumor allograft incidence. AIPs represent a novel class of simple fatty acid derivatives that are effective against liver tumors via diverse pathways. They show a low toxicity towards normal hepatocytes. The addition of AIPs may represent a new avenue towards the management of chronic liver injury and, ultimately, the prevention and treatment of HCC.

Further insight into reproductive incompatibility between putative cryptic species of the Bemisia tabaci whitefly complex.

PubMed

Qin, Li; Pan, Li-Long; Liu, Shu-Sheng

2016-04-01

The whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), with its global distribution and extensive genetic diversity, is now known to be a complex of over 35 cryptic species. However, a satisfactory resolution of the systematics of this species complex is yet to be achieved. Here, we designed experiments to examine reproductive compatibility among species with different levels of mitochondrial cytochrome oxidase I (mtCOI) divergence. The data show that putative species with mtCOI divergence of >8% between them consistently exhibited complete reproductive isolation. However, two of the putative species, Asia II 9 and Asia II 3, with mtCOI divergence of 4.47% between them, exhibited near complete reproductive compatibility in one direction of their cross, and partial reproductive compatibility in the other direction. Together with some recent reports on this topic from the literature, our data indicates that, while divergence in the mtCOI sequences provides a valid molecular marker for species delimitation in most clades, more genetic markers and more sophisticated molecular phylogeny will be required to achieve adequate delimitation of all species in this whitefly complex. While many attempts have been made to examine the reproductive compatibility among genetic groups of the B. tabaci complex, our study represents the first effort to conduct crossing experiments with putative species that were chosen with considerations of their genetic divergence. In light of the new data, we discuss the best strategy and protocols to conduct further molecular phylogenetic analysis and crossing trials, in order to reveal the overall pattern of reproductive incompatibility among species of this whitefly complex. © 2015 Institute of Zoology, Chinese Academy of Sciences.

A putative biomarker signature for clinically effective AKT inhibition: correlation of in vitro, in vivo and clinical data identifies the importance of modulation of the mTORC1 pathway.

PubMed

Cheraghchi-Bashi, Azadeh; Parker, Christine A; Curry, Ed; Salazar, Jean-Frederic; Gungor, Hatice; Saleem, Azeem; Cunnea, Paula; Rama, Nona; Salinas, Cristian; Mills, Gordon B; Morris, Shannon R; Kumar, Rakesh; Gabra, Hani; Stronach, Euan A

2015-12-08

Our identification of dysregulation of the AKT pathway in ovarian cancer as a platinum resistance specific event led to a comprehensive analysis of in vitro, in vivo and clinical behaviour of the AKT inhibitor GSK2141795. Proteomic biomarker signatures correlating with effects of GSK2141795 were developed using in vitro and in vivo models, well characterised for related molecular, phenotypic and imaging endpoints. Signatures were validated in temporally paired biopsies from patients treated with GSK2141795 in a clinical study. GSK2141795 caused growth-arrest as single agent in vitro, enhanced cisplatin-induced apoptosis in vitro and reduced tumour volume in combination with platinum in vivo. GSK2141795 treatment in vitro and in vivo resulted in ~50-90% decrease in phospho-PRAS40 and 20-80% decrease in fluoro-deoxyglucose (FDG) uptake. Proteomic analysis of GSK2141795 in vitro and in vivo identified a signature of pathway inhibition including changes in AKT and p38 phosphorylation and total Bim, IGF1R, AR and YB1 levels. In patient biopsies, prior to treatment with GSK2141795 in a phase 1 clinical trial, this signature was predictive of post-treatment changes in the response marker CA125. Development of this signature represents an opportunity to demonstrate the clinical importance of AKT inhibition for re-sensitisation of platinum resistant ovarian cancer to platinum.

Temperature controls on the basal emission rate of isoprene in a tropical tree Ficus septica: exploring molecular regulatory mechanisms.

PubMed

Mutanda, Ishmael; Inafuku, Masashi; Saitoh, Seikoh; Iwasaki, Hironori; Fukuta, Masakazu; Watanabe, Keiichi; Oku, Hirosuke

2016-10-01

Isoprene emission from plants is very sensitive to environmental temperature both at short-term and long-term scales. Our previous study demonstrated suppression of isoprene emission by cold temperatures in a high emitting tropical tree Ficus septica and revealed a strong correlation of emission to isoprene synthase (IspS) protein levels. When challenged with decreasing daily temperatures from 30 to 12 °C, F. septica completely stopped isoprene emission at 12 °C, only to recover on the second day after re-exposure to 30 °C. Here, we explored this regulation of isoprene emission in response to environmental temperature by a comprehensive analysis of transcriptome data, gene expressions and metabolite pools of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. MEP pathway genes and metabolites dynamics did not support substrate-level limitations as major control over observed basal emission, but transcriptome data, network inferences and putative regulatory elements on IspS promoter suggested transcriptional regulation of IspS gene through circadian rhythm and phytohormone signalling processes. Expression levels of 29 genes involved in these pathways were examined by quantitative real-time PCR. We propose that temperature controls over basal isoprene emission at a time-scale of hours to few days are regulated by phytohormone-mediated transcriptional modulation of IspS gene under synchronization by the circadian clock. © 2016 John Wiley & Sons Ltd.

Mathematical models of lignin biosynthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Faraji, Mojdeh; Fonseca, Luis; Escamilla-Trevino, Luis

Lignin is a natural polymer that is interwoven with cellulose and hemicellulose within plant cell walls. Due to this molecular arrangement, lignin is a major contributor to the recalcitrance of plant materials with respect to the extraction of sugars and their fermentation into ethanol, butanol, and other potential bioenergy crops. The lignin biosynthetic pathway is similar, but not identical in different plant species. It is in each case comprised of a moderate number of enzymatic steps, but its responses to manipulations, such as gene knock-downs, are complicated by the fact that several of the key enzymes are involved in severalmore » reaction steps. This feature poses a challenge to bioenergy production, as it renders it difficult to select the most promising combinations of genetic manipulations for the optimization of lignin composition and amount.Here, we present several computational models than can aid in the analysis of data characterizing lignin biosynthesis. While minimizing technical details, we focus on the questions of what types of data are particularly useful for modeling and what genuine benefits the biofuel researcher may gain from the resulting models. We demonstrate our analysis with mathematical models for black cottonwood (Populus trichocarpa), alfalfa (Medicago truncatula), switchgrass (Panicum virgatum) and the grass Brachypodium distachyon. Despite commonality in pathway structure, different plant species show different regulatory features and distinct spatial and topological characteristics. The putative lignin biosynthes pathway is not able to explain the plant specific laboratory data, and the necessity of plant specific modeling should be heeded.« less

Mathematical models of lignin biosynthesis

DOE PAGES

Faraji, Mojdeh; Fonseca, Luis; Escamilla-Trevino, Luis; ...

2018-02-09

Lignin is a natural polymer that is interwoven with cellulose and hemicellulose within plant cell walls. Due to this molecular arrangement, lignin is a major contributor to the recalcitrance of plant materials with respect to the extraction of sugars and their fermentation into ethanol, butanol, and other potential bioenergy crops. The lignin biosynthetic pathway is similar, but not identical in different plant species. It is in each case comprised of a moderate number of enzymatic steps, but its responses to manipulations, such as gene knock-downs, are complicated by the fact that several of the key enzymes are involved in severalmore » reaction steps. This feature poses a challenge to bioenergy production, as it renders it difficult to select the most promising combinations of genetic manipulations for the optimization of lignin composition and amount.Here, we present several computational models than can aid in the analysis of data characterizing lignin biosynthesis. While minimizing technical details, we focus on the questions of what types of data are particularly useful for modeling and what genuine benefits the biofuel researcher may gain from the resulting models. We demonstrate our analysis with mathematical models for black cottonwood (Populus trichocarpa), alfalfa (Medicago truncatula), switchgrass (Panicum virgatum) and the grass Brachypodium distachyon. Despite commonality in pathway structure, different plant species show different regulatory features and distinct spatial and topological characteristics. The putative lignin biosynthes pathway is not able to explain the plant specific laboratory data, and the necessity of plant specific modeling should be heeded.« less

Comparative Transcriptome Analysis Identifies Putative Genes Involved in the Biosynthesis of Xanthanolides in Xanthium strumarium L.

PubMed Central

Li, Yuanjun; Gou, Junbo; Chen, Fangfang; Li, Changfu; Zhang, Yansheng

2016-01-01

Xanthium strumarium L. is a traditional Chinese herb belonging to the Asteraceae family. The major bioactive components of this plant are sesquiterpene lactones (STLs), which include the xanthanolides. To date, the biogenesis of xanthanolides, especially their downstream pathway, remains largely unknown. In X. strumarium, xanthanolides primarily accumulate in its glandular trichomes. To identify putative gene candidates involved in the biosynthesis of xanthanolides, three X. strumarium transcriptomes, which were derived from the young leaves of two different cultivars and the purified glandular trichomes from one of the cultivars, were constructed in this study. In total, 157 million clean reads were generated and assembled into 91,861 unigenes, of which 59,858 unigenes were successfully annotated. All the genes coding for known enzymes in the upstream pathway to the biosynthesis of xanthanolides were present in the X. strumarium transcriptomes. From a comparative analysis of the X. strumarium transcriptomes, this study identified a number of gene candidates that are putatively involved in the downstream pathway to the synthesis of xanthanolides, such as four unigenes encoding CYP71 P450s, 50 unigenes for dehydrogenases, and 27 genes for acetyltransferases. The possible functions of these four CYP71 candidates are extensively discussed. In addition, 116 transcription factors that are highly expressed in X. strumarium glandular trichomes were also identified. Their possible regulatory roles in the biosynthesis of STLs are discussed. The global transcriptomic data for X. strumarium should provide a valuable resource for further research into the biosynthesis of xanthanolides. PMID:27625674

Bioinformatics analysis on molecular mechanism of rheum officinale in treatment of jaundice

NASA Astrophysics Data System (ADS)

Shan, Si; Tu, Jun; Nie, Peng; Yan, Xiaojun

2017-01-01

Objective: To study the molecular mechanism of Rheum officinale in the treatment of Jaundice by building molecular networks and comparing canonical pathways. Methods: Target proteins of Rheum officinale and related genes of Jaundice were searched from Pubchem and Gene databases online respectively. Molecular networks and canonical pathways comparison analyses were performed by Ingenuity Pathway Analysis (IPA). Results: The molecular networks of Rheum officinale and Jaundice were complex and multifunctional. The 40 target proteins of Rheum officinale and 33 Homo sapiens genes of Jaundice were found in databases. There were 19 common pathways both related networks. Rheum officinale could regulate endothelial differentiation, Interleukin-1B (IL-1B) and Tumor Necrosis Factor (TNF) in these pathways. Conclusions: Rheum officinale treat Jaundice by regulating many effective nodes of Apoptotic pathway and cellular immunity related pathways.

Isolation and molecular identification of Sunshine virus, a novel paramyxovirus found in Australian snakes.

PubMed

Hyndman, Timothy H; Marschang, Rachel E; Wellehan, James F X; Nicholls, Philip K

2012-10-01

This paper describes the isolation and molecular identification of a novel paramyxovirus found during an investigation of an outbreak of neurorespiratory disease in a collection of Australian pythons. Using Illumina® high-throughput sequencing, a 17,187 nucleotide sequence was assembled from RNA extracts from infected viper heart cells (VH2) displaying widespread cytopathic effects in the form of multinucleate giant cells. The sequence appears to contain all the coding regions of the genome, including the following predicted paramyxoviral open reading frames (ORFs): 3'--Nucleocapsid (N)--putative Phosphoprotein (P)--Matrix (M)--Fusion (F)--putative attachment protein--Polymerase (L)--5'. There is also a 540 nucleotide ORF between the N and putative P genes that may be an additional coding region. Phylogenetic analyses of the complete N, M, F and L genes support the clustering of this virus within the family Paramyxoviridae but outside both of the current subfamilies: Paramyxovirinae and Pneumovirinae. We propose to name this new virus, Sunshine virus, after the geographic origin of the first isolate--the Sunshine Coast of Queensland, Australia. Copyright © 2012 Elsevier B.V. All rights reserved.

miRnalyze: an interactive database linking tool to unlock intuitive microRNA regulation of cell signaling pathways

PubMed Central

Subhra Das, Sankha; James, Mithun; Paul, Sandip

2017-01-01

Abstract The various pathophysiological processes occurring in living systems are known to be orchestrated by delicate interplays and cross-talks between different genes and their regulators. Among the various regulators of genes, there is a class of small non-coding RNA molecules known as microRNAs. Although, the relative simplicity of miRNAs and their ability to modulate cellular processes make them attractive therapeutic candidates, their presence in large numbers make it challenging for experimental researchers to interpret the intricacies of the molecular processes they regulate. Most of the existing bioinformatic tools fail to address these challenges. Here, we present a new web resource ‘miRnalyze’ that has been specifically designed to directly identify the putative regulation of cell signaling pathways by miRNAs. The tool integrates miRNA-target predictions with signaling cascade members by utilizing TargetScanHuman 7.1 miRNA-target prediction tool and the KEGG pathway database, and thus provides researchers with in-depth insights into modulation of signal transduction pathways by miRNAs. miRnalyze is capable of identifying common miRNAs targeting more than one gene in the same signaling pathway—a feature that further increases the probability of modulating the pathway and downstream reactions when using miRNA modulators. Additionally, miRnalyze can sort miRNAs according to the seed-match types and TargetScan Context ++ score, thus providing a hierarchical list of most valuable miRNAs. Furthermore, in order to provide users with comprehensive information regarding miRNAs, genes and pathways, miRnalyze also links to expression data of miRNAs (miRmine) and genes (TiGER) and proteome abundance (PaxDb) data. To validate the capability of the tool, we have documented the correlation of miRnalyze’s prediction with experimental confirmation studies. Database URL: http://www.mirnalyze.in PMID:28365733

Identification of genomic variants putatively targeted by selection during dog domestication.

PubMed

Cagan, Alex; Blass, Torsten

2016-01-12

Dogs [Canis lupus familiaris] were the first animal species to be domesticated and continue to occupy an important place in human societies. Recent studies have begun to reveal when and where dog domestication occurred. While much progress has been made in identifying the genetic basis of phenotypic differences between dog breeds we still know relatively little about the genetic changes underlying the phenotypes that differentiate all dogs from their wild progenitors, wolves [Canis lupus]. In particular, dogs generally show reduced aggression and fear towards humans compared to wolves. Therefore, selection for tameness was likely a necessary prerequisite for dog domestication. With the increasing availability of whole-genome sequence data it is possible to try and directly identify the genetic variants contributing to the phenotypic differences between dogs and wolves. We analyse the largest available database of genome-wide polymorphism data in a global sample of dogs 69 and wolves 7. We perform a scan to identify regions of the genome that are highly differentiated between dogs and wolves. We identify putatively functional genomic variants that are segregating or at high frequency [> = 0.75 Fst] for alternative alleles between dogs and wolves. A biological pathways analysis of the genes containing these variants suggests that there has been selection on the 'adrenaline and noradrenaline biosynthesis pathway', well known for its involvement in the fight-or-flight response. We identify 11 genes with putatively functional variants fixed for alternative alleles between dogs and wolves. The segregating variants in these genes are strong candidates for having been targets of selection during early dog domestication. We present the first genome-wide analysis of the different categories of putatively functional variants that are fixed or segregating at high frequency between a global sampling of dogs and wolves. We find evidence that selection has been strongest around non-synonymous variants. Strong selection in the initial stages of dog domestication appears to have occurred on multiple genes involved in the fight-or-flight response, particularly in the catecholamine synthesis pathway. Different alleles in some of these genes have been associated with behavioral differences between modern dog breeds, suggesting an important role for this pathway at multiple stages in the domestication process.

Regulation of rat heme oxygenase-1 expression by interleukin-6 via the Jak/STAT pathway in hepatocytes.

PubMed

Tron, Kyrylo; Samoylenko, Anatoly; Musikowski, Gernot; Kobe, Fritz; Immenschuh, Stephan; Schaper, Fred; Ramadori, Giuliano; Kietzmann, Thomas

2006-07-01

Heme oxygenase-1 (HO-1) can be induced by various stimuli, one of which is interleukin-6 (IL-6). Therefore, the aim of this study was to elucidate the molecular mechanisms responsible for IL-6-dependent HO-1 induction in the liver. The IL-6-dependent HO-1 regulation in rat primary hepatocytes and HepG2 hepatoma cells was studied by Northern and Western blot analyses, HO-1 promoter reporter gene assays and EMSA. The HO-1 expression was transcriptionally induced by IL-6 in a time- and dose-dependent manner. Activation of signal transducers and activators of transcription (STAT) factors by the IL-6 receptor was crucial for HO-1 induction. By contrast, negative regulation of HO-1 expression appeared to be mediated through the SH2-domain-containing tyrosine phosphatase-2 (SHP2)/ suppressors of cytokine signaling-3 (SOCS3) binding site within the gp130 IL-6 receptor subunit. Among the three putative STAT binding elements (SBE) in the HO-1 promoter, only the distal one was functional and when deleted, the remaining Luc induction was completely obliterated by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. The HO-1 SBE3 mediates HO-1 gene induction by IL-6 mainly via activation of the Jak/STAT pathway.

Lignin, mitochondrial family, and photorespiratory transporter classification as case studies in using co-expression, co-response, and protein locations to aid in identifying transport functions

PubMed Central

Tohge, Takayuki; Fernie, Alisdair R.

2014-01-01

Whole genome sequencing and the relative ease of transcript profiling have facilitated the collection and data warehousing of immense quantities of expression data. However, a substantial proportion of genes are not yet functionally annotated a problem which is particularly acute for transport proteins. In Arabidopsis, for example, only a minor fraction of the estimated 700 intracellular transporters have been identified at the molecular genetic level. Furthermore it is only within the last couple of years that critical genes such as those encoding the final transport step required for the long distance transport of sucrose and the first transporter of the core photorespiratory pathway have been identified. Here we will describe how transcriptional coordination between genes of known function and non-annotated genes allows the identification of putative transporters on the premise that such co-expressed genes tend to be functionally related. We will additionally extend this to include the expansion of this approach to include phenotypic information from other levels of cellular organization such as proteomic and metabolomic data and provide case studies wherein this approach has successfully been used to fill knowledge gaps in important metabolic pathways and physiological processes. PMID:24672529

Innate immunity mediated longevity and longevity induced by germ cell removal converge on the C-type lectin domain protein IRG-7

PubMed Central

Yunger, Elad; Safra, Modi; Levi-Ferber, Mor; Haviv-Chesner, Anat

2017-01-01

In C. elegans, removal of the germline triggers molecular events in the neighboring intestine, which sends an anti-aging signal to the rest of the animal. In this study, we identified an innate immunity related gene, named irg-7, as a novel mediator of longevity in germlineless animals. We consider irg-7 to be an integral downstream component of the germline longevity pathway because its expression increases upon germ cell removal and its depletion interferes with the activation of the longevity-promoting transcription factors DAF-16 and DAF-12 in germlineless animals. Furthermore, irg-7 activation by itself sensitizes the animals' innate immune response and extends the lifespan of animals exposed to live bacteria. This lifespan-extending pathogen resistance relies on the somatic gonad as well as on many genes previously associated with the reproductive longevity pathway. This suggests that these genes are also relevant in animals with an intact gonad, and can affect their resistance to pathogens. Altogether, this study demonstrates the tight association between germline homeostasis and the immune response of animals, and raises the possibility that the reproductive system can act as a signaling center to divert resources towards defending against putative pathogen attacks. PMID:28196094

MicroRNAs: New Insight in Modulating Follicular Atresia: A Review.

PubMed

Worku, Tesfaye; Rehman, Zia Ur; Talpur, Hira Sajjad; Bhattarai, Dinesh; Ullah, Farman; Malobi, Ngabu; Kebede, Tesfaye; Yang, Liguo

2017-02-09

Our understanding of the post-transcriptional mechanisms involved in follicular atresia is limited; however, an important development has been made in understanding the biological regulatory networks responsible for mediating follicular atresia. MicroRNAs have come to be seen as a key regulatory actor in determining cell fate in a wide range of tissues in normal and pathological processes. Profiling studies of miRNAs during follicular atresia and development have identified several putative miRNAs enriched in apoptosis signaling pathways. Subsequent in vitro and/or in vivo studies of granulosa cells have elucidated the functional role of some miRNAs along with their molecular pathways. In particular, the regulatory roles of some miRNAs have been consistently observed during studies of follicular cellular apoptosis. Continued work should gradually lead to better understanding of the role of miRNAs in this field. Ultimately, we expect this understanding will have substantial benefits for fertility management at both the in vivo or/and in vitro levels. The stable nature of miRNA holds remarkable promise in clinical use as a diagnostic tool and in reproductive medicine to solve the ever-increasing fertility problem. In this review, we summarize current knowledge of the involvement of miRNAs in follicular atresia, discuss the challenges for further work and pinpoint areas for future research.

MicroRNAs: New Insight in Modulating Follicular Atresia: A Review

PubMed Central

Worku, Tesfaye; Rehman, Zia Ur; Talpur, Hira Sajjad; Bhattarai, Dinesh; Ullah, Farman; Malobi, Ngabu; Kebede, Tesfaye; Yang, Liguo

2017-01-01

Our understanding of the post-transcriptional mechanisms involved in follicular atresia is limited; however, an important development has been made in understanding the biological regulatory networks responsible for mediating follicular atresia. MicroRNAs have come to be seen as a key regulatory actor in determining cell fate in a wide range of tissues in normal and pathological processes. Profiling studies of miRNAs during follicular atresia and development have identified several putative miRNAs enriched in apoptosis signaling pathways. Subsequent in vitro and/or in vivo studies of granulosa cells have elucidated the functional role of some miRNAs along with their molecular pathways. In particular, the regulatory roles of some miRNAs have been consistently observed during studies of follicular cellular apoptosis. Continued work should gradually lead to better understanding of the role of miRNAs in this field. Ultimately, we expect this understanding will have substantial benefits for fertility management at both the in vivo or/and in vitro levels. The stable nature of miRNA holds remarkable promise in clinical use as a diagnostic tool and in reproductive medicine to solve the ever-increasing fertility problem. In this review, we summarize current knowledge of the involvement of miRNAs in follicular atresia, discuss the challenges for further work and pinpoint areas for future research. PMID:28208755

Proteomic characterization of Aspergillus fumigatus treated with an antifungal coumarin for identification of novel target molecules of key pathways.

PubMed

Singh, Seema; Gupta, Shilpi; Singh, Bharat; Sharma, Sunil K; Gupta, Vijay K; Sharma, Gainda L

2012-06-01

A synthetic coumarin, N,N,N-triethyl-11-(4-methyl-2-oxo-2H-chromen-7-yloxy)-11-oxoundecan-1-aminium bromide (SCD-1), having potent activity against pathogenic Aspergilli (MIC90 15.62 μg/mL), was investigated to identify its molecular targets in the pathogen. The proteome of Aspergillus fumigatus was developed after treatment with sublethal doses of compound and analyzed. The results demonstrated 143 differentially expressed proteins on treatment with SCD-1. The expression of four proteins, namely cell division control protein, ubiquitin-like activating enzyme, vacuolar ATP synthase catalytic subunit A, and UTP-glucose-1-phosphate uridylyltransferase of A. fumigatus, was completely inhibited, whereas there were 13 newly expressed and 96 overexpressed proteins, mainly belonging to stress pathway. The treatment of A. fumigatus with SCD-1 also led to attenuation of proteins involved in cell replication and other important biosynthetic processes, including riboflavin biosynthesis, which has been pathogen-specific. In addition to key enzymatic players and antioxidants, nine hypothetical proteins were also identified, seven of which have been novel, being described for the first time. As no cellular functions have yet been described for these hypothetical proteins, their alteration in response to SCD-1 provides significant information about their putative roles in pathogen defense.

Phosphoketolase pathway contributes to carbon metabolism in cyanobacteria.

PubMed

Xiong, Wei; Lee, Tai-Chi; Rommelfanger, Sarah; Gjersing, Erica; Cano, Melissa; Maness, Pin-Ching; Ghirardi, Maria; Yu, Jianping

2015-12-07

Central carbon metabolism in cyanobacteria comprises the Calvin-Benson-Bassham (CBB) cycle, glycolysis, the pentose phosphate (PP) pathway and the tricarboxylic acid (TCA) cycle. Redundancy in this complex metabolic network renders the rational engineering of cyanobacterial metabolism for the generation of biomass, biofuels and chemicals a challenge. Here we report the presence of a functional phosphoketolase pathway, which splits xylulose-5-phosphate (or fructose-6-phosphate) to acetate precursor acetyl phosphate, in an engineered strain of the model cyanobacterium Synechocystis (ΔglgC/xylAB), in which glycogen synthesis is blocked, and xylose catabolism enabled through the introduction of xylose isomerase and xylulokinase. We show that this mutant strain is able to metabolise xylose to acetate on nitrogen starvation. To see whether acetate production in the mutant is linked to the activity of phosphoketolase, we disrupted a putative phosphoketolase gene (slr0453) in the ΔglgC/xylAB strain, and monitored metabolic flux using (13)C labelling; acetate and 2-oxoglutarate production was reduced in the light. A metabolic flux analysis, based on isotopic data, suggests that the phosphoketolase pathway metabolises over 30% of the carbon consumed by ΔglgC/xylAB during photomixotrophic growth on xylose and CO2. Disruption of the putative phosphoketolase gene in wild-type Synechocystis also led to a deficiency in acetate production in the dark, indicative of a contribution of the phosphoketolase pathway to heterotrophic metabolism. We suggest that the phosphoketolase pathway, previously uncharacterized in photosynthetic organisms, confers flexibility in energy and carbon metabolism in cyanobacteria, and could be exploited to increase the efficiency of cyanobacterial carbon metabolism and photosynthetic productivity.

De Novo Assembly and Characterization of the Transcriptome of the Chinese Medicinal Herb, Gentiana rigescens

PubMed Central

Zhang, Xiaodong; Allan, Andrew C.; Li, Caixia; Wang, Yuanzhong; Yao, Qiuyang

2015-01-01

Gentiana rigescens is an important medicinal herb in China. The main validated medicinal component gentiopicroside is synthesized in shoots, but is mainly found in the plant’s roots. The gentiopicroside biosynthetic pathway and its regulatory control remain to be elucidated. Genome resources of gentian are limited. Next-generation sequencing (NGS) technologies can aid in supplying global gene expression profiles. In this study we present sequence and transcript abundance data for the root and leaf transcriptome of G. rigescens, obtained using the Illumina Hiseq2000. Over fifty million clean reads were obtained from leaf and root libraries. This yields 76,717 unigenes with an average length of 753 bp. Among these, 33,855 unigenes were identified as putative homologs of annotated sequences in public protein and nucleotide databases. Digital abundance analysis identified 3306 unigenes differentially enriched between leaf and root. Unigenes found in both tissues were categorized according to their putative functional categories. Of the differentially expressed genes, over 130 were annotated as related to terpenoid biosynthesis. This work is the first study of global transcriptome analyses in gentian. These sequences and putative functional data comprise a resource for future investigation of terpenoid biosynthesis in Gentianaceae species and annotation of the gentiopicroside biosynthetic pathway and its regulatory mechanisms. PMID:26006235

Brown Spider (Loxosceles genus) Venom Toxins: Tools for Biological Purposes

PubMed Central

Chaim, Olga Meiri; Trevisan-Silva, Dilza; Chaves-Moreira, Daniele; Wille, Ana Carolina M.; Ferrer, Valéria Pereira; Matsubara, Fernando Hitomi; Mangili, Oldemir Carlos; da Silveira, Rafael Bertoni; Gremski, Luiza Helena; Gremski, Waldemiro; Senff-Ribeiro, Andrea; Veiga, Silvio Sanches

2011-01-01

Venomous animals use their venoms as tools for defense or predation. These venoms are complex mixtures, mainly enriched of proteic toxins or peptides with several, and different, biological activities. In general, spider venom is rich in biologically active molecules that are useful in experimental protocols for pharmacology, biochemistry, cell biology and immunology, as well as putative tools for biotechnology and industries. Spider venoms have recently garnered much attention from several research groups worldwide. Brown spider (Loxosceles genus) venom is enriched in low molecular mass proteins (5–40 kDa). Although their venom is produced in minute volumes (a few microliters), and contain only tens of micrograms of protein, the use of techniques based on molecular biology and proteomic analysis has afforded rational projects in the area and permitted the discovery and identification of a great number of novel toxins. The brown spider phospholipase-D family is undoubtedly the most investigated and characterized, although other important toxins, such as low molecular mass insecticidal peptides, metalloproteases and hyaluronidases have also been identified and featured in literature. The molecular pathways of the action of these toxins have been reported and brought new insights in the field of biotechnology. Herein, we shall see how recent reports describing discoveries in the area of brown spider venom have expanded biotechnological uses of molecules identified in these venoms, with special emphasis on the construction of a cDNA library for venom glands, transcriptome analysis, proteomic projects, recombinant expression of different proteic toxins, and finally structural descriptions based on crystallography of toxins. PMID:22069711

Pyrosequencing the Bemisia tabaci Transcriptome Reveals a Highly Diverse Bacterial Community and a Robust System for Insecticide Resistance

PubMed Central

Wu, Qing-jun; Wang, Shao-li; Yang, Xin; Yang, Ni-na; Li, Ru-mei; Jiao, Xiao-guo; Pan, Hui-peng; Liu, Bai-ming; Su, Qi; Xu, Bao-yun; Hu, Song-nian; Zhou, Xu-guo; Zhang, You-jun

2012-01-01

Background Bemisia tabaci (Gennadius) is a phloem-feeding insect poised to become one of the major insect pests in open field and greenhouse production systems throughout the world. The high level of resistance to insecticides is a main factor that hinders continued use of insecticides for suppression of B. tabaci. Despite its prevalence, little is known about B. tabaci at the genome level. To fill this gap, an invasive B. tabaci B biotype was subjected to pyrosequencing-based transcriptome analysis to identify genes and gene networks putatively involved in various physiological and toxicological processes. Methodology and Principal Findings Using Roche 454 pyrosequencing, 857,205 reads containing approximately 340 megabases were obtained from the B. tabaci transcriptome. De novo assembly generated 178,669 unigenes including 30,980 from insects, 17,881 from bacteria, and 129,808 from the nohit. A total of 50,835 (28.45%) unigenes showed similarity to the non-redundant database in GenBank with a cut-off E-value of 10–5. Among them, 40,611 unigenes were assigned to one or more GO terms and 6,917 unigenes were assigned to 288 known pathways. De novo metatranscriptome analysis revealed highly diverse bacterial symbionts in B. tabaci, and demonstrated the host-symbiont cooperation in amino acid production. In-depth transcriptome analysis indentified putative molecular markers, and genes potentially involved in insecticide resistance and nutrient digestion. The utility of this transcriptome was validated by a thiamethoxam resistance study, in which annotated cytochrome P450 genes were significantly overexpressed in the resistant B. tabaci in comparison to its susceptible counterparts. Conclusions This transcriptome/metatranscriptome analysis sheds light on the molecular understanding of symbiosis and insecticide resistance in an agriculturally important phloem-feeding insect pest, and lays the foundation for future functional genomics research of the B. tabaci complex. Moreover, current pyrosequencing effort greatly enriched the existing whitefly EST database, and makes RNAseq a viable option for future genomic analysis. PMID:22558125

The association between local atherosclerosis of the prostatic artery and benign prostatic enlargement in humans: Putative mechanism of chronic ischemia for prostatic enlargement.

PubMed

Haga, Nobuhiro; Akaihata, Hidenori; Hata, Junya; Aikawa, Ken; Yanagida, Tomohiko; Matsuoka, Kanako; Koguchi, Tomoyuki; Hoshi, Seiji; Ogawa, Soichiro; Kataoka, Masao; Sato, Yuichi; Ishibashi, Kei; Suzuki, Osamu; Hashimoto, Yuko; Kojima, Yoshiyuki

2018-05-21

To investigate the possible pathogenesis of the benign prostatic enlargement (BPE) induced by local atherosclerosis, the association between local atherosclerosis and prostatic enlargement was investigated, and molecular biological analyses were performed using human prostatectomy specimens. A total of 69 consecutive patients who underwent robot-assisted radical prostatectomy (RARP) participated in this prospective study. To evaluate actual local atherosclerosis, prostatic arteries were removed during RARP. Microscopic assessment of local atherosclerosis was classified as one of three degrees of narrowing (minimal, moderate, and severe) according to the degree of obstruction of the inner cavity of the prostatic artery. The expressions of several mediators related to chronic ischemia and cell proliferation of the prostate were investigated by immunohistochemistry. The median age of the present cohort was 68 (range: 55-75) years. Although there was no relationship between local atherosclerosis and lower urinary symptoms evaluated by questionnaires, local atherosclerosis was significantly more severe in patients who had a history of treatment for benign prostatic hyperplasia (P = 0.02). Prostate size was significantly larger in the severe local atherosclerosis group than in the minimal and moderate local atherosclerosis groups (P < 0.001 and P = 0.03, respectively). Thepositive expression rates of hypoxia-inducible factor (HIF)-1α, malondialdehyde (MDA), transforming growth factor (TGF)-β 1 , and basic fibroblast growth factor (bFGF) in the prostate were significantly higher in patients with local atherosclerosis than in patients without local atherosclerosis (all P < 0.01, respectively). In human surgical specimens, there is evidence that local atherosclerosis of the prostatic artery is significantly associated with prostate size. Given the molecular evidence provided in this study, the putative mechanism for this relationship is that chronic ischemia induced upregulation of oxidative stress pathways, leading to BPE. © 2018 Wiley Periodicals, Inc.

Identification of Intermediate in Hepatitis B Virus CCC DNA Formation and Sensitive and Selective CCC DNA Detection.

PubMed

Luo, Jun; Cui, Xiuji; Gao, Lu; Hu, Jianming

2017-06-21

The hepatitis B virus (HBV) covalently closed circular (CCC) DNA functions as the only viral template capable of coding for all the viral RNA species and is thus essential to initiate and sustain viral replication. CCC DNA is converted, in a multi-step and ill-understood process, from a relaxed circular (RC) DNA, in which neither of the two DNA strands is covalently closed. To detect putative intermediates during RC to CCC DNA conversion, two 3' exonucleases Exo I and Exo III, in combination were used to degrade all DNA strands with a free 3' end, which would nevertheless preserve closed circular DNA, either single-stranded (SS) or double-stranded (DS). Indeed, a RC DNA species with a covalently closed minus strand but an open plus strand (closed minus-strand RC DNA or cM-RC DNA) was detected by this approach. Further analyses indicated that at least some of the plus strands in such a putative intermediate likely still retained the RNA primer that is attached to the 5' end of the plus strand in RC DNA, suggesting that minus strand closing can occur before plus strand processing. Furthermore, the same nuclease treatment proved to be useful for sensitive and specific detection of CCC DNA by removing all DNA species other than closed circular DNA. Application of these and similar approaches may allow the identification of additional intermediates during CCC DNA formation and facilitate specific and sensitive detection of CCC DNA, which should help elucidate the pathways of CCC DNA formation and factors involved. IMPORTANCE The hepatitis B virus (HBV) covalently closed circular (CCC) DNA is the molecular basis of viral persistence, by serving as the viral transcriptional template. CCC DNA is converted, in a multi-step and ill-understood process, from a relaxed circular (RC) DNA. Little is currently understood about the pathways or factors involved in CCC DNA formation. We have now detected a likely intermediate during the conversion of RC to CCC DNA, thus providing important clues to the pathways of CCC DNA formation. Furthermore, the same experimental approach that led to the detection of the intermediate could also facilitate specific and sensitive detection of CCC DNA, which has remained challenging. This and similar approaches will help identify additional intermediates during CCC DNA formation and elucidate the pathways and factors involved. Copyright © 2017 American Society for Microbiology.

Identification of an Intermediate in Hepatitis B Virus Covalently Closed Circular (CCC) DNA Formation and Sensitive and Selective CCC DNA Detection

PubMed Central

Luo, Jun; Cui, Xiuji; Gao, Lu

2017-01-01

ABSTRACT Hepatitis B virus (HBV) covalently closed circular (CCC) DNA functions as the only viral template capable of coding for all the viral RNA species and is thus essential to initiate and sustain viral replication. CCC DNA is converted, in a multistep and ill-understood process, from a relaxed circular (RC) DNA, in which neither of the two DNA strands is covalently closed. To detect putative intermediates during RC DNA to CCC DNA conversion, two 3′ exonucleases, exonuclease I (Exo I) and Exo III, were used in combination to degrade all DNA strands with a free 3′ end, which would nevertheless preserve closed circular DNA in either single-stranded (SS) or double-stranded (DS) form. Indeed, an RC DNA species with a covalently closed minus strand but an open plus strand (closed minus-strand RC DNA [cM-RC DNA]) was detected by this approach. Further analyses indicated that at least some of the plus strands in such a putative intermediate likely still retained the RNA primer that is attached to the 5′ end of the plus strand in RC DNA, suggesting that minus-strand closing can occur before plus-strand processing. Furthermore, the same nuclease treatment proved to be useful for sensitive and specific detection of CCC DNA by removing all DNA species other than closed circular DNA. Application of these and similar approaches may allow the identification of additional intermediates during CCC DNA formation and facilitate specific and sensitive detection of CCC DNA, which should help elucidate the pathways of CCC DNA formation and the factors involved. IMPORTANCE The hepatitis B virus (HBV) covalently closed circular (CCC) DNA, by serving as the viral transcriptional template, is the molecular basis of viral persistence. CCC DNA is converted, in a multistep and ill-understood process, from relaxed circular (RC) DNA. Little is currently understood about the pathways or factors involved in CCC DNA formation. We have now detected a likely intermediate during the conversion of RC DNA to CCC DNA, thus providing important clues to the pathways of CCC DNA formation. Furthermore, the same experimental approach that led to the detection of the intermediate could also facilitate specific and sensitive detection of CCC DNA, which has remained challenging. This and similar approaches will help identify additional intermediates during CCC DNA formation and elucidate the pathways and factors involved. PMID:28637752

Localization of the lysine epsilon-aminotransferase (lat) and delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (pcbAB) genes from Streptomyces clavuligerus and production of lysine epsilon-aminotransferase activity in Escherichia coli.

PubMed Central

Tobin, M B; Kovacevic, S; Madduri, K; Hoskins, J A; Skatrud, P L; Vining, L C; Stuttard, C; Miller, J R

1991-01-01

Lysine epsilon-aminotransferase (LAT) in the beta-lactam-producing actinomycetes is considered to be the first step in the antibiotic biosynthetic pathway. Cloning of restriction fragments from Streptomyces clavuligerus, a beta-lactam producer, into Streptomyces lividans, a nonproducer that lacks LAT activity, led to the production of LAT in the host. DNA sequencing of restriction fragments containing the putative lat gene revealed a single open reading frame encoding a polypeptide with an approximately Mr 49,000. Expression of this coding sequence in Escherichia coli led to the production of LAT activity. Hence, LAT activity in S. clavuligerus is derived from a single polypeptide. A second open reading frame began immediately downstream from lat. Comparison of this partial sequence with the sequences of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D valine (ACV) synthetases from Penicillium chrysogenum and Cephalosporium acremonium and with nonribosomal peptide synthetases (gramicidin S and tyrocidine synthetases) found similarities among the open reading frames. Since mapping of the putative N and C termini of S. clavuligerus pcbAB suggests that the coding region occupies approximately 12 kbp and codes for a polypeptide related in size to the fungal ACV synthetases, the molecular characterization of the beta-lactam biosynthetic cluster between pcbC and cefE (approximately 25 kbp) is nearly complete. Images PMID:1917855

Search for Partner Proteins of A. thaliana Immunophilins Involved in the Control of Plant Immunity.

PubMed

Abdeeva, Inna A; Pogorelko, Gennady V; Maloshenok, Liliya G; Mokrykova, Maria V; Fursova, Oksana V; Bruskin, Sergey A

2018-04-19

The involvement of plant immunophilins in multiple essential processes such as development, various ways of adapting to biotic and abiotic stresses, and photosynthesis has already been established. Previously, research has demonstrated the involvement of three immunophilin genes ( AtCYP19-1/ROC3 , AtFKBP65/ROF2 , and AtCYP57 ) in the control of plant response to invasion by various pathogens. Current research attempts to identify host target proteins for each of the selected immunophilins. As a result, candidate interactors have been determined and confirmed using a yeast 2-hybrid (Y2H) system for protein⁻protein interaction assays. The generation of mutant isoforms of ROC3 and AtCYP57 harboring substituted amino acids in the in silico-predicted active sites became essential to achieving significant binding to its target partners. This data shows that ROF2 targets calcium-dependent lipid-binding domain-containing protein (At1g70790; AT1) and putative protein phosphatase (At2g30020; АТ2), whereas ROC3 interacts with GTP-binding protein (At1g30580; ENGD-1) and RmlC-like cupin (At5g39120). The immunophilin AtCYP57 binds to putative pyruvate decarboxylase-1 (Pdc1) and clathrin adaptor complex-related protein (At5g05010). Identified interactors confirm our previous findings that immunophilins ROC3 , ROF2 , and AtCYP57 are directly involved with stress response control. Further, these findings extend our understanding of the molecular functional pathways of these immunophilins.

The putative roles of the ubiquitin/proteasome pathway in resistance to anticancer therapy.

PubMed

Smith, Laura; Lind, Michael J; Drew, Philip J; Cawkwell, Lynn

2007-11-01

The ubiquitin/proteasome (UP) pathway plays a significant role in many important biological functions and alterations in this pathway have been shown to contribute to the pathology of many human diseases, including cancer. Proteasome inhibition has been well established as a rational strategy for the treatment of multiple myeloma and is currently under investigation for the treatment of other haematological malignancies and solid tumours. Recent evidence suggests that proteasome inhibition may also sensitise tumour cells to the actions of both conventional chemotherapy and radiotherapy, suggesting that this pathway may modify clinical response to anticancer therapy. However, conflicting evidence exists as to the roles of the UP pathway in resistance to treatment. This review endeavours to discuss such roles.

MiR-592 Promotes Gastric Cancer Proliferation, Migration, and Invasion Through the PI3K/AKT and MAPK/ERK Signaling Pathways by Targeting Spry2.

PubMed

He, Yu; Ge, Yugang; Jiang, Mingkun; Zhou, Jundong; Luo, Dakui; Fan, Hao; Shi, Liang; Lin, Linling; Yang, Li

2018-06-21

Gastric cancer (GC) is one of the most prevalent digestive malignancies. MicroRNAs (miRNAs) are involved in multiple cellular processes, including oncogenesis, and miR-592 itself participates in many malignancies; however, its role in GC remains unknown. In this study, we investigated the expression and molecular mechanisms of miR-592 in GC. Quantitative real-time PCR and immunohistochemistry were performed to determine the expression of miR-592 and its putative targets in human tissues and cell lines. Proliferation, migration, and invasion were evaluated by Cell Counting Kit-8, population doubling time, colony formation, Transwell, and wound-healing assays in transfected GC cells in vitro. A dual-luciferase reporter assay was used to determine whether miR-592 could directly bind its target. A tumorigenesis assay was used to study whether miR-592 affected GC growth in vivo. Proteins involved in signaling pathways and the epithelial-mesenchymal transition (EMT) were detected with western blot. The ectopic expression of miR-592 promoted GC proliferation, migration, and invasion in vitro and facilitated tumorigenesis in vivo. Spry2 was a direct target of miR-592 and Spry2 overexpression partially counteracted the effects of miR-592. miR-592 induced the EMT and promoted its progression in GC via the PI3K/AKT and MAPK/ERK signaling pathways by inhibiting Spry2. Overexpression of miR-592 promotes GC proliferation, migration, and invasion and induces the EMT via the PI3K/AKT and MAPK/ERK signaling pathways by inhibiting Spry2, suggesting a potential therapeutic target for GC. © 2018 The Author(s). Published by S. Karger AG, Basel.

Jasmonate signalling pathway in strawberry: Genome-wide identification, molecular characterization and expression of JAZs and MYCs during fruit development and ripening.

PubMed

Garrido-Bigotes, Adrián; Figueroa, Nicolás E; Figueroa, Pablo M; Figueroa, Carlos R

2018-01-01

Jasmonates (JAs) are signalling molecules involved in stress responses, development and secondary metabolism biosynthesis, although their roles in fleshy-fruit development and ripening processes are not well known. In strawberry fruit, it has been proposed that JAs could regulate the early development through the activation of the JAs biosynthesis. Moreover, it has been reported that JA treatment increases anthocyanin content in strawberry fruit involving the bioactive jasmonate biosynthesis. Nevertheless, JA signalling pathway, of which main components are the COI1-JAZ co-receptor and the MYC transcription factors (TFs), has not been characterized in strawberry until now. Here we identified and characterized the woodland strawberry (Fragaria vesca) JAZ and MYC genes as well as studied their expression during development and ripening stages in commercial strawberry (Fragaria × ananassa) fruit. We described twelve putative JAZ proteins and two MYC TFs, which showed high conservation with respect to their orthologs in Arabidopsis thaliana and in other fleshy-fruit species such as Malus × domestica, Vitis vinifera and Solanum lycopersicum as revealed by gene synteny and phylogenetic analyses. Noteworthy, their expression levels exhibited a significant decrease from fruit development to ripening stages in F. × ananassa, along with others of the JA signalling-related genes such as FaNINJA and FaJAMs, encoding for negative regulators of JA responses. Moreover, we found that main JA signalling-related genes such as FaMYC2, and FaJAZ1 are promptly induced by JA treatment at early times in F. × ananassa fruit. These results suggest the conservation of the canonical JA signalling pathway in strawberry and a possible role of this pathway in early strawberry fruit development, which also correlates negatively with the beginning of the ripening process.

Jasmonate signalling pathway in strawberry: Genome-wide identification, molecular characterization and expression of JAZs and MYCs during fruit development and ripening

PubMed Central

Figueroa, Nicolás E.; Figueroa, Pablo M.

2018-01-01

Jasmonates (JAs) are signalling molecules involved in stress responses, development and secondary metabolism biosynthesis, although their roles in fleshy-fruit development and ripening processes are not well known. In strawberry fruit, it has been proposed that JAs could regulate the early development through the activation of the JAs biosynthesis. Moreover, it has been reported that JA treatment increases anthocyanin content in strawberry fruit involving the bioactive jasmonate biosynthesis. Nevertheless, JA signalling pathway, of which main components are the COI1-JAZ co-receptor and the MYC transcription factors (TFs), has not been characterized in strawberry until now. Here we identified and characterized the woodland strawberry (Fragaria vesca) JAZ and MYC genes as well as studied their expression during development and ripening stages in commercial strawberry (Fragaria × ananassa) fruit. We described twelve putative JAZ proteins and two MYC TFs, which showed high conservation with respect to their orthologs in Arabidopsis thaliana and in other fleshy-fruit species such as Malus × domestica, Vitis vinifera and Solanum lycopersicum as revealed by gene synteny and phylogenetic analyses. Noteworthy, their expression levels exhibited a significant decrease from fruit development to ripening stages in F. × ananassa, along with others of the JA signalling-related genes such as FaNINJA and FaJAMs, encoding for negative regulators of JA responses. Moreover, we found that main JA signalling-related genes such as FaMYC2, and FaJAZ1 are promptly induced by JA treatment at early times in F. × ananassa fruit. These results suggest the conservation of the canonical JA signalling pathway in strawberry and a possible role of this pathway in early strawberry fruit development, which also correlates negatively with the beginning of the ripening process. PMID:29746533

Prestroke Proteomic Changes in Cerebral Microvessels in Stroke-Prone, Transgenic[hCETP]-Hyperlipidemic, Dahl Salt-Sensitive Hypertensive Rats

PubMed Central

Bergerat, Agnes; Decano, Julius; Wu, Chang-Jiun; Choi, Hyungwon; Nesvizhskii, Alexey I; Moran, Ann Marie; Ruiz-Opazo, Nelson; Steffen, Martin; Herrera, Victoria LM

2011-01-01

Stroke is the third leading cause of death in the United States with high rates of morbidity among survivors. The search to fill the unequivocal need for new therapeutic approaches would benefit from unbiased proteomic analyses of animal models of spontaneous stroke in the prestroke stage. Since brain microvessels play key roles in neurovascular coupling, we investigated prestroke microvascular proteome changes. Proteomic analysis of cerebral cortical microvessels (cMVs) was done by tandem mass spectrometry comparing two prestroke time points. Metaprotein-pathway analyses of proteomic spectral count data were done to identify risk factor–induced changes, followed by QSPEC-analyses of individual protein changes associated with increased stroke susceptibility. We report 26 cMV proteome profiles from male and female stroke-prone and non–stroke-prone rats at 2 months and 4.5 months of age prior to overt stroke events. We identified 1,934 proteins by two or more peptides. Metaprotein pathway analysis detected age-associated changes in energy metabolism and cell-to-microenvironment interactions, as well as sex-specific changes in energy metabolism and endothelial leukocyte transmigration pathways. Stroke susceptibility was associated independently with multiple protein changes associated with ischemia, angiogenesis or involved in blood brain barrier (BBB) integrity. Immunohistochemical analysis confirmed aquaporin-4 and laminin-α1 induction in cMVs, representative of proteomic changes with >65 Bayes factor (BF), associated with stroke susceptibility. Altogether, proteomic analysis demonstrates significant molecular changes in ischemic cerebral microvasculature in the prestroke stage, which could contribute to the observed model phenotype of microhemorrhages and postischemic hemorrhagic transformation. These pathways comprise putative targets for translational research of much needed novel diagnostic and therapeutic approaches for stroke. PMID:21519634

Identification of MicroRNAs and Target Genes in the Fruit and Shoot Tip of Lycium chinense: A Traditional Chinese Medicinal Plant

PubMed Central

Khaldun, A. B. M.; Huang, Wenjun; Liao, Sihong; Lv, Haiyan; Wang, Ying

2015-01-01

Although Lycium chinense (goji berry) is an important traditional Chinese medicinal plant, little genome information is available for this plant, particularly at the small-RNA level. Recent findings indicate that the evolutionary role of miRNAs is very important for a better understanding of gene regulation in different plant species. To elucidate small RNAs and their potential target genes in fruit and shoot tissues, high-throughput RNA sequencing technology was used followed by qRT-PCR and RLM 5’-RACE experiments. A total of 60 conserved miRNAs belonging to 31 families and 30 putative novel miRNAs were identified. A total of 62 significantly differentially expressed miRNAs were identified, of which 15 (14 known and 1 novel) were shoot-specific, and 12 (7 known and 5 novel) were fruit-specific. Additionally, 28 differentially expressed miRNAs were recorded as up-regulated in fruit tissues. The predicted potential targets were involved in a wide range of metabolic and regulatory pathways. GO (Gene Ontology) enrichment analysis and the KEGG (Kyoto Encyclopedia of Genes and Genomes) database revealed that “metabolic pathways” is the most significant pathway with respect to the rich factor and gene numbers. Moreover, five miRNAs were related to fruit maturation, lycopene biosynthesis and signaling pathways, which might be important for the further study of fruit molecular biology. This study is the first, to detect known and novel miRNAs, and their potential targets, of L. chinense. The data and findings that are presented here might be a good source for the functional genomic study of medicinal plants and for understanding the links among diversified biological pathways. PMID:25587984

Cryptochromes and Hormone Signal Transduction under Near-Zero Magnetic Fields: New Clues to Magnetic Field Effects in a Rice Planthopper.

PubMed

Wan, Gui-Jun; Wang, Wen-Jing; Xu, Jing-Jing; Yang, Quan-Feng; Dai, Ming-Jiang; Zhang, Feng-Jiao; Sword, Gregory A; Pan, Wei-Dong; Chen, Fa-Jun

2015-01-01

Although there are considerable reports of magnetic field effects (MFE) on organisms, very little is known so far about the MFE-related signal transduction pathways. Here we establish a manipulative near-zero magnetic field (NZMF) to investigate the potential signal transduction pathways involved in MFE. We show that exposure of migratory white-backed planthopper, Sogatella furcifera, to the NZMF results in delayed egg and nymphal development, increased frequency of brachypterous females, and reduced longevity of macropterous female adults. To understand the changes in gene expression underlying these phenotypes, we examined the temporal patterns of gene expression of (i) CRY1 and CRY2 as putative magnetosensors, (ii) JHAMT, FAMeT and JHEH in the juvenile hormone pathway, (iii) CYP307A1 in the ecdysone pathway, and (iv) reproduction-related Vitellogenin (Vg). The significantly altered gene expression of CRY1 and CRY2 under the NZMF suggest their developmental stage-specific patterns and potential upstream location in magnetic response. Gene expression patterns of JHAMT, JHEH and CYP307A1 were consistent with the NZMF-triggered delay in nymphal development, higher proportion of brachypterous female adults, and the shortened longevity of macropterous female adults, which show feasible links between hormone signal transduction and phenotypic MFE. By conducting manipulative NZMF experiments, our study suggests an important role of the geomagnetic field (GMF) in modulating development and physiology of insects, provides new insights into the complexity of MFE-magnetosensitivity interactions, and represents an initial but crucial step forward in understanding the molecular basis of cryptochromes and hormone signal transduction involved in MFE.

Enzymes involved in the anaerobic degradation of ortho-phthalate by the nitrate-reducing bacterium Azoarcus sp. strain PA01.

PubMed

Junghare, Madan; Spiteller, Dieter; Schink, Bernhard

2016-09-01

The pathway of anaerobic degradation of o-phthalate was studied in the nitrate-reducing bacterium Azoarcus sp. strain PA01. Differential two-dimensional protein gel profiling allowed the identification of specifically induced proteins in o-phthalate-grown compared to benzoate-grown cells. The genes encoding o-phthalate-induced proteins were found in a 9.9 kb gene cluster in the genome of Azoarcus sp. strain PA01. The o-phthalate-induced gene cluster codes for proteins homologous to a dicarboxylic acid transporter, putative CoA-transferases and a UbiD-like decarboxylase that were assigned to be specifically involved in the initial steps of anaerobic o-phthalate degradation. We propose that o-phthalate is first activated to o-phthalyl-CoA by a putative succinyl-CoA-dependent succinyl-CoA:o-phthalate CoA-transferase, and o-phthalyl-CoA is subsequently decarboxylated to benzoyl-CoA by a putative o-phthalyl-CoA decarboxylase. Results from in vitro enzyme assays with cell-free extracts of o-phthalate-grown cells demonstrated the formation of o-phthalyl-CoA from o-phthalate and succinyl-CoA as CoA donor, and its subsequent decarboxylation to benzoyl-CoA. The putative succinyl-CoA:o-phthalate CoA-transferase showed high substrate specificity for o-phthalate and did not accept isophthalate, terephthalate or 3-fluoro-o-phthalate whereas the putative o-phthalyl-CoA decarboxylase converted fluoro-o-phthalyl-CoA to fluoro-benzoyl-CoA. No decarboxylase activity was observed with isophthalyl-CoA or terephthalyl-CoA. Both enzyme activities were oxygen-insensitive and inducible only after growth with o-phthalate. Further degradation of benzoyl-CoA proceeds analogous to the well-established anaerobic benzoyl-CoA degradation pathway of nitrate-reducing bacteria. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Molecular cloning of a novel receptor tyrosine kinase, tif, highly expressed in human ovary and testis.

PubMed

Dai, W; Pan, H; Hassanain, H; Gupta, S L; Murphy, M J

1994-03-01

Using a combination of polymerase chain reaction and conventional cDNA library screening approaches, we have cloned and characterized a putative receptor tyrosine kinase termed tif. The extracellular domain of tif has an immunoglobulin-like loop and a fibronectin type III structure. The intracellular domain contains a tyrosine kinase domain. Compared with ryk, a ubiquitously expressed receptor tyrosine kinase, tif expression is tissue-specific with human ovary and testis containing the highest amount of tif mRNA. Many other tested human tissues such as heart, liver, pancreas and thymus do not contain detectable levels of tif mRNA. The molecular cloning and characterization of tif cDNA will facilitate the identification of a potential ligand(s) for the putative receptor and the study of its biological role.

An experimental study of the putative mechanism of a synthetic autonomous rotary DNA nanomotor

NASA Astrophysics Data System (ADS)

Dunn, K. E.; Leake, M. C.; Wollman, A. J. M.; Trefzer, M. A.; Johnson, S.; Tyrrell, A. M.

2017-03-01

DNA has been used to construct a wide variety of nanoscale molecular devices. Inspiration for such synthetic molecular machines is frequently drawn from protein motors, which are naturally occurring and ubiquitous. However, despite the fact that rotary motors such as ATP synthase and the bacterial flagellar motor play extremely important roles in nature, very few rotary devices have been constructed using DNA. This paper describes an experimental study of the putative mechanism of a rotary DNA nanomotor, which is based on strand displacement, the phenomenon that powers many synthetic linear DNA motors. Unlike other examples of rotary DNA machines, the device described here is designed to be capable of autonomous operation after it is triggered. The experimental results are consistent with operation of the motor as expected, and future work on an enhanced motor design may allow rotation to be observed at the single-molecule level. The rotary motor concept presented here has potential applications in molecular processing, DNA computing, biosensing and photonics.

Coriandrum sativum L. (Coriander) Essential Oil: Antifungal Activity and Mode of Action on Candida spp., and Molecular Targets Affected in Human Whole-Genome Expression

PubMed Central

Freires, Irlan de Almeida; Murata, Ramiro Mendonça; Furletti, Vivian Fernandes; Sartoratto, Adilson; de Alencar, Severino Matias; Figueira, Glyn Mara; de Oliveira Rodrigues, Janaina Aparecida; Duarte, Marta Cristina Teixeira; Rosalen, Pedro Luiz

2014-01-01

Oral candidiasis is an opportunistic fungal infection of the oral cavity with increasingly worldwide prevalence and incidence rates. Novel specifically-targeted strategies to manage this ailment have been proposed using essential oils (EO) known to have antifungal properties. In this study, we aim to investigate the antifungal activity and mode of action of the EO from Coriandrum sativum L. (coriander) leaves on Candida spp. In addition, we detected the molecular targets affected in whole-genome expression in human cells. The EO phytochemical profile indicates monoterpenes and sesquiterpenes as major components, which are likely to negatively impact the viability of yeast cells. There seems to be a synergistic activity of the EO chemical compounds as their isolation into fractions led to a decreased antimicrobial effect. C. sativum EO may bind to membrane ergosterol, increasing ionic permeability and causing membrane damage leading to cell death, but it does not act on cell wall biosynthesis-related pathways. This mode of action is illustrated by photomicrographs showing disruption in biofilm integrity caused by the EO at varied concentrations. The EO also inhibited Candida biofilm adherence to a polystyrene substrate at low concentrations, and decreased the proteolytic activity of Candida albicans at minimum inhibitory concentration. Finally, the EO and its selected active fraction had low cytotoxicity on human cells, with putative mechanisms affecting gene expression in pathways involving chemokines and MAP-kinase (proliferation/apoptosis), as well as adhesion proteins. These findings highlight the potential antifungal activity of the EO from C. sativum leaves and suggest avenues for future translational toxicological research. PMID:24901768

In silico analysis of stomach lineage specific gene set expression pattern in gastric cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pandi, Narayanan Sathiya, E-mail: sathiyapandi@gmail.com; Suganya, Sivagurunathan; Rajendran, Suriliyandi

Highlights: •Identified stomach lineage specific gene set (SLSGS) was found to be under expressed in gastric tumors. •Elevated expression of SLSGS in gastric tumor is a molecular predictor of metabolic type gastric cancer. •In silico pathway scanning identified estrogen-α signaling is a putative regulator of SLSGS in gastric cancer. •Elevated expression of SLSGS in GC is associated with an overall increase in the survival of GC patients. -- Abstract: Stomach lineage specific gene products act as a protective barrier in the normal stomach and their expression maintains the normal physiological processes, cellular integrity and morphology of the gastric wall. However,more » the regulation of stomach lineage specific genes in gastric cancer (GC) is far less clear. In the present study, we sought to investigate the role and regulation of stomach lineage specific gene set (SLSGS) in GC. SLSGS was identified by comparing the mRNA expression profiles of normal stomach tissue with other organ tissue. The obtained SLSGS was found to be under expressed in gastric tumors. Functional annotation analysis revealed that the SLSGS was enriched for digestive function and gastric epithelial maintenance. Employing a single sample prediction method across GC mRNA expression profiles identified the under expression of SLSGS in proliferative type and invasive type gastric tumors compared to the metabolic type gastric tumors. Integrative pathway activation prediction analysis revealed a close association between estrogen-α signaling and SLSGS expression pattern in GC. Elevated expression of SLSGS in GC is associated with an overall increase in the survival of GC patients. In conclusion, our results highlight that estrogen mediated regulation of SLSGS in gastric tumor is a molecular predictor of metabolic type GC and prognostic factor in GC.« less

Molecular Dynamics Simulations of Amyloid β-Peptide (1-42): Tetramer Formation and Membrane Interactions.

PubMed

Brown, Anne M; Bevan, David R

2016-09-06

The aggregation cascade and peptide-membrane interactions of the amyloid β-peptide (Aβ) have been implicated as toxic events in the development and progression of Alzheimer's disease. Aβ42 forms oligomers and ultimately plaques, and it has been hypothesized that these oligomeric species are the main toxic species contributing to neuronal cell death. To better understand oligomerization events and subsequent oligomer-membrane interactions of Aβ42, we performed atomistic molecular-dynamics (MD) simulations to characterize both interpeptide interactions and perturbation of model membranes by the peptides. MD simulations were utilized to first show the formation of a tetramer unit by four separate Aβ42 peptides. Aβ42 tetramers adopted an oblate ellipsoid shape and showed a significant increase in β-strand formation in the final tetramer unit relative to the monomers, indicative of on-pathway events for fibril formation. The Aβ42 tetramer unit that formed in the initial simulations was used in subsequent MD simulations in the presence of a pure POPC or cholesterol-rich raft model membrane. Tetramer-membrane simulations resulted in elongation of the tetramer in the presence of both model membranes, with tetramer-raft interactions giving rise to the rearrangement of key hydrophobic regions in the tetramer and the formation of a more rod-like structure indicative of a fibril-seeding aggregate. Membrane perturbation by the tetramer was manifested in the form of more ordered, rigid membranes, with the pure POPC being affected to a greater extent than the raft membrane. These results provide critical atomistic insight into the aggregation pathway of Aβ42 and a putative toxic mechanism in the pathogenesis of Alzheimer's disease. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Reduced Ssy1-Ptr3-Ssy5 (SPS) signaling extends replicative life span by enhancing NAD+ homeostasis in Saccharomyces cerevisiae.

PubMed

Tsang, Felicia; James, Christol; Kato, Michiko; Myers, Victoria; Ilyas, Irtqa; Tsang, Matthew; Lin, Su-Ju

2015-05-15

Attenuated nutrient signaling extends the life span in yeast and higher eukaryotes; however, the mechanisms are not completely understood. Here we identify the Ssy1-Ptr3-Ssy5 (SPS) amino acid sensing pathway as a novel longevity factor. A null mutation of SSY5 (ssy5Δ) increases replicative life span (RLS) by ∼50%. Our results demonstrate that several NAD(+) homeostasis factors play key roles in this life span extension. First, expression of the putative malate-pyruvate NADH shuttle increases in ssy5Δ cells, and deleting components of this shuttle, MAE1 and OAC1, largely abolishes RLS extension. Next, we show that Stp1, a transcription factor of the SPS pathway, directly binds to the promoter of MAE1 and OAC1 to regulate their expression. Additionally, deletion of SSY5 increases nicotinamide riboside (NR) levels and phosphate-responsive (PHO) signaling activity, suggesting that ssy5Δ increases NR salvaging. This increase contributes to NAD(+) homeostasis, partially ameliorating the NAD(+) deficiency and rescuing the short life span of the npt1Δ mutant. Moreover, we observed that vacuolar phosphatase, Pho8, is partially required for ssy5Δ-mediated NR increase and RLS extension. Together, our studies present evidence that supports SPS signaling is a novel NAD(+) homeostasis factor and ssy5Δ-mediated life span extension is likely due to concomitantly increased mitochondrial and vacuolar function. Our findings may contribute to understanding the molecular basis of NAD(+) metabolism, cellular life span, and diseases associated with NAD(+) deficiency and aging. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of a Functional Plasmodesmal Localization Signal in a Plant Viral Cell-To-Cell-Movement Protein.

PubMed

Yuan, Cheng; Lazarowitz, Sondra G; Citovsky, Vitaly

2016-01-19

Our fundamental knowledge of the protein-sorting pathways required for plant cell-to-cell trafficking and communication via the intercellular connections termed plasmodesmata has been severely limited by the paucity of plasmodesmal targeting sequences that have been identified to date. To address this limitation, we have identified the plasmodesmal localization signal (PLS) in the Tobacco mosaic virus (TMV) cell-to-cell-movement protein (MP), which has emerged as the paradigm for dissecting the molecular details of cell-to-cell transport through plasmodesmata. We report here the identification of a bona fide functional TMV MP PLS, which encompasses amino acid residues between positions 1 and 50, with residues Val-4 and Phe-14 potentially representing critical sites for PLS function that most likely affect protein conformation or protein interactions. We then demonstrated that this PLS is both necessary and sufficient for protein targeting to plasmodesmata. Importantly, as TMV MP traffics to plasmodesmata by a mechanism that is distinct from those of the three plant cell proteins in which PLSs have been reported, our findings provide important new insights to expand our understanding of protein-sorting pathways to plasmodesmata. The science of virology began with the discovery of Tobacco mosaic virus (TMV). Since then, TMV has served as an experimental and conceptual model for studies of viruses and dissection of virus-host interactions. Indeed, the TMV cell-to-cell-movement protein (MP) has emerged as the paradigm for dissecting the molecular details of cell-to-cell transport through the plant intercellular connections termed plasmodesmata. However, one of the most fundamental and key functional features of TMV MP, its putative plasmodesmal localization signal (PLS), has not been identified. Here, we fill this gap in our knowledge and identify the TMV MP PLS. Copyright © 2016 Yuan et al.

De novo transcriptome sequencing and comprehensive analysis of the heat stress response genes in the basidiomycetes fungus Ganoderma lucidum.

PubMed

Tan, Xiaoyan; Sun, Junshe; Ning, Huijuan; Qin, Zifang; Miao, Yuxin; Sun, Tian; Zhang, Xiuqing

2018-06-30

Ganoderma lucidum is a valuable basidiomycete with numerous pharmacological compounds, which is widely consumed throughout China. We previously found that the polysaccharide content of Ganoderma lucidum fruiting bodies could be significantly improved by 45.63% with treatment of 42 °C heat stress (HS) for 2 h. To further investigate genes involved in HS response and explore the mechanisms of HS regulating the carbohydrate metabolism in Ganoderma lucidum, high-throughput RNA-Seq was conducted to analyse the difference between control and heat-treated mycelia at transcriptome level. We sequenced six cDNA libraries with three from control group (mycelia cultivated at 28 °C) and three from heat-treated group (mycelia subjected to 42 °C for 2 h). A total of 99,899 transcripts were generated using Trinity method and 59,136 unigenes were annotated by seven public databases. Among them, 2790 genes were identified to be differential expressed genes (DEGs) under HS condition, which included 1991 up-regulated and 799 down-regulated. 176 DEGs were then manually classified into five main responsive-related categories according to their putative functions and possible metabolic pathways. These groups include stress resistance-related factors; protein assembly, transportation and degradation; signal transduction; carbohydrate metabolism and energy provision-related process; other related functions, suggesting that a series of metabolic pathways in Ganoderma lucidum are activated by HS and the response mechanism involves a complex molecular network which needs further study. Remarkably, 48 DEGs were found to regulate carbohydrate metabolism, both in carbohydrate hydrolysis for energy provision and polysaccharide synthesis. In summary, this comprehensive transcriptome analysis will provide enlarged resource for further investigation into the molecular mechanisms of basidiomycete under HS condition. Copyright © 2018 Elsevier B.V. All rights reserved.

Research Resource: Preovulatory LH Surge Effects on Follicular Theca and Granulosa Transcriptomes

PubMed Central

Gunewardena, Sumedha; Hong, Xiaoman; Spitschak, Marion; Baufeld, Anja

2013-01-01

The molecular mechanisms that regulate the pivotal transformation processes observed in the follicular wall following the preovulatory LH surge, are still not established, particularly for cells of the thecal layer. To elucidate thecal cell (TC) and granulosa cell (GC) type-specific biologic functions and signaling pathways, large dominant bovine follicles were collected before and 21 hours after an exogenous GnRH-induced LH surge. Antral GCs (aGCs; aspirated by follicular puncture) and membrane-associated GCs (mGCs; scraped from the follicular wall) were compared with TC expression profiles determined by mRNA microarrays. Of the approximately 11 000 total genes expressed in the periovulatory follicle, only 2% of thecal vs 25% of the granulosa genes changed in response to the LH surge. The majority of the 203 LH-regulated thecal genes were also LH regulated in GCs, leaving a total of 57 genes as LH-regulated TC-specific genes. Of the 57 thecal-specific LH-regulated genes, 74% were down-regulated including CYP17A1 and NR5A1, whereas most other genes are being identified for the first time within theca. Many of the newly identified up-regulated thecal genes (eg, PTX3, RND3, PPP4R4) were also up-regulated in granulosa. Minimal expression differences were observed between aGCs and mGCs; however, transcripts encoding extracellular proteins (NID2) and matrix modulators (ADAMTS1, SASH1) dominated these differences. We also identified large numbers of unknown LH-regulated GC genes and discuss their putative roles in ovarian function. This Research Resource provides an easy-to-access global evaluation of LH regulation in TCs and GCs that implicates numerous molecular pathways heretofore unknown within the follicle. PMID:23716604

Research resource: preovulatory LH surge effects on follicular theca and granulosa transcriptomes.

PubMed

Christenson, Lane K; Gunewardena, Sumedha; Hong, Xiaoman; Spitschak, Marion; Baufeld, Anja; Vanselow, Jens

2013-07-01

The molecular mechanisms that regulate the pivotal transformation processes observed in the follicular wall following the preovulatory LH surge, are still not established, particularly for cells of the thecal layer. To elucidate thecal cell (TC) and granulosa cell (GC) type-specific biologic functions and signaling pathways, large dominant bovine follicles were collected before and 21 hours after an exogenous GnRH-induced LH surge. Antral GCs (aGCs; aspirated by follicular puncture) and membrane-associated GCs (mGCs; scraped from the follicular wall) were compared with TC expression profiles determined by mRNA microarrays. Of the approximately 11 000 total genes expressed in the periovulatory follicle, only 2% of thecal vs 25% of the granulosa genes changed in response to the LH surge. The majority of the 203 LH-regulated thecal genes were also LH regulated in GCs, leaving a total of 57 genes as LH-regulated TC-specific genes. Of the 57 thecal-specific LH-regulated genes, 74% were down-regulated including CYP17A1 and NR5A1, whereas most other genes are being identified for the first time within theca. Many of the newly identified up-regulated thecal genes (eg, PTX3, RND3, PPP4R4) were also up-regulated in granulosa. Minimal expression differences were observed between aGCs and mGCs; however, transcripts encoding extracellular proteins (NID2) and matrix modulators (ADAMTS1, SASH1) dominated these differences. We also identified large numbers of unknown LH-regulated GC genes and discuss their putative roles in ovarian function. This Research Resource provides an easy-to-access global evaluation of LH regulation in TCs and GCs that implicates numerous molecular pathways heretofore unknown within the follicle.

Neurogenetics of developmental dyslexia: from genes to behavior through brain neuroimaging and cognitive and sensorial mechanisms

PubMed Central

Mascheretti, S; De Luca, A; Trezzi, V; Peruzzo, D; Nordio, A; Marino, C; Arrigoni, F

2017-01-01

Developmental dyslexia (DD) is a complex neurodevelopmental deficit characterized by impaired reading acquisition, in spite of adequate neurological and sensorial conditions, educational opportunities and normal intelligence. Despite the successful characterization of DD-susceptibility genes, we are far from understanding the molecular etiological pathways underlying the development of reading (dis)ability. By focusing mainly on clinical phenotypes, the molecular genetics approach has yielded mixed results. More optimally reduced measures of functioning, that is, intermediate phenotypes (IPs), represent a target for researching disease-associated genetic variants and for elucidating the underlying mechanisms. Imaging data provide a viable IP for complex neurobehavioral disorders and have been extensively used to investigate both morphological, structural and functional brain abnormalities in DD. Performing joint genetic and neuroimaging studies in humans is an emerging strategy to link DD-candidate genes to the brain structure and function. A limited number of studies has already pursued the imaging–genetics integration in DD. However, the results are still not sufficient to unravel the complexity of the reading circuit due to heterogeneous study design and data processing. Here, we propose an interdisciplinary, multilevel, imaging–genetic approach to disentangle the pathways from genes to behavior. As the presence of putative functional genetic variants has been provided and as genetic associations with specific cognitive/sensorial mechanisms have been reported, new hypothesis-driven imaging–genetic studies must gain momentum. This approach would lead to the optimization of diagnostic criteria and to the early identification of ‘biologically at-risk’ children, supporting the definition of adequate and well-timed prevention strategies and the implementation of novel, specific remediation approach. PMID:28045463

Biosynthesis of the salinosporamide A polyketide synthase substrate chloroethylmalonyl-coenzyme A from S-adenosyl-L-methionine.

PubMed

Eustáquio, Alessandra S; McGlinchey, Ryan P; Liu, Yuan; Hazzard, Christopher; Beer, Laura L; Florova, Galina; Alhamadsheh, Mamoun M; Lechner, Anna; Kale, Andrew J; Kobayashi, Yoshihisa; Reynolds, Kevin A; Moore, Bradley S

2009-07-28

Polyketides are among the major classes of bioactive natural products used to treat microbial infections, cancer, and other diseases. Here we describe a pathway to chloroethylmalonyl-CoA as a polyketide synthase building block in the biosynthesis of salinosporamide A, a marine microbial metabolite whose chlorine atom is crucial for potent proteasome inhibition and anticancer activity. S-adenosyl-L-methionine (SAM) is converted to 5'-chloro-5'-deoxyadenosine (5'-ClDA) in a reaction catalyzed by a SAM-dependent chlorinase as previously reported. By using a combination of gene deletions, biochemical analyses, and chemical complementation experiments with putative intermediates, we now provide evidence that 5'-ClDA is converted to chloroethylmalonyl-CoA in a 7-step route via the penultimate intermediate 4-chlorocrotonyl-CoA. Because halogenation often increases the bioactivity of drugs, the availability of a halogenated polyketide building block may be useful in molecular engineering approaches toward polyketide scaffolds.

New genetic loci link adipose and insulin biology to body fat distribution.

PubMed

Shungin, Dmitry; Winkler, Thomas W; Croteau-Chonka, Damien C; Ferreira, Teresa; Locke, Adam E; Mägi, Reedik; Strawbridge, Rona J; Pers, Tune H; Fischer, Krista; Justice, Anne E; Workalemahu, Tsegaselassie; Wu, Joseph M W; Buchkovich, Martin L; Heard-Costa, Nancy L; Roman, Tamara S; Drong, Alexander W; Song, Ci; Gustafsson, Stefan; Day, Felix R; Esko, Tonu; Fall, Tove; Kutalik, Zoltán; Luan, Jian'an; Randall, Joshua C; Scherag, André; Vedantam, Sailaja; Wood, Andrew R; Chen, Jin; Fehrmann, Rudolf; Karjalainen, Juha; Kahali, Bratati; Liu, Ching-Ti; Schmidt, Ellen M; Absher, Devin; Amin, Najaf; Anderson, Denise; Beekman, Marian; Bragg-Gresham, Jennifer L; Buyske, Steven; Demirkan, Ayse; Ehret, Georg B; Feitosa, Mary F; Goel, Anuj; Jackson, Anne U; Johnson, Toby; Kleber, Marcus E; Kristiansson, Kati; Mangino, Massimo; Leach, Irene Mateo; Medina-Gomez, Carolina; Palmer, Cameron D; Pasko, Dorota; Pechlivanis, Sonali; Peters, Marjolein J; Prokopenko, Inga; Stančáková, Alena; Sung, Yun Ju; Tanaka, Toshiko; Teumer, Alexander; Van Vliet-Ostaptchouk, Jana V; Yengo, Loïc; Zhang, Weihua; Albrecht, Eva; Ärnlöv, Johan; Arscott, Gillian M; Bandinelli, Stefania; Barrett, Amy; Bellis, Claire; Bennett, Amanda J; Berne, Christian; Blüher, Matthias; Böhringer, Stefan; Bonnet, Fabrice; Böttcher, Yvonne; Bruinenberg, Marcel; Carba, Delia B; Caspersen, Ida H; Clarke, Robert; Daw, E Warwick; Deelen, Joris; Deelman, Ewa; Delgado, Graciela; Doney, Alex Sf; Eklund, Niina; Erdos, Michael R; Estrada, Karol; Eury, Elodie; Friedrich, Nele; Garcia, Melissa E; Giedraitis, Vilmantas; Gigante, Bruna; Go, Alan S; Golay, Alain; Grallert, Harald; Grammer, Tanja B; Gräßler, Jürgen; Grewal, Jagvir; Groves, Christopher J; Haller, Toomas; Hallmans, Goran; Hartman, Catharina A; Hassinen, Maija; Hayward, Caroline; Heikkilä, Kauko; Herzig, Karl-Heinz; Helmer, Quinta; Hillege, Hans L; Holmen, Oddgeir; Hunt, Steven C; Isaacs, Aaron; Ittermann, Till; James, Alan L; Johansson, Ingegerd; Juliusdottir, Thorhildur; Kalafati, Ioanna-Panagiota; Kinnunen, Leena; Koenig, Wolfgang; Kooner, Ishminder K; Kratzer, Wolfgang; Lamina, Claudia; Leander, Karin; Lee, Nanette R; Lichtner, Peter; Lind, Lars; Lindström, Jaana; Lobbens, Stéphane; Lorentzon, Mattias; Mach, François; Magnusson, Patrik Ke; Mahajan, Anubha; McArdle, Wendy L; Menni, Cristina; Merger, Sigrun; Mihailov, Evelin; Milani, Lili; Mills, Rebecca; Moayyeri, Alireza; Monda, Keri L; Mooijaart, Simon P; Mühleisen, Thomas W; Mulas, Antonella; Müller, Gabriele; Müller-Nurasyid, Martina; Nagaraja, Ramaiah; Nalls, Michael A; Narisu, Narisu; Glorioso, Nicola; Nolte, Ilja M; Olden, Matthias; Rayner, Nigel W; Renstrom, Frida; Ried, Janina S; Robertson, Neil R; Rose, Lynda M; Sanna, Serena; Scharnagl, Hubert; Scholtens, Salome; Sennblad, Bengt; Seufferlein, Thomas; Sitlani, Colleen M; Smith, Albert Vernon; Stirrups, Kathleen; Stringham, Heather M; Sundström, Johan; Swertz, Morris A; Swift, Amy J; Syvänen, Ann-Christine; Tayo, Bamidele O; Thorand, Barbara; Thorleifsson, Gudmar; Tomaschitz, Andreas; Troffa, Chiara; van Oort, Floor Va; Verweij, Niek; Vonk, Judith M; Waite, Lindsay L; Wennauer, Roman; Wilsgaard, Tom; Wojczynski, Mary K; Wong, Andrew; Zhang, Qunyuan; Zhao, Jing Hua; Brennan, Eoin P; Choi, Murim; Eriksson, Per; Folkersen, Lasse; Franco-Cereceda, Anders; Gharavi, Ali G; Hedman, Åsa K; Hivert, Marie-France; Huang, Jinyan; Kanoni, Stavroula; Karpe, Fredrik; Keildson, Sarah; Kiryluk, Krzysztof; Liang, Liming; Lifton, Richard P; Ma, Baoshan; McKnight, Amy J; McPherson, Ruth; Metspalu, Andres; Min, Josine L; Moffatt, Miriam F; Montgomery, Grant W; Murabito, Joanne M; Nicholson, George; Nyholt, Dale R; Olsson, Christian; Perry, John Rb; Reinmaa, Eva; Salem, Rany M; Sandholm, Niina; Schadt, Eric E; Scott, Robert A; Stolk, Lisette; Vallejo, Edgar E; Westra, Harm-Jan; Zondervan, Krina T; Amouyel, Philippe; Arveiler, Dominique; Bakker, Stephan Jl; Beilby, John; Bergman, Richard N; Blangero, John; Brown, Morris J; Burnier, Michel; Campbell, Harry; Chakravarti, Aravinda; Chines, Peter S; Claudi-Boehm, Simone; Collins, Francis S; Crawford, Dana C; Danesh, John; de Faire, Ulf; de Geus, Eco Jc; Dörr, Marcus; Erbel, Raimund; Eriksson, Johan G; Farrall, Martin; Ferrannini, Ele; Ferrières, Jean; Forouhi, Nita G; Forrester, Terrence; Franco, Oscar H; Gansevoort, Ron T; Gieger, Christian; Gudnason, Vilmundur; Haiman, Christopher A; Harris, Tamara B; Hattersley, Andrew T; Heliövaara, Markku; Hicks, Andrew A; Hingorani, Aroon D; Hoffmann, Wolfgang; Hofman, Albert; Homuth, Georg; Humphries, Steve E; Hyppönen, Elina; Illig, Thomas; Jarvelin, Marjo-Riitta; Johansen, Berit; Jousilahti, Pekka; Jula, Antti M; Kaprio, Jaakko; Kee, Frank; Keinanen-Kiukaanniemi, Sirkka M; Kooner, Jaspal S; Kooperberg, Charles; Kovacs, Peter; Kraja, Aldi T; Kumari, Meena; Kuulasmaa, Kari; Kuusisto, Johanna; Lakka, Timo A; Langenberg, Claudia; Le Marchand, Loic; Lehtimäki, Terho; Lyssenko, Valeriya; Männistö, Satu; Marette, André; Matise, Tara C; McKenzie, Colin A; McKnight, Barbara; Musk, Arthur W; Möhlenkamp, Stefan; Morris, Andrew D; Nelis, Mari; Ohlsson, Claes; Oldehinkel, Albertine J; Ong, Ken K; Palmer, Lyle J; Penninx, Brenda W; Peters, Annette; Pramstaller, Peter P; Raitakari, Olli T; Rankinen, Tuomo; Rao, D C; Rice, Treva K; Ridker, Paul M; Ritchie, Marylyn D; Rudan, Igor; Salomaa, Veikko; Samani, Nilesh J; Saramies, Jouko; Sarzynski, Mark A; Schwarz, Peter Eh; Shuldiner, Alan R; Staessen, Jan A; Steinthorsdottir, Valgerdur; Stolk, Ronald P; Strauch, Konstantin; Tönjes, Anke; Tremblay, Angelo; Tremoli, Elena; Vohl, Marie-Claude; Völker, Uwe; Vollenweider, Peter; Wilson, James F; Witteman, Jacqueline C; Adair, Linda S; Bochud, Murielle; Boehm, Bernhard O; Bornstein, Stefan R; Bouchard, Claude; Cauchi, Stéphane; Caulfield, Mark J; Chambers, John C; Chasman, Daniel I; Cooper, Richard S; Dedoussis, George; Ferrucci, Luigi; Froguel, Philippe; Grabe, Hans-Jörgen; Hamsten, Anders; Hui, Jennie; Hveem, Kristian; Jöckel, Karl-Heinz; Kivimaki, Mika; Kuh, Diana; Laakso, Markku; Liu, Yongmei; März, Winfried; Munroe, Patricia B; Njølstad, Inger; Oostra, Ben A; Palmer, Colin Na; Pedersen, Nancy L; Perola, Markus; Pérusse, Louis; Peters, Ulrike; Power, Chris; Quertermous, Thomas; Rauramaa, Rainer; Rivadeneira, Fernando; Saaristo, Timo E; Saleheen, Danish; Sinisalo, Juha; Slagboom, P Eline; Snieder, Harold; Spector, Tim D; Stefansson, Kari; Stumvoll, Michael; Tuomilehto, Jaakko; Uitterlinden, André G; Uusitupa, Matti; van der Harst, Pim; Veronesi, Giovanni; Walker, Mark; Wareham, Nicholas J; Watkins, Hugh; Wichmann, H-Erich; Abecasis, Goncalo R; Assimes, Themistocles L; Berndt, Sonja I; Boehnke, Michael; Borecki, Ingrid B; Deloukas, Panos; Franke, Lude; Frayling, Timothy M; Groop, Leif C; Hunter, David J; Kaplan, Robert C; O'Connell, Jeffrey R; Qi, Lu; Schlessinger, David; Strachan, David P; Thorsteinsdottir, Unnur; van Duijn, Cornelia M; Willer, Cristen J; Visscher, Peter M; Yang, Jian; Hirschhorn, Joel N; Zillikens, M Carola; McCarthy, Mark I; Speliotes, Elizabeth K; North, Kari E; Fox, Caroline S; Barroso, Inês; Franks, Paul W; Ingelsson, Erik; Heid, Iris M; Loos, Ruth Jf; Cupples, L Adrienne; Morris, Andrew P; Lindgren, Cecilia M; Mohlke, Karen L

2015-02-12

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms.

Silencing the alarms: innate immune antagonism by rotavirus NSP1 and VP3

PubMed Central

Morelli, Marco; Ogden, Kristen M.; Patton, John T.

2016-01-01

The innate immune response involves a broad array of pathogen sensors that stimulate the production of interferons (IFN) to induce an antiviral state. Rotavirus, a significant cause of childhood gastroenteritis and a member of the Reoviridae family of segmented, double-stranded RNA viruses, encodes at least two direct antagonists of host innate immunity: NSP1 and VP3. NSP1, a putative E3 ubiquitin ligase, mediates the degradation of cellular factors involved in both IFN induction and downstream signaling. VP3, the viral capping enzyme, utilizes a 2H-phosphodiesterase domain to prevent activation of the cellular oligoadenylate synthase (OAS)-RNase L pathway. Computational, molecular, and biochemical studies have provided key insights into the structural and mechanistic basis of innate immune antagonism by NSP1 and VP3 of group A rotaviruses (RVA). Future studies with non-RVA isolates will be essential to understand how other RV species evade host innate immune responses. PMID:25724417

Ethnic diversity in the genetics of venous thromboembolism.

PubMed

Tang, Liang; Hu, Yu

2015-11-01

Genetic susceptibility is considered as a crucial factor for the development of venous thromboembolism (VTE). Epidemiologic and genetic studies have revealed clear disparities in the incidence of VTE and the distribution of genetic factors for VTE in populations stratified by ethnicity worldwide. While gain-of-function polymorphisms in the procoagulant genes are common inherited factors in European-origin populations, the most prevalent molecular basis for venous thrombosis in Asians is confirmed to be dysfunctional variants in the anticoagulant genes. With the breakthrough of genomic technologies, a set of novel common alleles and rare mutations associated with VTE have also been identified, in different ethnic groups. Several putative pathways contributing to the pathogenesis of thrombophilia in populations of African-ancestry are largely unknown, as current knowledge of hereditary and acquired risk factors do not fully explain the highest risk of VTE in Black groups. In-depth studies across diverse ethnic populations are needed to unravel the whole genetics of VTE, which will help developing individual risk prediction models and strategies to minimise VTE in all populations.

Physiological recordings and RNA sequencing of the gustatory appendages of the yellow-fever mosquito Aedes aegypti.

PubMed

Sparks, Jackson T; Dickens, Joseph C

2014-12-30

Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.

Commonly Used Antioxidant Botanicals: Active Constituents and their Potential Role in Cardiovascular Illness

PubMed Central

Wang, Chong-Zhi; Mehendale, Sangeeta R.; Yuan, Chun-Su

2009-01-01

Cardiovascular disease continues to be the leading cause of death in the US. Recent studies found that reactive oxygen species (ROS) have been incriminated in the pathogenesis of both acute and chronic heart disease. Many botanicals possess antioxidant properties, and these herbal antioxidants may protect against cardiovascular diseases by contributing to the total antioxidant defense system of the human body. In this article, we reviewed the antioxidant components and properties of four putative antioxidant botanicals (i.e., grape seeds, green tea, Scutellaria baicalensis, and American ginseng), and their potential role in treating cardiovascular illness. The antioxidant activities of the herbal active constituents, and the relationship between their chemical structures and biological functions were also discussed. Further investigations are needed on the mechanisms of action of these botanicals as they affect salient cellular and molecular pathways involved in major diseases. Data obtained from future studies will have the potential for translation into practical benefits for human health. PMID:17708622

Genomic identification of direct target genes of LEAFY

PubMed Central

William, Dilusha A.; Su, Yanhui; Smith, Michael R.; Lu, Meina; Baldwin, Don A.; Wagner, Doris

2004-01-01

The switch from vegetative to reproductive development in plants necessitates a switch in the developmental program of the descendents of the stem cells in the shoot apical meristem. Genetic and molecular investigations have demonstrated that the plant-specific transcription factor and meristem identity regulator LEAFY (LFY) controls this developmental transition by inducing expression of a second transcription factor, APETALA1, and by regulating the expression of additional, as yet unknown, genes. Here we show that the additional LFY targets include the APETALA1-related factor, CAULI-FLOWER, as well as three transcription factors and two putative signal transduction pathway components. These genes are up-regulated by LFY even when protein synthesis is inhibited and, hence, appear to be direct targets of LFY. Supporting this conclusion, cis-regulatory regions upstream of these genes are bound by LFY in vivo. The newly identified LFY targets likely initiate the transcriptional changes that are required for the switch from vegetative to reproductive development in Arabidopsis. PMID:14736918

New genetic loci link adipose and insulin biology to body fat distribution

PubMed Central

Strawbridge, Rona J; Pers, Tune H; Fischer, Krista; Justice, Anne E; Workalemahu, Tsegaselassie; Wu, Joseph M.W.; Buchkovich, Martin L; Heard-Costa, Nancy L; Roman, Tamara S; Drong, Alexander W; Song, Ci; Gustafsson, Stefan; Day, Felix R; Esko, Tonu; Fall, Tove; Kutalik, Zoltán; Luan, Jian’an; Randall, Joshua C; Scherag, André; Vedantam, Sailaja; Wood, Andrew R; Chen, Jin; Fehrmann, Rudolf; Karjalainen, Juha; Kahali, Bratati; Liu, Ching-Ti; Schmidt, Ellen M; Absher, Devin; Amin, Najaf; Anderson, Denise; Beekman, Marian; Bragg-Gresham, Jennifer L; Buyske, Steven; Demirkan, Ayse; Ehret, Georg B; Feitosa, Mary F; Goel, Anuj; Jackson, Anne U; Johnson, Toby; Kleber, Marcus E; Kristiansson, Kati; Mangino, Massimo; Leach, Irene Mateo; Medina-Gomez, Carolina; Palmer, Cameron D; Pasko, Dorota; Pechlivanis, Sonali; Peters, Marjolein J; Prokopenko, Inga; Stančáková, Alena; Sung, Yun Ju; Tanaka, Toshiko; Teumer, Alexander; Van Vliet-Ostaptchouk, Jana V; Yengo, Loïc; Zhang, Weihua; Albrecht, Eva; Ärnlöv, Johan; Arscott, Gillian M; Bandinelli, Stefania; Barrett, Amy; Bellis, Claire; Bennett, Amanda J; Berne, Christian; Blüher, Matthias; Böhringer, Stefan; Bonnet, Fabrice; Böttcher, Yvonne; Bruinenberg, Marcel; Carba, Delia B; Caspersen, Ida H; Clarke, Robert; Daw, E Warwick; Deelen, Joris; Deelman, Ewa; Delgado, Graciela; Doney, Alex SF; Eklund, Niina; Erdos, Michael R; Estrada, Karol; Eury, Elodie; Friedrich, Nele; Garcia, Melissa E; Giedraitis, Vilmantas; Gigante, Bruna; Go, Alan S; Golay, Alain; Grallert, Harald; Grammer, Tanja B; Gräßler, Jürgen; Grewal, Jagvir; Groves, Christopher J; Haller, Toomas; Hallmans, Goran; Hartman, Catharina A; Hassinen, Maija; Hayward, Caroline; Heikkilä, Kauko; Herzig, Karl-Heinz; Helmer, Quinta; Hillege, Hans L; Holmen, Oddgeir; Hunt, Steven C; Isaacs, Aaron; Ittermann, Till; James, Alan L; Johansson, Ingegerd; Juliusdottir, Thorhildur; Kalafati, Ioanna-Panagiota; Kinnunen, Leena; Koenig, Wolfgang; Kooner, Ishminder K; Kratzer, Wolfgang; Lamina, Claudia; Leander, Karin; Lee, Nanette R; Lichtner, Peter; Lind, Lars; Lindström, Jaana; Lobbens, Stéphane; Lorentzon, Mattias; Mach, François; Magnusson, Patrik KE; Mahajan, Anubha; McArdle, Wendy L; Menni, Cristina; Merger, Sigrun; Mihailov, Evelin; Milani, Lili; Mills, Rebecca; Moayyeri, Alireza; Monda, Keri L; Mooijaart, Simon P; Mühleisen, Thomas W; Mulas, Antonella; Müller, Gabriele; Müller-Nurasyid, Martina; Nagaraja, Ramaiah; Nalls, Michael A; Narisu, Narisu; Glorioso, Nicola; Nolte, Ilja M; Olden, Matthias; Rayner, Nigel W; Renstrom, Frida; Ried, Janina S; Robertson, Neil R; Rose, Lynda M; Sanna, Serena; Scharnagl, Hubert; Scholtens, Salome; Sennblad, Bengt; Seufferlein, Thomas; Sitlani, Colleen M; Smith, Albert Vernon; Stirrups, Kathleen; Stringham, Heather M; Sundström, Johan; Swertz, Morris A; Swift, Amy J; Syvänen, Ann-Christine; Tayo, Bamidele O; Thorand, Barbara; Thorleifsson, Gudmar; Tomaschitz, Andreas; Troffa, Chiara; van Oort, Floor VA; Verweij, Niek; Vonk, Judith M; Waite, Lindsay L; Wennauer, Roman; Wilsgaard, Tom; Wojczynski, Mary K; Wong, Andrew; Zhang, Qunyuan; Zhao, Jing Hua; Brennan, Eoin P.; Choi, Murim; Eriksson, Per; Folkersen, Lasse; Franco-Cereceda, Anders; Gharavi, Ali G; Hedman, Åsa K; Hivert, Marie-France; Huang, Jinyan; Kanoni, Stavroula; Karpe, Fredrik; Keildson, Sarah; Kiryluk, Krzysztof; Liang, Liming; Lifton, Richard P; Ma, Baoshan; McKnight, Amy J; McPherson, Ruth; Metspalu, Andres; Min, Josine L; Moffatt, Miriam F; Montgomery, Grant W; Murabito, Joanne M; Nicholson, George; Nyholt, Dale R; Olsson, Christian; Perry, John RB; Reinmaa, Eva; Salem, Rany M; Sandholm, Niina; Schadt, Eric E; Scott, Robert A; Stolk, Lisette; Vallejo, Edgar E.; Westra, Harm-Jan; Zondervan, Krina T; Amouyel, Philippe; Arveiler, Dominique; Bakker, Stephan JL; Beilby, John; Bergman, Richard N; Blangero, John; Brown, Morris J; Burnier, Michel; Campbell, Harry; Chakravarti, Aravinda; Chines, Peter S; Claudi-Boehm, Simone; Collins, Francis S; Crawford, Dana C; Danesh, John; de Faire, Ulf; de Geus, Eco JC; Dörr, Marcus; Erbel, Raimund; Eriksson, Johan G; Farrall, Martin; Ferrannini, Ele; Ferrières, Jean; Forouhi, Nita G; Forrester, Terrence; Franco, Oscar H; Gansevoort, Ron T; Gieger, Christian; Gudnason, Vilmundur; Haiman, Christopher A; Harris, Tamara B; Hattersley, Andrew T; Heliövaara, Markku; Hicks, Andrew A; Hingorani, Aroon D; Hoffmann, Wolfgang; Hofman, Albert; Homuth, Georg; Humphries, Steve E; Hyppönen, Elina; Illig, Thomas; Jarvelin, Marjo-Riitta; Johansen, Berit; Jousilahti, Pekka; Jula, Antti M; Kaprio, Jaakko; Kee, Frank; Keinanen-Kiukaanniemi, Sirkka M; Kooner, Jaspal S; Kooperberg, Charles; Kovacs, Peter; Kraja, Aldi T; Kumari, Meena; Kuulasmaa, Kari; Kuusisto, Johanna; Lakka, Timo A; Langenberg, Claudia; Le Marchand, Loic; Lehtimäki, Terho; Lyssenko, Valeriya; Männistö, Satu; Marette, André; Matise, Tara C; McKenzie, Colin A; McKnight, Barbara; Musk, Arthur W; Möhlenkamp, Stefan; Morris, Andrew D; Nelis, Mari; Ohlsson, Claes; Oldehinkel, Albertine J; Ong, Ken K; Palmer, Lyle J; Penninx, Brenda W; Peters, Annette; Pramstaller, Peter P; Raitakari, Olli T; Rankinen, Tuomo; Rao, DC; Rice, Treva K; Ridker, Paul M; Ritchie, Marylyn D.; Rudan, Igor; Salomaa, Veikko; Samani, Nilesh J; Saramies, Jouko; Sarzynski, Mark A; Schwarz, Peter EH; Shuldiner, Alan R; Staessen, Jan A; Steinthorsdottir, Valgerdur; Stolk, Ronald P; Strauch, Konstantin; Tönjes, Anke; Tremblay, Angelo; Tremoli, Elena; Vohl, Marie-Claude; Völker, Uwe; Vollenweider, Peter; Wilson, James F; Witteman, Jacqueline C; Adair, Linda S; Bochud, Murielle; Boehm, Bernhard O; Bornstein, Stefan R; Bouchard, Claude; Cauchi, Stéphane; Caulfield, Mark J; Chambers, John C; Chasman, Daniel I; Cooper, Richard S; Dedoussis, George; Ferrucci, Luigi; Froguel, Philippe; Grabe, Hans-Jörgen; Hamsten, Anders; Hui, Jennie; Hveem, Kristian; Jöckel, Karl-Heinz; Kivimaki, Mika; Kuh, Diana; Laakso, Markku; Liu, Yongmei; März, Winfried; Munroe, Patricia B; Njølstad, Inger; Oostra, Ben A; Palmer, Colin NA; Pedersen, Nancy L; Perola, Markus; Pérusse, Louis; Peters, Ulrike; Power, Chris; Quertermous, Thomas; Rauramaa, Rainer; Rivadeneira, Fernando; Saaristo, Timo E; Saleheen, Danish; Sinisalo, Juha; Slagboom, P Eline; Snieder, Harold; Spector, Tim D; Stefansson, Kari; Stumvoll, Michael; Tuomilehto, Jaakko; Uitterlinden, André G; Uusitupa, Matti; van der Harst, Pim; Veronesi, Giovanni; Walker, Mark; Wareham, Nicholas J; Watkins, Hugh; Wichmann, H-Erich; Abecasis, Goncalo R; Assimes, Themistocles L; Berndt, Sonja I; Boehnke, Michael; Borecki, Ingrid B; Deloukas, Panos; Franke, Lude; Frayling, Timothy M; Groop, Leif C; Hunter, David J.; Kaplan, Robert C; O’Connell, Jeffrey R; Qi, Lu; Schlessinger, David; Strachan, David P; Thorsteinsdottir, Unnur; van Duijn, Cornelia M; Willer, Cristen J; Visscher, Peter M; Yang, Jian; Hirschhorn, Joel N; Zillikens, M Carola; McCarthy, Mark I; Speliotes, Elizabeth K; North, Kari E; Fox, Caroline S; Barroso, Inês; Franks, Paul W; Ingelsson, Erik; Heid, Iris M; Loos, Ruth JF; Cupples, L Adrienne; Morris, Andrew P; Lindgren, Cecilia M; Mohlke, Karen L

2014-01-01

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, we conducted genome-wide association meta-analyses of waist and hip circumference-related traits in up to 224,459 individuals. We identified 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (WHRadjBMI) and an additional 19 loci newly associated with related waist and hip circumference measures (P<5×10−8). Twenty of the 49 WHRadjBMI loci showed significant sexual dimorphism, 19 of which displayed a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation, and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms. PMID:25673412

Proteomic data analysis of glioma cancer stem-cell lines based on novel nonlinear dimensional data reduction techniques

NASA Astrophysics Data System (ADS)

Lespinats, Sylvain; Pinker-Domenig, Katja; Wengert, Georg; Houben, Ivo; Lobbes, Marc; Stadlbauer, Andreas; Meyer-Bäse, Anke

2016-05-01

Glioma-derived cancer stem cells (GSCs) are tumor-initiating cells and may be refractory to radiation and chemotherapy and thus have important implications for tumor biology and therapeutics. The analysis and interpretation of large proteomic data sets requires the development of new data mining and visualization approaches. Traditional techniques are insufficient to interpret and visualize these resulting experimental data. The emphasis of this paper lies in the application of novel approaches for the visualization, clustering and projection representation to unveil hidden data structures relevant for the accurate interpretation of biological experiments. These qualitative and quantitative methods are applied to the proteomic analysis of data sets derived from the GSCs. The achieved clustering and visualization results provide a more detailed insight into the protein-level fold changes and putative upstream regulators for the GSCs. However the extracted molecular information is insufficient in classifying GSCs and paving the pathway to an improved therapeutics of the heterogeneous glioma.

Chronic intermittent hypoxia activates nuclear factor-{kappa}B in cardiovascular tissues in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Greenberg, Harly; Ye Xiaobing; Wilson, David

2006-05-05

Obstructive sleep apnea (OSA) is an important risk factor for cardiovascular morbidity and mortality. The mechanisms through which OSA promotes the development of cardiovascular disease are poorly understood. In this study, we tested the hypotheses that chronic exposure to intermittent hypoxia and reoxygenation (CIH) is a major pathologic factor causing cardiovascular inflammation, and that CIH-induces cardiovascular inflammation and pathology by activating the NF-{kappa}B pathway. We demonstrated that exposure of mice to CIH activated NF-{kappa}B in cardiovascular tissues, and that OSA patients had markedly elevated monocyte NF-{kappa}B activity, which was significantly decreased when obstructive apneas and their resultant CIH were eliminatedmore » by nocturnal CPAP therapy. The elevated NF-{kappa}B activity induced by CIH is accompanied by and temporally correlated to the increased expression of iNOS protein, a putative and important NF-{kappa}B-dependent gene product. Thus, CIH-mediated NF-{kappa}B activation may be a molecular mechanism linking OSA and cardiovascular pathologies seen in OSA patients.« less

Expression of Putative Immune Response Genes during Early Ontogeny in the Coral Acropora millepora

PubMed Central

Puill-Stephan, Eneour; Seneca, François O.; Miller, David J.; van Oppen, Madeleine J. H.; Willis, Bette L.

2012-01-01

Background Corals, like many other marine invertebrates, lack a mature allorecognition system in early life history stages. Indeed, in early ontogeny, when corals acquire and establish associations with various surface microbiota and dinoflagellate endosymbionts, they do not efficiently distinguish between closely and distantly related individuals from the same population. However, very little is known about the molecular components that underpin allorecognition and immunity responses or how they change through early ontogeny in corals. Methodology/Principal Findings Patterns in the expression of four putative immune response genes (apextrin, complement C3, and two CELIII type lectin genes) were examined in juvenile colonies of Acropora millepora throughout a six-month post-settlement period using quantitative real-time PCR (qPCR). Expression of a CELIII type lectin gene peaked in the fourth month for most of the coral juveniles sampled and was significantly higher at this time than at any other sampling time during the six months following settlement. The timing of this increase in expression levels of putative immune response genes may be linked to allorecognition maturation which occurs around this time in A.millepora. Alternatively, the increase may represent a response to immune challenges, such as would be involved in the recognition of symbionts (such as Symbiodinium spp. or bacteria) during winnowing processes as symbioses are fine-tuned. Conclusions/Significance Our data, although preliminary, are consistent with the hypothesis that lectins may play an important role in the maturation of allorecognition responses in corals. The co-expression of lectins with apextrin during development of coral juveniles also raises the possibility that these proteins, which are components of innate immunity in other invertebrates, may influence the innate immune systems of corals through a common pathway or system. However, further studies investigating the expression of these genes in alloimmune-challenged corals are needed to further clarify emerging evidence of a complex innate immunity system in corals. PMID:22792163

Expression of putative immune response genes during early ontogeny in the coral Acropora millepora.

PubMed

Puill-Stephan, Eneour; Seneca, François O; Miller, David J; van Oppen, Madeleine J H; Willis, Bette L

2012-01-01

Corals, like many other marine invertebrates, lack a mature allorecognition system in early life history stages. Indeed, in early ontogeny, when corals acquire and establish associations with various surface microbiota and dinoflagellate endosymbionts, they do not efficiently distinguish between closely and distantly related individuals from the same population. However, very little is known about the molecular components that underpin allorecognition and immunity responses or how they change through early ontogeny in corals. Patterns in the expression of four putative immune response genes (apextrin, complement C3, and two CELIII type lectin genes) were examined in juvenile colonies of Acropora millepora throughout a six-month post-settlement period using quantitative real-time PCR (qPCR). Expression of a CELIII type lectin gene peaked in the fourth month for most of the coral juveniles sampled and was significantly higher at this time than at any other sampling time during the six months following settlement. The timing of this increase in expression levels of putative immune response genes may be linked to allorecognition maturation which occurs around this time in A. millepora. Alternatively, the increase may represent a response to immune challenges, such as would be involved in the recognition of symbionts (such as Symbiodinium spp. or bacteria) during winnowing processes as symbioses are fine-tuned. Our data, although preliminary, are consistent with the hypothesis that lectins may play an important role in the maturation of allorecognition responses in corals. The co-expression of lectins with apextrin during development of coral juveniles also raises the possibility that these proteins, which are components of innate immunity in other invertebrates, may influence the innate immune systems of corals through a common pathway or system. However, further studies investigating the expression of these genes in alloimmune-challenged corals are needed to further clarify emerging evidence of a complex innate immunity system in corals.

Stable Isotope Metabolic Labeling-based Quantitative Phosphoproteomic Analysis of Arabidopsis Mutants Reveals Ethylene-regulated Time-dependent Phosphoproteins and Putative Substrates of Constitutive Triple Response 1 Kinase*

PubMed Central

Yang, Zhu; Guo, Guangyu; Zhang, Manyu; Liu, Claire Y.; Hu, Qin; Lam, Henry; Cheng, Han; Xue, Yu; Li, Jiayang; Li, Ning

2013-01-01

Ethylene is an important plant hormone that regulates numerous cellular processes and stress responses. The mode of action of ethylene is both dose- and time-dependent. Protein phosphorylation plays a key role in ethylene signaling, which is mediated by the activities of ethylene receptors, constitutive triple response 1 (CTR1) kinase, and phosphatase. To address how ethylene alters the cellular protein phosphorylation profile in a time-dependent manner, differential and quantitative phosphoproteomics based on 15N stable isotope labeling in Arabidopsis was performed on both one-minute ethylene-treated Arabidopsis ethylene-overly-sensitive loss-of-function mutant rcn1-1, deficient in PP2A phosphatase activity, and a pair of long-term ethylene-treated wild-type and loss-of-function ethylene signaling ctr1-1 mutants, deficient in mitogen-activated kinase kinase kinase activity. In total, 1079 phosphopeptides were identified, among which 44 were novel. Several one-minute ethylene-regulated phosphoproteins were found from the rcn1-1. Bioinformatic analysis of the rcn1-1 phosphoproteome predicted nine phosphoproteins as the putative substrates for PP2A phosphatase. In addition, from CTR1 kinase-enhanced phosphosites, we also found putative CTR1 kinase substrates including plastid transcriptionally active protein and calcium-sensing receptor. These regulatory proteins are phosphorylated in the presence of ethylene. Analysis of ethylene-regulated phosphosites using the group-based prediction system with a protein–protein interaction filter revealed a total of 14 kinase–substrate relationships that may function in both CTR1 kinase- and PP2A phosphatase-mediated phosphor-relay pathways. Finally, several ethylene-regulated post-translational modification network models have been built using molecular systems biology tools. It is proposed that ethylene regulates the phosphorylation of arginine/serine-rich splicing factor 41, plasma membrane intrinsic protein 2A, light harvesting chlorophyll A/B binding protein 1.1, and flowering bHLH 3 proteins in a dual-and-opposing fashion. PMID:24043427

A Review of Selected Candidate Endophenotypes for Depression

PubMed Central

Goldstein, Brandon L.; Klein, Daniel N.

2014-01-01

Endophenotypes are proposed to occupy an intermediate position in the pathway between genotype and phenotype in genetically complex disorders such as depression. To be considered an endophenotype, a construct must meet a set of criteria proposed by Gottesman and Gould (2003). In this qualitative review, we summarize evidence for each criterion for several putative endophenotypes for depression: neuroticism, morning cortisol, frontal asymmetry of cortical electrical activity, reward learning, and biases of attention and memory. Our review indicates that while there is strong support for some depression endophenotypes, other putative endophenotypes lack data or have inconsistent findings for core criteria. PMID:25006008

RACK1 and the microRNA pathway: is it déjà-vu all over again?

PubMed

Speth, Corinna; Laubinger, Sascha

2014-01-01

MicroRNAs (miRNAs) control many aspects of development and adaption in plants and in animals by post-transcriptional control of mRNA stability and translatability. Over the last years numerous proteins have been identified in the miRNA pathway. The versatile scaffold protein RACK1 has been associated with efficient miRNA production and function in plants and metazoans. Here, we briefly summarize the differences of RACK1 function in the plant and animal miRNA pathways and discuss putative mechanisms and functional roles of RACK1 in miRNA biogenesis and action.

Disentangling the multigenic and pleiotropic nature of molecular function

PubMed Central

2015-01-01

Background Biological processes at the molecular level are usually represented by molecular interaction networks. Function is organised and modularity identified based on network topology, however, this approach often fails to account for the dynamic and multifunctional nature of molecular components. For example, a molecule engaging in spatially or temporally independent functions may be inappropriately clustered into a single functional module. To capture biologically meaningful sets of interacting molecules, we use experimentally defined pathways as spatial/temporal units of molecular activity. Results We defined functional profiles of Saccharomyces cerevisiae based on a minimal set of Gene Ontology terms sufficient to represent each pathway's genes. The Gene Ontology terms were used to annotate 271 pathways, accounting for pathway multi-functionality and gene pleiotropy. Pathways were then arranged into a network, linked by shared functionality. Of the genes in our data set, 44% appeared in multiple pathways performing a diverse set of functions. Linking pathways by overlapping functionality revealed a modular network with energy metabolism forming a sparse centre, surrounded by several denser clusters comprised of regulatory and metabolic pathways. Signalling pathways formed a relatively discrete cluster connected to the centre of the network. Genetic interactions were enriched within the clusters of pathways by a factor of 5.5, confirming the organisation of our pathway network is biologically significant. Conclusions Our representation of molecular function according to pathway relationships enables analysis of gene/protein activity in the context of specific functional roles, as an alternative to typical molecule-centric graph-based methods. The pathway network demonstrates the cooperation of multiple pathways to perform biological processes and organises pathways into functionally related clusters with interdependent outcomes. PMID:26678917

The Myb-domain protein ULTRAPETALA1 INTERACTING FACTOR 1 controls floral meristem activities in Arabidopsis.

PubMed

Moreau, Fanny; Thévenon, Emmanuel; Blanvillain, Robert; Lopez-Vidriero, Irene; Franco-Zorrilla, Jose Manuel; Dumas, Renaud; Parcy, François; Morel, Patrice; Trehin, Christophe; Carles, Cristel C

2016-04-01

Higher plants continuously and iteratively produce new above-ground organs in the form of leaves, stems and flowers. These organs arise from shoot apical meristems whose homeostasis depends on coordination between self-renewal of stem cells and their differentiation into organ founder cells. This coordination is stringently controlled by the central transcription factor WUSCHEL (WUS), which is both necessary and sufficient for stem cell specification in Arabidopsis thaliana ULTRAPETALA1 (ULT1) was previously identified as a plant-specific, negative regulator of WUS expression. However, molecular mechanisms underlying this regulation remain unknown. ULT1 protein contains a SAND putative DNA-binding domain and a B-box, previously proposed as a protein interaction domain in eukaryotes. Here, we characterise a novel partner of ULT1, named ULT1 INTERACTING FACTOR 1 (UIF1), which contains a Myb domain and an EAR motif. UIF1 and ULT1 function in the same pathway for regulation of organ number in the flower. Moreover, UIF1 displays DNA-binding activity and specifically binds to WUS regulatory elements. We thus provide genetic and molecular evidence that UIF1 and ULT1 work together in floral meristem homeostasis, probably by direct repression of WUS expression. © 2016. Published by The Company of Biologists Ltd.

Binding Mode and Structure-Activity Relationships of ITE as an Aryl Hydrocarbon Receptor (AhR) Agonist.

PubMed

Dolciami, Daniela; Gargaro, Marco; Cerra, Bruno; Scalisi, Giulia; Bagnoli, Luana; Servillo, Giuseppe; Fazia, Maria Agnese Della; Puccetti, Paolo; Quintana, Francisco J; Fallarino, Francesca; Macchiarulo, Antonio

2018-02-06

Discovered as a modulator of the toxic response to environmental pollutants, aryl hydrocarbon receptor (AhR) has recently gained attention for its involvement in various physiological and pathological pathways. AhR is a ligand-dependent transcription factor activated by a large array of chemical compounds, which include metabolites of l-tryptophan (l-Trp) catabolism as endogenous ligands of the receptor. Among these, 2-(1'H-indole-3'-carbonyl)thiazole-4-carboxylic acid methyl ester (ITE) has attracted interest in the scientific community, being endowed with nontoxic, immunomodulatory, and anticancer AhR-mediated functions. So far, no information about the binding mode and interactions of ITE with AhR is available. In this study, we used docking and molecular dynamics to propose a putative binding mode of ITE into the ligand binding pocket of AhR. Mutagenesis studies were then instrumental in validating the proposed binding mode, identifying His 285 and Tyr 316 as important key residues for ligand-dependent receptor activation. Finally, a set of ITE analogues was synthesized and tested to further probe molecular interactions of ITE to AhR and characterize the relevance of specific functional groups in the chemical structure for receptor activity. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Increased Expression of a myo-Inositol Methyl Transferase in Mesembryanthemum crystallinum Is Part of a Stress Response Distinct from Crassulacean Acid Metabolism Induction 1

PubMed Central

Vernon, Daniel M.; Bohnert, Hans J.

1992-01-01

The facultative halophyte Mesembryanthemum crystallinum responds to osmotic stress by switching from C3 photosynthesis to Crassulacean acid metabolism (CAM). This shift to CAM involves the stress-initiated up-regulation of mRNAs encoding CAM enzymes. The capability of the plants to induce a key CAM enzyme, phosphoenolpyruvate carboxylase, is influenced by plant age, and it has been suggested that adaptation to salinity in M. crystallinum may be modulated by a developmental program that controls molecular responses to stress. We have compared the effects of plant age on the expression of two salinity-induced genes: Gpdl, which encodes the photosynthesis-related enzyme glyceraldehyde 3-phosphate dehydrogenase, and Imtl, which encodes a methyl transferase involved in the biosynthesis of a putative osmoprotectant, pinitol. Imtl mRNA accumulation and the accompanying increase in pinitol in stressed Mesembryanthemum exhibit a pattern of induction distinct from that observed for CAM-related genes. We conclude that the molecular mechanisms that trigger Imtl and pinitol accumulation in response to salt stress in M. crystallinum differ in some respects from those that lead to CAM induction. There may be multiple signals or pathways that regulate inducible components of salinity tolerance in this facultative halophyte. ImagesFigure 1Figure 2 PMID:16669095

Mapping of Human FOXP2 Enhancers Reveals Complex Regulation.

PubMed

Becker, Martin; Devanna, Paolo; Fisher, Simon E; Vernes, Sonja C

2018-01-01

Mutations of the FOXP2 gene cause a severe speech and language disorder, providing a molecular window into the neurobiology of language. Individuals with FOXP2 mutations have structural and functional alterations affecting brain circuits that overlap with sites of FOXP2 expression, including regions of the cortex, striatum, and cerebellum. FOXP2 displays complex patterns of expression in the brain, as well as in non-neuronal tissues, suggesting that sophisticated regulatory mechanisms control its spatio-temporal expression. However, to date, little is known about the regulation of FOXP2 or the genomic elements that control its expression. Using chromatin conformation capture (3C), we mapped the human FOXP2 locus to identify putative enhancer regions that engage in long-range interactions with the promoter of this gene. We demonstrate the ability of the identified enhancer regions to drive gene expression. We also show regulation of the FOXP2 promoter and enhancer regions by candidate regulators - FOXP family and TBR1 transcription factors. These data point to regulatory elements that may contribute to the temporal- or tissue-specific expression patterns of human FOXP2 . Understanding the upstream regulatory pathways controlling FOXP2 expression will bring new insight into the molecular networks contributing to human language and related disorders.

Mapping of Human FOXP2 Enhancers Reveals Complex Regulation

PubMed Central

Becker, Martin; Devanna, Paolo; Fisher, Simon E.; Vernes, Sonja C.

2018-01-01

Mutations of the FOXP2 gene cause a severe speech and language disorder, providing a molecular window into the neurobiology of language. Individuals with FOXP2 mutations have structural and functional alterations affecting brain circuits that overlap with sites of FOXP2 expression, including regions of the cortex, striatum, and cerebellum. FOXP2 displays complex patterns of expression in the brain, as well as in non-neuronal tissues, suggesting that sophisticated regulatory mechanisms control its spatio-temporal expression. However, to date, little is known about the regulation of FOXP2 or the genomic elements that control its expression. Using chromatin conformation capture (3C), we mapped the human FOXP2 locus to identify putative enhancer regions that engage in long-range interactions with the promoter of this gene. We demonstrate the ability of the identified enhancer regions to drive gene expression. We also show regulation of the FOXP2 promoter and enhancer regions by candidate regulators – FOXP family and TBR1 transcription factors. These data point to regulatory elements that may contribute to the temporal- or tissue-specific expression patterns of human FOXP2. Understanding the upstream regulatory pathways controlling FOXP2 expression will bring new insight into the molecular networks contributing to human language and related disorders. PMID:29515369

Developing Hypothetical Inhibition Mechanism of Novel Urea Transporter B Inhibitor

NASA Astrophysics Data System (ADS)

Li, Min; Tou, Weng Ieong; Zhou, Hong; Li, Fei; Ren, Huiwen; Chen, Calvin Yu-Chian; Yang, Baoxue

2014-07-01

Urea transporter B (UT-B) is a membrane channel protein that specifically transports urea. UT-B null mouse exhibited urea selective urine concentrating ability deficiency, which suggests the potential clinical applications of the UT-B inhibitors as novel diuretics. Primary high-throughput virtual screening (HTVS) of 50000 small-molecular drug-like compounds identified 2319 hit compounds. These 2319 compounds were screened by high-throughput screening using an erythrocyte osmotic lysis assay. Based on the pharmacological data, putative UT-B binding sites were identified by structure-based drug design and validated by ligand-based and QSAR model. Additionally, UT-B structural and functional characteristics under inhibitors treated and untreated conditions were simulated by molecular dynamics (MD). As the result, we identified four classes of compounds with UT-B inhibitory activity and predicted a human UT-B model, based on which computative binding sites were identified and validated. A novel potential mechanism of UT-B inhibitory activity was discovered by comparing UT-B from different species. Results suggest residue PHE198 in rat and mouse UT-B might block the inhibitor migration pathway. Inhibitory mechanisms of UT-B inhibitors and the functions of key residues in UT-B were proposed. The binding site analysis provides a structural basis for lead identification and optimization of UT-B inhibitors.

Molecular Mechanisms Underlying the Cardiovascular Benefits of SGLT2i and GLP-1RA.

PubMed

Khat, Dorrin Zarrin; Husain, Mansoor

2018-06-09

In addition to their effects on glycemic control, two specific classes of relatively new anti-diabetic drugs, namely the sodium glucose co-transporter-2 inhibitors (SGLT2i) and glucagon-like peptide-1 receptor agonists (GLP-1RA) have demonstrated reduced rates of major adverse cardiovascular events (MACE) in subjects with type 2 diabetes (T2D) at high risk for cardiovascular disease (CVD). This review summarizes recent experimental results that inform putative molecular mechanisms underlying these benefits. SGLT2i and GLP-1RA exert cardiovascular effects by targeting in both common and distinctive ways (A) several mediators of macro- and microvascular pathophysiology: namely (A1) inflammation and atherogenesis, (A2) oxidative stress-induced endothelial dysfunction, (A3) vascular smooth muscle cell reactive oxygen species (ROS) production and proliferation, and (A4) thrombosis. These agents also exhibit (B) hemodynamic effects through modulation of (B1) natriuresis/diuresis and (B2) the renin-angiotensin-aldosterone system. This review highlights that while GLP-1RA exert direct effects on vascular (endothelial and smooth muscle) cells, the effects of SGLT2i appear to include the activation of signaling pathways that prevent adverse vascular remodeling. Both SGLT2i and GLP-1RA confer hemodynamic effects that counter adverse cardiac remodeling.

Somatostatin, neuronal vulnerability and behavioral emotionality.

PubMed

Lin, L C; Sibille, E

2015-03-01

Somatostatin (SST) deficits are common pathological features in depression and other neurological disorders with mood disturbances, but little is known about the contribution of SST deficits to mood symptoms or causes of these deficits. Here we show that mice lacking SST (Sst(KO)) exhibit elevated behavioral emotionality, high basal plasma corticosterone and reduced gene expression of Bdnf, Cortistatin and Gad67, together recapitulating behavioral, neuroendocrine and molecular features of human depression. Studies in Sst(KO) and heterozygous (Sst(HZ)) mice show that elevated corticosterone is not sufficient to reproduce the behavioral phenotype, suggesting a putative role for Sst cell-specific molecular changes. Using laser capture microdissection, we show that cortical SST-positive interneurons display significantly greater transcriptome deregulations after chronic stress compared with pyramidal neurons. Protein translation through eukaryotic initiation factor 2 (EIF2) signaling, a pathway previously implicated in neurodegenerative diseases, was most affected and suppressed in stress-exposed SST neurons. We then show that activating EIF2 signaling through EIF2 kinase inhibition mitigated stress-induced behavioral emotionality in mice. Taken together, our data suggest that (1) low SST has a causal role in mood-related phenotypes, (2) deregulated EIF2-mediated protein translation may represent a mechanism for vulnerability of SST neurons and (3) that global EIF2 signaling has antidepressant/anxiolytic potential.

Somatostatin, neuronal vulnerability and behavioral emotionality

PubMed Central

Lin, LC; Sibille, E

2014-01-01

Somatostatin (SST) deficits are common pathological features in depression and other neurological disorders with mood disturbances, but little is known about the contribution of SST deficits to mood symptoms or causes of these deficits. Here we show that mice lacking Sst (SstKO) exhibit elevated behavioral emotionality, high basal plasma corticosterone and reduced gene expression of Bdnf, Cortistatin, and Gad67, together recapitulating behavioral, neuroendocrine and molecular features of human depression. Studies in SstKO and heterozygous (SstHZ) mice show that elevated corticosterone is not sufficient to reproduce the behavioral phenotype, suggesting a putative role for Sst cell-specific molecular changes. Using laser-capture microdissection, we show that cortical SST-positive interneurons display significantly greater transcriptome deregulations after chronic stress compared to pyramidal neurons. Protein translation through eukaryotic initiation factor 2 (EIF2) signaling, a pathway previously implicated in neurodegenerative diseases, was most affected and suppressed in stress-exposed SST neurons. We then show that activating EIF2 signaling through EIF2 kinase inhibition mitigated stress-induced behavioral emotionality in mice. Together, our data suggest that (1) low SST plays a causal role in mood-related phenotypes, (2) deregulated EIF2-mediated protein translation may represent a mechanism for vulnerability of SST neurons, and (3) that global EIF2 signaling has antidepressant/anxiolytic potential. PMID:25600109

The SUD1 gene encodes a putative E3 ubiquitin ligase and is a positive regulator of 3-hydroxy-3-methylglutaryl coenzyme a reductase activity in Arabidopsis.

PubMed

Doblas, Verónica G; Amorim-Silva, Vítor; Posé, David; Rosado, Abel; Esteban, Alicia; Arró, Montserrat; Azevedo, Herlander; Bombarely, Aureliano; Borsani, Omar; Valpuesta, Victoriano; Ferrer, Albert; Tavares, Rui M; Botella, Miguel A

2013-02-01

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the major rate-limiting step of the mevalonic acid (MVA) pathway from which sterols and other isoprenoids are synthesized. In contrast with our extensive knowledge of the regulation of HMGR in yeast and animals, little is known about this process in plants. To identify regulatory components of the MVA pathway in plants, we performed a genetic screen for second-site suppressor mutations of the Arabidopsis thaliana highly drought-sensitive drought hypersensitive2 (dry2) mutant that shows decreased squalene epoxidase activity. We show that mutations in SUPPRESSOR OF DRY2 DEFECTS1 (SUD1) gene recover most developmental defects in dry2 through changes in HMGR activity. SUD1 encodes a putative E3 ubiquitin ligase that shows sequence and structural similarity to yeast Degradation of α factor (Doα10) and human TEB4, components of the endoplasmic reticulum-associated degradation C (ERAD-C) pathway. While in yeast and animals, the alternative ERAD-L/ERAD-M pathway regulates HMGR activity by controlling protein stability, SUD1 regulates HMGR activity without apparent changes in protein content. These results highlight similarities, as well as important mechanistic differences, among the components involved in HMGR regulation in plants, yeast, and animals.

The SUD1 Gene Encodes a Putative E3 Ubiquitin Ligase and Is a Positive Regulator of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity in Arabidopsis[C][W

PubMed Central

Doblas, Verónica G.; Amorim-Silva, Vítor; Posé, David; Rosado, Abel; Esteban, Alicia; Arró, Montserrat; Azevedo, Herlander; Bombarely, Aureliano; Borsani, Omar; Valpuesta, Victoriano; Ferrer, Albert; Tavares, Rui M.; Botella, Miguel A.

2013-01-01

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the major rate-limiting step of the mevalonic acid (MVA) pathway from which sterols and other isoprenoids are synthesized. In contrast with our extensive knowledge of the regulation of HMGR in yeast and animals, little is known about this process in plants. To identify regulatory components of the MVA pathway in plants, we performed a genetic screen for second-site suppressor mutations of the Arabidopsis thaliana highly drought-sensitive drought hypersensitive2 (dry2) mutant that shows decreased squalene epoxidase activity. We show that mutations in SUPPRESSOR OF DRY2 DEFECTS1 (SUD1) gene recover most developmental defects in dry2 through changes in HMGR activity. SUD1 encodes a putative E3 ubiquitin ligase that shows sequence and structural similarity to yeast Degradation of α factor (Doα10) and human TEB4, components of the endoplasmic reticulum–associated degradation C (ERAD-C) pathway. While in yeast and animals, the alternative ERAD-L/ERAD-M pathway regulates HMGR activity by controlling protein stability, SUD1 regulates HMGR activity without apparent changes in protein content. These results highlight similarities, as well as important mechanistic differences, among the components involved in HMGR regulation in plants, yeast, and animals. PMID:23404890

Conceptualizing neurodevelopmental disorders through a mechanistic understanding of fragile X syndrome and Williams syndrome.

PubMed

Fung, Lawrence K; Quintin, Eve-Marie; Haas, Brian W; Reiss, Allan L

2012-04-01

The overarching goal of this review is to compare and contrast the cognitive-behavioral features of fragile X syndrome (FraX) and Williams syndrome and to review the putative neural and molecular underpinnings of these features. Information is presented in a framework that provides guiding principles for conceptualizing gene-brain-behavior associations in neurodevelopmental disorders. Abnormalities, in particular cognitive-behavioral domains with similarities in underlying neurodevelopmental correlates, occur in both FraX and Williams syndrome including aberrant frontostriatal pathways leading to executive function deficits, and magnocellular/dorsal visual stream, superior parietal lobe, inferior parietal lobe, and postcentral gyrus abnormalities contributing to deficits in visuospatial function. Compelling cognitive-behavioral and neurodevelopmental contrasts also exist in these two disorders, for example, aberrant amygdala and fusiform cortex structure and function occurring in the context of contrasting social behavioral phenotypes, and temporal cortical and cerebellar abnormalities potentially underlying differences in language function. Abnormal dendritic development is a shared neurodevelopmental morphologic feature between FraX and Williams syndrome. Commonalities in molecular machinery and processes across FraX and Williams syndrome occur as well - microRNAs involved in translational regulation of major synaptic proteins; scaffolding proteins in excitatory synapses; and proteins involved in axonal development. Although the genetic variations leading to FraX and Williams syndrome are different, important similarities and contrasts in the phenotype, neurocircuitry, molecular machinery, and cellular processes in these two disorders allow for a unique approach to conceptualizing gene-brain-behavior links occurring in neurodevelopmental disorders.

The Role of Mitochondria in the Activation/Maintenance of SOCE: The Contribution of Mitochondrial Ca2+ Uptake, Mitochondrial Motility, and Location to Store-Operated Ca2+ Entry.

PubMed

Malli, Roland; Graier, Wolfgang F

2017-01-01

In most cell types, the depletion of internal Ca 2+ stores triggers the activation of Ca 2+ entry. This crucial phenomenon is known since the 1980s and referred to as store-operated Ca 2+ entry (SOCE). With the discoveries of the stromal-interacting molecules (STIMs) and the Ca 2+ -permeable Orai channels as the long-awaited molecular constituents of SOCE, the role of mitochondria in controlling the activity of this particular Ca 2+ entry pathway is kind of buried in oblivion. However, the capability of mitochondria to locally sequester Ca 2+ at sites of Ca 2+ release and entry was initially supposed to rule SOCE by facilitating the Ca 2+ depletion of the endoplasmic reticulum and removing entering Ca 2+ from the Ca 2+ -inhibitable channels, respectively. Moreover, the central role of these organelles in controlling the cellular energy metabolism has been linked to the activity of SOCE. Nevertheless, the exact molecular mechanisms by which mitochondria actually determine SOCE are still pretty obscure. In this essay we describe the complexity of the mitochondrial Ca 2+ uptake machinery and its regulation, molecular components, and properties, which open new ways for scrutinizing the contribution of mitochondria to SOCE. Moreover, data concerning the variability of the morphology and cellular distribution of mitochondria as putative determinants of SOCE activation, maintenance, and termination are summarized.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hill, David P.; D’Eustachio, Peter; Berardini, Tanya Z.

The concept of a biological pathway, an ordered sequence of molecular transformations, is used to collect and represent molecular knowledge for a broad span of organismal biology. Representations of biomedical pathways typically are rich but idiosyncratic presentations of organized knowledge about individual pathways. Meanwhile, biomedical ontologies and associated annotation files are powerful tools that organize molecular information in a logically rigorous form to support computational analysis. The Gene Ontology (GO), representing Molecular Functions, Biological Processes and Cellular Components, incorporates many aspects of biological pathways within its ontological representations. Here we present a methodology for extending and refining the classes inmore » the GO for more comprehensive, consistent and integrated representation of pathways, leveraging knowledge embedded in current pathway representations such as those in the Reactome Knowledgebase and MetaCyc. With carbohydrate metabolic pathways as a use case, we discuss how our representation supports the integration of variant pathway classes into a unified ontological structure that can be used for data comparison and analysis.« less

Subtype and pathway specific responses to anticancer compounds in breast cancer.

PubMed

Heiser, Laura M; Sadanandam, Anguraj; Kuo, Wen-Lin; Benz, Stephen C; Goldstein, Theodore C; Ng, Sam; Gibb, William J; Wang, Nicholas J; Ziyad, Safiyyah; Tong, Frances; Bayani, Nora; Hu, Zhi; Billig, Jessica I; Dueregger, Andrea; Lewis, Sophia; Jakkula, Lakshmi; Korkola, James E; Durinck, Steffen; Pepin, François; Guan, Yinghui; Purdom, Elizabeth; Neuvial, Pierre; Bengtsson, Henrik; Wood, Kenneth W; Smith, Peter G; Vassilev, Lyubomir T; Hennessy, Bryan T; Greshock, Joel; Bachman, Kurtis E; Hardwicke, Mary Ann; Park, John W; Marton, Laurence J; Wolf, Denise M; Collisson, Eric A; Neve, Richard M; Mills, Gordon B; Speed, Terence P; Feiler, Heidi S; Wooster, Richard F; Haussler, David; Stuart, Joshua M; Gray, Joe W; Spellman, Paul T

2012-02-21

Breast cancers are comprised of molecularly distinct subtypes that may respond differently to pathway-targeted therapies now under development. Collections of breast cancer cell lines mirror many of the molecular subtypes and pathways found in tumors, suggesting that treatment of cell lines with candidate therapeutic compounds can guide identification of associations between molecular subtypes, pathways, and drug response. In a test of 77 therapeutic compounds, nearly all drugs showed differential responses across these cell lines, and approximately one third showed subtype-, pathway-, and/or genomic aberration-specific responses. These observations suggest mechanisms of response and resistance and may inform efforts to develop molecular assays that predict clinical response.

Central projections of antennular chemosensory and mechanosensory afferents in the brain of the terrestrial hermit crab (Coenobita clypeatus; Coenobitidae, Anomura)

PubMed Central

Tuchina, Oksana; Koczan, Stefan; Harzsch, Steffen; Rybak, Jürgen; Wolff, Gabriella; Strausfeld, Nicholas J.; Hansson, Bill S.

2015-01-01

The Coenobitidae (Decapoda, Anomura, Paguroidea) is a taxon of hermit crabs that includes two genera with a fully terrestrial life style as adults. Previous studies have shown that Coenobitidae have evolved a sense of spatial odor localization that is behaviorally highly relevant. Here, we examined the central olfactory pathway of these animals by analyzing central projections of the antennular nerve of Coenobita clypeatus, combining backfilling of the nerve with dextran-coupled dye, Golgi impregnations and three-dimensional reconstruction of the primary olfactory center, the antennular lobe. The principal pattern of putative olfactory sensory afferents in C. clypeatus is in many aspects similar to what have been established for aquatic decapod crustaceans, such as the spiny lobster Panulirus argus. However, there are also obvious differences that may, or may not represent adaptations related to a terrestrial lifestyle. In C. clypeatus, the antennular lobe dominates the deutocerebrum, having more than one thousand allantoid-shaped subunits. We observed two distinct patterns of sensory neuron innervation: putative olfactory afferents from the aesthetascs either supply the cap/subcap region of the subunits or they extend through its full depth. Our data also demonstrate that any one sensory axon can supply input to several subunits. Putative chemosensory (non-aesthetasc) and mechanosensory axons represent a different pathway and innervate the lateral and median antennular neuropils. Hence, we suggest that the chemosensory input in C. clypeatus might be represented via a dual pathway: aesthetascs target the antennular lobe, and bimodal sensilla target the lateral antennular neuropil and median antennular neuropil. The present data is compared to related findings in other decapod crustaceans. PMID:26236202

Modeling biochemical pathways in the gene ontology

DOE PAGES

Hill, David P.; D’Eustachio, Peter; Berardini, Tanya Z.; ...

2016-09-01

The concept of a biological pathway, an ordered sequence of molecular transformations, is used to collect and represent molecular knowledge for a broad span of organismal biology. Representations of biomedical pathways typically are rich but idiosyncratic presentations of organized knowledge about individual pathways. Meanwhile, biomedical ontologies and associated annotation files are powerful tools that organize molecular information in a logically rigorous form to support computational analysis. The Gene Ontology (GO), representing Molecular Functions, Biological Processes and Cellular Components, incorporates many aspects of biological pathways within its ontological representations. Here we present a methodology for extending and refining the classes inmore » the GO for more comprehensive, consistent and integrated representation of pathways, leveraging knowledge embedded in current pathway representations such as those in the Reactome Knowledgebase and MetaCyc. With carbohydrate metabolic pathways as a use case, we discuss how our representation supports the integration of variant pathway classes into a unified ontological structure that can be used for data comparison and analysis.« less

ALMA 0.1–0.2 arcsec resolution imaging of the NGC 1068 Nucleus: compact dense molecular gas emission at the putative AGN location

DOE Office of Scientific and Technical Information (OSTI.GOV)

Imanishi, Masatoshi; Nakanishi, Kouichiro; Izumi, Takuma, E-mail: masa.imanishi@nao.ac.jp

2016-05-01

We present the results of our ALMA Cycle 2 high angular resolution (0.″1–0.″2) observations of the nuclear region of the nearby well-studied type-2 active galactic nucleus (AGN), NGC 1068, at HCN J = 3–2 and HCO{sup +} J = 3–2 emission lines. For the first time, due to a higher angular resolution than previous studies, we clearly detected dense molecular gas emission at the putative AGN location, identified as a ∼1.1 mm (∼266 GHz) continuum emission peak, by separating this emission from brighter emission located at 0.″5–2.″0 on the eastern and western sides of the AGN. The estimated intrinsic molecularmore » emission size and dense molecular mass, which are thought to be associated with the putative dusty molecular torus around an AGN, were ∼10 pc and ∼several × 10{sup 5} M {sub ⊙}, respectively. HCN-to-HCO{sup +} J = 3–2 flux ratios substantially higher than unity were found throughout the nuclear region of NGC 1068. The continuum emission displayed an elongated morphology along the direction of the radio jet located at the northern side of the AGN, as well as a weak spatially-resolved component at ∼2.″0 on the southwestern side of the AGN. The latter component most likely originated from star formation, with the estimated luminosity more than one order of magnitude lower than the luminosity of the central AGN. No vibrationally excited ( v {sub 2} = 1f) J = 3–2 emission lines were detected for HCN and HCO{sup +} across the field of view.« less

Molecular Pathology: Predictive, Prognostic, and Diagnostic Markers in Uterine Tumors.

PubMed

Ritterhouse, Lauren L; Howitt, Brooke E

2016-09-01

This article focuses on the diagnostic, prognostic, and predictive molecular biomarkers in uterine malignancies, in the context of morphologic diagnoses. The histologic classification of endometrial carcinomas is reviewed first, followed by the description and molecular classification of endometrial epithelial malignancies in the context of histologic classification. Taken together, the molecular and histologic classifications help clinicians to approach troublesome areas encountered in clinical practice and evaluate the utility of molecular alterations in the diagnosis and subclassification of endometrial carcinomas. Putative prognostic markers are reviewed. The use of molecular alterations and surrogate immunohistochemistry as prognostic and predictive markers is also discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

Complete genome sequence of Enterobacter sp. IIT-BT 08: A potential microbial strain for high rate hydrogen production.

PubMed

Khanna, Namita; Ghosh, Ananta Kumar; Huntemann, Marcel; Deshpande, Shweta; Han, James; Chen, Amy; Kyrpides, Nikos; Mavrommatis, Kostas; Szeto, Ernest; Markowitz, Victor; Ivanova, Natalia; Pagani, Ioanna; Pati, Amrita; Pitluck, Sam; Nolan, Matt; Woyke, Tanja; Teshima, Hazuki; Chertkov, Olga; Daligault, Hajnalka; Davenport, Karen; Gu, Wei; Munk, Christine; Zhang, Xiaojing; Bruce, David; Detter, Chris; Xu, Yan; Quintana, Beverly; Reitenga, Krista; Kunde, Yulia; Green, Lance; Erkkila, Tracy; Han, Cliff; Brambilla, Evelyne-Marie; Lang, Elke; Klenk, Hans-Peter; Goodwin, Lynne; Chain, Patrick; Das, Debabrata

2013-12-20

Enterobacter sp. IIT-BT 08 belongs to Phylum: Proteobacteria, Class: Gammaproteobacteria, Order: Enterobacteriales, Family: Enterobacteriaceae. The organism was isolated from the leaves of a local plant near the Kharagpur railway station, Kharagpur, West Bengal, India. It has been extensively studied for fermentative hydrogen production because of its high hydrogen yield. For further enhancement of hydrogen production by strain development, complete genome sequence analysis was carried out. Sequence analysis revealed that the genome was linear, 4.67 Mbp long and had a GC content of 56.01%. The genome properties encode 4,393 protein-coding and 179 RNA genes. Additionally, a putative pathway of hydrogen production was suggested based on the presence of formate hydrogen lyase complex and other related genes identified in the genome. Thus, in the present study we describe the specific properties of the organism and the generation, annotation and analysis of its genome sequence as well as discuss the putative pathway of hydrogen production by this organism.

Application of DEN refinement and automated model building to a difficult case of molecular-replacement phasing: the structure of a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum.

PubMed

Brunger, Axel T; Das, Debanu; Deacon, Ashley M; Grant, Joanna; Terwilliger, Thomas C; Read, Randy J; Adams, Paul D; Levitt, Michael; Schröder, Gunnar F

2012-04-01

Phasing by molecular replacement remains difficult for targets that are far from the search model or in situations where the crystal diffracts only weakly or to low resolution. Here, the process of determining and refining the structure of Cgl1109, a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum, at ∼3 Å resolution is described using a combination of homology modeling with MODELLER, molecular-replacement phasing with Phaser, deformable elastic network (DEN) refinement and automated model building using AutoBuild in a semi-automated fashion, followed by final refinement cycles with phenix.refine and Coot. This difficult molecular-replacement case illustrates the power of including DEN restraints derived from a starting model to guide the movements of the model during refinement. The resulting improved model phases provide better starting points for automated model building and produce more significant difference peaks in anomalous difference Fourier maps to locate anomalous scatterers than does standard refinement. This example also illustrates a current limitation of automated procedures that require manual adjustment of local sequence misalignments between the homology model and the target sequence.

Application of DEN refinement and automated model building to a difficult case of molecular-replacement phasing: the structure of a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum

PubMed Central

Brunger, Axel T.; Das, Debanu; Deacon, Ashley M.; Grant, Joanna; Terwilliger, Thomas C.; Read, Randy J.; Adams, Paul D.; Levitt, Michael; Schröder, Gunnar F.

2012-01-01

Phasing by molecular replacement remains difficult for targets that are far from the search model or in situations where the crystal diffracts only weakly or to low resolution. Here, the process of determining and refining the structure of Cgl1109, a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum, at ∼3 Å resolution is described using a combination of homology modeling with MODELLER, molecular-replacement phasing with Phaser, deformable elastic network (DEN) refinement and automated model building using AutoBuild in a semi-automated fashion, followed by final refinement cycles with phenix.refine and Coot. This difficult molecular-replacement case illustrates the power of including DEN restraints derived from a starting model to guide the movements of the model during refinement. The resulting improved model phases provide better starting points for automated model building and produce more significant difference peaks in anomalous difference Fourier maps to locate anomalous scatterers than does standard refinement. This example also illustrates a current limitation of automated procedures that require manual adjustment of local sequence misalignments between the homology model and the target sequence. PMID:22505259

Molecular characterization of putative Hepatozoon sp. from the sedge warbler (Acrocephalus schoenobaenus).

PubMed

Biedrzycka, Aleksandra; Kloch, Agnieszka; Migalska, Magdalena; Bielański, Wojciech

2013-05-01

We characterized partial sequences of 18S rDNA from sedge warblers infected with a parasite described previously as Hepatozoon kabeeni. Prevalence was 47% in sampled birds.We detected 3 parasite haplotypes in 62 sequenced samples from infected animals. In phylogenetic analyses, 2 of the putative Hepatozoon haplotypes closely resembled Lankesterella minima and L. valsainensis. The third haplotype grouped in a wider clade composed of Caryospora and Eimeria. None of the haplotypes showed resemblance to sequences of Hepatozoon from reptiles and mammals. Molecular detection results were consistent with those from microscopy of stained blood smears, confirming that the primers indeed amplified the parasite sequences. Here we provide evidence that the avian Hepatozoon-like parasites are most likely Lankesterella, supporting the suggestion that the systematic position of avian Hepatozoon-like species needs to be revised.

Morphology delimits more species than molecular genetic clusters of invasive Pilosella.

PubMed

Moffat, Chandra E; Ensing, David J; Gaskin, John F; De Clerck-Floate, Rosemarie A; Pither, Jason

2015-07-01

• Accurate assessments of biodiversity are paramount for understanding ecosystem processes and adaptation to change. Invasive species often contribute substantially to local biodiversity; correctly identifying and distinguishing invaders is thus necessary to assess their potential impacts. We compared the reliability of morphology and molecular sequences to discriminate six putative species of invasive Pilosella hawkweeds (syn. Hieracium, Asteraceae), known for unreliable identifications and historical introgression. We asked (1) which morphological traits dependably discriminate putative species, (2) if genetic clusters supported morphological species, and (3) if novel hybridizations occur in the invaded range.• We assessed 33 morphometric characters for their discriminatory power using the randomForest classifier and, using AFLPs, evaluated genetic clustering with the program structure and subsequently with an AMOVA. The strength of the association between morphological and genotypic dissimilarity was assessed with a Mantel test.• Morphometric analyses delimited six species while genetic analyses defined only four clusters. Specifically, we found (1) eight morphological traits could reliably distinguish species, (2) structure suggested strong genetic differentiation but for only four putative species clusters, and (3) genetic data suggest both novel hybridizations and multiple introductions have occurred.• (1) Traditional floristic techniques may resolve more species than molecular analyses in taxonomic groups subject to introgression. (2) Even within complexes of closely related species, relatively few but highly discerning morphological characters can reliably discriminate species. (3) By clarifying patterns of morphological and genotypic variation of invasive Pilosella, we lay foundations for further ecological study and mitigation. © 2015 Botanical Society of America, Inc.

Molecular characterization and genomic distribution of Isis: a new retrotransposon of Drosophila buzzatii.

PubMed

García Guerreiro, M P; Fontdevila, A

2007-01-01

A new transposable element, Isis, is identified as a LTR retrotransposon in Drosophila buzzatii. DNA sequence analysis shows that Isis contains three long ORFs similar to gag, pol and env genes of retroviruses. The ORF1 exhibits sequence homology to matrix, capsid and nucleocapsid gag proteins and ORF2 encodes a putative protease (PR), a reverse transcriptase (RT), an Rnase H (RH) and an integrase (IN) region. The analysis of a putative env product, encoded by the env ORF3, shows a degenerated protein containing several stop codons. The molecular study of the putative proteins coded by this new element shows striking similarities to both Ulysses and Osvaldo elements, two LTR retrotransposons, present in D. virilis and D. buzzatii, respectively. Comparisons of the predicted Isis RT to several known retrotransposons show strong phylogenetic relationships to gypsy-like elements, particulary to Ulysses retrotransposon. Studies of Isis chromosomal distribution show a strong hybridization signal in centromeric and pericentromeric regions, and a scattered distribution along all chromosomal arms. The existence of insertional polymorphisms between different strains and high molecular weight bands by Southern blot suggests the existence of full-sized copies that have been active recently. The presence of euchromatic insertion sites coincident between Isis and Osvaldo could indicate preferential insertion sites of Osvaldo element into Isis sequence or vice versa. Moreover, the presence of Isis in different species of the buzzatii complex indicates the ancient origin of this element.

[Isolation of ABA-regulated genes in Oryza sativa through fluorescent differential display PCR (FDD-PCR)].

PubMed

Xu, Shou Ling; Shen, Si Shi; Xu, Zhi Hong; Xue, Hong Wei

2002-12-01

Abscisic acid (ABA) was critical in plant seed development and response to environmental factors such as stress situations. To study the possible ABA related signaling transduction pathways, we tried to isolate the ABA-regulated genes through fluorescent differential display PCR (FDD-PCR) technology using rice seedling as materials (treated with ABA for 2, 4, 8 and 12h). In the 17 fragments isolated, 14 and 3 clones were up-and down-regulated respectively. Sequence analyses revealed that the encoded proteins were involved in photosynthesis (7 fragments), signal transduction (1 fragments), transcription (2 fragments), metabolism and resistance (6 fragments), and unknown protein (1 fragments). 3 clones, encoding putative alpha/beta hydrolase fold, putative vacuolar H+ -ATPase B subunit, putative tyrosine phosphatase, were confirmed to be regulated under ABA treatment by RT-PCR and northern blot analysis. FDD-PCR and possible functional mechanisms of ABA were discussed.

Proteogenomic Characterization of Monocyclic Aromatic Hydrocarbon Degradation Pathways in the Aniline-Degrading Bacterium Burkholderia sp. K24.

PubMed

Lee, Sang-Yeop; Kim, Gun-Hwa; Yun, Sung Ho; Choi, Chi-Won; Yi, Yoon-Sun; Kim, Jonghyun; Chung, Young-Ho; Park, Edmond Changkyun; Kim, Seung Il

2016-01-01

Burkholderia sp. K24, formerly known as Acinetobacter lwoffii K24, is a soil bacterium capable of utilizing aniline as its sole carbon and nitrogen source. Genomic sequence analysis revealed that this bacterium possesses putative gene clusters for biodegradation of various monocyclic aromatic hydrocarbons (MAHs), including benzene, toluene, and xylene (BTX), as well as aniline. We verified the proposed MAH biodegradation pathways by dioxygenase activity assays, RT-PCR, and LC/MS-based quantitative proteomic analyses. This proteogenomic approach revealed four independent degradation pathways, all converging into the citric acid cycle. Aniline and p-hydroxybenzoate degradation pathways converged into the β-ketoadipate pathway. Benzoate and toluene were degraded through the benzoyl-CoA degradation pathway. The xylene isomers, i.e., o-, m-, and p-xylene, were degraded via the extradiol cleavage pathways. Salicylate was degraded through the gentisate degradation pathway. Our results show that Burkholderia sp. K24 possesses versatile biodegradation pathways, which may be employed for efficient bioremediation of aniline and BTX.

Proteogenomic Characterization of Monocyclic Aromatic Hydrocarbon Degradation Pathways in the Aniline-Degrading Bacterium Burkholderia sp. K24

PubMed Central

Yun, Sung Ho; Choi, Chi-Won; Yi, Yoon-Sun; Kim, Jonghyun; Chung, Young-Ho; Park, Edmond Changkyun; Kim, Seung Il

2016-01-01

Burkholderia sp. K24, formerly known as Acinetobacter lwoffii K24, is a soil bacterium capable of utilizing aniline as its sole carbon and nitrogen source. Genomic sequence analysis revealed that this bacterium possesses putative gene clusters for biodegradation of various monocyclic aromatic hydrocarbons (MAHs), including benzene, toluene, and xylene (BTX), as well as aniline. We verified the proposed MAH biodegradation pathways by dioxygenase activity assays, RT-PCR, and LC/MS-based quantitative proteomic analyses. This proteogenomic approach revealed four independent degradation pathways, all converging into the citric acid cycle. Aniline and p-hydroxybenzoate degradation pathways converged into the β-ketoadipate pathway. Benzoate and toluene were degraded through the benzoyl-CoA degradation pathway. The xylene isomers, i.e., o-, m-, and p-xylene, were degraded via the extradiol cleavage pathways. Salicylate was degraded through the gentisate degradation pathway. Our results show that Burkholderia sp. K24 possesses versatile biodegradation pathways, which may be employed for efficient bioremediation of aniline and BTX. PMID:27124467

Identification of novel mutations in endometrial cancer patients by whole-exome sequencing.

PubMed

Chang, Ya-Sian; Huang, Hsien-Da; Yeh, Kun-Tu; Chang, Jan-Gowth

2017-05-01

The aim of the present study was to identify genomic alterations in Taiwanese endometrial cancer patients. This information is vitally important in Taiwan, where endometrial cancer is the second most common gynecological cancer. We performed whole-exome sequencing on DNA from 14 tumor tissue samples from Taiwanese endometrial cancer patients. We used the Genome Analysis Tool kit software package for data analysis, and the dbSNP, Catalogue of Somatic Mutations in Cancer (COSMIC) and The Cancer Genome Atlas (TCGA) databases for comparisons. Variants were validated via Sanger sequencing. We identified 143 non-synonymous mutations in 756 canonical cancer-related genes and 1,271 non-synonymous mutations in non-canonical cancer-related genes in 14 endometrial samples. PTEN, KRAS and PIK3R1 were the most frequently mutated canonical cancer-related genes. Our results revealed nine potential driver genes (MAPT, IL24, MCM6, TSC1, BIRC2, CIITA, DST, CASP8 and NOTCH2) and 21 potential passenger genes (ARMCX4, IGSF10, VPS13C, DCT, DNAH14, TLN1, ZNF605, ZSCAN29, MOCOS, CMYA5, PCDH17, UGT1A8, CYFIP2, MACF1, NUDT5, JAKMIP1, PCDHGB4, FAM178A, SNX6, IMP4 and PCMTD1). The detected molecular aberrations led to putative activation of the mTOR, Wnt, MAPK, VEGF and ErbB pathways, as well as aberrant DNA repair, cell cycle control and apoptosis pathways. We characterized the mutational landscape and genetic alterations in multiple cellular pathways of endometrial cancer in the Taiwanese population.

Genome-Wide Identification of Circular RNAs as a Novel Class of Putative Biomarkers for an Ocular Surface Disease.

PubMed

Li, Xiu-Miao; Ge, Hui-Min; Yao, Jin; Zhou, Yun-Fan; Yao, Mu-Di; Liu, Chang; Hu, Hai-Tao; Zhu, Yun-Xi; Shan, Kun; Yan, Biao; Jiang, Qin

2018-06-27

Pterygium is a common ocular surface disease with an unknown etiology and threatens vision as it invades into the cornea. Circular RNAs (circRNAs) are a novel class of RNA transcripts that participate in several physiological and pathological processes. However, the role of circRNAs in pathogenesis of pterygium remains largely unknown. Genome-wide circRNA expression profiling was performed to identify pterygium -related circRNAs. GO analysis, pathway analysis, and miRNA response elements analysis was performed to predict the function of differentially expressed circRNAs in pterygium. MTT assays, Ki67 staining, Transwell assay, Hoechst 33342 staining, and Calcein-AM/PI staining were performed to determine the effect of circRNA silencing on pterygium fibroblast and epithelial cell function. Approximately 669 circRNAs were identified to be abnormally expressed in pterygium tissues. GO analysis demonstrated that the host genes of differentially expressed circRNAs were targeted to extracellular matrix organization (ontology: biological process), cytoplasm (ontology: cellular component), and protein binding (ontology: molecular function). Pathway analysis showed that dysregulated circRNAs-mediated regulatory networks were mostly enriched in focal adhesion signaling pathway. Notably, circ_0085020 (circ-LAPTM4B) was shown as a potential biomarker for pterygium. circ_0085020 (circ-LAPTM4B) silencing affected the viability, proliferation, migration, and apoptosis of pterygium fibroblast and epithelial cells in vitro. This study provides evidence that circRNAs are involved in the pathogenesis of pterygium and might constitute promising targets for the therapeutic intervention of pterygium. © 2018 The Author(s). Published by S. Karger AG, Basel.

Flower bud transcriptome analysis of Sapium sebiferum (Linn.) Roxb. and primary investigation of drought induced flowering: pathway construction and G-quadruplex prediction based on transcriptome.

PubMed

Yang, Minglei; Wu, Ying; Jin, Shan; Hou, Jinyan; Mao, Yingji; Liu, Wenbo; Shen, Yangcheng; Wu, Lifang

2015-01-01

Sapium sebiferum (Linn.) Roxb. (Chinese Tallow Tree) is a perennial woody tree and its seeds are rich in oil which hold great potential for biodiesel production. Despite a traditional woody oil plant, our understanding on S. sebiferum genetics and molecular biology remains scant. In this study, the first comprehensive transcriptome of S. sebiferum flower has been generated by sequencing and de novo assembly. A total of 149,342 unigenes were generated from raw reads, of which 24,289 unigenes were successfully matched to public database. A total of 61 MADS box genes and putative pathways involved in S. sebiferum flower development have been identified. Abiotic stress response network was also constructed in this work, where 2,686 unigenes are involved in the pathway. As for lipid biosynthesis, 161 unigenes have been identified in fatty acid (FA) and triacylglycerol (TAG) biosynthesis. Besides, the G-Quadruplexes in RNA of S. sebiferum also have been predicted. An interesting finding is that the stress-induced flowering was observed in S. sebiferum for the first time. According to the results of semi-quantitative PCR, expression tendencies of flowering-related genes, GA1, AP2 and CRY2, accorded with stress-related genes, such as GRX50435 and PRXⅡ39562. This transcriptome provides functional genomic information for further research of S. sebiferum, especially for the genetic engineering to shorten the juvenile period and improve yield by regulating flower development. It also offers a useful database for the research of other Euphorbiaceae family plants.

De novo transcriptome sequencing and comparative analysis to discover genes involved in ovarian maturity in Strongylocentrotus nudus.

PubMed

Jia, Zhiying; Wang, Qiai; Wu, Kaikai; Wei, Zhenlin; Zhou, Zunchun; Liu, Xiaolin

2017-09-01

Strongylocentrotus nudus is an edible sea urchin, mainly harvested in China. Correlation studies indicated that S. nudus with larger diameter have a prolonged marketing time and better palatability owing to their precocious gonads and extended maturation process. However, the molecular mechanism underlying this phenomenon is still unknown. Here, transcriptome sequencing was applied to study the ovaries of adult S. nudus with different shell diameters to explore the possible mechanism. In this study, four independent cDNA libraries were constructed, including two from the big size urchins and two from the small ones using a HiSeq™2500 platform. A total of 88,581 unigenes were acquired with a mean length of 1354bp, of which 66,331 (74.88%) unigenes could be annotated using six major publicly available databases. Comparative analysis revealed that 353 unigenes were differentially expressed (with log2(ratio)≥1, FDR≤0.001) between the two groups. Of these, 20 differentially expressed genes (DEGs) were selected to confirm the accuracy of RNA-seq data by quantitative real-time RT-PCR. Furthermore, gene ontology and KEGG pathway enrichment analyses were performed to find the putative genes and pathways related to ovarian maturity. Eight unigenes were identified as significant DEGs involved in reproduction related pathways; these included Mos, Cdc20, Rec8, YP30, cytochrome P450 2U1, ovoperoxidase, proteoliaisin, and rendezvin. Our research fills the gap in the studies on the S. nudus ovaries using transcriptome analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

In vitro cell injury by oxidized low density lipoprotein involves lipid hydroperoxide-induced formation of alkoxyl, lipid, and peroxyl radicals.

PubMed Central

Coffey, M D; Cole, R A; Colles, S M; Chisolm, G M

1995-01-01

Mounting evidence supports current theories linking lipoprotein oxidation to atherosclerosis. We sought the cellular biochemical mechanism by which oxidized LDL inflicts cell injury. Inhibitors of candidate pathways of cell death were used to treat human fibroblast target cells exposed to oxidized LDL.. Ebselen, which degrades lipid hydroperoxides, inhibited oxidized LDL toxicity, consistent with our recent report that 7 beta-hydroperoxycholesterol (7 beta-OOH chol) is the major cytotoxin of oxidized LDL. Intracellular chelation of metal ions inhibited, while preloading cells with iron enhanced, toxicity, Inhibition of oxidized LDL and 7 beta-OOH chol toxicity by 2-keto-4-thiolmethyl butyric acid, a putative alkoxyl radical scavenger and by vitamin E, probucol and diphenylphenylenediamine, putative scavengers of peroxyl radicals was consistent with the involvement of these radicals in the lethal sequence. Cell death was thus postulated to occur due to lipid peroxidation via a sequence involving lipid hydroperoxide-induced, iron-mediated formation of alkoxyl, lipid, and peroxyl radicals. Pathways involving other reactive oxygen species, new protein synthesis, or altered cholesterol metabolism were considered less likely, since putative inhibitors failed to lessen toxicity. Understanding the mechanism of cell injury by oxidized LDL and its toxic moiety, 7 beta-OOH chol, may indicate specific interventions in the cell injury believed to accompany vascular lesion development. PMID:7560078

Mapping biological process relationships and disease perturbations within a pathway network.

PubMed

Stoney, Ruth; Robertson, David L; Nenadic, Goran; Schwartz, Jean-Marc

2018-01-01

Molecular interaction networks are routinely used to map the organization of cellular function. Edges represent interactions between genes, proteins, or metabolites. However, in living cells, molecular interactions are dynamic, necessitating context-dependent models. Contextual information can be integrated into molecular interaction networks through the inclusion of additional molecular data, but there are concerns about completeness and relevance of this data. We developed an approach for representing the organization of human cellular processes using pathways as the nodes in a network. Pathways represent spatial and temporal sets of context-dependent interactions, generating a high-level network when linked together, which incorporates contextual information without the need for molecular interaction data. Analysis of the pathway network revealed linked communities representing functional relationships, comparable to those found in molecular networks, including metabolism, signaling, immunity, and the cell cycle. We mapped a range of diseases onto this network and find that pathways associated with diseases tend to be functionally connected, highlighting the perturbed functions that result in disease phenotypes. We demonstrated that disease pathways cluster within the network. We then examined the distribution of cancer pathways and showed that cancer pathways tend to localize within the signaling, DNA processes and immune modules, although some cancer-associated nodes are found in other network regions. Altogether, we generated a high-confidence functional network, which avoids some of the shortcomings faced by conventional molecular models. Our representation provides an intuitive functional interpretation of cellular organization, which relies only on high-quality pathway and Gene Ontology data. The network is available at https://data.mendeley.com/datasets/3pbwkxjxg9/1.

MIMO: an efficient tool for molecular interaction maps overlap

PubMed Central

2013-01-01

Background Molecular pathways represent an ensemble of interactions occurring among molecules within the cell and between cells. The identification of similarities between molecular pathways across organisms and functions has a critical role in understanding complex biological processes. For the inference of such novel information, the comparison of molecular pathways requires to account for imperfect matches (flexibility) and to efficiently handle complex network topologies. To date, these characteristics are only partially available in tools designed to compare molecular interaction maps. Results Our approach MIMO (Molecular Interaction Maps Overlap) addresses the first problem by allowing the introduction of gaps and mismatches between query and template pathways and permits -when necessary- supervised queries incorporating a priori biological information. It then addresses the second issue by relying directly on the rich graph topology described in the Systems Biology Markup Language (SBML) standard, and uses multidigraphs to efficiently handle multiple queries on biological graph databases. The algorithm has been here successfully used to highlight the contact point between various human pathways in the Reactome database. Conclusions MIMO offers a flexible and efficient graph-matching tool for comparing complex biological pathways. PMID:23672344

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hiser, C.; Herdies, L.; McIntosh, L.

Higher plant mitochondria posses a cyanide-resistant, hydroxamate-sensitive alternative pathway of electron transport that does not conserve energy. Aging of potato tuber slices for 24 hours leads to the development of an alternative pathway capacity. We have shown that a monoclonal antibody raised against the alternative pathway terminal oxidase of Sauromatum guttatum crossreacts with a protein of similar size in aged potato slice mitochondria. This protein was partially purified and characterized by two-dimensional gel electrophoresis, and its relative levels parallel the rise in cyanide-resistant respiration. We are using a putative clone of the S. guttatum alternative oxidase gene to isolate themore » equivalent gene from potato and to examine its expression.« less

A review of selected candidate endophenotypes for depression.

PubMed

Goldstein, Brandon L; Klein, Daniel N

2014-07-01

Endophenotypes are proposed to occupy an intermediate position in the pathway between genotype and phenotype in genetically complex disorders such as depression. To be considered an endophenotype, a construct must meet a set of criteria proposed by Gottesman and Gould (2003). In this qualitative review, we summarize evidence for each criterion for several putative endophenotypes for depression: neuroticism, morning cortisol, frontal asymmetry of cortical electrical activity, reward learning, and biases of attention and memory. Our review indicates that while there is strong support for some depression endophenotypes, other putative endophenotypes lack data or have inconsistent findings for core criteria. Copyright © 2014 Elsevier Ltd. All rights reserved.

Expression patterns of ethylene biosynthesis genes from bananas during fruit ripening and in relationship with finger drop

PubMed Central

Hubert, Olivier; Mbéguié-A-Mbéguié, Didier

2012-01-01

Background and aims Banana finger drop is defined as dislodgement of individual fruits from the hand at the pedicel rupture area. For some banana varieties, this is a major feature of the ripening process, in addition to ethylene production and sugar metabolism. The few studies devoted to assessing the physiological and molecular basis of this process revealed (i) the similarity between this process and softening, (ii) the early onset of related molecular events, between the first and fourth day after ripening induction, and (iii) the putative involvement of ethylene as a regulatory factor. This study was conducted with the aim of identifying, through a candidate gene approach, a quality-related marker that could be used as a tool in breeding programmes. Here we examined the relationship between ripening ethylene biosynthesis (EB) and finger drop in order to gain further insight into the upstream regulatory steps of the banana finger drop process and to identify putative related candidate genes. Methods Postharvest ripening of green banana fruit was induced by acetylene treatment and fruit taken at 1–4 days after ripening induction, and total RNA extracted from the median area [control zone (CZ)] and the pedicel rupture area [drop zone (DZ)] of peel tissue. Then the expression patterns of EB genes (MaACO1, MaACO2, MaACS1, MaACS2, MaACS3 and MaACS4) were comparatively examined in CZ and DZ via real-time quantitative polymerase chain reaction. Principal results Differential expression of EB gene was observed in CZ and DZ during the postharvest period examined in this study. MaACO1, MaACS2 and MaACS1 were more highly induced in DZ than in the control, while a slight induction of the MaACS4 gene was observed. No marked differences between the two zones were observed for the MaACO2 gene. Conclusions The finger drop process enhanced EB gene expression including developmental- and ripening-induced genes (MaACO1), specific ripening-induced genes (MaACS1) and wound-induced genes (MaACS2). Thus, this process might be associated with a specific ethylene production in DZ of the pedicel area and the result of crosstalk between developmental, ripening and wound regulatory pathways. MaACO1, MaACS1, MaACS2, and to a lesser extent MaACS4 genes, which are more highly induced in DZ than in CZ, could be considered as putative candidates of the finger drop process. PMID:23267429

Haemocytes collected from experimentally infected Pacific oysters, Crassostrea gigas: Detection of ostreid herpesvirus 1 DNA, RNA, and proteins in relation with inhibition of apoptosis.

PubMed

Martenot, Claire; Gervais, Ophélie; Chollet, Bruno; Houssin, Maryline; Renault, Tristan

2017-01-01

Recent transcriptomic approaches focused on anti-viral immunity in molluscs lead to the assumption that the innate immune system, such as apoptosis, plays a crucial role against ostreid herpesvirus type 1 (OsHV-1), infecting Pacific cupped oyster, Crassostrea gigas. Apoptosis constitutes a major mechanism of anti-viral response by limiting viral spread and eliminating infected cells. In this way, an OsHV-1 challenge was performed and oysters were monitored at three times post injection to investigate viral infection and host response: 2h (early after viral injection in the adductor muscle), 24h (intermediate time), and 48h (just before first oyster mortality record). Virus infection, associated with high cumulative mortality rates (74% and 100%), was demonstrated in haemocytes by combining several detection techniques such as real-time PCR, real-time RT PCR, immunofluorescence assay, and transmission electron microscopy examination. High viral DNA amounts ranged from 5.46×104 to 3.68×105 DNA copies ng-1 of total DNA, were detected in dead oysters and an increase of viral transcripts was observed from 2, 24, and 48hpi for the five targeted OsHV-1 genes encoding three putative membrane proteins (ORFs 25, 41, and 72), a putative dUTPase (ORF 75), and a putative apoptosis inhibitor (ORF 87). Apoptosis was studied at molecular and cellular levels with an early marker (phosphatidyl-serine externalisation measured by flow cytometry and epifluorescence microscopy) and a later parameter (DNA fragmentation by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay (TUNEL)). The down-regulation of genes encoding proteins involved in the activation of the apoptotic pathway (TNF and caspase 3) and the up-regulation of genes encoding anti-apoptotic proteins (IAP-2, and Bcl-2) suggested an important anti-apoptosis phenomenon in haemocytes from OsHV-1 infected oysters at 24 and 48hpi. Additionally, more phosphatidyl-serines were externalized and more cells with DNA fragmentation were observed in haemocytes collected from artificial seawater injected oysters than in haemocytes collected from OsHV-1 infected oysters at 24 and 48hpi, suggesting an inhibition of the apoptotic process in presence of the virus. In conclusion, this study is the first to focus on C. gigas haemocytes, cells involved in the host immune defense, during an OsHV-1 challenge in controlled conditions by combining various and original approaches to investigate apoptosis at molecular and cellular levels.

Insights into the innate immunity of the Mediterranean mussel Mytilus galloprovincialis

PubMed Central

2011-01-01

Background Sessile bivalves of the genus Mytilus are suspension feeders relatively tolerant to a wide range of environmental changes, used as sentinels in ecotoxicological investigations and marketed worldwide as seafood. Mortality events caused by infective agents and parasites apparently occur less in mussels than in other bivalves but the molecular basis of such evidence is unknown. The arrangement of Mytibase, interactive catalogue of 7,112 transcripts of M. galloprovincialis, offered us the opportunity to look for gene sequences relevant to the host defences, in particular the innate immunity related genes. Results We have explored and described the Mytibase sequence clusters and singletons having a putative role in recognition, intracellular signalling, and neutralization of potential pathogens in M. galloprovincialis. Automatically assisted searches of protein signatures and manually cured sequence analysis confirmed the molecular diversity of recognition/effector molecules such as the antimicrobial peptides and many carbohydrate binding proteins. Molecular motifs identifying complement C1q, C-type lectins and fibrinogen-like transcripts emerged as the most abundant in the Mytibase collection whereas, conversely, sequence motifs denoting the regulatory cytokine MIF and cytokine-related transcripts represent singular and unexpected findings. Using a cross-search strategy, 1,820 putatively immune-related sequences were selected to design oligonucleotide probes and define a species-specific Immunochip (DNA microarray). The Immunochip performance was tested with hemolymph RNAs from mussels injected with Vibrio splendidus at 3 and 48 hours post-treatment. A total of 143 and 262 differentially expressed genes exemplify the early and late hemocyte response of the Vibrio-challenged mussels, respectively, with AMP trends confirmed by qPCR and clear modulation of interrelated signalling pathways. Conclusions The Mytibase collection is rich in gene transcripts modulated in response to antigenic stimuli and represents an interesting window for looking at the mussel immunome (transcriptomes mediating the mussel response to non-self or abnormal antigens). On this basis, we have defined a new microarray platform, a mussel Immunochip, as a flexible tool for the experimental validation of immune-candidate sequences, and tested its performance on Vibrio-activated mussel hemocytes. The microarray platform and related expression data can be regarded as a step forward in the study of the adaptive response of the Mytilus species to an evolving microbial world. PMID:21269501

Rapid Countermeasure Discovery against Francisella tularensis Based on a Metabolic Network Reconstruction

DTIC Science & Technology

2013-05-21

minimum inhibitory concentrations and mammalian cell cytotoxicities. The most promising compound had a low molecular weight, was non-toxic, and abolished... molecular weight, was non-toxic, and abolished bacterial growth at 13 mM, with putative activity against pantetheine-phosphate adenylyltransferase, an...time period. Metabolic genome-scale models of bacteria have provided a computational framework for in silico simulations to evaluate how metabolic

Cloning and characterization of the ONAC106 gene from Oryza sativa cultivar Kuku Belang

NASA Astrophysics Data System (ADS)

Basri, Khairunnisa; Sukiran, Noor Liyana; Zainal, Zamri

2016-11-01

Plants possess different mechanisms in stress response, where induction of stress-responsive genes provides tolerance to unfavorable conditions. Stress-responsive genes are characterized for functional and regulatory genes that help in overcoming stress by molecular, biochemical and morphological adaptations. NAC transcription factors are one of the regulatory proteins that involved in stress signaling pathway. A putative NAC transcription factor, ONAC016 was identified from drought transcriptomic data. Our data suggested that ONAC106 was induced by drought, but its function in abiotic stress is still unclear. In silico analysis of ONAC106 showed that this gene encodes 334 amino acids, and its protein consists of NAM (No Apical Meristem) domain. The orthologue of ONAC106 was present in several Poaceae family members, suggesting that ONAC106 is unique to monocot plants only. We found that ONAC106 was induced by salt and cold stresses, indicating that this gene involves in abiotic stress response. In addition, we also found that ONAC106 might function in defense response to pathogen invasion. The ABRE (Abscisic Acid Regulatory Element) cis-element was identified in the promoter region of ONAC106, suggesting that it may involve in the abscisic acid (ABA)-dependent signaling pathway. Based on this preliminary result, we hypothesize that ONAC106 may play a role in abiotic stress response by regulating ABA-responsive genes.

iTRAQ-based quantitative protein expression profiling and MRM verification of markers in type 2 diabetes.

PubMed

Kaur, Prabhjit; Rizk, Nasser M; Ibrahim, Sereen; Younes, Noura; Uppal, Arushi; Dennis, Kevin; Karve, Tejaswita; Blakeslee, Kenneth; Kwagyan, John; Zirie, Mahmoud; Ressom, Habtom W; Cheema, Amrita K

2012-11-02

The pathogenesis of Type 2 diabetes mellitus (T2DM) is complex owing to molecular heterogeneity in the afflicted population. Current diagnostic methods rely on blood glucose measurements, which are noninformative with respect to progression of the disease to other associated pathologies. Thus, predicting the risk and development of T2DM-related complications, such as cardiovascular disease, remains a major challenge. We have used a combination of quantitative methods for characterization of circulating serum biomarkers of T2DM using a cohort of nondiabetic control subjects (n = 76) and patients diagnosed with T2DM (n = 106). In this case-control study, the samples were randomly divided as training and validation data sets. In the first step, iTRAQ (isobaric tagging for relative and absolute quantification) based protein expression profiling was performed for identification of proteins displaying a significant differential expression in the two study groups. Five of these protein markers were selected for validation using multiple reaction-monitoring mass spectrometry (MRM-MS) and further confirmed with Western blot and QPCR analysis. Functional pathway analysis identified perturbations in lipid and small molecule metabolism as well as pathways that lead to disruption of glucose homeostasis and blood coagulation. These putative biomarkers may be clinically useful for subset stratification of T2DM patients as well as for the development of novel therapeutics targeting the specific pathology.

Hydrogen-rich water attenuates amyloid β-induced cytotoxicity through upregulation of Sirt1-FoxO3a by stimulation of AMP-activated protein kinase in SK-N-MC cells.

PubMed

Lin, Chih-Li; Huang, Wen-Nung; Li, Hsin-Hua; Huang, Chien-Ning; Hsieh, Sam; Lai, Copper; Lu, Fung-Jou

2015-10-05

Amyloid β (Aβ) peptides are identified in cause of neurodegenerative diseases such as Alzheimer's disease (AD). Previous evidence suggests Aβ-induced neurotoxicity is linked to the stimulation of reactive oxygen species (ROS) production. The accumulation of Aβ-induced ROS leads to increased mitochondrial dysfunction and triggers apoptotic cell death. This suggests antioxidant therapies may be beneficial for preventing ROS-related diseases such as AD. Recently, hydrogen-rich water (HRW) has been proven effective in treating oxidative stress-induced disorders because of its ROS-scavenging abilities. However, the precise molecular mechanisms whereby HRW prevents neuronal death are still unclear. In the present study, we evaluated the putative pathways by which HRW protects against Aβ-induced cytotoxicity. Our results indicated that HRW directly counteracts oxidative damage by neutralizing excessive ROS, leading to the alleviation of Aβ-induced cell death. In addition, HRW also stimulated AMP-activated protein kinase (AMPK) in a sirtuin 1 (Sirt1)-dependent pathway, which upregulates forkhead box protein O3a (FoxO3a) downstream antioxidant response and diminishes Aβ-induced mitochondrial potential loss and oxidative stress. Taken together, our findings suggest that HRW may have potential therapeutic value to inhibit Aβ-induced neurotoxicity. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Stem Cell Fate Determination during Development and Regeneration of Ectodermal Organs

PubMed Central

Jiménez-Rojo, Lucía; Granchi, Zoraide; Graf, Daniel; Mitsiadis, Thimios A.

2012-01-01

The development of ectoderm-derived appendages results in a large variety of highly specialized organs such as hair follicles, mammary glands, salivary glands, and teeth. Despite varying in number, shape, and function, all these ectodermal organs develop through continuous and reciprocal epithelial–mesenchymal interactions, sharing common morphological and molecular features especially during their embryonic development. Diseases such as ectodermal dysplasias can affect simultaneously these organs, suggesting that they may arise from common multipotent precursors residing in the embryonic ectoderm. During embryogenesis, these putative ectodermal stem cells may adopt different fates and consequently be able to generate a variety of tissue-specific stem cells, which are the sources for the various cell lineages that form the diverse organs. The specification of those common epithelial precursors, as well as their further lineage commitment to tissue-specific stem cells, might be controlled by specific signals. It has been well documented that Notch, Wnt, bone morphogenetic protein, and fibroblast growth factor signaling pathways regulate cell fate decisions during the various stages of ectodermal organ development. However, the in vivo spatial and temporal dynamics of these signaling pathways are not yet well understood. Improving the current knowledge on the mechanisms involved in stem cell fate determination during organogenesis and homeostasis of ectodermal organs is crucial to develop effective stem cell-based therapies in order to regenerate or replace pathological and damaged tissues. PMID:22539926

TRACING CO-REGULATORY NETWORK DYNAMICS IN NOISY, SINGLE-CELL TRANSCRIPTOME TRAJECTORIES.

PubMed

Cordero, Pablo; Stuart, Joshua M

2017-01-01

The availability of gene expression data at the single cell level makes it possible to probe the molecular underpinnings of complex biological processes such as differentiation and oncogenesis. Promising new methods have emerged for reconstructing a progression 'trajectory' from static single-cell transcriptome measurements. However, it remains unclear how to adequately model the appreciable level of noise in these data to elucidate gene regulatory network rewiring. Here, we present a framework called Single Cell Inference of MorphIng Trajectories and their Associated Regulation (SCIMITAR) that infers progressions from static single-cell transcriptomes by employing a continuous parametrization of Gaussian mixtures in high-dimensional curves. SCIMITAR yields rich models from the data that highlight genes with expression and co-expression patterns that are associated with the inferred progression. Further, SCIMITAR extracts regulatory states from the implicated trajectory-evolvingco-expression networks. We benchmark the method on simulated data to show that it yields accurate cell ordering and gene network inferences. Applied to the interpretation of a single-cell human fetal neuron dataset, SCIMITAR finds progression-associated genes in cornerstone neural differentiation pathways missed by standard differential expression tests. Finally, by leveraging the rewiring of gene-gene co-expression relations across the progression, the method reveals the rise and fall of co-regulatory states and trajectory-dependent gene modules. These analyses implicate new transcription factors in neural differentiation including putative co-factors for the multi-functional NFAT pathway.

Stem Cell Fate Determination during Development and Regeneration of Ectodermal Organs.

PubMed

Jiménez-Rojo, Lucía; Granchi, Zoraide; Graf, Daniel; Mitsiadis, Thimios A

2012-01-01

The development of ectoderm-derived appendages results in a large variety of highly specialized organs such as hair follicles, mammary glands, salivary glands, and teeth. Despite varying in number, shape, and function, all these ectodermal organs develop through continuous and reciprocal epithelial-mesenchymal interactions, sharing common morphological and molecular features especially during their embryonic development. Diseases such as ectodermal dysplasias can affect simultaneously these organs, suggesting that they may arise from common multipotent precursors residing in the embryonic ectoderm. During embryogenesis, these putative ectodermal stem cells may adopt different fates and consequently be able to generate a variety of tissue-specific stem cells, which are the sources for the various cell lineages that form the diverse organs. The specification of those common epithelial precursors, as well as their further lineage commitment to tissue-specific stem cells, might be controlled by specific signals. It has been well documented that Notch, Wnt, bone morphogenetic protein, and fibroblast growth factor signaling pathways regulate cell fate decisions during the various stages of ectodermal organ development. However, the in vivo spatial and temporal dynamics of these signaling pathways are not yet well understood. Improving the current knowledge on the mechanisms involved in stem cell fate determination during organogenesis and homeostasis of ectodermal organs is crucial to develop effective stem cell-based therapies in order to regenerate or replace pathological and damaged tissues.

Neuroprotection by Curcumin in Ischemic Brain Injury Involves the Akt/Nrf2 Pathway

PubMed Central

Wu, Jingxian; Li, Qiong; Wang, Xiaoyan; Yu, Shanshan; Li, Lan; Wu, Xuemei; Chen, Yanlin; Zhao, Jing; Zhao, Yong

2013-01-01

Oxidative damage plays a critical role in many diseases of the central nervous system. This study was conducted to determine the molecular mechanisms involved in the putative anti-oxidative effects of curcumin against experimental stroke. Oxygen and glucose deprivation/reoxygenation (OGD/R) was used to mimic ischemic insult in primary cultured cortical neurons. A rapid increase in the intracellular expression of NAD(P)H: quinone oxidoreductase1 (NQO1) induced by OGD was counteracted by curcumin post-treatment, which paralleled attenuated cell injury. The reduction of phosphorylation Akt induced by OGD was restored by curcumin. Consequently, NQO1 expression and the binding activity of nuclear factor-erythroid 2-related factor 2 (Nrf2) to antioxidant response element (ARE) were increased. LY294002 blocked the increase in phospho-Akt evoked by curcumin and abolished the associated protective effect. Adult male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion for 60 minutes. Curcumin administration significantly reduced infarct size. Curcumin also markedly reduced oxidative stress levels in middle cerebral artery occlusion (MCAO) rats; hence, these effects were all suppressed by LY294002. Taken together, these findings provide evidence that curcumin protects neurons against ischemic injury, and this neuroprotective effect involves the Akt/Nrf2 pathway. In addition, Nrf2 is involved in the neuroprotective effects of curcumin against oxidative damage. PMID:23555802

Acetylome Profiling Reveals Extensive Lysine Acetylation of the Fatty Acid Metabolism Pathway in the Diatom Phaeodactylum tricornutum.

PubMed

Chen, Zhuo; Luo, Ling; Chen, Runfa; Hu, Hanhua; Pan, Yufang; Jiang, Haibo; Wan, Xia; Jin, Hu; Gong, Yangmin

2018-03-01

N ε -lysine acetylation represents a highly dynamic and reversibly regulated post-translational modification widespread in almost all organisms, and plays important roles for regulation of protein function in diverse metabolic pathways. However, little is known about the role of lysine acetylation in photosynthetic eukaryotic microalgae. We integrated proteomic approaches to comprehensively characterize the lysine acetylome in the model diatom Phaeodactylum tricornutum In total, 2324 acetylation sites from 1220 acetylated proteins were identified, representing the largest data set of the lysine acetylome in plants to date. Almost all enzymes involved in fatty acid synthesis were found to be lysine acetylated. Six putative lysine acetylation sites were identified in a plastid-localized long-chain acyl-CoA synthetase. Site-directed mutagenesis and site-specific incorporation of N-acetyllysine in acyl-CoA synthetase show that acetylation at K407 and K425 increases its enzyme activity. Moreover, the nonenzymatically catalyzed overall hyperacetylation of acyl-CoA synthetase by acetyl-phosphate can be effectively deacetylated and reversed by a sirtuin-type NAD + -dependent deacetylase with subcellular localization of both the plastid and nucleus in Phaeodactylum This work indicates the regulation of acyl-CoA synthetase activity by site-specific lysine acetylation and highlights the potential regulation of fatty acid metabolism by lysine actetylation in the plastid of the diatom Phaeodactylum . © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Autoregulation of glial cell line-derived neurotrophic factor expression: implications for the long-lasting actions of the anti-addiction drug, Ibogaine.

PubMed

He, Dao-Yao; Ron, Dorit

2006-11-01

We recently showed that the up-regulation of the glial cell line-derived neurotrophic factor (GDNF) pathway in the midbrain, is the molecular mechanism by which the putative anti-addiction drug Ibogaine mediates its desirable action of reducing alcohol consumption. Human reports and studies in rodents have shown that a single administration of Ibogaine results in a long-lasting reduction of drug craving (humans) and drug and alcohol intake (rodents). Here we determine whether, and how, Ibogaine exerts its long-lasting actions on GDNF expression and signaling. Using the dopaminergic-like SHSY5Y cell line as a culture model, we observed that short-term Ibogaine exposure results in a sustained increase in GDNF expression that is mediated via the induction of a long-lasting autoregulatory cycle by which GDNF positively regulates its own expression. We show that the initial exposure of cells to Ibogaine or GDNF results in an increase in GDNF mRNA, leading to protein expression and to the corresponding activation of the GDNF signaling pathway. This, in turn, leads to a further increase in the mRNA level of the growth factor. The identification of a GDNF-mediated, autoregulatory long-lasting feedback loop could have important implications for GDNF's potential value as a treatment for addiction and neurodegenerative diseases.

Unraveling the molecular mechanisms of nitrogenase conformational protection against oxygen in diazotrophic bacteria.

PubMed

Lery, Letícia M S; Bitar, Mainá; Costa, Mauricio G S; Rössle, Shaila C S; Bisch, Paulo M

2010-12-22

G. diazotrophicus and A. vinelandii are aerobic nitrogen-fixing bacteria. Although oxygen is essential for the survival of these organisms, it irreversibly inhibits nitrogenase, the complex responsible for nitrogen fixation. Both microorganisms deal with this paradox through compensatory mechanisms. In A. vinelandii a conformational protection mechanism occurs through the interaction between the nitrogenase complex and the FeSII protein. Previous studies suggested the existence of a similar system in G. diazotrophicus, but the putative protein involved was not yet described. This study intends to identify the protein coding gene in the recently sequenced genome of G. diazotrophicus and also provide detailed structural information of nitrogenase conformational protection in both organisms. Genomic analysis of G. diazotrophicus sequences revealed a protein coding ORF (Gdia0615) enclosing a conserved "fer2" domain, typical of the ferredoxin family and found in A. vinelandii FeSII. Comparative models of both FeSII and Gdia0615 disclosed a conserved beta-grasp fold. Cysteine residues that coordinate the 2[Fe-S] cluster are in conserved positions towards the metallocluster. Analysis of solvent accessible residues and electrostatic surfaces unveiled an hydrophobic dimerization interface. Dimers assembled by molecular docking presented a stable behaviour and a proper accommodation of regions possibly involved in binding of FeSII to nitrogenase throughout molecular dynamics simulations in aqueous solution. Molecular modeling of the nitrogenase complex of G. diazotrophicus was performed and models were compared to the crystal structure of A. vinelandii nitrogenase. Docking experiments of FeSII and Gdia0615 with its corresponding nitrogenase complex pointed out in both systems a putative binding site presenting shape and charge complementarities at the Fe-protein/MoFe-protein complex interface. The identification of the putative FeSII coding gene in G. diazotrophicus genome represents a large step towards the understanding of the conformational protection mechanism of nitrogenase against oxygen. In addition, this is the first study regarding the structural complementarities of FeSII-nitrogenase interactions in diazotrophic bacteria. The combination of bioinformatic tools for genome analysis, comparative protein modeling, docking calculations and molecular dynamics provided a powerful strategy for the elucidation of molecular mechanisms and structural features of FeSII-nitrogenase interaction.

High-throughput transcriptome sequencing analysis provides preliminary insights into the biotransformation mechanism of Rhodopseudomonas palustris treated with alpha-rhamnetin-3-rhamnoside.

PubMed

Bi, Lei; Guan, Chun-jie; Yang, Guan-e; Yang, Fei; Yan, Hong-yu; Li, Qing-shan

2016-04-01

The purple photosynthetic bacterium Rhodopseudomonas palustris has been widely applied to enhance the therapeutic effects of traditional Chinese medicine using novel biotransformation technology. However, comprehensive studies of the R. palustris biotransformation mechanism are rare. Therefore, investigation of the expression patterns of genes involved in metabolic pathways that are active during the biotransformation process is essential to elucidate this complicated mechanism. To promote further study of the biotransformation of R. palustris, we assembled all R. palustris transcripts using Trinity software and performed differential expression analysis of the resulting unigenes. A total of 9725, 7341 and 10,963 unigenes were obtained by assembling the alpha-rhamnetin-3-rhamnoside-treated R. palustris (RPB) reads, control R. palustris (RPS) reads and combined RPB&RPS reads, respectively. A total of 9971 unigenes assembled from the RPB&RPS reads were mapped to the nr, nt, Swiss-Prot, Gene Ontology (GO), Clusters of Orthologous Groups (COGs) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (E-value <0.00001) databases using BLAST software. A total of 3360 unique differentially expressed genes (DEGs) in RPB versus RPS were identified, among which 922 unigenes were up-regulated and 2438 were down-regulated. The unigenes were mapped to the KEGG database, resulting in the identification of 7676 pathways among all annotated unigenes and 2586 pathways among the DEGs. Some sets of functional unigenes annotated to important metabolic pathways and environmental information processing were differentially expressed between the RPS and RPB samples, including those involved in energy metabolism (18.4% of total DEGs), carbohydrate metabolism (36.0% of total DEGs), ABC transport (6.0% of total DEGs), the two-component system (8.6% of total DEGs), cell motility (4.3% of total DEGs) and the cell cycle (1.5% of total DEGs). We also identified 19 transcripts annotated as hydrolytic enzymes and other enzymes involved in ARR catabolism in R. palustris. We present the first comparative transcriptome profiles of RPB and RPS samples to facilitate elucidation of the molecular mechanism of biotransformation in R. palustris. Furthermore, we propose two putative ARR biotransformation mechanisms in R. palustris. These analytical results represent a useful genomic resource for in-depth research into the molecular basis of biotransformation and genetic modification in R. palustris. Copyright © 2016 Elsevier GmbH. All rights reserved.

Deep sequencing and in silico analysis of small RNA library reveals novel miRNA from leaf Persicaria minor transcriptome.

PubMed

Samad, Abdul Fatah A; Nazaruddin, Nazaruddin; Murad, Abdul Munir Abdul; Jani, Jaeyres; Zainal, Zamri; Ismail, Ismanizan

2018-03-01

In current era, majority of microRNA (miRNA) are being discovered through computational approaches which are more confined towards model plants. Here, for the first time, we have described the identification and characterization of novel miRNA in a non-model plant, Persicaria minor ( P . minor ) using computational approach. Unannotated sequences from deep sequencing were analyzed based on previous well-established parameters. Around 24 putative novel miRNAs were identified from 6,417,780 reads of the unannotated sequence which represented 11 unique putative miRNA sequences. PsRobot target prediction tool was deployed to identify the target transcripts of putative novel miRNAs. Most of the predicted target transcripts (mRNAs) were known to be involved in plant development and stress responses. Gene ontology showed that majority of the putative novel miRNA targets involved in cellular component (69.07%), followed by molecular function (30.08%) and biological process (0.85%). Out of 11 unique putative miRNAs, 7 miRNAs were validated through semi-quantitative PCR. These novel miRNAs discoveries in P . minor may develop and update the current public miRNA database.

Reporter Gene-Facilitated Detection of Compounds in Arabidopsis Leaf Extracts that Activate the Karrikin Signaling Pathway.

PubMed

Sun, Yueming K; Flematti, Gavin R; Smith, Steven M; Waters, Mark T

2016-01-01

Karrikins are potent germination stimulants generated by the combustion of plant matter. Treatment of Arabidopsis with karrikins triggers a signaling process that is dependent upon a putative receptor protein KARRIKIN INSENSITIVE 2 (KAI2). KAI2 is a homolog of DWARF 14 (D14), the receptor for endogenous strigolactone hormones. Genetic analyses suggest that KAI2 also perceives endogenous signal(s) that are not strigolactones. Activation of KAI2 by addition of karrikins to Arabidopsis plants induces expression of transcripts including D14-LIKE 2 ( DLK2 ). We constructed the synthetic reporter gene DLK2 : LUC in Arabidopsis , which comprises the firefly luciferase gene ( LUC ) driven by the DLK2 promoter. Here we describe a luminescence-based reporter assay with Arabidopsis seeds to detect chemical signals that can activate the KAI2 signaling pathway. We demonstrate that the DLK2 : LUC assay can selectively and sensitively detect karrikins and a functionally similar synthetic strigolactone analog. Crucially we show that crude extracts from Arabidopsis leaves can also activate DLK2 : LUC in a KAI2-dependent manner. Our work provides the first direct evidence for the existence of endogenous chemical signals that can activate the KAI2-mediated signaling pathway in Arabidopsis . This sensitive reporter system can now be used for the bioassay-guided purification and identification of putative endogenous KAI2 ligands or their precursors, and endogenous compounds that might modulate the KAI2 signaling pathway.

Reporter Gene-Facilitated Detection of Compounds in Arabidopsis Leaf Extracts that Activate the Karrikin Signaling Pathway

PubMed Central

Sun, Yueming K.; Flematti, Gavin R.; Smith, Steven M.; Waters, Mark T.

2016-01-01

Karrikins are potent germination stimulants generated by the combustion of plant matter. Treatment of Arabidopsis with karrikins triggers a signaling process that is dependent upon a putative receptor protein KARRIKIN INSENSITIVE 2 (KAI2). KAI2 is a homolog of DWARF 14 (D14), the receptor for endogenous strigolactone hormones. Genetic analyses suggest that KAI2 also perceives endogenous signal(s) that are not strigolactones. Activation of KAI2 by addition of karrikins to Arabidopsis plants induces expression of transcripts including D14-LIKE 2 (DLK2). We constructed the synthetic reporter gene DLK2:LUC in Arabidopsis, which comprises the firefly luciferase gene (LUC) driven by the DLK2 promoter. Here we describe a luminescence-based reporter assay with Arabidopsis seeds to detect chemical signals that can activate the KAI2 signaling pathway. We demonstrate that the DLK2:LUC assay can selectively and sensitively detect karrikins and a functionally similar synthetic strigolactone analog. Crucially we show that crude extracts from Arabidopsis leaves can also activate DLK2:LUC in a KAI2-dependent manner. Our work provides the first direct evidence for the existence of endogenous chemical signals that can activate the KAI2-mediated signaling pathway in Arabidopsis. This sensitive reporter system can now be used for the bioassay-guided purification and identification of putative endogenous KAI2 ligands or their precursors, and endogenous compounds that might modulate the KAI2 signaling pathway. PMID:27994609

VIRTUAL LIVER: AN IN SILICO FRAMEWORK FOR ANALYZING CHEMICAL-INDUCED HEPATOTOXICITY

EPA Science Inventory

The US EPA Virtual Liver (v-LiverTM) is an in silico framework for the dose-dependent perturbation of normal hepatic functions by chemicals using in vitro data. The framework consists of a computable knowledge-base (KB) to infer putative pathways in hepatotoxicity and a cellular...

Derivation and evaluation of putative adverse outcome pathways for the effects of cyclooxygenase inhibitors on reproductive processes in female fish

EPA Science Inventory

Cyclooxygenase (COX) inhibition is of concern in fish because COX inhibitors (e.g., ibuprofen) are ubiquitous in aquatic systems/fish tissues, and can disrupt synthesis of prostaglandins that modulate a variety of essential biological functions including reproduction. High conten...

Enhanced resistance to soybean cyst nematode Heterodera glycines in transgenic soybean by silencing putative CLE receptors

USDA-ARS?s Scientific Manuscript database

CLE peptides are small extracellular proteins important in regulating plant meristematic activity through the CLE-receptor kinase-WOX signaling module. Stem cell pools in the SAM (shoot apical meristem), RAM (root apical meristem), and vascular cambium are tightly controlled by CLE signaling pathway...

Functional comparison of three transformer gene introns regulating conditional female lethality

USDA-ARS?s Scientific Manuscript database

The trasformer gene plays a critical role in the sex determination pathways of many insects. We cloned two transformer gene introns from Anastrepha suspensa, the Caribbean fruit fly. These introns have sequences that putatively have a role in sex-specific splicing patterns that affect sex determinat...

The Anisakis Transcriptome Provides a Resource for Fundamental and Applied Studies on Allergy-Causing Parasites.

PubMed

Baird, Fiona J; Su, Xiaopei; Aibinu, Ibukun; Nolan, Matthew J; Sugiyama, Hiromu; Otranto, Domenico; Lopata, Andreas L; Cantacessi, Cinzia

2016-07-01

Food-borne nematodes of the genus Anisakis are responsible for a wide range of illnesses (= anisakiasis), from self-limiting gastrointestinal forms to severe systemic allergic reactions, which are often misdiagnosed and under-reported. In order to enhance and refine current diagnostic tools for anisakiasis, knowledge of the whole spectrum of parasite molecules transcribed and expressed by this parasite, including those acting as potential allergens, is necessary. In this study, we employ high-throughput (Illumina) sequencing and bioinformatics to characterise the transcriptomes of two Anisakis species, A. simplex and A. pegreffii, and utilize this resource to compile lists of potential allergens from these parasites. A total of ~65,000,000 reads were generated from cDNA libraries for each species, and assembled into ~34,000 transcripts (= Unigenes); ~18,000 peptides were predicted from each cDNA library and classified based on homology searches, protein motifs and gene ontology and biological pathway mapping. Using comparative analyses with sequence data available in public databases, 36 (A. simplex) and 29 (A. pegreffii) putative allergens were identified, including sequences encoding 'novel' Anisakis allergenic proteins (i.e. cyclophilins and ABA-1 domain containing proteins). This study represents a first step towards providing the research community with a curated dataset to use as a molecular resource for future investigations of the biology of Anisakis, including molecules putatively acting as allergens, using functional genomics, proteomics and immunological tools. Ultimately, an improved knowledge of the biological functions of these molecules in the parasite, as well as of their immunogenic properties, will assist the development of comprehensive, reliable and robust diagnostic tools.

Novel insights into the response of Atlantic salmon (Salmo salar) to Piscirickettsia salmonis: Interplay of coding genes and lncRNAs during bacterial infection.

PubMed

Valenzuela-Miranda, Diego; Gallardo-Escárate, Cristian

2016-12-01

Despite the high prevalence and impact to Chilean salmon aquaculture of the intracellular bacterium Piscirickettsia salmonis, the molecular underpinnings of host-pathogen interactions remain unclear. Herein, the interplay of coding and non-coding transcripts has been proposed as a key mechanism involved in immune response. Therefore, the aim of this study was to evidence how coding and non-coding transcripts are modulated during the infection process of Atlantic salmon with P. salmonis. For this, RNA-seq was conducted in brain, spleen, and head kidney samples, revealing different transcriptional profiles according to bacterial load. Additionally, while most of the regulated genes annotated for diverse biological processes during infection, a common response associated with clathrin-mediated endocytosis and iron homeostasis was present in all tissues. Interestingly, while endocytosis-promoting factors and clathrin inductions were upregulated, endocytic receptors were mainly downregulated. Furthermore, the regulation of genes related to iron homeostasis suggested an intracellular accumulation of iron, a process in which heme biosynthesis/degradation pathways might play an important role. Regarding the non-coding response, 918 putative long non-coding RNAs were identified, where 425 were newly characterized for S. salar. Finally, co-localization and co-expression analyses revealed a strong correlation between the modulations of long non-coding RNAs and genes associated with endocytosis and iron homeostasis. These results represent the first comprehensive study of putative interplaying mechanisms of coding and non-coding RNAs during bacterial infection in salmonids. Copyright © 2016 Elsevier Ltd. All rights reserved.

NemaPath: online exploration of KEGG-based metabolic pathways for nematodes

PubMed Central

Wylie, Todd; Martin, John; Abubucker, Sahar; Yin, Yong; Messina, David; Wang, Zhengyuan; McCarter, James P; Mitreva, Makedonka

2008-01-01

Background Nematode.net is a web-accessible resource for investigating gene sequences from parasitic and free-living nematode genomes. Beyond the well-characterized model nematode C. elegans, over 500,000 expressed sequence tags (ESTs) and nearly 600,000 genome survey sequences (GSSs) have been generated from 36 nematode species as part of the Parasitic Nematode Genomics Program undertaken by the Genome Center at Washington University School of Medicine. However, these sequencing data are not present in most publicly available protein databases, which only include sequences in Swiss-Prot. Swiss-Prot, in turn, relies on GenBank/Embl/DDJP for predicted proteins from complete genomes or full-length proteins. Description Here we present the NemaPath pathway server, a web-based pathway-level visualization tool for navigating putative metabolic pathways for over 30 nematode species, including 27 parasites. The NemaPath approach consists of two parts: 1) a backend tool to align and evaluate nematode genomic sequences (curated EST contigs) against the annotated Kyoto Encyclopedia of Genes and Genomes (KEGG) protein database; 2) a web viewing application that displays annotated KEGG pathway maps based on desired confidence levels of primary sequence similarity as defined by a user. NemaPath also provides cross-referenced access to nematode genome information provided by other tools available on Nematode.net, including: detailed NemaGene EST cluster information; putative translations; GBrowse EST cluster views; links from nematode data to external databases for corresponding synonymous C. elegans counterparts, subject matches in KEGG's gene database, and also KEGG Ontology (KO) identification. Conclusion The NemaPath server hosts metabolic pathway mappings for 30 nematode species and is available on the World Wide Web at . The nematode source sequences used for the metabolic pathway mappings are available via FTP , as provided by the Genome Center at Washington University School of Medicine. PMID:18983679

Integration of genetic, genomic and transcriptomic information identifies putative regulators of adventitious root formation in Populus

DOE PAGES

Ribeiro, Cintia L.; Silva, Cynthia M.; Drost, Derek R.; ...

2016-03-16

In this study, adventitious roots (AR) develop from tissues other than the primary root, in a process physiologically regulated by phytohormones. Adventitious roots provide structural support and contribute to water and nutrient absorption, and are critical for commercial vegetative propagation of several crops. Here we quantified the number of AR, root architectural traits and root biomass in cuttings from a pseudo-backcross population of Populus deltoides and Populus trichocarpa. Quantitative trait loci (QTL) mapping and whole-transcriptome analysis of individuals with alternative QTL alleles for AR number were used to identify putative regulators of AR development. As a result, parental individuals andmore » progeny showed extensive segregation for AR developmental traits. Quantitative trait loci for number of AR mapped consistently in the same interval of linkage group (LG) II and LG XIV, explaining 7–10 % of the phenotypic variation. A time series transcriptome analysis identified 26,121 genes differentially expressed during AR development, particularly during the first 24 h after cuttings were harvested. Of those, 1929 genes were differentially regulated between individuals carrying alternative alleles for the two QTL for number of AR, in one or more time point. Eighty-one of these genes were physically located within the QTL intervals for number of AR, including putative homologs of the Arabidopsis genes SUPERROOT2 (SUR2) and TRYPTOPHAN SYNTHASE ALPHA CHAIN (TSA1), both of which are involved in the auxin indole-3-acetic acid (IAA) biosynthesis pathway. In conclusion, this study suggests the involvement of two genes of the tryptophan-dependent auxin biosynthesis pathway, SUR2 and TSA1, in the regulation of a critical trait for the clonal propagation of woody species. A possible model for this regulation is that poplar individuals that have poor AR formation synthesize auxin indole-3-acetic acid (IAA) primarily through the tryptophan (Trp) pathway. Much of the Trp pathway flux appears to be directed to the synthesis of indole glucosinolates (IG), as suggested by the over-expression of SUR2. Individuals that are efficient in AR formation may utilize alternative (non-Trp) pathways to synthesize IAA, based on the observation that they down-regulate the expression of TSA1, one of the critical steps in the synthesis of tryptophan.« less

Altered Placental Tryptophan Metabolism: A Crucial Molecular Pathway for the Fetal Programming of Neurodevelopmental Disorders

DTIC Science & Technology

2014-07-01

Molecular Pathway for the Fetal Programming of Neurodevelopmental Disorders PRINCIPAL INVESTIGATOR: Alexandre Bonnin, PhD CONTRACTING...Fetal Programming of Neurodevelopmental Disorders 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Alexandre Bonnin, PhD; Betty...metabolism by maternal inflammation during early gestation constitutes a new molecular pathway for the fetal programming of neurodevelopmental

Serrated colorectal cancer: Molecular classification, prognosis, and response to chemotherapy

PubMed Central

Murcia, Oscar; Juárez, Miriam; Hernández-Illán, Eva; Egoavil, Cecilia; Giner-Calabuig, Mar; Rodríguez-Soler, María; Jover, Rodrigo

2016-01-01

Molecular advances support the existence of an alternative pathway of colorectal carcinogenesis that is based on the hypermethylation of specific DNA regions that silences tumor suppressor genes. This alternative pathway has been called the serrated pathway due to the serrated appearance of tumors in histological analysis. New classifications for colorectal cancer (CRC) were proposed recently based on genetic profiles that show four types of molecular alterations: BRAF gene mutations, KRAS gene mutations, microsatellite instability, and hypermethylation of CpG islands. This review summarizes what is known about the serrated pathway of CRC, including CRC molecular and clinical features, prognosis, and response to chemotherapy. PMID:27053844

Metabolic Environments and Genomic Features Associated with Pathogenic and Mutualistic Interactions between Bacteria and Plants is accepted for publication in MPMI

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karpinets, Tatiana V; Park, Byung H; Syed, Mustafa H

Most bacterial symbionts of plants are phenotypically characterized by their parasitic or matualistic relationship with the host; however, the genomic characteristics that likely discriminate mutualistic symbionts from pathogens of plants are poorly understood. This study comparatively analyzed the genomes of 54 plant-symbiontic bacteria, 27 mutualists and 27 pathogens, to discover genomic determinants of their parasitic and mutualistic nature in terms of protein family domains, KEGG orthologous groups, metabolic pathways and families of carbohydrate-active enzymes (CAZymes). We further used all bacteria with sequenced genomesl, published microarrays and transcriptomics experimental datasets, and literature to validate and to explore results of the comparison.more » The analysis revealed that genomes of mutualists are larger in size and higher in GC content and encode greater molecular, functional and metabolic diversity than the investigated genomes of pathogens. This enriched molecular and functional enzyme diversity included constructive biosynthetic signatures of CAZymes and metabolic pathways in genomes of mutualists compared with catabolic signatures dominant in the genomes of pathogens. Another discriminative characteristic of mutualists is the co-occurence of gene clusters required for the expression and function of nitrogenase and RuBisCO. Analysis of previously published experimental data indicate that nitrogen-fixing mutualists may employ Rubisco to fix CO2 not in the canonical Calvin-Benson-Basham cycle but in a novel metabolic pathway, here called Rubisco-based glycolysis , to increase efficiency of sugar utilization during the symbiosis with plants. An important discriminative characteristic of plant pathogenic bacteria is two groups of genes likely encoding effector proteins involved in host invasion and a genomic locus encoding a putative secretion system that includes a DUF1525 domain protein conserved in pathogens of plants and of other organisms. The protein belongs to the same clan of thioredoxins as the circadian clock protein kaiB found in many mutualistic symbionts and highly abundant in blood cells colonized by a human pathogen, Salmonella enterica serotype Typhi, the cause of typhoid fever.« less

Molecular mechanism of TGF-β signaling pathway in colon carcinogenesis and status of curcumin as chemopreventive strategy.

PubMed

Ramamoorthi, Ganesan; Sivalingam, Nageswaran

2014-08-01

Colon cancer is one of the third most common cancer in man, the second most common cancer in women worldwide, and the second leading cause of mortality in the USA. There are a number of molecular pathways that have been implicated in colon carcinogenesis, including TGF-β/Smad signaling pathway. TGF-β (transforming growth factor-beta) signaling pathway has the potential to regulate various biological processes including cell growth, differentiation, apoptosis, extracellular matrix modeling, and immune response. TGF-β signaling pathway acts as a tumor suppressor, but alterations in TGF-β signaling pathway promotes colon cancer cell growth, migration, invasion, angiogenesis, and metastasis. Here we review the role of TGF-β signaling cascade in colon carcinogenesis and multiple molecular targets of curcumin in colon carcinogenesis. Elucidation of the molecular mechanism of curcumin on TGF-β signaling pathway-induced colon carcinogenesis may ultimately lead to novel and more effective treatments for colon cancer.

A comparative genomics perspective on the genetic content of the alkaliphilic haloarchaeon Natrialba magadii ATCC 43099T

PubMed Central

2012-01-01

Background Natrialba magadii is an aerobic chemoorganotrophic member of the Euryarchaeota and is a dual extremophile requiring alkaline conditions and hypersalinity for optimal growth. The genome sequence of Nab. magadii type strain ATCC 43099 was deciphered to obtain a comprehensive insight into the genetic content of this haloarchaeon and to understand the basis of some of the cellular functions necessary for its survival. Results The genome of Nab. magadii consists of four replicons with a total sequence of 4,443,643 bp and encodes 4,212 putative proteins, some of which contain peptide repeats of various lengths. Comparative genome analyses facilitated the identification of genes encoding putative proteins involved in adaptation to hypersalinity, stress response, glycosylation, and polysaccharide biosynthesis. A proton-driven ATP synthase and a variety of putative cytochromes and other proteins supporting aerobic respiration and electron transfer were encoded by one or more of Nab. magadii replicons. The genome encodes a number of putative proteases/peptidases as well as protein secretion functions. Genes encoding putative transcriptional regulators, basal transcription factors, signal perception/transduction proteins, and chemotaxis/phototaxis proteins were abundant in the genome. Pathways for the biosynthesis of thiamine, riboflavin, heme, cobalamin, coenzyme F420 and other essential co-factors were deduced by in depth sequence analyses. However, approximately 36% of Nab. magadii protein coding genes could not be assigned a function based on Blast analysis and have been annotated as encoding hypothetical or conserved hypothetical proteins. Furthermore, despite extensive comparative genomic analyses, genes necessary for survival in alkaline conditions could not be identified in Nab. magadii. Conclusions Based on genomic analyses, Nab. magadii is predicted to be metabolically versatile and it could use different carbon and energy sources to sustain growth. Nab. magadii has the genetic potential to adapt to its milieu by intracellular accumulation of inorganic cations and/or neutral organic compounds. The identification of Nab. magadii genes involved in coenzyme biosynthesis is a necessary step toward further reconstruction of the metabolic pathways in halophilic archaea and other extremophiles. The knowledge gained from the genome sequence of this haloalkaliphilic archaeon is highly valuable in advancing the applications of extremophiles and their enzymes. PMID:22559199

Identification and characterization of the polyketide synthase involved in ochratoxin A biosynthesis in Aspergillus carbonarius

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gallo, Antonia; Knox, Benjamin P.; Bruno, Kenneth S.

2014-06-02

Ochratoxin A (OTA) is a potent mycotoxin produced by Aspergillus and Penicillium species and is a common contaminant of a wide variety of food commodities, with Aspergillus carbonarius being the main producer of OTA contamination in grapes and wine. The molecular structure of OTA is composed of a dihydroisocoumarin ring linked to phenylalanine and, as shown in different producing fungal species, a polyketide synthase (PKS) is a component of the OTA biosynthetic pathway. Similar to observations in other filamentous ascomycetes, the genome sequence of A. carbonarius contains a large number of genes predicted to encode PKSs. In this work amore » pks gene identified within the putative OTA cluster of A. carbonarius, designated as AcOTApks, was inactivated and the resulting mutant strain was unable to produce OTA, confirming the role of AcOTApks in this biosynthetic pathway. AcOTApks protein is characteristic of the highly reduced (HR)-PKS family, and also contains a putative methyltransferase domain likely responsible for the addition of the methyl group to the OTA polyketide structure. AcOTApks is different from the ACpks protein that we previously described which showed an expression profile compatible with OTA production. We performed phylogenetic analyses of the β-ketosynthase and acyl-transferase domains of the OTA PKSs which had been identified and characterized in different OTA producing fungal species. The phylogenetic results were similar for both the two domains analyzed and showed that OTA PKS of A. carbonarius, Aspergillus niger, and Aspergillus ochraceus clustered in a monophyletic group with 100% bootstrap support suggesting a common origin, while the other OTA PKSs analyzed were phylogenetically distant. A qRT-PCR assay monitored AcOTApks expression during fungal growth and concomitant production of OTA by A. carbonarius in synthetic grape medium. A clear correlation between the expression profile of AcOTApks and kinetics of OTA production was observed with AcOTApks which reached its maximum level of transcription before OTA accumulation in mycelium reached its highest level, confirming the fact that gene transcription always precedes phenotypic production.« less

The use of high-throughput small RNA sequencing reveals differentially expressed microRNAs in response to aster yellows phytoplasma-infection in Vitis vinifera cv. ‘Chardonnay’

PubMed Central

Solofoharivelo, Marie-Chrystine; Souza-Richards, Rose; Stephan, Dirk; Murray, Shane; Burger, Johan T.

2017-01-01

Phytoplasmas are cell wall-less plant pathogenic bacteria responsible for major crop losses throughout the world. In grapevine they cause grapevine yellows, a detrimental disease associated with a variety of symptoms. The high economic impact of this disease has sparked considerable interest among researchers to understand molecular mechanisms related to pathogenesis. Increasing evidence exist that a class of small non-coding endogenous RNAs, known as microRNAs (miRNAs), play an important role in post-transcriptional gene regulation during plant development and responses to biotic and abiotic stresses. Thus, we aimed to dissect complex high-throughput small RNA sequencing data for the genome-wide identification of known and novel differentially expressed miRNAs, using read libraries constructed from healthy and phytoplasma-infected Chardonnay leaf material. Furthermore, we utilised computational resources to predict putative miRNA targets to explore the involvement of possible pathogen response pathways. We identified multiple known miRNA sequence variants (isomiRs), likely generated through post-transcriptional modifications. Sequences of 13 known, canonical miRNAs were shown to be differentially expressed. A total of 175 novel miRNA precursor sequences, each derived from a unique genomic location, were predicted, of which 23 were differentially expressed. A homology search revealed that some of these novel miRNAs shared high sequence similarity with conserved miRNAs from other plant species, as well as known grapevine miRNAs. The relative expression of randomly selected known and novel miRNAs was determined with real-time RT-qPCR analysis, thereby validating the trend of expression seen in the normalised small RNA sequencing read count data. Among the putative miRNA targets, we identified genes involved in plant morphology, hormone signalling, nutrient homeostasis, as well as plant stress. Our results may assist in understanding the role that miRNA pathways play during plant pathogenesis, and may be crucial in understanding disease symptom development in aster yellows phytoplasma-infected grapevines. PMID:28813447

Identification and functional analyses of sex determination genes in the sexually dimorphic stag beetle Cyclommatus metallifer.

PubMed

Gotoh, Hiroki; Zinna, Robert A; Warren, Ian; DeNieu, Michael; Niimi, Teruyuki; Dworkin, Ian; Emlen, Douglas J; Miura, Toru; Lavine, Laura C

2016-03-22

Genes in the sex determination pathway are important regulators of sexually dimorphic animal traits, including the elaborate and exaggerated male ornaments and weapons of sexual selection. In this study, we identified and functionally analyzed members of the sex determination gene family in the golden metallic stag beetle Cyclommatus metallifer, which exhibits extreme differences in mandible size between males and females. We constructed a C. metallifer transcriptomic database from larval and prepupal developmental stages and tissues of both males and females. Using Roche 454 pyrosequencing, we generated a de novo assembled database from a total of 1,223,516 raw reads, which resulted in 14,565 isotigs (putative transcript isoforms) contained in 10,794 isogroups (putative identified genes). We queried this database for C. metallifer conserved sex determination genes and identified 14 candidate sex determination pathway genes. We then characterized the roles of several of these genes in development of extreme sexual dimorphic traits in this species. We performed molecular expression analyses with RT-PCR and functional analyses using RNAi on three C. metallifer candidate genes--Sex-lethal (CmSxl), transformer-2 (Cmtra2), and intersex (Cmix). No differences in expression pattern were found between the sexes for any of these three genes. In the RNAi gene-knockdown experiments, we found that only the Cmix had any effect on sexually dimorphic morphology, and these mimicked the effects of Cmdsx knockdown in females. Knockdown of CmSxl had no measurable effects on stag beetle phenotype, while knockdown of Cmtra2 resulted in complete lethality at the prepupal period. These results indicate that the roles of CmSxl and Cmtra2 in the sex determination cascade are likely to have diverged in stag beetles when compared to Drosophila. Our results also suggest that Cmix has a conserved role in this pathway. In addition to those three genes, we also performed a more complete functional analysis of the C. metallifer dsx gene (Cmdsx) to identify the isoforms that regulate dimorphism more fully using exon-specific RNAi. We identified a total of 16 alternative splice variants of the Cmdsx gene that code for up to 14 separate exons. Despite the variation in RNA splice products of the Cmdsx gene, only four protein isoforms are predicted. The results of our exon-specific RNAi indicated that the essential CmDsx isoform for postembryonic male differentiation is CmDsxB, whereas postembryonic female specific differentiation is mainly regulated by CmDsxD. Taken together, our results highlight the importance of studying the function of highly conserved sex determination pathways in numerous insect species, especially those with dramatic and exaggerated sexual dimorphism, because conservation in protein structure does not always translate into conservation in downstream function.

Conceptualizing neurodevelopmental disorders through a mechanistic understanding of fragile X syndrome and Williams syndrome

PubMed Central

Fung, Lawrence K.; Quintin, Eve-Marie; Haas, Brian W.

2013-01-01

Purpose of review The overarching goal of this review is to compare and contrast the cognitive-behavioral features of fragile X syndrome (FraX) and Williams syndrome and to review the putative neural and molecular underpinnings of these features. Information is presented in a framework that provides guiding principles for conceptualizing gene-brain-behavior associations in neurodevelopmental disorders. Recent findings Abnormalities, in particular cognitive-behavioral domains with similarities in underlying neurodevelopmental correlates, occur in both FraX and Williams syndrome including aberrant frontostriatal pathways leading to executive function deficits, and magnocellular/dorsal visual stream, superior parietal lobe, inferior parietal lobe, and postcentral gyrus abnormalities contributing to deficits in visuospatial function. Compelling cognitive–behavioral and neurodevelopmental contrasts also exist in these two disorders, for example, aberrant amygdala and fusiform cortex structure and function occurring in the context of contrasting social behavioral phenotypes, and temporal cortical and cerebellar abnormalities potentially underlying differences in language function. Abnormal dendritic development is a shared neurodevelopmental morphologic feature between FraX and Williams syndrome. Commonalities in molecular machinery and processes across FraX and Williams syndrome occur as well – microRNAs involved in translational regulation of major synaptic proteins; scaffolding proteins in excitatory synapses; and proteins involved in axonal development. Summary Although the genetic variations leading to FraX and Williams syndrome are different, important similarities and contrasts in the phenotype, neurocircuitry, molecular machinery, and cellular processes in these two disorders allow for a unique approach to conceptualizing gene–brain–behavior links occurring in neurodevelopmental disorders. PMID:22395002

Electrostatic and Structural Bases of Fe2+ Translocation through Ferritin Channels.

PubMed

Chandramouli, Balasubramanian; Bernacchioni, Caterina; Di Maio, Danilo; Turano, Paola; Brancato, Giuseppe

2016-12-02

Ferritin molecular cages are marvelous 24-mer supramolecular architectures that enable massive iron storage (>2000 iron atoms) within their inner cavity. This cavity is connected to the outer environment by two channels at C3 and C4 symmetry axes of the assembly. Ferritins can also be exploited as carriers for in vivo imaging and therapeutic applications, owing to their capability to effectively protect synthetic non-endogenous agents within the cage cavity and deliver them to targeted tissue cells without stimulating adverse immune responses. Recently, X-ray crystal structures of Fe 2+ -loaded ferritins provided important information on the pathways followed by iron ions toward the ferritin cavity and the catalytic centers within the protein. However, the specific mechanisms enabling Fe 2+ uptake through wild-type and mutant ferritin channels is largely unknown. To shed light on this question, we report extensive molecular dynamics simulations, site-directed mutagenesis, and kinetic measurements that characterize the transport properties and translocation mechanism of Fe 2+ through the two ferritin channels, using the wild-type bullfrog Rana catesbeiana H' protein and some of its variants as case studies. We describe the structural features that determine Fe 2+ translocation with atomistic detail, and we propose a putative mechanism for Fe 2+ transport through the channel at the C3 symmetry axis, which is the only iron-permeable channel in vertebrate ferritins. Our findings have important implications for understanding how ion permeation occurs, and further how it may be controlled via purposely engineered channels for novel biomedical applications based on ferritin. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Cavity hydration dynamics in cytochrome c oxidase and functional implications

PubMed Central

Son, Chang Yun; Cui, Qiang

2017-01-01

Cytochrome c oxidase (CcO) is a transmembrane protein that uses the free energy of O2 reduction to generate the proton concentration gradient across the membrane. The regulation of competitive proton transfer pathways has been established to be essential to the vectorial transport efficiency of CcO, yet the underlying mechanism at the molecular level remains lacking. Recent studies have highlighted the potential importance of hydration-level change in an internal cavity that connects the proton entrance channel, the site of O2 reduction, and the putative proton exit route. In this work, we use atomistic molecular dynamics simulations to investigate the energetics and timescales associated with the volume fluctuation and hydration-level change in this central cavity. Extensive unrestrained molecular dynamics simulations (accumulatively ∼4 μs) and free energy computations for different chemical states of CcO support a model in which the volume and hydration level of the cavity are regulated by the protonation state of a propionate group of heme a3 and, to a lesser degree, the redox state of heme a and protonation state of Glu286. Markov-state model analysis of ∼2-μs trajectories suggests that hydration-level change occurs on the timescale of 100–200 ns before the proton-loading site is protonated. The computed energetic and kinetic features for the cavity wetting transition suggest that reversible hydration-level change of the cavity can indeed be a key factor that regulates the branching of proton transfer events and therefore contributes to the vectorial efficiency of proton transport. PMID:28973914

Molecular Phenotyping Combines Molecular Information, Biological Relevance, and Patient Data to Improve Productivity of Early Drug Discovery.

PubMed

Drawnel, Faye Marie; Zhang, Jitao David; Küng, Erich; Aoyama, Natsuyo; Benmansour, Fethallah; Araujo Del Rosario, Andrea; Jensen Zoffmann, Sannah; Delobel, Frédéric; Prummer, Michael; Weibel, Franziska; Carlson, Coby; Anson, Blake; Iacone, Roberto; Certa, Ulrich; Singer, Thomas; Ebeling, Martin; Prunotto, Marco

2017-05-18

Today, novel therapeutics are identified in an environment which is intrinsically different from the clinical context in which they are ultimately evaluated. Using molecular phenotyping and an in vitro model of diabetic cardiomyopathy, we show that by quantifying pathway reporter gene expression, molecular phenotyping can cluster compounds based on pathway profiles and dissect associations between pathway activities and disease phenotypes simultaneously. Molecular phenotyping was applicable to compounds with a range of binding specificities and triaged false positives derived from high-content screening assays. The technique identified a class of calcium-signaling modulators that can reverse disease-regulated pathways and phenotypes, which was validated by structurally distinct compounds of relevant classes. Our results advocate for application of molecular phenotyping in early drug discovery, promoting biological relevance as a key selection criterion early in the drug development cascade. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structural Insights into the Quadruplex-Duplex 3' Interface Formed from a Telomeric Repeat: A Potential Molecular Target.

PubMed

Russo Krauss, Irene; Ramaswamy, Sneha; Neidle, Stephen; Haider, Shozeb; Parkinson, Gary N

2016-02-03

We report here on an X-ray crystallographic and molecular modeling investigation into the complex 3' interface formed between putative parallel stranded G-quadruplexes and a duplex DNA sequence constructed from the human telomeric repeat sequence TTAGGG. Our crystallographic approach provides a detailed snapshot of a telomeric 3' quadruplex-duplex junction: a junction that appears to have the potential to form a unique molecular target for small molecule binding and interference with telomere-related functions. This unique target is particularly relevant as current high-affinity compounds that bind putative G-quadruplex forming sequences only rarely have a high degree of selectivity for a particular quadruplex. Here DNA junctions were assembled using different putative quadruplex-forming scaffolds linked at the 3' end to a telomeric duplex sequence and annealed to a complementary strand. We successfully generated a series of G-quadruplex-duplex containing crystals, both alone and in the presence of ligands. The structures demonstrate the formation of a parallel folded G-quadruplex and a B-form duplex DNA stacked coaxially. Most strikingly, structural data reveals the consistent formation of a TAT triad platform between the two motifs. This triad allows for a continuous stack of bases to link the quadruplex motif with the duplex region. For these crystal structures formed in the absence of ligands, the TAT triad interface occludes ligand binding at the 3' quadruplex-duplex interface, in agreement with in silico docking predictions. However, with the rearrangement of a single nucleotide, a stable pocket can be produced, thus providing an opportunity for the binding of selective molecules at the interface.

Divalent cations and molecular crowding buffers stabilize G-triplex at physiologically relevant temperatures

PubMed Central

Jiang, Hong-Xin; Cui, Yunxi; Zhao, Ting; Fu, Hai-Wei; Koirala, Deepak; Punnoose, Jibin Abraham; Kong, De-Ming; Mao, Hanbin

2015-01-01

G-triplexes are non-canonical DNA structures formed by G-rich sequences with three G-tracts. Putative G-triplex-forming sequences are expected to be more prevalent than putative G-quadruplex-forming sequences. However, the research on G-triplexes is rare. In this work, the effects of molecular crowding and several physiologically important metal ions on the formation and stability of G-triplexes were examined using a combination of circular dichroism, thermodynamics, optical tweezers and calorimetry techniques. We determined that molecular crowding conditions and cations, such as Na+, K+, Mg2+ and Ca2+, promote the formation of G-triplexes and stabilize these structures. Of these four metal cations, Ca2+ has the strongest stabilizing effect, followed by K+, Mg2+, and Na+ in a decreasing order. The binding of K+ to G-triplexes is accompanied by exothermic heats, and the binding of Ca2+ with G-triplexes is characterized by endothermic heats. G-triplexes formed from two G-triad layers are not stable at physiological temperatures; however, G-triplexes formed from three G-triads exhibit melting temperatures higher than 37°C, especially under the molecular crowding conditions and in the presence of K+ or Ca2+. These observations imply that stable G-triplexes may be formed under physiological conditions. PMID:25787838

Toward Repurposing Metformin as a Precision Anti-Cancer Therapy Using Structural Systems Pharmacology

PubMed Central

Hart, Thomas; Dider, Shihab; Han, Weiwei; Xu, Hua; Zhao, Zhongming; Xie, Lei

2016-01-01

Metformin, a drug prescribed to treat type-2 diabetes, exhibits anti-cancer effects in a portion of patients, but the direct molecular and genetic interactions leading to this pleiotropic effect have not yet been fully explored. To repurpose metformin as a precision anti-cancer therapy, we have developed a novel structural systems pharmacology approach to elucidate metformin’s molecular basis and genetic biomarkers of action. We integrated structural proteome-scale drug target identification with network biology analysis by combining structural genomic, functional genomic, and interactomic data. Through searching the human structural proteome, we identified twenty putative metformin binding targets and their interaction models. We experimentally verified the interactions between metformin and our top-ranked kinase targets. Notably, kinases, particularly SGK1 and EGFR were identified as key molecular targets of metformin. Subsequently, we linked these putative binding targets to genes that do not directly bind to metformin but whose expressions are altered by metformin through protein-protein interactions, and identified network biomarkers of phenotypic response of metformin. The molecular targets and the key nodes in genetic networks are largely consistent with the existing experimental evidence. Their interactions can be affected by the observed cancer mutations. This study will shed new light into repurposing metformin for safe, effective, personalized therapies. PMID:26841718

Whole exome sequencing in recurrent early pregnancy loss.

PubMed

Qiao, Ying; Wen, Jiadi; Tang, Flamingo; Martell, Sally; Shomer, Naomi; Leung, Peter C K; Stephenson, Mary D; Rajcan-Separovic, Evica

2016-05-01

Exome sequencing can identify genetic causes of idiopathic recurrent pregnancy loss (RPL). We identified compound heterozygous deleterious mutations affecting DYNC2H1 and ALOX15 in two out of four families with RPL. Both genes have a role in early development. Bioinformatics analysis of all genes with rare and putatively pathogenic mutations in miscarriages and couples showed enrichment in pathways relevant to pregnancy loss, including the complement and coagulation cascades pathways. Next generation sequencing (NGS) is increasingly being used to identify known and novel gene mutations in children with developmental delay and in fetuses with ultrasound-detected anomalies. In contrast, NGS is rarely used to study pregnancy loss. Chromosome microarray analysis detects putatively causative DNA copy number variants (CNVs) in ∼2% of miscarriages and CNVs of unknown significance (predominantly parental in origin) in up to 40% of miscarriages. Therefore, a large number of miscarriages still have an unknown cause. Whole exome sequencing (WES) was performed using Illumina HiSeq 2000 platform on seven euploid miscarriages from four families with RPL. Golden Helix SVS v8.1.5 was used for data assessment and inheritance analysis for deleterious DNA variants predicted to severely disrupt protein-coding genes by introducing a frameshift, loss of the stop codon, gain of the stop codon, changes in splicing or the initial codon. Webgestalt (http://bioinfo.vanderbilt.edu/webgestalt/) was used for pathway and disease association enrichment analysis of a gene pool containing putatively pathogenic variants in miscarriages and couples in comparison to control gene pools. Compound heterozygous mutations in DYNC2H1 and ALOX15 were identified in miscarriages from two families with RPL. DYNC2H1 is involved in cilia biogenesis and has been associated with fetal lethality in humans. ALOX15 is expressed in placenta and its dysregulation has been associated with inflammation, placental, dysfunction, abnormal oxidative stress response and angiogenesis. The pool of putatively pathogenic single nucleotide variants (SNVs) and small insertions and deletions (indels) detected in the miscarriages showed enrichment in 'complement and coagulation cascades pathway', and 'ciliary motility disorders'. We conclude that CNVs, individual SNVs and pool of deleterious gene mutations identified by exome sequencing could contribute to RPL. The size of our sample cohort is small. The functional effect of candidate mutations should be evaluated to determine whether the mutations are causative. This is the first study to assess whether SNVs may contribute to the pathogenesis of miscarriage. Furthermore, our findings suggest that collective effect of mutations in relevant biological pathways could be implicated in RPL. The study was funded by Canadian Institutes of Health Research (grant MOP 106467) and Michael Smith Foundation of Health Research Career Scholar salary award to ERS. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

PKC-epsilon activation is required for recognition memory in the rat.

PubMed

Zisopoulou, Styliani; Asimaki, Olga; Leondaritis, George; Vasilaki, Anna; Sakellaridis, Nikos; Pitsikas, Nikolaos; Mangoura, Dimitra

2013-09-15

Activation of PKCɛ, an abundant and developmentally regulated PKC isoform in the brain, has been implicated in memory throughout life and across species. Yet, direct evidence for a mechanistic role for PKCɛ in memory is still lacking. Hence, we sought to evaluate this in rats, using short-term treatments with two PKCɛ-selective peptides, the inhibitory ɛV1-2 and the activating ψɛRACK, and the novel object recognition task (NORT). Our results show that the PKCɛ-selective activator ψɛRACK, did not have a significant effect on recognition memory. In the short time frames used, however, inhibition of PKCɛ activation with the peptide inhibitor ɛV1-2 significantly impaired recognition memory. Moreover, when we addressed at the molecular level the immediate proximal signalling events of PKCɛ activation in acutely dissected rat hippocampi, we found that ψɛRACK increased in a time-dependent manner phosphorylation of MARCKS and activation of Src, Raf, and finally ERK1/2, whereas ɛV1-2 inhibited all basal activity of this pathway. Taken together, these findings present the first direct evidence that PKCɛ activation is an essential molecular component of recognition memory and point toward the use of systemically administered PKCɛ-regulating peptides as memory study tools and putative therapeutic agents. Copyright © 2013 Elsevier B.V. All rights reserved.

Transcriptome Analysis of the Scleractinian Coral Stylophora pistillata

PubMed Central

Salmon-Divon, Mali; Katzenellenbogen, Mark; Tambutté, Sylvie; Bertucci, Anthony; Hoegh-Guldberg, Ove; Deleury, Emeline; Allemand, Denis; Levy, Oren

2014-01-01

The principal architects of coral reefs are the scleractinian corals; these species are divided in two major clades referred to as “robust” and “complex” corals. Although the molecular diversity of the “complex” clade has received considerable attention, with several expressed sequence tag (EST) libraries and a complete genome sequence having been constructed, the “robust” corals have received far less attention, despite the fact that robust corals have been prominent focal points for ecological and physiological studies. Filling this gap affords important opportunities to extend these studies and to improve our understanding of the differences between the two major clades. Here, we present an EST library from Stylophora pistillata (Esper 1797) and systematically analyze the assembled transcripts compared to putative homologs from the complete proteomes of six well-characterized metazoans: Nematostella vectensis, Hydra magnipapillata, Caenorhabditis elegans, Drosophila melanogaster, Strongylocentrotus purpuratus, Ciona intestinalis and Homo sapiens. Furthermore, comparative analyses of the Stylophora pistillata ESTs were performed against several Cnidaria from the Scleractinia, Actiniaria and Hydrozoa, as well as against other stony corals separately. Functional characterization of S. pistillata transcripts into KOG/COG categories and further description of Wnt and bone morphogenetic protein (BMP) signaling pathways showed that the assembled EST library provides sufficient data and coverage. These features of this new library suggest considerable opportunities for extending our understanding of the molecular and physiological behavior of “robust” corals. PMID:24551124

Conversion to Sirolimus Ameliorates Cyclosporine-Induced Nephropathy in the Rat: Focus on Serum, Urine, Gene, and Protein Renal Expression Biomarkers

PubMed Central

Sereno, José; Nunes, Sara; Rodrigues-Santos, Paulo; Rocha-Pereira, Petronila; Fernandes, João; Teixeira, Frederico; Reis, Flávio

2014-01-01

Protocols of conversion from cyclosporin A (CsA) to sirolimus (SRL) have been widely used in immunotherapy after transplantation to prevent CsA-induced nephropathy, but the molecular mechanisms underlying these protocols remain nuclear. This study aimed to identify the molecular pathways and putative biomarkers of CsA-to-SRL conversion in a rat model. Four animal groups (n = 6) were tested during 9 weeks: control, CsA, SRL, and conversion (CsA for 3 weeks followed by SRL for 6 weeks). Classical and emergent serum, urinary, and kidney tissue (gene and protein expression) markers were assessed. Renal lesions were analyzed in hematoxylin and eosin, periodic acid-Schiff, and Masson's trichrome stains. SRL-treated rats presented proteinuria and NGAL (serum and urinary) as the best markers of renal impairment. Short CsA treatment presented slight or even absent kidney lesions and TGF-β, NF-κ β, mTOR, PCNA, TP53, KIM-1, and CTGF as relevant gene and protein changes. Prolonged CsA exposure aggravated renal damage, without clear changes on the traditional markers, but with changes in serums TGF-β and IL-7, TBARs clearance, and kidney TGF-β and mTOR. Conversion to SRL prevented CsA-induced renal damage evolution (absent/mild grade lesions), while NGAL (serum versus urine) seems to be a feasible biomarker of CsA replacement to SRL. PMID:24971338

Antifungal activity, kinetics and molecular mechanism of action of garlic oil against Candida albicans

PubMed Central

Li, Wen-Ru; Shi, Qing-Shan; Dai, Huan-Qin; Liang, Qing; Xie, Xiao-Bao; Huang, Xiao-Mo; Zhao, Guang-Ze; Zhang, Li-Xin

2016-01-01

The antifungal activity, kinetics, and molecular mechanism of action of garlic oil against Candida albicans were investigated in this study using multiple methods. Using the poisoned food technique, we determined that the minimum inhibitory concentration of garlic oil was 0.35 μg/mL. Observation by transmission electron microscopy indicated that garlic oil could penetrate the cellular membrane of C. albicans as well as the membranes of organelles such as the mitochondria, resulting in organelle destruction and ultimately cell death. RNA sequencing analysis showed that garlic oil induced differential expression of critical genes including those involved in oxidation-reduction processes, pathogenesis, and cellular response to drugs and starvation. Moreover, the differentially expressed genes were mainly clustered in 19 KEGG pathways, representing vital cellular processes such as oxidative phosphorylation, the spliceosome, the cell cycle, and protein processing in the endoplasmic reticulum. In addition, four upregulated proteins selected after two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) analysis were identified with high probability by mass spectrometry as putative cytoplasmic adenylate kinase, pyruvate decarboxylase, hexokinase, and heat shock proteins. This is suggestive of a C. albicans stress responses to garlic oil treatment. On the other hand, a large number of proteins were downregulated, leading to significant disruption of the normal metabolism and physical functions of C. albicans. PMID:26948845

The chemotaxis regulator pilG of Xylella fastidiosa is required for virulence in Vitis vinifera grapevines

USDA-ARS?s Scientific Manuscript database

Type IV pili of X. fastidiosa are regulated by pilG, a response regulator protein putatively involved in chemotaxis-like operon sensing stimuli through signal transduction pathways. To elucidate roles of pilG in pathogenicity of X. fastidiosa, the pilG-deletion mutant and complementary strain contai...

Enhancing high throughput toxicology - development of putative adverse outcome pathways linking US EPA ToxCast screening targets to relevant apical hazards.

EPA Science Inventory

High throughput toxicology programs, such as ToxCast and Tox21, have provided biological effects data for thousands of chemicals at multiple concentrations. Compared to traditional, whole-organism approaches, high throughput assays are rapid and cost-effective, yet they generall...

A preliminary crystallographic analysis of the putative mevalonate diphosphate decarboxylase from Trypanosoma brucei

DOE Office of Scientific and Technical Information (OSTI.GOV)

Byres, Emma; Martin, David M. A.; Hunter, William N., E-mail: w.n.hunter@dundee.ac.uk

2005-06-01

The gene encoding the putative mevalonate diphosphate decarboxylase, an enzyme from the mevalonate pathway of isoprenoid precursor biosynthesis, has been cloned from T. brucei. Recombinant protein has been expressed, purified and highly ordered crystals obtained and characterized to aid the structure–function analysis of this enzyme. Mevalonate diphosphate decarboxylase catalyses the last and least well characterized step in the mevalonate pathway for the biosynthesis of isopentenyl pyrophosphate, an isoprenoid precursor. A gene predicted to encode the enzyme from Trypanosoma brucei has been cloned, a highly efficient expression system established and a purification protocol determined. The enzyme gives monoclinic crystals in spacemore » group P2{sub 1}, with unit-cell parameters a = 51.5, b = 168.7, c = 54.9 Å, β = 118.8°. A Matthews coefficient V{sub M} of 2.5 Å{sup 3} Da{sup −1} corresponds to two monomers, each approximately 42 kDa (385 residues), in the asymmetric unit with 50% solvent content. These crystals are well ordered and data to high resolution have been recorded using synchrotron radiation.« less

De novo leaf and root transcriptome analysis to identify putative genes involved in triterpenoid saponins biosynthesis in Hedera helix L.

PubMed Central

Li, Fang; Xu, Zijian; Sun, Mengli; Cong, Hanqing; Qiao, Fei; Zhong, Xiaohong

2017-01-01

Hedera helix L. is an important traditional medicinal plant in Europe. The main active components are triterpenoid saponins, but none of the potential enzymes involved in triterpenoid saponins biosynthesis have been discovered and annotated. Here is reported the first study of global transcriptome analyses using the Illumina HiSeq™ 2500 platform for H. helix. In total, over 24 million clean reads were produced and 96,333 unigenes were assembled, with an average length of 1385 nt; more than 79,085 unigenes had at least one significant match to an existing gene model. Differentially Expressed Gene analysis identified 6,222 and 7,012 unigenes which were expressed either higher or lower in leaf samples when compared with roots. After functional annotation and classification, two pathways and 410 unigenes related to triterpenoid saponins biosynthesis were discovered. The accuracy of these de novo sequences was validated by RT-qPCR analysis and a RACE clone. These data will enrich our knowledge of triterpenoid saponin biosynthesis and provide a theoretical foundation for molecular research on H. helix. PMID:28771546

Identification of 28 cytochrome P450 genes from the transcriptome of the marine rotifer Brachionus plicatilis and analysis of their expression.

PubMed

Kim, Hui-Su; Han, Jeonghoon; Kim, Hee-Jin; Hagiwara, Atsushi; Lee, Jae-Seong

2017-09-01

Whole transcriptomes of the rotifer Brachionus plicatilis were analyzed using an Illumina sequencer. De novo assembly was performed with 49,122,780 raw reads using Trinity software. Among the assembled 42,820 contigs, 27,437 putative open reading frame contigs were identified (average length 1235bp; N50=1707bp). Functional gene annotation with Gene Ontology and InterProScan, in addition to Kyoto Encyclopedia of Genes and Genomes pathway analysis, highlighted the metabolism of xenobiotics by cytochrome P450 (CYP). In addition, 28 CYP genes were identified, and their transcriptional responses to benzo[α]pyrene (B[α]P) were investigated. Most of the CYPs were significantly upregulated or downregulated (P<0.05) in response to B[α]P, suggesting that Bp-CYP genes play a crucial role in detoxification mechanisms in response to xenobiotics. This study sheds light on the molecular defense mechanisms of the rotifer B. plicatilis in response to exposure to various chemicals. Copyright © 2017 Elsevier Inc. All rights reserved.

The oncogenic tyrosine kinase Lyn impairs the pro-apoptotic function of Bim.

PubMed

Aira, Lazaro E; Villa, Elodie; Colosetti, Pascal; Gamas, Parvati; Signetti, Laurie; Obba, Sandrine; Proics, Emma; Gautier, Fabien; Bailly-Maitre, Béatrice; Jacquel, Arnaud; Robert, Guillaume; Luciano, Frédéric; Juin, Philippe P; Ricci, Jean-Ehrland; Auberger, Patrick; Marchetti, Sandrine

2018-04-01

Phosphorylation of Ser/Thr residues is a well-established modulating mechanism of the pro-apoptotic function of the BH3-only protein Bim. However, nothing is known about the putative tyrosine phosphorylation of this Bcl-2 family member and its potential impact on Bim function and subsequent Bax/Bak-mediated cytochrome c release and apoptosis. As we have previously shown that the tyrosine kinase Lyn could behave as an anti-apoptotic molecule, we investigated whether this Src family member could directly regulate the pro-apoptotic function of Bim. In the present study, we show that Bim is phosphorylated onto tyrosine residues 92 and 161 by Lyn, which results in an inhibition of its pro-apoptotic function. Mechanistically, we show that Lyn-dependent tyrosine phosphorylation of Bim increases its interaction with anti-apoptotic members such as Bcl-xL, therefore limiting mitochondrial outer membrane permeabilization and subsequent apoptosis. Collectively, our data uncover one molecular mechanism through which the oncogenic tyrosine kinase Lyn negatively regulates the mitochondrial apoptotic pathway, which may contribute to the transformation and/or the chemotherapeutic resistance of cancer cells.

Integrated genome sequence and linkage map of physic nut (Jatropha curcas L.), a biodiesel plant.

PubMed

Wu, Pingzhi; Zhou, Changpin; Cheng, Shifeng; Wu, Zhenying; Lu, Wenjia; Han, Jinli; Chen, Yanbo; Chen, Yan; Ni, Peixiang; Wang, Ying; Xu, Xun; Huang, Ying; Song, Chi; Wang, Zhiwen; Shi, Nan; Zhang, Xudong; Fang, Xiaohua; Yang, Qing; Jiang, Huawu; Chen, Yaping; Li, Meiru; Wang, Ying; Chen, Fan; Wang, Jun; Wu, Guojiang

2015-03-01

The family Euphorbiaceae includes some of the most efficient biomass accumulators. Whole genome sequencing and the development of genetic maps of these species are important components in molecular breeding and genetic improvement. Here we report the draft genome of physic nut (Jatropha curcas L.), a biodiesel plant. The assembled genome has a total length of 320.5 Mbp and contains 27,172 putative protein-coding genes. We established a linkage map containing 1208 markers and anchored the genome assembly (81.7%) to this map to produce 11 pseudochromosomes. After gene family clustering, 15,268 families were identified, of which 13,887 existed in the castor bean genome. Analysis of the genome highlighted specific expansion and contraction of a number of gene families during the evolution of this species, including the ribosome-inactivating proteins and oil biosynthesis pathway enzymes. The genomic sequence and linkage map provide a valuable resource not only for fundamental and applied research on physic nut but also for evolutionary and comparative genomics analysis, particularly in the Euphorbiaceae. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

A putative octopamine/tyramine receptor mediating appetite in a hungry fly

NASA Astrophysics Data System (ADS)

Ishida, Yuko; Ozaki, Mamiko

2011-07-01

In the blowfly Phormia regina, experience of simultaneous feeding with d-limonene exposure inhibits proboscis extension reflex (PER) due to decreased tyramine (TA) titer in the brain. To elucidate the molecular mechanism of TA signaling pathway related to the associated feeding behavior, we cloned cDNA encoding the octopamine/TA receptor (PregOAR/TAR). The deduced protein is composed of 607 amino acid residues and has 7 predicted transmembrane domains. Based on homology and phylogenetic analyses, this protein belongs to the OAR/TAR family. The PregOAR/TAR was mainly expressed in head, with low levels of expression in other tissues at adult stages. Gene expression profile is in agreement with a plethora of functions ascribed to TA in various insect tissues. The immunolabeled cell bodies and processes were localized in the medial protocerebrum, outer layer of lobula, antennal lobe, and subesophageal ganglion. These results suggest that decrease of TA level in the brain likely affects neurons expressing PregOAR/TAR, causing mediation of the sensitivity in the sensillum and/or output of motor neurons for PER.

A structure-based approach to ligand discovery for 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase: a target for antimicrobial therapy.

PubMed

Ramsden, Nicola L; Buetow, Lori; Dawson, Alice; Kemp, Lauris A; Ulaganathan, Venkatsubramanian; Brenk, Ruth; Klebe, Gerhard; Hunter, William N

2009-04-23

The nonmevalonate route to isoprenoid biosynthesis is essential in Gram-negative bacteria and apicomplexan parasites. The enzymes of this pathway are absent from mammals, contributing to their appeal as chemotherapeutic targets. One enzyme, 2C-methyl-d-erythritol-2,4-cyclodiphosphate synthase (IspF), has been validated as a target by genetic approaches in bacteria. Virtual screening against Escherichia coli IspF (EcIspF) was performed by combining a hierarchical filtering methodology with molecular docking. Docked compounds were inspected and 10 selected for experimental validation. A surface plasmon resonance assay was developed and two weak ligands identified. Crystal structures of EcIspF complexes were determined to support rational ligand development. Cytosine analogues and Zn(2+)-binding moieties were characterized. One of the putative Zn(2+)-binding compounds gave the lowest measured K(D) to date (1.92 +/- 0.18 muM). These data provide a framework for the development of IspF inhibitors to generate lead compounds of therapeutic potential against microbial pathogens.

Signal transduction in the development of pulmonary arterial hypertension

PubMed Central

Malenfant, Simon; Neyron, Anne-Sophie; Paulin, Roxane; Potus, François; Meloche, Jolyane; Provencher, Steeve; Bonnet, Sébastien

2013-01-01

Pulmonary arterial hypertension (PAH) is a unique disease. Properly speaking, it is not a disease of the lung. It can be seen more as a microvascular disease occurring mainly in the lungs and affecting the heart. At the cellular level, the PAH paradigm is characterized by inflammation, vascular tone imbalance, pulmonary arterial smooth muscle cell proliferation and resistance to apoptosis and the presence of in situ thrombosis. At a clinical level, the aforementioned abnormal vascular properties alter physically the pulmonary circulation and ventilation, which greatly influence the right ventricle function as it highly correlates with disease severity. Consequently, right heart failure remains the principal cause of death within this cohort of patients. While current treatment modestly improve patients’ conditions, none of them are curative and, as of today, new therapies are lacking. However, the future holds potential new therapies that might have positive influence on the quality of life of the patient. This article will first review the clinical presentation of the disease and the different molecular pathways implicated in the pathobiology of PAH. The second part will review tomorrow's future putative therapies for PAH. PMID:24015329

Genome-wide DNA methylation patterns in wild samples of two morphotypes of threespine stickleback (Gasterosteus aculeatus).

PubMed

Smith, Gilbert; Smith, Carl; Kenny, John G; Chaudhuri, Roy R; Ritchie, Michael G

2015-04-01

Epigenetic marks such as DNA methylation play important biological roles in gene expression regulation and cellular differentiation during development. To examine whether DNA methylation patterns are potentially associated with naturally occurring phenotypic differences, we examined genome-wide DNA methylation within Gasterosteus aculeatus, using reduced representation bisulfite sequencing. First, we identified highly methylated regions of the stickleback genome, finding such regions to be located predominantly within genes, and associated with genes functioning in metabolism and biosynthetic processes, cell adhesion, signaling pathways, and blood vessel development. Next, we identified putative differentially methylated regions (DMRs) of the genome between complete and low lateral plate morphs of G. aculeatus. We detected 77 DMRs that were mainly located in intergenic regions. Annotations of genes associated with these DMRs revealed potential functions in a number of known divergent adaptive phenotypes between G. aculeatus ecotypes, including cardiovascular development, growth, and neuromuscular development. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Gene expression in the scleractinian Acropora microphthalma exposed to high solar irradiance reveals elements of photoprotection and coral bleaching.

PubMed

Starcevic, Antonio; Dunlap, Walter C; Cullum, John; Shick, J Malcolm; Hranueli, Daslav; Long, Paul F

2010-11-12

The success of tropical reef-building corals depends on the metabolic co-operation between the animal host and the photosynthetic performance of endosymbiotic algae residing within its cells. To examine the molecular response of the coral Acropora microphthalma to high levels of solar irradiance, a cDNA library was constructed by PCR-based suppression subtractive hybridisation (PCR-SSH) from mRNA obtained by transplantation of a colony from a depth of 12.7 m to near-surface solar irradiance, during which the coral became noticeably paler from loss of endosymbionts in sun-exposed tissues. A novel approach to sequence annotation of the cDNA library gave genetic evidence for a hypothetical biosynthetic pathway branching from the shikimic acid pathway that leads to the formation of 4-deoxygadusol. This metabolite is a potent antioxidant and expected precursor of the UV-protective mycosporine-like amino acids (MAAs), which serve as sunscreens in coral phototrophic symbiosis. Empirical PCR based evidence further upholds the contention that the biosynthesis of these MAA sunscreens is a 'shared metabolic adaptation' between the symbiotic partners. Additionally, gene expression induced by enhanced solar irradiance reveals a cellular mechanism of light-induced coral bleaching that invokes a Ca(2+)-binding synaptotagmin-like regulator of SNARE protein assembly of phagosomal exocytosis, whereby algal partners are lost from the symbiosis. Bioinformatics analyses of DNA sequences obtained by differential gene expression of a coral exposed to high solar irradiance has revealed the identification of putative genes encoding key steps of the MAA biosynthetic pathway. Revealed also by this treatment are genes that implicate exocytosis as a cellular process contributing to a breakdown in the metabolically essential partnership between the coral host and endosymbiotic algae, which manifests as coral bleaching.

The GPRC6A receptor displays constitutive internalization and sorting to the slow recycling pathway.

PubMed

Jacobsen, Stine Engesgaard; Ammendrup-Johnsen, Ina; Jansen, Anna Mai; Gether, Ulrik; Madsen, Kenneth Lindegaard; Bräuner-Osborne, Hans

2017-04-28

The class C G protein-coupled receptor GPRC6A is a putative nutrient-sensing receptor and represents a possible new drug target in metabolic disorders. However, the specific physiological role of this receptor has yet to be identified, and the mechanisms regulating its activity and cell surface availability also remain enigmatic. In the present study, we investigated the trafficking properties of GPRC6A by use of both a classical antibody feeding internalization assay in which cells were visualized using confocal microscopy and a novel internalization assay that is based on real-time measurements of fluorescence resonance energy transfer. Both assays revealed that GPRC6A predominantly undergoes constitutive internalization, whereas the agonist-induced effects were imperceptible. Moreover, postendocytic sorting was investigated by assessing the co-localization of internalized GPRC6A with selected Rab protein markers. Internalized GPRC6A was mainly co-localized with the early endosome marker Rab5 and the long loop recycling endosome marker Rab11 and to a much lesser extent with the late endosome marker Rab7. This suggests that upon agonist-independent internalization, GPRC6A is recycled via the Rab11-positive slow recycling pathway, which may be responsible for ensuring a persistent pool of GPRC6A receptors at the cell surface despite chronic agonist exposure. Distinct trafficking pathways have been reported for several of the class C receptors, and our results thus substantiate that non-canonical trafficking mechanisms are a common feature for the nutrient-sensing class C family that ensure functional receptors in the cell membrane despite prolonged agonist exposure. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Relevance of the Axis Spermidine/eIF5A for Plant Growth and Development

PubMed Central

Belda-Palazón, Borja; Almendáriz, Carla; Martí, Esmeralda; Carbonell, Juan; Ferrando, Alejandro

2016-01-01

One key role of the essential polyamine spermidine in eukaryotes is to provide the 4-aminobutyl moiety group destined to the post-translational modification of a lysine in the highly conserved translation factor eIF5A. This modification is catalyzed by two sequential enzymatic steps leading to the activation of eIF5A by the conversion of one conserved lysine to the unusual amino acid hypusine. The active translation factor facilitates the sequence-specific translation of polyproline sequences that otherwise cause ribosome stalling. In spite of the well-characterized involvement of active eIF5A in the translation of proline repeat-rich proteins, its biological role has been recently elucidated only in mammals, and it is poorly described at the functional level in plants. Here we describe the alterations in plant growth and development caused by RNAi-mediated conditional genetic inactivation of the hypusination pathway in Arabidopsis thaliana by knocking-down the enzyme deoxyhypusine synthase. We have uncovered that spermidine-mediated activation of eIF5A by hypusination is involved in several aspects of plant biology such as the control of flowering time, the aerial and root architecture, and root hair growth. In addition this pathway is required for adaptation to challenging growth conditions such as high salt and high glucose medium and to elevated concentrations of the plant hormone ABA. We have also performed a bioinformatic analysis of polyproline-rich containing proteins as putative eIF5A targets to uncover their organization in clusters of protein networks to find molecular culprits for the disclosed phenotypes. This study represents a first attempt to provide a holistic view of the biological relevance of the spermidine-dependent hypusination pathway for plant growth and development. PMID:26973686

Proteome profiling in the aorta and kidney of type 1 diabetic rats

PubMed Central

Zhu, Rui; Jaffa, Miran A.; Zhao, Jingfu; Mirzaei, Parvin; Ahmed, Adnan; Kobeissy, Firas; Ziyadeh, Fuad N.; Mechref, Yehia

2017-01-01

Diabetes is associated with a number of metabolic and cardiovascular risk factors that contribute to a high rate of microvascular and macrovascular complications. The risk factors and mechanisms that contribute to the development of micro- and macrovascular disease in diabetes are not fully explained. In this study, we employed mass spectrometric analysis using tandem LC-MS/MS to generate a proteomic profile of protein abundance and post-translational modifications (PTM) in the aorta and kidney of diabetic rats. In addition, systems biology analyses were employed to identify key protein markers that can provide insights into molecular pathways and processes that are differentially regulated in the aorta and kidney of type 1 diabetic rats. Our results indicated that 188 (111 downregulated and 77 upregulated) proteins were significantly identified in the aorta of diabetic rats compared to normal controls. A total of 223 (109 downregulated and 114 upregulated) proteins were significantly identified in the kidney of diabetic rats compared to normal controls. When the protein profiles from the kidney and aorta of diabetic and control rats were analyzed by principal component analysis, a distinct separation of the groups was observed. In addition, diabetes resulted in a significant increase in PTM (oxidation, phosphorylation, and acetylation) of proteins in the kidney and aorta and this effect was partially reversed by insulin treatment. Ingenuity pathway analysis performed on the list of differentially expressed proteins depicted mitochondrial dysfunction, oxidative phosphorylation and acute phase response signaling to be among the altered canonical pathways by diabetes in both tissues. The findings of the present study provide a global proteomics view of markers that highlight the mechanisms and putative processes that modulate renal and vascular injury in diabetes. PMID:29121074

Proteome profiling in the aorta and kidney of type 1 diabetic rats.

PubMed

Al Hariri, Moustafa; Elmedawar, Mohamad; Zhu, Rui; Jaffa, Miran A; Zhao, Jingfu; Mirzaei, Parvin; Ahmed, Adnan; Kobeissy, Firas; Ziyadeh, Fuad N; Mechref, Yehia; Jaffa, Ayad A

2017-01-01

Diabetes is associated with a number of metabolic and cardiovascular risk factors that contribute to a high rate of microvascular and macrovascular complications. The risk factors and mechanisms that contribute to the development of micro- and macrovascular disease in diabetes are not fully explained. In this study, we employed mass spectrometric analysis using tandem LC-MS/MS to generate a proteomic profile of protein abundance and post-translational modifications (PTM) in the aorta and kidney of diabetic rats. In addition, systems biology analyses were employed to identify key protein markers that can provide insights into molecular pathways and processes that are differentially regulated in the aorta and kidney of type 1 diabetic rats. Our results indicated that 188 (111 downregulated and 77 upregulated) proteins were significantly identified in the aorta of diabetic rats compared to normal controls. A total of 223 (109 downregulated and 114 upregulated) proteins were significantly identified in the kidney of diabetic rats compared to normal controls. When the protein profiles from the kidney and aorta of diabetic and control rats were analyzed by principal component analysis, a distinct separation of the groups was observed. In addition, diabetes resulted in a significant increase in PTM (oxidation, phosphorylation, and acetylation) of proteins in the kidney and aorta and this effect was partially reversed by insulin treatment. Ingenuity pathway analysis performed on the list of differentially expressed proteins depicted mitochondrial dysfunction, oxidative phosphorylation and acute phase response signaling to be among the altered canonical pathways by diabetes in both tissues. The findings of the present study provide a global proteomics view of markers that highlight the mechanisms and putative processes that modulate renal and vascular injury in diabetes.

Integrated High Throughput Analysis Identifies GSK3 as a Crucial Determinant of p53-Mediated Apoptosis in Lung Cancer Cells.

PubMed

Zhang, Yu; Zhu, Chenyang; Sun, Bangyao; Lv, Jiawei; Liu, Zhonghua; Liu, Shengwang; Li, Hai

2017-01-01

p53 dysfunction is frequently observed in lung cancer. Although restoring the tumour suppressor function of p53 is recently approved as a putative strategy for combating cancers, the lack of understanding of the molecular mechanism underlying p53-mediated lung cancer suppression has limited the application of p53-based therapies in lung cancer. Using RNA sequencing, we determined the transcriptional profile of human non-small cell lung carcinoma A549 cells after treatment with two p53-activating chemical compounds, nutlin and RITA, which could induce A549 cell cycle arrest and apoptosis, respectively. Bioinformatics analysis of genome-wide gene expression data showed that distinct transcription profiles were induced by nutlin and RITA and 66 pathways were differentially regulated by these two compounds. However, only two of these pathways, 'Adherens junction' and 'Axon guidance', were found to be synthetic lethal with p53 re-activation, as determined via integrated analysis of genome-wide gene expression profile and short hairpin RNA (shRNA) screening. Further functional protein association analysis of significantly regulated genes associated with these two synthetic lethal pathways indicated that GSK3 played a key role in p53-mediated A549 cell apoptosis, and then gene function study was performed, which revealed that GSK3 inhibition promoted p53-mediated A549 cell apoptosis in a p53 post-translational activity-dependent manner. Our findings provide us with new insights regarding the mechanism by which p53 mediates A549 apoptosis and may cast light on the development of more efficient p53-based strategies for treating lung cancer. © 201 The Author(s). Published by S. Karger AG, Basel.

Brucella Modulates Secretory Trafficking via Multiple Type IV Secretion Effector Proteins

PubMed Central

Myeni, Sebenzile; Child, Robert; Ng, Tony W.; Kupko, John J.; Wehrly, Tara D.; Porcella, Stephen F.; Knodler, Leigh A.; Celli, Jean

2013-01-01

The intracellular pathogenic bacterium Brucella generates a replicative vacuole (rBCV) derived from the endoplasmic reticulum via subversion of the host cell secretory pathway. rBCV biogenesis requires the expression of the Type IV secretion system (T4SS) VirB, which is thought to translocate effector proteins that modulate membrane trafficking along the endocytic and secretory pathways. To date, only a few T4SS substrates have been identified, whose molecular functions remain unknown. Here, we used an in silico screen to identify putative T4SS effector candidate proteins using criteria such as limited homology in other bacterial genera, the presence of features similar to known VirB T4SS effectors, GC content and presence of eukaryotic-like motifs. Using β-lactamase and CyaA adenylate cyclase reporter assays, we identified eleven proteins translocated into host cells by Brucella, five in a VirB T4SS-dependent manner, namely BAB1_0678 (BspA), BAB1_0712 (BspB), BAB1_0847 (BspC), BAB1_1671 (BspE) and BAB1_1948 (BspF). A subset of the translocated proteins targeted secretory pathway compartments when ectopically expressed in HeLa cells, and the VirB effectors BspA, BspB and BspF inhibited protein secretion. Brucella infection also impaired host protein secretion in a process requiring BspA, BspB and BspF. Single or combined deletions of bspA, bspB and bspF affected Brucella ability to replicate in macrophages and persist in the liver of infected mice. Taken together, these findings demonstrate that Brucella modulates secretory trafficking via multiple T4SS effector proteins that likely act coordinately to promote Brucella pathogenesis. PMID:23950720

Transcriptomics of In Vitro Immune-Stimulated Hemocytes from the Manila Clam Ruditapes philippinarum Using High-Throughput Sequencing

PubMed Central

Moreira, Rebeca; Balseiro, Pablo; Planas, Josep V.; Fuste, Berta; Beltran, Sergi; Novoa, Beatriz; Figueras, Antonio

2012-01-01

Background The Manila clam (Ruditapes philippinarum) is a worldwide cultured bivalve species with important commercial value. Diseases affecting this species can result in large economic losses. Because knowledge of the molecular mechanisms of the immune response in bivalves, especially clams, is scarce and fragmentary, we sequenced RNA from immune-stimulated R. philippinarum hemocytes by 454-pyrosequencing to identify genes involved in their immune defense against infectious diseases. Methodology and Principal Findings High-throughput deep sequencing of R. philippinarum using 454 pyrosequencing technology yielded 974,976 high-quality reads with an average read length of 250 bp. The reads were assembled into 51,265 contigs and the 44.7% of the translated nucleotide sequences into protein were annotated successfully. The 35 most frequently found contigs included a large number of immune-related genes, and a more detailed analysis showed the presence of putative members of several immune pathways and processes like the apoptosis, the toll like signaling pathway and the complement cascade. We have found sequences from molecules never described in bivalves before, especially in the complement pathway where almost all the components are present. Conclusions This study represents the first transcriptome analysis using 454-pyrosequencing conducted on R. philippinarum focused on its immune system. Our results will provide a rich source of data to discover and identify new genes, which will serve as a basis for microarray construction and the study of gene expression as well as for the identification of genetic markers. The discovery of new immune sequences was very productive and resulted in a large variety of contigs that may play a role in the defense mechanisms of Ruditapes philippinarum. PMID:22536348

Characterization of CpSte11, a MAPKKK gene of Cryphonectria parasitica, and initial evidence of its involvement in the pheromone response pathway.

PubMed

Park, Jin-Ah; Kim, Jung-Mi; Park, Seung-Moon; Kim, Dae-Hyuk

2012-04-01

The gene CpSte11 of Cryphonectria parasitica, which encodes a yeast Ste11 homologue, was cloned and characterized. Gene replacement analysis revealed a high frequency of CpSte11 null mutants. When compared with the wild-type parent strain, CpSte11 null mutants showed no difference in terms of growth rate or pigmentation. However, CpSte11 null mutants showed a marked decrease in both the number and size of stromal pustules on chestnut twigs. The virulence test showed that, in comparison with those of the wild-type and virus-infected hypovirulent strains, CpSte11 null mutants produced necrotic areas of intermediate size. Disruption of the CpSte11 gene also resulted in defects in female fertility. Down-regulation of transcripts for the mating pheromone precursor gene, Mf2/2, and mating response transcription factors, such as cpst12 and pro1, was observed in CpSte11 null mutants. The down-regulation of Mf2/2, cpst12 and pro1 was also observed in the mutant phenotype of Cpmk2, a mating response Fus3-like mitogen-activated protein kinase (MAPK) gene, but not in the mutant of Cpmk1, a high-osmolarity glycerol Hog1-like MAPK gene. These results indicate that the cloned CpSte11 gene is functionally involved in the mating response pathway and acts through downstream targets, including Cpmk2, cpst12, pro1 and Mf2/2. However, the characteristics of the CpSte11 null mutant were fully phenocopied only in the cpst12 null mutant, but not in other studied null mutants of components of the putative mating response pathway. © 2011 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2011 BSPP AND BLACKWELL PUBLISHING LTD.

The role of the Hippo pathway in human disease and tumorigenesis

PubMed Central

2014-01-01

Understanding the molecular nature of human cancer is essential to the development of effective and personalized therapies. Several different molecular signal transduction pathways drive tumorigenesis when deregulated and respond to different types of therapeutic interventions. The Hippo signaling pathway has been demonstrated to play a central role in the regulation of tissue and organ size during development. The deregulation of Hippo signaling leads to a concurrent combination of uncontrolled cellular proliferation and inhibition of apoptosis, two key hallmarks in cancer development. The molecular nature of this pathway was first uncovered in Drosophila melanogaster through genetic screens to identify regulators of cell growth and cell division. The pathway is strongly conserved in humans, rendering Drosophila a suitable and efficient model system to better understand the molecular nature of this pathway. In the present study, we review the current understanding of the molecular mechanism and clinical impact of the Hippo pathway. Current studies have demonstrated that a variety of deregulated molecules can alter Hippo signaling, leading to the constitutive activation of the transcriptional activator YAP or its paralog TAZ. Additionally, the Hippo pathway integrates inputs from a number of growth signaling pathways, positioning the Hippo pathway in a central role in the regulation of tissue size. Importantly, deregulated Hippo signaling is frequently observed in human cancers. YAP is commonly activated in a number of in vitro and in vivo models of tumorigenesis, as well as a number of human cancers. The common activation of YAP in many different tumor types provides an attractive target for potential therapeutic intervention. PMID:25097728

Molecular Genetic Analysis of Activation-tagged Transcription Factors Thought to be Involved in Photomorphogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Neff, Michael

2011-06-23

Plants utilize light as a source of information via families of photoreceptors such as the red/far-red absorbing phytochromes (PHY) and the blue/UVA absorbing cryptochromes (CRY). The main goal of the Neff lab is to use molecular-genetic mutant screens to elucidate signaling components downstream of these photoreceptors. Activation-tagging mutagenesis led to the identification of two putative transcription factors that may be involved in both photomorphogenesis and hormone signaling pathways. sob1-D (suppressor of phyB-dominant) mutant phenotypes are caused by the over-expression of a Dof transcription factor previously named OBP3. Our previous studies indicate that OBP3 is a negative regulator of light-mediated cotyledonmore » expansion and may be involved in modulating responsiveness to the growth-regulating hormone auxin. The sob2-D mutant uncovers a role for LEP, a putative AP2/EREBP-like transcription factor, in seed germination, hypocotyl elongation and responsiveness to the hormone abscisic acid. Based on photobiological and genetic analysis of OBP3-knockdown and LEP-null mutations, we hypothesize that these transcription factors are involved in both light-mediated seedling development and hormone signaling. To examine the role that these genes play in photomorphogenesis we will: 1) Further explore the genetic role of OBP3 in cotyledon/leaf expansion and other photomorphogenic processes as well as examine potential physical interactions between OBP3 and CRY1 or other signaling components that genetically interact with this transcription factor 2) Test the hypothesis that OBP3 is genetically involved in auxin signaling and root development as well as examine the affects of this hormone and light on OBP3 protein accumulation. 3) Test the hypothesis that LEP is involved in seed germination, seedling photomorphogenesis and hormone signaling. Together these experiments will lead to a greater understanding of the complexity of interactions between photoreceptors and DNA-interacting proteins during photomorphogenesis. These studies also address the roles OBP3 and LEP may play as points of intersection between hormone signaling and photomorphogenesis. In the future, phenotypes caused by altered expression of these genes may generate useful traits for improving crop yield.« less

Multifidus Muscle Changes After Back Injury Are Characterized by Structural Remodeling of Muscle, Adipose and Connective Tissue, but Not Muscle Atrophy: Molecular and Morphological Evidence.

PubMed

Hodges, Paul W; James, Gregory; Blomster, Linda; Hall, Leanne; Schmid, Annina; Shu, Cindy; Little, Chris; Melrose, James

2015-07-15

Longitudinal case-controlled animal study. To investigate putative cellular mechanisms to explain structural changes in muscle and adipose and connective tissues of the back muscles after intervertebral disc (IVD) injury. Structural back muscle changes are ubiquitous with back pain/injury and considered relevant for outcome, but their exact nature, time course, and cellular mechanisms remain elusive. We used an animal model that produces phenotypic back muscle changes after IVD injury to study these issues at the cellular/molecular level. Multifidus muscle was harvested from both sides of the spine at L1-L2 and L3-L4 IVDs in 27 castrated male sheep at 3 (n = 10) or 6 (n = 17) months after a surgical anterolateral IVD injury at both levels. Ten control sheep underwent no surgery (3 mo, n = 4; 6 mo, n = 6). Tissue was harvested at L4 for histological analysis of cross-sectional area of muscle and adipose and connective tissue (whole muscle), plus immunohistochemistry to identify proportion and cross-sectional area of individual muscle fiber types in the deepest fascicle. Quantitative polymerase chain reaction measured gene expression of typical cytokines/signaling molecules at L2. Contrary to predictions, there was no multifidus muscle atrophy (whole muscle or individual fiber). There was increased adipose and connective tissue (fibrotic proliferation) cross-sectional area and slow-to-fast muscle fiber transition at 6 but not 3 months. Within the multifidus muscle, increases in the expression of several cytokines (tumor necrosis factor α and interleukin-1β) and molecules that signal trophic/atrophic processes for the 3 tissue types (e.g., growth factor pathway [IGF-1, PI3k, Akt1, mTOR], potent tissue modifiers [calcineurin, PCG-1α, and myostatin]) were present. This study provides cellular evidence that refutes the presence of multifidus muscle atrophy accompanying IVD degeneration at this intermediate time point. Instead, adipose/connective tissue increased in parallel with the expression of the genes that provide putative mechanisms for multifidus structural remodeling. This provides novel targets for pharmacological and physical interventions. N/A.

De novo metatranscriptome assembly and coral gene expression profile of Montipora capitata with growth anomaly.

PubMed

Frazier, Monika; Helmkampf, Martin; Bellinger, M Renee; Geib, Scott M; Takabayashi, Misaki

2017-09-11

Scleractinian corals are a vital component of coral reef ecosystems, and of significant cultural and economic value worldwide. As anthropogenic and natural stressors are contributing to a global decline of coral reefs, understanding coral health is critical to help preserve these ecosystems. Growth anomaly (GA) is a coral disease that has significant negative impacts on coral biology, yet our understanding of its etiology and pathology is lacking. In this study we used RNA-seq along with de novo metatranscriptome assembly and homology assignment to identify coral genes that are expressed in three distinct coral tissue types: tissue from healthy corals ("healthy"), GA lesion tissue from diseased corals ("GA-affected") and apparently healthy tissue from diseased corals ("GA-unaffected"). We conducted pairwise comparisons of gene expression among these three tissue types to identify genes and pathways that help us to unravel the molecular pathology of this coral disease. The quality-filtered de novo-assembled metatranscriptome contained 76,063 genes, of which 13,643 were identified as putative coral genes. Overall gene expression profiles of coral genes revealed high similarity between healthy tissue samples, in contrast to high variance among diseased samples. This indicates GA has a variety of genetic effects at the colony level, including on seemingly healthy (GA-unaffected) tissue. A total of 105 unique coral genes were found differentially expressed among tissue types. Pairwise comparisons revealed the greatest number of differentially expressed genes between healthy and GA-affected tissue (93 genes), followed by healthy and GA-unaffected tissue (33 genes), and GA-affected and -unaffected tissue (7 genes). The putative function of these genes suggests GA is associated with changes in the activity of genes involved in developmental processes and activation of the immune system. This is one of the first transcriptome-level studies to investigate coral GA, and the first metatranscriptome assembly for the M. capitata holobiont. The gene expression data, metatranscriptome assembly and methodology developed through this study represent a significant addition to the molecular information available to further our understanding of this coral disease.

Tangential migratory pathways of subpallial origin in the embryonic telencephalon of sharks: evolutionary implications.

PubMed

Quintana-Urzainqui, Idoia; Rodríguez-Moldes, Isabel; Mazan, Sylvie; Candal, Eva

2015-09-01

Tangential neuronal migration occurs along different axes from the axis demarcated by radial glia and it is thought to have evolved as a mechanism to increase the diversity of cell types in brain areas, which in turn resulted in increased complexity of functional networks. In the telencephalon of amniotes, different embryonic tangential pathways have been characterized. However, little is known about the exact routes of migrations in basal vertebrates. Cartilaginous fishes occupy a key phylogenetic position to assess the ancestral condition of vertebrate brain organization. In order to identify putative subpallial-derived tangential migratory pathways in the telencephalon of sharks, we performed a detailed analysis of the distribution pattern of GAD and Dlx2, two reliable markers of tangentially migrating interneurons of subpallial origin in the developing forebrain. We propose the existence of five tangential routes directed toward different telencephalic regions. We conclude that four of the five routes might have emerged in the common ancestor of jawed vertebrates. We have paid special attention to the characterization of the proposed migratory pathway directed towards the olfactory bulbs. Our results suggest that it may be equivalent to the "rostral migratory stream" of mammals and led us to propose a hypothesis about its evolution. The analysis of the final destinations of two other streams allowed us to identify the putative dorsal and medial pallium of sharks, the regions from which the neocortex and hippocampus might have, respectively, evolved. Derived features were also reported and served to explain some distinctive traits in the morphology of the telencephalon of cartilaginous fishes.

De Novo Transcriptome Assembly and Characterization of Lithospermum officinale to Discover Putative Genes Involved in Specialized Metabolites Biosynthesis.

PubMed

Rai, Amit; Nakaya, Taiki; Shimizu, Yohei; Rai, Megha; Nakamura, Michimi; Suzuki, Hideyuki; Saito, Kazuki; Yamazaki, Mami

2018-05-29

Lithospermum officinale is a valuable source of bioactive metabolites with medicinal and industrial values. However, little is known about genes involved in the biosynthesis of these metabolites, primarily due to the lack of genome or transcriptome resources. This study presents the first effort to establish and characterize de novo transcriptome assembly resource for L. officinale and expression analysis for three of its tissues, namely leaf, stem, and root. Using over 4Gbps of RNA-sequencing datasets, we obtained de novo transcriptome assembly of L. officinale , consisting of 77,047 unigenes with assembly N50 value as 1524 bps. Based on transcriptome annotation and functional classification, 52,766 unigenes were assigned with putative genes functions, gene ontology terms, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. KEGG pathway and gene ontology enrichment analysis using highly expressed unigenes across three tissues and targeted metabolome analysis showed active secondary metabolic processes enriched specifically in the root of L. officinale . Using co-expression analysis, we also identified 20 and 48 unigenes representing different enzymes of lithospermic/chlorogenic acid and shikonin biosynthesis pathways, respectively. We further identified 15 candidate unigenes annotated as cytochrome P450 with the highest expression in the root of L. officinale as novel genes with a role in key biochemical reactions toward shikonin biosynthesis. Thus, through this study, we not only generated a high-quality genomic resource for L. officinale but also propose candidate genes to be involved in shikonin biosynthesis pathways for further functional characterization. Georg Thieme Verlag KG Stuttgart · New York.

Analysis of expressed sequence tags from Maize mosaic rhabdovirus-infected gut tissues of Peregrinus maidis reveals the presence of key components of insect innate immunity.

PubMed

Whitfield, A E; Rotenberg, D; Aritua, V; Hogenhout, S A

2011-04-01

The corn planthopper, Peregrinus maidis, causes direct feeding damage to plants and transmits Maize mosaic rhabdovirus (MMV) in a persistent-propagative manner. MMV must cross several insect tissue layers for successful transmission to occur, and the gut serves as an important barrier for rhabdovirus transmission. In order to facilitate the identification of proteins that may interact with MMV either by facilitating acquisition or responding to virus infection, we generated and analysed the gut transcriptome of P. maidis. From two normalized cDNA libraries, we generated a P. maidis gut transcriptome composed of 20,771 expressed sequence tags (ESTs). Assembly of the sequences yielded 1860 contigs and 14,032 singletons, and biological roles were assigned to 5793 (36%). Comparison of P. maidis ESTs with other insect amino acid sequences revealed that P. maidis shares greatest sequence similarity with another hemipteran, the brown planthopper Nilaparvata lugens. We identified 202 P. maidis transcripts with putative homology to proteins associated with insect innate immunity, including those implicated in the Toll, Imd, JAK/STAT, Jnk and the small-interfering RNA-mediated pathways. Sequence comparisons between our P. maidis gut EST collection and the currently available National Center for Biotechnology Information EST database collection for Ni. lugens revealed that a pathogen recognition receptor in the Imd pathway, peptidoglycan recognition protein-long class (PGRP-LC), is present in these two members of the family Delphacidae; however, these recognition receptors are lacking in the model hemipteran Acyrthosiphon pisum. In addition, we identified sequences in the P. maidis gut transcriptome that share significant amino acid sequence similarities with the rhabdovirus receptor molecule, acetylcholine receptor (AChR), found in other hosts. This EST analysis sheds new light on immune response pathways in hemipteran guts that will be useful for further dissecting innate defence response pathways to rhabdovirus infection. © 2011 The Authors. Insect Molecular Biology © 2011 The Royal Entomological Society.

Evidence of activation of the Toll-like receptor-4 proinflammatory pathway in patients with schizophrenia

PubMed Central

García-Bueno, Borja; Gassó, Patricia; MacDowell, Karina S.; Callado, Luis F.; Mas, Sergi; Bernardo, Miguel; Lafuente, Amalia; Meana, J. Javier; Leza, Juan C.

2016-01-01

Background Alterations in the innate immune/inflammatory system may underlie the pathophysiology of schizophrenia, but we do not understand the mechanisms involved. The main agents of innate immunity are the Toll-like receptors (TLRs), which detect molecular patterns associated with damage and pathogens. The TLR first reported was TLR4, and it is still the most studied one. Methods We aimed to describe putative modifications to the TLR4 proinflammatory pathway using 2 different strategies in 2 cohorts of patients with schizophrenia and matched controls: 1) quantification of protein and mRNA expression in postmortem prefrontal cortex samples from 30 patients with schizophrenia and 30 controls, and 2) identification of single nucleotide polymorphisms associated with the risk of schizophrenia using whole blood samples from 214 patients with schizophrenia and 216 controls. Results We found evidence of alterations in the expression of the initial elements of the TLR4 signalling pathway (TLR4, Myeloid differentiation primary response gene 88 [MyD88] and nuclear factor-κ B [NF-κB]) in the PFC of patients with schizophrenia. These alterations seem to depend on the presence/absence of antipsychotic treatment at death. Moreover, a polymorphism within the MyD88 gene was significantly associated with schizophrenia risk. Limitations The use of 2 different approaches in 2 different cohorts, the lack of a complementary neuropsychiatric group, the possible confounding effects of antipsychotic treatment and suicide are the main limitations of our study. Conclusion The evidence from this dual approach suggests there is an altered innate immune response in patients with chronic schizophrenia in which the TLR4 proinflammatory pathway could be affected. Improved understanding of the stimuli and mechanisms responsible for this response could lead to improved schizophrenia treatment and better control of the side effects of current antipsychotics. PMID:27070349

De Novo Assembly of Mud Loach (Misgurnus anguillicaudatus) Skin Transcriptome to Identify Putative Genes Involved in Immunity and Epidermal Mucus Secretion

PubMed Central

Long, Yong; Li, Qing; Zhou, Bolan; Song, Guili; Li, Tao; Cui, Zongbin

2013-01-01

Fish skin serves as the first line of defense against a wide variety of chemical, physical and biological stressors. Secretion of mucus is among the most prominent characteristics of fish skin and numerous innate immune factors have been identified in the epidermal mucus. However, molecular mechanisms underlying the mucus secretion and immune activities of fish skin remain largely unclear due to the lack of genomic and transcriptomic data for most economically important fish species. In this study, we characterized the skin transcriptome of mud loach using Illumia paired-end sequencing. A total of 40364 unigenes were assembled from 86.6 million (3.07 gigabases) filtered reads. The mean length, N50 size and maximum length of assembled transcripts were 387, 611 and 8670 bp, respectively. A total of 17336 (43.76%) unigenes were annotated by blast searches against the NCBI non-redundant protein database. Gene ontology mapping assigned a total of 108513 GO terms to 15369 (38.08%) unigenes. KEGG orthology mapping annotated 9337 (23.23%) unigenes. Among the identified KO categories, immune system is the largest category that contains various components of multiple immune pathways such as chemokine signaling, leukocyte transendothelial migration and T cell receptor signaling, suggesting the complexity of immune mechanisms in fish skin. As for mucin biosynthesis, 37 unigenes were mapped to 7 enzymes of the mucin type O-glycan biosynthesis pathway and 8 members of the polypeptide N-acetylgalactosaminyltransferase family were identified. Additionally, 38 unigenes were mapped to 23 factors of the SNARE interactions in vesicular transport pathway, indicating that the activity of this pathway is required for the processes of epidermal mucus storage and release. Moreover, 1754 simple sequence repeats (SSRs) were detected in 1564 unigenes and dinucleotide repeats represented the most abundant type. These findings have laid the foundation for further understanding the secretary processes and immune functions of loach skin mucus. PMID:23437293

De novo assembly of mud loach (Misgurnus anguillicaudatus) skin transcriptome to identify putative genes involved in immunity and epidermal mucus secretion.

PubMed

Long, Yong; Li, Qing; Zhou, Bolan; Song, Guili; Li, Tao; Cui, Zongbin

2013-01-01

Fish skin serves as the first line of defense against a wide variety of chemical, physical and biological stressors. Secretion of mucus is among the most prominent characteristics of fish skin and numerous innate immune factors have been identified in the epidermal mucus. However, molecular mechanisms underlying the mucus secretion and immune activities of fish skin remain largely unclear due to the lack of genomic and transcriptomic data for most economically important fish species. In this study, we characterized the skin transcriptome of mud loach using Illumia paired-end sequencing. A total of 40364 unigenes were assembled from 86.6 million (3.07 gigabases) filtered reads. The mean length, N50 size and maximum length of assembled transcripts were 387, 611 and 8670 bp, respectively. A total of 17336 (43.76%) unigenes were annotated by blast searches against the NCBI non-redundant protein database. Gene ontology mapping assigned a total of 108513 GO terms to 15369 (38.08%) unigenes. KEGG orthology mapping annotated 9337 (23.23%) unigenes. Among the identified KO categories, immune system is the largest category that contains various components of multiple immune pathways such as chemokine signaling, leukocyte transendothelial migration and T cell receptor signaling, suggesting the complexity of immune mechanisms in fish skin. As for mucin biosynthesis, 37 unigenes were mapped to 7 enzymes of the mucin type O-glycan biosynthesis pathway and 8 members of the polypeptide N-acetylgalactosaminyltransferase family were identified. Additionally, 38 unigenes were mapped to 23 factors of the SNARE interactions in vesicular transport pathway, indicating that the activity of this pathway is required for the processes of epidermal mucus storage and release. Moreover, 1754 simple sequence repeats (SSRs) were detected in 1564 unigenes and dinucleotide repeats represented the most abundant type. These findings have laid the foundation for further understanding the secretary processes and immune functions of loach skin mucus.

MetaboLyzer: A Novel Statistical Workflow for Analyzing Post-Processed LC/MS Metabolomics Data

PubMed Central

Mak, Tytus D.; Laiakis, Evagelia C.; Goudarzi, Maryam; Fornace, Albert J.

2014-01-01

Metabolomics, the global study of small molecules in a particular system, has in the last few years risen to become a primary –omics platform for the study of metabolic processes. With the ever-increasing pool of quantitative data yielded from metabolomic research, specialized methods and tools with which to analyze and extract meaningful conclusions from these data are becoming more and more crucial. Furthermore, the depth of knowledge and expertise required to undertake a metabolomics oriented study is a daunting obstacle to investigators new to the field. As such, we have created a new statistical analysis workflow, MetaboLyzer, which aims to both simplify analysis for investigators new to metabolomics, as well as provide experienced investigators the flexibility to conduct sophisticated analysis. MetaboLyzer’s workflow is specifically tailored to the unique characteristics and idiosyncrasies of postprocessed liquid chromatography/mass spectrometry (LC/MS) based metabolomic datasets. It utilizes a wide gamut of statistical tests, procedures, and methodologies that belong to classical biostatistics, as well as several novel statistical techniques that we have developed specifically for metabolomics data. Furthermore, MetaboLyzer conducts rapid putative ion identification and putative biologically relevant analysis via incorporation of four major small molecule databases: KEGG, HMDB, Lipid Maps, and BioCyc. MetaboLyzer incorporates these aspects into a comprehensive workflow that outputs easy to understand statistically significant and potentially biologically relevant information in the form of heatmaps, volcano plots, 3D visualization plots, correlation maps, and metabolic pathway hit histograms. For demonstration purposes, a urine metabolomics data set from a previously reported radiobiology study in which samples were collected from mice exposed to gamma radiation was analyzed. MetaboLyzer was able to identify 243 statistically significant ions out of a total of 1942. Numerous putative metabolites and pathways were found to be biologically significant from the putative ion identification workflow. PMID:24266674

Transcriptome Analysis of Blunt Snout Bream (Megalobrama amblycephala) Reveals Putative Differential Expression Genes Related to Growth and Hypoxia

PubMed Central

Li, Fu-Gui; Chen, Jie; Jiang, Xia-Yun; Zou, Shu-Ming

2015-01-01

The blunt snout bream (Megalobrama amblycephala) is an important freshwater aquaculture species, but it is sensitive to hypoxia. No transcriptome data related to growth and hypoxia response are available for this species. In this study, we performed de novo transcriptome sequencing for the liver and gills of the fast-growth family and slow-growth family derived from ‘Pujiang No.1’ F10 blunt snout bream that were under hypoxic stress and normoxia, respectively. The fish were divided into the following 4 groups: fast-growth family under hypoxic stress, FH; slow-growth family under hypoxic stress, SH; fast-growth family under normoxia, FN; and slow-growth family under normoxia, SN. A total of 185 million high-quality reads were obtained from the normalized cDNA of the pooled samples, which were assembled into 465,582 contigs and 237,172 transcripts. A total of 31,338 transcripts from the same locus (unigenes) were annotated and assigned to 104 functional groups, and 23,103 unigenes were classified into seven main categories, including 45 secondary KEGG pathways. A total of 22,255 (71%) known putative unigenes were found to be shared across the genomes of five model fish species and mammals, and a substantial number (9.4%) of potentially novel genes were identified. When 6,639 unigenes were used in the analysis of differential expression (DE) genes, the number of putative DE genes related to growth pathways in FH, SH, SN and FN was 159, 118, 92 and 65 in both the liver and gills, respectively, and the number of DE genes related to hypoxic response was 57, 33, 23 and 21 in FH, FN, SH and SN, respectively. Our results suggest that growth performance of the fast-growth family should be due to complex mutual gene regulatory mechanisms of these putative DE genes between growth and hypoxia. PMID:26554582

Identification of druggable cancer driver genes amplified across TCGA datasets.

PubMed

Chen, Ying; McGee, Jeremy; Chen, Xianming; Doman, Thompson N; Gong, Xueqian; Zhang, Youyan; Hamm, Nicole; Ma, Xiwen; Higgs, Richard E; Bhagwat, Shripad V; Buchanan, Sean; Peng, Sheng-Bin; Staschke, Kirk A; Yadav, Vipin; Yue, Yong; Kouros-Mehr, Hosein

2014-01-01

The Cancer Genome Atlas (TCGA) projects have advanced our understanding of the driver mutations, genetic backgrounds, and key pathways activated across cancer types. Analysis of TCGA datasets have mostly focused on somatic mutations and translocations, with less emphasis placed on gene amplifications. Here we describe a bioinformatics screening strategy to identify putative cancer driver genes amplified across TCGA datasets. We carried out GISTIC2 analysis of TCGA datasets spanning 16 cancer subtypes and identified 486 genes that were amplified in two or more datasets. The list was narrowed to 75 cancer-associated genes with potential "druggable" properties. The majority of the genes were localized to 14 amplicons spread across the genome. To identify potential cancer driver genes, we analyzed gene copy number and mRNA expression data from individual patient samples and identified 42 putative cancer driver genes linked to diverse oncogenic processes. Oncogenic activity was further validated by siRNA/shRNA knockdown and by referencing the Project Achilles datasets. The amplified genes represented a number of gene families, including epigenetic regulators, cell cycle-associated genes, DNA damage response/repair genes, metabolic regulators, and genes linked to the Wnt, Notch, Hedgehog, JAK/STAT, NF-KB and MAPK signaling pathways. Among the 42 putative driver genes were known driver genes, such as EGFR, ERBB2 and PIK3CA. Wild-type KRAS was amplified in several cancer types, and KRAS-amplified cancer cell lines were most sensitive to KRAS shRNA, suggesting that KRAS amplification was an independent oncogenic event. A number of MAP kinase adapters were co-amplified with their receptor tyrosine kinases, such as the FGFR adapter FRS2 and the EGFR family adapters GRB2 and GRB7. The ubiquitin-like ligase DCUN1D1 and the histone methyltransferase NSD3 were also identified as novel putative cancer driver genes. We discuss the patient tailoring implications for existing cancer drug targets and we further discuss potential novel opportunities for drug discovery efforts.

Identification of Druggable Cancer Driver Genes Amplified across TCGA Datasets

PubMed Central

Chen, Ying; McGee, Jeremy; Chen, Xianming; Doman, Thompson N.; Gong, Xueqian; Zhang, Youyan; Hamm, Nicole; Ma, Xiwen; Higgs, Richard E.; Bhagwat, Shripad V.; Buchanan, Sean; Peng, Sheng-Bin; Staschke, Kirk A.; Yadav, Vipin; Yue, Yong; Kouros-Mehr, Hosein

2014-01-01

The Cancer Genome Atlas (TCGA) projects have advanced our understanding of the driver mutations, genetic backgrounds, and key pathways activated across cancer types. Analysis of TCGA datasets have mostly focused on somatic mutations and translocations, with less emphasis placed on gene amplifications. Here we describe a bioinformatics screening strategy to identify putative cancer driver genes amplified across TCGA datasets. We carried out GISTIC2 analysis of TCGA datasets spanning 14 cancer subtypes and identified 461 genes that were amplified in two or more datasets. The list was narrowed to 73 cancer-associated genes with potential “druggable” properties. The majority of the genes were localized to 14 amplicons spread across the genome. To identify potential cancer driver genes, we analyzed gene copy number and mRNA expression data from individual patient samples and identified 40 putative cancer driver genes linked to diverse oncogenic processes. Oncogenic activity was further validated by siRNA/shRNA knockdown and by referencing the Project Achilles datasets. The amplified genes represented a number of gene families, including epigenetic regulators, cell cycle-associated genes, DNA damage response/repair genes, metabolic regulators, and genes linked to the Wnt, Notch, Hedgehog, JAK/STAT, NF-KB and MAPK signaling pathways. Among the 40 putative driver genes were known driver genes, such as EGFR, ERBB2 and PIK3CA. Wild-type KRAS was amplified in several cancer types, and KRAS-amplified cancer cell lines were most sensitive to KRAS shRNA, suggesting that KRAS amplification was an independent oncogenic event. A number of MAP kinase adapters were co-amplified with their receptor tyrosine kinases, such as the FGFR adapter FRS2 and the EGFR family adapter GRB7. The ubiquitin-like ligase DCUN1D1 and the histone methyltransferase NSD3 were also identified as novel putative cancer driver genes. We discuss the patient tailoring implications for existing cancer drug targets and we further discuss potential novel opportunities for drug discovery efforts. PMID:24874471

Comparative transcriptome analysis of Methylibium petroleiphilum PM1 exposed to the fuel oxygenates methyl tert-butyl ether and ethanol.

PubMed

Hristova, Krassimira R; Schmidt, Radomir; Chakicherla, Anu Y; Legler, Tina C; Wu, Janice; Chain, Patrick S; Scow, Kate M; Kane, Staci R

2007-11-01

High-density whole-genome cDNA microarrays were used to investigate substrate-dependent gene expression of Methylibium petroleiphilum PM1, one of the best-characterized aerobic methyl tert-butyl ether (MTBE)-degrading bacteria. Differential gene expression profiling was conducted with PM1 grown on MTBE and ethanol as sole carbon sources. Based on microarray high scores and protein similarity analysis, an MTBE regulon located on the megaplasmid was identified for further investigation. Putative functions for enzymes encoded in this regulon are described with relevance to the predicted MTBE degradation pathway. A new unique dioxygenase enzyme system that carries out the hydroxylation of tert-butyl alcohol to 2-methyl-2-hydroxy-1-propanol in M. petroleiphilum PM1 was discovered. Hypotheses regarding the acquisition and evolution of MTBE genes as well as the involvement of IS elements in these complex processes were formulated. The pathways for toluene, phenol, and alkane oxidation via toluene monooxygenase, phenol hydroxylase, and propane monooxygenase, respectively, were upregulated in MTBE-grown cells compared to ethanol-grown cells. Four out of nine putative cyclohexanone monooxygenases were also upregulated in MTBE-grown cells. The expression data allowed prediction of several hitherto-unknown enzymes of the upper MTBE degradation pathway in M. petroleiphilum PM1 and aided our understanding of the regulation of metabolic processes that may occur in response to pollutant mixtures and perturbations in the environment.

Use of the adverse outcome pathway framework to represent cross-species consequences of specific pathway perturbations

EPA Science Inventory

The adverse outcome pathway (AOP) framework has been developed as a means for assembling scientifically defensible descriptions of how particular molecular perturbations, termed molecular initiating events (MIEs), can evoke a set of predictable responses at different levels of bi...

Prioritizing and modelling of putative drug target proteins of Candida albicans by systems biology approach.

PubMed

Ismail, Tariq; Fatima, Nighat; Muhammad, Syed Aun; Zaidi, Syed Saoud; Rehman, Nisar; Hussain, Izhar; Tariq, Najam Us Sahr; Amirzada, Imran; Mannan, Abdul

2018-01-01

Candida albicans (Candida albicans) is one of the major sources of nosocomial infections in humans which may prove fatal in 30% of cases. The hospital acquired infection is very difficult to treat affectively due to the presence of drug resistant pathogenic strains, therefore there is a need to find alternative drug targets to cure this infection. In silico and computational level frame work was used to prioritize and establish antifungal drug targets of Candida albicans. The identification of putative drug targets was based on acquiring 5090 completely annotated genes of Candida albicans from available databases which were categorized into essential and non-essential genes. The result indicated that 9% of proteins were essential and could become potential candidates for intervention which might result in pathogen eradication. We studied cluster of orthologs and the subtractive genomic analysis of these essential proteins against human genome was made as a reference to minimize the side effects. It was seen that 14% of Candida albicans proteins were evolutionary related to the human proteins while 86% are non-human homologs. In the next step of compatible drug target selections, the non-human homologs were sequentially compared to the human microbiome data to minimize the potential effects against gut flora which accumulated to 38% of the essential genome. The sub-cellular localization of these candidate proteins in fungal cellular systems indicated that 80% of them are cytoplasmic, 10% are mitochondrial and the remaining 10% are associated with the cell wall. The role of these non-human and non-gut flora putative target proteins in Candida albicans biological pathways was studied. Due to their integrated and critical role in Candida albicans replication cycle, four proteins were selected for molecular modeling. For drug designing and development, four high quality and reliable protein models with more than 70% sequence identity were constructed. These proteins are used for the docking studies of the known and new ligands (unpublished data). Our study will be an effective framework for drug target identifications of pathogenic microbial strains and development of new therapies against the infections they cause.

Metallofullerenol Gd@C82(OH)22 distracts the proline-rich-motif from putative binding on the SH3 domain

NASA Astrophysics Data System (ADS)

Kang, Seung-Gu; Huynh, Tien; Zhou, Ruhong

2013-03-01

Biocompatibility is often regarded as one important aspect of de novo designed nanomaterials for biosafety. However, the toxicological effect, appearing along with its latency, is much more difficult to address by linearly mapping physicochemical properties of related nanomaterials with biological effects such as immune or cellular regulatory responses due to the complicated protein-protein interactions. Here, we investigate a potential interference of a metallofullerenol, Gd@C82(OH)22, on the function of SH3 domain, a highly promiscuous protein-protein interaction mediator involved in signaling and regulatory pathways through its binding with the proline-rich motif (PRM) peptides, using the atomistic molecular dynamics simulation. Our study shows that when only Gd@C82(OH)22 and the SH3 domain are present (without the PRM ligand), Gd@C82(OH)22 can interact with the SH3 domain by either directly blocking the hydrophobic active site or binding with a hydrophilic off-site with almost equal probability, which can be understood from its intrinsic amphiphilic nature. In a binding competition with the PRM onto the SH3 domain, however, the on-site binding mode is depleted while Gd@C82(OH)22 effectively intercepts the PRM from the putative binding site of the SH3 domain, implying that Gd@C82(OH)22 can disturb protein-protein interactions mediated by the SH3 domain. Despite a successful surface modification in an aqueous biological medium and a more recent demonstration as potential de novo cancer therapeutics, our study indicates that greater attention is needed in assessing the potential cytotoxicity of these nanomaterials.Biocompatibility is often regarded as one important aspect of de novo designed nanomaterials for biosafety. However, the toxicological effect, appearing along with its latency, is much more difficult to address by linearly mapping physicochemical properties of related nanomaterials with biological effects such as immune or cellular regulatory responses due to the complicated protein-protein interactions. Here, we investigate a potential interference of a metallofullerenol, Gd@C82(OH)22, on the function of SH3 domain, a highly promiscuous protein-protein interaction mediator involved in signaling and regulatory pathways through its binding with the proline-rich motif (PRM) peptides, using the atomistic molecular dynamics simulation. Our study shows that when only Gd@C82(OH)22 and the SH3 domain are present (without the PRM ligand), Gd@C82(OH)22 can interact with the SH3 domain by either directly blocking the hydrophobic active site or binding with a hydrophilic off-site with almost equal probability, which can be understood from its intrinsic amphiphilic nature. In a binding competition with the PRM onto the SH3 domain, however, the on-site binding mode is depleted while Gd@C82(OH)22 effectively intercepts the PRM from the putative binding site of the SH3 domain, implying that Gd@C82(OH)22 can disturb protein-protein interactions mediated by the SH3 domain. Despite a successful surface modification in an aqueous biological medium and a more recent demonstration as potential de novo cancer therapeutics, our study indicates that greater attention is needed in assessing the potential cytotoxicity of these nanomaterials. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr33756a

Transcriptional landscapes of Axolotl (Ambystoma mexicanum).

PubMed

Caballero-Pérez, Juan; Espinal-Centeno, Annie; Falcon, Francisco; García-Ortega, Luis F; Curiel-Quesada, Everardo; Cruz-Hernández, Andrés; Bako, Laszlo; Chen, Xuemei; Martínez, Octavio; Alberto Arteaga-Vázquez, Mario; Herrera-Estrella, Luis; Cruz-Ramírez, Alfredo

2018-01-15

The axolotl (Ambystoma mexicanum) is the vertebrate model system with the highest regeneration capacity. Experimental tools established over the past 100 years have been fundamental to start unraveling the cellular and molecular basis of tissue and limb regeneration. In the absence of a reference genome for the Axolotl, transcriptomic analysis become fundamental to understand the genetic basis of regeneration. Here we present one of the most diverse transcriptomic data sets for Axolotl by profiling coding and non-coding RNAs from diverse tissues. We reconstructed a population of 115,906 putative protein coding mRNAs as full ORFs (including isoforms). We also identified 352 conserved miRNAs and 297 novel putative mature miRNAs. Systematic enrichment analysis of gene expression allowed us to identify tissue-specific protein-coding transcripts. We also found putative novel and conserved microRNAs which potentially target mRNAs which are reported as important disease candidates in heart and liver. Copyright © 2017 Elsevier Inc. All rights reserved.

Discovery-2: an interactive resource for the rational selection and comparison of putative drug target proteins in malaria

PubMed Central

2013-01-01

Background Drug resistance to anti-malarial compounds remains a serious problem, with resistance to newer pharmaceuticals developing at an alarming rate. The development of new anti-malarials remains a priority, and the rational selection of putative targets is a key element of this process. Discovery-2 is an update of the original Discovery in silico resource for the rational selection of putative drug target proteins, enabling researchers to obtain information for a protein which may be useful for the selection of putative drug targets, and to perform advanced filtering of proteins encoded by the malaria genome based on a series of molecular properties. Methods An updated in silico resource has been developed where researchers are able to mine information on malaria proteins and predicted ligands, as well as perform comparisons to the human and mosquito host characteristics. Protein properties used include: domains, motifs, EC numbers, GO terms, orthologs, protein-protein interactions, protein-ligand interactions. Newly added features include drugability measures from ChEMBL, automated literature relations and links to clinical trial information. Searching by chemical structure is also available. Results The updated functionality of the Discovery-2 resource is presented, together with a detailed case study of the Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase (PfSAHH) protein. A short example of a chemical search with pyrimethamine is also illustrated. Conclusion The updated Discovery-2 resource allows researchers to obtain detailed properties of proteins from the malaria genome, which may be of interest in the target selection process, and to perform advanced filtering and selection of proteins based on a relevant range of molecular characteristics. PMID:23537208

Molecular cloning and characterization of a gene encoding glutaminase from Aspergillus oryzae.

PubMed

Koibuchi, K; Nagasaki, H; Yuasa, A; Kataoka, J; Kitamoto, K

2000-07-01

A glutaminase from Aspergillus oryzae was purified and its molecular weight was determined to be 82,091 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Purified glutaminase catalysed the hydrolysis not only of L-glutamine but also of D-glutamine. Both the molecular weight and the substrate specificity of this glutaminase were different from those reported previously [Yano et al. (1998) J Ferment Technol 66: 137-143]. On the basis of its internal amino acid sequences, we have isolated and characterized the glutaminase gene (gtaA) from A. oryzae. The gtaA gene had an open reading frame coding for 690 amino acid residues, including a signal peptide of 20 amino acid residues and a mature protein of 670 amino acid residues. In the 5'-flanking region of the gene, there were three putative CreAp binding sequences and one putative AreAp binding sequence. The gtaA structural gene was introduced into A. oryzae NS4 and a marked increase in activity was detected in comparison with the control strain. The gtaA gene was also isolated from Aspergillus nidulans on the basis of the determined nucleotide sequence of the gtaA gene from A. oryzae.

Learning cellular sorting pathways using protein interactions and sequence motifs.

PubMed

Lin, Tien-Ho; Bar-Joseph, Ziv; Murphy, Robert F

2011-11-01

Proper subcellular localization is critical for proteins to perform their roles in cellular functions. Proteins are transported by different cellular sorting pathways, some of which take a protein through several intermediate locations until reaching its final destination. The pathway a protein is transported through is determined by carrier proteins that bind to specific sequence motifs. In this article, we present a new method that integrates protein interaction and sequence motif data to model how proteins are sorted through these sorting pathways. We use a hidden Markov model (HMM) to represent protein sorting pathways. The model is able to determine intermediate sorting states and to assign carrier proteins and motifs to the sorting pathways. In simulation studies, we show that the method can accurately recover an underlying sorting model. Using data for yeast, we show that our model leads to accurate prediction of subcellular localization. We also show that the pathways learned by our model recover many known sorting pathways and correctly assign proteins to the path they utilize. The learned model identified new pathways and their putative carriers and motifs and these may represent novel protein sorting mechanisms. Supplementary results and software implementation are available from http://murphylab.web.cmu.edu/software/2010_RECOMB_pathways/.

Characterization of the venom from the Australian scorpion Urodacus yaschenkoi: Molecular mass analysis of components, cDNA sequences and peptides with antimicrobial activity.

PubMed

Luna-Ramírez, Karen; Quintero-Hernández, Veronica; Vargas-Jaimes, Leonel; Batista, Cesar V F; Winkel, Kenneth D; Possani, Lourival D

2013-03-01

The Urodacidae scorpions are the most widely distributed of the four families in Australia and represent half of the species in the continent, yet their venoms remain largely unstudied. This communication reports the first results of a proteome analysis of the venom of the scorpion Urodacus yaschenkoi performed by mass fingerprinting, after high performance liquid chromatography (HPLC) separation. A total of 74 fractions were obtained by HPLC separation allowing the identification of approximately 274 different molecular masses with molecular weights varying from 287 to 43,437 Da. The most abundant peptides were those from 1 K Da and 4-5 K Da representing antimicrobial peptides and putative potassium channel toxins, respectively. Three such peptides were chemically synthesized and tested against Gram-positive and Gram-negative bacteria showing minimum inhibitory concentration in the low micromolar range, but with moderate hemolytic activity. It also reports a transcriptome analysis of the venom glands of the same scorpion species, undertaken by constructing a cDNA library and conducting random sequencing screening of the transcripts. From the resultant cDNA library 172 expressed sequence tags (ESTs) were analyzed. These transcripts were further clustered into 120 unique sequences (23 contigs and 97 singlets). The identified putative proteins can be assorted in several groups, such as those implicated in common cellular processes, putative neurotoxins and antimicrobial peptides. The scorpion U. yaschenkoi is not known to be dangerous to humans and its venom contains peptides similar to those of Opisthacanthus cayaporum (antibacterial), Scorpio maurus palmatus (maurocalcin), Opistophthalmus carinatus (opistoporines) and Hadrurus gerstchi (scorpine-like molecules), amongst others. Copyright © 2012 Elsevier Ltd. All rights reserved.

The putative serine protease inhibitor Api m 6 from Apis mellifera venom: recombinant and structural evaluation.

PubMed

Michel, Y; McIntyre, M; Ginglinger, H; Ollert, M; Cifuentes, L; Blank, S; Spillner, E

2012-01-01

Immunoglobulin (Ig) E-mediated reactions to honeybee venom can cause severe anaphylaxis, sometimes with fatal consequences. Detailed knowledge of the allergic potential of all venom components is necessary to ensure proper diagnosis and treatment of allergy and to gain a better understanding of the allergological mechanisms of insect venoms. Our objective was to undertake an immunochemical and structural evaluation of the putative low-molecular-weight serine protease inhibitor Api m 6, a component of honeybee venom. We recombinantly produced Api m 6 as a soluble protein in Escherichia coli and in Spodoptera frugiperda (Sf9) insect cells.We also assessed specific IgE reactivity of venom-sensitized patients with 2 prokaryotically produced Api m 6 variants using enzyme-linked immunosorbent assay. Moreover, we built a structural model ofApi m 6 and compared it with other protease inhibitor structures to gain insights into the function of Api m 6. In a population of 31 honeybee venom-allergic patients, 26% showed specific IgE reactivity with prokaryotically produced Api m 6, showing it to be a minor but relevant allergen. Molecular modeling of Api m 6 revealed a typical fold of canonical protease inhibitors, supporting the putative function of this venom allergen. Although Api m 6 has a highly variant surface charge, its epitope distribution appears to be similar to that of related proteins. Api m 6 is a honeybee venom component with IgE-sensitizing potential in a fraction of venom-allergic patients. Recombinant Api m 6 can help elucidate individual component-resolved reactivity profiles and increase our understanding of immune responses to low-molecular-weight allergens

Bayesian species delimitation in Pleophylla chafers (Coleoptera) - the importance of prior choice and morphology.

PubMed

Eberle, Jonas; Warnock, Rachel C M; Ahrens, Dirk

2016-05-05

Defining species units can be challenging, especially during the earliest stages of speciation, when phylogenetic inference and delimitation methods may be compromised by incomplete lineage sorting (ILS) or secondary gene flow. Integrative approaches to taxonomy, which combine molecular and morphological evidence, have the potential to be valuable in such cases. In this study we investigated the South African scarab beetle genus Pleophylla using data collected from 110 individuals of eight putative morphospecies. The dataset included four molecular markers (cox1, 16S, rrnL, ITS1) and morphometric data based on male genital morphology. We applied a suite of molecular and morphological approaches to species delimitation, and implemented a novel Bayesian approach in the software iBPP, which enables continuous morphological trait and molecular data to be combined. Traditional morphology-based species assignments were supported quantitatively by morphometric analyses of the male genitalia (eigenshape analysis, CVA, LDA). While the ITS1-based delineation was also broadly congruent with the morphospecies, the cox1 data resulted in over-splitting (GMYC modelling, haplotype networks, PTP, ABGD). In the most extreme case morphospecies shared identical haplotypes, which may be attributable to ILS based on statistical tests performed using the software JML. We found the strongest support for putative morphospecies based on phylogenetic evidence using the combined approach implemented in iBPP. However, support for putative species was sensitive to the use of alternative guide trees and alternative combinations of priors on the population size (θ) and rootage (τ 0 ) parameters, especially when the analysis was based on molecular or morphological data alone. We demonstrate that continuous morphological trait data can be extremely valuable in assessing competing hypotheses to species delimitation. In particular, we show that the inclusion of morphological data in an integrative Bayesian framework can improve the resolution of inferred species units. However, we also demonstrate that this approach is extremely sensitive to guide tree and prior parameter choice. These parameters should be chosen with caution - if possible - based on independent empirical evidence, or careful sensitivity analyses should be performed to assess the robustness of results. Young species provide exemplars for investigating the mechanisms of speciation and for assessing the performance of tools used to delimit species on the basis of molecular and/or morphological evidence.

[Pharmacodynamic evaluation and molecular mechanism research of Huanshao capsule on irregular menstruation].

PubMed

Sun, Jian-Hui; Huo, Hai-Ru; Li, Xiao-Qin; Li, Hong-Mei; Qin, De-Huai; Wu, Chun

2018-04-01

Huanshao capsule is widely used in irregular menstruation and has achieved a good effect. Huanshao capsule can promote gonad development in mice, significantly improve the ovarian index in mice, increase estrogen level and reduce FSH level in rats, inhibit the pain response induced by oxytocin and estrogen, inhibit writhing reaction induced by acetic acid pain in mice. Due to the complexity of traditional Chinese medical formula, the pharmacological mechanism of the treatment on the irregular menstruation of the Huanshao capsule is unclear. In this study, the internet-based computation platform (www.tcmip.cn）was used to explore the molecular mechanism of Huanshao capsule on the menstrual. The aim of this study was to find the molecular mechanism of Huanshao capsule in treating menstrual. In the study of the molecular mechanism of Huanshao capsule in the treatment of menstrual by using the internet-based computation platform, Huanshao capsule maybe treat the menstrual by the pathway of endocrine system, GnRH signal transduction pathway, estrogen signal transduction pathway, oxytocin signaling pathway, thyroid hormone signaling pathway, VEGF signaling pathway, FCεRI signaling pathway and purine metabolism and nucleotide metabolism. The early pharmacological study confirmed Huanshao capsule could increase the serum estradiol level and decrease follicle stimulating hormone level and the traditional Chinese medicine pharmacology coincide with the prediction result of internet-based computation platform which roles as the pathway of GnRH signaling pathway and estrogen signal transduction pathway. Other pathway needs further experimental verification. Copyright© by the Chinese Pharmaceutical Association.

Gene variants associated with antisocial behaviour: A latent variable approach

PubMed Central

Bentley, Mary Jane; Lin, Haiqun; Fernandez, Thomas V.; Lee, Maria; Yrigollen, Carolyn M.; Pakstis, Andrew J.; Katsovich, Liliya; Olds, David L.; Grigorenko, Elena L.; Leckman, James F.

2013-01-01

Objective The aim of this study was to determine if a latent variable approach might be useful in identifying shared variance across genetic risk alleles that is associated with antisocial behaviour at age 15 years. Methods Using a conventional latent variable approach, we derived an antisocial phenotype in 328 adolescents utilizing data from a 15-year follow-up of a randomized trial of a prenatal and infancy nurse-home visitation program in Elmira, New York. We then investigated, via a novel latent variable approach, 450 informative genetic polymorphisms in 71 genes previously associated with antisocial behaviour, drug use, affiliative behaviours, and stress response in 241 consenting individuals for whom DNA was available. Haplotype and Pathway analyses were also performed. Results Eight single-nucleotide polymorphisms (SNPs) from 8 genes contributed to the latent genetic variable that in turn accounted for 16.0% of the variance within the latent antisocial phenotype. The number of risk alleles was linearly related to the latent antisocial variable scores. Haplotypes that included the putative risk alleles for all 8 genes were also associated with higher latent antisocial variable scores. In addition, 33 SNPs from 63 of the remaining genes were also significant when added to the final model. Many of these genes interact on a molecular level, forming molecular networks. The results support a role for genes related to dopamine, norepinephrine, serotonin, glutamate, opioid, and cholinergic signaling as well as stress response pathways in mediating susceptibility to antisocial behaviour. Conclusions This preliminary study supports use of relevant behavioural indicators and latent variable approaches to study the potential “co-action” of gene variants associated with antisocial behaviour. It also underscores the cumulative relevance of common genetic variants for understanding the etiology of complex behaviour. If replicated in future studies, this approach may allow the identification of a ‘shared’ variance across genetic risk alleles associated with complex neuropsychiatric dimensional phenotypes using relatively small numbers of well-characterized research participants. PMID:23822756

Genome-wide analysis of ivermectin response by Onchocerca volvulus reveals that genetic drift and soft selective sweeps contribute to loss of drug sensitivity

PubMed Central

Nana-Djeunga, Hugues C.; Kengne-Ouafo, Jonas A.; Pion, Sébastien D. S.; Bopda, Jean; Kamgno, Joseph; Wanji, Samuel; Che, Hua; Kuesel, Annette C.; Walker, Martin; Basáñez, Maria-Gloria; Boakye, Daniel A.; Osei-Atweneboana, Mike Y.; Boussinesq, Michel; Prichard, Roger K.; Grant, Warwick N.

2017-01-01

Background Treatment of onchocerciasis using mass ivermectin administration has reduced morbidity and transmission throughout Africa and Central/South America. Mass drug administration is likely to exert selection pressure on parasites, and phenotypic and genetic changes in several Onchocerca volvulus populations from Cameroon and Ghana—exposed to more than a decade of regular ivermectin treatment—have raised concern that sub-optimal responses to ivermectin's anti-fecundity effect are becoming more frequent and may spread. Methodology/Principal findings Pooled next generation sequencing (Pool-seq) was used to characterise genetic diversity within and between 108 adult female worms differing in ivermectin treatment history and response. Genome-wide analyses revealed genetic variation that significantly differentiated good responder (GR) and sub-optimal responder (SOR) parasites. These variants were not randomly distributed but clustered in ~31 quantitative trait loci (QTLs), with little overlap in putative QTL position and gene content between the two countries. Published candidate ivermectin SOR genes were largely absent in these regions; QTLs differentiating GR and SOR worms were enriched for genes in molecular pathways associated with neurotransmission, development, and stress responses. Finally, single worm genotyping demonstrated that geographic isolation and genetic change over time (in the presence of drug exposure) had a significantly greater role in shaping genetic diversity than the evolution of SOR. Conclusions/Significance This study is one of the first genome-wide association analyses in a parasitic nematode, and provides insight into the genomics of ivermectin response and population structure of O. volvulus. We argue that ivermectin response is a polygenically-determined quantitative trait (QT) whereby identical or related molecular pathways but not necessarily individual genes are likely to determine the extent of ivermectin response in different parasite populations. Furthermore, we propose that genetic drift rather than genetic selection of SOR is the underlying driver of population differentiation, which has significant implications for the emergence and potential spread of SOR within and between these parasite populations. PMID:28746337

Exploring O2 Diffusion in A-Type Cytochrome c Oxidases: Molecular Dynamics Simulations Uncover Two Alternative Channels towards the Binuclear Site

PubMed Central

Oliveira, A. Sofia F.; Damas, João M.; Baptista, António M.; Soares, Cláudio M.

2014-01-01

Cytochrome c oxidases (Ccoxs) are the terminal enzymes of the respiratory chain in mitochondria and most bacteria. These enzymes couple dioxygen (O2) reduction to the generation of a transmembrane electrochemical proton gradient. Despite decades of research and the availability of a large amount of structural and biochemical data available for the A-type Ccox family, little is known about the channel(s) used by O2 to travel from the solvent/membrane to the heme a3-CuB binuclear center (BNC). Moreover, the identification of all possible O2 channels as well as the atomic details of O2 diffusion is essential for the understanding of the working mechanisms of the A-type Ccox. In this work, we determined the O2 distribution within Ccox from Rhodobacter sphaeroides, in the fully reduced state, in order to identify and characterize all the putative O2 channels leading towards the BNC. For that, we use an integrated strategy combining atomistic molecular dynamics (MD) simulations (with and without explicit O2 molecules) and implicit ligand sampling (ILS) calculations. Based on the 3D free energy map for O2 inside Ccox, three channels were identified, all starting in the membrane hydrophobic region and connecting the surface of the protein to the BNC. One of these channels corresponds to the pathway inferred from the X-ray data available, whereas the other two are alternative routes for O2 to reach the BNC. Both alternative O2 channels start in the membrane spanning region and terminate close to Y288I. These channels are a combination of multiple transiently interconnected hydrophobic cavities, whose opening and closure is regulated by the thermal fluctuations of the lining residues. Furthermore, our results show that, in this Ccox, the most likely (energetically preferred) routes for O2 to reach the BNC are the alternative channels, rather than the X-ray inferred pathway. PMID:25474152

Biased Gs versus Gq proteins and β-arrestin signaling in the NK1 receptor determined by interactions in the water hydrogen bond network.

PubMed

Valentin-Hansen, Louise; Frimurer, Thomas M; Mokrosinski, Jacek; Holliday, Nicholas D; Schwartz, Thue W

2015-10-02

X-ray structures, molecular dynamics simulations, and mutational analysis have previously indicated that an extended water hydrogen bond network between trans-membranes I-III, VI, and VII constitutes an allosteric interface essential for stabilizing different active and inactive helical constellations during the seven-trans-membrane receptor activation. The neurokinin-1 receptor signals efficiently through Gq, Gs, and β-arrestin when stimulated by substance P, but it lacks any sign of constitutive activity. In the water hydrogen bond network the neurokinin-1 has a unique Glu residue instead of the highly conserved AspII:10 (2.50). Here, we find that this GluII:10 occupies the space of a putative allosteric modulating Na(+) ion and makes direct inter-helical interactions in particular with SerIII:15 (3.39) and AsnVII:16 (7.49) of the NPXXY motif. Mutational changes in the interface between GluII:10 and AsnVII:16 created receptors that selectively signaled through the following: 1) Gq only; 2) β-arrestin only; and 3) Gq and β-arrestin but not through Gs. Interestingly, increased constitutive Gs but not Gq signaling was observed by Ala substitution of four out of the six core polar residues of the network, in particular SerIII:15. Three residues were essential for all three signaling pathways, i.e. the water-gating micro-switch residues TrpVI:13 (6.48) of the CWXP motif and TyrVII:20 (7.53) of the NPXXY motif plus the totally conserved AsnI:18 (1.50) stabilizing the kink in trans-membrane VII. It is concluded that the interface between position II:10 (2.50), III:15 (3.39), and VII:16 (7.49) in the center of the water hydrogen bond network constitutes a focal point for fine-tuning seven trans-membrane receptor conformations activating different signal transduction pathways. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Snakes on the Balearic islands: an invasion tale with implications for native biodiversity conservation.

PubMed

Silva-Rocha, Iolanda; Salvi, Daniele; Sillero, Neftalí; Mateo, Jose A; Carretero, Miguel A

2015-01-01

Biological invasions are a major conservation threat for biodiversity worldwide. Islands are particularly vulnerable to invasive species, especially Mediterranean islands which have suffered human pressure since ancient times. In the Balearic archipelago, reptiles represent an outstanding case with more alien than native species. Moreover, in the last decade a new wave of alien snakes landed in the main islands of the archipelago, some of which were originally snake-free. The identification of the origin and colonization pathways of alien species, as well as the prediction of their expansion, is crucial to develop effective conservation strategies. In this study, we used molecular markers to assess the allochthonous status and the putative origin of the four introduced snake species (Hemorrhois hippocrepis, Malpolon monspessulanus, Macroprotodon mauritanicus and Rhinechis scalaris) as well as ecological niche models to infer their patterns of invasion and expansion based on current and future habitat suitability. For most species, DNA sequence data suggested the Iberian Peninsula as the potential origin of the allochthonous populations, although the shallow phylogeographic structure of these species prevented the identification of a restricted source-area. For all of them, the ecological niche models showed a current low habitat suitability in the Balearic, which is however predicted to increase significantly in the next few decades under climate change scenarios. Evidence from direct observations and spatial distribution of the first-occurrence records of alien snakes (but also lizards and worm lizards) suggest the nursery trade, and in particular olive tree importation from Iberian Peninsula, as the main pathway of introduction of alien reptiles in the Balearic islands. This trend has been reported also for recent invasions in NE Spain, thus showing that olive trees transplantation may be an effective vector for bioinvasion across the Mediterranean. The combination of molecular and ecological tools used in this study reveals a promising approach for the understanding of the complex invasion process, hence guiding conservation management actions.

Snakes on the Balearic Islands: An Invasion Tale with Implications for Native Biodiversity Conservation

PubMed Central

Sillero, Neftalí; Mateo, Jose A.; Carretero, Miguel A.

2015-01-01

Biological invasions are a major conservation threat for biodiversity worldwide. Islands are particularly vulnerable to invasive species, especially Mediterranean islands which have suffered human pressure since ancient times. In the Balearic archipelago, reptiles represent an outstanding case with more alien than native species. Moreover, in the last decade a new wave of alien snakes landed in the main islands of the archipelago, some of which were originally snake-free. The identification of the origin and colonization pathways of alien species, as well as the prediction of their expansion, is crucial to develop effective conservation strategies. In this study, we used molecular markers to assess the allochthonous status and the putative origin of the four introduced snake species (Hemorrhois hippocrepis, Malpolon monspessulanus, Macroprotodon mauritanicus and Rhinechis scalaris) as well as ecological niche models to infer their patterns of invasion and expansion based on current and future habitat suitability. For most species, DNA sequence data suggested the Iberian Peninsula as the potential origin of the allochthonous populations, although the shallow phylogeographic structure of these species prevented the identification of a restricted source-area. For all of them, the ecological niche models showed a current low habitat suitability in the Balearic, which is however predicted to increase significantly in the next few decades under climate change scenarios. Evidence from direct observations and spatial distribution of the first-occurrence records of alien snakes (but also lizards and worm lizards) suggest the nursery trade, and in particular olive tree importation from Iberian Peninsula, as the main pathway of introduction of alien reptiles in the Balearic islands. This trend has been reported also for recent invasions in NE Spain, thus showing that olive trees transplantation may be an effective vector for bioinvasion across the Mediterranean. The combination of molecular and ecological tools used in this study reveals a promising approach for the understanding of the complex invasion process, hence guiding conservation management actions. PMID:25853711

Pederin-type pathways of uncultivated bacterial symbionts: analysis of o-methyltransferases and generation of a biosynthetic hybrid.

PubMed

Zimmermann, Katrin; Engeser, Marianne; Blunt, John W; Munro, Murray H G; Piel, Jörn

2009-03-04

The complex polyketide pederin is a potent antitumor agent isolated from Paederus spp. rove beetles. We have previously isolated a set of genes from a bacterial endosymbiont that are good candidates for pederin biosynthesis. To biochemically study this pathway, we expressed three methyltransferases from the putative pederin pathway and used the partially unmethylated analogue mycalamide A from the marine sponge Mycale hentscheli as test substrate. Analysis by high-resolution MS/MS and NMR revealed that PedO regiospecifically methylates the marine compound to generate the nonnatural hybrid compound 18-O-methylmycalamide A with increased cytotoxicity. To our knowledge, this is the first biochemical evidence that invertebrates can obtain defensive complex polyketides from bacterial symbionts.

Structural investigation of C4b-binding protein by molecular modeling: localization of putative binding sites.

PubMed

Villoutreix, B O; Härdig, Y; Wallqvist, A; Covell, D G; García de Frutos, P; Dahlbäck, B

1998-06-01

C4b-binding protein (C4BP) contributes to the regulation of the classical pathway of the complement system and plays an important role in blood coagulation. The main human C4BP isoform is composed of one beta-chain and seven alpha-chains essentially built from three and eight complement control protein (CCP) modules, respectively, followed by a nonrepeat carboxy-terminal region involved in polymerization of the chains. C4BP is known to interact with heparin, C4b, complement factor I, serum amyloid P component, streptococcal Arp and Sir proteins, and factor VIII/VIIIa via its alpha-chains and with protein S through its beta-chain. The principal aim of the present study was to localize regions of C4BP involved in the interaction with C4b, Arp, and heparin. For this purpose, a computer model of the 8 CCP modules of C4BP alpha-chain was constructed, taking into account data from previous electron microscopy (EM) studies. This structure was investigated in the context of known and/or new experimental data. Analysis of the alpha-chain model, together with monoclonal antibody studies and heparin binding experiments, suggests that a patch of positively charged residues, at the interface between the first and second CCP modules, plays an important role in the interaction between C4BP and C4b/Arp/Sir/heparin. Putative binding sites, secondary-structure prediction for the central core, and an overall reevaluation of the size of the C4BP molecule are also presented. An understanding of these intermolecular interactions should contribute to the rational design of potential therapeutic agents aiming at interfering specifically some of these protein-protein interactions.

First molecular cloning and characterisation of caspase-9 gene in fish and its involvement in a gram negative septicaemia.

PubMed

Reis, Marta I R; do Vale, Ana; Pinto, Cristina; Nascimento, Diana S; Costa-Ramos, Carolina; Silva, Daniela S P; Silva, Manuel T; Dos Santos, Nuno M S

2007-03-01

Caspase-9 is an initiator caspase in the apoptotic process whose function is to activate effector caspases that are downstream in the mitochondrial pathway of apoptosis. This work reports for the first time the complete sequencing and characterisation of caspase-9 in fish. A 1924bp cDNA of sea bass caspase-9 was obtained, consisting of 1308bp open reading frame coding for 435 amino acids, 199bp of the 5'-UTR and 417bp of the 3'-UTR including a canonical polyadenilation signal 10 nucleotides upstream the polyadenilation tail. The sequence retains the pentapeptide active-site motif (QACGG) and the putative cleavage sites at Asp(121), Asp(325) and Asp(343). The sequence of sea bass caspase-9 exhibits a very close homology to the sequences of caspase-9 from other vertebrates, particularly with the putative caspases-9 of Danio rerio and Tetraodon nigroviridis (77.5 and 75.4% similarity, respectively), justifying the fact that the phylogenetic analysis groups these species together with sea bass. The sea bass caspase-9 gene exists as a single copy gene and is organised in 9 introns and 10 exons. The sea bass caspase-9 showed a basal expression in all the organs analysed, although weaker in spleen. The expression of sea bass caspase-9 in the head kidney of sea bass infected with the Photobacterium damselae ssp. piscicida (Phdp) strain PP3, showed increased expression from 0 to 12h returning to control levels at 24h. Caspase-9 activity was detected in Phdp infected sea bass head kidney from 18 to 48h post-infection, when the fish were with advanced septicaemia.

Molecular cloning and heterologous expression of a biosynthetic gene cluster for the antitubercular agent D-cycloserine produced by Streptomyces lavendulae.

PubMed

Kumagai, Takanori; Koyama, Yusuke; Oda, Kosuke; Noda, Masafumi; Matoba, Yasuyuki; Sugiyama, Masanori

2010-03-01

In the present study, we successfully cloned a 21-kb DNA fragment containing a d-cycloserine (DCS) biosynthetic gene cluster from a DCS-producing Streptomyces lavendulae strain, ATCC 11924. The putative gene cluster consists of 10 open reading frames (ORFs), designated dcsA to dcsJ. This cluster includes two ORFs encoding D-alanyl-D-alanine ligase (dcsI) and a putative membrane protein (dcsJ) as the self-resistance determinants of the producer organism, indicated by our previous work. When the 10 ORFs were introduced into DCS-nonproducing Streptomyces lividans 66 as a heterologous host cell, the transformant acquired DCS productivity. This reveals that the introduced genes are responsible for the biosynthesis of DCS. As anticipated, the disruption of dcsG, seen in the DCS biosynthetic gene cluster, made it possible for the strain ATCC 11924 to lose its DCS production. We here propose the DCS biosynthetic pathway. First, L-serine is O acetylated by a dcsE-encoded enzyme homologous to homoserine O-acetyltransferase. Second, O-acetyl-L-serine accepts hydroxyurea via an O-acetylserine sulfhydrylase homolog (dcsD product) and forms O-ureido-L-serine. The hydroxyurea must be supplied by the catalysis of a dcsB-encoded arginase homolog using the L-arginine derivative, N(G)-hydroxy-L-arginine. The resulting O-ureido-L-serine is then racemized to O-ureido-D-serine by a homolog of diaminopimelate epimerase. Finally, O-ureido-D-serine is cyclized to form DCS with the release of ammonia and carbon dioxide. The cyclization must be done by the dcsG or dcsH product, which belongs to the ATP-grasp fold family of protein.

Assembly and Analysis of Differential Transcriptome Responses of Hevea brasiliensis on Interaction with Microcyclus ulei

PubMed Central

Restrepo Restrepo, Silvia; Aristizábal Gutiérrez, Fabio Ancizar; Montoya Castaño, Dolly

2015-01-01

Natural rubber (Hevea brasiliensis) is a tropical tree used commercially for the production of latex, from which 40,000 products are generated. The fungus Microcyclus ulei infects this tree, causing South American leaf blight (SALB) disease. This disease causes developmental delays and significant crop losses, thereby decreasing the production of latex. Currently several groups are working on obtaining clones of rubber tree with durable resistance to SALB through the use of extensive molecular biology techniques. In this study, we used a secondary clone that was resistant to M. ulei isolate GCL012. This clone, FX 3864 was obtained by crossing between clones PB 86 and B 38 (H. brasiliensis x H. brasiliensis). RNA-Seq high-throughput sequencing technology was used to analyze the differential expression of the FX 3864 clone transcriptome at 0 and 48 h post infection (hpi) with the M. ulei isolate GCL012. A total of 158,134,220 reads were assembled using the de novo assembly strategy to generate 90,775 contigs with an N50 of 1672. Using a reference-based assembly, 76,278 contigs were generated with an N50 of 1324. We identified 86 differentially expressed genes associated with the defense response of FX 3864 to GCL012. Seven putative genes members of the AP2/ERF ethylene (ET)-dependent superfamily were found to be down-regulated. An increase in salicylic acid (SA) was associated with the up-regulation of three genes involved in cell wall synthesis and remodeling, as well as in the down-regulation of the putative gene CPR5. The defense response of FX 3864 against the GCL012 isolate was associated with the antagonistic SA, ET and jasmonic acid (JA) pathways. These responses are characteristic of plant resistance to biotrophic pathogens. PMID:26287380

Capitate glandular trichomes in Aldama discolor (Heliantheae - Asteraceae): morphology, metabolite profile and sesquiterpene biosynthesis.

PubMed

Bombo, A B; Appezzato-da-Glória, B; Aschenbrenner, A-K; Spring, O

2016-05-01

The capitate glandular trichome is the most common type described in Asteraceae species. It is known for its ability to produce various plant metabolites of ecological and economic importance, among which sesquiterpene lactones are predominant. In this paper, we applied microscopy, phytochemical and molecular genetics techniques to characterise the capitate glandular trichome in Aldama discolor, a native Brazilian species of Asteraceae, with pharmacological potential. It was found that formation of trichomes on leaf primordia of germinating seeds starts between 24 h and 48 h after radicle growth indicates germination. The start of metabolic activity of trichomes was indicated by separation of the cuticle from the cell wall of secretory cells at the trichome tip after 72 h. This coincided with the accumulation of budlein A, the major sesquiterpene lactone of A. discolor capitate glandular trichomes, in extracts of leaf primordia after 96 h. In the same timeframe of 72-96 h post-germination, gene expression studies showed up-regulation of the putative germacrene A synthase (pGAS2) and putative germacrene A oxidase (pGAO) of A. discolor in the transcriptome of these samples, indicating the start of sesquiterpene lactone biosynthesis. Sequencing of the two genes revealed high similarity to HaGAS and HaGAO from sunflower, which shows that key steps of this pathway are highly conserved. The processes of trichome differentiation, metabolic activity and genetic regulation in A. discolor and in sunflower appear to be typical for other species of the subtribe Helianthinae. © 2015 German Botanical Society and The Royal Botanical Society of the Netherlands.

MiR-145 regulates PAK4 via the MAPK pathway and exhibits an antitumor effect in human colon cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Zhigang; Zhang, Xiaoping; Yang, Zhili

2012-10-26

Highlights: Black-Right-Pointing-Pointer MiR-145 targets a putative binding site in the 3 Prime UTR of PAK4. Black-Right-Pointing-Pointer MiR-145 played an important role in inhibiting cell growth by directly targeting PAK4. Black-Right-Pointing-Pointer MiR-145 may function as tumor suppressors. -- Abstract: MicroRNAs (miRNAs) are regulators of numerous cellular events; accumulating evidence indicates that miRNAs play a key role in a wide range of biological functions, such as cellular proliferation, differentiation, and apoptosis in cancer. Down-regulated expression of miR-145 has been reported in colon cancer tissues and cell lines. The molecular mechanisms underlying miR-145 and the regulation of colon carcinogenesis remain unclear. In thismore » study, we investigated the levels of miR-145 in human colon cancer cells using qRT-PCR and found markedly decreased levels compared to normal epithelial cells. We identified PAK4 as a novel target of miR-145 using informatics screening. Additionally, we demonstrated that miR-145 targets a putative binding site in the 3 Prime UTR of PAK4 and that its abundance is inversely associated with miR-145 expression in colon cancer cells; we confirmed this relationship using the luciferase reporter assay. Furthermore, restoration of miR-145 by mimics in SW620 cells significantly attenuated cell growth in vitro, in accordance with the inhibitory effects induced by siRNA mediated knockdown of PAK4. Taken together, these findings demonstrate that miR-145 downregulates P-ERK expression by targeting PAK4 and leads to inhibition of tumor growth.« less

The Anisakis Transcriptome Provides a Resource for Fundamental and Applied Studies on Allergy-Causing Parasites

PubMed Central

Baird, Fiona J.; Su, Xiaopei; Aibinu, Ibukun; Nolan, Matthew J.; Sugiyama, Hiromu; Otranto, Domenico

2016-01-01

Background Food-borne nematodes of the genus Anisakis are responsible for a wide range of illnesses (= anisakiasis), from self-limiting gastrointestinal forms to severe systemic allergic reactions, which are often misdiagnosed and under-reported. In order to enhance and refine current diagnostic tools for anisakiasis, knowledge of the whole spectrum of parasite molecules transcribed and expressed by this parasite, including those acting as potential allergens, is necessary. Methodology/Principal Findings In this study, we employ high-throughput (Illumina) sequencing and bioinformatics to characterise the transcriptomes of two Anisakis species, A. simplex and A. pegreffii, and utilize this resource to compile lists of potential allergens from these parasites. A total of ~65,000,000 reads were generated from cDNA libraries for each species, and assembled into ~34,000 transcripts (= Unigenes); ~18,000 peptides were predicted from each cDNA library and classified based on homology searches, protein motifs and gene ontology and biological pathway mapping. Using comparative analyses with sequence data available in public databases, 36 (A. simplex) and 29 (A. pegreffii) putative allergens were identified, including sequences encoding ‘novel’ Anisakis allergenic proteins (i.e. cyclophilins and ABA-1 domain containing proteins). Conclusions/Significance This study represents a first step towards providing the research community with a curated dataset to use as a molecular resource for future investigations of the biology of Anisakis, including molecules putatively acting as allergens, using functional genomics, proteomics and immunological tools. Ultimately, an improved knowledge of the biological functions of these molecules in the parasite, as well as of their immunogenic properties, will assist the development of comprehensive, reliable and robust diagnostic tools. PMID:27472517

Transcriptional profiling of epigenetic regulators in somatic embryos during temperature induced formation of an epigenetic memory in Norway spruce.

PubMed

Yakovlev, Igor A; Carneros, Elena; Lee, YeonKyeong; Olsen, Jorunn E; Fossdal, Carl Gunnar

2016-05-01

A significant number of epigenetic regulators were differentially expressed during embryogenesis at different epitype-inducing conditions. Our results support that methylation of DNA and histones, as well as sRNAs, are pivotal for the establishment of the epigenetic memory. As a forest tree species with long generation times, Norway spruce is remarkably well adapted to local environmental conditions despite having recently, from an evolutionary perspective, recolonized large areas following the last glaciation. In this species, there is an enigmatic epigenetic memory of the temperature conditions during embryogenesis that allows rapid adaptation to changing environment. We used a transcriptomic approach to investigate the molecular mechanisms underlying the formation of the epigenetic memory during somatic embryogenesis in Norway spruce. Nine mRNA libraries were prepared from three epitypes of the same genotype resulting from exposure to epitype-inducing temperatures of 18, 23 and 28 °C. RNA-Seq analysis revealed more than 10,000 differentially expressed genes (DEGs). The epitype-inducing conditions during SE were accompanied by marked transcriptomic changes for multiple gene models related to the epigenetic machinery. Out of 735 putative orthologs of epigenetic regulators, 329 were affected by the epitype-inducing temperatures and differentially expressed. The majority of DEGs among the epigenetic regulators was related to DNA and histone methylation, along with sRNA pathways and a range of putative thermosensing and signaling genes. These genes could be the main epigenetic regulators involved in formation of the epigenetic memory. We suggest considerable expansion of gene families of epigenetic regulators in Norway spruce compared to orthologous gene families in Populus and Arabidopsis. Obtained results provide a solid basis for further genome annotation and studies focusing on the importance of these candidate genes for the epigenetic memory formation.