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Sample records for acid analysis revealed

  1. Trophic hierarchies revealed via amino acid isotopic analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Despite the potential of isotopic methods to illuminate trophic function, accurate estimates of lifetime feeding tendencies have remained elusive. A relatively new approach—referred to as compound-specific isotopic analysis (CSIA)—has emerged, centering on the measurement of 15N:14N ratios in amino ...

  2. Evolution and Functional Implications of the Tricarboxylic Acid Cycle as Revealed by Phylogenetic Analysis

    PubMed Central

    Cavalcanti, João Henrique Frota; Esteves-Ferreira, Alberto A.; Quinhones, Carla G.S.; Pereira-Lima, Italo A.; Nunes-Nesi, Adriano; Fernie, Alisdair R.; Araújo, Wagner L.

    2014-01-01

    The tricarboxylic acid (TCA) cycle, a crucial component of respiratory metabolism, is composed of a set of eight enzymes present in the mitochondrial matrix. However, most of the TCA cycle enzymes are encoded in the nucleus in higher eukaryotes. In addition, evidence has accumulated demonstrating that nuclear genes were acquired from the mitochondrial genome during the course of evolution. For this reason, we here analyzed the evolutionary history of all TCA cycle enzymes in attempt to better understand the origin of these nuclear-encoded proteins. Our results indicate that prior to endosymbiotic events the TCA cycle seemed to operate only as isolated steps in both the host (eubacterial cell) and mitochondria (alphaproteobacteria). The origin of isoforms present in different cell compartments might be associated either with gene-transfer events which did not result in proper targeting of the protein to mitochondrion or with duplication events. Further in silico analyses allow us to suggest new insights into the possible roles of TCA cycle enzymes in different tissues. Finally, we performed coexpression analysis using mitochondrial TCA cycle genes revealing close connections among these genes most likely related to the higher efficiency of oxidative phosphorylation in this specialized organelle. Moreover, these analyses allowed us to identify further candidate genes which might be used for metabolic engineering purposes given the importance of the TCA cycle during development and/or stress situations. PMID:25274566

  3. Analysis of TP53 mutation spectra reveals the fingerprint of the potent environmental carcinogen, aristolochic acid

    PubMed Central

    Hollstein, M; Moriya, M.; Grollman, AP; Olivier, M

    2013-01-01

    Genetic alterations in cancer tissues may reflect the mutational fingerprint of environmental carcinogens. Here we review the evidence that support the role of aristolochic acid (AA) in inducing a mutational fingerprint in the tumor suppressor gene TP53 in urothelial carcinomas of the upper urinary tract (UUT). Exposure to AA, a nitrophenathrene carboxylic acid present in certain herbal remedies and in flour prepared from wheat grain contaminated with seeds of Aristolochia clematitis, has been linked to chronic nephropathy and UUT. TP53 mutations in UUT of individuals exposed to AA reveal a unique pattern of mutations characterised by A to T transversions on the non-transcribed strand, which cluster at hotspots rarely mutated in other cancers. This unusual pattern, originally discovered in UUTs from two different populations, one in Taiwan, and one in the Balkans, has been reproduced experimentally by treating mouse cells that harbour human TP53 sequences with AA. The convergence of molecular epidemiological and experimental data establishes a clear causal association between exposure to the human carcinogen AA and UUT cancer. Despite bans on the sale of herbs containing AA, their use continues, raising global public health concern and an urgent need to identify populations at risk. PMID:23422071

  4. Proteomic analysis reveals dynamic regulation of fruit development and sugar and acid accumulation in apple.

    PubMed

    Li, Mingjun; Li, Dongxia; Feng, Fengjuan; Zhang, Sheng; Ma, Fengwang; Cheng, Lailiang

    2016-09-01

    Understanding the fruit developmental process is critical for fruit quality improvement. Here, we report a comprehensive proteomic analysis of apple fruit development over five growth stages, from young fruit to maturity, coupled with metabolomic profiling. A tandem mass tag (TMT)-based comparative proteomics approach led to the identification and quantification of 7098 and 6247 proteins, respectively. This large-scale proteomic dataset presents a global view of the critical pathways involved in fruit development and metabolism. When linked with metabolomics data, these results provide new insights into the modulation of fruit development, the metabolism and storage of sugars and organic acids (mainly malate), and events within the energy-related pathways for respiration and glycolysis. We suggest that the key steps identified here (e.g. those involving the FK2, TST, EDR6, SPS, mtME and mtMDH switches), can be further targeted to confirm their roles in accumulation and balance of fructose, sucrose and malate. Moreover, our findings imply that the primary reason for decreases in amino acid concentrations during fruit development is related to a reduction in substrate flux via glycolysis, which is mainly regulated by fructose-bisphosphate aldolase and bisphosphoglycerate mutase. PMID:27535992

  5. Transcriptomic Analysis Reveals the Metabolic Mechanism of L-Ascorbic Acid in Ziziphus jujuba Mill.

    PubMed Central

    Zhang, Chunmei; Huang, Jian; Li, Xingang

    2016-01-01

    Chinese jujube (Ziziphus jujuba Mill.) is the most economically important member of the Rhamnaceae family and contains a high concentration of ascorbic acid (AsA). To explore the metabolic mechanism of AsA accumulation, we investigated the abundance of AsA in the fruit development stages, the leaf and flower of Z. jujuba cv Junzao, and the mature fruit of one type of wild jujube (Z. jujuba var. spinosa Hu, Yanchuan sour jujube). And the expression patterns of genes involved in AsA biosynthesis, degradation, and recycling were analyzed. The result showed that AsA biosynthesis during early fruit development (the enlargement stage) is the main reason for jujube high accumulation. The L-galactose pathway plays a predominant role in the biosynthesis of AsA during jujube fruit development, and the genes GMP1, GME1, GGP, and GaLDH involved in the determination of AsA concentration during fruit development and in different genotypes; the myo-inositol pathway along with the genes GME2 and GMP2 in the L-galactose pathway play a compensatory role in maintaining AsA accumulation during the ripening stage. These findings enhance our understanding of the molecular mechanism in regulating AsA accumulation for jujube. PMID:26913041

  6. Comparative Transcriptome Analysis Reveals the Influence of Abscisic Acid on the Metabolism of Pigments, Ascorbic Acid and Folic Acid during Strawberry Fruit Ripening

    PubMed Central

    Luo, Zisheng; Mou, Wangshu; Mao, Linchun; Ying, Tiejin

    2015-01-01

    A comprehensive investigation of abscisic acid (ABA) biosynthesis and its influence on other important phytochemicals is critical for understanding the versatile roles that ABA plays during strawberry fruit ripening. Using RNA-seq technology, we sampled strawberry fruit in response to ABA or nordihydroguaiaretic acid (NDGA; an ABA biosynthesis blocker) treatment during ripening and assessed the expression changes of genes involved in the metabolism of pigments, ascorbic acid (AsA) and folic acid in the receptacles. The transcriptome analysis identified a lot of genes differentially expressed in response to ABA or NDGA treatment. In particular, genes in the anthocyanin biosynthesis pathway were actively regulated by ABA, with the exception of the gene encoding cinnamate 4-hydroxylase. Chlorophyll degradation was accelerated by ABA mainly owing to the higher expression of gene encoding pheide a oxygenase. The decrease of β-carotene content was accelerated by ABA treatment and delayed by NDGA. A high negative correlation rate was found between ABA and β-carotene content, indicating the importance of the requirement for ABA synthesis during fruit ripening. In addition, evaluation on the folate biosynthetic pathway indicate that ABA might have minor function in this nutrient’s biosynthesis process, however, it might be involved in its homeostasis. Surprisingly, though AsA content accumulated during fruit ripening, expressions of genes involved in its biosynthesis in the receptacles were significantly lower in ABA-treated fruits. This transcriptome analysis expands our understanding of ABA’s role in phytochemical metabolism during strawberry fruit ripening and the regulatory mechanisms of ABA on these pathways were discussed. Our study provides a wealth of genetic information in the metabolism pathways and may be helpful for molecular manipulation in the future. PMID:26053069

  7. Comparative Transcriptome Analysis Reveals the Influence of Abscisic Acid on the Metabolism of Pigments, Ascorbic Acid and Folic Acid during Strawberry Fruit Ripening.

    PubMed

    Li, Dongdong; Li, Li; Luo, Zisheng; Mou, Wangshu; Mao, Linchun; Ying, Tiejin

    2015-01-01

    A comprehensive investigation of abscisic acid (ABA) biosynthesis and its influence on other important phytochemicals is critical for understanding the versatile roles that ABA plays during strawberry fruit ripening. Using RNA-seq technology, we sampled strawberry fruit in response to ABA or nordihydroguaiaretic acid (NDGA; an ABA biosynthesis blocker) treatment during ripening and assessed the expression changes of genes involved in the metabolism of pigments, ascorbic acid (AsA) and folic acid in the receptacles. The transcriptome analysis identified a lot of genes differentially expressed in response to ABA or NDGA treatment. In particular, genes in the anthocyanin biosynthesis pathway were actively regulated by ABA, with the exception of the gene encoding cinnamate 4-hydroxylase. Chlorophyll degradation was accelerated by ABA mainly owing to the higher expression of gene encoding pheide a oxygenase. The decrease of β-carotene content was accelerated by ABA treatment and delayed by NDGA. A high negative correlation rate was found between ABA and β-carotene content, indicating the importance of the requirement for ABA synthesis during fruit ripening. In addition, evaluation on the folate biosynthetic pathway indicate that ABA might have minor function in this nutrient's biosynthesis process, however, it might be involved in its homeostasis. Surprisingly, though AsA content accumulated during fruit ripening, expressions of genes involved in its biosynthesis in the receptacles were significantly lower in ABA-treated fruits. This transcriptome analysis expands our understanding of ABA's role in phytochemical metabolism during strawberry fruit ripening and the regulatory mechanisms of ABA on these pathways were discussed. Our study provides a wealth of genetic information in the metabolism pathways and may be helpful for molecular manipulation in the future. PMID:26053069

  8. Quantitative proteomics analysis reveals glutamine deprivation activates fatty acid β-oxidation pathway in HepG2 cells.

    PubMed

    Long, Baisheng; Muhamad, Rodiallah; Yan, Guokai; Yu, Jie; Fan, Qiwen; Wang, Zhichang; Li, Xiuzhi; Purnomoadi, Agung; Achmadi, Joelal; Yan, Xianghua

    2016-05-01

    Glutamine, a multifunctional amino acid, functions in nutrient metabolism, energy balance, apoptosis, and cell proliferation. Lipid is an important nutrient and controls a broad range of physiological processes. Previous studies have demonstrated that glutamine can affect lipolysis and lipogenesis, but the effect of glutamine on the detailed lipid metabolism remains incompletely understood. Here, we applied the quantitative proteomics approach to estimate the relative abundance of proteins in HepG2 cells treated by glutamine deprivation. The results showed that there were 212 differentially abundant proteins in response to glutamine deprivation, including 150 significantly increased proteins and 62 significantly decreased proteins. Interestingly, functional classification showed that 43 differentially abundant proteins were related to lipid metabolism. Further bioinformatics analysis and western blotting validation revealed that lipid accumulation may be affected by β-oxidation of fatty acid induced by glutamine deprivation in HepG2 cells. Together, our results may provide the potential for regulating lipid metabolism by glutamine in animal production and human nutrition. The MS data have been deposited to the ProteomeXchange Consortium with identifier PXD003387. PMID:26837383

  9. Comparative Proteomic Analysis Reveals the Effects of Exogenous Calcium against Acid Rain Stress in Liquidambar formosana Hance Leaves.

    PubMed

    Hu, Wen-Jun; Wu, Qian; Liu, Xiang; Shen, Zhi-Jun; Chen, Juan; Liu, Ting-Wu; Chen, Juan; Zhu, Chun-Quan; Wu, Fei-Hua; Chen, Lin; Wei, Jia; Qiu, Xiao-Yun; Shen, Guo-Xin; Zheng, Hai-Lei

    2016-01-01

    Acid rain (AR) impacts forest health by leaching calcium (Ca) away from soils and plants. Ca is an essential element and participates in various plant physiological responses. In the present study, the protective role of exogenous Ca in alleviating AR stress in Liquidambar formosana Hance at the physiological and proteomic levels was examined. Our results showed that low Ca condition resulted in the chlorophyll content and photosynthesis decreasing significantly in L. formosana leaves; however, these effects could be reversed by high Ca supplementation. Further proteomic analyses successfully identified 81 differentially expressed proteins in AR-treated L. formosana under different Ca levels. In particular, some of the proteins are involved in primary metabolism, photosynthesis, energy production, antioxidant defense, transcription, and translation. Moreover, quantitative real time polymerase chain reaction (qRT-PCR) results indicated that low Ca significantly increased the expression level of the investigated Ca-related genes, which can be reversed by high Ca supplementation under AR stress. Further, Western blotting analysis revealed that exogenous Ca supply reduced AR damage by elevating the expression of proteins involved in the Calvin cycle, reactive oxygen species (ROS) scavenging system. These findings allowed us to better understand how woody plants respond to AR stress at various Ca levels and the protective role of exogenous Ca against AR stress in forest tree species. PMID:26616104

  10. Crystallographic analysis reveals the structural basis of the high-affinity binding of iophenoxic acid to human serum albumin

    PubMed Central

    2011-01-01

    Background Iophenoxic acid is an iodinated radiocontrast agent that was withdrawn from clinical use because of its exceptionally long half-life in the body, which was due in part to its high-affinity binding to human serum albumin (HSA). It was replaced by Iopanoic acid, which has an amino rather than a hydroxyl group at position 3 on the iodinated benzyl ring and, as a result, binds to albumin with lower affinity and is excreted more rapidly from the body. To understand how iophenoxic acid binds so tightly to albumin, we wanted to examine the structural basis of its interaction with HSA. Results We have determined the co-crystal structure of HSA in complex with iophenoxic acid at 2.75 Å resolution, revealing a total of four binding sites, two of which - in drugs sites 1 and 2 on the protein - are likely to be occupied at clinical doses. High-affinity binding of iophenoxic acid occurs at drug site 1. The structure reveals that polar and apolar groups on the compound are involved in its interactions with drug site 1. In particular, the 3-hydroxyl group makes three hydrogen bonds with the side-chains of Tyr 150 and Arg 257. The mode of binding to drug site 2 is similar except for the absence of a binding partner for the hydroxyl group on the benzyl ring of the compound. Conclusions The HSA-iophenoxic acid structure indicates that high-affinity binding to drug site 1 is likely to be due to extensive desolvation of the compound, coupled with the ability of the binding pocket to provide a full set of salt-bridging or hydrogen bonding partners for its polar groups. Consistent with this interpretation, the structure also suggests that the lower-affinity binding of iopanoic acid arises because replacement of the 3-hydroxyl by an amino group eliminates hydrogen bonding to Arg 257. This finding underscores the importance of polar interactions in high-affinity binding to albumin. PMID:21501503

  11. Metabolomics analysis reveals that bile acids and phospholipids contribute to variable responses to low-temperature-induced ascites syndrome.

    PubMed

    Shen, Yiru; Shi, Shourong; Tong, Haibing; Guo, Yuming; Zou, Jianmin

    2014-06-01

    Ascites is a major problem for both human health and animal production, due to its association with high rates of morbidity and mortality, low efficiency of nutrient utilization, and permanent adverse effects on performance. Although it is one of the three major metabolic diseases in poultry production, the underlying mechanisms are largely unknown. In this study, six ascites syndrome (AS) chickens and six normal chickens were obtained from each group (108 chickens) at 21 and 35 days. A liver metabolomics method based on ultra-performance liquid chromatography/quadruple time-of-flight mass spectrometry (UPLC/Q-TOF/MS) was used to explore the metabolic pattern of low molecular mass metabolites in chickens with low-temperature-induced AS. Coupled with blood biochemistry and histopathology results, the significant difference in metabolic profiling between the AS group and the control group, as determined through pattern recognition analysis, indicated changes in global tissue metabolites. The results showed that a primary bile acid synthesis disorder and inflammation had occurred by 21 days and that lysophospholipid metabolism was disrupted by 35 days with the continuation of low temperatures. Several metabolites, including taurodeoxycholic acid, cholic acid glucuronide, glycocholic acid, LysoPC(15 : 0) and taurocholic acid, were identified as the potential and proper biomarkers of AS. These biochemical changes in tissue metabolites are related to perturbations of lipid metabolism, which may be helpful to further understand the AS mechanisms. This work shows that the metabolomics is a valuable tool for studying metabolic diseases. PMID:24700147

  12. A Causal Network Analysis of the Fatty Acid Metabolome in African-Americans Reveals a Critical Role for Palmitoleate and Margarate

    PubMed Central

    Yazdani, Akram; Boerwinkle, Eric

    2016-01-01

    Abstract Fatty acids are important sources of energy and possible predictors and etiologic factors in many common complex pathologies such as cardiovascular disease, diabetes, and certain forms of cancers. While fatty acids are thought to covary with each other, their underlying causal networks have not been fully elucidated. This study reports the identification and analysis of a statistical causal network among 15 mostly long-chain fatty acids. In an African-American population sample and using the Genome granularity-Directed Acyclic Graph (GDAG) algorithm, we determined directions or causal relationships in the fatty acid metabolome. A directed causal network was constructed that revealed 29 significant edges among the 15 nodes (p < 0.001). We report that two fatty acid metabolites, palmitoleate and margarate, which originate from dietary intake, together influence every other fatty acid in the network. On the other hand, despite its high connectivity, dihomo-linoleate did not appear to play an important role over the whole fatty acid network. These findings collectively suggest possible strategic entry points for new treatments or preventive modalities against diseases affected by fatty acid metabolites such as cardiovascular disease, diabetes, and obesity. Further studies examining the embedded substructure of the fatty acid metabolite networks in independent population samples would be timely and warranted as we move toward novel postgenomic diagnostics and therapeutics. PMID:27501297

  13. A Causal Network Analysis of the Fatty Acid Metabolome in African-Americans Reveals a Critical Role for Palmitoleate and Margarate.

    PubMed

    Yazdani, Azam; Yazdani, Akram; Boerwinkle, Eric

    2016-08-01

    Fatty acids are important sources of energy and possible predictors and etiologic factors in many common complex pathologies such as cardiovascular disease, diabetes, and certain forms of cancers. While fatty acids are thought to covary with each other, their underlying causal networks have not been fully elucidated. This study reports the identification and analysis of a statistical causal network among 15 mostly long-chain fatty acids. In an African-American population sample and using the Genome granularity-Directed Acyclic Graph (GDAG) algorithm, we determined directions or causal relationships in the fatty acid metabolome. A directed causal network was constructed that revealed 29 significant edges among the 15 nodes (p < 0.001). We report that two fatty acid metabolites, palmitoleate and margarate, which originate from dietary intake, together influence every other fatty acid in the network. On the other hand, despite its high connectivity, dihomo-linoleate did not appear to play an important role over the whole fatty acid network. These findings collectively suggest possible strategic entry points for new treatments or preventive modalities against diseases affected by fatty acid metabolites such as cardiovascular disease, diabetes, and obesity. Further studies examining the embedded substructure of the fatty acid metabolite networks in independent population samples would be timely and warranted as we move toward novel postgenomic diagnostics and therapeutics. PMID:27501297

  14. New insights into the diets of harbor seals in the Salish Sea revealed by quantitative fatty acid signature analysis

    USGS Publications Warehouse

    Bromaghin, Jeffrey F.; Lance, Monique M.; Elliott, Elizabeth W.; Jeffries, Steven J.; Acevedo-Gutiérrez, Alejandro; Kennish, John M.

    2012-01-01

    Harbor seals (Phoca vitulina) are an abundant predator along the west coast of North America, and there is considerable interest in their diet composition, especially in regard to predation on valued fish stocks. Available information on harbor seal diets, primarily derived from scat analysis, suggests that adult salmon (Oncorhynchus spp.), Pacific Herring (Clupea pallasii), and gadids predominate. Because diet assessments based on scat analysis may be biased, we investigated diet composition through quantitative analysis of fatty acid signatures. Blubber samples from 49 harbor seals captured in western North America from haul-outs within the area of the San Juan Islands and southern Strait of Georgia in the Salish Sea were analyzed for fatty acid composition, along with 269 fish and squid specimens representing 27 potential prey classes. Diet estimates varied spatially, demographically, and among individual harbor seals. Findings confirmed the prevalence of previously identified prey species in harbor seal diets, but other species also contributed significantly. In particular, Black (Sebastes melanops) and Yellowtail (S. flavidus) Rockfish were estimated to compose up to 50% of some individual seal diets. Specialization and high predation rates on Black and Yellowtail Rockfish by a subset of harbor seals may play a role in the population dynamics of these regional rockfish stocks that is greater than previously realized.

  15. Analysis of olfactory sensitivity in rainbow trout (Oncorhynchus mykiss) reveals their ability to detect lactic acid, pyruvic acid and four B vitamins.

    PubMed

    Valdés, Joaquín; Olivares, Jesús; Ponce, Daniela; Schmachtenberg, Oliver

    2015-08-01

    Salmonid fishes like the rainbow trout Oncorhynchus mykiss have a highly developed olfactory sense that allows them to perceive some odorants at very low concentrations, such as certain amino acids and bile salts. Previous behavioral and electrophysiological studies in salmonids have shown strong responses to human skin odor. Because this stimulus represents a complex and heterogeneous mixture of components, we sought to determine which odorants contribute to the sensitive detection of human skin odor by salmonids. In vivo electroolfactogram recordings in O. mykiss revealed lactic acid, pyruvic acid and two B vitamins, thiamine and riboflavin, as novel, potent odorants which triggered responses at nanomolar concentrations. Two more B vitamins, nicotinic and pantothenic acid, were detected at micromolar concentrations. These compounds share important roles in cellular energy metabolism, supporting an original role in food search and feeding behavior of this species and most likely other fishes. The olfactory detection of B vitamins by salmonids represents a new paradigm in chemosensation, warranting further investigation in other teleosts. PMID:25864178

  16. A phylogenetic analysis of the boreal lichen Mycoblastus sanguinarius (Mycoblastaceae, lichenized Ascomycota) reveals cryptic clades correlated with fatty acid profiles

    PubMed Central

    Spribille, Toby; Klug, Barbara; Mayrhofer, Helmut

    2011-01-01

    Lichens are a prominent feature of northern conifer forests and a large number of species are thought to be circumboreal. Whether or not circumboreal lichen species really constitute monophyletic groups has seldom been tested. We investigated molecular phylogenetic patterns in the mycobiont of Mycoblastus sanguinarius, a well known epiphytic lichen species of the boreal forest, based on material collected from across the high latitude northern hemisphere. A three-locus dataset of internal transcribed spacer rDNA, translation elongation factor 1-α and replication licensing factor Mcm7 DNA sequences revealed that material treated until now as belonging to M. sanguinarius does indeed form a monophyletic group within the genus and is distinct from a strongly supported Mycoblastus affinis. The M. sanguinarius complex appears closely related to the rare Mycoblastus glabrescens, which is currently known only from the Pacific Northwest and was rediscovered during the present study. However, within M. sanguinarius s.lat. in the northern hemisphere, two deeply divergent and morphologically coherent species can be recovered, one of which matches the southern hemisphere species Mycoblastus sanguinarioides and turns out to be widespread in North America and Asia, and one of which corresponds to M. sanguinarius s.str. Both M. sanguinarius and M. sanguinarioides exhibit additional low-level genetic differentiation into geographically structured clades, the most prominent of which are distributed in East Asia/eastern North America and western North America/Europe, respectively. Individuals from these lowest-level clades are morphologically indistinguishable but chemical analyses by thin layer chromatography revealed that each clade possesses its own fatty acid profile, suggesting that chemical differentiation precedes morphological differentiation and may be a precursor to speciation. PMID:21443957

  17. Analysis of porcine adipose tissue transcriptome reveals differences in de novo fatty acid synthesis in pigs with divergent muscle fatty acid composition

    PubMed Central

    2013-01-01

    Background In pigs, adipose tissue is one of the principal organs involved in the regulation of lipid metabolism. It is particularly involved in the overall fatty acid synthesis with consequences in other lipid-target organs such as muscles and the liver. With this in mind, we have used massive, parallel high-throughput sequencing technologies to characterize the porcine adipose tissue transcriptome architecture in six Iberian x Landrace crossbred pigs showing extreme phenotypes for intramuscular fatty acid composition (three per group). Results High-throughput RNA sequencing was used to generate a whole characterization of adipose tissue (backfat) transcriptome. A total of 4,130 putative unannotated protein-coding sequences were identified in the 20% of reads which mapped in intergenic regions. Furthermore, 36% of the unmapped reads were represented by interspersed repeats, SINEs being the most abundant elements. Differential expression analyses identified 396 candidate genes among divergent animals for intramuscular fatty acid composition. Sixty-two percent of these genes (247/396) presented higher expression in the group of pigs with higher content of intramuscular SFA and MUFA, while the remaining 149 showed higher expression in the group with higher content of PUFA. Pathway analysis related these genes to biological functions and canonical pathways controlling lipid and fatty acid metabolisms. In concordance with the phenotypic classification of animals, the major metabolic pathway differentially modulated between groups was de novo lipogenesis, the group with more PUFA being the one that showed lower expression of lipogenic genes. Conclusions These results will help in the identification of genetic variants at loci that affect fatty acid composition traits. The implications of these results range from the improvement of porcine meat quality traits to the application of the pig as an animal model of human metabolic diseases. PMID:24289474

  18. Transcriptomic and proteomic analysis of a compatible tomato-aphid interaction reveals a predominant salicylic acid-dependent plant response

    PubMed Central

    2013-01-01

    Background Aphids are among the most destructive pests in temperate climates, causing significant damage on several crops including tomato. We carried out a transcriptomic and proteomic study to get insights into the molecular mechanisms and dynamics of the tomato response to the Macrosyphum euphorbiae aphid. Results The time course analysis of aphid infestation indicated a complex, dynamic pattern of gene expression. Several biological functions were affected and genes related to the stress and defence response were the most represented. The Gene Ontology categories of the differentially expressed genes (899) and identified proteins (57) indicated that the tomato response is characterized by an increased oxidative stress accompanied by the production of proteins involved in the detoxification of oxygen radicals. Aphids elicit a defense reaction based on the cross-communication of different hormone-related signaling pathways such as those related to the salicylic acid (SA), jasmonic acid (JA), ethylene and brassinosteroids. Among them, the SA-signaling pathway and stress-responsive SA-dependent genes play a dominant role. Furthermore, tomato response is characterized by a reduced accumulation of photosynthetic proteins and a modification of the expression of various cell wall related genes. Conclusions Our work allowed a more comprehensive understanding of the signaling events and the defense dynamics of the tomato response to aphids in a compatible interaction and, based on experimental data, a model of the tomato–aphid molecular interaction was proposed. Considering the rapid advancement of tomato genomics, this information will be important for the development of new protection strategies. PMID:23895395

  19. Analysis of Hydroxycinnamic Acid Degradation in Agrobacterium fabrum Reveals a Coenzyme A-Dependent, Beta-Oxidative Deacetylation Pathway

    PubMed Central

    Campillo, Tony; Renoud, Sébastien; Kerzaon, Isabelle; Vial, Ludovic; Baude, Jessica; Gaillard, Vincent; Bellvert, Floriant; Chamignon, Cécile; Comte, Gilles; Lavire, Céline; Hommais, Florence

    2014-01-01

    The soil- and rhizosphere-inhabiting bacterium Agrobacterium fabrum (genomospecies G8 of the Agrobacterium tumefaciens species complex) is known to have species-specific genes involved in ferulic acid degradation. Here, we characterized, by genetic and analytical means, intermediates of degradation as feruloyl coenzyme A (feruloyl-CoA), 4-hydroxy-3-methoxyphenyl-β-hydroxypropionyl–CoA, 4-hydroxy-3-methoxyphenyl-β-ketopropionyl–CoA, vanillic acid, and protocatechuic acid. The genes atu1416, atu1417, and atu1420 have been experimentally shown to be necessary for the degradation of ferulic acid. Moreover, the genes atu1415 and atu1421 have been experimentally demonstrated to be essential for this degradation and are proposed to encode a phenylhydroxypropionyl-CoA dehydrogenase and a 4-hydroxy-3-methoxyphenyl-β-ketopropionic acid (HMPKP)–CoA β-keto-thiolase, respectively. We thus demonstrated that the A. fabrum hydroxycinnamic degradation pathway is an original coenzyme A-dependent β-oxidative deacetylation that could also transform p-coumaric and caffeic acids. Finally, we showed that this pathway enables the metabolism of toxic compounds from plants and their use for growth, likely providing the species an ecological advantage in hydroxycinnamic-rich environments, such as plant roots or decaying plant materials. PMID:24657856

  20. Raman Spectroscopic Analysis Reveals Abnormal Fatty Acid Composition in Tumor Micro- and Macroenvironments in Human Breast and Rat Mammary Cancer

    PubMed Central

    You, Sixian; Tu, Haohua; Zhao, Youbo; Liu, Yuan; Chaney, Eric J.; Marjanovic, Marina; Boppart, Stephen A.

    2016-01-01

    Fatty acids play essential roles in the growth and metastasis of cancer cells. To facilitate their avid growth and proliferation, cancer cells not only alter the fatty acid synthesis and metabolism intracellularly and extracellularly, but also in the macroenvironment via direct or indirect pathways. We report here, using Raman micro-spectroscopy, that an increase in the production of polyunsaturated fatty acids (PUFAs) was identified in both cancerous and normal appearing breast tissue obtained from breast cancer patients and tumor-bearing rats. By minimizing confounding effects from mixed chemicals and optimizing the signal-to-noise ratio of Raman spectra, we observed a large-scale transition from monounsaturated fatty acids to PUFAs in the tumor while only a small subset of fatty acids transitioned to PUFAs in the tumor micro- and macroenvironment. These data have important implications for further clarifying the macroenvironmental effect of cancer progression and provide new potential approaches for characterizing the tumor micro- and macroenvironment of breast cancer in both pre-clinical animal studies and clinical applications. PMID:27596041

  1. Raman Spectroscopic Analysis Reveals Abnormal Fatty Acid Composition in Tumor Micro- and Macroenvironments in Human Breast and Rat Mammary Cancer.

    PubMed

    You, Sixian; Tu, Haohua; Zhao, Youbo; Liu, Yuan; Chaney, Eric J; Marjanovic, Marina; Boppart, Stephen A

    2016-01-01

    Fatty acids play essential roles in the growth and metastasis of cancer cells. To facilitate their avid growth and proliferation, cancer cells not only alter the fatty acid synthesis and metabolism intracellularly and extracellularly, but also in the macroenvironment via direct or indirect pathways. We report here, using Raman micro-spectroscopy, that an increase in the production of polyunsaturated fatty acids (PUFAs) was identified in both cancerous and normal appearing breast tissue obtained from breast cancer patients and tumor-bearing rats. By minimizing confounding effects from mixed chemicals and optimizing the signal-to-noise ratio of Raman spectra, we observed a large-scale transition from monounsaturated fatty acids to PUFAs in the tumor while only a small subset of fatty acids transitioned to PUFAs in the tumor micro- and macroenvironment. These data have important implications for further clarifying the macroenvironmental effect of cancer progression and provide new potential approaches for characterizing the tumor micro- and macroenvironment of breast cancer in both pre-clinical animal studies and clinical applications. PMID:27596041

  2. Transcriptomic Analysis of Escherichia coli O157:H7 and K-12 Cultures Exposed to Inorganic and Organic Acids in Stationary Phase Reveals Acidulant- and Strain-Specific Acid Tolerance Responses ▿ †

    PubMed Central

    King, Thea; Lucchini, Sacha; Hinton, Jay C. D.; Gobius, Kari

    2010-01-01

    The food-borne pathogen Escherichia coli O157:H7 is commonly exposed to organic acid in processed and preserved foods, allowing adaptation and the development of tolerance to pH levels otherwise lethal. Since little is known about the molecular basis of adaptation of E. coli to organic acids, we studied K-12 MG1655 and O157:H7 Sakai during exposure to acetic, lactic, and hydrochloric acid at pH 5.5. This is the first analysis of the pH-dependent transcriptomic response of stationary-phase E. coli. Thirty-four genes and three intergenic regions were upregulated by both strains during exposure to all acids. This universal acid response included genes involved in oxidative, envelope, and cold stress resistance and iron and manganese uptake, as well as 10 genes of unknown function. Acidulant- and strain-specific responses were also revealed. The acidulant-specific response reflects differences in the modes of microbial inactivation, even between weak organic acids. The two strains exhibited similar responses to lactic and hydrochloric acid, while the response to acetic acid was distinct. Acidulant-dependent differences between the strains involved induction of genes involved in the heat shock response, osmoregulation, inorganic ion and nucleotide transport and metabolism, translation, and energy production. E. coli O157:H7-specific acid-inducible genes were identified, suggesting that the enterohemorrhagic E. coli strain possesses additional molecular mechanisms contributing to acid resistance that are absent in K-12. While E. coli K-12 was most resistant to lactic and hydrochloric acid, O157:H7 may have a greater ability to survive in more complex acidic environments, such as those encountered in the host and during food processing. PMID:20709847

  3. Transcriptomic Analysis of Shiga-Toxigenic Bacteriophage Carriage Reveals a Profound Regulatory Effect on Acid Resistance in Escherichia coli

    PubMed Central

    Veses-Garcia, Marta; Liu, Xuan; Rigden, Daniel J.; Kenny, John G.; McCarthy, Alan J.

    2015-01-01

    Shiga-toxigenic bacteriophages are converting lambdoid phages that impart the ability to produce Shiga toxin to their hosts. Little is known about the function of most of the genes carried by these phages or the impact that lysogeny has on the Escherichia coli host. Here we use next-generation sequencing to compare the transcriptomes of E. coli strains infected with an Stx phage, before and after triggering of the bacterial SOS response that initiates the lytic cycle of the phage. We were able to discriminate between bacteriophage genes expressed in the lysogenic and lytic cycles, and we describe transcriptional changes that occur in the bacterial host as a consequence of Stx phage carriage. Having identified upregulation of the glutamic acid decarboxylase (GAD) operon, confirmed by reverse transcription-quantitative PCR (RT-qPCR), we used phenotypic assays to establish the ability of the Stx prophage to confer a greater acid resistance phenotype on the E. coli host. Known phage regulators were overexpressed in E. coli, and the acid resistance of the recombinant strains was tested. The phage-encoded transcriptional regulator CII was identified as the controller of the acid response in the lysogen. Infection of an E. coli O157 strain, from which integrated Stx prophages were previously removed, showed increased acid resistance following infection with a nontoxigenic phage, ϕ24B. In addition to demonstrating this link between Stx phage carriage and E. coli acid resistance, with its implications for survival postingestion, the data set provides a number of other potential insights into the impact of lambdoid phage carriage on the biology of E. coli. PMID:26386055

  4. Transcriptomic analysis of Shiga-toxigenic bacteriophage carriage reveals a profound regulatory effect on acid resistance in Escherichia coli.

    PubMed

    Veses-Garcia, Marta; Liu, Xuan; Rigden, Daniel J; Kenny, John G; McCarthy, Alan J; Allison, Heather E

    2015-12-01

    Shiga-toxigenic bacteriophages are converting lambdoid phages that impart the ability to produce Shiga toxin to their hosts. Little is known about the function of most of the genes carried by these phages or the impact that lysogeny has on the Escherichia coli host. Here we use next-generation sequencing to compare the transcriptomes of E. coli strains infected with an Stx phage, before and after triggering of the bacterial SOS response that initiates the lytic cycle of the phage. We were able to discriminate between bacteriophage genes expressed in the lysogenic and lytic cycles, and we describe transcriptional changes that occur in the bacterial host as a consequence of Stx phage carriage. Having identified upregulation of the glutamic acid decarboxylase (GAD) operon, confirmed by reverse transcription-quantitative PCR (RT-qPCR), we used phenotypic assays to establish the ability of the Stx prophage to confer a greater acid resistance phenotype on the E. coli host. Known phage regulators were overexpressed in E. coli, and the acid resistance of the recombinant strains was tested. The phage-encoded transcriptional regulator CII was identified as the controller of the acid response in the lysogen. Infection of an E. coli O157 strain, from which integrated Stx prophages were previously removed, showed increased acid resistance following infection with a nontoxigenic phage, ϕ24B. In addition to demonstrating this link between Stx phage carriage and E. coli acid resistance, with its implications for survival postingestion, the data set provides a number of other potential insights into the impact of lambdoid phage carriage on the biology of E. coli. PMID:26386055

  5. Catalytic Analysis of APOBEC3G Involving Real-Time NMR Spectroscopy Reveals Nucleic Acid Determinants for Deamination

    PubMed Central

    Kamba, Keisuke; Nagata, Takashi; Katahira, Masato

    2015-01-01

    APOBEC3G (A3G) is a single-stranded DNA-specific cytidine deaminase that preferentially converts cytidine to uridine at the third position of triplet cytosine (CCC) hotspots. A3G restricts the infectivity of viruses, such as HIV-1, by targeting CCC hotspots scattered through minus DNA strands, reverse-transcribed from genomic RNA. Previously, we developed a real-time NMR method and elucidated the origin of the 3'→5' polarity of deamination of DNA by the C-terminal domain of A3G (CD2), which is a phenomenon by which a hotspot located closer to the 5'-end is deaminated more effectively than one less close to the 5'-end, through quantitative analysis involving nonspecific binding to and sliding along DNA. In the present study we applied the real-time NMR method to analyze the catalytic activity of CD2 toward DNA oligonucleotides containing a nucleotide analog at a single or multiple positions. Analyses revealed the importance of the sugar and base moieties throughout the consecutive 5 nucleotides, the CCC hotspot being positioned at the center. It was also shown that the sugar or base moieties of the nucleotides outside this 5 nucleotide recognition sequence are also relevant as to CD2's activity. Analyses involving DNA oligonucleotides having two CCC hotspots linked by a long sequence of either deoxyribonucleotides, ribonucleotides or abasic deoxyribonucleotides suggested that the phosphate backbone is required for CD2 to slide along the DNA strand and to exert the 3'→5' polarity. Examination of the effects of different salt concentrations on the 3'→5' polarity indicated that the higher the salt concentration, the less prominent the 3'→5' polarity. This is most likely the result of alleviation of sliding due to a decrease in the affinity of CD2 with the phosphate backbone at high salt concentrations. We also investigated the reactivity of substrates containing 5-methylcytidine (5mC) or 5-hydroxymethylcytidine, and found that A3G exhibited low activity toward

  6. Metagenomic analysis reveals adaptations to a cold-adapted lifestyle in a low-temperature acid mine drainage stream.

    PubMed

    Liljeqvist, Maria; Ossandon, Francisco J; González, Carolina; Rajan, Sukithar; Stell, Adam; Valdes, Jorge; Holmes, David S; Dopson, Mark

    2015-04-01

    An acid mine drainage (pH 2.5-2.7) stream biofilm situated 250 m below ground in the low-temperature (6-10°C) Kristineberg mine, northern Sweden, contained a microbial community equipped for growth at low temperature and acidic pH. Metagenomic sequencing of the biofilm and planktonic fractions identified the most abundant microorganism to be similar to the psychrotolerant acidophile, Acidithiobacillus ferrivorans. In addition, metagenome contigs were most similar to other Acidithiobacillus species, an Acidobacteria-like species, and a Gallionellaceae-like species. Analyses of the metagenomes indicated functional characteristics previously characterized as related to growth at low temperature including cold-shock proteins, several pathways for the production of compatible solutes and an anti-freeze protein. In addition, genes were predicted to encode functions related to pH homeostasis and metal resistance related to growth in the acidic metal-containing mine water. Metagenome analyses identified microorganisms capable of nitrogen fixation and exhibiting a primarily autotrophic lifestyle driven by the oxidation of the ferrous iron and inorganic sulfur compounds contained in the sulfidic mine waters. The study identified a low diversity of abundant microorganisms adapted to a low-temperature acidic environment as well as identifying some of the strategies the microorganisms employ to grow in this extreme environment. PMID:25764459

  7. Metabolomic Analysis Reveals Increased Aerobic Glycolysis and Amino Acid Deficit in a Cellular Model of Amyotrophic Lateral Sclerosis.

    PubMed

    Valbuena, Gabriel N; Rizzardini, Milena; Cimini, Sara; Siskos, Alexandros P; Bendotti, Caterina; Cantoni, Lavinia; Keun, Hector C

    2016-05-01

    Defects in energy metabolism are potential pathogenic mechanisms in amyotrophic lateral sclerosis (ALS), a rapidly fatal disease with no cure. The mechanisms through which this occurs remain elusive and their understanding may prove therapeutically useful. We used metabolomics and stable isotope tracers to examine metabolic changes in a well-characterized cell model of familial ALS, the motor neuronal NSC-34 line stably expressing human wild-type Cu/Zn superoxide dismutase (wtSOD1) or mutant G93A (G93ASOD1). Our findings indicate that wt and G93ASOD1 expression both enhanced glucose metabolism under serum deprivation. However, in wtSOD1 cells, this phenotype increased supply of amino acids for protein and glutathione synthesis, while in G93ASOD1 cells it was associated with death, aerobic glycolysis, and a broad dysregulation of amino acid homeostasis. Aerobic glycolysis was mainly due to induction of pyruvate dehydrogenase kinase 1. Our study thus provides novel insight into the role of deranged energy metabolism as a cause of poor adaptation to stress and a promoter of neural cell damage in the presence of mutant SOD1. Furthermore, the metabolic alterations we report may help explain why mitochondrial dysfunction and impairment of the endoplasmic reticulum stress response are frequently seen in ALS. PMID:25963727

  8. Chemical Genetic Analysis and Functional Characterization of Staphylococcal Wall Teichoic Acid 2-Epimerases Reveals Unconventional Antibiotic Drug Targets.

    PubMed

    Mann, Paul A; Müller, Anna; Wolff, Kerstin A; Fischmann, Thierry; Wang, Hao; Reed, Patricia; Hou, Yan; Li, Wenjin; Müller, Christa E; Xiao, Jianying; Murgolo, Nicholas; Sher, Xinwei; Mayhood, Todd; Sheth, Payal R; Mirza, Asra; Labroli, Marc; Xiao, Li; McCoy, Mark; Gill, Charles J; Pinho, Mariana G; Schneider, Tanja; Roemer, Terry

    2016-05-01

    Here we describe a chemical biology strategy performed in Staphylococcus aureus and Staphylococcus epidermidis to identify MnaA, a 2-epimerase that we demonstrate interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA) biosynthesis enzymes. Genetic inactivation of mnaA results in complete loss of WTA and dramatic in vitro β-lactam hypersensitivity in methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE). Likewise, the β-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSA and MRSE infection. Interestingly, whereas MnaA serves as the sole 2-epimerase required for WTA biosynthesis in S. epidermidis, MnaA and Cap5P provide compensatory WTA functional roles in S. aureus. We also demonstrate that MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSA and MRSE. We further determine the 1.9Å crystal structure of S. aureus MnaA and identify critical residues for enzymatic dimerization, stability, and substrate binding. Finally, the natural product antibiotic tunicamycin is shown to physically bind MnaA and Cap5P and inhibit 2-epimerase activity, demonstrating that it inhibits a previously unanticipated step in WTA biosynthesis. In summary, MnaA serves as a new Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore β-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation. PMID:27144276

  9. Chemical Genetic Analysis and Functional Characterization of Staphylococcal Wall Teichoic Acid 2-Epimerases Reveals Unconventional Antibiotic Drug Targets

    PubMed Central

    Mann, Paul A.; Müller, Anna; Wolff, Kerstin A.; Fischmann, Thierry; Wang, Hao; Reed, Patricia; Hou, Yan; Li, Wenjin; Müller, Christa E.; Xiao, Jianying; Murgolo, Nicholas; Sher, Xinwei; Mayhood, Todd; Sheth, Payal R.; Mirza, Asra; Labroli, Marc; Xiao, Li; McCoy, Mark; Gill, Charles J.; Pinho, Mariana G.; Schneider, Tanja; Roemer, Terry

    2016-01-01

    Here we describe a chemical biology strategy performed in Staphylococcus aureus and Staphylococcus epidermidis to identify MnaA, a 2-epimerase that we demonstrate interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA) biosynthesis enzymes. Genetic inactivation of mnaA results in complete loss of WTA and dramatic in vitro β-lactam hypersensitivity in methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE). Likewise, the β-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSA and MRSE infection. Interestingly, whereas MnaA serves as the sole 2-epimerase required for WTA biosynthesis in S. epidermidis, MnaA and Cap5P provide compensatory WTA functional roles in S. aureus. We also demonstrate that MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSA and MRSE. We further determine the 1.9Å crystal structure of S. aureus MnaA and identify critical residues for enzymatic dimerization, stability, and substrate binding. Finally, the natural product antibiotic tunicamycin is shown to physically bind MnaA and Cap5P and inhibit 2-epimerase activity, demonstrating that it inhibits a previously unanticipated step in WTA biosynthesis. In summary, MnaA serves as a new Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore β-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation. PMID:27144276

  10. DNA microarray analysis reveals a role for lysophosphatidic acid in the regulation of anti-inflammatory genes in MC3T3-E1 cells

    SciTech Connect

    Waters, Katrina M.; Tan, Ruimin; Genetos, Damian C.; Verma, Seema; Yellowley, Clare E.; Karin, Norm J.

    2007-11-01

    DNA microarray analysis revealed that treatment of bone cells with a lipid growth factor led to extensive changes in gene expression. Particular relevance to fracture healing and inflammation was revealed.

  11. Integrated Systems Biology Analysis of Transcriptomes Reveals Candidate Genes for Acidity Control in Developing Fruits of Sweet Orange (Citrus sinensis L. Osbeck)

    PubMed Central

    Huang, Dingquan; Zhao, Yihong; Cao, Minghao; Qiao, Liang; Zheng, Zhi-Liang

    2016-01-01

    Organic acids, such as citrate and malate, are important contributors for the sensory traits of fleshy fruits. Although their biosynthesis has been illustrated, regulatory mechanisms of acid accumulation remain to be dissected. To provide transcriptional architecture and identify candidate genes for citrate accumulation in fruits, we have selected for transcriptome analysis four varieties of sweet orange (Citrus sinensis L. Osbeck) with varying fruit acidity, Succari (acidless), Bingtang (low acid), and Newhall and Xinhui (normal acid). Fruits of these varieties at 45 days post anthesis (DPA), which corresponds to Stage I (cell division), had similar acidity, but they displayed differential acid accumulation at 142 DPA (Stage II, cell expansion). Transcriptomes of fruits at 45 and 142 DPA were profiled using RNA sequencing and analyzed with three different algorithms (Pearson correlation, gene coexpression network and surrogate variable analysis). Our network analysis shows that the acid-correlated genes belong to three distinct network modules. Several of these candidate fruit acidity genes encode regulatory proteins involved in transport (such as AHA10), degradation (such as APD2) and transcription (such as AIL6) and act as hubs in the citrate accumulation gene networks. Taken together, our integrated systems biology analysis has provided new insights into the fruit citrate accumulation gene network and led to the identification of candidate genes likely associated with the fruit acidity control. PMID:27092171

  12. Analysis of Amino Acid Variation in the P2 Domain of the GII-4 Norovirus VP1 Protein Reveals Putative Variant-Specific Epitopes

    PubMed Central

    Allen, David J.; Gray, Jim J.; Gallimore, Chris I.; Xerry, Jacqueline; Iturriza-Gómara, Miren

    2008-01-01

    Background Human noroviruses are a highly diverse group of viruses classified into three of the five currently recognised Norovirus genogroups, and contain numerous genotypes or genetic clusters. Noroviruses are the major aetiological agent of endemic gastroenteritis in all age groups, as well as the cause of periodic epidemic gastroenteritis. The noroviruses most commonly associated with outbreaks of gastroenteritis are genogroup II genotype 4 (GII-4) strains. The relationship between genotypes of noroviruses with their phenotypes and antigenic profile remains poorly understood through an inability to culture these viruses and the lack of a suitable animal model. Methodology/Principal Findings Here we describe a study of the diversity of amino acid sequences of the highly variable P2 region in the major capsid protein, VP1, of the GII-4 human noroviruses strains using sequence analysis and homology modelling techniques. Conclusions/Significance Our data identifies two sites in this region, which show significant amino acid substitutions associated with the appearance of variant strains responsible for epidemics with major public health impact. Homology modelling studies revealed the exposed nature of these sites on the capsid surface, providing supportive structural data that these two sites are likely to be associated with putative variant-specific epitopes. Furthermore, the patterns in the evolution of these viruses at these sites suggests that noroviruses follow a neutral network pattern of evolution. PMID:18213393

  13. Comparative Transcriptome Analysis Reveals Different Molecular Mechanisms of Bacillus coagulans 2-6 Response to Sodium Lactate and Calcium Lactate during Lactic Acid Production

    PubMed Central

    Qin, Jiayang; Wang, Xiuwen; Wang, Landong; Zhu, Beibei; Zhang, Xiaohua; Yao, Qingshou; Xu, Ping

    2015-01-01

    Lactate production is enhanced by adding calcium carbonate or sodium hydroxide during fermentation. However, Bacillus coagulans 2-6 can produce more than 180 g/L L-lactic acid when calcium lactate is accumulated, but less than 120 g/L L-lactic acid when sodium lactate is formed. The molecular mechanisms by which B. coagulans responds to calcium lactate and sodium lactate remain unclear. In this study, comparative transcriptomic methods based on high-throughput RNA sequencing were applied to study gene expression changes in B. coagulans 2-6 cultured in non-stress, sodium lactate stress and calcium lactate stress conditions. Gene expression profiling identified 712 and 1213 significantly regulated genes in response to calcium lactate stress and sodium lactate stress, respectively. Gene ontology assignments of the differentially expressed genes were performed. KEGG pathway enrichment analysis revealed that ‘ATP-binding cassette transporters’ were significantly affected by calcium lactate stress, and ‘amino sugar and nucleotide sugar metabolism’ was significantly affected by sodium lactate stress. It was also found that lactate fermentation was less affected by calcium lactate stress than by sodium lactate stress. Sodium lactate stress had negative effect on the expression of ‘glycolysis/gluconeogenesis’ genes but positive effect on the expression of ‘citrate cycle (TCA cycle)’ genes. However, calcium lactate stress had positive influence on the expression of ‘glycolysis/gluconeogenesis’ genes and had minor influence on ‘citrate cycle (TCA cycle)’ genes. Thus, our findings offer new insights into the responses of B. coagulans to different lactate stresses. Notably, our RNA-seq dataset constitute a robust database for investigating the functions of genes induced by lactate stress in the future and identify potential targets for genetic engineering to further improve L-lactic acid production by B. coagulans. PMID:25875592

  14. Pseudomonas lini Strain ZBG1 Revealed Carboxylic Acid Utilization and Copper Resistance Features Required for Adaptation to Vineyard Soil Environment: A Draft Genome Analysis

    PubMed Central

    Chan, Kok-Gan; Chong, Teik-Min; Adrian, Tan-Guan-Sheng; Kher, Heng Leong; Grandclément, Catherine; Faure, Denis; Yin, Wai-Fong; Dessaux, Yves; Hong, Kar-Wai

    2016-01-01

    Pseudomonas lini strain ZBG1 was isolated from the soil of vineyard in Zellenberg, France and the draft genome was reported in this study. Bioinformatics analyses of the genome revealed presence of genes encoding tartaric and malic acid utilization as well as copper resistance that correspond to the adaptation this strain in vineyard soil environment. PMID:27512520

  15. Transcriptional analysis of porcine intestinal mucosa infected with Salmonella Typhimurium revealed a massive inflammatory response and disruption of bile acid absorption in ileum.

    PubMed

    Uribe, Juber Herrera; Collado-Romero, Melania; Zaldívar-López, Sara; Arce, Cristina; Bautista, Rocío; Carvajal, Ana; Cirera, Susanna; Claros, M Gonzalo; Garrido, Juan J

    2016-01-01

    Infected pork meat is an important source of non-typhoidal human salmonellosis. Understanding of molecular mechanisms involved in disease pathogenesis is important for the development of therapeutic and preventive strategies. Thus, hereby we study the transcriptional profiles along the porcine intestine during infection with Salmonella Typhimurium, as well as post-transcriptional gene modulation by microRNAs (miRNA). Sixteen piglets were orally challenged with S. Typhimurium. Samples from jejunum, ileum and colon, collected 1, 2 and 6 days post infection (dpi) were hybridized to mRNA and miRNA expression microarrays and analyzed. Jejunum showed a reduced transcriptional response indicating mild inflammation only at 2 dpi. In ileum inflammatory genes were overexpressed (e.g., IL-1B, IL-6, IL-8, IL1RAP, TNFα), indicating a strong immune response at all times of infection. Infection also down-regulated genes of the FXR pathway (e.g., NR1H4, FABP6, APOA1, SLC10A2), indicating disruption of the bile acid absorption in ileum. This result was confirmed by decreased high-density lipoprotein cholesterol in serum of infected pigs. Ileal inflammatory gene expression changes peaked at 2 dpi and tended to resolve at 6 dpi. Furthermore, miRNA analysis of ileum at 2 dpi revealed 62 miRNAs potentially regulating target genes involved in this inflammatory process (e.g., miR-374 and miR-451). In colon, genes involved in epithelial adherence, proliferation and cellular reorganization were down-regulated at 2 and 6 dpi. In summary, here we show the transcriptional changes occurring at the intestine at different time points of the infection, which are mainly related to inflammation and disruption of the bile acid metabolism. PMID:26738723

  16. Microarray Analysis Reveals Higher Gestational Folic Acid Alters Expression of Genes in the Cerebellum of Mice Offspring—A Pilot Study

    PubMed Central

    Barua, Subit; Kuizon, Salomon; Chadman, Kathryn K.; Brown, W. Ted; Junaid, Mohammed A.

    2015-01-01

    Folate is a water-soluble vitamin that is critical for nucleotide synthesis and can modulate methylation of DNA by altering one-carbon metabolism. Previous studies have shown that folate status during pregnancy is associated with various congenital defects including the risk of aberrant neural tube closure. Maternal exposure to a methyl supplemented diet also can alter DNA methylation and gene expression, which may influence the phenotype of offspring. We investigated if higher gestational folic acid (FA) in the diet dysregulates the expression of genes in the cerebellum of offspring in C57BL/6 J mice. One week before gestation and throughout the pregnancy, groups of dams were supplemented with FA either at 2 mg/kg or 20 mg/kg of diet. Microarray analysis was used to investigate the genome wide gene expression profile in the cerebellum from day old pups. Our results revealed that exposure to the higher dose FA diet during gestation dysregulated expression of several genes in the cerebellum of both male and female pups. Several transcription factors, imprinted genes, neuro-developmental genes and genes associated with autism spectrum disorder exhibited altered expression levels. These findings suggest that higher gestational FA potentially dysregulates gene expression in the offspring brain and such changes may adversely alter fetal programming and overall brain development. PMID:25629700

  17. Fatty acids and small organic compounds bind to mineralo-organic nanoparticles derived from human body fluids as revealed by metabolomic analysis

    NASA Astrophysics Data System (ADS)

    Martel, Jan; Wu, Cheng-Yeu; Hung, Cheng-Yu; Wong, Tsui-Yin; Cheng, Ann-Joy; Cheng, Mei-Ling; Shiao, Ming-Shi; Young, John D.

    2016-03-01

    Nanoparticles entering the human body instantly become coated with a ``protein corona'' that influences the effects and distribution of the particles in vivo. Yet, whether nanoparticles may bind to other organic compounds remains unclear. Here we use an untargeted metabolomic approach based on ultra-performance liquid chromatography and quadruple time-of-flight mass spectrometry to identify the organic compounds that bind to mineral nanoparticles formed in human body fluids (serum, plasma, saliva, and urine). A wide range of organic compounds is identified, including fatty acids, glycerophospholipids, amino acids, sugars, and amides. Our results reveal that, in addition to the proteins identified previously, nanoparticles harbor an ``organic corona'' containing several fatty acids which may affect particle-cell interactions in vivo. This study provides a platform to study the organic corona of biological and synthetic nanoparticles found in the human body.Nanoparticles entering the human body instantly become coated with a ``protein corona'' that influences the effects and distribution of the particles in vivo. Yet, whether nanoparticles may bind to other organic compounds remains unclear. Here we use an untargeted metabolomic approach based on ultra-performance liquid chromatography and quadruple time-of-flight mass spectrometry to identify the organic compounds that bind to mineral nanoparticles formed in human body fluids (serum, plasma, saliva, and urine). A wide range of organic compounds is identified, including fatty acids, glycerophospholipids, amino acids, sugars, and amides. Our results reveal that, in addition to the proteins identified previously, nanoparticles harbor an ``organic corona'' containing several fatty acids which may affect particle-cell interactions in vivo. This study provides a platform to study the organic corona of biological and synthetic nanoparticles found in the human body. Electronic supplementary information (ESI) available. See

  18. Metabolomics analysis reveals elevation of 3-indoxyl sulfate in plasma and brain during chemically-induced acute kidney injury in mice: Investigation of nicotinic acid receptor agonists

    SciTech Connect

    Zgoda-Pols, Joanna R.; Chowdhury, Swapan; Wirth, Mark; Milburn, Michael V.; Alexander, Danny C.; Alton, Kevin B.

    2011-08-15

    An investigative renal toxicity study using metabolomics was conducted with a potent nicotinic acid receptor (NAR) agonist, SCH 900424. Liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) techniques were used to identify small molecule biomarkers of acute kidney injury (AKI) that could aid in a better mechanistic understanding of SCH 900424-induced AKI in mice. The metabolomics study revealed 3-indoxyl sulfate (3IS) as a more sensitive marker of SCH 900424-induced renal toxicity than creatinine or urea. An LC-MS assay for quantitative determination of 3IS in mouse matrices was also developed. Following treatment with SCH 900424, 3IS levels were markedly increased in murine plasma and brain, thereby potentially contributing to renal- and central nervous system (CNS)-related rapid onset of toxicities. Furthermore, significant decrease in urinary excretion of 3IS in those animals due to compromised renal function may be associated with the elevation of 3IS in plasma and brain. These data suggest that 3IS has a potential to be a marker of renal and CNS toxicities during chemically-induced AKI in mice. In addition, based on the metabolomic analysis other statistically significant plasma markers including p-cresol-sulfate and tryptophan catabolites (kynurenate, kynurenine, 3-indole-lactate) might be of toxicological importance but have not been studied in detail. This comprehensive approach that includes untargeted metabolomic and targeted bioanalytical sample analyses could be used to investigate toxicity of other compounds that pose preclinical or clinical development challenges in a pharmaceutical discovery and development. - Research Highlights: > Nicotinic acid receptor agonist, SCH 900424, caused acute kidney injury in mice. > MS-based metabolomics was conducted to identify potential small molecule markers of renal toxicity. > 3-indoxyl-sulfate was found to be as a more sensitive marker of renal toxicity than creatinine

  19. Amino acid analysis

    NASA Technical Reports Server (NTRS)

    Winitz, M.; Graff, J. (Inventor)

    1974-01-01

    The process and apparatus for qualitative and quantitative analysis of the amino acid content of a biological sample are presented. The sample is deposited on a cation exchange resin and then is washed with suitable solvents. The amino acids and various cations and organic material with a basic function remain on the resin. The resin is eluted with an acid eluant, and the eluate containing the amino acids is transferred to a reaction vessel where the eluant is removed. Final analysis of the purified acylated amino acid esters is accomplished by gas-liquid chromatographic techniques.

  20. Transcriptome Profiling and Functional Analysis of Agrobacterium tumefaciens Reveals a General Conserved Response to Acidic Conditions (pH 5.5) and a Complex Acid-Mediated Signaling Involved in Agrobacterium-Plant Interactions▿

    PubMed Central

    Yuan, Ze-Chun; Liu, Pu; Saenkham, Panatda; Kerr, Kathleen; Nester, Eugene W.

    2008-01-01

    Agrobacterium tumefaciens transferred DNA (T-DNA) transfer requires that the virulence genes (vir regulon) on the tumor-inducing (Ti) plasmid be induced by plant phenolic signals in an acidic environment. Using transcriptome analysis, we found that these acidic conditions elicit two distinct responses: (i) a general and conserved response through which Agrobacterium modulates gene expression patterns to adapt to environmental acidification and (ii) a highly specialized acid-mediated signaling response involved in Agrobacterium-plant interactions. Overall, 78 genes were induced and 74 genes were repressed significantly under acidic conditions (pH 5.5) compared to neutral conditions (pH 7.0). Microarray analysis not only confirmed previously identified acid-inducible genes but also uncovered many new acid-induced genes which may be directly involved in Agrobacterium-plant interactions. These genes include virE0, virE1, virH1, and virH2. Further, the chvG-chvI two-component system, previously shown to be critical for virulence, was also induced under acid conditions. Interestingly, acidic conditions induced a type VI secretion system and a putative nonheme catalase. We provide evidence suggesting that acid-induced gene expression was independent of the VirA-VirG two-component system. Our results, together with previous data, support the hypothesis that there is three-step sequential activation of the vir regulon. This process involves a cascade regulation and hierarchical signaling pathway featuring initial direct activation of the VirA-VirG system by the acid-activated ChvG-ChvI system. Our data strengthen the notion that Agrobacterium has evolved a mechanism to perceive and subvert the acidic conditions of the rhizosphere to an important signal that initiates and directs the early virulence program, culminating in T-DNA transfer. PMID:17993523

  1. Metabolomics analysis reveals the association between lipid abnormalities and oxidative stress, inflammation, fibrosis, and Nrf2 dysfunction in aristolochic acid-induced nephropathy

    PubMed Central

    Zhao, Ying-Yong; Wang, Hui-Ling; Cheng, Xian-Long; Wei, Feng; Bai, Xu; Lin, Rui-Chao; Vaziri, Nosratola D.

    2015-01-01

    Alternative medicines are commonly used for the disease prevention and treatment worldwide. Aristolochic acid (AAI) nephropathy (AAN) is a common and rapidly progressive interstitial nephropathy caused by ingestion of Aristolochia herbal medications. Available data on pathophysiology and molecular mechanisms of AAN are limited and were explored here. SD rats were randomized to AAN and control groups. AAN group was treated with AAI by oral gavage for 12 weeks and observed for additional 12 weeks. Kidneys were processed for histological evaluation, Western blotting, and metabolomics analyses using UPLC-QTOF/HDMS. The concentrations of two phosphatidylcholines, two diglycerides and two acyl-carnitines were significantly altered in AAI treated rats at week 4 when renal function and histology were unchanged. Data obtained on weeks 8 to 24 revealed progressive tubulointerstitial fibrosis, inflammation, renal dysfunction, activation of NF-κB, TGF-β, and oxidative pathways, impaired Nrf2 system, and profound changes in lipid metabolites including numerous PC, lysoPC, PE, lysoPE, ceramides and triglycerides. In conclusion, exposure to AAI results in dynamic changes in kidney tissue fatty acid, phospholipid, and glycerolipid metabolisms prior to and after the onset of detectable changes in renal function or histology. These findings point to participation of altered tissue lipid metabolism in the pathogenesis of AAN. PMID:26251179

  2. Amino acid analysis.

    PubMed

    Crabb, J W; West, K A; Dodson, W S; Hulmes, J D

    2001-05-01

    Amino acid analysis (AAA) is one of the best methods to quantify peptides and proteins. Two general approaches to quantitative AAA exist, namely, classical postcolumn derivatization following ion-exchange chromatography and precolumn derivatization followed by reversed-phase HPLC (RP-HPLC). Excellent instrumentation and several specific methodologies are available for both approaches, and both have advantages and disadvantages. This unit focuses on picomole-level AAA of peptides and proteins using the most popular precolumn-derivatization method, namely, phenylthiocarbamyl amino acid analysis (PTC-AAA). It is directed primarily toward those interested in establishing the technology with a modest budget. PTC derivatization and analysis conditions are described, and support and alternate protocols describe additional techniques necessary or useful for most any AAA method--e.g., sample preparation, hydrolysis, instrument calibration, data interpretation, and analysis of difficult or unusual residues such as cysteine, tryptophan, phosphoamino acids, and hydroxyproline. PMID:18429107

  3. Proteomic Analysis of Plasma from California Sea Lions (Zalophus californianus) Reveals Apolipoprotein E as a Candidate Biomarker of Chronic Domoic Acid Toxicosis

    PubMed Central

    Neely, Benjamin A.; Ferrante, Jason A.; Chaves, J. Mauro; Soper, Jennifer L.; Almeida, Jonas S.; Arthur, John M.; Gulland, Frances M. D.; Janech, Michael G.

    2015-01-01

    Domoic acid toxicosis (DAT) in California sea lions (Zalophus californianus) is caused by exposure to the marine biotoxin domoic acid and has been linked to massive stranding events and mortality. Diagnosis is based on clinical signs in addition to the presence of domoic acid in body fluids. Chronic DAT further is characterized by reoccurring seizures progressing to status epilepticus. Diagnosis of chronic DAT is often slow and problematic, and minimally invasive tests for DAT have been the focus of numerous recent biomarker studies. The goal of this study was to retrospectively profile plasma proteins in a population of sea lions with chronic DAT and those without DAT using two dimensional gel electrophoresis to discover whether individual, multiple, or combinations of protein and clinical data could be utilized to identify sea lions with DAT. Using a training set of 32 sea lion sera, 20 proteins and their isoforms were identified that were significantly different between the two groups (p<0.05). Interestingly, 11 apolipoprotein E (ApoE) charge forms were decreased in DAT samples, indicating that ApoE charge form distributions may be important in the progression of DAT. In order to develop a classifier of chronic DAT, an independent blinded test set of 20 sea lions, seven with chronic DAT, was used to validate models utilizing ApoE charge forms and eosinophil counts. The resulting support vector machine had high sensitivity (85.7% with 92.3% negative predictive value) and high specificity (92.3% with 85.7% positive predictive value). These results suggest that ApoE and eosinophil counts along with machine learning can perform as a robust and accurate tool to diagnose chronic DAT. Although this analysis is specifically focused on blood biomarkers and routine clinical data, the results demonstrate promise for future studies combining additional variables in multidimensional space to create robust classifiers. PMID:25919366

  4. Proteomic Analysis of Plasma from California Sea Lions (Zalophus californianus) Reveals Apolipoprotein E as a Candidate Biomarker of Chronic Domoic Acid Toxicosis.

    PubMed

    Neely, Benjamin A; Ferrante, Jason A; Chaves, J Mauro; Soper, Jennifer L; Almeida, Jonas S; Arthur, John M; Gulland, Frances M D; Janech, Michael G

    2014-01-01

    Domoic acid toxicosis (DAT) in California sea lions (Zalophus californianus) is caused by exposure to the marine biotoxin domoic acid and has been linked to massive stranding events and mortality. Diagnosis is based on clinical signs in addition to the presence of domoic acid in body fluids. Chronic DAT further is characterized by reoccurring seizures progressing to status epilepticus. Diagnosis of chronic DAT is often slow and problematic, and minimally invasive tests for DAT have been the focus of numerous recent biomarker studies. The goal of this study was to retrospectively profile plasma proteins in a population of sea lions with chronic DAT and those without DAT using two dimensional gel electrophoresis to discover whether individual, multiple, or combinations of protein and clinical data could be utilized to identify sea lions with DAT. Using a training set of 32 sea lion sera, 20 proteins and their isoforms were identified that were significantly different between the two groups (p<0.05). Interestingly, 11 apolipoprotein E (ApoE) charge forms were decreased in DAT samples, indicating that ApoE charge form distributions may be important in the progression of DAT. In order to develop a classifier of chronic DAT, an independent blinded test set of 20 sea lions, seven with chronic DAT, was used to validate models utilizing ApoE charge forms and eosinophil counts. The resulting support vector machine had high sensitivity (85.7% with 92.3% negative predictive value) and high specificity (92.3% with 85.7% positive predictive value). These results suggest that ApoE and eosinophil counts along with machine learning can perform as a robust and accurate tool to diagnose chronic DAT. Although this analysis is specifically focused on blood biomarkers and routine clinical data, the results demonstrate promise for future studies combining additional variables in multidimensional space to create robust classifiers. PMID:25919366

  5. Real-time analysis of endogenous protoporphyrin IX fluorescence from δ-aminolevulinic acid and its derivatives reveals distinct time- and dose-dependent characteristics in vitro

    NASA Astrophysics Data System (ADS)

    Kiesslich, Tobias; Helander, Linda; Illig, Romana; Oberdanner, Christian; Wagner, Andrej; Lettner, Herbert; Jakab, Martin; Plaetzer, Kristjan

    2014-08-01

    Photodynamic therapy (PDT) and photodiagnosis based on the intracellular production of the photosensitizer protoporphyrin IX (PPIX) by administration of its metabolic precursor δ-aminolevulinic acid (ALA) achieved their breakthrough upon the clinical approval of MAL (ALA methyl ester) and HAL (ALA hexyl ester). For newly developed ALA derivatives or application in new tumor types, in vitro determination of PPIX formation involves multiparametric experiments covering variable pro-drug concentrations, medium composition, time points of analysis, and cell type(s). This study uses a fluorescence microplate reader with a built-in temperature and atmosphere control to investigate the high-resolution long-term kinetics (72 h) of cellular PPIX fueled by administration of either ALA, MAL, or HAL for each 10 different concentrations. For simultaneous proliferation correction, A431 cells were stably transfected with green fluorescent protein. The results indicate that the peak PPIX level is a function of both, incubation concentration and period: maximal PPIX is generated with 1 to 2-mM ALA/MAL or 0.125-mM HAL; also, the PPIX peak shifts to longer incubation periods with increasing pro-drug concentrations. The results underline the need for detailed temporal analysis of PPIX formation to optimize ALA (derivative)-based PDT or photodiagnosis and highlight the value of environment-controlled microplate readers for automated in vitro analysis.

  6. Proteomic Analysis Reveals Differences in Tolerance to Acid Rain in Two Broad-Leaf Tree Species, Liquidambar formosana and Schima superba

    PubMed Central

    Wang, Chao; Liu, Ting-Wu; Chalifour, Annie; Chen, Juan; Shen, Zhi-Jun; Liu, Xiang; Wang, Wen-Hua; Zheng, Hai-Lei

    2014-01-01

    Acid rain (AR) is a serious environmental issue inducing harmful impacts on plant growth and development. It has been reported that Liquidambar formosana, considered as an AR-sensitive tree species, was largely injured by AR, compared with Schima superba, an AR-tolerant tree species. To clarify the different responses of these two species to AR, a comparative proteomic analysis was conducted in this study. More than 1000 protein spots were reproducibly detected on two-dimensional electrophoresis gels. Among them, 74 protein spots from L. formosana gels and 34 protein spots from S. superba gels showed significant changes in their abundances under AR stress. In both L. formosana and S. superba, the majority proteins with more than 2 fold changes were involved in photosynthesis and energy production, followed by material metabolism, stress and defense, transcription, post-translational and modification, and signal transduction. In contrast with L. formosana, no hormone response-related protein was found in S. superba. Moreover, the changes of proteins involved in photosynthesis, starch synthesis, and translation were distinctly different between L. formosana and S. superba. Protein expression analysis of three proteins (ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, ascorbate peroxidase and glutathione-S-transferase) by Western blot was well correlated with the results of proteomics. In conclusion, our study provides new insights into AR stress responses in woody plants and clarifies the differences in strategies to cope with AR between L. formosana and S. superba. PMID:25025692

  7. Transcriptome Analysis Reveals the Mechanism Underlying the Production of a High Quantity of Chlorogenic Acid in Young Leaves of Lonicera macranthoides Hand.-Mazz

    PubMed Central

    Chen, Zexiong; Tang, Ning; You, Yuming; Lan, Jianbin; Liu, Yiqing; Li, Zhengguo

    2015-01-01

    Lonicera macranthoides Hand.-Mazz (L. macranthoides) is a medicinal herb that is widely distributed in southern China. The biosynthetic and metabolic pathways for a core secondary metabolite in L. macranthoides, chlorogenic acid (CGA), have been elucidated in many species. However, the mechanisms of CGA biosynthesis and the related gene regulatory network in L. macranthoides are still not well understood. In this study, CGA content was quantified by high performance liquid chromatography (HPLC), and CGA levels differed significantly among three tissues; specifically, the CGA content in young leaves (YL) was greater than that in young stems (YS), which was greater than that in mature flowers (MF). Transcriptome analysis of L. macranthoides yielded a total of 53,533,014 clean reads (average length 90 bp) and 76,453 unigenes (average length 703 bp). A total of 3,767 unigenes were involved in biosynthesis pathways of secondary metabolites. Of these unigenes, 80 were possibly related to CGA biosynthesis. Furthermore, differentially expressed genes (DEGs) were screened in different tissues including YL, MF and YS. In these tissues, 24 DEGs were found to be associated with CGA biosynthesis, including six phenylalanine ammonia lyase (PAL) genes, six 4-coumarate coenzyme A ligase (4CL) genes, four cinnamate 4-Hydroxylase (C4H) genes, seven hydroxycinnamoyl transferase/hydroxycinnamoyl-CoA quinate transferase HCT/HQT genes and one coumarate 3-hydroxylase (C3H) gene.These results further the understanding of CGA biosynthesis and the related regulatory network in L. macranthoides. PMID:26381882

  8. Analysis of HSD3B7 knockout mice reveals that a 3α-hydroxyl stereochemistry is required for bile acid function

    PubMed Central

    Shea, Heidi C.; Head, Daphne D.; Setchell, Kenneth D. R.; Russell, David W.

    2007-01-01

    Primary bile acids are synthesized from cholesterol in the liver and thereafter are secreted into the bile and small intestine. Gut flora modify primary bile acids to produce secondary bile acids leading to a chemically diverse bile acid pool that is circulated between the small intestine and liver. A majority of primary and secondary bile acids in higher vertebrates have a 3α-hydroxyl group. Here, we characterize a line of knockout mice that cannot epimerize the 3β-hydroxyl group of cholesterol and as a consequence synthesize a bile acid pool in which 3β-hydroxylated bile acids predominate. This alteration causes death in 90% of newborn mice and decreases the absorption of dietary cholesterol in surviving adults. Negative feedback regulation of bile acid synthesis mediated by the farnesoid X receptor (FXR) is disrupted in the mutant mice. We conclude that the correct stereochemistry of a single hydroxyl group at carbon 3 in bile acids is required to maintain their physiologic and regulatory functions in mammals. PMID:17601774

  9. Genetic analysis of two OsLpa1-like genes in Arabidopsis reveals that only one is required for wild-type seed phytic acid levels

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phytic acid (inositol-1,2,3,4,5,6-hexakisphosphate or InsP6) is the primary storage form of phosphorus in plant seeds. The rice OsLpa1 encodes a novel protein required for wild-type levels of seed InsP6 and was identified from a low phytic acid (lpa) mutant exhibiting a 45-50% reduction in seed InsP...

  10. Analysis of Δ12-fatty acid desaturase function revealed that two distinct pathways are active for the synthesis of PUFAs in T. aureum ATCC 34304

    PubMed Central

    Matsuda, Takanori; Sakaguchi, Keishi; Hamaguchi, Rie; Kobayashi, Takumi; Abe, Eriko; Hama, Yoichiro; Hayashi, Masahiro; Honda, Daiske; Okita, Yuji; Sugimoto, Shinichi; Okino, Nozomu; Ito, Makoto

    2012-01-01

    Thraustochytrids are known to synthesize PUFAs such as docosahexaenoic acid (DHA). Accumulating evidence suggests the presence of two synthetic pathways of PUFAs in thraustochytrids: the polyketide synthase-like (PUFA synthase) and desaturase/elongase (standard) pathways. It remains unclear whether the latter pathway functions in thraustochytrids. In this study, we report that the standard pathway produces PUFA in Thraustochytrium aureum ATCC 34304. We isolated a gene encoding a putative Δ12-fatty acid desaturase (TauΔ12des) from T. aureum. Yeasts transformed with the tauΔ12des converted endogenous oleic acid (OA) into linoleic acid (LA). The disruption of the tauΔ12des in T. aureum by homologous recombination resulted in the accumulation of OA and a decrease in the levels of LA and its downstream PUFAs. However, the DHA content was increased slightly in tauΔ12des-disruption mutants, suggesting that DHA is primarily produced in T. aureum via the PUFA synthase pathway. The transformation of the tauΔ12des-disruption mutants with a tauΔ12des expression cassette restored the wild-type fatty acid profiles. These data clearly indicate that TauΔ12des functions as Δ12-fatty acid desaturase in the standard pathway of T. aureum and demonstrate that this thraustochytrid produces PUFAs via both the PUFA synthase and the standard pathways. PMID:22368282

  11. Analysis of Δ12-fatty acid desaturase function revealed that two distinct pathways are active for the synthesis of PUFAs in T. aureum ATCC 34304.

    PubMed

    Matsuda, Takanori; Sakaguchi, Keishi; Hamaguchi, Rie; Kobayashi, Takumi; Abe, Eriko; Hama, Yoichiro; Hayashi, Masahiro; Honda, Daiske; Okita, Yuji; Sugimoto, Shinichi; Okino, Nozomu; Ito, Makoto

    2012-06-01

    Thraustochytrids are known to synthesize PUFAs such as docosahexaenoic acid (DHA). Accumulating evidence suggests the presence of two synthetic pathways of PUFAs in thraustochytrids: the polyketide synthase-like (PUFA synthase) and desaturase/elongase (standard) pathways. It remains unclear whether the latter pathway functions in thraustochytrids. In this study, we report that the standard pathway produces PUFA in Thraustochytrium aureum ATCC 34304. We isolated a gene encoding a putative Δ12-fatty acid desaturase (TauΔ12des) from T. aureum. Yeasts transformed with the tauΔ12des converted endogenous oleic acid (OA) into linoleic acid (LA). The disruption of the tauΔ12des in T. aureum by homologous recombination resulted in the accumulation of OA and a decrease in the levels of LA and its downstream PUFAs. However, the DHA content was increased slightly in tauΔ12des-disruption mutants, suggesting that DHA is primarily produced in T. aureum via the PUFA synthase pathway. The transformation of the tauΔ12des-disruption mutants with a tauΔ12des expression cassette restored the wild-type fatty acid profiles. These data clearly indicate that TauΔ12des functions as Δ12-fatty acid desaturase in the standard pathway of T. aureum and demonstrate that this thraustochytrid produces PUFAs via both the PUFA synthase and the standard pathways. PMID:22368282

  12. Kinetic analysis of the antibacterial activity of probiotic lactobacilli towards Salmonella enterica serovar Typhimurium reveals a role for lactic acid and other inhibitory compounds.

    PubMed

    Makras, Lefteris; Triantafyllou, Vagelis; Fayol-Messaoudi, Domitille; Adriany, Tom; Zoumpopoulou, Georgia; Tsakalidou, Effie; Servin, Alain; De Vuyst, Luc

    2006-04-01

    Six Lactobacillus strains including commercial probiotic ones (L. acidophilus IBB 801, L. amylovorus DCE 471, L. casei Shirota, L. johnsonii La1, L. plantarum ACA-DC 287 and L. rhamnosus GG) were investigated, through batch fermentations under controlled conditions, for their capacity to inhibit Salmonella enterica serovar Typhimurium SL1344. All lactobacilli displayed strong antibacterial activity toward this Gram-negative pathogen and significantly inhibited invasion of the pathogen into cultured human enterocyte-like Caco-2/TC7 cells. By studying the production kinetics of antibacterial activity and applying the appropriate acid and pH control samples during a killing assay, we were able to distinguish between the effect of lactic acid and other inhibitory compounds produced. The antibacterial activity of L. acidophilus IBB 801, L. amylovorus DCE 471, L. casei Shirota and L. rhamnosus GG was solely due to the production of lactic acid. The antibacterial activity of L. johnsonii La1 and L. plantarum ACA-DC 287 was due to the production of lactic acid and (an) unknown inhibitory substance(s). The latter was (were) only active in the presence of lactic acid. In addition, the lactic acid produced was responsible for significant inhibitory activity upon invasion of Salmonella into Caco-2/TC7 cells. PMID:16266797

  13. Innovations in host and microbial sialic acid biosynthesis revealed by phylogenomic prediction of nonulosonic acid structure

    PubMed Central

    Lewis, Amanda L.; Desa, Nolan; Hansen, Elizabeth E.; Knirel, Yuriy A.; Gordon, Jeffrey I.; Gagneux, Pascal; Nizet, Victor; Varki, Ajit

    2009-01-01

    Sialic acids (Sias) are nonulosonic acid (NulO) sugars prominently displayed on vertebrate cells and occasionally mimicked by bacterial pathogens using homologous biosynthetic pathways. It has been suggested that Sias were an animal innovation and later emerged in pathogens by convergent evolution or horizontal gene transfer. To better illuminate the evolutionary processes underlying the phenomenon of Sia molecular mimicry, we performed phylogenomic analyses of biosynthetic pathways for Sias and related higher sugars derived from 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acids. Examination of ≈1,000 sequenced microbial genomes indicated that such biosynthetic pathways are far more widely distributed than previously realized. Phylogenetic analysis, validated by targeted biochemistry, was used to predict NulO types (i.e., neuraminic, legionaminic, or pseudaminic acids) expressed by various organisms. This approach uncovered previously unreported occurrences of Sia pathways in pathogenic and symbiotic bacteria and identified at least one instance in which a human archaeal symbiont tentatively reported to express Sias in fact expressed the related pseudaminic acid structure. Evaluation of targeted phylogenies and protein domain organization revealed that the “unique” Sia biosynthetic pathway of animals was instead a much more ancient innovation. Pathway phylogenies suggest that bacterial pathogens may have acquired Sia expression via adaptation of pathways for legionaminic acid biosynthesis, one of at least 3 evolutionary paths for de novo Sia synthesis. Together, these data indicate that some of the long-standing paradigms in Sia biology should be reconsidered in a wider evolutionary context of the extended family of NulO sugars. PMID:19666579

  14. Highly expressed amino acid biosynthesis genes revealed by global gene expression analysis of Salmonella enterica serovar Enteritidis during growth in whole egg are not essential for this growth.

    PubMed

    Jakočiūnė, Džiuginta; Herrero-Fresno, Ana; Jelsbak, Lotte; Olsen, John Elmerdahl

    2016-05-01

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is the most common cause of egg borne salmonellosis in many parts of the world. This study analyzed gene expression of this bacterium during growth in whole egg, and whether highly expressed genes were essential for the growth. High quality RNA was extracted from S. Enteritidis using a modified RNA-extraction protocol. Global gene expression during growth in whole egg was compared to growth in LB-medium using DNA array method. Twenty-six genes were significantly upregulated during growth in egg; these belonged to amino acid biosynthesis, di/oligopeptide transport system, biotin synthesis, ferrous iron transport system, and type III secretion system. Significant downregulation of 15 genes related to formate hydrogenlyase (FHL) and trehalose metabolism was observed. The results suggested that S. Enteritidis is starved for amino-acids, biotin and iron when growing in egg. However, site specific mutation of amino acid biosynthesis genes asnA (17.3 fold upregulated), asnB (18.6 fold upregulated), asnA/asnB and, serA (12.0 fold upregulated) and gdhA (3.7 fold upregulated), did not result in growth attenuation, suggesting that biosynthesis using the enzymes encoded from these genes may represent the first choice for S. Enteritidis when growing in egg, but when absent, the bacterium could use alternative ways to obtain the amino acids. PMID:26945769

  15. Cannabinoid acids analysis.

    PubMed

    Lercker, G; Bocci, F; Frega, N; Bortolomeazzi, R

    1992-03-01

    The cannabinoid pattern of vegetable preparations from Cannabis sativa (hashish, marijuana) allows to recognize the phenotype of the plants, to be used as drug or for fiber. Cannabinoid determination by analytical point of view has represented some problems caused by the complex composition of the hexane extract. Capillary gas chromatography of the hexane extracts of vegetable samples, shows the presence of rather polar constituents that eluted, with noticeable interactions, only on polar phase. The compounds can be methylated by diazomethane and silanized (TMS) by silylating reagents. The methyl and methyl-TMS derivatives are analyzed by high resolution gas chromatography (HRGC) and by gas chromatography-mass spectrometry (GC-MS). The identification of the compounds shows their nature of cannabinoid acids, which the main by quantitative point of view results the cannabidiolic acid (CBDA). It is known that the cannabinoid acids are thermally unstable and are transformed in the corresponding cannabinoids by decarboxilation. This is of interest in forensic analysis with the aim to establish the total amount of THC in the Cannabis preparations, as the active component. PMID:1503600

  16. The hsp 16 gene of the probiotic Lactobacillus acidophilus is differently regulated by salt, high temperature and acidic stresses, as revealed by reverse transcription quantitative PCR (qRT-PCR) analysis.

    PubMed

    Capozzi, Vittorio; Arena, Mattia Pia; Crisetti, Elisabetta; Spano, Giuseppe; Fiocco, Daniela

    2011-01-01

    Small heat shock proteins (sHsps) are ubiquitous conserved chaperone-like proteins involved in cellular proteins protection under stressful conditions. In this study, a reverse transcription quantitative PCR (RT-qPCR) procedure was developed and used to quantify the transcript level of a small heat shock gene (shs) in the probiotic bacterium Lactobacillus acidophilus NCFM, under stress conditions such as heat (45 °C and 53 °C), bile (0.3% w/v), hyperosmosis (1 M and 2.5 M NaCl), and low pH value (pH 4). The shs gene of L. acidophilus NCFM was induced by salt, high temperature and acidic stress, while repression was observed upon bile stress. Analysis of the 5' noncoding region of the hsp16 gene reveals the presence of an inverted repeat (IR) sequence (TTAGCACTC-N9-GAGTGCTAA) homologue to the controlling IR of chaperone expression (CIRCE) elements found in the upstream regulatory region of Gram-positive heat shock operons, suggesting that the hsp16 gene of L. acidophilus might be transcriptionally controlled by HrcA. In addition, the alignment of several small heat shock proteins identified so far in lactic acid bacteria, reveals that the Hsp16 of L. acidophilus exhibits a strong evolutionary relationship with members of the Lactobacillus acidophilus group. PMID:21954366

  17. Structural and biochemical analyses reveal how ornithine acetyl transferase binds acidic and basic amino acid substrates.

    PubMed

    Iqbal, Aman; Clifton, Ian J; Chowdhury, Rasheduzzaman; Ivison, David; Domene, Carmen; Schofield, Christopher J

    2011-09-21

    Structural and biochemical analyses reveal how ornithine acetyl-transferases catalyse the reversible transfer of an acetyl-group from a basic (ornithine) to an acidic (glutamate) amino acid by employing a common mechanism involving an acetyl-enzyme intermediate but using different side chain binding modes. PMID:21796301

  18. Structural and Enzymatic Analysis of TarM Glycosyltransferase from Staphylococcus aureus Reveals an Oligomeric Protein Specific for the Glycosylation of Wall Teichoic Acid*

    PubMed Central

    Koç, Cengiz; Gerlach, David; Beck, Sebastian; Peschel, Andreas; Xia, Guoqing; Stehle, Thilo

    2015-01-01

    Anionic glycopolymers known as wall teichoic acids (WTAs) functionalize the peptidoglycan layers of many Gram-positive bacteria. WTAs play central roles in many fundamental aspects of bacterial physiology, and they are important determinants of pathogenesis and antibiotic resistance. A number of enzymes that glycosylate WTA in Staphylococcus aureus have recently been identified. Among these is the glycosyltransferase TarM, a component of the WTA de novo biosynthesis pathway. TarM performs the synthesis of α-O-N-acetylglycosylated poly-5′-phosphoribitol in the WTA structure. We have solved the crystal structure of TarM at 2.4 Å resolution, and we have also determined a structure of the enzyme in complex with its substrate UDP-GlcNAc at 2.8 Å resolution. The protein assembles into a propeller-like homotrimer in which each blade contains a GT-B-type glycosyltransferase domain with a typical Rossmann fold. The enzymatic reaction retains the stereochemistry of the anomeric center of the transferred GlcNAc-moiety on the polyribitol backbone. TarM assembles into a trimer using a novel trimerization domain, here termed the HUB domain. Structure-guided mutagenesis experiments of TarM identify residues critical for enzyme activity, assign a putative role for the HUB in TarM function, and allow us to propose a likely reaction mechanism. PMID:25697358

  19. Microarray and genetic analysis reveals that csa-miR159b plays a critical role in abscisic acid-mediated heat tolerance in grafted cucumber plants.

    PubMed

    Li, Hao; Wang, Yu; Wang, Ze; Guo, Xie; Wang, Feng; Xia, Xiao-Jian; Zhou, Jie; Shi, Kai; Yu, Jing-Quan; Zhou, Yan-Hong

    2016-08-01

    Root-shoot communication plays a vital role in plant growth, development and adaptation to environmental stimuli. Grafting-induced stress tolerance is associated with the induction of plentiful stress-related genes and proteins; the mechanism involved, however, remains obscure. Here, we show that the enhanced tolerance against heat stress in cucumber plants with luffa as rootstock was accompanied with an increased accumulation of abscisic acid (ABA), down-regulation of a subset of microRNAs (miRNAs) but up-regulation of their target genes and CsHSP70 accumulation in the shoots. Significantly, luffa rootstock and foliar application of ABA both down-regulated csa-miR159b and up-regulated its target mRNAs CsGAMYB1 and CsMYB29-like and CsHSP70 accumulation in cucumber, while ectopic expression of csa-miR159b led to decreased heat tolerance, AtMYB33 transcript and AtHSP70 accumulation in Arabidopsis plants. Taken together, our results suggest that root-originated signals such as ABA could alter miRNAs in the shoots, which have a major role in the post-transcriptional regulation of the stress-responsive genes. PMID:27037862

  20. Molecular interactions between the specialist herbivore Manduca sexta (lepidoptera, sphingidae) and its natural host Nicotiana attenuata. VI. Microarray analysis reveals that most herbivore-specific transcriptional changes are mediated by fatty acid-amino acid conjugates.

    PubMed

    Halitschke, Rayko; Gase, Klaus; Hui, Dequan; Schmidt, Dominik D; Baldwin, Ian T

    2003-04-01

    Evidence is accumulating that insect-specific plant responses are mediated by constituents in the oral secretions and regurgitants (R) of herbivores, however the relative importance of the different potentially active constituents remains unclear. Fatty acid-amino acid conjugates (FACs) are found in the R of many insect herbivores and have been shown to be necessary and sufficient to elicit a set of herbivore-specific responses when the native tobacco plant Nicotiana attenuata is attacked by the tobacco hornworm, Manduca sexta. Attack by this specialist herbivore results in a large transcriptional reorganization in N. attenuata, and 161 genes have been cloned from previous cDNA differential display-polymerase chain reaction and subtractive hybridization with magnetic beads analysis. cDNAs of these genes, in addition to those of 73 new R-responsive genes identified by cDNA-amplified fragment-length polymorphism display of R-elicited plants, were spotted on polyepoxide coated glass slides to create microarrays highly enriched in Manduca spp.- and R-induced genes. With these microarrays, we compare transcriptional responses in N. attenuata treated with R from the two most damaging lepidopteran herbivores of this plant in nature, M. sexta and Manduca quinquemaculata, which have very similar FAC compositions in their R, and with the two most abundant FACs in Manduca spp. R. More than 68% of the genes up- and down-regulated by M. sexta R were similarly regulated by M. quinquemaculata R. A majority of genes up-regulated (64%) and down-regulated (49%) by M. sexta R were similarly regulated by treatment with the two FACs. In contrast, few genes showed similar transcriptional changes after H(2)O(2)- and R-treatment. These results demonstrate that the two most abundant FACs in Manduca spp. R can account for the majority of Manduca spp.-induced alterations of the wound response of N. attenuata. PMID:12692348

  1. Molecular Dynamic Simulations Reveal the Structural Determinants of Fatty Acid Binding to Oxy-Myoglobin

    PubMed Central

    Chintapalli, Sree V.; Bhardwaj, Gaurav; Patel, Reema; Shah, Natasha; Patterson, Randen L.; van Rossum, Damian B.; Anishkin, Andriy; Adams, Sean H.

    2015-01-01

    The mechanism(s) by which fatty acids are sequestered and transported in muscle have not been fully elucidated. A potential key player in this process is the protein myoglobin (Mb). Indeed, there is a catalogue of empirical evidence supporting direct interaction of globins with fatty acid metabolites; however, the binding pocket and regulation of the interaction remains to be established. In this study, we employed a computational strategy to elucidate the structural determinants of fatty acids (palmitic & oleic acid) binding to Mb. Sequence analysis and docking simulations with a horse (Equus caballus) structural Mb reference reveals a fatty acid-binding site in the hydrophobic cleft near the heme region in Mb. Both palmitic acid and oleic acid attain a “U” shaped structure similar to their conformation in pockets of other fatty acid-binding proteins. Specifically, we found that the carboxyl head group of palmitic acid coordinates with the amino group of Lys45, whereas the carboxyl group of oleic acid coordinates with both the amino groups of Lys45 and Lys63. The alkyl tails of both fatty acids are supported by surrounding hydrophobic residues Leu29, Leu32, Phe33, Phe43, Phe46, Val67, Val68 and Ile107. In the saturated palmitic acid, the hydrophobic tail moves freely and occasionally penetrates deeper inside the hydrophobic cleft, making additional contacts with Val28, Leu69, Leu72 and Ile111. Our simulations reveal a dynamic and stable binding pocket in which the oxygen molecule and heme group in Mb are required for additional hydrophobic interactions. Taken together, these findings support a mechanism in which Mb acts as a muscle transporter for fatty acid when it is in the oxygenated state and releases fatty acid when Mb converts to deoxygenated state. PMID:26030763

  2. Molecular dynamic simulations reveal the structural determinants of Fatty Acid binding to oxy-myoglobin.

    PubMed

    Chintapalli, Sree V; Bhardwaj, Gaurav; Patel, Reema; Shah, Natasha; Patterson, Randen L; van Rossum, Damian B; Anishkin, Andriy; Adams, Sean H

    2015-01-01

    The mechanism(s) by which fatty acids are sequestered and transported in muscle have not been fully elucidated. A potential key player in this process is the protein myoglobin (Mb). Indeed, there is a catalogue of empirical evidence supporting direct interaction of globins with fatty acid metabolites; however, the binding pocket and regulation of the interaction remains to be established. In this study, we employed a computational strategy to elucidate the structural determinants of fatty acids (palmitic & oleic acid) binding to Mb. Sequence analysis and docking simulations with a horse (Equus caballus) structural Mb reference reveals a fatty acid-binding site in the hydrophobic cleft near the heme region in Mb. Both palmitic acid and oleic acid attain a "U" shaped structure similar to their conformation in pockets of other fatty acid-binding proteins. Specifically, we found that the carboxyl head group of palmitic acid coordinates with the amino group of Lys45, whereas the carboxyl group of oleic acid coordinates with both the amino groups of Lys45 and Lys63. The alkyl tails of both fatty acids are supported by surrounding hydrophobic residues Leu29, Leu32, Phe33, Phe43, Phe46, Val67, Val68 and Ile107. In the saturated palmitic acid, the hydrophobic tail moves freely and occasionally penetrates deeper inside the hydrophobic cleft, making additional contacts with Val28, Leu69, Leu72 and Ile111. Our simulations reveal a dynamic and stable binding pocket in which the oxygen molecule and heme group in Mb are required for additional hydrophobic interactions. Taken together, these findings support a mechanism in which Mb acts as a muscle transporter for fatty acid when it is in the oxygenated state and releases fatty acid when Mb converts to deoxygenated state. PMID:26030763

  3. Analysis of Organic Acids.

    ERIC Educational Resources Information Center

    Griswold, John R.; Rauner, Richard A.

    1990-01-01

    Presented are the procedures and a discussion of the results for an experiment in which students select unknown carboxylic acids, determine their melting points, and investigate their solubility behavior in water and ethanol. A table of selected carboxylic acids is included. (CW)

  4. Microfluidics in amino acid analysis.

    PubMed

    Pumera, Martin

    2007-07-01

    Microfluidic devices have been widely used to derivatize, separate, and detect amino acids employing many different strategies. Virtually zero-dead volume interconnections and fast mass transfer in small volume microchannels enable dramatic increases in on-chip derivatization reaction speed, while only minute amounts of sample and reagent are needed. Due to short channel path, fast subsecond separations can be carried out. With sophisticated miniaturized detectors, the whole analytical process can be integrated on one platform. This article reviews developments of lab-on-chip technology in amino acid analysis, it shows important design features such as sample preconcentration, precolumn and postcolumn amino acid derivatization, and unlabeled and labeled amino acid detection with focus on advanced designs. The review also describes important biomedical and space exploration applications of amino acid analysis on microfluidic devices. PMID:17542043

  5. Analysis of Cytokinin Mutants and Regulation of Cytokinin Metabolic Genes Reveals Important Regulatory Roles of Cytokinins in Drought, Salt and Abscisic Acid Responses, and Abscisic Acid Biosynthesis[C][W

    PubMed Central

    Nishiyama, Rie; Watanabe, Yasuko; Fujita, Yasunari; Le, Dung Tien; Kojima, Mikiko; Werner, Tomás; Vankova, Radomira; Yamaguchi-Shinozaki, Kazuko; Shinozaki, Kazuo; Kakimoto, Tatsuo; Sakakibara, Hitoshi; Schmülling, Thomas; Tran, Lam-Son Phan

    2011-01-01

    Cytokinins (CKs) regulate plant growth and development via a complex network of CK signaling. Here, we perform functional analyses with CK-deficient plants to provide direct evidence that CKs negatively regulate salt and drought stress signaling. All CK-deficient plants with reduced levels of various CKs exhibited a strong stress-tolerant phenotype that was associated with increased cell membrane integrity and abscisic acid (ABA) hypersensitivity rather than stomatal density and ABA-mediated stomatal closure. Expression of the Arabidopsis thaliana ISOPENTENYL-TRANSFERASE genes involved in the biosynthesis of bioactive CKs and the majority of the Arabidopsis CYTOKININ OXIDASES/DEHYDROGENASES genes was repressed by stress and ABA treatments, leading to a decrease in biologically active CK contents. These results demonstrate a novel mechanism for survival under abiotic stress conditions via the homeostatic regulation of steady state CK levels. Additionally, under normal conditions, although CK deficiency increased the sensitivity of plants to exogenous ABA, it caused a downregulation of key ABA biosynthetic genes, leading to a significant reduction in endogenous ABA levels in CK-deficient plants relative to the wild type. Taken together, this study provides direct evidence that mutual regulation mechanisms exist between the CK and ABA metabolism and signals underlying different processes regulating plant adaptation to stressors as well as plant growth and development. PMID:21719693

  6. Effects of a blend of essential oil compounds and benzoic acid on performance of broiler chickens as revealed by a meta-analysis of 4 growth trials in various locations.

    PubMed

    Weber, G M; Michalczuk, M; Huyghebaert, G; Juin, H; Kwakernaak, C; Gracia, M I

    2012-11-01

    A series of growth trials with broiler chicks was conducted in various geographical locations to evaluate the efficacy of a novel eubiotic feed additive (EFA) at various dietary inclusion levels on performance of growing chicks. The EFA product consisted of a blend of essential oil compounds (thymol, eugenol, piperine) with benzoic acid, all belonging to the group of flavoring substances. Although variable in responses, the overall results indicated that 300 mg/kg of this EFA represented an optimum supplementation dose for generation of beneficial performance effects in broilers. A meta-analysis with all data from the 300 mg/kg EFA-supplemented treatments in comparison with the non-supplemented controls revealed that the eubiotic product significantly improved BW on d 21 (+2.0%; P = 0.0021) and on d 42 (+1.4%; P = 0.0151). Furthermore, the birds on the EFA 300 mg/kg treatment expressed a higher average daily gain in the starter phase (d 1-21; +2.1%; P = 0.0023) and over the entire experimental period (d 1-42; +1.5%; P = 0.0154). Feed conversion ratio was more favorable with dietary EFA supplementation (-0.6%; P = 0.0414), when compared with the control birds. Mortality was considered normal and was not affected by the dietary treatment (control = 3.09%; EFA 300 mg/kg = 3.26%). In conclusion, 300 mg/kg of this new eubiotic product demonstrated to effectively improve performance of broiler chicks under various husbandry conditions. PMID:23091138

  7. Analysis of a pair of END+ and END− viruses derived from the same bovine viral diarrhea virus stock reveals the amino acid determinants in Npro responsible for inhibition of type I interferon production

    PubMed Central

    KOZASA, Takashi; ABE, Yuri; MITSUHASHI, Kazuya; TAMURA, Tomokazu; AOKI, Hiroshi; ISHIMARU, Masatoshi; NAKAMURA, Shigeyuki; OKAMATSU, Masatoshi; KIDA, Hiroshi; SAKODA, Yoshihiro

    2014-01-01

    The Exaltation of Newcastle disease virus (END) phenomenon is induced by the inhibition of type I interferon in pestivirus-infected cells in vitro, via proteasomal degradation of cellular interferon regulatory factor (IRF)-3 with the property of the viral autoprotease protein Npro. Reportedly, the amino acid residues in the zinc-binding TRASH motif of Npro determine the difference in characteristics between END-phenomenon-positive (END+) and END-phenomenon-negative (END−) classical swine fever viruses (CSFVs). However, the basic mechanism underlying this function in bovine viral diarrhea virus (BVDV) has not been elucidated from the genomic differences between END+ and END− viruses using reverse genetics till date. In the present study, comparison of complete genome sequences of a pair of END+ and END− viruses isolated from the same virus stock revealed that there were only four amino acid substitutions (D136G, I2623V, D3148G and D3502Y) between two viruses. Based on these differences, viruses with and without mutations at these positions were generated using reverse genetics. The END assay, measurements of induced type I interferon and IRF-3 detection in cells infected with these viruses revealed that the aspartic acid at position 136 in the zinc-binding TRASH motif of Npro was required to inhibit the production of type I interferon via the degradation of cellular IRF-3, consistently with CSFV. PMID:25648277

  8. Analysis of a pair of END+ and END- viruses derived from the same bovine viral diarrhea virus stock reveals the amino acid determinants in Npro responsible for inhibition of type I interferon production.

    PubMed

    Kozasa, Takashi; Abe, Yuri; Mitsuhashi, Kazuya; Tamura, Tomokazu; Aoki, Hiroshi; Ishimaru, Masatoshi; Nakamura, Shigeyuki; Okamatsu, Masatoshi; Kida, Hiroshi; Sakoda, Yoshihiro

    2015-05-01

    The Exaltation of Newcastle disease virus (END) phenomenon is induced by the inhibition of type I interferon in pestivirus-infected cells in vitro, via proteasomal degradation of cellular interferon regulatory factor (IRF)-3 with the property of the viral autoprotease protein N(pro). Reportedly, the amino acid residues in the zinc-binding TRASH motif of N(pro) determine the difference in characteristics between END-phenomenon-positive (END(+)) and END-phenomenon-negative (END(-)) classical swine fever viruses (CSFVs). However, the basic mechanism underlying this function in bovine viral diarrhea virus (BVDV) has not been elucidated from the genomic differences between END(+) and END(-) viruses using reverse genetics till date. In the present study, comparison of complete genome sequences of a pair of END(+) and END(-) viruses isolated from the same virus stock revealed that there were only four amino acid substitutions (D136G, I2623V, D3148G and D3502Y) between two viruses. Based on these differences, viruses with and without mutations at these positions were generated using reverse genetics. The END assay, measurements of induced type I interferon and IRF-3 detection in cells infected with these viruses revealed that the aspartic acid at position 136 in the zinc-binding TRASH motif of N(pro) was required to inhibit the production of type I interferon via the degradation of cellular IRF-3, consistently with CSFV. PMID:25648277

  9. Elastoviscous Transitions of Articular Cartilage Reveal a Mechanism of Synergy between Lubricin and Hyaluronic Acid

    PubMed Central

    Bonnevie, Edward D.; Galesso, Devis; Secchieri, Cynthia; Cohen, Itai; Bonassar, Lawrence J.

    2015-01-01

    When lubricated by synovial fluid, articular cartilage provides some of the lowest friction coefficients found in nature. While it is known that macromolecular constituents of synovial fluid provide it with its lubricating ability, it is not fully understood how two of the main molecules, lubricin and hyaluronic acid, lubricate and interact with one another. Here, we develop a novel framework for cartilage lubrication based on the elastoviscous transition to show that lubricin and hyaluronic acid lubricate by distinct mechanisms. Such analysis revealed nonspecific interactions between these molecules in which lubricin acts to concentrate hyaluronic acid near the tissue surface and promotes a transition to a low friction regime consistent with the theory of viscous boundary lubrication. Understanding the mechanics of synovial fluid not only provides insight into the progression of diseases such as arthritis, but also may be applicable to the development of new biomimetic lubricants. PMID:26599797

  10. Metabolomic Analyses of Leishmania Reveal Multiple Species Differences and Large Differences in Amino Acid Metabolism

    PubMed Central

    Wang, Lijie; Zhang, Tong; Watson, David G.; Silva, Ana Marta; Coombs, Graham H.

    2015-01-01

    Comparative genomic analyses of Leishmania species have revealed relatively minor heterogeneity amongst recognised housekeeping genes and yet the species cause distinct infections and pathogenesis in their mammalian hosts. To gain greater information on the biochemical variation between species, and insights into possible metabolic mechanisms underpinning visceral and cutaneous leishmaniasis, we have undertaken in this study a comparative analysis of the metabolomes of promastigotes of L. donovani, L. major and L. mexicana. The analysis revealed 64 metabolites with confirmed identity differing 3-fold or more between the cell extracts of species, with 161 putatively identified metabolites differing similarly. Analysis of the media from cultures revealed an at least 3-fold difference in use or excretion of 43 metabolites of confirmed identity and 87 putatively identified metabolites that differed to a similar extent. Strikingly large differences were detected in their extent of amino acid use and metabolism, especially for tryptophan, aspartate, arginine and proline. Major pathways of tryptophan and arginine catabolism were shown to be to indole-3-lactate and arginic acid, respectively, which were excreted. The data presented provide clear evidence on the value of global metabolomic analyses in detecting species-specific metabolic features, thus application of this technology should be a major contributor to gaining greater understanding of how pathogens are adapted to infecting their hosts. PMID:26368322

  11. Fatty acid profiling reveals seasonal and spatial shifts in zooplankton diet in a temperate estuary

    NASA Astrophysics Data System (ADS)

    Gonçalves, A. M. M.; Azeiteiro, U. M.; Pardal, M. A.; De Troch, M.

    2012-08-01

    Fatty acids composition of copepod and cladoceran species and their possible food sources was investigated in the Mondego estuary (southern Europe) in order to explain the seasonal variation of the small copepods Acartia clausi, Acartia tonsa, Copidodiaptomus numidicus, Temora longicornis and the freshwater cladoceran Daphnia longispina. A total of 12 zooplankton species (7 marine, 2 estuarine and 3 freshwater species) were studied. A multivariate analysis revealed a clear seasonal distribution of zooplankton species in terms of fatty acids composition and abundance, with winter and spring zooplankton species showing maximal concentrations and diversity of total fatty acids. These findings underline the role of lipids as storage during the colder seasons in a highly variable environment like an estuary. Estuarine and freshwater species showed a more diverse array of saturated and unsaturated fatty acids rather than marine species, except for Centropages typicus. Fatty acids markers of trophic position indicated the presence of two trophic levels: copepod species were primarily omnivorous, whereas cladocerans showed to be herbivorous. Our results suggest that feeding patterns of plankton change spatially and temporally, reflecting the shifts in dominance between diatoms and flagellates as well as between dinoflagellates/diatoms and small animals.

  12. Functionalised carboxylic acids in atmospheric particles: An annual cycle revealing seasonal trends and possible sources

    NASA Astrophysics Data System (ADS)

    Teich, Monique; van Pinxteren, Dominik; Herrmann, Hartmut

    2013-04-01

    Carboxylic acids represent a major fraction of the water soluble organic carbon (WSOC) in atmospheric particles. Among the particle phase carboxylic acids, straight-chain monocarboxylic acids (MCA) and dicarboxylic acids (DCA) with 2-10 carbon atoms have extensively been studied in the past. However, only a few studies exist dealing with functionalised carboxylic acids, i.e. having additional hydroxyl-, oxo- or nitro-groups. Regarding atmospheric chemistry, these functionalised carboxylic acids are of particular interest as they are supposed to be formed during atmospheric oxidation processes, e.g. through radical reactions. Therefore they can provide insights into the tropospheric multiphase chemistry. During this work 28 carboxylic acids (4 functionalised aliphatic MCAs, 5 aromatic MCAs, 3 nitroaromatic MCAs, 6 aliphatic DCAs, 6 functionalised aliphatic DCAs, 4 aromatic DCAs) were quantitatively determined in 256 filter samples taken at the rural research station Melpitz (Saxony, Germany) with a PM10 Digitel DHA-80 filter sampler. All samples were taken in 2010 covering a whole annual cycle. The resulting dataset was examined for a possible seasonal dependency of the acid concentrations. Furthermore the influence of the air mass origin on the acid concentrations was studied based on a simple two-sector classification (western or eastern sector) using a back trajectory analysis. Regarding the annual average, adipic acid was found to be the most abundant compound with a mean concentration of 7.8 ng m-3 followed by 4-oxopimelic acid with 6.1 ng m-3. The sum of all acid concentrations showed two maxima during the seasonal cycle; one in summer and one in winter, whereas the highest overall acid concentrations were found in summer. In general the target acids could be divided into two different groups, where one group has its maximum concentration in summer and the other group during winter. The first group contains all investigated aliphatic mono- and dicarboxylic

  13. Characterization of Drosophila CMP-sialic acid synthetase activity reveals unusual enzymatic properties.

    PubMed

    Mertsalov, Ilya B; Novikov, Boris N; Scott, Hilary; Dangott, Lawrence; Panin, Vladislav M

    2016-07-01

    CMP-sialic acid synthetase (CSAS) is a key enzyme of the sialylation pathway. CSAS produces the activated sugar donor, CMP-sialic acid, which serves as a substrate for sialyltransferases to modify glycan termini with sialic acid. Unlike other animal CSASs that normally localize in the nucleus, Drosophila melanogaster CSAS (DmCSAS) localizes in the cell secretory compartment, predominantly in the Golgi, which suggests that this enzyme has properties distinct from those of its vertebrate counterparts. To test this hypothesis, we purified recombinant DmCSAS and characterized its activity in vitro Our experiments revealed several unique features of this enzyme. DmCSAS displays specificity for N-acetylneuraminic acid as a substrate, shows preference for lower pH and can function with a broad range of metal cofactors. When tested at a pH corresponding to the Golgi compartment, the enzyme showed significant activity with several metal cations, including Zn(2+), Fe(2+), Co(2+) and Mn(2+), whereas the activity with Mg(2+) was found to be low. Protein sequence analysis and site-specific mutagenesis identified an aspartic acid residue that is necessary for enzymatic activity and predicted to be involved in co-ordinating a metal cofactor. DmCSAS enzymatic activity was found to be essential in vivo for rescuing the phenotype of DmCSAS mutants. Finally, our experiments revealed a steep dependence of the enzymatic activity on temperature. Taken together, our results indicate that DmCSAS underwent evolutionary adaptation to pH and ionic environment different from that of counterpart synthetases in vertebrates. Our data also suggest that environmental temperatures can regulate Drosophila sialylation, thus modulating neural transmission. PMID:27114558

  14. Quantitative three-dimensional analysis of poly (lactic-co-glycolic acid) microsphere using hard X-ray nano-tomography revealed correlation between structural parameters and drug burst release.

    PubMed

    Huang, Xiaozhou; Li, Na; Wang, Dajiang; Luo, Yuyan; Wu, Ziyu; Guo, Zhefei; Jin, Qixing; Liu, Zhuying; Huang, Yafei; Zhang, Yongming; Wu, Chuanbin

    2015-08-10

    The objective of this study was to investigate the use of transmission hard X-ray nano-computed-tomography (nano-CT) for characterization of the pore structure and drug distribution in poly (lactic-co-glycolic acid) (PLGA) microspheres encapsulating bovine serum albumin and to study the correlation between drug distribution and burst release. The PLGA microspheres were fabricated using a double-emulsion method. The results of pore structure analysis accessed with nano-CT were compared with those acquired by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Surface pore interconnectivity and surface protein interconnectivity were obtained using combined nano-CT and pixel analysis. The correlation between surface protein interconnectivity with the initial burst release across various tested formulations was also analyzed. The size, shape, and distribution of the pores and protein could be clearly observed in the whole microsphere using nano-CT, whereas only the sectional information was observed using SEM or CLSM. Interconnected pores and surface connected pores could be clearly distinguished in nano-CT, which enables the quantitative analysis of surface pore interconnectivity and surface protein interconnectivity. The surface protein interconnectivity in different formulations correlated well with the burst release at 5-10h. Nano-CT provided a nondestructive, high-resolution, and three-dimensional analysis method to characterize the porous microsphere. PMID:25951620

  15. Antiparasitic evaluation of betulinic acid derivatives reveals effective and selective anti-Trypanosoma cruzi inhibitors.

    PubMed

    Meira, Cássio Santana; Barbosa-Filho, José Maria; Lanfredi-Rangel, Adriana; Guimarães, Elisalva Teixeira; Moreira, Diogo Rodrigo Magalhães; Soares, Milena Botelho Pereira

    2016-07-01

    Betulinic acid is a pentacyclic triterpenoid with several biological properties already described, including antiparasitic activity. Here, the anti-Trypanosoma cruzi activity of betulinic acid and its semi-synthetic amide derivatives (BA1-BA8) was investigated. The anti-Trypanosoma cruzi activity and selectivity were enhanced in semi-synthetic derivatives, specially on derivatives BA5, BA6 and BA8. To understand the mechanism of action underlying betulinic acid anti-T. cruzi activity, we investigated ultrastructural changes by electron microscopy. Ultrastructural studies showed that trypomastigotes incubated with BA5 had membrane blebling, flagella retraction, atypical cytoplasmic vacuoles and Golgi cisternae dilatation. Flow cytometry analysis showed that parasite death is mainly caused by necrosis. Treatment with derivatives BA5, BA6 or BA8 reduced the invasion process, as well as intracellular parasite development in host cells, with a potency and selectivity similar to that observed in benznidazole-treated cells. More importantly, the combination of BA5 and benznidazole revealed synergistic effects on trypomastigote and amastigote forms of T. cruzi. In conclusion, we demonstrated that BA5 compound is an effective and selective anti-T. cruzi agent. PMID:27080160

  16. Quantitative interactome analysis reveals a chemoresistant edgotype

    PubMed Central

    Chavez, Juan D.; Schweppe, Devin K.; Eng, Jimmy K.; Zheng, Chunxiang; Taipale, Alex; Zhang, Yiyi; Takara, Kohji; Bruce, James E.

    2015-01-01

    Chemoresistance is a common mode of therapy failure for many cancers. Tumours develop resistance to chemotherapeutics through a variety of mechanisms, with proteins serving pivotal roles. Changes in protein conformations and interactions affect the cellular response to environmental conditions contributing to the development of new phenotypes. The ability to understand how protein interaction networks adapt to yield new function or alter phenotype is limited by the inability to determine structural and protein interaction changes on a proteomic scale. Here, chemical crosslinking and mass spectrometry were employed to quantify changes in protein structures and interactions in multidrug-resistant human carcinoma cells. Quantitative analysis of the largest crosslinking-derived, protein interaction network comprising 1,391 crosslinked peptides allows for ‘edgotype' analysis in a cell model of chemoresistance. We detect consistent changes to protein interactions and structures, including those involving cytokeratins, topoisomerase-2-alpha, and post-translationally modified histones, which correlate with a chemoresistant phenotype. PMID:26235782

  17. Quantitative interactome analysis reveals a chemoresistant edgotype.

    PubMed

    Chavez, Juan D; Schweppe, Devin K; Eng, Jimmy K; Zheng, Chunxiang; Taipale, Alex; Zhang, Yiyi; Takara, Kohji; Bruce, James E

    2015-01-01

    Chemoresistance is a common mode of therapy failure for many cancers. Tumours develop resistance to chemotherapeutics through a variety of mechanisms, with proteins serving pivotal roles. Changes in protein conformations and interactions affect the cellular response to environmental conditions contributing to the development of new phenotypes. The ability to understand how protein interaction networks adapt to yield new function or alter phenotype is limited by the inability to determine structural and protein interaction changes on a proteomic scale. Here, chemical crosslinking and mass spectrometry were employed to quantify changes in protein structures and interactions in multidrug-resistant human carcinoma cells. Quantitative analysis of the largest crosslinking-derived, protein interaction network comprising 1,391 crosslinked peptides allows for 'edgotype' analysis in a cell model of chemoresistance. We detect consistent changes to protein interactions and structures, including those involving cytokeratins, topoisomerase-2-alpha, and post-translationally modified histones, which correlate with a chemoresistant phenotype. PMID:26235782

  18. Analysis of the structure of mycolic acids of Mycobacterium simiae reveals a particular composition of alpha-mycolates in strain 'habana' TMC 5135, considered as immunogenic in tuberculosis and leprosy.

    PubMed

    Mederos, Lilian; Valdivia, José A; Valero-Guillén, Pedro L

    2007-12-01

    Structural analysis of mycolic acids from Mycobacterium simiae (including some 'habana' strains) was carried out using (1)H-NMR and MS. Results indicated that this species presents a general pattern of alpha-, alpha'- and keto-mycolates. alpha-Mycolates were composed of a complex mixture of 82 to 89 carbon atoms (C82-C89), with the predominant molecular species containing two di-substituted cyclopropane rings. Among keto-mycolates (C84-C89), those containing one trans di-substituted cyclopropane ring were the most abundant. The alpha'-mycolates were monounsaturated (C64, C66). According to MS and (1)H-NMR data, the strains studied differed in fine structural details of alpha-mycolates and keto-mycolates. Notably, strain 'habana' TMC 5135 (belonging to the 'habana' group, and considered as highly immunogenic in tuberculosis and leprosy) presented a particular composition of alpha-mycolates, with a major component (C87) containing one cis plus one trans di-substituted cyclopropane ring, unlike the type strain of M. simiae and other strains of the 'habana' group (IPK-220 and IPK-337R), in which the major component (C84) contained two cis di-substituted cyclopropane rings. In spite of this finding, the 'habana' strains were closely related to each other and mainly differed from the type strain of M. simiae in some details of the fine structure of keto-mycolates. The present work indicated that within an identical general pattern of mycolic acids, there is a complex composition in M. simiae and structural variation among different strains, as reported for pathogenic species of the genus. Noteworthy was the particular composition of alpha-mycolates in strain 'habana' TMC 5135. PMID:18048929

  19. Phylogenomic analysis of 16S rRNA:(guanine-N2) methyltransferases suggests new family members and reveals highly conserved motifs and a domain structure similar to other nucleic acid amino-methyltransferases.

    PubMed

    Bujnicki, J M

    2000-11-01

    The sequences of known Escherichia coli 16S rRNA:m2G1207 methyltransferase (MTase) RsmC and hypothetical 16S rRNA:m2G966 MTase encoded by the ygjo open reading frame were used to carry out a database search of other putative m2G-generating enzymes in finished and unfinished genomic sequences. Sequence comparison and phylogenetic analysis of 21 close homologs of RsmC and YgjO revealed the presence of the third paralogous lineage in E. coli and other gamma-Proteobacteria, which might correspond to the subfamily of MTases specific for G1516 in 16S rRNA. In addition, the comparative sequence analysis supported by sequence/structure threading suggests that rRNA:m2G MTases are very closely related to RNA and DNA:m6A MTases and that these two enzyme families share common architecture of the active site and presumably a similar mechanism of methyl group transfer onto the exocyclic amino group of their target bases. PMID:11053259

  20. Comparative Transcriptomics Reveals Jasmonic Acid-Associated Metabolism Related to Cotton Fiber Initiation.

    PubMed

    Wang, Liman; Zhu, Youmin; Hu, Wenjing; Zhang, Xueying; Cai, Caiping; Guo, Wangzhen

    2015-01-01

    Analysis of mutants and gene expression patterns provides a powerful approach for investigating genes involved in key stages of plant fiber development. In this study, lintless-fuzzless XinWX and linted-fuzzless XinFLM with a single genetic locus difference for lint were used to identify differentially expressed genes. Scanning electron microscopy showed fiber initiation in XinFLM at 0 days post anthesis (DPA). Fiber transcriptional profiling of the lines at three initiation developmental stages (-1, 0, 1 DPA) was performed using an oligonucleotide microarray. Loop comparisons of the differentially expressed genes within and between the lines was carried out, and functional classification and enrichment analysis showed that gene expression patterns during fiber initiation were heavily associated with hormone metabolism, transcription factor regulation, lipid transport, and asparagine biosynthetic processes, as previously reported. Further, four members of the allene-oxide cyclase (AOC) family that function in jasmonate biosynthesis were parallel up-regulation in fiber initiation, especially at -1 DPA, compared to other tissues and organs in linted-fuzzed TM-1. Real time-quantitative PCR (RT-qPCR) analysis in different fiber mutant lines revealed that AOCs were up-regulated higher at -1 DPA in lintless-fuzzless than that in linted-fuzzless and linted-fuzzed materials, and transcription of the AOCs was increased under jasmonic acid (JA) treatment. Expression analysis of JA biosynthesis-associated genes between XinWX and XinFLM showed that they were up-regulated during fiber initiation in the fuzzless-lintless mutant. Taken together, jasmonic acid-associated metabolism was related to cotton fiber initiation. Parallel up-regulation of AOCs expression may be important for normal fiber initiation development, while overproduction of AOCs might disrupt normal fiber development. PMID:26079621

  1. Comparative Transcriptomics Reveals Jasmonic Acid-Associated Metabolism Related to Cotton Fiber Initiation

    PubMed Central

    Wang, Liman; Zhu, Youmin; Hu, Wenjing; Zhang, Xueying; Cai, Caiping; Guo, Wangzhen

    2015-01-01

    Analysis of mutants and gene expression patterns provides a powerful approach for investigating genes involved in key stages of plant fiber development. In this study, lintless-fuzzless XinWX and linted-fuzzless XinFLM with a single genetic locus difference for lint were used to identify differentially expressed genes. Scanning electron microscopy showed fiber initiation in XinFLM at 0 days post anthesis (DPA). Fiber transcriptional profiling of the lines at three initiation developmental stages (-1, 0, 1 DPA) was performed using an oligonucleotide microarray. Loop comparisons of the differentially expressed genes within and between the lines was carried out, and functional classification and enrichment analysis showed that gene expression patterns during fiber initiation were heavily associated with hormone metabolism, transcription factor regulation, lipid transport, and asparagine biosynthetic processes, as previously reported. Further, four members of the allene-oxide cyclase (AOC) family that function in jasmonate biosynthesis were parallel up-regulation in fiber initiation, especially at -1 DPA, compared to other tissues and organs in linted-fuzzed TM-1. Real time-quantitative PCR (RT-qPCR) analysis in different fiber mutant lines revealed that AOCs were up-regulated higher at -1 DPA in lintless-fuzzless than that in linted-fuzzless and linted-fuzzed materials, and transcription of the AOCs was increased under jasmonic acid (JA) treatment. Expression analysis of JA biosynthesis-associated genes between XinWX and XinFLM showed that they were up-regulated during fiber initiation in the fuzzless-lintless mutant. Taken together, jasmonic acid-associated metabolism was related to cotton fiber initiation. Parallel up-regulation of AOCs expression may be important for normal fiber initiation development, while overproduction of AOCs might disrupt normal fiber development. PMID:26079621

  2. The Fatty Acid Profile Analysis of Cyperus laxus Used for Phytoremediation of Soils from Aged Oil Spill-Impacted Sites Revealed That This Is a C18:3 Plant Species.

    PubMed

    Rivera Casado, Noemí Araceli; Montes Horcasitas, María del Carmen; Rodríguez Vázquez, Refugio; Esparza García, Fernando José; Pérez Vargas, Josefina; Ariza Castolo, Armando; Ferrera-Cerrato, Ronald; Gómez Guzmán, Octavio; Calva Calva, Graciano

    2015-01-01

    The effect of recalcitrant hydrocarbons on the fatty acid profile from leaf, basal corm, and roots of Cyperus laxus plants cultivated in greenhouse phytoremediation systems of soils from aged oil spill-impacted sites containing from 16 to 340 g/Kg total hydrocarbons (THC) was assessed to investigate if this is a C18:3 species and if the hydrocarbon removal during the phytoremediation process has a relationship with the fatty acid profile of this plant. The fatty acid profile was specific to each vegetative organ and was strongly affected by the hydrocarbons level in the impacted sites. Leaf extracts of plants from uncontaminated soil produced palmitic acid (C16), octadecanoic acid (C18:0), unsaturated oleic acids (C18:1-C18:3), and unsaturated eichosanoic (C20:2-C20:3) acids with a noticeable absence of the unsaturated hexadecatrienoic acid (C16:3); this finding demonstrates, for the first time, that C. laxus is a C18:3 plant. In plants from the phytoremediation systems, the total fatty acid contents in the leaf and the corm were negatively affected by the hydrocarbons presence; however, the effect was positive in root. Interestingly, under contaminated conditions, unusual fatty acids such as odd numbered carbons (C15, C17, C21, and C23) and uncommon unsaturated chains (C20:3n6 and C20:4) were produced together with a remarkable quantity of C22:2 and C24:0 chains in the corm and the leaf. These results demonstrate that weathered hydrocarbons may drastically affect the lipidic composition of C. laxus at the fatty acid level, suggesting that this species adjusts the cover lipid composition in its vegetative organs, mainly in roots, in response to the weathered hydrocarbon presence and uptake during the phytoremediation process. PMID:26473488

  3. The Fatty Acid Profile Analysis of Cyperus laxus Used for Phytoremediation of Soils from Aged Oil Spill-Impacted Sites Revealed That This Is a C18:3 Plant Species

    PubMed Central

    Montes Horcasitas, María del Carmen; Rodríguez Vázquez, Refugio; Esparza García, Fernando José; Pérez Vargas, Josefina; Ariza Castolo, Armando; Ferrera-Cerrato, Ronald; Gómez Guzmán, Octavio

    2015-01-01

    The effect of recalcitrant hydrocarbons on the fatty acid profile from leaf, basal corm, and roots of Cyperus laxus plants cultivated in greenhouse phytoremediation systems of soils from aged oil spill-impacted sites containing from 16 to 340 g/Kg total hydrocarbons (THC) was assessed to investigate if this is a C18:3 species and if the hydrocarbon removal during the phytoremediation process has a relationship with the fatty acid profile of this plant. The fatty acid profile was specific to each vegetative organ and was strongly affected by the hydrocarbons level in the impacted sites. Leaf extracts of plants from uncontaminated soil produced palmitic acid (C16), octadecanoic acid (C18:0), unsaturated oleic acids (C18:1-C18:3), and unsaturated eichosanoic (C20:2-C20:3) acids with a noticeable absence of the unsaturated hexadecatrienoic acid (C16:3); this finding demonstrates, for the first time, that C. laxus is a C18:3 plant. In plants from the phytoremediation systems, the total fatty acid contents in the leaf and the corm were negatively affected by the hydrocarbons presence; however, the effect was positive in root. Interestingly, under contaminated conditions, unusual fatty acids such as odd numbered carbons (C15, C17, C21, and C23) and uncommon unsaturated chains (C20:3n6 and C20:4) were produced together with a remarkable quantity of C22:2 and C24:0 chains in the corm and the leaf. These results demonstrate that weathered hydrocarbons may drastically affect the lipidic composition of C. laxus at the fatty acid level, suggesting that this species adjusts the cover lipid composition in its vegetative organs, mainly in roots, in response to the weathered hydrocarbon presence and uptake during the phytoremediation process. PMID:26473488

  4. Comparative genomics of lactic acid bacteria reveals a niche-specific gene set

    PubMed Central

    2009-01-01

    Background The recently sequenced genome of Lactobacillus helveticus DPC4571 [1] revealed a dairy organism with significant homology (75% of genes are homologous) to a probiotic bacteria Lb. acidophilus NCFM [2]. This led us to hypothesise that a group of genes could be determined which could define an organism's niche. Results Taking 11 fully sequenced lactic acid bacteria (LAB) as our target, (3 dairy LAB, 5 gut LAB and 3 multi-niche LAB), we demonstrated that the presence or absence of certain genes involved in sugar metabolism, the proteolytic system, and restriction modification enzymes were pivotal in suggesting the niche of a strain. We identified 9 niche specific genes, of which 6 are dairy specific and 3 are gut specific. The dairy specific genes identified in Lactobacillus helveticus DPC4571 were lhv_1161 and lhv_1171, encoding components of the proteolytic system, lhv_1031 lhv_1152, lhv_1978 and lhv_0028 encoding restriction endonuclease genes, while bile salt hydrolase genes lba_0892 and lba_1078, and the sugar metabolism gene lba_1689 from Lb. acidophilus NCFM were identified as gut specific genes. Conclusion Comparative analysis revealed that if an organism had homologs to the dairy specific geneset, it probably came from a dairy environment, whilst if it had homologs to gut specific genes, it was highly likely to be of intestinal origin. We propose that this "barcode" of 9 genes will be a useful initial guide to researchers in the LAB field to indicate an organism's ability to occupy a specific niche. PMID:19265535

  5. Polyploid genome of Camelina sativa revealed by isolation of fatty acid synthesis genes

    PubMed Central

    2010-01-01

    Background Camelina sativa, an oilseed crop in the Brassicaceae family, has inspired renewed interest due to its potential for biofuels applications. Little is understood of the nature of the C. sativa genome, however. A study was undertaken to characterize two genes in the fatty acid biosynthesis pathway, fatty acid desaturase (FAD) 2 and fatty acid elongase (FAE) 1, which revealed unexpected complexity in the C. sativa genome. Results In C. sativa, Southern analysis indicates the presence of three copies of both FAD2 and FAE1 as well as LFY, a known single copy gene in other species. All three copies of both CsFAD2 and CsFAE1 are expressed in developing seeds, and sequence alignments show that previously described conserved sites are present, suggesting that all three copies of both genes could be functional. The regions downstream of CsFAD2 and upstream of CsFAE1 demonstrate co-linearity with the Arabidopsis genome. In addition, three expressed haplotypes were observed for six predicted single-copy genes in 454 sequencing analysis and results from flow cytometry indicate that the DNA content of C. sativa is approximately three-fold that of diploid Camelina relatives. Phylogenetic analyses further support a history of duplication and indicate that C. sativa and C. microcarpa might share a parental genome. Conclusions There is compelling evidence for triplication of the C. sativa genome, including a larger chromosome number and three-fold larger measured genome size than other Camelina relatives, three isolated copies of FAD2, FAE1, and the KCS17-FAE1 intergenic region, and three expressed haplotypes observed for six predicted single-copy genes. Based on these results, we propose that C. sativa be considered an allohexaploid. The characterization of fatty acid synthesis pathway genes will allow for the future manipulation of oil composition of this emerging biofuel crop; however, targeted manipulations of oil composition and general development of C. sativa should

  6. Widespread occurrence of the tfd-II genes in soil bacteria revealed by nucleotide sequence analysis of 2,4-dichlorophenoxyacetic acid degradative plasmids pDB1 and p712.

    PubMed

    Kim, Dong-Uk; Kim, Min-Sun; Lim, Jong-Sung; Ka, Jong-Ok

    2013-05-01

    Variovorax sp. strain DB1 and Pseudomonas pickettii strain 712 are 2,4-dicholorophenoxy-acetic acid (2,4-D)-degrading bacteria, which were isolated from agricultural soils in Republic of Korea and USA, respectively. Each strain harbors a 2,4-D degradative plasmid and is able to utilize 2,4-D as the sole source of carbon for its growth. The 2,4-D degradative plasmid pDB1 of strain DB1 consisted of a 65,269-bp circular molecule with a G+C content of 66.23% and had 68 ORFs. The 2,4-D degradative plasmid p712 of strain 712 was composed of a 62,798-bp circular molecule with a 62.11% G+C content and had 62 ORFs. The plasmids pDB1 and p712 share significantly homologous 2,4-D degradative genes with high similarity to the tfdR, tfdB-II, tfdC-II, tfdD-II, tfdE-II, tfdF-II, tfdK and tfdA genes of plasmid pJP4 of Alcaligenes eutrophus isolated from Australia. In a phylogenetic analysis with trfA, traL, and trbA genes, pDB1 belonged to IncP-1β with pJP4, while p712 belonged to IncP-1ε with pKJK5 and pEMT3. The results indicated that, in spite of the differences in their backbone regions, the 2,4-D catabolic genes of the two plasmids were closely related and also related to the well-known 2,4-D degradative plasmid pJP4 even though all were isolated from different geographic regions. Other similarities in the genetic organization and the presence of IS1071 suggested that these catabolic genes may be on a transposable element, leading to widespread occurrence in soil bacteria. PMID:23376020

  7. Amino Acid Proximities in Two Sup35 Prion Strains Revealed by Chemical Cross-linking.

    PubMed

    Wong, Shenq-Huey; King, Chih-Yen

    2015-10-01

    Strains of the yeast prion [PSI] are different folding patterns of the same Sup35 protein, which stacks up periodically to form a prion fiber. Chemical cross-linking is employed here to probe different fiber structures assembled with a mutant Sup35 fragment. The photo-reactive cross-linker, p-benzoyl-l-phenylalanine (pBpa), was biosynthetically incorporated into bacterially prepared recombinant Sup(1-61)-GFP, containing the first 61 residues of Sup35, followed by the green fluorescent protein. Four methionine substitutions and two alanine substitutions were introduced at fixed positions in Sup(1-61) to allow cyanogen bromide cleavage to facilitate subsequent mass spectrometry analysis. Amyloid fibers of pBpa and Met/Ala-substituted Sup(1-61)-GFP were nucleated from purified yeast prion particles of two different strains, namely VK and VL, and shown to faithfully transmit specific strain characteristics to yeast expressing the wild type Sup35 protein. Intra- and intermolecular cross-linking were distinguished by tandem mass spectrometry analysis on fibers seeded from solutions containing equal amounts of (14)N- and (15)N-labeled protein. Fibers propagating the VL strain type exhibited intra- and intermolecular cross-linking between amino acid residues 3 and 28, as well as intra- and intermolecular linking between 32 and 55. Inter- and intramolecular cross-linking between residues 32 and 55 were detected in fibers propagating the VK strain type. Adjacencies of amino acid residues in space revealed by cross-linking were used to constrain possible chain folds of different [PSI] strains. PMID:26265470

  8. Phenolic Profiling of Caffeic Acid O-Methyltransferase-Deficient Poplar Reveals Novel Benzodioxane Oligolignols1

    PubMed Central

    Morreel, Kris; Ralph, John; Lu, Fachuang; Goeminne, Geert; Busson, Roger; Herdewijn, Piet; Goeman, Jan L.; Van der Eycken, Johan; Boerjan, Wout; Messens, Eric

    2004-01-01

    Caffeic acid O-methyltransferase (COMT) catalyzes preferentially the methylation of 5-hydroxyconiferaldehyde to sinapaldehyde in monolignol biosynthesis. Here, we have compared HPLC profiles of the methanol-soluble phenolics fraction of xylem tissue from COMT-deficient and control poplars (Populus spp.), using statistical analysis of the peak heights. COMT down-regulation results in significant concentration differences for 25 of the 91 analyzed peaks. Eight peaks were exclusively detected in COMT-deficient poplar, of which four could be purified for further identification using mass spectrometry/mass spectrometry, nuclear magnetic resonance, and spiking of synthesized reference compounds. These new compounds were derived from 5-hydroxyconiferyl alcohol or 5-hydroxyconiferaldehyde and were characterized by benzodioxane moieties, a structural type that is also increased in the lignins of COMT-deficient plants. One of these four benzodioxanes amounted to the most abundant oligolignol in the HPLC profile. Furthermore, all of the differentially accumulating oligolignols involving sinapyl units were either reduced in abundance or undetectable. The concentration levels of all identified oligolignols were in agreement with the relative supply of monolignols and with their chemical coupling propensities, which supports the random coupling hypothesis. Chiral HPLC analysis of the most abundant benzodioxane dimer revealed the presence of both enantiomers in equal amounts, indicating that they were formed by radical coupling reactions under simple chemical control rather than guided by dirigent proteins. PMID:15563622

  9. Simultaneous analysis of biologically active aminoalkanephosphonic acids.

    PubMed

    Kudzin, Zbigniew H; Gralak, Dorota K; Andrijewski, Grzegorz; Drabowicz, Józef; Luczak, Jerzy

    2003-05-23

    A new approach for simultaneous analysis of biologically active aminoalkanephosphonic acids, namely glyphosate, phosphonoglycine, phosphonosarcosine, phosphonoalanine, phosphono-beta-alanine, phosphonohomoalanine, phosphono-gamma-homoalanine and glufosinate, is presented. This includes a preliminary 31p NMR analysis of these amino acids, their further derivatization to volatile phosphonates (phosphinates) by means of trifluoroacetic acid-trifluoroacetic anhydride-trimethyl orthoacetate reagent and subsequent analysis of derivatization products using MS and/or GC-MS (chemical ionization and/or electron impact ionization). PMID:12862383

  10. Quantitative flux analysis reveals folate-dependent NADPH production

    NASA Astrophysics Data System (ADS)

    Fan, Jing; Ye, Jiangbin; Kamphorst, Jurre J.; Shlomi, Tomer; Thompson, Craig B.; Rabinowitz, Joshua D.

    2014-06-01

    ATP is the dominant energy source in animals for mechanical and electrical work (for example, muscle contraction or neuronal firing). For chemical work, there is an equally important role for NADPH, which powers redox defence and reductive biosynthesis. The most direct route to produce NADPH from glucose is the oxidative pentose phosphate pathway, with malic enzyme sometimes also important. Although the relative contribution of glycolysis and oxidative phosphorylation to ATP production has been extensively analysed, similar analysis of NADPH metabolism has been lacking. Here we demonstrate the ability to directly track, by liquid chromatography-mass spectrometry, the passage of deuterium from labelled substrates into NADPH, and combine this approach with carbon labelling and mathematical modelling to measure NADPH fluxes. In proliferating cells, the largest contributor to cytosolic NADPH is the oxidative pentose phosphate pathway. Surprisingly, a nearly comparable contribution comes from serine-driven one-carbon metabolism, in which oxidation of methylene tetrahydrofolate to 10-formyl-tetrahydrofolate is coupled to reduction of NADP+ to NADPH. Moreover, tracing of mitochondrial one-carbon metabolism revealed complete oxidation of 10-formyl-tetrahydrofolate to make NADPH. As folate metabolism has not previously been considered an NADPH producer, confirmation of its functional significance was undertaken through knockdown of methylenetetrahydrofolate dehydrogenase (MTHFD) genes. Depletion of either the cytosolic or mitochondrial MTHFD isozyme resulted in decreased cellular NADPH/NADP+ and reduced/oxidized glutathione ratios (GSH/GSSG) and increased cell sensitivity to oxidative stress. Thus, although the importance of folate metabolism for proliferating cells has been long recognized and attributed to its function of producing one-carbon units for nucleic acid synthesis, another crucial function of this pathway is generating reducing power.

  11. Principal component analysis of phenolic acid spectra

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phenolic acids are common plant metabolites that exhibit bioactive properties and have applications in functional food and animal feed formulations. The ultraviolet (UV) and infrared (IR) spectra of four closely related phenolic acid structures were evaluated by principal component analysis (PCA) to...

  12. Amino acid isotopic analysis in agricultural systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A relatively new approach to stable isotopic analysis—referred to as compound-specific isotopic analysis (CSIA)—has emerged, centering on the measurement of 15N:14N ratios in amino acids (glutamic acid and phenylalanine). CSIA has recently been used to generate trophic position estimates among anima...

  13. Boric Acid in Kjeldahl Analysis

    ERIC Educational Resources Information Center

    Cruz, Gregorio

    2013-01-01

    The use of boric acid in the Kjeldahl determination of nitrogen is a variant of the original method widely applied in many laboratories all over the world. Its use is recommended by control organizations such as ISO, IDF, and EPA because it yields reliable and accurate results. However, the chemical principles the method is based on are not…

  14. The Crystal Structure of the Adenylation Enzyme VinN Reveals a Unique β-Amino Acid Recognition Mechanism*

    PubMed Central

    Miyanaga, Akimasa; Cieślak, Jolanta; Shinohara, Yuji; Kudo, Fumitaka; Eguchi, Tadashi

    2014-01-01

    Adenylation enzymes play important roles in the biosynthesis and degradation of primary and secondary metabolites. Mechanistic insights into the recognition of α-amino acid substrates have been obtained for α-amino acid adenylation enzymes. The Asp residue is invariant and is essential for the stabilization of the α-amino group of the substrate. In contrast, the β-amino acid recognition mechanism of adenylation enzymes is still unclear despite the importance of β-amino acid activation for the biosynthesis of various natural products. Herein, we report the crystal structure of the stand-alone adenylation enzyme VinN, which specifically activates (2S,3S)-3-methylaspartate (3-MeAsp) in vicenistatin biosynthesis. VinN has an overall structure similar to that of other adenylation enzymes. The structure of the complex with 3-MeAsp revealed that a conserved Asp230 residue is used in the recognition of the β-amino group of 3-MeAsp similar to α-amino acid adenylation enzymes. A mutational analysis and structural comparison with α-amino acid adenylation enzymes showed that the substrate-binding pocket of VinN has a unique architecture to accommodate 3-MeAsp as a β-amino acid substrate. Thus, the VinN structure allows the first visualization of the interaction of an adenylation enzyme with a β-amino acid and provides new mechanistic insights into the selective recognition of β-amino acids in this family of enzymes. PMID:25246523

  15. The crystal structure of the adenylation enzyme VinN reveals a unique β-amino acid recognition mechanism.

    PubMed

    Miyanaga, Akimasa; Cieślak, Jolanta; Shinohara, Yuji; Kudo, Fumitaka; Eguchi, Tadashi

    2014-11-01

    Adenylation enzymes play important roles in the biosynthesis and degradation of primary and secondary metabolites. Mechanistic insights into the recognition of α-amino acid substrates have been obtained for α-amino acid adenylation enzymes. The Asp residue is invariant and is essential for the stabilization of the α-amino group of the substrate. In contrast, the β-amino acid recognition mechanism of adenylation enzymes is still unclear despite the importance of β-amino acid activation for the biosynthesis of various natural products. Herein, we report the crystal structure of the stand-alone adenylation enzyme VinN, which specifically activates (2S,3S)-3-methylaspartate (3-MeAsp) in vicenistatin biosynthesis. VinN has an overall structure similar to that of other adenylation enzymes. The structure of the complex with 3-MeAsp revealed that a conserved Asp(230) residue is used in the recognition of the β-amino group of 3-MeAsp similar to α-amino acid adenylation enzymes. A mutational analysis and structural comparison with α-amino acid adenylation enzymes showed that the substrate-binding pocket of VinN has a unique architecture to accommodate 3-MeAsp as a β-amino acid substrate. Thus, the VinN structure allows the first visualization of the interaction of an adenylation enzyme with a β-amino acid and provides new mechanistic insights into the selective recognition of β-amino acids in this family of enzymes. PMID:25246523

  16. Phylogenetic analysis based on the PKS gene involved in fusaric acid biosynthesis production reveals close relationship between US race 1 lineage isolates & Australian biotype isolates of Fusarium Oxysporum f. sp. Vasinfectum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Isolates of Fusarium oxysporum f. sp. vasinfectum, the causal agent of fusarium wilt of cotton, vary significantly in their virulence. Isolates have been further subcategorized into pathogenic races based on their differential interaction with host genotypes. Phylogenetic analysis based on three n...

  17. Ecological roles of dominant and rare prokaryotes in acid mine drainage revealed by metagenomics and metatranscriptomics.

    PubMed

    Hua, Zheng-Shuang; Han, Yu-Jiao; Chen, Lin-Xing; Liu, Jun; Hu, Min; Li, Sheng-Jin; Kuang, Jia-Liang; Chain, Patrick S G; Huang, Li-Nan; Shu, Wen-Sheng

    2015-06-01

    High-throughput sequencing is expanding our knowledge of microbial diversity in the environment. Still, understanding the metabolic potentials and ecological roles of rare and uncultured microbes in natural communities remains a major challenge. To this end, we applied a 'divide and conquer' strategy that partitioned a massive metagenomic data set (>100 Gbp) into subsets based on K-mer frequency in sequence assembly to a low-diversity acid mine drainage (AMD) microbial community and, by integrating with an additional metatranscriptomic assembly, successfully obtained 11 draft genomes most of which represent yet uncultured and/or rare taxa (relative abundance <1%). We report the first genome of a naturally occurring Ferrovum population (relative abundance >90%) and its metabolic potentials and gene expression profile, providing initial molecular insights into the ecological role of these lesser known, but potentially important, microorganisms in the AMD environment. Gene transcriptional analysis of the active taxa revealed major metabolic capabilities executed in situ, including carbon- and nitrogen-related metabolisms associated with syntrophic interactions, iron and sulfur oxidation, which are key in energy conservation and AMD generation, and the mechanisms of adaptation and response to the environmental stresses (heavy metals, low pH and oxidative stress). Remarkably, nitrogen fixation and sulfur oxidation were performed by the rare taxa, indicating their critical roles in the overall functioning and assembly of the AMD community. Our study demonstrates the potential of the 'divide and conquer' strategy in high-throughput sequencing data assembly for genome reconstruction and functional partitioning analysis of both dominant and rare species in natural microbial assemblages. PMID:25361395

  18. Ecological roles of dominant and rare prokaryotes in acid mine drainage revealed by metagenomics and metatranscriptomics

    DOE PAGESBeta

    Hua, Zheng-Shuang; Han, Yu-Jiao; Chen, Lin-Xing; Liu, Jun; Hu, Min; Li, Sheng-Jin; Kuang, Jia-Liang; Chain, Patrick SG; Huang, Li-Nan; Shu, Wen-Sheng

    2014-11-07

    Here we report that high-throughput sequencing is expanding our knowledge of microbial diversity in the environment. Still, understanding the metabolic potentials and ecological roles of rare and uncultured microbes in natural communities remains a major challenge. To this end, we applied a ‘divide and conquer’ strategy that partitioned a massive metagenomic data set (>100 Gbp) into subsets based on K-mer frequency in sequence assembly to a low-diversity acid mine drainage (AMD) microbial community and, by integrating with an additional metatranscriptomic assembly, successfully obtained 11 draft genomes most of which represent yet uncultured and/or rare taxa (relative abundance <1%). We reportmore » the first genome of a naturally occurring Ferrovum population (relative abundance >90%) and its metabolic potentials and gene expression profile, providing initial molecular insights into the ecological role of these lesser known, but potentially important, microorganisms in the AMD environment. Gene transcriptional analysis of the active taxa revealed major metabolic capabilities executed in situ, including carbon- and nitrogen-related metabolisms associated with syntrophic interactions, iron and sulfur oxidation, which are key in energy conservation and AMD generation, and the mechanisms of adaptation and response to the environmental stresses (heavy metals, low pH and oxidative stress). Remarkably, nitrogen fixation and sulfur oxidation were performed by the rare taxa, indicating their critical roles in the overall functioning and assembly of the AMD community. Finally, our study demonstrates the potential of the ‘divide and conquer’ strategy in high-throughput sequencing data assembly for genome reconstruction and functional partitioning analysis of both dominant and rare species in natural microbial assemblages.« less

  19. Ecological roles of dominant and rare prokaryotes in acid mine drainage revealed by metagenomics and metatranscriptomics

    PubMed Central

    Hua, Zheng-Shuang; Han, Yu-Jiao; Chen, Lin-Xing; Liu, Jun; Hu, Min; Li, Sheng-Jin; Kuang, Jia-Liang; Chain, Patrick SG; Huang, Li-Nan; Shu, Wen-Sheng

    2015-01-01

    High-throughput sequencing is expanding our knowledge of microbial diversity in the environment. Still, understanding the metabolic potentials and ecological roles of rare and uncultured microbes in natural communities remains a major challenge. To this end, we applied a ‘divide and conquer' strategy that partitioned a massive metagenomic data set (>100 Gbp) into subsets based on K-mer frequency in sequence assembly to a low-diversity acid mine drainage (AMD) microbial community and, by integrating with an additional metatranscriptomic assembly, successfully obtained 11 draft genomes most of which represent yet uncultured and/or rare taxa (relative abundance <1%). We report the first genome of a naturally occurring Ferrovum population (relative abundance >90%) and its metabolic potentials and gene expression profile, providing initial molecular insights into the ecological role of these lesser known, but potentially important, microorganisms in the AMD environment. Gene transcriptional analysis of the active taxa revealed major metabolic capabilities executed in situ, including carbon- and nitrogen-related metabolisms associated with syntrophic interactions, iron and sulfur oxidation, which are key in energy conservation and AMD generation, and the mechanisms of adaptation and response to the environmental stresses (heavy metals, low pH and oxidative stress). Remarkably, nitrogen fixation and sulfur oxidation were performed by the rare taxa, indicating their critical roles in the overall functioning and assembly of the AMD community. Our study demonstrates the potential of the ‘divide and conquer' strategy in high-throughput sequencing data assembly for genome reconstruction and functional partitioning analysis of both dominant and rare species in natural microbial assemblages. PMID:25361395

  20. Ecological roles of dominant and rare prokaryotes in acid mine drainage revealed by metagenomics and metatranscriptomics

    SciTech Connect

    Hua, Zheng-Shuang; Han, Yu-Jiao; Chen, Lin-Xing; Liu, Jun; Hu, Min; Li, Sheng-Jin; Kuang, Jia-Liang; Chain, Patrick SG; Huang, Li-Nan; Shu, Wen-Sheng

    2014-11-07

    Here we report that high-throughput sequencing is expanding our knowledge of microbial diversity in the environment. Still, understanding the metabolic potentials and ecological roles of rare and uncultured microbes in natural communities remains a major challenge. To this end, we applied a ‘divide and conquer’ strategy that partitioned a massive metagenomic data set (>100 Gbp) into subsets based on K-mer frequency in sequence assembly to a low-diversity acid mine drainage (AMD) microbial community and, by integrating with an additional metatranscriptomic assembly, successfully obtained 11 draft genomes most of which represent yet uncultured and/or rare taxa (relative abundance <1%). We report the first genome of a naturally occurring Ferrovum population (relative abundance >90%) and its metabolic potentials and gene expression profile, providing initial molecular insights into the ecological role of these lesser known, but potentially important, microorganisms in the AMD environment. Gene transcriptional analysis of the active taxa revealed major metabolic capabilities executed in situ, including carbon- and nitrogen-related metabolisms associated with syntrophic interactions, iron and sulfur oxidation, which are key in energy conservation and AMD generation, and the mechanisms of adaptation and response to the environmental stresses (heavy metals, low pH and oxidative stress). Remarkably, nitrogen fixation and sulfur oxidation were performed by the rare taxa, indicating their critical roles in the overall functioning and assembly of the AMD community. Finally, our study demonstrates the potential of the ‘divide and conquer’ strategy in high-throughput sequencing data assembly for genome reconstruction and functional partitioning analysis of both dominant and rare species in natural microbial assemblages.

  1. Analysis of Chiral Carboxylic Acids in Meteorites

    NASA Technical Reports Server (NTRS)

    Burton, A. S.; Elsila, J. E.; Hein, J. E.; Aponte, J. C.; Parker, E. T.; Glavin, D. P.; Dworkin, J. P.

    2015-01-01

    our efforts to develop highly sensitive LC-MS methods for the analysis of chiral carboxylic acids including hydroxy acids.

  2. Amino acid analysis for pharmacopoeial purposes.

    PubMed

    Wahl, Oliver; Holzgrabe, Ulrike

    2016-07-01

    The impurity profile of amino acids depends strongly on the production process. Since there are many different production methods (e.g. fermentation, protein hydrolysis or chemical synthesis) universal, state of the art methods are required to determine the impurity profile of amino acids produced by all relevant competitors. At the moment TLC tests provided by the Ph. Eur. are being replaced by a very specific amino acid analysis procedure possibly missing out on currently unknown process related impurities. Production methods and possible impurities as well as separation and detection methods suitable for said impurities are subject to this review. PMID:27154660

  3. Re-examination of Dietary Amino Acid Sensing Reveals a GCN2-Independent Mechanism.

    PubMed

    Leib, David E; Knight, Zachary A

    2015-11-10

    Animals cannot synthesize nine essential amino acids (EAAs) and must therefore obtain them from food. Mice reportedly reject food lacking a single EAA within the first hour of feeding. This remarkable phenomenon is proposed to involve post-ingestive sensing of amino acid imbalance by the protein kinase GCN2 in the brain. Here, we systematically re-examine dietary amino acid sensing in mice. In contrast to previous results, we find that mice cannot rapidly identify threonine- or leucine-deficient food in common feeding paradigms. However, mice attain the ability to identify EAA-deficient food following 2 days of EAA deprivation, suggesting a requirement for physiologic need. In addition, we report that mice can rapidly identify lysine-deficient food without prior EAA deficit, revealing a distinct sensing mechanism for this amino acid. These behaviors are independent of the proposed amino acid sensor GCN2, pointing to the existence of an undescribed mechanism for rapid sensing of dietary EAAs. PMID:26526991

  4. Re-examination of Dietary Amino Acid Sensing Reveals a GCN2-Independent Mechanism

    PubMed Central

    Leib, David E.; Knight, Zachary A.

    2016-01-01

    SUMMARY Animals cannot synthesize nine essential amino acids (EAAs) and must therefore obtain them from food. Mice reportedly reject food lacking a single EAA within the first hour of feeding. This remarkable phenomenon is proposed to involve post-ingestive sensing of amino acid imbalance by the protein kinase GCN2 in the brain. Here, we systematically re-examine dietary amino acid sensing in mice. In contrast to previous results, we find that mice cannot rapidly identify threonine- or leucine-deficient food in common feeding paradigms. However, mice attain the ability to identify EAA-deficient food following 2 days of EAA deprivation, suggesting a requirement for physiologic need. In addition, we report that mice can rapidly identify lysine-deficient food without prior EAA deficit, revealing a distinct sensing mechanism for this amino acid. These behaviors are independent of the proposed amino acid sensor GCN2, pointing to the existence of an undescribed mechanism for rapid sensing of dietary EAAs. PMID:26526991

  5. CPAG: software for leveraging pleiotropy in GWAS to reveal similarity between human traits links plasma fatty acids and intestinal inflammation.

    PubMed

    Wang, Liuyang; Oehlers, Stefan H; Espenschied, Scott T; Rawls, John F; Tobin, David M; Ko, Dennis C

    2015-01-01

    Meta-analyses of genome-wide association studies (GWAS) have demonstrated that the same genetic variants can be associated with multiple diseases and other complex traits. We present software called CPAG (Cross-Phenotype Analysis of GWAS) to look for similarities between 700 traits, build trees with informative clusters, and highlight underlying pathways. Clusters are consistent with pre-defined groups and literature-based validation but also reveal novel connections. We report similarity between plasma palmitoleic acid and Crohn's disease and find that specific fatty acids exacerbate enterocolitis in zebrafish. CPAG will become increasingly powerful as more genetic variants are uncovered, leading to a deeper understanding of complex traits. CPAG is freely available at www.sourceforge.net/projects/CPAG/. PMID:26374098

  6. Untangling the Effect of Fatty Acid Addition at Species Level Revealed Different Transcriptional Responses of the Biogas Microbial Community Members.

    PubMed

    Treu, Laura; Campanaro, Stefano; Kougias, Panagiotis G; Zhu, Xinyu; Angelidaki, Irini

    2016-06-01

    In the present study, RNA-sequencing was used to elucidate the change of anaerobic digestion metatranscriptome after long chain fatty acids (oleate) exposure. To explore the general transcriptional behavior of the microbiome, the analysis was first performed on shotgun reads without considering a reference metagenome. As a second step, RNA reads were aligned on the genes encoded by the microbial community, revealing the expression of more than 51 000 different transcripts. The present study is the first research which was able to dissect the transcriptional behavior at a single species level by considering the 106 microbial genomes previously identified. The exploration of the metabolic pathways confirmed the importance of Syntrophomonas species in fatty acids degradation, and also highlighted the presence of protective mechanisms toward the long chain fatty acid effects in bacteria belonging to Clostridiales, Rykenellaceae, and in species of the genera Halothermothrix and Anaerobaculum. Additionally, an interesting transcriptional activation of the chemotaxis genes was evidenced in seven species belonging to Clostridia, Halothermothrix, and Tepidanaerobacter. Surprisingly, methanogens revealed a very versatile behavior different from each other, even among similar species of the Methanoculleus genus, while a strong increase of the expression level in Methanosarcina sp. was evidenced after oleate addition. PMID:27154312

  7. Vibrational analysis of α-cyanohydroxycinnamic acid

    NASA Astrophysics Data System (ADS)

    Mojica, Elmer-Rico E.; Vedad, Jayson; Desamero, Ruel Z. B.

    2015-08-01

    In the present study, a comparative Raman vibrational analysis of alpha-cyano-4-hydroxycinnamic acid (4CHCA) and its derivative, alpha-cyano-3-hydroxycinnamic acid (3CHCA), was performed. The Raman spectra of the 4CHCA and 3CHCA in solid form were obtained and analyzed to determine differences between the two structurally similar derivatives. For comparison, the CHCA derivatives cyanocinnamic acid (CCA) and coumaric acid (CA) were also studied. The plausible vibrational assignments were made and matched with those obtained theoretically using density functional theory (DFT) based method employing a 6-31 g basis set. The computational wavenumbers obtained were in good agreement with the observed experimental results. This was the first reported Raman study of CCA, 3CHCA and 4CHCA.

  8. Water Stress Responses of Tomato Mutants Impaired in Hormone Biosynthesis Reveal Abscisic Acid, Jasmonic Acid and Salicylic Acid Interactions.

    PubMed

    Muñoz-Espinoza, Valeria A; López-Climent, María F; Casaretto, José A; Gómez-Cadenas, Aurelio

    2015-01-01

    To investigate the putative crosstalk between JA and ABA in Solanum lycopersicum plants in response to drought, suppressor of prosystemin-mediated responses2 (spr2, JA-deficient) and flacca (flc, ABA-deficient) mutants together with the naphthalene/salicylate hydroxylase (NahG) transgenic (SA-deficient) line were used. Hormone profiling and gene expression of key enzymes in ABA, JA and SA biosynthesis were analyzed during early stages of drought. ABA accumulation was comparable in spr2 and wild type (WT) plants whereas expression of 9-cis-epoxycarotenoid dioxygenase 1 (NCED1) and NCED2 was different, implying a compensation mechanism between NCED genes and an organ-specific regulation of NCED1 expression. JA levels and 12-oxo-phytodienoic acid reductase 3 (OPR3) expression in flc plants suggest that ABA regulates the induction of the OPR3 gene in roots. By contrast, ABA treatment to flc plants leads to a reduction of JA and SA contents. Furthermore, different pattern of SA accumulation (and expression of isochorismate synthase and phenylalanine ammonia lyase 1) was observed between WT seedlings and mutants, suggesting that SA plays an important role on the early response of tomato plants to drought and also that JA and ABA modulate its biosynthesis. Finally, hormone profiling in spr2 and NahG plants indicate a crosstalk between JA and SA that could enhance tolerance of tomato to water stress. PMID:26635826

  9. Water Stress Responses of Tomato Mutants Impaired in Hormone Biosynthesis Reveal Abscisic Acid, Jasmonic Acid and Salicylic Acid Interactions

    PubMed Central

    Muñoz-Espinoza, Valeria A.; López-Climent, María F.; Casaretto, José A.; Gómez-Cadenas, Aurelio

    2015-01-01

    To investigate the putative crosstalk between JA and ABA in Solanum lycopersicum plants in response to drought, suppressor of prosystemin-mediated responses2 (spr2, JA-deficient) and flacca (flc, ABA-deficient) mutants together with the naphthalene/salicylate hydroxylase (NahG) transgenic (SA-deficient) line were used. Hormone profiling and gene expression of key enzymes in ABA, JA and SA biosynthesis were analyzed during early stages of drought. ABA accumulation was comparable in spr2 and wild type (WT) plants whereas expression of 9-cis-epoxycarotenoid dioxygenase 1 (NCED1) and NCED2 was different, implying a compensation mechanism between NCED genes and an organ-specific regulation of NCED1 expression. JA levels and 12-oxo-phytodienoic acid reductase 3 (OPR3) expression in flc plants suggest that ABA regulates the induction of the OPR3 gene in roots. By contrast, ABA treatment to flc plants leads to a reduction of JA and SA contents. Furthermore, different pattern of SA accumulation (and expression of isochorismate synthase and phenylalanine ammonia lyase 1) was observed between WT seedlings and mutants, suggesting that SA plays an important role on the early response of tomato plants to drought and also that JA and ABA modulate its biosynthesis. Finally, hormone profiling in spr2 and NahG plants indicate a crosstalk between JA and SA that could enhance tolerance of tomato to water stress. PMID:26635826

  10. Lipidomic profiling reveals protective function of fatty acid oxidation in cocaine-induced hepatotoxicity[S

    PubMed Central

    Shi, Xiaolei; Yao, Dan; Gosnell, Blake A.; Chen, Chi

    2012-01-01

    During cocaine-induced hepatotoxicity, lipid accumulation occurs prior to necrotic cell death in the liver. However, the exact influences of cocaine on the homeostasis of lipid metabolism remain largely unknown. In this study, the progression of subacute hepatotoxicity, including centrilobular necrosis in the liver and elevation of transaminase activity in serum, was observed in a three-day cocaine treatment, accompanying the disruption of triacylglycerol (TAG) turnover. Serum TAG level increased on day 1 of cocaine treatment but remained unchanged afterwards. In contrast, hepatic TAG level was elevated continuously during three days of cocaine treatment and was better correlated with the development of hepatotoxicity. Lipidomic analyses of serum and liver samples revealed time-dependent separation of the control and cocaine-treated mice in multivariate models, which was due to the accumulation of long-chain acylcarnitines together with the disturbances of many bioactive phospholipid species in the cocaine-treated mice. An in vitro function assay confirmed the progressive inhibition of mitochondrial fatty acid oxidation after the cocaine treatment. Cotreatment of fenofibrate significantly increased the expression of peroxisome proliferator-activated receptor α (PPARα)-targeted genes and the mitochondrial fatty acid oxidation activity in the cocaine-treated mice, resulting in the inhibition of cocaine-induced acylcarnitine accumulation and other hepatotoxic effects. Overall, the results from this lipidomics-guided study revealed that the inhibition of fatty acid oxidation plays an important role in cocaine-induced liver injury. PMID:22904346

  11. A transcriptomic study reveals differentially expressed genes and pathways respond to simulated acid rain in Arabidopsis thaliana.

    PubMed

    Liu, Ting-Wu; Niu, Li; Fu, Bin; Chen, Juan; Wu, Fei-Hua; Chen, Juan; Wang, Wen-Hua; Hu, Wen-Jun; He, Jun-Xian; Zheng, Hai-Lei

    2013-01-01

    Acid rain, as a worldwide environmental issue, can cause serious damage to plants. In this study, we provided the first case study on the systematic responses of arabidopsis (Arabidopsis thaliana (L.) Heynh.) to simulated acid rain (SiAR) by transcriptome approach. Transcriptomic analysis revealed that the expression of a set of genes related to primary metabolisms, including nitrogen, sulfur, amino acid, photosynthesis, and reactive oxygen species metabolism, were altered under SiAR. In addition, transport and signal transduction related pathways, especially calcium-related signaling pathways, were found to play important roles in the response of arabidopsis to SiAR stress. Further, we compared our data set with previously published data sets on arabidopsis transcriptome subjected to various stresses, including wound, salt, light, heavy metal, karrikin, temperature, osmosis, etc. The results showed that many genes were overlapped in several stresses, suggesting that plant response to SiAR is a complex process, which may require the participation of multiple defense-signaling pathways. The results of this study will help us gain further insights into the response mechanisms of plants to acid rain stress. PMID:23379338

  12. Decoding genome-wide GadEWX-transcriptional regulatory networks reveals multifaceted cellular responses to acid stress in Escherichia coli.

    PubMed

    Seo, Sang Woo; Kim, Donghyuk; O'Brien, Edward J; Szubin, Richard; Palsson, Bernhard O

    2015-01-01

    The regulators GadE, GadW and GadX (which we refer to as GadEWX) play a critical role in the transcriptional regulation of the glutamate-dependent acid resistance (GDAR) system in Escherichia coli K-12 MG1655. However, the genome-wide regulatory role of GadEWX is still unknown. Here we comprehensively reconstruct the genome-wide GadEWX transcriptional regulatory network and RpoS involvement in E. coli K-12 MG1655 under acidic stress. Integrative data analysis reveals that GadEWX regulons consist of 45 genes in 31 transcription units and 28 of these genes were associated with RpoS-binding sites. We demonstrate that GadEWX directly and coherently regulate several proton-generating/consuming enzymes with pairs of negative-feedback loops for pH homeostasis. In addition, GadEWX regulate genes with assorted functions, including molecular chaperones, acid resistance, stress response and other regulatory activities. These results show how GadEWX simultaneously coordinate many cellular processes to produce the overall response of E. coli to acid stress. PMID:26258987

  13. Decoding genome-wide GadEWX-transcriptional regulatory networks reveals multifaceted cellular responses to acid stress in Escherichia coli

    PubMed Central

    Seo, Sang Woo; Kim, Donghyuk; O'Brien, Edward J.; Szubin, Richard; Palsson, Bernhard O.

    2015-01-01

    The regulators GadE, GadW and GadX (which we refer to as GadEWX) play a critical role in the transcriptional regulation of the glutamate-dependent acid resistance (GDAR) system in Escherichia coli K-12 MG1655. However, the genome-wide regulatory role of GadEWX is still unknown. Here we comprehensively reconstruct the genome-wide GadEWX transcriptional regulatory network and RpoS involvement in E. coli K-12 MG1655 under acidic stress. Integrative data analysis reveals that GadEWX regulons consist of 45 genes in 31 transcription units and 28 of these genes were associated with RpoS-binding sites. We demonstrate that GadEWX directly and coherently regulate several proton-generating/consuming enzymes with pairs of negative-feedback loops for pH homeostasis. In addition, GadEWX regulate genes with assorted functions, including molecular chaperones, acid resistance, stress response and other regulatory activities. These results show how GadEWX simultaneously coordinate many cellular processes to produce the overall response of E. coli to acid stress. PMID:26258987

  14. Acid retardation method in analysis of strongly acidic solutions by inductively coupled plasma mass-spectrometry.

    PubMed

    Seregina, I F; Perevoznik, O A; Bolshov, M A

    2016-10-01

    Acid retardation on the sorbents as a technique for reduction of the acidity of the solutions prior to their analysis by inductively coupled plasma mass spectrometry was proposed and investigated. The proposed scheme provides substantial separation of the analytes and nitric acid, which allows direct introduction of the eluates in plasma without dilution. Two sorbents were examined - AV-17 anion-exchange resin and the Stirosorb 584 sorbent. Sorption and desorption of 38 elements on these sorbents were investigated. The efficiencies of the REEs' sorption on the anion-exchange and neutral sorbents were compared. The higher efficiency of the REEs and HNO3 separation was revealed for the neutral Stirosorb 584 sorbent. It was also found that most elements come out quantitatively of the column filled with the AV-17 resin after pumping 2-4mL of the solution. Wherein, the concentration of nitric acid decreased by 20 times. The anomalous behaviour of Ag, Pb, Th and U on the AV-17 resin was found. These analytes were eluted only after pumping 4 column volumes of deionized water. Na, K, Fe, Al and Li in concentrations within (50-1000mgL(-1)) range did not affect the recovery of REEs. The potential of ARM technique was demonstrate by the analysis of puriss. HNO3 and silverware. ARM enables to avoid dilution of highly acidic solutions prior to their introduction in ICP-MS. PMID:27474322

  15. Mechanistic Modeling Reveals the Critical Knowledge Gaps in Bile Acid-Mediated DILI.

    PubMed

    Woodhead, J L; Yang, K; Brouwer, K L R; Siler, S Q; Stahl, S H; Ambroso, J L; Baker, D; Watkins, P B; Howell, B A

    2014-01-01

    Bile salt export pump (BSEP) inhibition has been proposed to be an important mechanism for drug-induced liver injury (DILI). Modeling can prioritize knowledge gaps concerning bile acid (BA) homeostasis and thus help guide experimentation. A submodel of BA homeostasis in rats and humans was constructed within DILIsym, a mechanistic model of DILI. In vivo experiments in rats with glibenclamide were conducted, and data from these experiments were used to validate the model. The behavior of DILIsym was analyzed in the presence of a simulated theoretical BSEP inhibitor. BSEP inhibition in humans is predicted to increase liver concentrations of conjugated chenodeoxycholic acid (CDCA) and sulfate-conjugated lithocholic acid (LCA) while the concentration of other liver BAs remains constant or decreases. On the basis of a sensitivity analysis, the most important unknowns are the level of BSEP expression, the amount of intestinal synthesis of LCA, and the magnitude of farnesoid-X nuclear receptor (FXR)-mediated regulation. PMID:25006780

  16. Transcriptome Profiling of Tomato Fruit Development Reveals Transcription Factors Associated with Ascorbic Acid, Carotenoid and Flavonoid Biosynthesis

    PubMed Central

    Ye, Jie; Hu, Tixu; Yang, Congmei; Li, Hanxia; Yang, Mingze; Ijaz, Raina; Ye, Zhibiao; Zhang, Yuyang

    2015-01-01

    Tomato (Solanum lycopersicum) serves as a research model for fruit development; however, while it is an important dietary source of antioxidant nutrients, the transcriptional regulation of genes that determine nutrient levels remains poorly understood. Here, the transcriptomes of fruit at seven developmental stages (7, 14, 21, 28, 35, 42 and 49 days after flowering) from two tomato cultivars (Ailsa Craig and HG6-61) were evaluated using the Illumina sequencing platform. A total of 26,397 genes, which were expressed in at least one developmental stage, were detected in the two cultivars, and the expression patterns of those genes could be divided into 20 groups using a K-mean cluster analysis. Gene Ontology term enrichment analysis indicated that genes involved in RNA regulation, secondary metabolism, hormone metabolism and cell wall metabolism were the most highly differentially expressed genes during fruit development and ripening. A co-expression analysis revealed several transcription factors whose expression patterns correlated with those of genes associated with ascorbic acid, carotenoid and flavonoid biosynthesis. This transcriptional correlation was confirmed by agroinfiltration mediated transient expression, which showed that most of the enzymatic genes in the ascorbic acid biosynthesis were regulated by the overexpression of each of the three transcription factors that were tested. The metabolic dynamics of ascorbic acid, carotenoid and flavonoid were investigated during fruit development and ripening, and some selected transcription factors showed transcriptional correlation with the accumulation of ascorbic acid, carotenoid and flavonoid. This transcriptome study provides insight into the regulatory mechanism of fruit development and presents candidate transcription factors involved in secondary metabolism. PMID:26133783

  17. Lipidomic analysis of Toxoplasma gondii reveals unusual polar lipids†

    PubMed Central

    Welti, Ruth; Mui, Ernie; Sparks, Alexis; Wernimont, Sarah; Isaac, Giorgis; Kirisits, Michael; Roth, Mary; Roberts, Craig W.; Botté, Cyrille; Maréchal, Eric; McLeod, Rima

    2008-01-01

    Analysis of the polar lipids of Toxoplasma gondii by electrospray ionization tandem mass spectrometry provides a detailed picture of the lipid molecular species of this parasitic protozoan. Most notably, T. gondii contains a relatively high level, estimated to about 2% of the total polar lipid, of ceramide phosphoethanolamine. The ceramide phosphoethanolamine has a fatty amide profile with only 16- and 18-carbon species. Compared with the host fibroblasts in which it was grown, T. gondii also has higher levels of phosphatidylcholine, but lower levels of sphingomyelin and phosphatidylserine. Analysis at the molecular species level indicated that T. gondii has greater amounts of shorter-chain fatty acid in its polar lipid molecular species than the host fibroblasts. Shorter-chain fatty acids with a combined total of 30 or fewer acyl carbons make up 21% of Toxoplasma’s, but only 3% of the host’s, diacyl phosphatidylcholine. Furthermore, diacyl phosphatidylcholine with two saturated acyl chains with 12, 14, or 16 carbons make up over 11% of parasite phosphatidylcholine, but less than 3% of the host phosphatidylcholine molecular species. The distinctive T. gondii tachyzoite lipid profile may be particularly suited to the function of parasitic membranes and the interaction of the parasite with the host cell and the host’s immune system. Combined with T. gondii genomic data, these lipidomic data will assist in elucidation of metabolic pathways for lipid biosynthesis in this important human pathogen. PMID:17988103

  18. Global Metabolomic Profiling Reveals an Association of Metal Fume Exposure and Plasma Unsaturated Fatty Acids

    PubMed Central

    Chang, Chiung-yu; Fan, Tianteng; Su, Li; Chen, Feng; Christiani, David C.

    2013-01-01

    Background Welding-associated air pollutants negatively affect the health of exposed workers; however, their molecular mechanisms in causing disease remain largely unclear. Few studies have systematically investigated the systemic toxic effects of welding fumes on humans. Objectives To explore the effects of welding fumes on the plasma metabolome, and to identify biomarkers for risk assessment of welding fume exposure. Methods The two-stage, self-controlled exploratory study included 11 boilermakers from a 2011 discovery panel and 8 boilermakers from a 2012 validation panel. Plasma samples were collected pre- and post-welding fume exposure and analyzed by chromatography/mass spectrometry. Results Eicosapentaenoic or docosapentaenoic acid metabolic changes post-welding were significantly associated with particulate (PM2.5) exposure (p<0.05). The combined analysis by linear mixed-effects model showed that exposure was associated with a statistically significant decline in metabolite change of eicosapentaenoic acid [(95% CI) = −0.013(−0.022∼−0.004); p = 0.005], docosapentaenoic acid n3 [(95% CI) = −0.010(−0.018∼−0.002); p = 0.017], and docosapentaenoic acid n6 [(95% CI) = −0.007(−0.013∼−0.001); p = 0.021]. Pathway analysis identified an association of the unsaturated fatty acid pathway with exposure (pStudy−2011 = 0.025; pStudy−2012 = 0.021; pCombined = 0.009). The functional network built by these fatty acids and their interactive genes contained significant enrichment of genes associated with various diseases, including neoplasms, cardiovascular diseases, and lipid metabolism disorders. Conclusions High-dose exposure of metal welding fumes decreases unsaturated fatty acids with an exposure-response relationship. This alteration in fatty acids is a potential biological mediator and biomarker for exposure-related health disorders. PMID:24143234

  19. Mathematical Analysis of Biomolecular Network Reveals Connections Between Diseases

    NASA Astrophysics Data System (ADS)

    Wang, Guanyu

    2012-02-01

    Connections between cancer and metabolic diseases may consist in the complex network of interactions among a common set of biomolecules. By applying singularity and bifurcation analysis, the phenotypes constrained by the AKT signaling pathway are identified and mapped onto the parameter space, which include cancer and certain metabolic diseases. By considering physiologic properties (sensitivity, robustness and adaptivity) the AKT pathway must possess in order to efficiently sense growth factors and nutrients, the region of normal responses is located. The analysis illuminates the parameter space and reveals system-level mechanisms in regulating biological functions (cell growth, survival, proliferation and metabolism) and how their deregulation may lead to the development of diseases. The analytical expressions summarize the synergistic interactions among many molecules, which provides valuable insights into therapeutic interventions.

  20. Geometric morphometric analysis reveals sexual dimorphism in the distal femur.

    PubMed

    Cavaignac, Etienne; Savall, Frederic; Faruch, Marie; Reina, Nicolas; Chiron, Philippe; Telmon, Norbert

    2016-02-01

    An individual's sex can be determined by the shape of their distal femur. The goal of this study was to show that differences in distal femur shape related to sexual dimorphism could be identified, visualized, and quantified using 3D geometric morphometric analysis. Geometric morphometric analysis was carried out on CT scans of the distal femur of 256 subjects living in the south of France. Ten landmarks were defined on 3D reconstructions of the distal femur. Both traditional metric and geometric morphometric analyses were carried out on these bone reconstructions; these analyses identified trends in bone shape in sex-based subgroups. Sex-related differences in shape were statistically significant. The subject's sex was correctly assigned in 77.3% of cases using geometric morphometric analysis. This study has shown that geometric morphometric analysis of the distal femur is feasible and has revealed sexual dimorphism differences in this bone segment. This reliable, accurate method could be used for virtual autopsy and be used to perform diachronic and interethnic comparisons. Moreover, this study provides updated morphometric data for a modern population in the south of France. PMID:26743712

  1. Integration of Metabolomics and Transcriptomics Revealed a Fatty Acid Network Exerting Growth Inhibitory Effects in Human Pancreatic Cancer

    PubMed Central

    Zhang, Geng; He, Peijun; Tan, Hanson; Budhu, Anuradha; Gaedcke, Jochen; Ghadimi, B. Michael; Ried, Thomas; Yfantis, Harris G.; Lee, Dong H.; Maitra, Anirban; Hanna, Nader; Alexander, H. Richard; Hussain, S. Perwez

    2013-01-01

    Purpose To identify metabolic pathways that are perturbed in pancreatic ductal adenocarcinoma (PDAC), we investigated gene-metabolite networks with integration of metabolomics and transcriptomics. Experimental design We have performed global metabolite profiling analysis on two independent cohorts of resected PDAC cases to identify critical metabolites alteration that may contribute to the progression of pancreatic cancer. We then searched for gene surrogates that were significantly correlated with the key metabolites by integrating metabolite and gene expression profiles. Results 55 metabolites were consistently altered in tumors as compared with adjacent nontumor tissues in a test cohort (N=33) and an independent validation cohort (N=31). Weighted network analysis revealed a unique set of free fatty acids (FFAs) that were highly co-regulated and decreased in PDAC. Pathway analysis of 157 differentially expressed gene surrogates revealed a significantly altered lipid metabolism network, including key lipolytic enzymes PNLIP, CLPS, PNLIPRP1, and PNLIPRP2. Gene expressions of these lipases were significantly decreased in pancreatic tumors as compared with nontumor tissues, leading to reduced FFAs. More importantly, a lower gene expression of PNLIP in tumors was associated with poorer survival in two independent cohorts. We further demonstrated that two saturated FFAs, palmitate and stearate significantly induced TRAIL expression, triggered apoptosis, and inhibited proliferation in pancreatic cancer cells. Conclusions Our results suggest that impairment in a lipolytic pathway involving lipases and a unique set of FFAs, may play an important role in the development and progression of pancreatic cancer and provide potential targets for therapeutic intervention. PMID:23918603

  2. DNA Methylation Profiling at Single-Base Resolution Reveals Gestational Folic Acid Supplementation Influences the Epigenome of Mouse Offspring Cerebellum

    PubMed Central

    Barua, Subit; Kuizon, Salomon; Brown, W. Ted; Junaid, Mohammed A.

    2016-01-01

    It is becoming increasingly more evident that lifestyle, environmental factors, and maternal nutrition during gestation can influence the epigenome of the developing fetus and thus modulate the physiological outcome. Variations in the intake of maternal nutrients affecting one-carbon metabolism may influence brain development and exert long-term effects on the health of the progeny. In this study, we investigated whether supplementation with high maternal folic acid during gestation alters DNA methylation and gene expression in the cerebellum of mouse offspring. We used reduced representation bisulfite sequencing to analyze the DNA methylation profile at the single-base resolution level. The genome-wide DNA methylation analysis revealed that supplementation with higher maternal folic acid resulted in distinct methylation patterns (P < 0.05) of CpG and non-CpG sites in the cerebellum of offspring. Such variations of methylation and gene expression in the cerebellum of offspring were highly sex-specific, including several genes of the neuronal pathways. These findings demonstrate that alterations in the level of maternal folic acid during gestation can influence methylation and gene expression in the cerebellum of offspring. Such changes in the offspring epigenome may alter neurodevelopment and influence the functional outcome of neurologic and psychiatric diseases. PMID:27199632

  3. Cation–Anion Interactions within the Nucleic Acid Ion Atmosphere Revealed by Ion Counting

    PubMed Central

    Gebala, Magdalena; Giambasu, George M.; Lipfert, Jan; Bisaria, Namita; Bonilla, Steve; Li, Guangchao; York, Darrin M.; Herschlag, Daniel

    2016-01-01

    The ion atmosphere is a critical structural, dynamic, and energetic component of nucleic acids that profoundly affects their interactions with proteins and ligands. Experimental methods that “count” the number of ions thermodynamically associated with the ion atmosphere allow dissection of energetic properties of the ion atmosphere, and thus provide direct comparison to theoretical results. Previous experiments have focused primarily on the cations that are attracted to nucleic acid polyanions, but have also showed that anions are excluded from the ion atmosphere. Herein, we have systematically explored the properties of anion exclusion, testing the zeroth-order model that anions of different identity are equally excluded due to electrostatic repulsion. Using a series of monovalent salts, we find, surprisingly, that the extent of anion exclusion and cation inclusion significantly depends on salt identity. The differences are prominent at higher concentrations and mirror trends in mean activity coefficients of the electrolyte solutions. Salts with lower activity coefficients exhibit greater accumulation of both cations and anions within the ion atmosphere, strongly suggesting that cation–anion correlation effects are present in the ion atmosphere and need to be accounted for to understand electrostatic interactions of nucleic acids. To test whether the effects of cation–anion correlations extend to nucleic acid kinetics and thermodynamics, we followed the folding of P4–P6, a domain of the Tetrahymena group I ribozyme, via single-molecule fluorescence resonance energy transfer in solutions with different salts. Solutions of identical concentration but lower activity gave slower and less favorable folding. Our results reveal hitherto unknown properties of the ion atmosphere and suggest possible roles of oriented ion pairs or anion-bridged cations in the ion atmosphere for electrolyte solutions of salts with reduced activity. Consideration of these new

  4. Molecular Analysis of Sarcoidosis Granulomas Reveals Antimicrobial Targets.

    PubMed

    Rotsinger, Joseph E; Celada, Lindsay J; Polosukhin, Vasiliy V; Atkinson, James B; Drake, Wonder P

    2016-07-01

    Sarcoidosis is a granulomatous disease of unknown cause. Prior molecular and immunologic studies have confirmed the presence of mycobacterial virulence factors, such as catalase peroxidase and superoxide dismutase A, within sarcoidosis granulomas. Molecular analysis of granulomas can identify targets of known antibiotics classes. Currently, major antibiotics are directed against DNA synthesis, protein synthesis, and cell wall formation. We conducted molecular analysis of 40 sarcoidosis diagnostic specimens and compared them with 33 disease control specimens for the presence of mycobacterial genes that encode antibiotic targets. We assessed for genes involved in DNA synthesis (DNA gyrase A [gyrA] and DNA gyrase B), protein synthesis (RNA polymerase subunit β), cell wall synthesis (embCAB operon and enoyl reductase), and catalase peroxidase. Immunohistochemical analysis was conducted to investigate the locale of mycobacterial genes such as gyrA within 12 sarcoidosis specimens and 12 disease controls. Mycobacterial DNA was detected in 33 of 39 sarcoidosis specimens by quantitative real-time polymerase chain reaction compared with 2 of 30 disease control specimens (P < 0.001, two-tailed Fisher's test). Twenty of 39 were positive for three or more mycobacterial genes, compared with 1 of 30 control specimens (P < 0.001, two-tailed Fisher's test). Immunohistochemistry analysis localized mycobacterial gyrA nucleic acids to sites of granuloma formation in 9 of 12 sarcoidosis specimens compared with 1 of 12 disease controls (P < 0.01). Microbial genes encoding enzymes that can be targeted by currently available antimycobacterial antibiotics are present in sarcoidosis specimens and localize to sites of granulomatous inflammation. Use of antimicrobials directed against target enzymes may be an innovative treatment alternative. PMID:26807608

  5. Interactome Analysis Reveals Ezrin Can Adopt Multiple Conformational States*

    PubMed Central

    Viswanatha, Raghuvir; Wayt, Jessica; Ohouo, Patrice Y.; Smolka, Marcus B.; Bretscher, Anthony

    2013-01-01

    Ezrin, a member of the ezrin-radixin-moesin family (ERM), is an essential regulator of the structure of microvilli on the apical aspect of epithelial cells. Ezrin provides a linkage between membrane-associated proteins and F-actin, oscillating between active/open and inactive/closed states, and is regulated in part by phosphorylation of a C-terminal threonine. In the open state, ezrin can bind a number of ligands, but in the closed state the ligand-binding sites are inaccessible. In vitro analysis has proposed that there may be a third hyperactivated form of ezrin. To gain a better understanding of ezrin, we conducted an unbiased proteomic analysis of ezrin-binding proteins in an epithelial cell line, Jeg-3. We refined our list of interactors by comparing the interactomes using quantitative mass spectrometry between wild-type ezrin, closed ezrin, open ezrin, and hyperactivated ezrin. The analysis reveals several novel interactors confirmed by their localization to microvilli, as well as a significant class of proteins that bind closed ezrin. Taken together, the data indicate that ezrin can exist in three different conformational states, and different ligands “perceive” ezrin conformational states differently. PMID:24151071

  6. Genomic analysis of primordial dwarfism reveals novel disease genes.

    PubMed

    Shaheen, Ranad; Faqeih, Eissa; Ansari, Shinu; Abdel-Salam, Ghada; Al-Hassnan, Zuhair N; Al-Shidi, Tarfa; Alomar, Rana; Sogaty, Sameera; Alkuraya, Fowzan S

    2014-02-01

    Primordial dwarfism (PD) is a disease in which severely impaired fetal growth persists throughout postnatal development and results in stunted adult size. The condition is highly heterogeneous clinically, but the use of certain phenotypic aspects such as head circumference and facial appearance has proven helpful in defining clinical subgroups. In this study, we present the results of clinical and genomic characterization of 16 new patients in whom a broad definition of PD was used (e.g., 3M syndrome was included). We report a novel PD syndrome with distinct facies in two unrelated patients, each with a different homozygous truncating mutation in CRIPT. Our analysis also reveals, in addition to mutations in known PD disease genes, the first instance of biallelic truncating BRCA2 mutation causing PD with normal bone marrow analysis. In addition, we have identified a novel locus for Seckel syndrome based on a consanguineous multiplex family and identified a homozygous truncating mutation in DNA2 as the likely cause. An additional novel PD disease candidate gene XRCC4 was identified by autozygome/exome analysis, and the knockout mouse phenotype is highly compatible with PD. Thus, we add a number of novel genes to the growing list of PD-linked genes, including one which we show to be linked to a novel PD syndrome with a distinct facial appearance. PD is extremely heterogeneous genetically and clinically, and genomic tools are often required to reach a molecular diagnosis. PMID:24389050

  7. Extensive exometabolome analysis reveals extended overflow metabolism in various microorganisms

    PubMed Central

    2012-01-01

    Overflow metabolism is well known for yeast, bacteria and mammalian cells. It typically occurs under glucose excess conditions and is characterized by excretions of by-products such as ethanol, acetate or lactate. This phenomenon, also denoted the short-term Crabtree effect, has been extensively studied over the past few decades, however, its basic regulatory mechanism and functional role in metabolism is still unknown. Here we present a comprehensive quantitative and time-dependent analysis of the exometabolome of Escherichia coli, Corynebacterium glutamicum, Bacillus licheniformis, and Saccharomyces cerevisiae during well-controlled bioreactor cultivations. Most surprisingly, in all cases a great diversity of central metabolic intermediates and amino acids is found in the culture medium with extracellular concentrations varying in the micromolar range. Different hypotheses for these observations are formulated and experimentally tested. As a result, the intermediates in the culture medium during batch growth must originate from passive or active transportation due to a new phenomenon termed “extended” overflow metabolism. Moreover, we provide broad evidence that this could be a common feature of all microorganism species when cultivated under conditions of carbon excess and non-inhibited carbon uptake. In turn, this finding has consequences for metabolite balancing and, particularly, for intracellular metabolite quantification and 13C-metabolic flux analysis. PMID:22963408

  8. Graph analysis of cortical networks reveals complex anatomical communication substrate

    NASA Astrophysics Data System (ADS)

    Zamora-López, Gorka; Zhou, Changsong; Kurths, Jürgen

    2009-03-01

    Sensory information entering the nervous system follows independent paths of processing such that specific features are individually detected. However, sensory perception, awareness, and cognition emerge from the combination of information. Here we have analyzed the corticocortical network of the cat, looking for the anatomical substrate which permits the simultaneous segregation and integration of information in the brain. We find that cortical communications are mainly governed by three topological factors of the underlying network: (i) a large density of connections, (ii) segregation of cortical areas into clusters, and (iii) the presence of highly connected hubs aiding the multisensory processing and integration. Statistical analysis of the shortest paths reveals that, while information is highly accessible to all cortical areas, the complexity of cortical information processing may arise from the rich and intricate alternative paths in which areas can influence each other.

  9. Single cell transcriptional analysis reveals novel innate immune cell types.

    PubMed

    Kippner, Linda E; Kim, Jinhee; Gibson, Greg; Kemp, Melissa L

    2014-01-01

    Single-cell analysis has the potential to provide us with a host of new knowledge about biological systems, but it comes with the challenge of correctly interpreting the biological information. While emerging techniques have made it possible to measure inter-cellular variability at the transcriptome level, no consensus yet exists on the most appropriate method of data analysis of such single cell data. Methods for analysis of transcriptional data at the population level are well established but are not well suited to single cell analysis due to their dependence on population averages. In order to address this question, we have systematically tested combinations of methods for primary data analysis on single cell transcription data generated from two types of primary immune cells, neutrophils and T lymphocytes. Cells were obtained from healthy individuals, and single cell transcript expression data was obtained by a combination of single cell sorting and nanoscale quantitative real time PCR (qRT-PCR) for markers of cell type, intracellular signaling, and immune functionality. Gene expression analysis was focused on hierarchical clustering to determine the existence of cellular subgroups within the populations. Nine combinations of criteria for data exclusion and normalization were tested and evaluated. Bimodality in gene expression indicated the presence of cellular subgroups which were also revealed by data clustering. We observed evidence for two clearly defined cellular subtypes in the neutrophil populations and at least two in the T lymphocyte populations. When normalizing the data by different methods, we observed varying outcomes with corresponding interpretations of the biological characteristics of the cell populations. Normalization of the data by linear standardization taking into account technical effects such as plate effects, resulted in interpretations that most closely matched biological expectations. Single cell transcription profiling provides

  10. Transcriptional analysis of the effect of exogenous decanoic acid stress on Streptomyces roseosporus

    PubMed Central

    2013-01-01

    Backgroud Daptomycin is an important antibiotic against infections caused by drug-resistant pathogens. Its production critically depends on the addition of decanoic acid during fermentation. Unfortunately, decanoic acid (>2.5 mM) is toxic to daptomycin producer, Streptomyces roseosporus. Results To understand the mechanism underlying decanoic tolerance or toxicity, the responses of S. roseosporus was determined by a combination of phospholipid fatty acid analysis, reactive oxygen species (ROS) measurement and RNA sequencing. Assays using fluorescent dyes indicated a sharp increase in reactive oxygen species during decanoic acid stress; fatty acid analysis revealed a marked increase in the composition of branched-chain fatty acids by approximately 10%, with a corresponding decrease in straight-chain fatty acids; functional analysis indicated decanoic acid stress has components common to other stress response, including perturbation of respiratory functions (nuo and cyd operons), oxidative stress, and heat shock. Interestingly, our transcriptomic analysis revealed that genes coding for components of proteasome and related to treholase synthesis were up-regulated in the decanoic acid –treated cells. Conclusion These findings represent an important first step in understanding mechanism of decanoic acid toxicity and provide a basis for engineering microbial tolerance. PMID:23432849

  11. beta-Keratins in crocodiles reveal amino acid homology with avian keratins.

    PubMed

    Ye, Changjiang; Wu, Xiaobing; Yan, Peng; Amato, George

    2010-03-01

    The DNA sequences encoding beta-keratin have been obtained from Marsh Mugger (Crocodylus palustris) and Orinoco Crocodiles (Crocodylus intermedius). Through the deduced amino acid sequence, these proteins are rich in glycine, proline and serine. The central region of the proteins are composed of two beta-folded regions and show a high degree of identity with beta-keratins of aves and squamates. This central part is thought to be the site of polymerization to build the framework of beta-keratin filaments. It is believed that the beta-keratins in reptiles and birds share a common ancestry. Near the C-terminal, these beta-keratins contain a peptide rich in glycine-X and glycine-X-X, and the distinctive feature of the region is some 12-amino acid repeats, which are similar to the 13-amino acid repeats in chick scale keratin but absent from avian feather keratin. From our phylogenetic analysis, the beta-keratins in crocodile have a closer relationship with avian keratins than the other keratins in reptiles. PMID:19266314

  12. Nucleic Acid Aptamers for Living Cell Analysis

    NASA Astrophysics Data System (ADS)

    Xiong, Xiangling; Lv, Yifan; Chen, Tao; Zhang, Xiaobing; Wang, Kemin; Tan, Weihong

    2014-06-01

    Cells as the building blocks of life determine the basic functions and properties of a living organism. Understanding the structure and components of a cell aids in the elucidation of its biological functions. Moreover, knowledge of the similarities and differences between diseased and healthy cells is essential to understanding pathological mechanisms, identifying diagnostic markers, and designing therapeutic molecules. However, monitoring the structures and activities of a living cell remains a challenging task in bioanalytical and life science research. To meet the requirements of this task, aptamers, as “chemical antibodies,” have become increasingly powerful tools for cellular analysis. This article reviews recent advances in the development of nucleic acid aptamers in the areas of cell membrane analysis, cell detection and isolation, real-time monitoring of cell secretion, and intracellular delivery and analysis with living cell models. Limitations of aptamers and possible solutions are also discussed.

  13. Transcriptome meta-analysis reveals dysregulated pathways in nasopharyngeal carcinoma.

    PubMed

    Tulalamba, Warut; Larbcharoensub, Noppadol; Sirachainan, Ekaphop; Tantiwetrueangdet, Aunchalee; Janvilisri, Tavan

    2015-08-01

    Nasopharyngeal carcinoma (NPC) is a malignant cancer arising from the epithelial surface of the nasopharynx that mostly appears in advanced stages of the disease, leading to a poor prognosis. To date, a number of mRNA profiling investigations on NPC have been reported in order to identify suitable biomarkers for early detection. However, the results may be specific to each study with distinct sample types. In this study, an integrative meta-analysis of NPC transcriptome data was performed to determine dysregulated pathways, potentially leading to identification of molecular markers. Ten independent NPC gene expression profiling microarray datasets, including 135 samples from NPC cell lines, primary cell lines, and tissues were assimilated into a meta-analysis and cross-validation to identify a cohort of genes that were significantly dysregulated in NPC. Bioinformatics analyses of these genes revealed the significant pathways and individual players involving in cellular metabolism, cell cycle regulation, DNA repair, as well as ErbB pathway. Altogether, we propose that dysregulation of these molecular pathways in NPC might play a role in the NPC pathogenesis, providing clues, which could eventually translate into diagnostic and therapeutic approaches. PMID:25724187

  14. Proteomics Analysis Reveals Overlapping Functions of Clustered Protocadherins*

    PubMed Central

    Han, Meng-Hsuan; Lin, Chengyi; Meng, Shuxia; Wang, Xiaozhong

    2010-01-01

    The three tandem-arrayed protocadherin (Pcdh) gene clusters, namely Pcdh-α, Pcdh-β, and Pcdh-γ, play important roles in the development of the vertebrate central nervous system. To gain insight into the molecular action of PCDHs, we performed a systematic proteomics analysis of PCDH-γ-associated protein complexes. We identified a list of 154 non-redundant proteins in the PCDH-γ complexes. This list includes nearly 30 members of clustered Pcdh-α, -β, and -γ families as core components of the complexes and additionally over 120 putative PCDH-associated proteins. We validated a selected subset of PCDH-γ-associated proteins using specific antibodies. Analysis of the identities of PCDH-associated proteins showed that the majority of them overlap with the proteomic profile of postsynaptic density preparations. Further analysis of membrane protein complexes revealed that several validated PCDH-γ-associated proteins exhibit reduced levels in Pcdh-γ-deficient brain tissues. Therefore, PCDH-γs are required for the integrity of the complexes. However, the size of the overall complexes and the abundance of many other proteins remained unchanged, raising a possibility that PCDH-αs and PCDH-βs might compensate for PCDH-γ function in complex formation. As a test of this idea, RNA interference knockdown of both PCDH-αs and PCDH-γs showed that PCDHs have redundant functions in regulating neuronal survival in the chicken spinal cord. Taken together, our data provide evidence that clustered PCDHs coexist in large protein complexes and have overlapping functions during vertebrate neural development. PMID:19843561

  15. Microsporidian genome analysis reveals evolutionary strategies for obligate intracellular growth

    PubMed Central

    Cuomo, Christina A.; Desjardins, Christopher A.; Bakowski, Malina A.; Goldberg, Jonathan; Ma, Amy T.; Becnel, James J.; Didier, Elizabeth S.; Fan, Lin; Heiman, David I.; Levin, Joshua Z.; Young, Sarah; Zeng, Qiandong; Troemel, Emily R.

    2012-01-01

    Microsporidia comprise a large phylum of obligate intracellular eukaryotes that are fungal-related parasites responsible for widespread disease, and here we address questions about microsporidia biology and evolution. We sequenced three microsporidian genomes from two species, Nematocida parisii and Nematocida sp1, which are natural pathogens of Caenorhabditis nematodes and provide model systems for studying microsporidian pathogenesis. We performed deep sequencing of transcripts from a time course of N. parisii infection. Examination of pathogen gene expression revealed compact transcripts and a dramatic takeover of host cells by Nematocida. We also performed phylogenomic analyses of Nematocida and other microsporidian genomes to refine microsporidian phylogeny and identify evolutionary events of gene loss, acquisition, and modification. In particular, we found that all microsporidia lost the tumor-suppressor gene retinoblastoma, which we speculate could accelerate the parasite cell cycle and increase the mutation rate. We also found that microsporidia acquired transporters that could import nucleosides to fuel rapid growth. In addition, microsporidian hexokinases gained secretion signal sequences, and in a functional assay these were sufficient to export proteins out of the cell; thus hexokinase may be targeted into the host cell to reprogram it toward biosynthesis. Similar molecular changes appear during formation of cancer cells and may be evolutionary strategies adopted independently by microsporidia to proliferate rapidly within host cells. Finally, analysis of genome polymorphisms revealed evidence for a sexual cycle that may provide genetic diversity to alleviate problems caused by clonal growth. Together these events may explain the emergence and success of these diverse intracellular parasites. PMID:22813931

  16. Microsporidian genome analysis reveals evolutionary strategies for obligate intracellular growth.

    PubMed

    Cuomo, Christina A; Desjardins, Christopher A; Bakowski, Malina A; Goldberg, Jonathan; Ma, Amy T; Becnel, James J; Didier, Elizabeth S; Fan, Lin; Heiman, David I; Levin, Joshua Z; Young, Sarah; Zeng, Qiandong; Troemel, Emily R

    2012-12-01

    Microsporidia comprise a large phylum of obligate intracellular eukaryotes that are fungal-related parasites responsible for widespread disease, and here we address questions about microsporidia biology and evolution. We sequenced three microsporidian genomes from two species, Nematocida parisii and Nematocida sp1, which are natural pathogens of Caenorhabditis nematodes and provide model systems for studying microsporidian pathogenesis. We performed deep sequencing of transcripts from a time course of N. parisii infection. Examination of pathogen gene expression revealed compact transcripts and a dramatic takeover of host cells by Nematocida. We also performed phylogenomic analyses of Nematocida and other microsporidian genomes to refine microsporidian phylogeny and identify evolutionary events of gene loss, acquisition, and modification. In particular, we found that all microsporidia lost the tumor-suppressor gene retinoblastoma, which we speculate could accelerate the parasite cell cycle and increase the mutation rate. We also found that microsporidia acquired transporters that could import nucleosides to fuel rapid growth. In addition, microsporidian hexokinases gained secretion signal sequences, and in a functional assay these were sufficient to export proteins out of the cell; thus hexokinase may be targeted into the host cell to reprogram it toward biosynthesis. Similar molecular changes appear during formation of cancer cells and may be evolutionary strategies adopted independently by microsporidia to proliferate rapidly within host cells. Finally, analysis of genome polymorphisms revealed evidence for a sexual cycle that may provide genetic diversity to alleviate problems caused by clonal growth. Together these events may explain the emergence and success of these diverse intracellular parasites. PMID:22813931

  17. Acidic Properties and Structure-Activity Correlations of Solid Acid Catalysts Revealed by Solid-State NMR Spectroscopy.

    PubMed

    Zheng, Anmin; Li, Shenhui; Liu, Shang-Bin; Deng, Feng

    2016-04-19

    Solid acid materials with tunable structural and acidic properties are promising heterogeneous catalysts for manipulating and/or emulating the activity and selectivity of industrially important catalytic reactions. On the other hand, the performances of acid-catalyzed reactions are mostly dictated by the acidic features, namely, type (Brønsted vs Lewis acidity), amount, strength, and local environment of acid sites. The latter is relevant to their location (intra- vs extracrystalline), and possible confinement and Brønsted-Lewis acid synergy effects that may strongly affect the host-guest interactions, reaction mechanism, and shape selectivity of the catalytic system. This account aims to highlight some important applications of state-of-the-art solid-state NMR (SSNMR) techniques for exploring the structural and acidic properties of solid acid catalysts as well as their catalytic performances and relevant reaction pathway invoked. In addition, density functional theory (DFT) calculations may be exploited in conjunction with experimental SSNMR studies to verify the structure-activity correlations of the catalytic system at a microscopic scale. We describe in this Account the developments and applications of advanced ex situ and/or in situ SSNMR techniques, such as two-dimensional (2D) double-quantum magic-angle spinning (DQ MAS) homonuclear correlation spectroscopy for structural investigation of solid acids as well as study of their acidic properties. Moreover, the energies and electronic structures of the catalysts and detailed catalytic reaction processes, including the identification of reaction species, elucidation of reaction mechanism, and verification of structure-activity correlations, made available by DFT theoretical calculations were also discussed. Relevant discussions will focus primarily on results obtained from our laboratories in the past decade, including (i) quantitative and qualitative acidity characterization utilizing assorted probe molecules

  18. Metabonomics Reveals Drastic Changes in Anti-Inflammatory/Pro-Resolving Polyunsaturated Fatty Acids-Derived Lipid Mediators in Leprosy Disease

    PubMed Central

    Amaral, Julio J.; Antunes, Luis Caetano M.; de Macedo, Cristiana S.; Mattos, Katherine A.; Han, Jun; Pan, Jingxi; Candéa, André L. P.; Henriques, Maria das Graças M. O.; Ribeiro-Alves, Marcelo; Borchers, Christoph H.; Sarno, Euzenir N.; Bozza, Patrícia T.; Finlay, B. Brett; Pessolani, Maria Cristina V.

    2013-01-01

    Despite considerable efforts over the last decades, our understanding of leprosy pathogenesis remains limited. The complex interplay between pathogens and hosts has profound effects on host metabolism. To explore the metabolic perturbations associated with leprosy, we analyzed the serum metabolome of leprosy patients. Samples collected from lepromatous and tuberculoid patients before and immediately after the conclusion of multidrug therapy (MDT) were subjected to high-throughput metabolic profiling. Our results show marked metabolic alterations during leprosy that subside at the conclusion of MDT. Pathways showing the highest modulation were related to polyunsaturated fatty acid (PUFA) metabolism, with emphasis on anti-inflammatory, pro-resolving omega-3 fatty acids. These results were confirmed by eicosanoid measurements through enzyme-linked immunoassays. Corroborating the repertoire of metabolites altered in sera, metabonomic analysis of skin specimens revealed alterations in the levels of lipids derived from lipase activity, including PUFAs, suggesting a high lipid turnover in highly-infected lesions. Our data suggest that omega-6 and omega-3, PUFA-derived, pro-resolving lipid mediators contribute to reduced tissue damage irrespectively of pathogen burden during leprosy disease. Our results demonstrate the utility of a comprehensive metabonomic approach for identifying potential contributors to disease pathology that may facilitate the development of more targeted treatments for leprosy and other inflammatory diseases. PMID:23967366

  19. Lipidome analysis reveals antifungal polyphenol curcumin affects membrane lipid homeostasis.

    PubMed

    Sharma, Monika; Dhamgaye, Sanjiveeni; Singh, Ashutosh; Prasad, Rajendra

    2012-01-01

    This study shows that antifungal curcumin (CUR), significantly depletes ergosterol levels in Candida albicans. CUR while displaying synergy with fluconazole (FLC) lowers ergosterol. However, CUR alone at its synergistic concentration (lower than MIC50), could not affect ergosterol contents. For deeper insight of CUR effects on lipids, we performed high throughput mass spectroscopy (MS) based lipid profiling of C. albicans cells. The lipidome analysis revealed that there were no major changes in phosphoglycerides (PGLs) composition following CUR treatment of Candida, however, significant differences in molecular species of PGLs were detected. Among major SPLs, CUR treatment resulted in the reduction of ceramide and accumulation of IPCs levels. The lipidome of CUR treated cells confirmed a dramatic drop in the ergosterol levels with a simultaneous accumulation of its biosynthetic precursors. This was further supported by the fact that the mutants defective in ergosterol biosynthesis (ERG2 and ERG11) and those lacking the transcription factor regulating ergosterol biosynthesis, UPC2, were highly susceptible to CUR. Our study first time shows that CUR, for its antifungal activity, targets and down regulates delta 5, 6 desaturase (ERG3) resulting in depletion of ergosterol. This results in parallel accumulation of ergosterol biosynthetic precursors, generation of reactive oxygen species (ROS) and cell death. PMID:22201946

  20. Phosphoproteomic analysis reveals regulatory mechanisms at the kidney filtration barrier.

    PubMed

    Rinschen, Markus M; Wu, Xiongwu; König, Tim; Pisitkun, Trairak; Hagmann, Henning; Pahmeyer, Caroline; Lamkemeyer, Tobias; Kohli, Priyanka; Schnell, Nicole; Schermer, Bernhard; Dryer, Stuart; Brooks, Bernard R; Beltrao, Pedro; Krueger, Marcus; Brinkkoetter, Paul T; Benzing, Thomas

    2014-07-01

    Diseases of the kidney filtration barrier are a leading cause of ESRD. Most disorders affect the podocytes, polarized cells with a limited capacity for self-renewal that require tightly controlled signaling to maintain their integrity, viability, and function. Here, we provide an atlas of in vivo phosphorylated, glomerulus-expressed proteins, including podocyte-specific gene products, identified in an unbiased tandem mass spectrometry-based approach. We discovered 2449 phosphorylated proteins corresponding to 4079 identified high-confidence phosphorylated residues and performed a systematic bioinformatics analysis of this dataset. We discovered 146 phosphorylation sites on proteins abundantly expressed in podocytes. The prohibitin homology domain of the slit diaphragm protein podocin contained one such site, threonine 234 (T234), located within a phosphorylation motif that is mutated in human genetic forms of proteinuria. The T234 site resides at the interface of podocin dimers. Free energy calculation through molecular dynamic simulations revealed a role for T234 in regulating podocin dimerization. We show that phosphorylation critically regulates formation of high molecular weight complexes and that this may represent a general principle for the assembly of proteins containing prohibitin homology domains. PMID:24511133

  1. Sequential analysis of the numerical Stroop effect reveals response suppression.

    PubMed

    Cohen Kadosh, Roi; Gevers, Wim; Notebaert, Wim

    2011-09-01

    Automatic processing of irrelevant stimulus dimensions has been demonstrated in a variety of tasks. Previous studies have shown that conflict between relevant and irrelevant dimensions can be reduced when a feature of the irrelevant dimension is repeated. The specific level at which the automatic process is suppressed (e.g., perceptual repetition, response repetition), however, is less understood. In the current experiment we used the numerical Stroop paradigm, in which the processing of irrelevant numerical values of 2 digits interferes with the processing of their physical size, to pinpoint the precise level of the suppression. Using a sequential analysis, we dissociated perceptual repetition from response repetition of the relevant and irrelevant dimension. Our analyses of reaction times, error rates, and diffusion modeling revealed that the congruity effect is significantly reduced or even absent when the response sequence of the irrelevant dimension, rather than the numerical value or the physical size, is repeated. These results suggest that automatic activation of the irrelevant dimension is suppressed at the response level. The current results shed light on the level of interaction between numerical magnitude and physical size as well as the effect of variability of responses and stimuli on automatic processing. PMID:21500951

  2. Point-of-gaze analysis reveals visual search strategies

    NASA Astrophysics Data System (ADS)

    Rajashekar, Umesh; Cormack, Lawrence K.; Bovik, Alan C.

    2004-06-01

    Seemingly complex tasks like visual search can be analyzed using a cognition-free, bottom-up framework. We sought to reveal strategies used by observers in visual search tasks using accurate eye tracking and image analysis at point of gaze. Observers were instructed to search for simple geometric targets embedded in 1/f noise. By analyzing the stimulus at the point of gaze using the classification image (CI) paradigm, we discovered CI templates that indeed resembled the target. No such structure emerged for a random-searcher. We demonstrate, qualitatively and quantitatively, that these CI templates are useful in predicting stimulus regions that draw human fixations in search tasks. Filtering a 1/f noise stimulus with a CI results in a 'fixation prediction map'. A qualitative evaluation of the prediction was obtained by overlaying k-means clusters of observers' fixations on the prediction map. The fixations clustered around the local maxima in the prediction map. To obtain a quantitative comparison, we computed the Kullback-Leibler distance between the recorded fixations and the prediction. Using random-searcher CIs in Monte Carlo simulations, a distribution of this distance was obtained. The z-scores for the human CIs and the original target were -9.70 and -9.37 respectively indicating that even in noisy stimuli, observers deploy their fixations efficiently to likely targets rather than casting them randomly hoping to fortuitously find the target.

  3. Using Willie's Acid-Base Box for Blood Gas Analysis

    ERIC Educational Resources Information Center

    Dietz, John R.

    2011-01-01

    In this article, the author describes a method developed by Dr. William T. Lipscomb for teaching blood gas analysis of acid-base status and provides three examples using Willie's acid-base box. Willie's acid-base box is constructed using three of the parameters of standard arterial blood gas analysis: (1) pH; (2) bicarbonate; and (3) CO[subscript…

  4. Carbon and nitrogen isotopes from top predator amino acids reveal rapidly shifting ocean biochemistry in the outer California Current.

    PubMed

    Ruiz-Cooley, Rocio I; Koch, Paul L; Fiedler, Paul C; McCarthy, Matthew D

    2014-01-01

    Climatic variation alters biochemical and ecological processes, but it is difficult both to quantify the magnitude of such changes, and to differentiate long-term shifts from inter-annual variability. Here, we simultaneously quantify decade-scale isotopic variability at the lowest and highest trophic positions in the offshore California Current System (CCS) by measuring δ15N and δ13C values of amino acids in a top predator, the sperm whale (Physeter macrocephalus). Using a time series of skin tissue samples as a biological archive, isotopic records from individual amino acids (AAs) can reveal the proximate factors driving a temporal decline we observed in bulk isotope values (a decline of ≥1 ‰) by decoupling changes in primary producer isotope values from those linked to the trophic position of this toothed whale. A continuous decline in baseline (i.e., primary producer) δ15N and δ13C values was observed from 1993 to 2005 (a decrease of ∼4‰ for δ15N source-AAs and 3‰ for δ13C essential-AAs), while the trophic position of whales was variable over time and it did not exhibit directional trends. The baseline δ15N and δ13C shifts suggest rapid ongoing changes in the carbon and nitrogen biogeochemical cycling in the offshore CCS, potentially occurring at faster rates than long-term shifts observed elsewhere in the Pacific. While the mechanisms forcing these biogeochemical shifts remain to be determined, our data suggest possible links to natural climate variability, and also corresponding shifts in surface nutrient availability. Our study demonstrates that isotopic analysis of individual amino acids from a top marine mammal predator can be a powerful new approach to reconstructing temporal variation in both biochemical cycling and trophic structure. PMID:25329915

  5. Carbon and Nitrogen Isotopes from Top Predator Amino Acids Reveal Rapidly Shifting Ocean Biochemistry in the Outer California Current

    PubMed Central

    Ruiz-Cooley, Rocio I.; Koch, Paul L.; Fiedler, Paul C.; McCarthy, Matthew D.

    2014-01-01

    Climatic variation alters biochemical and ecological processes, but it is difficult both to quantify the magnitude of such changes, and to differentiate long-term shifts from inter-annual variability. Here, we simultaneously quantify decade-scale isotopic variability at the lowest and highest trophic positions in the offshore California Current System (CCS) by measuring δ15N and δ13C values of amino acids in a top predator, the sperm whale (Physeter macrocephalus). Using a time series of skin tissue samples as a biological archive, isotopic records from individual amino acids (AAs) can reveal the proximate factors driving a temporal decline we observed in bulk isotope values (a decline of ≥1 ‰) by decoupling changes in primary producer isotope values from those linked to the trophic position of this toothed whale. A continuous decline in baseline (i.e., primary producer) δ15N and δ13C values was observed from 1993 to 2005 (a decrease of ∼4‰ for δ15N source-AAs and 3‰ for δ13C essential-AAs), while the trophic position of whales was variable over time and it did not exhibit directional trends. The baseline δ15N and δ13C shifts suggest rapid ongoing changes in the carbon and nitrogen biogeochemical cycling in the offshore CCS, potentially occurring at faster rates than long-term shifts observed elsewhere in the Pacific. While the mechanisms forcing these biogeochemical shifts remain to be determined, our data suggest possible links to natural climate variability, and also corresponding shifts in surface nutrient availability. Our study demonstrates that isotopic analysis of individual amino acids from a top marine mammal predator can be a powerful new approach to reconstructing temporal variation in both biochemical cycling and trophic structure. PMID:25329915

  6. Urinary Metabolomics Reveals Alterations of Aromatic Amino Acid Metabolism of Alzheimer's Disease in the Transgenic CRND8 Mice.

    PubMed

    Tang, Zhi; Liu, Liangfeng; Li, Yongle; Dong, Jiyang; Li, Min; Huang, Jiandong; Lin, Shuhai; Cai, Zongwei

    2016-01-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disorder, with amyloid plaques accumulation as the key feature involved in its pathology. To date, however, the biochemical changes in AD have not been clearly characterized. Here, we present that urinary metabolomics based on high resolution mass spectrometry was employed for delineation of metabolic alterations in transgenic CRND8 mice. In this noninvasive approach, urinary metabolome reveals the biochemical changes in early onset of this AD mouse model. In virtue of comprehensive metabolite profiling and multivariate statistical analysis, a total of 73 differential metabolites of urine sample sets was identified in 12-week and 18-week transgenic mice compared to wild-type littermates, covering perturbations of aromatic amino acid metabolism, the Krebs cycle and one-carbon metabolism. Of particular interest is that divergent tryptophan metabolism, such as upregulation of serotonin pathway while downregulation of kynurenine pathway, was observed. Meanwhile, the accumulation of both N-acetylvanilalanine and 3-methoxytyrosine indicated aromatic L-amino acid decarboxylase deficiency. And the microbial metabolites derived from aromatic amino acid metabolism and drug-like phase II metabolic response via the glycine conjugation reactions were also highlighted, indicating that genetic modification in mouse brain not only alters genotype but also perturbs the gut microbiome. Together, our study demonstrated that the integrative approach employing mass spectrometry-based metabolomics and a transgenic mouse model for AD may provide new evidence for distinct metabolic signatures. The perturbations of metabolic pathways may have far-reaching implications for early diagnosis and intervention in AD. PMID:26825095

  7. Proteomic Analysis of MG132-Treated Germinating Pollen Reveals Expression Signatures Associated with Proteasome Inhibition

    PubMed Central

    Vannini, Candida; Bracale, Marcella; Crinelli, Rita; Marconi, Valerio; Campomenosi, Paola; Marsoni, Milena; Scoccianti, Valeria

    2014-01-01

    Chemical inhibition of the proteasome has been previously found to effectively impair pollen germination and tube growth in vitro. However, the mediators of these effects at the molecular level are unknown. By performing 2DE proteomic analysis, 24 differentially expressed protein spots, representing 14 unique candidate proteins, were identified in the pollen of kiwifruit (Actinidia deliciosa) germinated in the presence of the MG132 proteasome inhibitor. qPCR analysis revealed that 11 of these proteins are not up-regulated at the mRNA level, but are most likely stabilized by proteasome inhibition. These differentially expressed proteins are predicted to function in various pathways including energy and lipid metabolism, cell wall synthesis, protein synthesis/degradation and stress responses. In line with this evidence, the MG132-induced changes in the proteome were accompanied by an increase in ATP and ROS content and by an alteration in fatty acid composition. PMID:25265451

  8. Amino acid coevolution reveals three-dimensional structure and functional domains of insect odorant receptors

    PubMed Central

    Hopf, Thomas A.; Morinaga, Satoshi; Ihara, Sayoko; Touhara, Kazushige; Marks, Debora S.; Benton, Richard

    2015-01-01

    Insect Odorant Receptors (ORs) comprise an enormous protein family that translates environmental chemical signals into neuronal electrical activity. These heptahelical receptors are proposed to function as ligand-gated ion channels and/or to act metabotropically as G protein-coupled receptors (GPCRs). Resolving their signalling mechanism has been hampered by the lack of tertiary structural information and primary sequence similarity to other proteins. We use amino acid evolutionary covariation across these ORs to define restraints on structural proximity of residue pairs, which permit de novo generation of three-dimensional models. The validity of our analysis is supported by the location of functionally important residues in highly constrained regions of the protein. Importantly, insect OR models exhibit a distinct transmembrane domain packing arrangement to that of canonical GPCRs, establishing the structural unrelatedness of these receptor families. The evolutionary couplings and models predict odour binding and ion conduction domains, and provide a template for rationale structure-activity dissection. PMID:25584517

  9. Neuroinformatics analyses reveal GABAt and SSADH as major proteins involved in anticonvulsant activity of valproic acid.

    PubMed

    Piplani, Sakshi; Verma, Prabhakar Kumar; Kumar, Ajit

    2016-07-01

    The unequivocal hypotheses about anticonvulsant activity of valproic acid (VPA) have always been a basic hurdle in designing next generation neurotherapeutics, particularly the anti-epileptic drugs. The present study reports about a comprehensive in-silico investigation into qualitative and quantitative binding of VPA and corresponding natural ligands of four major enzymes involved in neurotransmissions, namely-GABA transaminase (GABAt), α-keto glutarate dehydrogenase (α-KGDH), Succinate Semialdehyde dehydrogenase (SSADH) and Glutamate Decarboxylase (GAD), respectively. The molecular docking analyses revealed that VPA inhibits GABAt and α-KGDH through allosteric while SSADH through competitive mode of binding. There is an observed elevation in binding of glutamate over GAD in the presence of VPA. The docking inhibition constant (Ki) of VPA to all the studied enzymatic receptors were observed to be well below the therapeutic concentration of VPA in blood, except for α-KGDH, thus favouring GABAergic over glutamatergic mode of anticonvulsant activity of VPA. The report is probably the first comprehensive in-silico molecular study about VPA action. PMID:27261619

  10. Genetic heterogeneity in rhabdomyosarcoma revealed by SNP array analysis.

    PubMed

    Walther, Charles; Mayrhofer, Markus; Nilsson, Jenny; Hofvander, Jakob; Jonson, Tord; Mandahl, Nils; Øra, Ingrid; Gisselsson, David; Mertens, Fredrik

    2016-01-01

    Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and adolescents. Alveolar (ARMS) and embryonal (ERMS) histologies predominate, but rare cases are classified as spindle cell/sclerosing (SRMS). For treatment stratification, RMS is further subclassified as fusion-positive (FP-RMS) or fusion-negative (FN-RMS), depending on whether a gene fusion involving PAX3 or PAX7 is present or not. We investigated 19 cases of pediatric RMS using high resolution single-nucleotide polymorphism (SNP) array. FP-ARMS displayed, on average, more structural rearrangements than ERMS; the single FN-ARMS had a genomic profile similar to ERMS. Apart from previously known amplification (e.g., MYCN, CDK4, and MIR17HG) and deletion (e.g., NF1, CDKN2A, and CDKN2B) targets, amplification of ERBB2 and homozygous loss of ASCC3 or ODZ3 were seen. Combining SNP array with cytogenetic data revealed that most cases were polyploid, with at least one case having started as a near-haploid tumor. Further bioinformatic analysis of the SNP array data disclosed genetic heterogeneity, in the form of subclonal chromosomal imbalances, in five tumors. The outcome was worse for patients with FP-ARMS than ERMS or FN-ARMS (6/8 vs. 1/9 dead of disease), and the only children with ERMS showing intratumor diversity or with MYOD1 mutation-positive SRMS also died of disease. High resolution SNP array can be useful in evaluating genomic imbalances in pediatric RMS. PMID:26482321

  11. Isotope Analysis Reveals Foraging Area Dichotomy for Atlantic Leatherback Turtles

    PubMed Central

    Angulo, Elena; Das, Krishna; Girondot, Marc

    2008-01-01

    Background The leatherback turtle (Dermochelys coriacea) has undergone a dramatic decline over the last 25 years, and this is believed to be primarily the result of mortality associated with fisheries bycatch followed by egg and nesting female harvest. Atlantic leatherback turtles undertake long migrations across ocean basins from subtropical and tropical nesting beaches to productive frontal areas. Migration between two nesting seasons can last 2 or 3 years, a time period termed the remigration interval (RI). Recent satellite transmitter data revealed that Atlantic leatherbacks follow two major dispersion patterns after nesting season, through the North Gulf Stream area or more eastward across the North Equatorial Current. However, information on the whole RI is lacking, precluding the accurate identification of feeding areas where conservation measures may need to be applied. Methodology/Principal Findings Using stable isotopes as dietary tracers we determined the characteristics of feeding grounds of leatherback females nesting in French Guiana. During migration, 3-year RI females differed from 2-year RI females in their isotope values, implying differences in their choice of feeding habitats (offshore vs. more coastal) and foraging latitude (North Atlantic vs. West African coasts, respectively). Egg-yolk and blood isotope values are correlated in nesting females, indicating that egg analysis is a useful tool for assessing isotope values in these turtles, including adults when not available. Conclusions/Significance Our results complement previous data on turtle movements during the first year following the nesting season, integrating the diet consumed during the year before nesting. We suggest that the French Guiana leatherback population segregates into two distinct isotopic groupings, and highlight the urgent need to determine the feeding habitats of the turtle in the Atlantic in order to protect this species from incidental take by commercial fisheries. Our

  12. Analysis of lipids reveals differences between 'Mycobacterium habana' and Mycobacterium simiae.

    PubMed

    Mederos, L M; Valdivia, J A; Sempere, M A; Valero-Guillén, P L

    1998-05-01

    Fatty and mycolic acids and the pattern of glycolipids were studied in a collection of 34 strains of 'Mycobacterium habana' and in two strains of Mycobacterium simiae. Major glycolipids of these micro-organisms were assigned to the glycopeptidolipid (GPL) structural type, but both mycobacteria differed in the patterns obtained by TLC. The strains of 'M. habana' were separated into four groups (A-D), taking into account the presence or absence of several polar GPLs: group A contained GPL-I, GPL-II and GPL-III; group B contained GPL-I, GPL-II', GPL-II and GPL-III; group C contained GPL-II', GPL-II and GPL-III; group D did not contain any of these compounds. Fatty acids of both bacteria were similar, and ranged from 14 to 26 carbon atoms, hexadecanoic, octadecenoic and tuberculostearic acids being predominant. Mycolic acids were also similar by TLC and HPLC, and consisted of alpha-, alpha'- and ketomycolates. Partial structural analysis by MS carried out in strains 'M. habana' TMC 5135 and M. simiae ATCC 25275T revealed that alpha- and ketomycolates ranged, in general, from 79 to 87 carbon atoms, and alpha'-mycolates from 58 to 67 carbon atoms. The alpha- and ketomycolates belonged to several structural series, and minor variations were found between the two strain examined. The data obtained justified the synonymy between 'M. habana' and M. simiae but indicated, in turn, that the former can be distinguished on the basis of GPL analysis. Most strains of 'M. habana' can be defined by the presence of GPL-II and GPL-III, a finding that could be useful in the quality control of potential vaccine strains. PMID:9611792

  13. Analysis of the Transcriptional Differences between Indigenous and Invasive Whiteflies Reveals Possible Mechanisms of Whitefly Invasion

    PubMed Central

    Wang, Yong-Liang; Wang, Yu-Jun; Luan, Jun-Bo; Yan, Gen-Hong; Liu, Shu-Sheng; Wang, Xiao-Wei

    2013-01-01

    Background The whitefly Bemisa tabaci is a species complex of more than 31 cryptic species which include some of the most destructive invasive pests of crops worldwide. Among them, Middle East-Asia Minor 1 (MEAM1) and Mediterranean have invaded many countries and displaced the native whitefly species. The successful invasion of the two species is largely due to their wide range of host plants, high resistance to insecticides and remarkable tolerance to environmental stresses. However, the molecular differences between invasive and indigenous whiteflies remain largely unknown. Methodology/Principal Findings Here the global transcriptional difference between the two invasive whitefly species (MEAM1, MED) and one indigenous whitefly species (Asia II 3) were analyzed using the Illumina sequencing. Our analysis indicated that 2,422 genes between MEAM1 and MED; 3,073 genes between MEAM1 and Asia II 3; and 3,644 genes between MED and Asia II 3 were differentially expressed. Gene Ontology enrichment analysis revealed that the differently expressed genes between the invasive and indigenous whiteflies were significantly enriched in the term of ‘oxidoreductase activity’. Pathway enrichment analysis showed that carbohydrate, amino acid and glycerolipid metabolisms were more active in MEAM1 and MED than in Asia II 3, which may contribute to their differences in biological characteristics. Our analysis also illustrated that the majority of genes involved in ‘drug metabolic pathway’ were expressed at a higher level in MEAM1 and MED than in Asia II 3. Taken together, these results revealed that the genes related to basic metabolism and detoxification were expressed at an elevated level in the invasive whiteflies, which might be responsible for their higher resistance to insecticides and environmental stresses. Conclusions/Significance The extensive comparison of MEAM1, MED and Asia II 3 gene expression may serve as an invaluable resource for revealing the molecular

  14. An analysis of issues concerning acid rain

    SciTech Connect

    Not Available

    1984-01-01

    GAO examines the implications of current scientific knowledge for policy decisions on acid rain and offers a series of observations on the following issues involved in the debate: to what extent has it been scientifically demonstrated that acid rain is resulting in damage to the environment. What are the causes of acid rain and where is it most prevalent. What alternatives exist for controlling acid rain and what are their economic effects.

  15. Recent trends in the advanced analysis of bioactive fatty acids.

    PubMed

    Ruiz-Rodriguez, Alejandro; Reglero, Guillermo; Ibañez, Elena

    2010-01-20

    The consumption of dietary fats have been long associated to chronic diseases such as obesity, diabetes, cancer, arthritis, asthma, and cardiovascular disease; although some controversy still exists in the role of dietary fats in human health, certain fats have demonstrated their positive effect in the modulation of abnormal fatty acid and eicosanoid metabolism, both of them associated to chronic diseases. Among the different fats, some fatty acids can be used as functional ingredients such as alpha-linolenic acid (ALA), arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), gamma-linolenic acid (GLA), stearidonic acid (STA) and conjugated linoleic acid (CLA), among others. The present review is focused on recent developments in FAs analysis, covering sample preparation methods such as extraction, fractionation and derivatization as well as new advances in chromatographic methods such as GC and HPLC. Special attention is paid to trans fatty acids due its increasing interest for the food industry. PMID:19525080

  16. GENETIC ANALYSIS OF ABSCISIC ACID BIOSYNTHESIS

    SciTech Connect

    MCCARTY D R

    2012-01-10

    The carotenoid cleavage dioxygenases (CCD) catalyze synthesis of a variety of apo-carotenoid secondary metabolites in plants, animals and bacteria. In plants, the reaction catalyzed by the 11, 12, 9-cis-epoxy carotenoid dioxygenase (NCED) is the first committed and key regulated step in synthesis of the plant hormone, abscisic acid (ABA). ABA is a key regulator of plant stress responses and has critical functions in normal root and seed development. The molecular mechanisms responsible for developmental control of ABA synthesis in plant tissues are poorly understood. Five of the nine CCD genes present in the Arabidopsis genome encode NCED's involved in control of ABA synthesis in the plant. This project is focused on functional analysis of these five AtNCED genes as a key to understanding developmental regulation of ABA synthesis and dissecting the role of ABA in plant development. For this purpose, the project developed a comprehensive set of gene knockouts in the AtNCED genes that facilitate genetic dissection of ABA synthesis. These mutants were used in combination with key molecular tools to address the following specific objectives: (1) the role of ABA synthesis in root development; (2) developmental control of ABA synthesis in seeds; (3) analysis of ATNCED over-expressers; (4) preliminary crystallography of the maize VP14 protein.

  17. Metagenomic analysis of the rhizosphere soil microbiome with respect to phytic acid utilization.

    PubMed

    Unno, Yusuke; Shinano, Takuro

    2013-01-01

    While phytic acid is a major form of organic phosphate in many soils, plant utilization of phytic acid is normally limited; however, culture trials of Lotus japonicus using experimental field soil that had been managed without phosphate fertilizer for over 90 years showed significant usage of phytic acid applied to soil for growth and flowering and differences in the degree of growth, even in the same culture pot. To understand the key metabolic processes involved in soil phytic acid utilization, we analyzed rhizosphere soil microbial communities using molecular ecological approaches. Although molecular fingerprint analysis revealed changes in the rhizosphere soil microbial communities from bulk soil microbial community, no clear relationship between the microbiome composition and flowering status that might be related to phytic acid utilization of L. japonicus could be determined. However, metagenomic analysis revealed changes in the relative abundance of the classes Bacteroidetes, Betaproteobacteria, Chlorobi, Dehalococcoidetes and Methanobacteria, which include strains that potentially promote plant growth and phytic acid utilization, and some gene clusters relating to phytic acid utilization, such as alkaline phosphatase and citrate synthase, with the phytic acid utilization status of the plant. This study highlights phylogenetic and metabolic features of the microbial community of the L. japonicus rhizosphere and provides a basic understanding of how rhizosphere microbial communities affect the phytic acid status in soil. PMID:23257911

  18. Phylogenomic Analysis of Oenococcus oeni Reveals Specific Domestication of Strains to Cider and Wines

    PubMed Central

    Campbell-Sills, Hugo; El Khoury, Mariette; Favier, Marion; Romano, Andrea; Biasioli, Franco; Spano, Giuseppe; Sherman, David J.; Bouchez, Olivier; Coton, Emmanuel; Coton, Monika; Okada, Sanae; Tanaka, Naoto; Dols-Lafargue, Marguerite; Lucas, Patrick M.

    2015-01-01

    Oenococcus oeni is a lactic acid bacteria species encountered particularly in wine, where it achieves the malolactic fermentation. Molecular typing methods have previously revealed that the species is made of several genetic groups of strains, some being specific to certain types of wines, ciders or regions. Here, we describe 36 recently released O. oeni genomes and the phylogenomic analysis of these 36 plus 14 previously reported genomes. We also report three genome sequences of the sister species Oenococcus kitaharae that were used for phylogenomic reconstructions. Phylogenomic and population structure analyses performed revealed that the 50 O. oeni genomes delineate two major groups of 12 and 37 strains, respectively, named A and B, plus a putative group C, consisting of a single strain. A study on the orthologs and single nucleotide polymorphism contents of the genetic groups revealed that the domestication of some strains to products such as cider, wine, or champagne, is reflected at the genetic level. While group A strains proved to be predominant in wine and to form subgroups adapted to specific types of wine such as champagne, group B strains were found in wine and cider. The strain from putative group C was isolated from cider and genetically closer to group B strains. The results suggest that ancestral O. oeni strains were adapted to low-ethanol containing environments such as overripe fruits, and that they were domesticated to cider and wine, with group A strains being naturally selected in a process of further domestication to specific wines such as champagne. PMID:25977455

  19. Quantitative Proteomics Reveals the Flooding-Tolerance Mechanism in Mutant and Abscisic Acid-Treated Soybean.

    PubMed

    Yin, Xiaojian; Nishimura, Minoru; Hajika, Makita; Komatsu, Setsuko

    2016-06-01

    Flooding negatively affects the growth of soybean, and several flooding-specific stress responses have been identified; however, the mechanisms underlying flooding tolerance in soybean remain unclear. To explore the initial flooding tolerance mechanisms in soybean, flooding-tolerant mutant and abscisic acid (ABA)-treated plants were analyzed. In the mutant and ABA-treated soybeans, 146 proteins were commonly changed at the initial flooding stress. Among the identified proteins, protein synthesis-related proteins, including nascent polypeptide-associated complex and chaperonin 20, and RNA regulation-related proteins were increased in abundance both at protein and mRNA expression. However, these proteins identified at the initial flooding stress were not significantly changed during survival stages under continuous flooding. Cluster analysis indicated that glycolysis- and cell wall-related proteins, such as enolase and polygalacturonase inhibiting protein, were increased in abundance during survival stages. Furthermore, lignification of root tissue was improved even under flooding stress. Taken together, these results suggest that protein synthesis- and RNA regulation-related proteins play a key role in triggering tolerance to the initial flooding stress in soybean. Furthermore, the integrity of cell wall and balance of glycolysis might be important factors for promoting tolerance of soybean root to flooding stress during survival stages. PMID:27132649

  20. Trophic spectra under the lens of amino acid isotopic analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recent advances in compound specific isotopic ratio analysis (CSIRA) have allowed researchers to measure trophic fractionation of 15N in specific amino acids, namely glutamic acid and phenylalanine. These amino acids have proven useful in food web studies because of the wide and consistent disparity...

  1. Lipidomic profiling of Saccharomyces cerevisiae and Zygosaccharomyces bailii reveals critical changes in lipid composition in response to acetic acid stress.

    PubMed

    Lindberg, Lina; Santos, Aline Xs; Riezman, Howard; Olsson, Lisbeth; Bettiga, Maurizio

    2013-01-01

    When using microorganisms as cell factories in the production of bio-based fuels or chemicals from lignocellulosic hydrolysate, inhibitory concentrations of acetic acid, released from the biomass, reduce the production rate. The undissociated form of acetic acid enters the cell by passive diffusion across the lipid bilayer, mediating toxic effects inside the cell. In order to elucidate a possible link between lipid composition and acetic acid stress, the present study presents detailed lipidomic profiling of the major lipid species found in the plasma membrane, including glycerophospholipids, sphingolipids and sterols, in Saccharomyces cerevisiae (CEN.PK 113_7D) and Zygosaccharomyces bailii (CBS7555) cultured with acetic acid. Detailed physiological characterization of the response of the two yeasts to acetic acid has also been performed in aerobic batch cultivations using bioreactors. Physiological characterization revealed, as expected, that Z. bailii is more tolerant to acetic acid than S. cerevisiae. Z. bailii grew at acetic acid concentrations above 24 g L(-1), while limited growth of S. cerevisiae was observed after 11 h when cultured with only 12 g L(-1) acetic acid. Detailed lipidomic profiling using electrospray ionization, multiple-reaction-monitoring mass spectrometry (ESI-MRM-MS) showed remarkable changes in the glycerophospholipid composition of Z. bailii, including an increase in saturated glycerophospholipids and considerable increases in complex sphingolipids in both S. cerevisiae (IPC 6.2×, MIPC 9.1×, M(IP)2C 2.2×) and Z. bailii (IPC 4.9×, MIPC 2.7×, M(IP)2C 2.7×), when cultured with acetic acid. In addition, the basal level of complex sphingolipids was significantly higher in Z. bailii than in S. cerevisiae, further emphasizing the proposed link between lipid saturation, high sphingolipid levels and acetic acid tolerance. The results also suggest that acetic acid tolerance is associated with the ability of a given strain to generate large

  2. Method comparison study for weak acid dissociation cyanide analysis.

    PubMed

    Evans, Joseph D; Thompson, Leslie; Clark, Patrick J; Beckman, Scott W

    2003-02-01

    Method comparison studies of two different methods for the analysis of weak acid dissociable (WAD) cyanide revealed analytical flaws and/or matrix interference problems with both procedures. EPA "draft" method 1677 using a Perstorp 3202 CN analyzer was compared to Standard Method 4500 CN I. It was discovered that the Perstorp analyzer produced more precise and more accurate results once appropriate and necessary procedural steps from the EPA draft method were modified. Comparison of these two methods, was based on "real world" samples collected from a mine-tailing solution. The mine-tailing solution contained high concentrations of cyanide and metals. Inconsistencies in method procedures were traced to sulfide interferences and high concentrations of WAD metals. Conclusions were based upon a large sample base collected from a mine site over a 90-day period. PMID:12630477

  3. Compound-Specific Isotope Analysis of Amino Acids for Stardust-Returned Samples

    NASA Technical Reports Server (NTRS)

    Cook, Jamie; Elsila, Jamie E.; Stern J. C.; Glavin, D. P.; Dworkin, J. P.

    2008-01-01

    Significant portions of the early Earth's prebiotic organic inventory , including amino acids, could have been delivered to the Earth's sur face by comets and their fragments. Analysis of comets via spectrosc opic observations has identified many organic molecules, including me thane, ethane, arnmonia, cyanic acid, formaldehyde, formamide, acetal ehyde, acetonitrile, and methanol. Reactions between these identifie d molecules could allow the formation of more complex organics such a s amino acids. Isotopic analysis could reveal whether an extraterrest rial signature is present in the Stardust-exposed amines and amino ac ids. Although bulk isotopic analysis would be dominated by the EACA contaminant's terrestrial signature, compoundspecific isotope analysi s (CSIA) could determine the signature of each of the other individua l amines. Here, we report on progress made towards CSIA of the amino acids glycine and EACA in Stardustreturned samples.

  4. Effects of Perfluorooctanoic Acid on Metabolic Profiles in Brain and Liver of Mouse Revealed by a High-throughput Targeted Metabolomics Approach

    NASA Astrophysics Data System (ADS)

    Yu, Nanyang; Wei, Si; Li, Meiying; Yang, Jingping; Li, Kan; Jin, Ling; Xie, Yuwei; Giesy, John P.; Zhang, Xiaowei; Yu, Hongxia

    2016-04-01

    Perfluorooctanoic acid (PFOA), a perfluoroalkyl acid, can result in hepatotoxicity and neurobehavioral effects in animals. The metabolome, which serves as a connection among transcriptome, proteome and toxic effects, provides pathway-based insights into effects of PFOA. Since understanding of changes in the metabolic profile during hepatotoxicity and neurotoxicity were still incomplete, a high-throughput targeted metabolomics approach (278 metabolites) was used to investigate effects of exposure to PFOA for 28 d on brain and liver of male Balb/c mice. Results of multivariate statistical analysis indicated that PFOA caused alterations in metabolic pathways in exposed individuals. Pathway analysis suggested that PFOA affected metabolism of amino acids, lipids, carbohydrates and energetics. Ten and 18 metabolites were identified as potential unique biomarkers of exposure to PFOA in brain and liver, respectively. In brain, PFOA affected concentrations of neurotransmitters, including serotonin, dopamine, norepinephrine, and glutamate in brain, which provides novel insights into mechanisms of PFOA-induced neurobehavioral effects. In liver, profiles of lipids revealed involvement of β-oxidation and biosynthesis of saturated and unsaturated fatty acids in PFOA-induced hepatotoxicity, while alterations in metabolism of arachidonic acid suggesting potential of PFOA to cause inflammation response in liver. These results provide insight into the mechanism and biomarkers for PFOA-induced effects.

  5. Effects of Perfluorooctanoic Acid on Metabolic Profiles in Brain and Liver of Mouse Revealed by a High-throughput Targeted Metabolomics Approach

    PubMed Central

    Yu, Nanyang; Wei, Si; Li, Meiying; Yang, Jingping; Li, Kan; Jin, Ling; Xie, Yuwei; Giesy, John P.; Zhang, Xiaowei; Yu, Hongxia

    2016-01-01

    Perfluorooctanoic acid (PFOA), a perfluoroalkyl acid, can result in hepatotoxicity and neurobehavioral effects in animals. The metabolome, which serves as a connection among transcriptome, proteome and toxic effects, provides pathway-based insights into effects of PFOA. Since understanding of changes in the metabolic profile during hepatotoxicity and neurotoxicity were still incomplete, a high-throughput targeted metabolomics approach (278 metabolites) was used to investigate effects of exposure to PFOA for 28 d on brain and liver of male Balb/c mice. Results of multivariate statistical analysis indicated that PFOA caused alterations in metabolic pathways in exposed individuals. Pathway analysis suggested that PFOA affected metabolism of amino acids, lipids, carbohydrates and energetics. Ten and 18 metabolites were identified as potential unique biomarkers of exposure to PFOA in brain and liver, respectively. In brain, PFOA affected concentrations of neurotransmitters, including serotonin, dopamine, norepinephrine, and glutamate in brain, which provides novel insights into mechanisms of PFOA-induced neurobehavioral effects. In liver, profiles of lipids revealed involvement of β-oxidation and biosynthesis of saturated and unsaturated fatty acids in PFOA-induced hepatotoxicity, while alterations in metabolism of arachidonic acid suggesting potential of PFOA to cause inflammation response in liver. These results provide insight into the mechanism and biomarkers for PFOA-induced effects. PMID:27032815

  6. Effects of Perfluorooctanoic Acid on Metabolic Profiles in Brain and Liver of Mouse Revealed by a High-throughput Targeted Metabolomics Approach.

    PubMed

    Yu, Nanyang; Wei, Si; Li, Meiying; Yang, Jingping; Li, Kan; Jin, Ling; Xie, Yuwei; Giesy, John P; Zhang, Xiaowei; Yu, Hongxia

    2016-01-01

    Perfluorooctanoic acid (PFOA), a perfluoroalkyl acid, can result in hepatotoxicity and neurobehavioral effects in animals. The metabolome, which serves as a connection among transcriptome, proteome and toxic effects, provides pathway-based insights into effects of PFOA. Since understanding of changes in the metabolic profile during hepatotoxicity and neurotoxicity were still incomplete, a high-throughput targeted metabolomics approach (278 metabolites) was used to investigate effects of exposure to PFOA for 28 d on brain and liver of male Balb/c mice. Results of multivariate statistical analysis indicated that PFOA caused alterations in metabolic pathways in exposed individuals. Pathway analysis suggested that PFOA affected metabolism of amino acids, lipids, carbohydrates and energetics. Ten and 18 metabolites were identified as potential unique biomarkers of exposure to PFOA in brain and liver, respectively. In brain, PFOA affected concentrations of neurotransmitters, including serotonin, dopamine, norepinephrine, and glutamate in brain, which provides novel insights into mechanisms of PFOA-induced neurobehavioral effects. In liver, profiles of lipids revealed involvement of β-oxidation and biosynthesis of saturated and unsaturated fatty acids in PFOA-induced hepatotoxicity, while alterations in metabolism of arachidonic acid suggesting potential of PFOA to cause inflammation response in liver. These results provide insight into the mechanism and biomarkers for PFOA-induced effects. PMID:27032815

  7. Molecular dynamic simulations reveal the structural determinants of fatty acid binding to oxy-myoglobin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The mechanism(s) by which fatty acids are sequestered and transported in muscle have not been fully elucidated. A potential key player in this process is the protein myoglobin (Mb). Indeed, there is a catalogue of empirical evidence supporting direct interaction of globins with fatty acid metabolite...

  8. Exploratory Metabolomics Profiling in the Kainic Acid Rat Model Reveals Depletion of 25-Hydroxyvitamin D3 during Epileptogenesis

    PubMed Central

    Heischmann, Svenja; Quinn, Kevin; Cruickshank-Quinn, Charmion; Liang, Li-Ping; Reisdorph, Rick; Reisdorph, Nichole; Patel, Manisha

    2016-01-01

    Currently, no reliable markers are available to evaluate the epileptogenic potential of a brain injury. The electroencephalogram is the standard method of diagnosis of epilepsy; however, it is not used to predict the risk of developing epilepsy. Biomarkers that indicate an individual’s risk to develop epilepsy, especially those measurable in the periphery are urgently needed. Temporal lobe epilepsy (TLE), the most common form of acquired epilepsy, is characterized by spontaneous recurrent seizures following brain injury and a seizure-free “latent” period. Elucidation of mechanisms at play during epilepsy development (epileptogenesis) in animal models of TLE could enable the identification of predictive biomarkers. Our pilot study using liquid chromatography-mass spectrometry metabolomics analysis revealed changes (p-value ≤ 0.05, ≥1.5-fold change) in lipid, purine, and sterol metabolism in rat plasma and hippocampus during epileptogenesis and chronic epilepsy in the kainic acid model of TLE. Notably, disease development was associated with dysregulation of vitamin D3 metabolism at all stages and plasma 25-hydroxyvitamin D3 depletion in the acute and latent phase of injury-induced epileptogenesis. These data suggest that plasma VD3 metabolites reflect the severity of an epileptogenic insult and that a panel of plasma VD3 metabolites may be able to serve as a marker of epileptogenesis. PMID:27526857

  9. Revealing Nucleic Acid Mutations Using Förster Resonance Energy Transfer-Based Probes.

    PubMed

    Junager, Nina P L; Kongsted, Jacob; Astakhova, Kira

    2016-01-01

    Nucleic acid mutations are of tremendous importance in modern clinical work, biotechnology and in fundamental studies of nucleic acids. Therefore, rapid, cost-effective and reliable detection of mutations is an object of extensive research. Today, Förster resonance energy transfer (FRET) probes are among the most often used tools for the detection of nucleic acids and in particular, for the detection of mutations. However, multiple parameters must be taken into account in order to create efficient FRET probes that are sensitive to nucleic acid mutations. In this review; we focus on the design principles for such probes and available computational methods that allow for their rational design. Applications of advanced, rationally designed FRET probes range from new insights into cellular heterogeneity to gaining new knowledge of nucleic acid structures directly in living cells. PMID:27472344

  10. RNA-Seq Analysis Reveals MAPKKK Family Members Related to Drought Tolerance in Maize

    PubMed Central

    Ren, Wen; Yang, Fengling; He, Hang; Zhao, Jiuran

    2015-01-01

    The mitogen-activated protein kinase (MAPK) cascade is an evolutionarily conserved signal transduction pathway that is involved in plant development and stress responses. As the first component of this phosphorelay cascade, mitogen-activated protein kinase kinase kinases (MAPKKKs) act as adaptors linking upstream signaling steps to the core MAPK cascade to promote the appropriate cellular responses; however, the functions of MAPKKKs in maize are unclear. Here, we identified 71 MAPKKK genes, of which 14 were novel, based on a computational analysis of the maize (Zea mays L.) genome. Using an RNA-seq analysis in the leaf, stem and root of maize under well-watered and drought-stress conditions, we identified 5,866 differentially expressed genes (DEGs), including 8 MAPKKK genes responsive to drought stress. Many of the DEGs were enriched in processes such as drought stress, abiotic stimulus, oxidation-reduction, and metabolic processes. The other way round, DEGs involved in processes such as oxidation, photosynthesis, and starch, proline, ethylene, and salicylic acid metabolism were clearly co-expressed with the MAPKKK genes. Furthermore, a quantitative real-time PCR (qRT-PCR) analysis was performed to assess the relative expression levels of MAPKKKs. Correlation analysis revealed that there was a significant correlation between expression levels of two MAPKKKs and relative biomass responsive to drought in 8 inbred lines. Our results indicate that MAPKKKs may have important regulatory functions in drought tolerance in maize. PMID:26599013

  11. RNA-Seq Analysis Reveals MAPKKK Family Members Related to Drought Tolerance in Maize.

    PubMed

    Liu, Ya; Zhou, Miaoyi; Gao, Zhaoxu; Ren, Wen; Yang, Fengling; He, Hang; Zhao, Jiuran

    2015-01-01

    The mitogen-activated protein kinase (MAPK) cascade is an evolutionarily conserved signal transduction pathway that is involved in plant development and stress responses. As the first component of this phosphorelay cascade, mitogen-activated protein kinase kinase kinases (MAPKKKs) act as adaptors linking upstream signaling steps to the core MAPK cascade to promote the appropriate cellular responses; however, the functions of MAPKKKs in maize are unclear. Here, we identified 71 MAPKKK genes, of which 14 were novel, based on a computational analysis of the maize (Zea mays L.) genome. Using an RNA-seq analysis in the leaf, stem and root of maize under well-watered and drought-stress conditions, we identified 5,866 differentially expressed genes (DEGs), including 8 MAPKKK genes responsive to drought stress. Many of the DEGs were enriched in processes such as drought stress, abiotic stimulus, oxidation-reduction, and metabolic processes. The other way round, DEGs involved in processes such as oxidation, photosynthesis, and starch, proline, ethylene, and salicylic acid metabolism were clearly co-expressed with the MAPKKK genes. Furthermore, a quantitative real-time PCR (qRT-PCR) analysis was performed to assess the relative expression levels of MAPKKKs. Correlation analysis revealed that there was a significant correlation between expression levels of two MAPKKKs and relative biomass responsive to drought in 8 inbred lines. Our results indicate that MAPKKKs may have important regulatory functions in drought tolerance in maize. PMID:26599013

  12. Pretreatment and integrated analysis of spectral data reveal seaweed similarities based on chemical diversity.

    PubMed

    Wei, Feifei; Ito, Kengo; Sakata, Kenji; Date, Yasuhiro; Kikuchi, Jun

    2015-03-01

    Extracting useful information from high dimensionality and large data sets is a major challenge for data-driven approaches. The present study was aimed at developing novel integrated analytical strategies for comprehensively characterizing seaweed similarities based on chemical diversity. The chemical compositions of 107 seaweed and 2 seagrass samples were analyzed using multiple techniques, including Fourier transform infrared (FT-IR) and solid- and solution-state nuclear magnetic resonance (NMR) spectroscopy, thermogravimetry-differential thermal analysis (TG-DTA), inductively coupled plasma-optical emission spectrometry (ICP-OES), CHNS/O total elemental analysis, and isotope ratio mass spectrometry (IR-MS). The spectral data were preprocessed using non-negative matrix factorization (NMF) and NMF combined with multivariate curve resolution-alternating least-squares (MCR-ALS) methods in order to separate individual component information from the overlapping and/or broad spectral peaks. Integrated analysis of the preprocessed chemical data demonstrated distinct discrimination of differential seaweed species. Further network analysis revealed a close correlation between the heavy metal elements and characteristic components of brown algae, such as cellulose, alginic acid, and sulfated mucopolysaccharides, providing a componential basis for its metal-sorbing potential. These results suggest that this integrated analytical strategy is useful for extracting and identifying the chemical characteristics of diverse seaweeds based on large chemical data sets, particularly complicated overlapping spectral data. PMID:25647718

  13. Metabolite profiling and network analysis reveal coordinated changes in grapevine water stress response

    PubMed Central

    2013-01-01

    Background Grapevine metabolism in response to water deficit was studied in two cultivars, Shiraz and Cabernet Sauvignon, which were shown to have different hydraulic behaviors (Hochberg et al. Physiol. Plant. 147:443–453, 2012). Results Progressive water deficit was found to effect changes in leaf water potentials accompanied by metabolic changes. In both cultivars, but more intensively in Shiraz than Cabernet Sauvignon, water deficit caused a shift to higher osmolality and lower C/N ratios, the latter of which was also reflected in marked increases in amino acids, e.g., Pro, Val, Leu, Thr and Trp, reductions of most organic acids, and changes in the phenylpropanoid pathway. PCA analysis showed that changes in primary metabolism were mostly associated with water stress, while diversification of specialized metabolism was mostly linked to the cultivars. In the phloem sap, drought was characterized by higher ABA concentration and major changes in benzoate levels coinciding with lower stomatal conductance and suberinization of vascular bundles. Enhanced suberin biosynthesis in Shiraz was reflected by the higher abundance of sap hydroxybenzoate derivatives. Correlation-based network analysis revealed that compared to Cabernet Sauvignon, Shiraz had considerably larger and highly coordinated stress-related changes, reflected in its increased metabolic network connectivity under stress. Network analysis also highlighted the structural role of major stress related metabolites, e.g., Pro, quercetin and ascorbate, which drastically altered their connectedness in the Shiraz network under water deficit. Conclusions Taken together, the results showed that Vitis vinifera cultivars possess a common metabolic response to water deficit. Central metabolism, and specifically N metabolism, plays a significant role in stress response in vine. At the cultivar level, Cabernet Sauvignon was characterized by milder metabolic perturbations, likely due to a tighter regulation of stomata

  14. Analysis of issues concerning acid rain

    SciTech Connect

    Bowsher, C.A.

    1984-12-11

    Although science has largely determined that man-made emissions cause acid rain, there is uncertainty concerning the extent and timing of its anticipated effects. Thus, at the present time scientific information alone does not lead unequivocally to a conclusion on whether it is appropriate to begin control actions now or to await better understanding. Given this uncertainty, decisionmakers must weigh the risks of further, potentially avoidable environmental damage against the risks of economic impacts from acid rain control actions which may ultimately prove to be unwarranted. GAO examines the implications of current scientific knowledge for policy decisions on acid rain and offers a series of observations on the following issues involved in the debate: To what extent has it been scientifically demonstrated that acid rain is resulting in damage to the environment. What are the causes of acid rain and where is it most prevalent. What alternatives exist for controlling acid rain and what are their economic effects. 5 figures, 20 tables.

  15. Trophic Hierarchies Illuminated via Amino Acid Isotopic Analysis

    PubMed Central

    Steffan, Shawn A.; Chikaraishi, Yoshito; Horton, David R.; Ohkouchi, Naohiko; Singleton, Merritt E.; Miliczky, Eugene; Hogg, David B.; Jones, Vincent P.

    2013-01-01

    Food web ecologists have long sought to characterize the trophic niches of animals using stable isotopic analysis. However, distilling trophic position from isotopic composition has been difficult, largely because of the variability associated with trophic discrimination factors (inter-trophic isotopic fractionation and routing). We circumvented much of this variability using compound-specific isotopic analysis (CSIA). We examined the 15N signatures of amino acids extracted from organisms reared in pure culture at four discrete trophic levels, across two model communities. We calculated the degree of enrichment at each trophic level and found there was a consistent trophic discrimination factor (~7.6‰). The constancy of the CSIA-derived discrimination factor permitted unprecedented accuracy in the measurement of animal trophic position. Conversely, trophic position estimates generated via bulk-15N analysis significantly underestimated trophic position, particularly among higher-order consumers. We then examined the trophic hierarchy of a free-roaming arthropod community, revealing the highest trophic position (5.07) and longest food chain ever reported using CSIA. High accuracy in trophic position estimation brings trophic function into sharper focus, providing greater resolution to the analysis of food webs. PMID:24086703

  16. [Evaluation on hepatotoxicity caused by Dioscorea bulbifera based on analysis of bile acids].

    PubMed

    Xu, Ying; Chen, Chong-Chong; Yang, Li; Wang, Jun-Ming; Ji, Li-Li; Wang, Zheng-Tao; Hu, Zhi-Bi

    2011-01-01

    Metabolic profile of bile acids was used to evaluate hepatotoxicity of mice caused by ethanol extraction of Dioscorea bulbifera L. (ethanol extraction, ET) and diosbulbin B (DB), separately. Ultra-performance liquid chromatography coupled with quadrupole mass spectrometry (UPLC-MS) was applied to determine the contents of all kinds of endogenous bile acids including free bile acids, taurine conjugates and glycine conjugates. Obvious liver injuries could be observed in mice after administrated with ET and DB. Based on the analysis using principle components analysis (PCA), toxic groups could be distinguished from their control groups, which suggested that the variance of the contents of bile acids could evaluate hepatotoxicity caused by ET and DB. Meanwhile, ET and DB toxic groups were classified in the same trends comparing to control groups in the loading plot, and difference between the two toxic groups could also be observed. DB proved to be one of the toxic components in Dioscorea bulbifera L. Bile acids of tauroursodeoxycholic acid (TUDCA), taurochenodeoxycholic acid (TCDCA), taurocholic acid (TCA), taurodeoxycholic acid (TDCA), cholic acid (CA) and others proved to be important corresponds to ET and DB induced liver injury according to analysis of partial least square-discriminant analysis (PLS-DA) and the statistical analysis showed that there were significant differences between the control groups and toxic groups (P < 0.01). Furthermore, good correlation could be revealed between the foregoing bile acids and ALT, AST. It indicated that taurine conjugated bile acids as TUDCA, TCDCA, TCA and TDCA along with CA could be considered as sensitive biomarkers of ET and DB induced liver injury. This work can provide the base for the further research on the evaluation and mechanism of hepatotoxicity caused by Dioscorea bulbifera L. PMID:21465807

  17. Effect of Pre-Stressing on the Acid-Stress Response in Bifidobacterium Revealed Using Proteomic and Physiological Approaches.

    PubMed

    Jin, Junhua; Qin, Qian; Guo, Huiyuan; Liu, Songling; Ge, Shaoyang; Zhang, Hongxing; Cui, Jianyun; Ren, Fazheng

    2015-01-01

    Weak acid resistance limits the application of Bifidobacteria as a probiotic in food. The acid tolerance response (ATR), caused by pre-stressing cells at a sublethal pH, could improve the acid resistance of Bifidobacteria to subsequent acid stress. In this study, we used Bifidobacterium longum sub. longum BBMN68 to investigate the effect of the ATR on the acid stress response (ASR), and compared the difference between the ATR and the ASR by analyzing the two-dimensional-PAGE protein profiles and performing physiological tests. The results revealed that a greater abundance of proteins involved in carbohydrate metabolism and protein protection was present after the ASR than after the ATR in Bifidobacterium. Pre-stressing cells increased the abundance of proteins involved in energy production, amino acid metabolism, and peptidoglycan synthesis during the ASR of Bifidobacterium. Moreover, after the ASR, the content of ATP, NH3, thiols, and peptidoglycan, the activity of H+-ATPase, and the maintenance of the intracellular pH in the pre-stressed Bifidobacterium cells was significantly higher than in the uninduced cells. These results provide the first explanation as to why the resistance of Bifidobacterium to acid stress improved after pre-stressing. PMID:25689631

  18. Plasticity of photoreceptor-generating retinal progenitors revealed by prolonged retinoic acid exposure

    PubMed Central

    2011-01-01

    Background Retinoic acid (RA) is important for vertebrate eye morphogenesis and is a regulator of photoreceptor development in the retina. In the zebrafish, RA treatment of postmitotic photoreceptor precursors has been shown to promote the differentiation of rods and red-sensitive cones while inhibiting the differentiation of blue- and UV-sensitive cones. The roles played by RA and its receptors in modifying photoreceptor fate remain to be determined. Results Treatment of zebrafish embryos with RA, beginning at the time of retinal progenitor cell proliferation and prior to photoreceptor terminal mitosis, resulted in a significant alteration of rod and cone mosaic patterns, suggesting an increase in the production of rods at the expense of red cones. Quantitative pattern analyses documented increased density of rod photoreceptors and reduced local spacing between rod cells, suggesting rods were appearing in locations normally occupied by cone photoreceptors. Cone densities were correspondingly reduced and cone photoreceptor mosaics displayed expanded and less regular spacing. These results were consistent with replacement of approximately 25% of positions normally occupied by red-sensitive cones, with additional rods. Analysis of embryos from a RA-signaling reporter line determined that multiple retinal cell types, including mitotic cells and differentiating rods and cones, are capable of directly responding to RA. The RA receptors RXRγ and RARαb are expressed in patterns consistent with mediating the effects of RA on photoreceptors. Selective knockdown of RARαb expression resulted in a reduction in endogenous RA signaling in the retina. Knockdown of RARαb also caused a reduced production of rods that was not restored by simultaneous treatments with RA. Conclusions These data suggest that developing retinal cells have a dynamic sensitivity to RA during retinal neurogenesis. In zebrafish RA may influence the rod vs. cone cell fate decision. The RARαb receptor

  19. Molecular annotation of ketol-acid reductoisomerases from Streptomyces reveals a novel amino acid biosynthesis interlock mediated by enzyme promiscuity.

    PubMed

    Verdel-Aranda, Karina; López-Cortina, Susana T; Hodgson, David A; Barona-Gómez, Francisco

    2015-03-01

    The 6-phosphogluconate dehydrogenase superfamily oxidize and reduce a wide range of substrates, making their functional annotation challenging. Ketol-acid reductoisomerase (KARI), encoded by the ilvC gene in branched-chain amino acids biosynthesis, is a promiscuous reductase enzyme within this superfamily. Here, we obtain steady-state enzyme kinetic parameters for 10 IlvC homologues from the genera Streptomyces and Corynebacterium, upon eight selected chemically diverse substrates, including some not normally recognized by enzymes of this superfamily. This biochemical data suggested a Streptomyces biosynthetic interlock between proline and the branched-chain amino acids, mediated by enzyme substrate promiscuity, which was confirmed via mutagenesis and complementation analyses of the proC, ilvC1 and ilvC2 genes in Streptomyces coelicolor. Moreover, both ilvC orthologues and paralogues were analysed, such that the relationship between gene duplication and functional diversification could be explored. The KARI paralogues present in S. coelicolor and Streptomyces lividans, despite their conserved high sequence identity (97%), were shown to be more promiscuous, suggesting a recent functional diversification. In contrast, the KARI paralogue from Streptomyces viridifaciens showed selectivity towards the synthesis of valine precursors, explaining its recruitment within the biosynthetic gene cluster of valanimycin. These results allowed us to assess substrate promiscuity indices as a tool to annotate new molecular functions with metabolic implications. PMID:25296650

  20. Molecular annotation of ketol-acid reductoisomerases from Streptomyces reveals a novel amino acid biosynthesis interlock mediated by enzyme promiscuity

    PubMed Central

    Verdel-Aranda, Karina; López-Cortina, Susana T; Hodgson, David A; Barona-Gómez, Francisco

    2015-01-01

    The 6-phosphogluconate dehydrogenase superfamily oxidize and reduce a wide range of substrates, making their functional annotation challenging. Ketol-acid reductoisomerase (KARI), encoded by the ilvC gene in branched-chain amino acids biosynthesis, is a promiscuous reductase enzyme within this superfamily. Here, we obtain steady-state enzyme kinetic parameters for 10 IlvC homologues from the genera Streptomyces and Corynebacterium, upon eight selected chemically diverse substrates, including some not normally recognized by enzymes of this superfamily. This biochemical data suggested a Streptomyces biosynthetic interlock between proline and the branched-chain amino acids, mediated by enzyme substrate promiscuity, which was confirmed via mutagenesis and complementation analyses of the proC, ilvC1 and ilvC2 genes in Streptomyces coelicolor. Moreover, both ilvC orthologues and paralogues were analysed, such that the relationship between gene duplication and functional diversification could be explored. The KARI paralogues present in S. coelicolor and Streptomyces lividans, despite their conserved high sequence identity (97%), were shown to be more promiscuous, suggesting a recent functional diversification. In contrast, the KARI paralogue from Streptomyces viridifaciens showed selectivity towards the synthesis of valine precursors, explaining its recruitment within the biosynthetic gene cluster of valanimycin. These results allowed us to assess substrate promiscuity indices as a tool to annotate new molecular functions with metabolic implications. PMID:25296650

  1. Metatranscriptome analysis reveals host-microbiome interactions in traps of carnivorous Genlisea species

    PubMed Central

    Cao, Hieu X.; Schmutzer, Thomas; Scholz, Uwe; Pecinka, Ales; Schubert, Ingo; Vu, Giang T. H.

    2015-01-01

    In the carnivorous plant genus Genlisea a unique lobster pot trapping mechanism supplements nutrition in nutrient-poor habitats. A wide spectrum of microbes frequently occurs in Genlisea's leaf-derived traps without clear relevance for Genlisea carnivory. We sequenced the metatranscriptomes of subterrestrial traps vs. the aerial chlorophyll-containing leaves of G. nigrocaulis and of G. hispidula. Ribosomal RNA assignment revealed soil-borne microbial diversity in Genlisea traps, with 92 genera of 19 phyla present in more than one sample. Microbes from 16 of these phyla including proteobacteria, green algae, amoebozoa, fungi, ciliates and metazoans, contributed additionally short-lived mRNA to the metatranscriptome. Furthermore, transcripts of 438 members of hydrolases (e.g., proteases, phosphatases, lipases), mainly resembling those of metazoans, ciliates and green algae, were found. Compared to aerial leaves, Genlisea traps displayed a transcriptional up-regulation of endogenous NADH oxidases generating reactive oxygen species as well as of acid phosphatases for prey digestion. A leaf-vs.-trap transcriptome comparison reflects that carnivory provides inorganic P- and different forms of N-compounds (ammonium, nitrate, amino acid, oligopeptides) and implies the need to protect trap cells against oxidative stress. The analysis elucidates a complex food web inside the Genlisea traps, and suggests ecological relationships between this plant genus and its entrapped microbiome. PMID:26236284

  2. Metatranscriptome analysis reveals host-microbiome interactions in traps of carnivorous Genlisea species.

    PubMed

    Cao, Hieu X; Schmutzer, Thomas; Scholz, Uwe; Pecinka, Ales; Schubert, Ingo; Vu, Giang T H

    2015-01-01

    In the carnivorous plant genus Genlisea a unique lobster pot trapping mechanism supplements nutrition in nutrient-poor habitats. A wide spectrum of microbes frequently occurs in Genlisea's leaf-derived traps without clear relevance for Genlisea carnivory. We sequenced the metatranscriptomes of subterrestrial traps vs. the aerial chlorophyll-containing leaves of G. nigrocaulis and of G. hispidula. Ribosomal RNA assignment revealed soil-borne microbial diversity in Genlisea traps, with 92 genera of 19 phyla present in more than one sample. Microbes from 16 of these phyla including proteobacteria, green algae, amoebozoa, fungi, ciliates and metazoans, contributed additionally short-lived mRNA to the metatranscriptome. Furthermore, transcripts of 438 members of hydrolases (e.g., proteases, phosphatases, lipases), mainly resembling those of metazoans, ciliates and green algae, were found. Compared to aerial leaves, Genlisea traps displayed a transcriptional up-regulation of endogenous NADH oxidases generating reactive oxygen species as well as of acid phosphatases for prey digestion. A leaf-vs.-trap transcriptome comparison reflects that carnivory provides inorganic P- and different forms of N-compounds (ammonium, nitrate, amino acid, oligopeptides) and implies the need to protect trap cells against oxidative stress. The analysis elucidates a complex food web inside the Genlisea traps, and suggests ecological relationships between this plant genus and its entrapped microbiome. PMID:26236284

  3. Salts of phenylacetic acid and 4-hydroxyphenylacetic acid with Cinchona alkaloids: Crystal structures, thermal analysis and FTIR spectroscopy

    NASA Astrophysics Data System (ADS)

    Amombo Noa, Francoise M.; Jacobs, Ayesha

    2016-06-01

    Seven salts were formed with phenylacetic acid (PAA), 4-hydroxyphenylacetic acid (HPAA) and the Cinchona alkaloids; cinchonidine (CIND), quinidine (QUID) and quinine (QUIN). For all the structures the proton was transferred from the carboxylic acid of the PAA/HPAA to the quinuclidine nitrogen of the respective Cinchona alkaloid. For six of the salts, water was included in the crystal structures with one of these also incorporating an isopropanol solvent molecule. However HPAA co-crystallised with quinine to form an anhydrous salt, (HPAA-)(QUIN+). The thermal stability of the salts were determined and differential scanning calorimetry revealed that the (HPAA-)(QUIN+) salt had the highest thermal stability compared to the other salt hydrates. The salts were also characterized using Fourier transform infrared spectroscopy. (PAA-)(QUID+)·H2O and (PAA-)(QUIN+)·H2O are isostructural and Hirshfeld surface analysis was completed to compare the intermolecular interactions in these two structures.

  4. Subfield profitability analysis reveals an economic case for cropland diversification

    NASA Astrophysics Data System (ADS)

    Brandes, E.; McNunn, G. S.; Schulte, L. A.; Bonner, I. J.; Muth, D. J.; Babcock, B. A.; Sharma, B.; Heaton, E. A.

    2016-01-01

    Public agencies and private enterprises increasingly desire to achieve ecosystem service outcomes in agricultural systems, but are limited by perceived conflicts between economic and ecosystem service goals and a lack of tools enabling effective operational management. Here we use Iowa—an agriculturally homogeneous state representative of the Maize Belt—to demonstrate an economic rationale for cropland diversification at the subfield scale. We used a novel computational framework that integrates disparate but publicly available data to map ˜3.3 million unique potential management polygons (9.3 Mha) and reveal subfield opportunities to increase overall field profitability. We analyzed subfield profitability for maize/soybean fields during 2010-2013—four of the most profitable years in recent history—and projected results for 2015. While cropland operating at a loss of US 250 ha-1 or more was negligible between 2010 and 2013 at 18 000-190 000 ha (<2% of row-crop land), the extent of highly unprofitable land increased to 2.5 Mha, or 27% of row-crop land, in the 2015 projection. Aggregation of these areas to the township level revealed ‘hotspots’ for potential management change in Western, Central, and Northeast Iowa. In these least profitable areas, incorporating conservation management that breaks even (e.g., planting low-input perennials), into low-yielding portions of fields could increase overall cropland profitability by 80%. This approach is applicable to the broader region and differs substantially from the status quo of ‘top-down’ land management for conservation by harnessing private interest to align profitability with the production of ecosystem services.

  5. Analysis of copy number variations reveals differences among cattle breeds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genomic structural variation is an important and abundant source of genetic and phenotypic variation. Here we describe the first systematic and genome-wide analysis of copy number variations (CNVs) in the modern domesticated cattle using array comparative genomic hybridization (array CGH) and quanti...

  6. An evolutionary analysis of flightin reveals a conserved motif unique and widespread in Pancrustacea.

    PubMed

    Soto-Adames, Felipe N; Alvarez-Ortiz, Pedro; Vigoreaux, Jim O

    2014-01-01

    Flightin is a thick filament protein that in Drosophila melanogaster is uniquely expressed in the asynchronous, indirect flight muscles (IFM). Flightin is required for the structure and function of the IFM and is indispensable for flight in Drosophila. Given the importance of flight acquisition in the evolutionary history of insects, here we study the phylogeny and distribution of flightin. Flightin was identified in 69 species of hexapods in classes Collembola (springtails), Protura, Diplura, and insect orders Thysanura (silverfish), Dictyoptera (roaches), Orthoptera (grasshoppers), Pthiraptera (lice), Hemiptera (true bugs), Coleoptera (beetles), Neuroptera (green lacewing), Hymenoptera (bees, ants, and wasps), Lepidoptera (moths), and Diptera (flies and mosquitoes). Flightin was also found in 14 species of crustaceans in orders Anostraca (water flea), Cladocera (brine shrimp), Isopoda (pill bugs), Amphipoda (scuds, sideswimmers), and Decapoda (lobsters, crabs, and shrimps). Flightin was not identified in representatives of chelicerates, myriapods, or any species outside Pancrustacea (Tetraconata, sensu Dohle). Alignment of amino acid sequences revealed a conserved region of 52 amino acids, referred herein as WYR, that is bound by strictly conserved tryptophan (W) and arginine (R) and an intervening sequence with a high content of tyrosines (Y). This motif has no homologs in GenBank or PROSITE and is unique to flightin and paraflightin, a putative flightin paralog identified in decapods. A third motif of unclear affinities to pancrustacean WYR was observed in chelicerates. Phylogenetic analysis of amino acid sequences of the conserved motif suggests that paraflightin originated before the divergence of amphipods, isopods, and decapods. We conclude that flightin originated de novo in the ancestor of Pancrustacea > 500 MYA, well before the divergence of insects (~400 MYA) and the origin of flight (~325 MYA), and that its IFM-specific function in Drosophila is a more

  7. Transcriptomic analysis of the oleaginous microalga Neochloris oleoabundans reveals metabolic insights into triacylglyceride accumulation

    PubMed Central

    2012-01-01

    Background The lack of sequenced genomes for oleaginous microalgae limits our understanding of the mechanisms these organisms utilize to become enriched in triglycerides. Here we report the de novo transcriptome assembly and quantitative gene expression analysis of the oleaginous microalga Neochloris oleoabundans, with a focus on the complex interaction of pathways associated with the production of the triacylglycerol (TAG) biofuel precursor. Results After growth under nitrogen replete and nitrogen limiting conditions, we quantified the cellular content of major biomolecules including total lipids, triacylglycerides, starch, protein, and chlorophyll. Transcribed genes were sequenced, the transcriptome was assembled de novo, and the expression of major functional categories, relevant pathways, and important genes was quantified through the mapping of reads to the transcriptome. Over 87 million, 77 base pair high quality reads were produced on the Illumina HiSeq sequencing platform. Metabolite measurements supported by genes and pathway expression results indicated that under the nitrogen-limiting condition, carbon is partitioned toward triglyceride production, which increased fivefold over the nitrogen-replete control. In addition to the observed overexpression of the fatty acid synthesis pathway, TAG production during nitrogen limitation was bolstered by repression of the β-oxidation pathway, up-regulation of genes encoding for the pyruvate dehydrogenase complex which funnels acetyl-CoA to lipid biosynthesis, activation of the pentose phosphate pathway to supply reducing equivalents to inorganic nitrogen assimilation and fatty acid biosynthesis, and the up-regulation of lipases—presumably to reconstruct cell membranes in order to supply additional fatty acids for TAG biosynthesis. Conclusions Our quantitative transcriptome study reveals a broad overview of how nitrogen stress results in excess TAG production in N. oleoabundans, and provides a variety of genetic

  8. Proteomics-Based Metabolic Modeling Reveals That Fatty Acid Oxidation (FAO) Controls Endothelial Cell (EC) Permeability*

    PubMed Central

    Patella, Francesca; Schug, Zachary T.; Persi, Erez; Neilson, Lisa J.; Erami, Zahra; Avanzato, Daniele; Maione, Federica; Hernandez-Fernaud, Juan R.; Mackay, Gillian; Zheng, Liang; Reid, Steven; Frezza, Christian; Giraudo, Enrico; Fiorio Pla, Alessandra; Anderson, Kurt; Ruppin, Eytan; Gottlieb, Eyal; Zanivan, Sara

    2015-01-01

    Endothelial cells (ECs) play a key role to maintain the functionality of blood vessels. Altered EC permeability causes severe impairment in vessel stability and is a hallmark of pathologies such as cancer and thrombosis. Integrating label-free quantitative proteomics data into genome-wide metabolic modeling, we built up a model that predicts the metabolic fluxes in ECs when cultured on a tridimensional matrix and organize into a vascular-like network. We discovered how fatty acid oxidation increases when ECs are assembled into a fully formed network that can be disrupted by inhibiting CPT1A, the fatty acid oxidation rate-limiting enzyme. Acute CPT1A inhibition reduces cellular ATP levels and oxygen consumption, which are restored by replenishing the tricarboxylic acid cycle. Remarkably, global phosphoproteomic changes measured upon acute CPT1A inhibition pinpointed altered calcium signaling. Indeed, CPT1A inhibition increases intracellular calcium oscillations. Finally, inhibiting CPT1A induces hyperpermeability in vitro and leakage of blood vessel in vivo, which were restored blocking calcium influx or replenishing the tricarboxylic acid cycle. Fatty acid oxidation emerges as central regulator of endothelial functions and blood vessel stability and druggable pathway to control pathological vascular permeability. PMID:25573745

  9. Proteomics-based metabolic modeling reveals that fatty acid oxidation (FAO) controls endothelial cell (EC) permeability.

    PubMed

    Patella, Francesca; Schug, Zachary T; Persi, Erez; Neilson, Lisa J; Erami, Zahra; Avanzato, Daniele; Maione, Federica; Hernandez-Fernaud, Juan R; Mackay, Gillian; Zheng, Liang; Reid, Steven; Frezza, Christian; Giraudo, Enrico; Fiorio Pla, Alessandra; Anderson, Kurt; Ruppin, Eytan; Gottlieb, Eyal; Zanivan, Sara

    2015-03-01

    Endothelial cells (ECs) play a key role to maintain the functionality of blood vessels. Altered EC permeability causes severe impairment in vessel stability and is a hallmark of pathologies such as cancer and thrombosis. Integrating label-free quantitative proteomics data into genome-wide metabolic modeling, we built up a model that predicts the metabolic fluxes in ECs when cultured on a tridimensional matrix and organize into a vascular-like network. We discovered how fatty acid oxidation increases when ECs are assembled into a fully formed network that can be disrupted by inhibiting CPT1A, the fatty acid oxidation rate-limiting enzyme. Acute CPT1A inhibition reduces cellular ATP levels and oxygen consumption, which are restored by replenishing the tricarboxylic acid cycle. Remarkably, global phosphoproteomic changes measured upon acute CPT1A inhibition pinpointed altered calcium signaling. Indeed, CPT1A inhibition increases intracellular calcium oscillations. Finally, inhibiting CPT1A induces hyperpermeability in vitro and leakage of blood vessel in vivo, which were restored blocking calcium influx or replenishing the tricarboxylic acid cycle. Fatty acid oxidation emerges as central regulator of endothelial functions and blood vessel stability and druggable pathway to control pathological vascular permeability. PMID:25573745

  10. Deciphering Carbamoylpolyoxamic Acid Biosynthesis Reveals Unusual Acetylation Cycle Associated with Tandem Reduction and Sequential Hydroxylation.

    PubMed

    Qi, Jianzhao; Wan, Dan; Ma, Hongmin; Liu, Yuanzhen; Gong, Rong; Qu, Xudong; Sun, Yuhui; Deng, Zixin; Chen, Wenqing

    2016-08-18

    Polyoxin, produced by Streptomcyes cacaoi var. asoensis and Streptomyces aureochromogenes, contains two non-proteinogenic amino acids, carbamoylpolyoxamic acid (CPOAA) and polyoximic acid. Although the CPOAA moiety is highly unusual, its biosynthetic logic has remained enigmatic for decades. Here, we address CPOAA biosynthesis by reconstitution of its pathway. We demonstrated that its biosynthesis is initiated by a versatile N-acetyltransferase, PolN, catalyzing L-glutamate (1) to N-acetyl glutamate (2). Remarkably, we verified that PolM, a previously annotated dehydrogenase, catalyzes an unprecedented tandem reduction of acyl-phosphate to aldehyde, and subsequently to alcohol. We also unveiled a distinctive acetylation cycle catalyzed by PolN to synthesize α-amino-δ-hydroxyvaleric acid (6). Finally, we report that PolL is capable of converting a rare sequential hydroxylation of α-amino-δ-carbamoylhydroxyvaleric acid (7) to CPOAA. PolL represents an intriguing family of Fe(II)-dependent α-ketoglutarate dioxygenase with a cupin fold. These data illustrate several novel enzymatic reactions, and also set a foundation for rational pathway engineering for polyoxin production. PMID:27541195

  11. Extraterrestrial material analysis: loss of amino acids during liquid-phase acid hydrolysis

    NASA Astrophysics Data System (ADS)

    Buch, Arnaud; Brault, Amaury; Szopa, Cyril; Freissinet, Caroline

    2015-04-01

    Searching for building blocks of life in extraterrestrial material is a way to learn more about how life could have appeared on Earth. With this aim, liquid-phase acid hydrolysis has been used, since at least 1970 , in order to extract amino acids and other organic molecules from extraterrestrial materials (e.g. meteorites, lunar fines) or Earth analogues (e.g. Atacama desert soil). This procedure involves drastic conditions such as heating samples in 6N HCl for 24 h, either under inert atmosphere/vacuum, or air. Analysis of the hydrolyzed part of the sample should give its total (free plus bound) amino acid content. The present work deals with the influence of the 6N HCl hydrolysis on amino acid degradation. Our experiments have been performed on a standard solution of 17 amino acids. After liquid-phase acid hydrolysis (6N HCl) under argon atmosphere (24 h at 100°C), the liquid phase was evaporated and the dry residue was derivatized with N-Methyl-N-(t-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) and dimethylformamide (DMF), followed by gas chromatography-mass spectrometry analysis. After comparison with derivatized amino acids from the standard solution, a significant reduction of the chromatographic peak areas was observed for most of the amino acids after liquid-phase acid hydrolysis. Furthermore, the same loss pattern was observed when the amino acids were exposed to cold 6N HCl for a short amount of time. The least affected amino acid, i.e. glycine, was found to be 73,93% percent less abundant compared to the non-hydrolyzed standard, while the most affected, i.e. histidine, was not found in the chromatograms after hydrolysis. Our experiments thereby indicate that liquid-phase acid hydrolysis, even under inert atmosphere, leads to a partial or total loss of all of the 17 amino acids present in the standard solution, and that a quick cold contact with 6N HCl is sufficient to lead to a loss of amino acids. Therefore, in the literature, the reported increase

  12. Hyperdimensional analysis of amino acid pair distributions in proteins.

    PubMed

    Henriksen, Svend B; Mortensen, Rasmus J; Geertz-Hansen, Henrik M; Neves-Petersen, Maria Teresa; Arnason, Omar; Söring, Jón; Petersen, Steffen B

    2011-01-01

    Our manuscript presents a novel approach to protein structure analyses. We have organized an 8-dimensional data cube with protein 3D-structural information from 8706 high-resolution non-redundant protein-chains with the aim of identifying packing rules at the amino acid pair level. The cube contains information about amino acid type, solvent accessibility, spatial and sequence distance, secondary structure and sequence length. We are able to pose structural queries to the data cube using program ProPack. The response is a 1, 2 or 3D graph. Whereas the response is of a statistical nature, the user can obtain an instant list of all PDB-structures where such pair is found. The user may select a particular structure, which is displayed highlighting the pair in question. The user may pose millions of different queries and for each one he will receive the answer in a few seconds. In order to demonstrate the capabilities of the data cube as well as the programs, we have selected well known structural features, disulphide bridges and salt bridges, where we illustrate how the queries are posed, and how answers are given. Motifs involving cysteines such as disulphide bridges, zinc-fingers and iron-sulfur clusters are clearly identified and differentiated. ProPack also reveals that whereas pairs of Lys residues virtually never appear in close spatial proximity, pairs of Arg are abundant and appear at close spatial distance, contrasting the belief that electrostatic repulsion would prevent this juxtaposition and that Arg-Lys is perceived as a conservative mutation. The presented programs can find and visualize novel packing preferences in proteins structures allowing the user to unravel correlations between pairs of amino acids. The new tools allow the user to view statistical information and visualize instantly the structures that underpin the statistical information, which is far from trivial with most other SW tools for protein structure analysis. PMID:22174733

  13. Fractal analysis reveals reduced complexity of retinal vessels in CADASIL.

    PubMed

    Cavallari, Michele; Falco, Teresa; Frontali, Marina; Romano, Silvia; Bagnato, Francesca; Orzi, Francesco

    2011-01-01

    The Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) affects mainly small cerebral arteries and leads to disability and dementia. The relationship between clinical expression of the disease and progression of the microvessel pathology is, however, uncertain as we lack tools for imaging brain vessels in vivo. Ophthalmoscopy is regarded as a window into the cerebral microcirculation. In this study we carried out an ophthalmoscopic examination in subjects with CADASIL. Specifically, we performed fractal analysis of digital retinal photographs. Data are expressed as mean fractal dimension (mean-D), a parameter that reflects complexity of the retinal vessel branching. Ten subjects with genetically confirmed diagnosis of CADASIL and 10 sex and age-matched control subjects were enrolled. Fractal analysis of retinal digital images was performed by means of a computer-based program, and the data expressed as mean-D. Brain MRI lesion volume in FLAIR and T1-weighted images was assessed using MIPAV software. Paired t-test was used to disclose differences in mean-D between CADASIL and control groups. Spearman rank analysis was performed to evaluate potential associations between mean-D values and both disease duration and disease severity, the latter expressed as brain MRI lesion volumes, in the subjects with CADASIL. The results showed that mean-D value of patients (1.42±0.05; mean±SD) was lower than control (1.50±0.04; p = 0.002). Mean-D did not correlate with disease duration nor with MRI lesion volumes of the subjects with CADASIL. The findings suggest that fractal analysis is a sensitive tool to assess changes of retinal vessel branching, likely reflecting early brain microvessel alterations, in CADASIL patients. PMID:21556373

  14. Amino acid racemization reveals differential protein turnover in osteoarthritic articular and meniscal cartilages

    PubMed Central

    Stabler, Thomas V; Byers, Samuel S; Zura, Robert D; Kraus, Virginia Byers

    2009-01-01

    Introduction Certain amino acids within proteins have been reported to change from the L form to the D form over time. This process is known as racemization and is most likely to occur in long-lived low-turnover tissues such as normal cartilage. We hypothesized that diseased tissue, as found in an osteoarthritic (OA) joint, would have increased turnover reflected by a decrease in the racemized amino acid content. Methods Using high-performance liquid chromatography methods, we quantified the L and D forms of amino acids reported to racemize in vivo on a biological timescale: alanine, aspartate (Asp), asparagine (Asn), glutamate, glutamine, isoleucine, leucine (Leu), and serine (Ser). Furthermore, using a metabolically inactive control material (tooth dentin) and a control material with normal metabolism (normal articular cartilage), we developed an age adjustment in order to make inferences about the state of protein turnover in cartilage and meniscus. Results In the metabolically inactive control material (n = 25, ages 13 to 80 years) and the normal metabolizing control material (n = 19, ages 17 to 83 years), only Asp + Asn (Asx), Ser, and Leu showed a significant change (increase) in racemization with age (P < 0.01). The age-adjusted proportions of racemized to total amino acid (D/D+L expressed as a percentage of the control material) for Asx, Ser, and Leu when compared with the normal articular cartilage control were 97%, 74%, and 73% in OA meniscal cartilage and 97%, 70%, and 78% in OA articular cartilage. We also observed lower amino acid content in OA articular and meniscal cartilages compared with normal articular cartilage as well as a loss of total amino acids with age in the OA meniscal but not the OA articular cartilage. Conclusions These data demonstrate comparable anabolic responses for non-lesioned OA articular cartilage and OA meniscal cartilage but an excess of catabolism over anabolism for the meniscal cartilage. PMID:19267899

  15. Integration of computational modeling with membrane transport studies reveals new insights into amino acid exchange transport mechanisms

    PubMed Central

    Widdows, Kate L.; Panitchob, Nuttanont; Crocker, Ian P.; Please, Colin P.; Hanson, Mark A.; Sibley, Colin P.; Johnstone, Edward D.; Sengers, Bram G.; Lewis, Rohan M.; Glazier, Jocelyn D.

    2015-01-01

    Uptake of system L amino acid substrates into isolated placental plasma membrane vesicles in the absence of opposing side amino acid (zero-trans uptake) is incompatible with the concept of obligatory exchange, where influx of amino acid is coupled to efflux. We therefore hypothesized that system L amino acid exchange transporters are not fully obligatory and/or that amino acids are initially present inside the vesicles. To address this, we combined computational modeling with vesicle transport assays and transporter localization studies to investigate the mechanisms mediating [14C]l-serine (a system L substrate) transport into human placental microvillous plasma membrane (MVM) vesicles. The carrier model provided a quantitative framework to test the 2 hypotheses that l-serine transport occurs by either obligate exchange or nonobligate exchange coupled with facilitated transport (mixed transport model). The computational model could only account for experimental [14C]l-serine uptake data when the transporter was not exclusively in exchange mode, best described by the mixed transport model. MVM vesicle isolates contained endogenous amino acids allowing for potential contribution to zero-trans uptake. Both L-type amino acid transporter (LAT)1 and LAT2 subtypes of system L were distributed to MVM, with l-serine transport attributed to LAT2. These findings suggest that exchange transporters do not function exclusively as obligate exchangers.—Widdows, K. L., Panitchob, N., Crocker, I. P., Please, C. P., Hanson, M. A., Sibley, C. P., Johnstone, E. D., Sengers, B. G., Lewis, R. M., Glazier, J. D. Integration of computational modeling with membrane transport studies reveals new insights into amino acid exchange transport mechanisms. PMID:25761365

  16. Efficient analysis of mouse genome sequences reveal many nonsense variants.

    PubMed

    Steeland, Sophie; Timmermans, Steven; Van Ryckeghem, Sara; Hulpiau, Paco; Saeys, Yvan; Van Montagu, Marc; Vandenbroucke, Roosmarijn E; Libert, Claude

    2016-05-17

    Genetic polymorphisms in coding genes play an important role when using mouse inbred strains as research models. They have been shown to influence research results, explain phenotypical differences between inbred strains, and increase the amount of interesting gene variants present in the many available inbred lines. SPRET/Ei is an inbred strain derived from Mus spretus that has ∼1% sequence difference with the C57BL/6J reference genome. We obtained a listing of all SNPs and insertions/deletions (indels) present in SPRET/Ei from the Mouse Genomes Project (Wellcome Trust Sanger Institute) and processed these data to obtain an overview of all transcripts having nonsynonymous coding sequence variants. We identified 8,883 unique variants affecting 10,096 different transcripts from 6,328 protein-coding genes, which is about 28% of all coding genes. Because only a subset of these variants results in drastic changes in proteins, we focused on variations that are nonsense mutations that ultimately resulted in a gain of a stop codon. These genes were identified by in silico changing the C57BL/6J coding sequences to the SPRET/Ei sequences, converting them to amino acid (AA) sequences, and comparing the AA sequences. All variants and transcripts affected were also stored in a database, which can be browsed using a SPRET/Ei M. spretus variants web tool (www.spretus.org), including a manual. We validated the tool by demonstrating the loss of function of three proteins predicted to be severely truncated, namely Fas, IRAK2, and IFNγR1. PMID:27147605

  17. Multiple etiologies for Alzheimer disease are revealed by segregation analysis

    SciTech Connect

    Rao, V.S.; Connor-Lacke, L.; Cupplies, L.A.; Growdon, J.H.; Farrer, L.A.; Duijn, C.M. van

    1994-11-01

    We have evaluated several transmission models for Alzheimer disease (AD), using the logistic regressive approach in 401 nuclear families of consecutively ascertained and rigorously diagnosed probands. Models postulating no major gene effect, random environmental transmission, recessive inheritance, and sporadic occurrence were rejected under varied assumptions regarding the associations among sex, age, and major gene susceptibility. Transmission of the disorder was not fully explained by a single Mendelian model for all families. Stratification of families as early- and late-onset by using the median of family mean onset ages showed that, regardless of the model studied, two groups of families fit better than a single group. AD in early-onset families is transmitted as an autosomal dominant trait with full penetrance in both sexes and has a gene frequency of 1.5%. Dominant inheritance also gave the best fit of the data in late-onset families, but this hypothesis was rejected, suggesting the presence of heterogeneity within this subset. Our study also revealed that genetically nonsusceptible males and females develop AD, indicating the presence of phenocopies within early-onset and late-onset groups. Moreover, our results suggest that the higher risk to females is not solely due to their increased longevity. 50 refs., 5 tabs.

  18. Proteomics Analysis Reveals Previously Uncharacterized Virulence Factors in Vibrio proteolyticus

    PubMed Central

    Ray, Ann; Kinch, Lisa N.; de Souza Santos, Marcela; Grishin, Nick V.

    2016-01-01

    ABSTRACT Members of the genus Vibrio include many pathogens of humans and marine animals that share genetic information via horizontal gene transfer. Hence, the Vibrio pan-genome carries the potential to establish new pathogenic strains by sharing virulence determinants, many of which have yet to be characterized. Here, we investigated the virulence properties of Vibrio proteolyticus, a Gram-negative marine bacterium previously identified as part of the Vibrio consortium isolated from diseased corals. We found that V. proteolyticus causes actin cytoskeleton rearrangements followed by cell lysis in HeLa cells in a contact-independent manner. In search of the responsible virulence factor involved, we determined the V. proteolyticus secretome. This proteomics approach revealed various putative virulence factors, including active type VI secretion systems and effectors with virulence toxin domains; however, these type VI secretion systems were not responsible for the observed cytotoxic effects. Further examination of the V. proteolyticus secretome led us to hypothesize and subsequently demonstrate that a secreted hemolysin, belonging to a previously uncharacterized clan of the leukocidin superfamily, was the toxin responsible for the V. proteolyticus-mediated cytotoxicity in both HeLa cells and macrophages. Clearly, there remains an armory of yet-to-be-discovered virulence factors in the Vibrio pan-genome that will undoubtedly provide a wealth of knowledge on how a pathogen can manipulate host cells. PMID:27460800

  19. Proteomics Analysis Reveals Novel RASSF2 Interaction Partners.

    PubMed

    Barnoud, Thibaut; Wilkey, Daniel W; Merchant, Michael L; Clark, Jennifer A; Donninger, Howard

    2016-01-01

    RASSF2 is a tumor suppressor that shares homology with other Ras-association domain (RASSF) family members. It is a powerful pro-apoptotic K-Ras effector that is frequently inactivated in many human tumors. The exact mechanism by which RASSF2 functions is not clearly defined, but it likely acts as a scaffolding protein, modulating the activity of other pro-apoptotic effectors, thereby regulating and integrating tumor suppressor pathways. However, only a limited number of RASSF2 interacting partners have been identified to date. We used a proteomics based approach to identify additional RASSF2 interactions, and thereby gain a better insight into the mechanism of action of RASSF2. We identified several proteins, including C1QBP, Vimentin, Protein phosphatase 1G and Ribonuclease inhibitor that function in diverse biological processes, including protein post-translational modifications, epithelial-mesenchymal transition, cell migration and redox homeostasis, which have not previously been reported to interact with RASSF2. We independently validated two of these novel interactions, C1QBP and Vimentin and found that the interaction with C1QBP was enhanced by K-Ras whereas, interestingly, the Vimentin interaction was reduced by K-Ras. Additionally, RASSF2/K-Ras regulated the acetylation of Vimentin. Our data thus reveal novel mechanisms by which RASSF2 may exert its functions, several of which may be Ras-regulated. PMID:26999212

  20. Tremor patches in Cascadia revealed by seismic array analysis

    NASA Astrophysics Data System (ADS)

    Ghosh, Abhijit; Vidale, John E.; Sweet, Justin R.; Creager, Kenneth C.; Wech, Aaron G.

    2009-09-01

    Episodic tremor and slip (ETS) events in Cascadia have recently been observed, illuminating the general area that radiates seismic energy in the form of non-volcanic tremor (NVT). However, the picture of the ETS zone remains fuzzy because of difficulties in tremor detection and location. To observe the intimate details of tremor, we deployed a dense 84-element small-aperture seismic array on the Olympic Peninsula, Washington, above the tremor migration path. It recorded the main ETS event in May 2008, as well as a weaker tremor episode two months earlier. Using a beamforming technique, we are able to capture and track tremor activity with an unprecedented resolution from southern Puget Sound to the Strait of Juan de Fuca. The array technique reveals up to four times more duration of tremor compared to the conventional envelope cross-correlation method. Our findings suggest that NVT is not uniformly distributed on the subduction interface, and unveils several distinct patches that release much of the tremor moment. The patches appear to be devoid of ordinary earthquakes, and may indicate the heterogeneity in fault strength that affects the modes of stress release within the ETS zone.

  1. Proteomics Analysis Reveals Novel RASSF2 Interaction Partners

    PubMed Central

    Barnoud, Thibaut; Wilkey, Daniel W.; Merchant, Michael L.; Clark, Jennifer A.; Donninger, Howard

    2016-01-01

    RASSF2 is a tumor suppressor that shares homology with other Ras-association domain (RASSF) family members. It is a powerful pro-apoptotic K-Ras effector that is frequently inactivated in many human tumors. The exact mechanism by which RASSF2 functions is not clearly defined, but it likely acts as a scaffolding protein, modulating the activity of other pro-apoptotic effectors, thereby regulating and integrating tumor suppressor pathways. However, only a limited number of RASSF2 interacting partners have been identified to date. We used a proteomics based approach to identify additional RASSF2 interactions, and thereby gain a better insight into the mechanism of action of RASSF2. We identified several proteins, including C1QBP, Vimentin, Protein phosphatase 1G and Ribonuclease inhibitor that function in diverse biological processes, including protein post-translational modifications, epithelial-mesenchymal transition, cell migration and redox homeostasis, which have not previously been reported to interact with RASSF2. We independently validated two of these novel interactions, C1QBP and Vimentin and found that the interaction with C1QBP was enhanced by K-Ras whereas, interestingly, the Vimentin interaction was reduced by K-Ras. Additionally, RASSF2/K-Ras regulated the acetylation of Vimentin. Our data thus reveal novel mechanisms by which RASSF2 may exert its functions, several of which may be Ras-regulated. PMID:26999212

  2. Fatty Acid Structure and Degradation Analysis in Fingerprint Residues.

    PubMed

    Pleik, Stefanie; Spengler, Bernhard; Schäfer, Thomas; Urbach, Dieter; Luhn, Steven; Kirsch, Dieter

    2016-09-01

    GC-MS investigations were carried out to elucidate the aging behavior of unsaturated fatty acids in fingerprint residues and to identify their degradation products in aged samples. For this purpose, a new sample preparation technique for fingerprint residues was developed that allows producing N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) derivatives of the analyzed unsaturated fatty acids and their degradation products. MSTFA derivatization catalyzed by iodotrimethylsilane enables the reliable identification of aldehydes and oxoacids as characteristic MSTFA derivatives in GCMS. The obtained results elucidate the degradation pathway of unsaturated fatty acids. Our study of aged fingerprint residues reveals that decanal is the main degradation product of the observed unsaturated fatty acids. Furthermore, oxoacids with different chain lengths are detected as specific degradation products of the unsaturated fatty acids. The detection of the degradation products and their chain length is a simple and effective method to determine the double bond position in unsaturated compounds. We can show that the hexadecenoic and octadecenoic acids found in fingerprint residues are not the pervasive fatty acids Δ9-hexadecenoic (palmitoleic acid) and Δ9-octadecenoic (oleic acid) acid but Δ6-hexadecenoic acid (sapienic acid) and Δ8-octadecenoic acid. The present study focuses on the structure identification of human sebum-specific unsaturated fatty acids in fingerprint residues based on the identification of their degradation products. These results are discussed for further investigations and method developments for age determination of fingerprints, which is still a tremendous challenge because of several factors affecting the aging behavior of individual compounds in fingerprints. Graphical Abstract ᅟ. PMID:27324649

  3. Fatty Acid Structure and Degradation Analysis in Fingerprint Residues

    NASA Astrophysics Data System (ADS)

    Pleik, Stefanie; Spengler, Bernhard; Schäfer, Thomas; Urbach, Dieter; Luhn, Steven; Kirsch, Dieter

    2016-09-01

    GC-MS investigations were carried out to elucidate the aging behavior of unsaturated fatty acids in fingerprint residues and to identify their degradation products in aged samples. For this purpose, a new sample preparation technique for fingerprint residues was developed that allows producing N-methyl- N-trimethylsilyl-trifluoroacetamide (MSTFA) derivatives of the analyzed unsaturated fatty acids and their degradation products. MSTFA derivatization catalyzed by iodotrimethylsilane enables the reliable identification of aldehydes and oxoacids as characteristic MSTFA derivatives in GCMS. The obtained results elucidate the degradation pathway of unsaturated fatty acids. Our study of aged fingerprint residues reveals that decanal is the main degradation product of the observed unsaturated fatty acids. Furthermore, oxoacids with different chain lengths are detected as specific degradation products of the unsaturated fatty acids. The detection of the degradation products and their chain length is a simple and effective method to determine the double bond position in unsaturated compounds. We can show that the hexadecenoic and octadecenoic acids found in fingerprint residues are not the pervasive fatty acids Δ9-hexadecenoic (palmitoleic acid) and Δ9-octadecenoic (oleic acid) acid but Δ6-hexadecenoic acid (sapienic acid) and Δ8-octadecenoic acid. The present study focuses on the structure identification of human sebum-specific unsaturated fatty acids in fingerprint residues based on the identification of their degradation products. These results are discussed for further investigations and method developments for age determination of fingerprints, which is still a tremendous challenge because of several factors affecting the aging behavior of individual compounds in fingerprints.

  4. Molecular analysis of Baylisascaris columnaris revealed mitochondrial and nuclear polymorphisms

    PubMed Central

    2013-01-01

    Background Baylisascaris species are intestinal nematodes of skunks, raccoons, badgers, and bears belonging to the genus Ascarididae. Oral uptake of embryonated Baylisascaris sp. eggs by a wide variety of mammals and birds can lead to visceral, ocular and neurological larva migrans. B. procyonis, the raccoon roundworm, is known to cause severe illness in intermediate hosts and in humans, whereas the skunk roundworm B. columnaris is probably less pathogenic. Skunks and raccoons are kept as pets in Europe, sometimes together with cats and dogs, living in close contact with humans. B. procyonis and B. columnaris are difficult to differentiate based on morphological criteria and molecular and phylogenetic information concerning B. columnaris is missing. This is the first study on the genetic characterisation of B. columnaris, based on mitochondrial and nuclear molecular markers. Methods B. columnaris worms were isolated from pet skunks, and used for molecular analysis. PCR primers targeted at mitochondrial cytochrome c oxidase 1 and 2 (CO1 and CO2), ribosomal ITS1-5.8S-ITS2 and ribosomal 28S genes were used. DNA sequences from B. columnaris, B. procyonis and B. transfuga from bears were analysed by cluster analysis. Results Four different multi-locus genotypes were found in B. columnaris, based on 14 single nucleotide polymorphisms (SNPs) and two insertions / deletions in CO1, CO2, ITS1-5.8S-ITS2 and 28S. Conclusions The genetic characteristics of B. columnaris show close resemblance to those of B. procyonis, but in contrast to B. procyonis, show several polymorphisms in both mitochondrial and nuclear markers. These polymorphisms could be used as a tool to differentiate B. columnaris from B. procyonis in molecular diagnostic assays, and to identify B. columnaris by PCR, in addition to or replacing morphometric analysis. This might lead to more insight into the zoonotic relevance of B. columnaris in humans. PMID:23627901

  5. Mutational Studies on Resurrected Ancestral Proteins Reveal Conservation of Site-Specific Amino Acid Preferences throughout Evolutionary History

    PubMed Central

    Risso, Valeria A.; Manssour-Triedo, Fadia; Delgado-Delgado, Asunción; Arco, Rocio; Barroso-delJesus, Alicia; Ingles-Prieto, Alvaro; Godoy-Ruiz, Raquel; Gavira, Jose A.; Gaucher, Eric A.; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M.

    2015-01-01

    Local protein interactions (“molecular context” effects) dictate amino acid replacements and can be described in terms of site-specific, energetic preferences for any different amino acid. It has been recently debated whether these preferences remain approximately constant during evolution or whether, due to coevolution of sites, they change strongly. Such research highlights an unresolved and fundamental issue with far-reaching implications for phylogenetic analysis and molecular evolution modeling. Here, we take advantage of the recent availability of phenotypically supported laboratory resurrections of Precambrian thioredoxins and β-lactamases to experimentally address the change of site-specific amino acid preferences over long geological timescales. Extensive mutational analyses support the notion that evolutionary adjustment to a new amino acid may occur, but to a large extent this is insufficient to erase the primitive preference for amino acid replacements. Generally, site-specific amino acid preferences appear to remain conserved throughout evolutionary history despite local sequence divergence. We show such preference conservation to be readily understandable in molecular terms and we provide crystallographic evidence for an intriguing structural-switch mechanism: Energetic preference for an ancestral amino acid in a modern protein can be linked to reorganization upon mutation to the ancestral local structure around the mutated site. Finally, we point out that site-specific preference conservation naturally leads to one plausible evolutionary explanation for the existence of intragenic global suppressor mutations. PMID:25392342

  6. Mutational studies on resurrected ancestral proteins reveal conservation of site-specific amino acid preferences throughout evolutionary history.

    PubMed

    Risso, Valeria A; Manssour-Triedo, Fadia; Delgado-Delgado, Asunción; Arco, Rocio; Barroso-delJesus, Alicia; Ingles-Prieto, Alvaro; Godoy-Ruiz, Raquel; Gavira, Jose A; Gaucher, Eric A; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M

    2015-02-01

    Local protein interactions ("molecular context" effects) dictate amino acid replacements and can be described in terms of site-specific, energetic preferences for any different amino acid. It has been recently debated whether these preferences remain approximately constant during evolution or whether, due to coevolution of sites, they change strongly. Such research highlights an unresolved and fundamental issue with far-reaching implications for phylogenetic analysis and molecular evolution modeling. Here, we take advantage of the recent availability of phenotypically supported laboratory resurrections of Precambrian thioredoxins and β-lactamases to experimentally address the change of site-specific amino acid preferences over long geological timescales. Extensive mutational analyses support the notion that evolutionary adjustment to a new amino acid may occur, but to a large extent this is insufficient to erase the primitive preference for amino acid replacements. Generally, site-specific amino acid preferences appear to remain conserved throughout evolutionary history despite local sequence divergence. We show such preference conservation to be readily understandable in molecular terms and we provide crystallographic evidence for an intriguing structural-switch mechanism: Energetic preference for an ancestral amino acid in a modern protein can be linked to reorganization upon mutation to the ancestral local structure around the mutated site. Finally, we point out that site-specific preference conservation naturally leads to one plausible evolutionary explanation for the existence of intragenic global suppressor mutations. PMID:25392342

  7. C-1s NEXAFS spectroscopy reveals chemical fractionation of humic acid by cation-induced coagulation

    SciTech Connect

    Christl,I.; Kretzschmar, R.

    2007-01-01

    The influence of cation-induced coagulation on the chemical composition of dissolved and coagulated fractions of humic acid was investigated in batch coagulation experiments for additions of aluminum at pH 4 and 5, iron at pH 4, and calcium and lead at pH 6. The partitioning of organic carbon and metals was determined by analyzing total organic carbon and total metal contents of the dissolved phase. Both the dissolved and the coagulated humic acid fractions were characterized using synchrotron scanning transmission X-ray microscopy (STXM) and C-1s near-edge X-ray absorption fine structure (NEXAFS) spectroscopy. Intensities of {pi}* transitions of carboxyl carbon and {sigma}* transitions of alkyl, O-alkyl, and carboxyl carbon decreased with increasing metal concentration for the dissolved humic acid fractions. This decrease was accompanied by an increase of the respective intensities in the coagulated fraction as shown for lead. Intensities of aromatic and phenolic carbon were affected to a larger extent only by aluminum and iron additions. The changes observed in the C-1s NEXAFS spectra coincided with an increasing removal of organic carbon from the dissolved phase with increasing total metal concentrations. We conclude that humic acid was chemically fractionated by cation-induced coagulation, which preferentially removed functional groups involved in metal-cation binding from solution.

  8. Genome-Based Metabolic Mapping and 13C Flux Analysis Reveal Systematic Properties of an Oleaginous Microalga Chlorella protothecoides

    DOE PAGESBeta

    Wu, Chao; Xiong, Wei; Dai, Junbiao; Wu, Qingyu

    2014-12-15

    We report that integrated and genome-based flux balance analysis, metabolomics, and 13C-label profiling of phototrophic and heterotrophic metabolism in Chlorella protothecoides, an oleaginous green alga for biofuel. The green alga Chlorella protothecoides, capable of autotrophic and heterotrophic growth with rapid lipid synthesis, is a promising candidate for biofuel production. Based on the newly available genome knowledge of the alga, we reconstructed the compartmentalized metabolic network consisting of 272 metabolic reactions, 270 enzymes, and 461 encoding genes and simulated the growth in different cultivation conditions with flux balance analysis. Phenotype-phase plane analysis shows conditions achieving theoretical maximum of the biomass andmore » corresponding fatty acid-producing rate for phototrophic cells (the ratio of photon uptake rate to CO2 uptake rate equals 8.4) and heterotrophic ones (the glucose uptake rate to O2 consumption rate reaches 2.4), respectively. Isotope-assisted liquid chromatography-mass spectrometry/mass spectrometry reveals higher metabolite concentrations in the glycolytic pathway and the tricarboxylic acid cycle in heterotrophic cells compared with autotrophic cells. We also observed enhanced levels of ATP, nicotinamide adenine dinucleotide (phosphate), reduced, acetyl-Coenzyme A, and malonyl-Coenzyme A in heterotrophic cells consistently, consistent with a strong activity of lipid synthesis. To profile the flux map in experimental conditions, we applied nonstationary 13C metabolic flux analysis as a complementing strategy to flux balance analysis. We found that the result reveals negligible photorespiratory fluxes and a metabolically low active tricarboxylic acid cycle in phototrophic C. protothecoides. In comparison, high throughput of amphibolic reactions and the tricarboxylic acid cycle with no glyoxylate shunt activities were measured for heterotrophic cells. Lastly, taken together, the metabolic network modeling assisted

  9. Genome-based metabolic mapping and 13C flux analysis reveal systematic properties of an oleaginous microalga Chlorella protothecoides.

    PubMed

    Wu, Chao; Xiong, Wei; Dai, Junbiao; Wu, Qingyu

    2015-02-01

    Integrated and genome-based flux balance analysis, metabolomics, and (13)C-label profiling of phototrophic and heterotrophic metabolism in Chlorella protothecoides, an oleaginous green alga for biofuel. The green alga Chlorella protothecoides, capable of autotrophic and heterotrophic growth with rapid lipid synthesis, is a promising candidate for biofuel production. Based on the newly available genome knowledge of the alga, we reconstructed the compartmentalized metabolic network consisting of 272 metabolic reactions, 270 enzymes, and 461 encoding genes and simulated the growth in different cultivation conditions with flux balance analysis. Phenotype-phase plane analysis shows conditions achieving theoretical maximum of the biomass and corresponding fatty acid-producing rate for phototrophic cells (the ratio of photon uptake rate to CO2 uptake rate equals 8.4) and heterotrophic ones (the glucose uptake rate to O2 consumption rate reaches 2.4), respectively. Isotope-assisted liquid chromatography-mass spectrometry/mass spectrometry reveals higher metabolite concentrations in the glycolytic pathway and the tricarboxylic acid cycle in heterotrophic cells compared with autotrophic cells. We also observed enhanced levels of ATP, nicotinamide adenine dinucleotide (phosphate), reduced, acetyl-Coenzyme A, and malonyl-Coenzyme A in heterotrophic cells consistently, consistent with a strong activity of lipid synthesis. To profile the flux map in experimental conditions, we applied nonstationary (13)C metabolic flux analysis as a complementing strategy to flux balance analysis. The result reveals negligible photorespiratory fluxes and a metabolically low active tricarboxylic acid cycle in phototrophic C. protothecoides. In comparison, high throughput of amphibolic reactions and the tricarboxylic acid cycle with no glyoxylate shunt activities were measured for heterotrophic cells. Taken together, the metabolic network modeling assisted by experimental metabolomics and (13)C

  10. Clostridium clariflavum: Key Cellulosome Players Are Revealed by Proteomic Analysis

    PubMed Central

    Artzi, Lior; Morag, Ely; Barak, Yoav; Lamed, Raphael

    2015-01-01

    ABSTRACT Clostridium clariflavum is an anaerobic, cellulosome-forming thermophile, containing in its genome genes for a large number of cellulosomal enzyme and a complex scaffoldin system. Previously, we described the major cohesin-dockerin interactions of the cellulosome components, and on this basis a model of diverse cellulosome assemblies was derived. In this work, we cultivated C. clariflavum on cellobiose-, microcrystalline cellulose-, and switchgrass-containing media and isolated cell-free cellulosome complexes from each culture. Gel filtration separation of the cellulosome samples revealed two major fractions, which were analyzed by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) in order to identify the key players of the cellulosome assemblies therein. From the 13 scaffoldins present in the C. clariflavum genome, 11 were identified, and a variety of enzymes from different glycoside hydrolase and carbohydrate esterase families were identified, including the glycoside hydrolase families GH48, GH9, GH5, GH30, GH11, and GH10. The expression level of the cellulosomal proteins varied as a function of the carbon source used for cultivation of the bacterium. In addition, the catalytic activity of each cellulosome was examined on different cellulosic substrates, xylan and switchgrass. The cellulosome isolated from the microcrystalline cellulose-containing medium was the most active of all the cellulosomes that were tested. The results suggest that the expression of the cellulosome proteins is regulated by the type of substrate in the growth medium. Moreover, both cell-free and cell-bound cellulosome complexes were produced which together may degrade the substrate in a synergistic manner. These observations are compatible with our previously published model of cellulosome assemblies in this bacterium. PMID:25991683

  11. A combined physiological and proteomic approach to reveal lactic-acid-induced alterations in Lactobacillus casei Zhang and its mutant with enhanced lactic acid tolerance.

    PubMed

    Wu, Chongde; Zhang, Juan; Chen, Wei; Wang, Miao; Du, Guocheng; Chen, Jian

    2012-01-01

    Lactobacillus casei has traditionally been recognized as a probiotic and frequently used as an adjunct culture in fermented dairy products, where acid stress is an environmental condition commonly encountered. In the present study, we carried out a comparative physiological and proteomic study to investigate lactic-acid-induced alterations in Lactobacillus casei Zhang (WT) and its acid-resistant mutant. Analysis of the physiological data showed that the mutant exhibited 33.8% higher glucose phosphoenolpyruvate:sugar phosphotransferase system activity and lower glycolytic pH compared with the WT under acidic conditions. In addition, significant differences were detected in both cells during acid stress between intracellular physiological state, including intracellular pH, H(+)-ATPase activity, and intracellular ATP pool. Comparison of the proteomic data based on 2D-DIGE and i-TRAQ indicated that acid stress invoked a global change in both strains. The mutant protected the cells against acid damage by regulating the expression of key proteins involved in cellular metabolism, DNA replication, RNA synthesis, translation, and some chaperones. Proteome results were validated by Lactobacillus casei displaying higher intracellular aspartate and arginine levels, and the survival at pH 3.3 was improved 1.36- and 2.10-fold by the addition of 50-mM aspartate and arginine, respectively. To our knowledge, this is the first demonstration that aspartate may be involved in acid tolerance in Lactobacillus casei. Results presented here may help us understand acid resistance mechanisms and help formulate new strategies to enhance the industrial applications of this species. PMID:22159611

  12. Genomic analysis of regulatory network dynamics reveals large topological changes

    NASA Astrophysics Data System (ADS)

    Luscombe, Nicholas M.; Madan Babu, M.; Yu, Haiyuan; Snyder, Michael; Teichmann, Sarah A.; Gerstein, Mark

    2004-09-01

    Network analysis has been applied widely, providing a unifying language to describe disparate systems ranging from social interactions to power grids. It has recently been used in molecular biology, but so far the resulting networks have only been analysed statically. Here we present the dynamics of a biological network on a genomic scale, by integrating transcriptional regulatory information and gene-expression data for multiple conditions in Saccharomyces cerevisiae. We develop an approach for the statistical analysis of network dynamics, called SANDY, combining well-known global topological measures, local motifs and newly derived statistics. We uncover large changes in underlying network architecture that are unexpected given current viewpoints and random simulations. In response to diverse stimuli, transcription factors alter their interactions to varying degrees, thereby rewiring the network. A few transcription factors serve as permanent hubs, but most act transiently only during certain conditions. By studying sub-network structures, we show that environmental responses facilitate fast signal propagation (for example, with short regulatory cascades), whereas the cell cycle and sporulation direct temporal progression through multiple stages (for example, with highly inter-connected transcription factors). Indeed, to drive the latter processes forward, phase-specific transcription factors inter-regulate serially, and ubiquitously active transcription factors layer above them in a two-tiered hierarchy. We anticipate that many of the concepts presented here-particularly the large-scale topological changes and hub transience-will apply to other biological networks, including complex sub-systems in higher eukaryotes.

  13. Proteogenomic analysis reveals exosomes are more oncogenic than ectosomes.

    PubMed

    Keerthikumar, Shivakumar; Gangoda, Lahiru; Liem, Michael; Fonseka, Pamali; Atukorala, Ishara; Ozcitti, Cemil; Mechler, Adam; Adda, Christopher G; Ang, Ching-Seng; Mathivanan, Suresh

    2015-06-20

    Extracellular vesicles (EVs) include the exosomes (30-100 nm) that are produced through the endocytic pathway via the multivesicular bodies and the ectosomes (100-1000 nm) that are released through the budding of the plasma membrane. Despite the differences in the mode of biogenesis and size, reliable markers that can distinguish between exosomes and ectosomes are non-existent. Moreover, the precise functional differences between exosomes and ectosomes remains poorly characterised. Here, using label-free quantitative proteomics, we highlight proteins that could be exploited as markers to discriminate between exosomes and ectosomes. For the first time, a global proteogenomics analysis unveiled the secretion of mutant proteins that are implicated in cancer progression through tumor-derived EVs. Follow up integrated bioinformatics analysis highlighted the enrichment of oncogenic cargo in exosomes and ectosomes. Interestingly, exosomes induced significant cell proliferation and migration in recipient cells compared to ectosomes confirming the oncogenic nature of exosomes. These findings ascertain that cancer cells facilitate oncogenesis by the secretion of mutant and oncoproteins into the tumor microenvironment via exosomes and ectosomes. The integrative proteogenomics approach utilized in this study has the potential to identify disease biomarker candidates which can be later assayed in liquid biopsies obtained from cancer patients. PMID:25944692

  14. Network analysis reveals potential markers for pediatric adrenocortical carcinoma

    PubMed Central

    Kulshrestha, Anurag; Suman, Shikha; Ranjan, Rakesh

    2016-01-01

    Pediatric adrenocortical carcinoma (ACC) is a rare malignancy with a poor outcome. Molecular mechanisms of pediatric ACC oncogenesis and advancement are not well understood. Accurate and timely diagnosis of the disease requires identification of new markers for pediatric ACC. Differentially expressed genes (DEGs) were identified from the gene expression profile of pediatric ACC and obtained from Gene Expression Omnibus. Gene Ontology functional and pathway enrichment analysis was implemented to recognize the functions of DEGs. A protein–protein interaction (PPI) and gene–gene functional interaction (GGI) network of DEGs was constructed. Hub gene detection and enrichment analysis of functional modules were performed. Furthermore, a gene regulatory network incorporating DEGs–microRNAs–transcription factors was constructed and analyzed. A total of 431 DEGs including 228 upregulated and 203 downregulated DEGs were screened. These genes were largely involved in cell cycle, steroid biosynthesis, and p53 signaling pathways. Upregulated genes, CDK1, CCNB1, CDC20, and BUB1B, were identified as the common hubs of PPI and GGI networks. All the four common hub genes were also part of modules of the PPI network. Moreover, all the four genes were also present in the largest module of GGI network. A gene regulatory network consisting of 82 microRNAs and 100 transcription factors was also constructed. CDK1, CCNB1, CDC20, and BUB1B may serve as potential biomarker of pediatric ACC and as potential targets for therapeutic approach, although experimental studies are required to authenticate our findings. PMID:27555782

  15. Single amino acid sequence polymorphisms in rat cardiac troponin revealed by top-down tandem mass spectrometry.

    PubMed

    Sancho Solis, Raquel; Ge, Ying; Walker, Jeffery W

    2008-01-01

    Heterotrimeric cardiac troponin (cTn) is a critical component of the thin filament regulatory complex in cardiac muscle. Two of the three subunits, cTnI and cTnT, are subject to post-translational modifications such as proteolysis and phosphorylation, but linking modification patterns to function remains a major challenge. To obtain a global view of the biochemical state of cTn in native tissue, we performed high resolution top-down mass spectrometry of cTn heterotrimers from healthy adult rat hearts. cTn heterotrimers were affinity purified, desalted and then directly subjected to mass spectrometry using a 7 Tesla Thermo LTQ-FT-ICR instrument equipped with an ESI source. Molecular ions for N-terminally processed and acetylated cTnI and cTnT were readily detected as were other post-translationally modified forms of these proteins. cTnI was phosphorylated with a distribution of un-, mono- and bisphosphorylated forms of 41 +/- 3%, 46 +/- 1%, 13 +/- 3%, respectively. cTnT was predominantly monophosphorylated and partially proteolyzed at the Glu(29)-Pro(30) peptide bond. Also observed in high resolution spectra were 'shadow' peaks of similar intensity to 'parent' peaks exhibiting masses of cTnI+16 Da and cTnT+128 Da, subsequently shown by tandem mass spectrometry (MS/MS) to be single amino acid polymorphisms. Intact and protease-digested cTn subunits were fragmented by electron capture dissociation or collision activated dissociation to localize an Ala/Ser polymorphism at residue 7 of cTnI. Similar analysis of cTnT localized an additional Gln within a three residue alternative splice site beginning at residue 192. Besides being able to provide unique insights into the global state of post-translational modification of cTn subunits, high resolution top-down mass spectrometry readily revealed naturally occurring single amino acid sequence variants including a genetic polymorphism at residue 7 in cTnI, and an alternative splice isoform that affects a putative hinge region

  16. Six Tissue Transcriptomics Reveals Specific Immune Suppression in Spleen by Dietary Polyunsaturated Fatty Acids

    PubMed Central

    Gabrielsson, Britt G.; Peris, Eduard; Nookaew, Intawat; Grahnemo, Louise; Sandberg, Ann-Sofie; Wernstedt Asterholm, Ingrid; Jansson, John-Olov; Nielsen, Jens

    2016-01-01

    Dietary polyunsaturated fatty acids (PUFA) are suggested to modulate immune function, but the effects of dietary fatty acids composition on gene expression patterns in immune organs have not been fully characterized. In the current study we investigated how dietary fatty acids composition affects the total transcriptome profile, and especially, immune related genes in two immune organs, spleen (SPL) and bone marrow cells (BMC). Four tissues with metabolic function, skeletal muscle (SKM), white adipose tissue (WAT), brown adipose tissue (BAT), and liver (LIV), were investigated as a comparison. Following 8 weeks on low fat diet (LFD), high fat diet (HFD) rich in saturated fatty acids (HFD-S), or HFD rich in PUFA (HFD-P), tissue transcriptomics were analyzed by microarray and metabolic health assessed by fasting blood glucose level, HOMA-IR index, oral glucose tolerance test as well as quantification of crown-like structures in WAT. HFD-P corrected the metabolic phenotype induced by HFD-S. Interestingly, SKM and BMC were relatively inert to the diets, whereas the two adipose tissues (WAT and BAT) were mainly affected by HFD per se (both HFD-S and HFD-P). In particular, WAT gene expression was driven closer to that of the immune organs SPL and BMC by HFDs. The LIV exhibited different responses to both of the HFDs. Surprisingly, the spleen showed a major response to HFD-P (82 genes differed from LFD, mostly immune genes), while it was not affected at all by HFD-S (0 genes differed from LFD). In conclusion, the quantity and composition of dietary fatty acids affected the transcriptome in distinct manners in different organs. Remarkably, dietary PUFA, but not saturated fat, prompted a specific regulation of immune related genes in the spleen, opening the possibility that PUFA can regulate immune function by influencing gene expression in this organ. PMID:27166587

  17. Six Tissue Transcriptomics Reveals Specific Immune Suppression in Spleen by Dietary Polyunsaturated Fatty Acids.

    PubMed

    Svahn, Sara L; Väremo, Leif; Gabrielsson, Britt G; Peris, Eduard; Nookaew, Intawat; Grahnemo, Louise; Sandberg, Ann-Sofie; Wernstedt Asterholm, Ingrid; Jansson, John-Olov; Nielsen, Jens; Johansson, Maria E

    2016-01-01

    Dietary polyunsaturated fatty acids (PUFA) are suggested to modulate immune function, but the effects of dietary fatty acids composition on gene expression patterns in immune organs have not been fully characterized. In the current study we investigated how dietary fatty acids composition affects the total transcriptome profile, and especially, immune related genes in two immune organs, spleen (SPL) and bone marrow cells (BMC). Four tissues with metabolic function, skeletal muscle (SKM), white adipose tissue (WAT), brown adipose tissue (BAT), and liver (LIV), were investigated as a comparison. Following 8 weeks on low fat diet (LFD), high fat diet (HFD) rich in saturated fatty acids (HFD-S), or HFD rich in PUFA (HFD-P), tissue transcriptomics were analyzed by microarray and metabolic health assessed by fasting blood glucose level, HOMA-IR index, oral glucose tolerance test as well as quantification of crown-like structures in WAT. HFD-P corrected the metabolic phenotype induced by HFD-S. Interestingly, SKM and BMC were relatively inert to the diets, whereas the two adipose tissues (WAT and BAT) were mainly affected by HFD per se (both HFD-S and HFD-P). In particular, WAT gene expression was driven closer to that of the immune organs SPL and BMC by HFDs. The LIV exhibited different responses to both of the HFDs. Surprisingly, the spleen showed a major response to HFD-P (82 genes differed from LFD, mostly immune genes), while it was not affected at all by HFD-S (0 genes differed from LFD). In conclusion, the quantity and composition of dietary fatty acids affected the transcriptome in distinct manners in different organs. Remarkably, dietary PUFA, but not saturated fat, prompted a specific regulation of immune related genes in the spleen, opening the possibility that PUFA can regulate immune function by influencing gene expression in this organ. PMID:27166587

  18. Zinc-induced oligomerization of zinc α2 glycoprotein reveals multiple fatty acid-binding sites.

    PubMed

    Zahid, Henna; Miah, Layeque; Lau, Andy M; Brochard, Lea; Hati, Debolina; Bui, Tam T T; Drake, Alex F; Gor, Jayesh; Perkins, Stephen J; McDermott, Lindsay C

    2016-01-01

    Zinc α2 glycoprotein (ZAG) is an adipokine with a class I MHC protein fold and is associated with obesity and diabetes. Although its intrinsic ligand remains unknown, ZAG binds the dansylated C11 fatty acid 11-(dansylamino)undecanoic acid (DAUDA) in the groove between the α1 and α2 domains. The surface of ZAG has approximately 15 weak zinc-binding sites deemed responsible for precipitation from human plasma. In the present study the functional significance of these metal sites was investigated. Analytical ultracentrifugation (AUC) and CD showed that zinc, but not other divalent metals, causes ZAG to oligomerize in solution. Thus ZAG dimers and trimers were observed in the presence of 1 and 2 mM zinc. Molecular modelling of X-ray scattering curves and sedimentation coefficients indicated a progressive stacking of ZAG monomers, suggesting that the ZAG groove may be occluded in these. Using fluorescence-detected sedimentation velocity, these ZAG-zinc oligomers were again observed in the presence of the fluorescent boron dipyrromethene fatty acid C16-BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid). Fluorescence spectroscopy confirmed that ZAG binds C16-BODIPY. ZAG binding to C16-BODIPY, but not to DAUDA, was reduced by increased zinc concentrations. We conclude that the lipid-binding groove in ZAG contains at least two distinct fatty acid-binding sites for DAUDA and C16-BODIPY, similar to the multiple lipid binding seen in the structurally related immune protein CD1c. In addition, because high concentrations of zinc occur in the pancreas, the perturbation of these multiple lipid-binding sites by zinc may be significant in Type 2 diabetes where dysregulation of ZAG and zinc homoeostasis occurs. PMID:26487699

  19. Genome analysis of the platypus reveals unique signatures of evolution.

    PubMed

    Warren, Wesley C; Hillier, LaDeana W; Marshall Graves, Jennifer A; Birney, Ewan; Ponting, Chris P; Grützner, Frank; Belov, Katherine; Miller, Webb; Clarke, Laura; Chinwalla, Asif T; Yang, Shiaw-Pyng; Heger, Andreas; Locke, Devin P; Miethke, Pat; Waters, Paul D; Veyrunes, Frédéric; Fulton, Lucinda; Fulton, Bob; Graves, Tina; Wallis, John; Puente, Xose S; López-Otín, Carlos; Ordóñez, Gonzalo R; Eichler, Evan E; Chen, Lin; Cheng, Ze; Deakin, Janine E; Alsop, Amber; Thompson, Katherine; Kirby, Patrick; Papenfuss, Anthony T; Wakefield, Matthew J; Olender, Tsviya; Lancet, Doron; Huttley, Gavin A; Smit, Arian F A; Pask, Andrew; Temple-Smith, Peter; Batzer, Mark A; Walker, Jerilyn A; Konkel, Miriam K; Harris, Robert S; Whittington, Camilla M; Wong, Emily S W; Gemmell, Neil J; Buschiazzo, Emmanuel; Vargas Jentzsch, Iris M; Merkel, Angelika; Schmitz, Juergen; Zemann, Anja; Churakov, Gennady; Kriegs, Jan Ole; Brosius, Juergen; Murchison, Elizabeth P; Sachidanandam, Ravi; Smith, Carly; Hannon, Gregory J; Tsend-Ayush, Enkhjargal; McMillan, Daniel; Attenborough, Rosalind; Rens, Willem; Ferguson-Smith, Malcolm; Lefèvre, Christophe M; Sharp, Julie A; Nicholas, Kevin R; Ray, David A; Kube, Michael; Reinhardt, Richard; Pringle, Thomas H; Taylor, James; Jones, Russell C; Nixon, Brett; Dacheux, Jean-Louis; Niwa, Hitoshi; Sekita, Yoko; Huang, Xiaoqiu; Stark, Alexander; Kheradpour, Pouya; Kellis, Manolis; Flicek, Paul; Chen, Yuan; Webber, Caleb; Hardison, Ross; Nelson, Joanne; Hallsworth-Pepin, Kym; Delehaunty, Kim; Markovic, Chris; Minx, Pat; Feng, Yucheng; Kremitzki, Colin; Mitreva, Makedonka; Glasscock, Jarret; Wylie, Todd; Wohldmann, Patricia; Thiru, Prathapan; Nhan, Michael N; Pohl, Craig S; Smith, Scott M; Hou, Shunfeng; Nefedov, Mikhail; de Jong, Pieter J; Renfree, Marilyn B; Mardis, Elaine R; Wilson, Richard K

    2008-05-01

    We present a draft genome sequence of the platypus, Ornithorhynchus anatinus. This monotreme exhibits a fascinating combination of reptilian and mammalian characters. For example, platypuses have a coat of fur adapted to an aquatic lifestyle; platypus females lactate, yet lay eggs; and males are equipped with venom similar to that of reptiles. Analysis of the first monotreme genome aligned these features with genetic innovations. We find that reptile and platypus venom proteins have been co-opted independently from the same gene families; milk protein genes are conserved despite platypuses laying eggs; and immune gene family expansions are directly related to platypus biology. Expansions of protein, non-protein-coding RNA and microRNA families, as well as repeat elements, are identified. Sequencing of this genome now provides a valuable resource for deep mammalian comparative analyses, as well as for monotreme biology and conservation. PMID:18464734

  20. Genome analysis of the platypus reveals unique signatures of evolution

    PubMed Central

    Warren, Wesley C.; Hillier, LaDeana W.; Marshall Graves, Jennifer A.; Birney, Ewan; Ponting, Chris P.; Grützner, Frank; Belov, Katherine; Miller, Webb; Clarke, Laura; Chinwalla, Asif T.; Yang, Shiaw-Pyng; Heger, Andreas; Locke, Devin P.; Miethke, Pat; Waters, Paul D.; Veyrunes, Frédéric; Fulton, Lucinda; Fulton, Bob; Graves, Tina; Wallis, John; Puente, Xose S.; López-Otín, Carlos; Ordóñez, Gonzalo R.; Eichler, Evan E.; Chen, Lin; Cheng, Ze; Deakin, Janine E.; Alsop, Amber; Thompson, Katherine; Kirby, Patrick; Papenfuss, Anthony T.; Wakefield, Matthew J.; Olender, Tsviya; Lancet, Doron; Huttley, Gavin A.; Smit, Arian F. A.; Pask, Andrew; Temple-Smith, Peter; Batzer, Mark A.; Walker, Jerilyn A.; Konkel, Miriam K.; Harris, Robert S.; Whittington, Camilla M.; Wong, Emily S. W.; Gemmell, Neil J.; Buschiazzo, Emmanuel; Vargas Jentzsch, Iris M.; Merkel, Angelika; Schmitz, Juergen; Zemann, Anja; Churakov, Gennady; Kriegs, Jan Ole; Brosius, Juergen; Murchison, Elizabeth P.; Sachidanandam, Ravi; Smith, Carly; Hannon, Gregory J.; Tsend-Ayush, Enkhjargal; McMillan, Daniel; Attenborough, Rosalind; Rens, Willem; Ferguson-Smith, Malcolm; Lefèvre, Christophe M.; Sharp, Julie A.; Nicholas, Kevin R.; Ray, David A.; Kube, Michael; Reinhardt, Richard; Pringle, Thomas H.; Taylor, James; Jones, Russell C.; Nixon, Brett; Dacheux, Jean-Louis; Niwa, Hitoshi; Sekita, Yoko; Huang, Xiaoqiu; Stark, Alexander; Kheradpour, Pouya; Kellis, Manolis; Flicek, Paul; Chen, Yuan; Webber, Caleb; Hardison, Ross; Nelson, Joanne; Hallsworth-Pepin, Kym; Delehaunty, Kim; Markovic, Chris; Minx, Pat; Feng, Yucheng; Kremitzki, Colin; Mitreva, Makedonka; Glasscock, Jarret; Wylie, Todd; Wohldmann, Patricia; Thiru, Prathapan; Nhan, Michael N.; Pohl, Craig S.; Smith, Scott M.; Hou, Shunfeng; Renfree, Marilyn B.; Mardis, Elaine R.; Wilson, Richard K.

    2009-01-01

    We present a draft genome sequence of the platypus, Ornithorhynchus anatinus. This monotreme exhibits a fascinating combination of reptilian and mammalian characters. For example, platypuses have a coat of fur adapted to an aquatic lifestyle; platypus females lactate, yet lay eggs; and males are equipped with venom similar to that of reptiles. Analysis of the first monotreme genome aligned these features with genetic innovations. We find that reptile and platypus venom proteins have been co-opted independently from the same gene families; milk protein genes are conserved despite platypuses laying eggs; and immune gene family expansions are directly related to platypus biology. Expansions of protein, non-protein-coding RNA and microRNA families, as well as repeat elements, are identified. Sequencing of this genome now provides a valuable resource for deep mammalian comparative analyses, as well as for monotreme biology and conservation. PMID:18464734

  1. Image analysis of weaverbird nests reveals signature weave textures.

    PubMed

    Bailey, Ida E; Backes, André; Walsh, Patrick T; Morgan, Kate V; Meddle, Simone L; Healy, Susan D

    2015-06-01

    In nature, many animals build structures that can be readily measured at the scale of their gross morphology (e.g. length, volume and weight). Capturing individuality as can be done with the structures designed and built by human architects or artists, however, is more challenging. Here, we tested whether computer-aided image texture classification approaches can be used to describe textural variation in the nests of weaverbirds (Ploceus species) in order to attribute nests to the individual weaverbird that built them. We found that a computer-aided texture analysis approach does allow the assignment of a signature to weaverbirds' nests. We suggest that this approach will be a useful tool with which to examine individual variation across a range of animal constructions, not just for nests. PMID:26543586

  2. Image analysis of weaverbird nests reveals signature weave textures

    PubMed Central

    Bailey, Ida E.; Backes, André; Walsh, Patrick T.; Morgan, Kate V.; Meddle, Simone L.; Healy, Susan D.

    2015-01-01

    In nature, many animals build structures that can be readily measured at the scale of their gross morphology (e.g. length, volume and weight). Capturing individuality as can be done with the structures designed and built by human architects or artists, however, is more challenging. Here, we tested whether computer-aided image texture classification approaches can be used to describe textural variation in the nests of weaverbirds (Ploceus species) in order to attribute nests to the individual weaverbird that built them. We found that a computer-aided texture analysis approach does allow the assignment of a signature to weaverbirds' nests. We suggest that this approach will be a useful tool with which to examine individual variation across a range of animal constructions, not just for nests. PMID:26543586

  3. Time-Frequency Analysis Reveals Pairwise Interactions in Insect Swarms

    NASA Astrophysics Data System (ADS)

    Puckett, James G.; Ni, Rui; Ouellette, Nicholas T.

    2015-06-01

    The macroscopic emergent behavior of social animal groups is a classic example of dynamical self-organization, and is thought to arise from the local interactions between individuals. Determining these interactions from empirical data sets of real animal groups, however, is challenging. Using multicamera imaging and tracking, we studied the motion of individual flying midges in laboratory mating swarms. By performing a time-frequency analysis of the midge trajectories, we show that the midge behavior can be segmented into two distinct modes: one that is independent and composed of low-frequency maneuvers, and one that consists of higher-frequency nearly harmonic oscillations conducted in synchrony with another midge. We characterize these pairwise interactions, and make a hypothesis as to their biological function.

  4. Time-Frequency Analysis Reveals Pairwise Interactions in Insect Swarms.

    PubMed

    Puckett, James G; Ni, Rui; Ouellette, Nicholas T

    2015-06-26

    The macroscopic emergent behavior of social animal groups is a classic example of dynamical self-organization, and is thought to arise from the local interactions between individuals. Determining these interactions from empirical data sets of real animal groups, however, is challenging. Using multicamera imaging and tracking, we studied the motion of individual flying midges in laboratory mating swarms. By performing a time-frequency analysis of the midge trajectories, we show that the midge behavior can be segmented into two distinct modes: one that is independent and composed of low-frequency maneuvers, and one that consists of higher-frequency nearly harmonic oscillations conducted in synchrony with another midge. We characterize these pairwise interactions, and make a hypothesis as to their biological function. PMID:26197145

  5. Comparative transcriptome analysis reveals vertebrate phylotypic period during organogenesis

    PubMed Central

    Irie, Naoki; Kuratani, Shigeru

    2011-01-01

    One of the central issues in evolutionary developmental biology is how we can formulate the relationships between evolutionary and developmental processes. Two major models have been proposed: the 'funnel-like' model, in which the earliest embryo shows the most conserved morphological pattern, followed by diversifying later stages, and the 'hourglass' model, in which constraints are imposed to conserve organogenesis stages, which is called the phylotypic period. Here we perform a quantitative comparative transcriptome analysis of several model vertebrate embryos and show that the pharyngula stage is most conserved, whereas earlier and later stages are rather divergent. These results allow us to predict approximate developmental timetables between different species, and indicate that pharyngula embryos have the most conserved gene expression profiles, which may be the source of the basic body plan of vertebrates. PMID:21427719

  6. Proteogenomic analysis reveals unanticipated adaptations of colorectal tumor cells to deficiencies in DNA mismatch repair

    PubMed Central

    Halvey, Patrick J.; Wang, Xiaojing; Wang, Jing; Bhat, Ajaz A.; Dhawan, Punita; Li, Ming; Zhang, Bing; Liebler, Daniel C.; Slebos, Robbert J.C.

    2014-01-01

    Summary A growing body of genomic data on human cancers poses the critical question of how genomic variations translate to cancer phenotypes. We employed standardized shotgun proteomics and targeted protein quantitation platforms to analyze a panel of 10 colon cancer cell lines differing by mutations in DNA mismatch repair (MMR) genes. In addition, we performed transcriptome sequencing (RNA-seq) to enable detection of protein sequence variants from the proteomic data. Biological replicate cultures yielded highly consistent proteomic inventories with a cumulative total of 6,513 protein groups with a protein FDR of 3.17% across all cell lines. Networks of co-expressed proteins with differential expression based on MMR status revealed impact on protein folding, turnover and transport, on cellular metabolism and on DNA and RNA synthesis and repair. Analysis of variant amino acid sequences suggested higher stability of proteins affected by naturally occurring germline polymorphisms than of proteins affected by somatic protein sequence changes. The data provide evidence for multi-system adaptation to MMR deficiency with a stress response that targets misfolded proteins for degradation through the ubiquitin-dependent proteasome pathway. Enrichment analysis suggested epithelial-to-mesenchymal transition (EMT) in RKO cells, as evidenced by increased mobility and invasion properties compared to SW480. The observed proteomic profiles demonstrate previously unknown consequences of altered DNA repair and provide an expanded basis for mechanistic interpretation of MMR phenotypes. PMID:24247723

  7. Integrative microbial community analysis reveals full-scale enhanced biological phosphorus removal under tropical conditions.

    PubMed

    Law, Yingyu; Kirkegaard, Rasmus Hansen; Cokro, Angel Anisa; Liu, Xianghui; Arumugam, Krithika; Xie, Chao; Stokholm-Bjerregaard, Mikkel; Drautz-Moses, Daniela I; Nielsen, Per Halkjær; Wuertz, Stefan; Williams, Rohan B H

    2016-01-01

    Management of phosphorus discharge from human waste is essential for the control of eutrophication in surface waters. Enhanced biological phosphorus removal (EBPR) is a sustainable, efficient way of removing phosphorus from waste water without employing chemical precipitation, but is assumed unachievable in tropical temperatures due to conditions that favour glycogen accumulating organisms (GAOs) over polyphosphate accumulating organisms (PAOs). Here, we show these assumptions are unfounded by studying comparative community dynamics in a full-scale plant following systematic perturbation of operational conditions, which modified community abundance, function and physicochemical state. A statistically significant increase in the relative abundance of the PAO Accumulibacter was associated with improved EBPR activity. GAO relative abundance also increased, challenging the assumption of competition. An Accumulibacter bin-genome was identified from a whole community metagenomic survey, and comparative analysis against extant Accumulibacter genomes suggests a close relationship to Type II. Analysis of the associated metatranscriptome data revealed that genes encoding proteins involved in the tricarboxylic acid cycle and glycolysis pathways were highly expressed, consistent with metabolic modelling results. Our findings show that tropical EBPR is indeed possible, highlight the translational potential of studying competition dynamics in full-scale waste water communities and carry implications for plant design in tropical regions. PMID:27193869

  8. Integrative microbial community analysis reveals full-scale enhanced biological phosphorus removal under tropical conditions

    PubMed Central

    Law, Yingyu; Kirkegaard, Rasmus Hansen; Cokro, Angel Anisa; Liu, Xianghui; Arumugam, Krithika; Xie, Chao; Stokholm-Bjerregaard, Mikkel; Drautz-Moses, Daniela I.; Nielsen, Per Halkjær; Wuertz, Stefan; Williams, Rohan B. H.

    2016-01-01

    Management of phosphorus discharge from human waste is essential for the control of eutrophication in surface waters. Enhanced biological phosphorus removal (EBPR) is a sustainable, efficient way of removing phosphorus from waste water without employing chemical precipitation, but is assumed unachievable in tropical temperatures due to conditions that favour glycogen accumulating organisms (GAOs) over polyphosphate accumulating organisms (PAOs). Here, we show these assumptions are unfounded by studying comparative community dynamics in a full-scale plant following systematic perturbation of operational conditions, which modified community abundance, function and physicochemical state. A statistically significant increase in the relative abundance of the PAO Accumulibacter was associated with improved EBPR activity. GAO relative abundance also increased, challenging the assumption of competition. An Accumulibacter bin-genome was identified from a whole community metagenomic survey, and comparative analysis against extant Accumulibacter genomes suggests a close relationship to Type II. Analysis of the associated metatranscriptome data revealed that genes encoding proteins involved in the tricarboxylic acid cycle and glycolysis pathways were highly expressed, consistent with metabolic modelling results. Our findings show that tropical EBPR is indeed possible, highlight the translational potential of studying competition dynamics in full-scale waste water communities and carry implications for plant design in tropical regions. PMID:27193869

  9. Integrative microbial community analysis reveals full-scale enhanced biological phosphorus removal under tropical conditions

    NASA Astrophysics Data System (ADS)

    Law, Yingyu; Kirkegaard, Rasmus Hansen; Cokro, Angel Anisa; Liu, Xianghui; Arumugam, Krithika; Xie, Chao; Stokholm-Bjerregaard, Mikkel; Drautz-Moses, Daniela I.; Nielsen, Per Halkjær; Wuertz, Stefan; Williams, Rohan B. H.

    2016-05-01

    Management of phosphorus discharge from human waste is essential for the control of eutrophication in surface waters. Enhanced biological phosphorus removal (EBPR) is a sustainable, efficient way of removing phosphorus from waste water without employing chemical precipitation, but is assumed unachievable in tropical temperatures due to conditions that favour glycogen accumulating organisms (GAOs) over polyphosphate accumulating organisms (PAOs). Here, we show these assumptions are unfounded by studying comparative community dynamics in a full-scale plant following systematic perturbation of operational conditions, which modified community abundance, function and physicochemical state. A statistically significant increase in the relative abundance of the PAO Accumulibacter was associated with improved EBPR activity. GAO relative abundance also increased, challenging the assumption of competition. An Accumulibacter bin-genome was identified from a whole community metagenomic survey, and comparative analysis against extant Accumulibacter genomes suggests a close relationship to Type II. Analysis of the associated metatranscriptome data revealed that genes encoding proteins involved in the tricarboxylic acid cycle and glycolysis pathways were highly expressed, consistent with metabolic modelling results. Our findings show that tropical EBPR is indeed possible, highlight the translational potential of studying competition dynamics in full-scale waste water communities and carry implications for plant design in tropical regions.

  10. Comparative Metagenomic Analysis Reveals Mechanisms for Stress Response in Hypoliths from Extreme Hyperarid Deserts.

    PubMed

    Le, Phuong Thi; Makhalanyane, Thulani P; Guerrero, Leandro D; Vikram, Surendra; Van de Peer, Yves; Cowan, Don A

    2016-01-01

    Understanding microbial adaptation to environmental stressors is crucial for interpreting broader ecological patterns. In the most extreme hot and cold deserts, cryptic niche communities are thought to play key roles in ecosystem processes and represent excellent model systems for investigating microbial responses to environmental stressors. However, relatively little is known about the genetic diversity underlying such functional processes in climatically extreme desert systems. This study presents the first comparative metagenome analysis of cyanobacteria-dominated hypolithic communities in hot (Namib Desert, Namibia) and cold (Miers Valley, Antarctica) hyperarid deserts. The most abundant phyla in both hypolith metagenomes were Actinobacteria, Proteobacteria, Cyanobacteria and Bacteroidetes with Cyanobacteria dominating in Antarctic hypoliths. However, no significant differences between the two metagenomes were identified. The Antarctic hypolithic metagenome displayed a high number of sequences assigned to sigma factors, replication, recombination and repair, translation, ribosomal structure, and biogenesis. In contrast, the Namib Desert metagenome showed a high abundance of sequences assigned to carbohydrate transport and metabolism. Metagenome data analysis also revealed significant divergence in the genetic determinants of amino acid and nucleotide metabolism between these two metagenomes and those of soil from other polar deserts, hot deserts, and non-desert soils. Our results suggest extensive niche differentiation in hypolithic microbial communities from these two extreme environments and a high genetic capacity for survival under environmental extremes. PMID:27503299

  11. Differential network analysis reveals dysfunctional regulatory networks in gastric carcinogenesis.

    PubMed

    Cao, Mu-Shui; Liu, Bing-Ya; Dai, Wen-Tao; Zhou, Wei-Xin; Li, Yi-Xue; Li, Yuan-Yuan

    2015-01-01

    Gastric Carcinoma is one of the most common cancers in the world. A large number of differentially expressed genes have been identified as being associated with gastric cancer progression, however, little is known about the underlying regulatory mechanisms. To address this problem, we developed a differential networking approach that is characterized by including a nascent methodology, differential coexpression analysis (DCEA), and two novel quantitative methods for differential regulation analysis. We first applied DCEA to a gene expression dataset of gastric normal mucosa, adenoma and carcinoma samples to identify gene interconnection changes during cancer progression, based on which we inferred normal, adenoma, and carcinoma-specific gene regulation networks by using linear regression model. It was observed that cancer genes and drug targets were enriched in each network. To investigate the dynamic changes of gene regulation during carcinogenesis, we then designed two quantitative methods to prioritize differentially regulated genes (DRGs) and gene pairs or links (DRLs) between adjacent stages. It was found that known cancer genes and drug targets are significantly higher ranked. The top 4% normal vs. adenoma DRGs (36 genes) and top 6% adenoma vs. carcinoma DRGs (56 genes) proved to be worthy of further investigation to explore their association with gastric cancer. Out of the 16 DRGs involved in two top-10 DRG lists of normal vs. adenoma and adenoma vs. carcinoma comparisons, 15 have been reported to be gastric cancer or cancer related. Based on our inferred differential networking information and known signaling pathways, we generated testable hypotheses on the roles of GATA6, ESRRG and their signaling pathways in gastric carcinogenesis. Compared with established approaches which build genome-scale GRNs, or sub-networks around differentially expressed genes, the present one proved to be better at enriching cancer genes and drug targets, and prioritizing

  12. Differential network analysis reveals dysfunctional regulatory networks in gastric carcinogenesis

    PubMed Central

    Cao, Mu-Shui; Liu, Bing-Ya; Dai, Wen-Tao; Zhou, Wei-Xin; Li, Yi-Xue; Li, Yuan-Yuan

    2015-01-01

    Gastric Carcinoma is one of the most common cancers in the world. A large number of differentially expressed genes have been identified as being associated with gastric cancer progression, however, little is known about the underlying regulatory mechanisms. To address this problem, we developed a differential networking approach that is characterized by including a nascent methodology, differential coexpression analysis (DCEA), and two novel quantitative methods for differential regulation analysis. We first applied DCEA to a gene expression dataset of gastric normal mucosa, adenoma and carcinoma samples to identify gene interconnection changes during cancer progression, based on which we inferred normal, adenoma, and carcinoma-specific gene regulation networks by using linear regression model. It was observed that cancer genes and drug targets were enriched in each network. To investigate the dynamic changes of gene regulation during carcinogenesis, we then designed two quantitative methods to prioritize differentially regulated genes (DRGs) and gene pairs or links (DRLs) between adjacent stages. It was found that known cancer genes and drug targets are significantly higher ranked. The top 4% normal vs. adenoma DRGs (36 genes) and top 6% adenoma vs. carcinoma DRGs (56 genes) proved to be worthy of further investigation to explore their association with gastric cancer. Out of the 16 DRGs involved in two top-10 DRG lists of normal vs. adenoma and adenoma vs. carcinoma comparisons, 15 have been reported to be gastric cancer or cancer related. Based on our inferred differential networking information and known signaling pathways, we generated testable hypotheses on the roles of GATA6, ESRRG and their signaling pathways in gastric carcinogenesis. Compared with established approaches which build genome-scale GRNs, or sub-networks around differentially expressed genes, the present one proved to be better at enriching cancer genes and drug targets, and prioritizing

  13. Sulphur Kβ emission spectra reveal protonation states of aqueous sulfuric acid

    NASA Astrophysics Data System (ADS)

    Niskanen, Johannes; Sahle, Christoph J.; Ruotsalainen, Kari O.; Müller, Harald; Kavčič, Matjaž; Žitnik, Matjaž; Bučar, Klemen; Petric, Marko; Hakala, Mikko; Huotari, Simo

    2016-02-01

    In this paper we report an X-ray emission study of bulk aqueous sulfuric acid. Throughout the range of molarities from 1 M to 18 M the sulfur Kβ emission spectra from H2SO4 (aq) depend on the molar fractions and related deprotonation of H2SO4. We compare the experimental results with results from emission spectrum calculations based on atomic structures of single molecules and structures from ab initio molecular dynamics simulations. We show that the S Kβ emission spectrum is a sensitive probe of the protonation state of the acid molecules. Using non-negative matrix factorization we are able to extract the fractions of different protonation states in the spectra, and the results are in good agreement with the simulation for the higher part of the concentration range.

  14. Sulphur Kβ emission spectra reveal protonation states of aqueous sulfuric acid.

    PubMed

    Niskanen, Johannes; Sahle, Christoph J; Ruotsalainen, Kari O; Müller, Harald; Kavčič, Matjaž; Žitnik, Matjaž; Bučar, Klemen; Petric, Marko; Hakala, Mikko; Huotari, Simo

    2016-01-01

    In this paper we report an X-ray emission study of bulk aqueous sulfuric acid. Throughout the range of molarities from 1 M to 18 M the sulfur Kβ emission spectra from H2SO4 (aq) depend on the molar fractions and related deprotonation of H2SO4. We compare the experimental results with results from emission spectrum calculations based on atomic structures of single molecules and structures from ab initio molecular dynamics simulations. We show that the S Kβ emission spectrum is a sensitive probe of the protonation state of the acid molecules. Using non-negative matrix factorization we are able to extract the fractions of different protonation states in the spectra, and the results are in good agreement with the simulation for the higher part of the concentration range. PMID:26888159

  15. Sulphur Kβ emission spectra reveal protonation states of aqueous sulfuric acid

    PubMed Central

    Niskanen, Johannes; Sahle, Christoph J.; Ruotsalainen, Kari O.; Müller, Harald; Kavčič, Matjaž; Žitnik, Matjaž; Bučar, Klemen; Petric, Marko; Hakala, Mikko; Huotari, Simo

    2016-01-01

    In this paper we report an X-ray emission study of bulk aqueous sulfuric acid. Throughout the range of molarities from 1 M to 18 M the sulfur Kβ emission spectra from H2SO4 (aq) depend on the molar fractions and related deprotonation of H2SO4. We compare the experimental results with results from emission spectrum calculations based on atomic structures of single molecules and structures from ab initio molecular dynamics simulations. We show that the S Kβ emission spectrum is a sensitive probe of the protonation state of the acid molecules. Using non-negative matrix factorization we are able to extract the fractions of different protonation states in the spectra, and the results are in good agreement with the simulation for the higher part of the concentration range. PMID:26888159

  16. Layered Social Network Analysis Reveals Complex Relationships in Kindergarteners.

    PubMed

    Golemiec, Mireille; Schneider, Jonathan; Boyce, W Thomas; Bush, Nicole R; Adler, Nancy; Levine, Joel D

    2016-01-01

    The interplay between individuals forms building blocks for social structure. Here, we examine the structure of behavioral interactions among kindergarten classroom with a hierarchy-neutral approach to examine all possible underlying patterns in the formation of layered networks of "reciprocal" interactions. To understand how these layers are coordinated, we used a layered motif approach. Our dual layered motif analysis can therefore be thought of as the dynamics of smaller groups that tile to create the group structure, or alternatively they provide information on what the average child would do in a given local social environment. When we examine the regulated motifs in layered networks, we find that transitivity is at least partially involved in the formation of these layered network structures. We also found complex combinations of the expected reciprocal interactions. The mechanisms used to understand social networks of kindergarten children here are also applicable on a more general scale to any group of individuals where interactions and identities can be readily observed and scored. PMID:26973572

  17. Layered Social Network Analysis Reveals Complex Relationships in Kindergarteners

    PubMed Central

    Golemiec, Mireille; Schneider, Jonathan; Boyce, W. Thomas; Bush, Nicole R.; Adler, Nancy; Levine, Joel D.

    2016-01-01

    The interplay between individuals forms building blocks for social structure. Here, we examine the structure of behavioral interactions among kindergarten classroom with a hierarchy-neutral approach to examine all possible underlying patterns in the formation of layered networks of “reciprocal” interactions. To understand how these layers are coordinated, we used a layered motif approach. Our dual layered motif analysis can therefore be thought of as the dynamics of smaller groups that tile to create the group structure, or alternatively they provide information on what the average child would do in a given local social environment. When we examine the regulated motifs in layered networks, we find that transitivity is at least partially involved in the formation of these layered network structures. We also found complex combinations of the expected reciprocal interactions. The mechanisms used to understand social networks of kindergarten children here are also applicable on a more general scale to any group of individuals where interactions and identities can be readily observed and scored. PMID:26973572

  18. Phylogenetic analysis reveals a scattered distribution of autumn colours

    PubMed Central

    Archetti, Marco

    2009-01-01

    Background and Aims Leaf colour in autumn is rarely considered informative for taxonomy, but there is now growing interest in the evolution of autumn colours and different hypotheses are debated. Research efforts are hindered by the lack of basic information: the phylogenetic distribution of autumn colours. It is not known when and how autumn colours evolved. Methods Data are reported on the autumn colours of 2368 tree species belonging to 400 genera of the temperate regions of the world, and an analysis is made of their phylogenetic relationships in order to reconstruct the evolutionary origin of red and yellow in autumn leaves. Key Results Red autumn colours are present in at least 290 species (70 genera), and evolved independently at least 25 times. Yellow is present independently from red in at least 378 species (97 genera) and evolved at least 28 times. Conclusions The phylogenetic reconstruction suggests that autumn colours have been acquired and lost many times during evolution. This scattered distribution could be explained by hypotheses involving some kind of coevolutionary interaction or by hypotheses that rely on the need for photoprotection. PMID:19126636

  19. Bioimage analysis of Shigella infection reveals targeting of colonic crypts

    PubMed Central

    Arena, Ellen T.; Campbell-Valois, Francois-Xavier; Tinevez, Jean-Yves; Nigro, Giulia; Sachse, Martin; Moya-Nilges, Maryse; Nothelfer, Katharina; Marteyn, Benoit; Shorte, Spencer L.; Sansonetti, Philippe J.

    2015-01-01

    Few studies within the pathogenic field have used advanced imaging and analytical tools to quantitatively measure pathogenicity in vivo. In this work, we present a novel approach for the investigation of host–pathogen processes based on medium-throughput 3D fluorescence imaging. The guinea pig model for Shigella flexneri invasion of the colonic mucosa was used to monitor the infectious process over time with GFP-expressing S. flexneri. A precise quantitative imaging protocol was devised to follow individual S. flexneri in a large tissue volume. An extensive dataset of confocal images was obtained and processed to extract specific quantitative information regarding the progression of S. flexneri infection in an unbiased and exhaustive manner. Specific parameters included the analysis of S. flexneri positions relative to the epithelial surface, S. flexneri density within the tissue, and volume of tissue destruction. In particular, at early time points, there was a clear association of S. flexneri with crypts, key morphological features of the colonic mucosa. Numerical simulations based on random bacterial entry confirmed the bias of experimentally measured S. flexneri for early crypt targeting. The application of a correlative light and electron microscopy technique adapted for thick tissue samples further confirmed the location of S. flexneri within colonocytes at the mouth of crypts. This quantitative imaging approach is a novel means to examine host–pathogen systems in a tailored and robust manner, inclusive of the infectious agent. PMID:26056271

  20. Bioimage analysis of Shigella infection reveals targeting of colonic crypts.

    PubMed

    Arena, Ellen T; Campbell-Valois, Francois-Xavier; Tinevez, Jean-Yves; Nigro, Giulia; Sachse, Martin; Moya-Nilges, Maryse; Nothelfer, Katharina; Marteyn, Benoit; Shorte, Spencer L; Sansonetti, Philippe J

    2015-06-23

    Few studies within the pathogenic field have used advanced imaging and analytical tools to quantitatively measure pathogenicity in vivo. In this work, we present a novel approach for the investigation of host-pathogen processes based on medium-throughput 3D fluorescence imaging. The guinea pig model for Shigella flexneri invasion of the colonic mucosa was used to monitor the infectious process over time with GFP-expressing S. flexneri. A precise quantitative imaging protocol was devised to follow individual S. flexneri in a large tissue volume. An extensive dataset of confocal images was obtained and processed to extract specific quantitative information regarding the progression of S. flexneri infection in an unbiased and exhaustive manner. Specific parameters included the analysis of S. flexneri positions relative to the epithelial surface, S. flexneri density within the tissue, and volume of tissue destruction. In particular, at early time points, there was a clear association of S. flexneri with crypts, key morphological features of the colonic mucosa. Numerical simulations based on random bacterial entry confirmed the bias of experimentally measured S. flexneri for early crypt targeting. The application of a correlative light and electron microscopy technique adapted for thick tissue samples further confirmed the location of S. flexneri within colonocytes at the mouth of crypts. This quantitative imaging approach is a novel means to examine host-pathogen systems in a tailored and robust manner, inclusive of the infectious agent. PMID:26056271

  1. Evaluation of Meterorite Amono Acid Analysis Data Using Multivariate Techniques

    NASA Technical Reports Server (NTRS)

    McDonald, G.; Storrie-Lombardi, M.; Nealson, K.

    1999-01-01

    The amino acid distributions in the Murchison carbonaceous chondrite, Mars meteorite ALH84001, and ice from the Allan Hills region of Antarctica are shown, using a multivariate technique known as Principal Component Analysis (PCA), to be statistically distinct from the average amino acid compostion of 101 terrestrial protein superfamilies.

  2. Structure of Human Acid Sphingomyelinase Reveals the Role of the Saposin Domain in Activating Substrate Hydrolysis.

    PubMed

    Xiong, Zi-Jian; Huang, Jingjing; Poda, Gennady; Pomès, Régis; Privé, Gilbert G

    2016-07-31

    Acid sphingomyelinase (ASM) is a lysosomal phosphodiesterase that catalyzes the hydrolysis of sphingomyelin to produce ceramide and phosphocholine. While other lysosomal sphingolipid hydrolases require a saposin activator protein for full activity, the ASM polypeptide incorporates a built-in N-terminal saposin domain and does not require an external activator protein. Here, we report the crystal structure of human ASM and describe the organization of the three main regions of the enzyme: the N-terminal saposin domain, the proline-rich connector, and the catalytic domain. The saposin domain is tightly associated along an edge of the large, bowl-shaped catalytic domain and adopts an open form that exposes a hydrophobic concave surface approximately 30Å from the catalytic center. The calculated electrostatic potential of the enzyme is electropositive at the acidic pH of the lysosome, consistent with the strict requirement for the presence of acidic lipids in target membranes. Docking studies indicate that sphingomyelin binds with the ceramide-phosphate group positioned at the binuclear zinc center and molecular dynamic simulations indicate that the intrinsic flexibility of the saposin domain is important for monomer-dimer exchange and for membrane interactions. Overall, ASM uses a combination of electrostatic and hydrophobic interactions to cause local disruptions of target bilayers in order to bring the lipid headgroup to the catalytic center in a membrane-bound reaction. PMID:27349982

  3. Single-Molecule Imaging Reveals That Argonaute Reshapes the Binding Properties of Its Nucleic Acid Guides

    PubMed Central

    Salomon, William E.; Jolly, Samson M.; Moore, Melissa J.; Zamore, Phillip D.; Serebrov, Victor

    2015-01-01

    SUMMARY Argonaute proteins repress gene expression and defend against foreign nucleic acids using short RNAs or DNAs to specify the correct target RNA or DNA sequence. We have developed single-molecule methods to analyze target binding and cleavage mediated by the Argonaute:guide complex, RISC. We find that both eukaryotic and prokaryotic Argonaute proteins reshape the fundamental properties of RNA:RNA, RNA:DNA, and DNA:DNA hybridization: a small RNA or DNA bound to Argonaute as a guide no longer follows the well-established rules by which oligonucleotides find, bind, and dissociate from complementary nucleic acid sequences. Argonautes distinguish substrates from targets with similar complementarity. Mouse AGO2, for example, binds tighter to miRNA targets than its RNAi cleavage product, even though the cleaved product contains more base pairs. By re-writing the rules for nucleic acid hybridization, Argonautes allow oligonucleotides to serve as specificity determinants with thermodynamic and kinetic properties more typical of RNA-binding proteins than of RNA or DNA. PMID:26140592

  4. THPP target assignment reveals EchA6 as an essential fatty acid shuttle in mycobacteria.

    PubMed

    Cox, Jonathan A G; Abrahams, Katherine A; Alemparte, Carlos; Ghidelli-Disse, Sonja; Rullas, Joaquín; Angulo-Barturen, Iñigo; Singh, Albel; Gurcha, Sudagar S; Nataraj, Vijayashankar; Bethell, Stephen; Remuiñán, Modesto J; Encinas, Lourdes; Jervis, Peter J; Cammack, Nicholas C; Bhatt, Apoorva; Kruse, Ulrich; Bantscheff, Marcus; Fütterer, Klaus; Barros, David; Ballell, Lluis; Drewes, Gerard; Besra, Gurdyal S

    2016-01-01

    Phenotypic screens for bactericidal compounds against drug-resistant tuberculosis are beginning to yield novel inhibitors. However, reliable target identification remains challenging. Here, we show that tetrahydropyrazo[1,5-a]pyrimidine-3-carboxamide (THPP) selectively pulls down EchA6 in a stereospecific manner, instead of the previously assigned target Mycobacterium tuberculosis MmpL3. While homologous to mammalian enoyl-coenzyme A (CoA) hydratases, EchA6 is non-catalytic yet essential and binds long-chain acyl-CoAs. THPP inhibitors compete with CoA-binding, suppress mycolic acid synthesis, and are bactericidal in a mouse model of chronic tuberculosis infection. A point mutation, W133A, abrogated THPP-binding and increased both the in vitro minimum inhibitory concentration and the in vivo effective dose 99 in mice. Surprisingly, EchA6 interacts with selected enzymes of fatty acid synthase II (FAS-II) in bacterial two-hybrid assays, suggesting essentiality may be linked to feeding long-chain fatty acids to FAS-II. Finally, our data show that spontaneous resistance-conferring mutations can potentially obscure the actual target or alternative targets of small molecule inhibitors. PMID:27571973

  5. Revealing the amino acid composition of proteins within an expanded genetic code

    PubMed Central

    Aerni, Hans R.; Shifman, Mark A.; Rogulina, Svetlana; O'Donoghue, Patrick; Rinehart, Jesse

    2015-01-01

    The genetic code can be manipulated to reassign codons for the incorporation of non-standard amino acids (NSAA). Deletion of release factor 1 in Escherichia coli enhances translation of UAG (Stop) codons, yet may also extended protein synthesis at natural UAG terminated messenger RNAs. The fidelity of protein synthesis at reassigned UAG codons and the purity of the NSAA containing proteins produced require careful examination. Proteomics would be an ideal tool for these tasks, but conventional proteomic analyses cannot readily identify the extended proteins and accurately discover multiple amino acid (AA) insertions at a single UAG. To address these challenges, we created a new proteomic workflow that enabled the detection of UAG readthrough in native proteins in E. coli strains in which UAG was reassigned to encode phosphoserine. The method also enabled quantitation of NSAA and natural AA incorporation at UAG in a recombinant reporter protein. As a proof-of-principle, we measured the fidelity and purity of the phosphoserine orthogonal translation system (OTS) and used this information to improve its performance. Our results show a surprising diversity of natural AAs at reassigned stop codons. Our method can be used to improve OTSs and to quantify amino acid purity at reassigned codons in organisms with expanded genetic codes. PMID:25378305

  6. Single-Molecule Imaging Reveals that Argonaute Reshapes the Binding Properties of Its Nucleic Acid Guides.

    PubMed

    Salomon, William E; Jolly, Samson M; Moore, Melissa J; Zamore, Phillip D; Serebrov, Victor

    2015-07-01

    Argonaute proteins repress gene expression and defend against foreign nucleic acids using short RNAs or DNAs to specify the correct target RNA or DNA sequence. We have developed single-molecule methods to analyze target binding and cleavage mediated by the Argonaute:guide complex, RISC. We find that both eukaryotic and prokaryotic Argonaute proteins reshape the fundamental properties of RNA:RNA, RNA:DNA, and DNA:DNA hybridization—a small RNA or DNA bound to Argonaute as a guide no longer follows the well-established rules by which oligonucleotides find, bind, and dissociate from complementary nucleic acid sequences. Argonautes distinguish substrates from targets with similar complementarity. Mouse AGO2, for example, binds tighter to miRNA targets than its RNAi cleavage product, even though the cleaved product contains more base pairs. By re-writing the rules for nucleic acid hybridization, Argonautes allow oligonucleotides to serve as specificity determinants with thermodynamic and kinetic properties more typical of RNA-binding proteins than of RNA or DNA. PMID:26140592

  7. Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides

    PubMed Central

    Gagne, Steve J.; Stout, Jake M.; Liu, Enwu; Boubakir, Zakia; Clark, Shawn M.; Page, Jonathan E.

    2012-01-01

    Δ9-Tetrahydrocannabinol (THC) and other cannabinoids are responsible for the psychoactive and medicinal properties of Cannabis sativa L. (marijuana). The first intermediate in the cannabinoid biosynthetic pathway is proposed to be olivetolic acid (OA), an alkylresorcinolic acid that forms the polyketide nucleus of the cannabinoids. OA has been postulated to be synthesized by a type III polyketide synthase (PKS) enzyme, but so far type III PKSs from cannabis have been shown to produce catalytic byproducts instead of OA. We analyzed the transcriptome of glandular trichomes from female cannabis flowers, which are the primary site of cannabinoid biosynthesis, and searched for polyketide cyclase-like enzymes that could assist in OA cyclization. Here, we show that a type III PKS (tetraketide synthase) from cannabis trichomes requires the presence of a polyketide cyclase enzyme, olivetolic acid cyclase (OAC), which catalyzes a C2–C7 intramolecular aldol condensation with carboxylate retention to form OA. OAC is a dimeric α+β barrel (DABB) protein that is structurally similar to polyketide cyclases from Streptomyces species. OAC transcript is present at high levels in glandular trichomes, an expression profile that parallels other cannabinoid pathway enzymes. Our identification of OAC both clarifies the cannabinoid pathway and demonstrates unexpected evolutionary parallels between polyketide biosynthesis in plants and bacteria. In addition, the widespread occurrence of DABB proteins in plants suggests that polyketide cyclases may play an overlooked role in generating plant chemical diversity. PMID:22802619

  8. Revealing the amino acid composition of proteins within an expanded genetic code.

    PubMed

    Aerni, Hans R; Shifman, Mark A; Rogulina, Svetlana; O'Donoghue, Patrick; Rinehart, Jesse

    2015-01-01

    The genetic code can be manipulated to reassign codons for the incorporation of non-standard amino acids (NSAA). Deletion of release factor 1 in Escherichia coli enhances translation of UAG (Stop) codons, yet may also extended protein synthesis at natural UAG terminated messenger RNAs. The fidelity of protein synthesis at reassigned UAG codons and the purity of the NSAA containing proteins produced require careful examination. Proteomics would be an ideal tool for these tasks, but conventional proteomic analyses cannot readily identify the extended proteins and accurately discover multiple amino acid (AA) insertions at a single UAG. To address these challenges, we created a new proteomic workflow that enabled the detection of UAG readthrough in native proteins in E. coli strains in which UAG was reassigned to encode phosphoserine. The method also enabled quantitation of NSAA and natural AA incorporation at UAG in a recombinant reporter protein. As a proof-of-principle, we measured the fidelity and purity of the phosphoserine orthogonal translation system (OTS) and used this information to improve its performance. Our results show a surprising diversity of natural AAs at reassigned stop codons. Our method can be used to improve OTSs and to quantify amino acid purity at reassigned codons in organisms with expanded genetic codes. PMID:25378305

  9. Acid Rain Analysis by Standard Addition Titration.

    ERIC Educational Resources Information Center

    Ophardt, Charles E.

    1985-01-01

    The standard addition titration is a precise and rapid method for the determination of the acidity in rain or snow samples. The method requires use of a standard buret, a pH meter, and Gran's plot to determine the equivalence point. Experimental procedures used and typical results obtained are presented. (JN)

  10. Network analysis reveals multiscale controls on streamwater chemistry

    USGS Publications Warehouse

    McGuire, Kevin J.; Torgersen, Christian E.; Likens, Gene E.; Buso, Donald C.; Lowe, Winsor H.; Bailey, Scott W.

    2014-01-01

    By coupling synoptic data from a basin-wide assessment of streamwater chemistry with network-based geostatistical analysis, we show that spatial processes differentially affect biogeochemical condition and pattern across a headwater stream network. We analyzed a high-resolution dataset consisting of 664 water samples collected every 100 m throughout 32 tributaries in an entire fifth-order stream network. These samples were analyzed for an exhaustive suite of chemical constituents. The fine grain and broad extent of this study design allowed us to quantify spatial patterns over a range of scales by using empirical semivariograms that explicitly incorporated network topology. Here, we show that spatial structure, as determined by the characteristic shape of the semivariograms, differed both among chemical constituents and by spatial relationship (flow-connected, flow-unconnected, or Euclidean). Spatial structure was apparent at either a single scale or at multiple nested scales, suggesting separate processes operating simultaneously within the stream network and surrounding terrestrial landscape. Expected patterns of spatial dependence for flow-connected relationships (e.g., increasing homogeneity with downstream distance) occurred for some chemical constituents (e.g., dissolved organic carbon, sulfate, and aluminum) but not for others (e.g., nitrate, sodium). By comparing semivariograms for the different chemical constituents and spatial relationships, we were able to separate effects on streamwater chemistry of (i) fine-scale versus broad-scale processes and (ii) in-stream processes versus landscape controls. These findings provide insight on the hierarchical scaling of local, longitudinal, and landscape processes that drive biogeochemical patterns in stream networks.

  11. Neuronal Networks during Burst Suppression as Revealed by Source Analysis

    PubMed Central

    Reinicke, Christine; Moeller, Friederike; Anwar, Abdul Rauf; Mideksa, Kidist Gebremariam; Pressler, Ronit; Deuschl, Günther; Stephani, Ulrich; Siniatchkin, Michael

    2015-01-01

    Introduction Burst-suppression (BS) is an electroencephalography (EEG) pattern consisting of alternant periods of slow waves of high amplitude (burst) and periods of so called flat EEG (suppression). It is generally associated with coma of various etiologies (hypoxia, drug-related intoxication, hypothermia, and childhood encephalopathies, but also anesthesia). Animal studies suggest that both the cortex and the thalamus are involved in the generation of BS. However, very little is known about mechanisms of BS in humans. The aim of this study was to identify the neuronal network underlying both burst and suppression phases using source reconstruction and analysis of functional and effective connectivity in EEG. Material/Methods Dynamic imaging of coherent sources (DICS) was applied to EEG segments of 13 neonates and infants with burst and suppression EEG pattern. The brain area with the strongest power in the analyzed frequency (1–4 Hz) range was defined as the reference region. DICS was used to compute the coherence between this reference region and the entire brain. The renormalized partial directed coherence (RPDC) was used to describe the informational flow between the identified sources. Results/Conclusion Delta activity during the burst phases was associated with coherent sources in the thalamus and brainstem as well as bilateral sources in cortical regions mainly frontal and parietal, whereas suppression phases were associated with coherent sources only in cortical regions. Results of the RPDC analyses showed an upwards informational flow from the brainstem towards the thalamus and from the thalamus to cortical regions, which was absent during the suppression phases. These findings may support the theory that a “cortical deafferentiation” between the cortex and sub-cortical structures exists especially in suppression phases compared to burst phases in burst suppression EEGs. Such a deafferentiation may play a role in the poor neurological outcome of

  12. Network analysis reveals multiscale controls on streamwater chemistry

    PubMed Central

    McGuire, Kevin J.; Torgersen, Christian E.; Likens, Gene E.; Buso, Donald C.; Lowe, Winsor H.; Bailey, Scott W.

    2014-01-01

    By coupling synoptic data from a basin-wide assessment of streamwater chemistry with network-based geostatistical analysis, we show that spatial processes differentially affect biogeochemical condition and pattern across a headwater stream network. We analyzed a high-resolution dataset consisting of 664 water samples collected every 100 m throughout 32 tributaries in an entire fifth-order stream network. These samples were analyzed for an exhaustive suite of chemical constituents. The fine grain and broad extent of this study design allowed us to quantify spatial patterns over a range of scales by using empirical semivariograms that explicitly incorporated network topology. Here, we show that spatial structure, as determined by the characteristic shape of the semivariograms, differed both among chemical constituents and by spatial relationship (flow-connected, flow-unconnected, or Euclidean). Spatial structure was apparent at either a single scale or at multiple nested scales, suggesting separate processes operating simultaneously within the stream network and surrounding terrestrial landscape. Expected patterns of spatial dependence for flow-connected relationships (e.g., increasing homogeneity with downstream distance) occurred for some chemical constituents (e.g., dissolved organic carbon, sulfate, and aluminum) but not for others (e.g., nitrate, sodium). By comparing semivariograms for the different chemical constituents and spatial relationships, we were able to separate effects on streamwater chemistry of (i) fine-scale versus broad-scale processes and (ii) in-stream processes versus landscape controls. These findings provide insight on the hierarchical scaling of local, longitudinal, and landscape processes that drive biogeochemical patterns in stream networks. PMID:24753575

  13. Network analysis reveals multiscale controls on streamwater chemistry.

    PubMed

    McGuire, Kevin J; Torgersen, Christian E; Likens, Gene E; Buso, Donald C; Lowe, Winsor H; Bailey, Scott W

    2014-05-13

    By coupling synoptic data from a basin-wide assessment of streamwater chemistry with network-based geostatistical analysis, we show that spatial processes differentially affect biogeochemical condition and pattern across a headwater stream network. We analyzed a high-resolution dataset consisting of 664 water samples collected every 100 m throughout 32 tributaries in an entire fifth-order stream network. These samples were analyzed for an exhaustive suite of chemical constituents. The fine grain and broad extent of this study design allowed us to quantify spatial patterns over a range of scales by using empirical semivariograms that explicitly incorporated network topology. Here, we show that spatial structure, as determined by the characteristic shape of the semivariograms, differed both among chemical constituents and by spatial relationship (flow-connected, flow-unconnected, or Euclidean). Spatial structure was apparent at either a single scale or at multiple nested scales, suggesting separate processes operating simultaneously within the stream network and surrounding terrestrial landscape. Expected patterns of spatial dependence for flow-connected relationships (e.g., increasing homogeneity with downstream distance) occurred for some chemical constituents (e.g., dissolved organic carbon, sulfate, and aluminum) but not for others (e.g., nitrate, sodium). By comparing semivariograms for the different chemical constituents and spatial relationships, we were able to separate effects on streamwater chemistry of (i) fine-scale versus broad-scale processes and (ii) in-stream processes versus landscape controls. These findings provide insight on the hierarchical scaling of local, longitudinal, and landscape processes that drive biogeochemical patterns in stream networks. PMID:24753575

  14. Demographic analysis reveals gradual senescence in the flatworm Macrostomum lignano

    PubMed Central

    Mouton, Stijn; Willems, Maxime; Back, Patricia; Braeckman, Bart P; Borgonie, Gaetan

    2009-01-01

    Free-living flatworms ("Turbellaria") are appropriate model organisms to gain better insight into the role of stem cells in ageing and rejuvenation. Ageing research in flatworms is, however, still scarce. This is partly due to culture difficulties and the lack of a complete set of demographic data, including parameters such as median lifespan and age-specific mortality rate. In this paper, we report on the first flatworm survival analysis. We used the species Macrostomum lignano, which is an emerging model for studying the reciprocal influence between stem cells, ageing and rejuvenation. This species has a median lifespan of 205 ± 13 days (average ± standard deviation [SD]) and a 90th percentile lifespan of 373 ± 32 days. The maximum lifespan, however, is more than 745 days, and the average survival curve is characterised by a long tail because a small number of individuals lives twice as long as 90% of the population. Similar to earlier observations in a wide range of animals, in M. lignano the age-specific mortality rate increases exponentially, but levels off at the oldest ages. To compare the senescence of M. lignano with that of other ageing models, we determined the mortality rate doubling time, which is 0.20 ± 0.02 years. As a result, we can conclude that M. lignano shows gradual senescence at a rate similar to the vertebrate ageing models Rattus norvegicus and Mus musculus. We argue that M. lignano is a suitable model for ageing and rejuvenation research, and especially for the role of stem cells in these processes, due to its accessible stem cell system and regeneration capacity, and the possibility of combining stem cell studies with demographic analyses. PMID:19642971

  15. The diversity of algal phospholipase D homologs revealed by biocomputational analysis.

    PubMed

    Beligni, María Verónica; Bagnato, Carolina; Prados, María Belén; Bondino, Hernán; Laxalt, Ana María; Munnik, Teun; Ten Have, Arjen

    2015-10-01

    Phospholipase D (PLD) participates in the formation of phosphatidic acid, a precursor in glycerolipid biosynthesis and a second messenger. PLDs are part of a superfamily of proteins that hydrolyze phosphodiesters and share a catalytic motif, HxKxxxxD, and hence a mechanism of action. Although HKD-PLDs have been thoroughly characterized in plants, animals and bacteria, very little is known about these enzymes in algae. To fill this gap in knowledge, we performed a biocomputational analysis by means of HMMER iterative profiling, using most eukaryotic algae genomes available. Phylogenetic analysis revealed that algae exhibit very few eukaryotic-type PLDs but possess, instead, many bacteria-like PLDs. Among algae eukaryotic-type PLDs, we identified C2-PLDs and PXPH-like PLDs. In addition, the dinoflagellate Alexandrium tamarense features several proteins phylogenetically related to oomycete PLDs. Our phylogenetic analysis also showed that algae bacteria-like PLDs (proteins with putative PLD activity) fall into five clades, three of which are novel lineages in eukaryotes, composed almost entirely of algae. Specifically, Clade II is almost exclusive to diatoms, whereas Clade I and IV are mainly represented by proteins from prasinophytes. The other two clades are composed of mitochondrial PLDs (Clade V or Mito-PLDs), previously found in mammals, and a subfamily of potentially secreted proteins (Clade III or SP-PLDs), which includes a homolog formerly characterized in rice. In addition, our phylogenetic analysis shows that algae have non-PLD members within the bacteria-like HKD superfamily with putative cardiolipin synthase and phosphatidylserine/phosphatidylglycerophosphate synthase activities. Altogether, our results show that eukaryotic algae possess a moderate number of PLDs that belong to very diverse phylogenetic groups. PMID:26986890

  16. Metatranscriptomic Analysis of Groundwater Reveals an Active Anammox Bacterial Population

    NASA Astrophysics Data System (ADS)

    Jewell, T. N. M.; Karaoz, U.; Thomas, B. C.; Banfield, J. F.; Brodie, E.; Williams, K. H.; Beller, H. R.

    2014-12-01

    Groundwater is a major natural resource, yet little is known about the contribution of microbial anaerobic ammonium oxidation (anammox) activity to subsurface nitrogen cycling. During anammox, energy is generated as ammonium is oxidized under anaerobic conditions to dinitrogen gas, using nitrite as the final electron acceptor. This process is a global sink for fixed nitrogen. Only a narrow range of monophyletic bacteria within the Planctomycetes carries out anammox, and the full extent of their metabolism, and subsequent impact on nitrogen cycling and microbial community structure, is still unknown. Here, we employ a metatranscriptomic analysis on enriched mRNA to identify the abundance and activity of a population of anammox bacteria within an aquifer at Rifle, CO. Planktonic biomass was collected over a two-month period after injection of up to 1.5 mM nitrate. Illumina-generated sequences were mapped to a phylogenetically binned Rifle metagenome database. We identified transcripts for genes with high protein sequence identities (81-98%) to those of anammox strain KSU-1 and to two of the five anammox bacteria genera, Brocadia and Kuenenia, suggesting an active, if not diverse, anammox population. Many of the most abundant anammox transcripts mapped to a single scaffold, indicative of a single dominant anammox species. Transcripts of the genes necessary for the anammox pathway were present, including an ammonium transporter (amtB), nitrite/formate transporter, nitrite reductase (nirK), and hydrazine oxidoreductase (hzoB). The form of nitrite reductase encoded by anammox is species-dependent, and we only identified nirK, with no evidence of anammox nirS. In addition to the anammox pathway we saw evidence of the anammox bacterial dissimilatory nitrate reduction to ammonium pathway (narH, putative nrfA, and nrfB), which provides an alternate means of generating substrates for anammox from nitrate, rather than relying on an external pool. Transcripts for hydroxylamine

  17. Metabolomics revealed diurnal heat stress and zinc supplementation-induced changes in amino acid, lipid, and microbial metabolism.

    PubMed

    Wang, Lei; Urriola, Pedro E; Luo, Zhao-Hui; Rambo, Zachary J; Wilson, Mark E; Torrison, Jerry L; Shurson, Gerald C; Chen, Chi

    2016-01-01

    Heat stress (HS) dramatically disrupts the events in energy and nutrient metabolism, many of which requires zinc (Zn) as a cofactor. In this study, metabolic effects of HS and Zn supplementation were evaluated by examining growth performance, blood chemistry, and metabolomes of crossbred gilts fed with ZnNeg (no Zn supplementation), ZnIO (120 ppm ZnSO4), or ZnAA (60 ppm ZnSO4 + 60 ppm zinc amino acid complex) diets under diurnal HS or thermal-neutral (TN) condition. The results showed that growth performance was reduced by HS but not by Zn supplementation. Among measured serum biochemicals, HS was found to increase creatinine but decrease blood urea nitrogen (BUN) level. Metabolomic analysis indicated that HS greatly affected diverse metabolites associated with amino acid, lipid, and microbial metabolism, including urea cycle metabolites, essential amino acids, phospholipids, medium-chain dicarboxylic acids, fatty acid amides, and secondary bile acids. More importantly, many changes in these metabolite markers were correlated with both acute and adaptive responses to HS. Relative to HS-induced metabolic effects, Zn supplementation-associated effects were much more limited. A prominent observation was that ZnIO diet, potentially through its influences on microbial metabolism, yielded different responses to HS compared with two other diets, which included higher levels of short-chain fatty acids (SCFAs) in cecal fluid and higher levels of lysine in the liver and feces. Overall, comprehensive metabolomic analysis identified novel metabolite markers associated with HS and Zn supplementation, which could guide further investigation on the mechanisms of these metabolic effects. PMID:26755737

  18. Insight toward epithelial Na+ channel mechanism revealed by the acid-sensing ion channel 1 structure.

    PubMed

    Stockand, James D; Staruschenko, Alexander; Pochynyuk, Oleh; Booth, Rachell E; Silverthorn, Dee U

    2008-09-01

    The epithelial Na(+) channel/degenerin (ENaC/DEG) protein family includes a diverse group of ion channels, including nonvoltage-gated Na(+) channels of epithelia and neurons, and the acid-sensing ion channel 1 (ASIC1). In mammalian epithelia, ENaC helps regulate Na(+) and associated water transport, making it a critical determinant of systemic blood pressure and pulmonary mucosal fluidity. In the nervous system, ENaC/DEG proteins are related to sensory transduction. While the importance and physiological function of these ion channels are established, less is known about their structure. One hallmark of the ENaC/DEG channel family is that each channel subunit has only two transmembrane domains connected by an exceedingly large extracellular loop. This subunit structure was recently confirmed when Jasti and colleagues determined the crystal structure of chicken ASIC1, a neuronal acid-sensing ENaC/DEG channel. By mapping ENaC to the structural coordinates of cASIC1, as we do here, we hope to provide insight toward ENaC structure. ENaC, like ASIC1, appears to be a trimeric channel containing 1alpha, 1beta, and 1gamma subunit. Heterotrimeric ENaC and monomeric ENaC subunits within the trimer possibly contain many of the major secondary, tertiary, and quaternary features identified in cASIC1 with a few subtle but critical differences. These differences are expected to have profound effects on channel behavior. In particular, they may contribute to ENaC insensitivity to acid and to its constitutive activity in the absence of time- and ligand-dependent inactivation. Experiments resulting from this comparison of cASIC1 and ENaC may help clarify unresolved issues related to ENaC architecture, and may help identify secondary structures and residues critical to ENaC function. PMID:18459164

  19. The acidic pH-induced structural changes in Pin1 as revealed by spectral methodologies

    NASA Astrophysics Data System (ADS)

    Wang, Jing-Zhang; Xi, Lei; Zhu, Guo-Fei; Han, Yong-Guang; Luo, Yue; Wang, Mei; Du, Lin-Fang

    2012-12-01

    Pin1 is closely associated with the pathogenesis of cancers and Alzheimer's disease (AD). Previously, we have shown the characteristics of the thermal denaturation of Pin1. Herein, the acid-induced denaturation of Pin1 was determined by means of fluorescence emission, synchronous fluorescence, far-UV CD, ANS fluorescence and RLS spectroscopies. The fluorescence emission spectra and the synchronous fluorescence spectra suggested the partially reversible unfolding (approximately from pH 7.0 to 4.0) and refolding (approximately from pH 4.0 to 1.0) of the structures around the chromophores in Pin1, apparently with an intermediate state at about pH 4.0-4.5. The far-UV CD spectra indicated that acidic pH (below pH 4.0) induced the structural transition from α-helix and random coils to β-sheet in Pin1. The ANS fluorescence and the RLS spectra further suggested the exposure of the hydrophobic side-chains of Pin1 and the aggregation of it especially below pH 2.3, and the aggregation possibly resulted in the formation of extra intermolecular β-sheet. The present work primarily shows that acidic pH can induce kinds of irreversible structural changes in Pin1, such as the exposure of the hydrophobic side-chains, the transition from α-helix to β-sheet and the aggregation of Pin1, and also explains why Pin1 loses most of its activity below pH 5.0. The results emphasize the important role of decreased pH in the pathogenesis of some Pin1-related diseases, and support the therapeutic approach for them by targeting acidosis and modifying the intracellular pH gradients.

  20. Tandem mass spectrometric analysis of glyphosate, glufosinate, aminomethylphosphonic acid and methylphosphinicopropionic acid.

    PubMed

    Goodwin, Lee; Startin, James R; Goodall, David M; Keely, Brendan J

    2003-01-01

    A detailed MS(n) study of glyphosate, glufosinate and their main metabolites, aminomethylphosphonic acid and methylphosphinicopropionic acid, using an ion trap mass spectrometer, was performed. The analytes show good response in negative ion electrospray mass spectrometry (ES-MS) as [M-H](-) ions. Tandem-MS spectra reveal a wealth of structurally specific ions, allowing characterisation of the fragmentation pathways of the four analytes in their native form for the first time. The ions formed at each stage of fragmentation reveal ions common to each analyte, such as phosphinate, as well as analyte specific transitions. Simplex optimisation allows optimum trapping and fragmentation parameters to be determined leading to improved response for particular transitions and transition sequences, and revealing previously unseen ions. PMID:12717770

  1. Metabolomics Analysis Reveals Specific Novel Tetrapeptide and Potential Anti-Inflammatory Metabolites in Pathogenic Aspergillus species

    PubMed Central

    Lee, Kim-Chung; Tam, Emily W. T.; Lo, Ka-Ching; Tsang, Alan K. L.; Lau, Candy C. Y.; To, Kelvin K. W.; Chan, Jasper F. W.; Lam, Ching-Wan; Yuen, Kwok-Yung; Lau, Susanna K. P.; Woo, Patrick C. Y.

    2015-01-01

    Infections related to Aspergillus species have emerged to become an important focus in infectious diseases, as a result of the increasing use of immunosuppressive agents and high fatality associated with invasive aspergillosis. However, laboratory diagnosis of Aspergillus infections remains difficult. In this study, by comparing the metabolomic profiles of the culture supernatants of 30 strains of six pathogenic Aspergillus species (A. fumigatus, A. flavus, A. niger, A. terreus, A. nomius and A. tamarii) and 31 strains of 10 non-Aspergillus fungi, eight compounds present in all strains of the six Aspergillus species but not in any strain of the non-Aspergillus fungi were observed. One of the eight compounds, Leu–Glu–Leu–Glu, is a novel tetrapeptide and represents the first linear tetrapeptide observed in Aspergillus species, which we propose to be named aspergitide. Two other closely related Aspergillus-specific compounds, hydroxy-(sulfooxy)benzoic acid and (sulfooxy)benzoic acid, may possess anti-inflammatory properties, as 2-(sulfooxy)benzoic acid possesses a structure similar to those of aspirin [2-(acetoxy)benzoic acid] and salicylic acid (2-hydroxybenzoic acid). Further studies to examine the potentials of these Aspergillus-specific compounds for laboratory diagnosis of aspergillosis are warranted and further experiments will reveal whether Leu–Glu–Leu–Glu, hydroxy-(sulfooxy)benzoic acid and (sulfooxy)benzoic acid are virulent factors of the pathogenic Aspergillus species. PMID:26090713

  2. Metabolomics Analysis Reveals Specific Novel Tetrapeptide and Potential Anti-Inflammatory Metabolites in Pathogenic Aspergillus species.

    PubMed

    Lee, Kim-Chung; Tam, Emily W T; Lo, Ka-Ching; Tsang, Alan K L; Lau, Candy C Y; To, Kelvin K W; Chan, Jasper F W; Lam, Ching-Wan; Yuen, Kwok-Yung; Lau, Susanna K P; Woo, Patrick C Y

    2015-01-01

    Infections related to Aspergillus species have emerged to become an important focus in infectious diseases, as a result of the increasing use of immunosuppressive agents and high fatality associated with invasive aspergillosis. However, laboratory diagnosis of Aspergillus infections remains difficult. In this study, by comparing the metabolomic profiles of the culture supernatants of 30 strains of six pathogenic Aspergillus species (A. fumigatus, A. flavus, A. niger, A. terreus, A. nomius and A. tamarii) and 31 strains of 10 non-Aspergillus fungi, eight compounds present in all strains of the six Aspergillus species but not in any strain of the non-Aspergillus fungi were observed. One of the eight compounds, Leu-Glu-Leu-Glu, is a novel tetrapeptide and represents the first linear tetrapeptide observed in Aspergillus species, which we propose to be named aspergitide. Two other closely related Aspergillus-specific compounds, hydroxy-(sulfooxy)benzoic acid and (sulfooxy)benzoic acid, may possess anti-inflammatory properties, as 2-(sulfooxy)benzoic acid possesses a structure similar to those of aspirin [2-(acetoxy)benzoic acid] and salicylic acid (2-hydroxybenzoic acid). Further studies to examine the potentials of these Aspergillus-specific compounds for laboratory diagnosis of aspergillosis are warranted and further experiments will reveal whether Leu-Glu-Leu-Glu, hydroxy-(sulfooxy)benzoic acid and (sulfooxy)benzoic acid are virulent factors of the pathogenic Aspergillus species. PMID:26090713

  3. Field-Based Stable Isotope Probing Reveals the Identities of Benzoic Acid-Metabolizing Microorganisms and Their In Situ Growth in Agricultural Soil▿

    PubMed Central

    Pumphrey, Graham M.; Madsen, Eugene L.

    2008-01-01

    We used a combination of stable isotope probing (SIP), gas chromatography-mass spectrometry-based respiration, isolation/cultivation, and quantitative PCR procedures to discover the identity and in situ growth of soil microorganisms that metabolize benzoic acid. We added [13C]benzoic acid or [12C]benzoic acid (100 μg) once, four times, or five times at 2-day intervals to agricultural field plots. After monitoring 13CO2 evolution from the benzoic acid-dosed soil, field soils were harvested and used for nucleic acid extraction and for cultivation of benzoate-degrading bacteria. Exposure of soil to benzoate increased the number of culturable benzoate degraders compared to unamended soil, and exposure to benzoate shifted the dominant culturable benzoate degraders from Pseudomonas species to Burkholderia species. Isopycnic separation of heavy [13C]DNA from the unlabeled fraction allowed terminal restriction fragment length polymorphism (T-RFLP) analyses to confirm that distinct 16S rRNA genes were localized in the heavy fraction. Phylogenetic analysis of sequenced 16S rRNA genes revealed a predominance (15 of 58 clones) of Burkholderia species in the heavy fraction. Burkholderia sp. strain EBA09 shared 99.5% 16S rRNA sequence similarity with a group of clones representing the dominant RFLP pattern, and the T-RFLP fragment for strain EBA09 and a clone from that cluster matched the fragment enriched in the [13C]DNA fraction. Growth of the population represented by EBA09 during the field-dosing experiment was demonstrated by using most-probable-number-PCR and primers targeting EBA09 and the closely related species Burkholderia hospita. Thus, the target population identified by SIP not only actively metabolized benzoic acid but reproduced in the field upon the addition of the substrate. PMID:18469130

  4. Differential RNA-seq, Multi-Network Analysis and Metabolic Regulation Analysis of Kluyveromyces marxianus Reveals a Compartmentalised Response to Xylose

    PubMed Central

    Schabort, Du Toit W. P.; Letebele, Precious K.; Steyn, Laurinda; Kilian, Stephanus G.; du Preez, James C.

    2016-01-01

    We investigated the transcriptomic response of a new strain of the yeast Kluyveromyces marxianus, in glucose and xylose media using RNA-seq. The data were explored in a number of innovative ways using a variety of networks types, pathway maps, enrichment statistics, reporter metabolites and a flux simulation model, revealing different aspects of the genome-scale response in an integrative systems biology manner. The importance of the subcellular localisation in the transcriptomic response is emphasised here, revealing new insights. As was previously reported by others using a rich medium, we show that peroxisomal fatty acid catabolism was dramatically up-regulated in a defined xylose mineral medium without fatty acids, along with mechanisms to activate fatty acids and transfer products of β-oxidation to the mitochondria. Notably, we observed a strong up-regulation of the 2-methylcitrate pathway, supporting capacity for odd-chain fatty acid catabolism. Next we asked which pathways would respond to the additional requirement for NADPH for xylose utilisation, and rationalised the unexpected results using simulations with Flux Balance Analysis. On a fundamental level, we investigated the contribution of the hierarchical and metabolic regulation levels to the regulation of metabolic fluxes. Metabolic regulation analysis suggested that genetic level regulation plays a major role in regulating metabolic fluxes in adaptation to xylose, even for the high capacity reactions, which is unexpected. In addition, isozyme switching may play an important role in re-routing of metabolic fluxes in subcellular compartments in K. marxianus. PMID:27315089

  5. Negative Ion Photoelectron Spectroscopy Reveals Thermodynamic Advantage of Organic Acids in Facilitating Formation of Bisulfate Ion Clusters: Atmospheric Implications

    SciTech Connect

    Hou, Gao-Lei; Lin, Wei; Deng, Shihu; Zhang, Jian; Zheng, Weijun; Paesani, Francesco; Wang, Xue B.

    2013-03-07

    Recent lab and field measurements have indicated critical roles of organic acids in enhancing new atmospheric aerosol formation. Such findings have stimulated theoretical studies with the aim of understanding interaction of organic acids with common aerosol nucleation precursors like bisulfate (HSO4-). In this Letter, we report a combined negative ion photoelectron spectroscopic and theoretical investigation of molecular clusters formed by HSO4- with succinic acid (SUA, HO2C(CH2)2CO2H), HSO4-(SUA)n (n = 0-2), along with HSO4-(H2O)n and HSO4-(H2SO4)n. It is found that one SUA molecule can stabilize HSO4- by ca. 39 kcal/mol, triple the corresponding value that one water molecule is capable of (ca. 13 kcal/mol). Molecular dynamics simulations and quantum chemical calculations reveal the most plausible structures of these clusters and attribute the stability of these clusters due to formation of strong hydrogen bonds. This work provides direct experimental evidence showing significant thermodynamic advantage by involving organic acid molecules to promote formation and growth in bisulfate clusters and aerosols.

  6. Down-regulation of the Caffeic acid O-methyltransferase Gene in Switchgrass Reveals a Novel Monolignol Analog

    SciTech Connect

    Tschaplinski, Timothy J; Standaert, Robert F; Engle, Nancy L; Martin, Madhavi Z; Sangha, Amandeep K; Parks, Jerry M; Smith, Jeremy C; Samuel, Reichel; Pu, Yunqiao; Ragauskas, A J; Hamilton, Choo Yieng; Fu, Chunxiang; Wang, Zeng-Yu; Davison, Brian H; Dixon, Richard A; Mielenz, Jonathan R

    2012-01-01

    Down-regulation of the caffeic acid 3-O-methyltransferase (COMT) gene in the lignin biosynthetic pathway of switchgrass (Panicum virgatum) resulted in cell walls of transgenic plants releasing more constituent sugars after pretreatment by dilute acid and treatment with glycosyl hydrolases from an added enzyme preparation and from Clostridium thermocellum. Fermentation of both wild-type and transgenic switchgrass after milder hot water pretreatment with no water washing showed that only the transgenic switchgrass inhibited C. thermocellum. Gas chromatography-mass spectrometry-based metabolomics were undertaken on cell wall aqueous extracts to determine the nature of the microbial inhibitors, confirming the increased concentration of a number of phenolic acids and aldehydes that are known inhibitors of fermentation. Metabolomic analyses of the transgenic biomass additionally revealed the presence of a novel monolignol-like metabolite, identified as trans-3, 4-dimethoxy-5-hydroxycinnamyl alcohol (iso-sinapyl alcohol) in both non-pretreated, as well as hot water pretreated samples. Although there was no indication that iso-sinapyl alcohol was integrated into the cell wall, diversion of substrates from sinapyl alcohol to free iso-sinapyl alcohol, its glucoside, and associated upstream lignin pathway changes, including increased phenolic aldehydes and acids, are associated with more facile cell wall deconstruction, and to the observed inhibitory effect on microbial growth.

  7. Analysis of fatty acid content and composition in microalgae.

    PubMed

    Breuer, Guido; Evers, Wendy A C; de Vree, Jeroen H; Kleinegris, Dorinde M M; Martens, Dirk E; Wijffels, René H; Lamers, Packo P

    2013-01-01

    A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (ω-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes. With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of. This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other. The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification. PMID:24121679

  8. Analysis of Fatty Acid Content and Composition in Microalgae

    PubMed Central

    Breuer, Guido; Evers, Wendy A. C.; de Vree, Jeroen H.; Kleinegris, Dorinde M. M.; Martens, Dirk E.; Wijffels, René H.; Lamers, Packo P.

    2013-01-01

    A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (ω-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes. With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of. This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other. The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification. PMID:24121679

  9. Co-expression network analysis reveals transcription factors associated to cell wall biosynthesis in sugarcane.

    PubMed

    Ferreira, Savio Siqueira; Hotta, Carlos Takeshi; Poelking, Viviane Guzzo de Carli; Leite, Debora Chaves Coelho; Buckeridge, Marcos Silveira; Loureiro, Marcelo Ehlers; Barbosa, Marcio Henrique Pereira; Carneiro, Monalisa Sampaio; Souza, Glaucia Mendes

    2016-05-01

    Sugarcane is a hybrid of Saccharum officinarum and Saccharum spontaneum, with minor contributions from other species in Saccharum and other genera. Understanding the molecular basis of cell wall metabolism in sugarcane may allow for rational changes in fiber quality and content when designing new energy crops. This work describes a comparative expression profiling of sugarcane ancestral genotypes: S. officinarum, S. spontaneum and S. robustum and a commercial hybrid: RB867515, linking gene expression to phenotypes to identify genes for sugarcane improvement. Oligoarray experiments of leaves, immature and intermediate internodes, detected 12,621 sense and 995 antisense transcripts. Amino acid metabolism was particularly evident among pathways showing natural antisense transcripts expression. For all tissues sampled, expression analysis revealed 831, 674 and 648 differentially expressed genes in S. officinarum, S. robustum and S. spontaneum, respectively, using RB867515 as reference. Expression of sugar transporters might explain sucrose differences among genotypes, but an unexpected differential expression of histones were also identified between high and low Brix° genotypes. Lignin biosynthetic genes and bioenergetics-related genes were up-regulated in the high lignin genotype, suggesting that these genes are important for S. spontaneum to allocate carbon to lignin, while S. officinarum allocates it to sucrose storage. Co-expression network analysis identified 18 transcription factors possibly related to cell wall biosynthesis while in silico analysis detected cis-elements involved in cell wall biosynthesis in their promoters. Our results provide information to elucidate regulatory networks underlying traits of interest that will allow the improvement of sugarcane for biofuel and chemicals production. PMID:26820137

  10. Pretreatment of Gymnema sylvestre revealed the protection against acetic acid-induced ulcerative colitis in rats

    PubMed Central

    2014-01-01

    Background Overproduction of free radicals and decreased antioxidant capacity are well-known risk factors for inflammatory bowel diseases. Gymnema sylvestre (GS) leaves extract is distinguished for its anti-diabetic, antioxidant and anti-inflammatory properties. Present study is designed to evaluate the preventative activities of GS against acetic acid (AA)-induced ulcerative colitis in Wistar rats. Methods Experimentally ulcerative colitis (UC) was induced by AA in animals pretreated with three different doses of GS leaves extract (50, 100, 200 mg/kg/day) and a single dose of mesalazine (MES, 300 mg/kg/day) for seven days. Twenty four hours later, animals were sacrificed and the colonic tissues were collected. Colonic mucus content was determined using Alcian blue dye binding technique. Levels of thiobarbituric acid reactive substances (TBARS), total glutathione sulfhydryl group (T-GSH) and non-protein sulfhydryl group (NPSH) as well as the activity of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) were estimated in colon tissues. Colonic nucleic acids (DNA and RNA) and total protein (TP) concentrations were also determined. Levels of pro-inflammatory cytokines including interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) as well as prostaglandin E2 (PGE2) and nitric oxide (NO) were estimated in colonic tissues. The histopathological changes of the colonic tissues were also observed. Results In AA administered group TBARS levels were increased, while colonic mucus content, T-GSH and NP-SH, SOD and CAT were reduced in colon. Pretreatment with GS inhibited TBARS elevation as well as mucus content, T-GSH and NP-SH reduction. Enzymatic activities of SOD and CAT were brought back to their normal levels in GS pretreated group. A significant reduction in DNA, RNA and TP levels was seen following AA administration and this inhibition was significantly eliminated by GS treatment. GS pretreatment also inhibited

  11. Individual and simultaneous determination of uric acid and ascorbic acid by flow injection analysis.

    PubMed

    Almuaibed, A M; Townshend, A

    1992-11-01

    Flow injection methods for the individual and simultaneous determination of ascorbic acid and uric acid are proposed. A spectrophotometer and a miniamperometric detector are connected in sequence. The calibration graphs for uric acid obtained by measuring its absorbance at 293 nm and its current at +0.6 V are linear up to at least 80 and 70 mug/ml, respectively, with an rsd (n = 10) of 1 % for both methods at mid-range concentrations. The calibration graph for ascorbic acid with amperometric detection is linear up to 80 mg/l. with an rsd (n = 10) of 0.8% at 30 mg/l. The simultaneous determination of uric acid and ascorbic acid is based on measurement of the absorbance of uric acid at 393 nm and amperometric determination of both analytes at +0.6 V. The average relative errors of the analysis of binary mixtures of uric acid and ascorbic acid are 2.2 and 4.2%, respectively. PMID:18965554

  12. Analysis of SSH library of rice variety Aganni reveals candidate gall midge resistance genes.

    PubMed

    Divya, Dhanasekar; Singh, Y Tunginba; Nair, Suresh; Bentur, J S

    2016-03-01

    The Asian rice gall midge, Orseolia oryzae, is a serious insect pest causing extensive yield loss. Interaction between the gall midge and rice genotypes is known to be on a gene-for-gene basis. Here, we report molecular basis of HR- (hypersensitive reaction-negative) type of resistance in Aganni (an indica rice variety possessing gall midge resistance gene Gm8) through the construction and analysis of a suppressive subtraction hybridization (SSH) cDNA library. In all, 2,800 positive clones were sequenced and analyzed. The high-quality ESTs were assembled into 448 non-redundant gene sequences. Homology search with the NCBI databases, using BlastX and BlastN, revealed that 73% of the clones showed homology to genes with known function and majority of ESTs belonged to the gene ontology category 'biological process'. Validation of 27 putative candidate gall midge resistance genes through real-time PCR, following gall midge infestation, in contrasting parents and their derived pre-NILs (near isogenic lines) revealed induction of specific genes related to defense and metabolism. Interestingly, four genes, belonging to families of leucine-rich repeat (LRR), heat shock protein (HSP), pathogenesis related protein (PR), and NAC domain-containing protein, implicated in conferring HR+ type of resistance, were found to be up-regulated in Aganni. Two of the reactive oxygen intermediates (ROI)-scavenging-enzyme-coding genes Cytosolic Ascorbate Peroxidase1, 2 (OsAPx1 and OsAPx2) were found up-regulated in Aganni in incompatible interaction possibly suppressing HR. We suggest that Aganni has a deviant form of inducible, salicylic acid (SA)-mediated resistance but without HR. PMID:26801786

  13. Acetic Acid Bacteria Genomes Reveal Functional Traits for Adaptation to Life in Insect Guts

    PubMed Central

    Chouaia, Bessem; Gaiarsa, Stefano; Crotti, Elena; Comandatore, Francesco; Degli Esposti, Mauro; Ricci, Irene; Alma, Alberto; Favia, Guido; Bandi, Claudio; Daffonchio, Daniele

    2014-01-01

    Acetic acid bacteria (AAB) live in sugar rich environments, including food matrices, plant tissues, and the gut of sugar-feeding insects. By comparing the newly sequenced genomes of Asaia platycodi and Saccharibacter sp., symbionts of Anopheles stephensi and Apis mellifera, respectively, with those of 14 other AAB, we provide a genomic view of the evolutionary pattern of this bacterial group and clues on traits that explain the success of AAB as insect symbionts. A specific pre-adaptive trait, cytochrome bo3 ubiquinol oxidase, appears ancestral in AAB and shows a phylogeny that is congruent with that of the genomes. The functional properties of this terminal oxidase might have allowed AAB to adapt to the diverse oxygen levels of arthropod guts. PMID:24682158

  14. Stable isotope tracer reveals that viviparous snakes transport amino acids to offspring during gestation.

    PubMed

    Van Dyke, James U; Beaupre, Steven J

    2012-03-01

    Viviparity and placentation have evolved from oviparity over 100 times in squamate reptiles (lizards and snakes). The independent origins of placentation have resulted in a variety of placental morphologies in different taxa, ranging from simple apposition of fetal and maternal tissues to endotheliochorial implantation that is homoplasious with mammalian placentation. Because the eggs of oviparous squamates transport gases and water from the environment and calcium from the eggshell, the placentae of viviparous squamates are thought to have initially evolved to accomplish these functions from within the maternal oviduct. Species with complex placentae have also been shown to rely substantially, or even primarily, on placental transport of organic nutrients for embryonic nutrition. However, it is unclear whether species with only simple placentae are also capable of transporting organic nutrients to offspring. Among viviparous squamates, all of the snakes that have been studied thus far have been shown to have simple placentae. However, most studies of snake placentation are limited to a single lineage, the North American Natricinae. We tested the abilities of four species of viviparous snakes - Agkistrodon contortrix (Viperidae), Boa constrictor (Boidae), Nerodia sipedon (Colubridae: Natricinae) and Thamnophis sirtalis (Colubridae: Natricinae) - to transport diet-derived amino acids to offspring during gestation. We fed [(15)N]leucine to pregnant snakes, and compared offspring (15)N content with that of unlabeled controls. Labeled females allocated significantly more (15)N to offspring than did controls, but (15)N allocation did not differ among species. Our results indicate that viviparous snakes are capable of transporting diet-derived amino acids to their offspring during gestation, possibly via placentation. PMID:22323198

  15. Global Phospholipidomics Analysis Reveals Selective Pulmonary Peroxidation Profiles Upon Inhalation of Single Walled Carbon Nanotubes

    PubMed Central

    Tyurina, Yulia Y.; Kisin, Elena R.; Murray, Ashley; Tyurin, Vladimir A.; Kapralova, Valentina I.; Sparvero, Louis J.; Amoscato, Andrew A.; Samhan-Arias, Alejandro K.; Swedin, Linda; Lahesmaa, Riitta; Fadeel, Bengt; Shvedova, Anna A.; Kagan, Valerian E.

    2011-01-01

    It is commonly believed that nanomaterials cause non-specific oxidative damage. Our mass spectrometry-based oxidative lipidomics analysis of all major phospholipid classes revealed highly selective patterns of pulmonary peroxidation after inhalation exposure of mice to single-walled carbon nanotubes. No oxidized molecular species were found in two most abundant phospholipid classes – phosphatidylcholine and phosphatidylethanolamine. Peroxidation products were identified in three relatively minor classes of anionic phospholipids, cardiolipin, phosphatidylserine and phosphatidylinositol whereby oxygenation of polyunsaturated fatty acid residues also showed unusual substrate specificity. This non-random peroxidation coincided with the accumulation of apoptotic cells in the lung. A similar selective phospholipid peroxidation profile was detected upon incubation of a mixture of total lung lipids with H2O2/cytochrome c known to catalyze cardiolipin and phosphatidylserine peroxidation in apoptotic cells. The characterized specific phospholipid peroxidation signaling pathways indicate new approaches to the development of mitochondria targeted regulators of cardiolipin peroxidation to protect against deleterious effects of pro-apoptotic effects of single-walled carbon nanotubes in the lung. PMID:21800898

  16. Global phospholipidomics analysis reveals selective pulmonary peroxidation profiles upon inhalation of single-walled carbon nanotubes.

    PubMed

    Tyurina, Yulia Y; Kisin, Elena R; Murray, Ashley; Tyurin, Vladimir A; Kapralova, Valentina I; Sparvero, Louis J; Amoscato, Andrew A; Samhan-Arias, Alejandro K; Swedin, Linda; Lahesmaa, Riitta; Fadeel, Bengt; Shvedova, Anna A; Kagan, Valerian E

    2011-09-27

    It is commonly believed that nanomaterials cause nonspecific oxidative damage. Our mass spectrometry-based oxidative lipidomics analysis of all major phospholipid classes revealed highly selective patterns of pulmonary peroxidation after inhalation exposure of mice to single-walled carbon nanotubes. No oxidized molecular species were found in the two most abundant phospholipid classes: phosphatidylcholine and phosphatidylethanolamine. Peroxidation products were identified in three relatively minor classes of anionic phospholipids, cardiolipin, phosphatidylserine, and phosphatidylinositol, whereby oxygenation of polyunsaturated fatty acid residues also showed unusual substrate specificity. This nonrandom peroxidation coincided with the accumulation of apoptotic cells in the lung. A similar selective phospholipid peroxidation profile was detected upon incubation of a mixture of total lung lipids with H(2)O(2)/cytochrome c known to catalyze cardiolipin and phosphatidylserine peroxidation in apoptotic cells. The characterized specific phospholipid peroxidation signaling pathways indicate new approaches to the development of mitochondria-targeted regulators of cardiolipin peroxidation to protect against deleterious effects of pro-apoptotic effects of single-walled carbon nanotubes in the lung. PMID:21800898

  17. Differential proteomic analysis of STAT6 knockout mice reveals new regulatory function in liver lipid homeostasis.

    PubMed

    Iff, Joël; Wang, Wei; Sajic, Tatjana; Oudry, Nathalie; Gueneau, Estelle; Hopfgartner, Gérard; Varesio, Emmanuel; Szanto, Ildiko

    2009-10-01

    Increased inflammatory signaling is a key feature of metabolic disorders. In this context, the role of increased pro-inflammatory signals has been extensively studied. By contrast, no efforts have been dedicated to study the contrasting scenario: the attenuation of anti-inflammatory signals and their role in metabolic homeostasis. IL-4 and IL-13 are anti-inflammatory cytokines signaling through the Signal Transducer and Activator of Transcription 6 (STAT6). Our study was aimed at evaluating the lack of STAT6 signaling on liver homeostasis. To this end we analyzed the liver proteome of wild type and STAT6 knock-out mice using 2D nanoscale LC-MS/MS with iTRAQ labeling technique. The coordinated changes in proteins identified by this quantitative proteome analysis indicated disturbed lipid homeostasis and a state of hepatocellular stress. Most significantly, the expression of the liver fatty acid binding protein (FABP1) was increased in the knock-out mice. In line with the elevated FABP1 expression we found latent liver lipid accumulation in the STAT6-deficient mice which was further aggravated when mice were challenged by a high fat diet. In conclusion, our study revealed a so far uncharacterized role for STAT6 in regulating liver lipid homeostasis and demonstrates the importance of anti-inflammatory signaling in the defense against the development of liver steatosis. PMID:19663508

  18. Genomic Analysis Reveals the Molecular Basis for Capsule Loss in the Group B Streptococcus Population

    PubMed Central

    Rosini, Roberto; Campisi, Edmondo; De Chiara, Matteo; Tettelin, Hervé; Rinaudo, Daniela; Toniolo, Chiara; Metruccio, Matteo; Guidotti, Silvia; Sørensen, Uffe B. Skov; Kilian, Mogens; Ramirez, Mario; Janulczyk, Robert; Donati, Claudio; Grandi, Guido; Margarit, Immaculada

    2015-01-01

    The human and bovine bacterial pathogen Streptococcus agalactiae (Group B Streptococcus, GBS) expresses a thick polysaccharide capsule that constitutes a major virulence factor and vaccine target. GBS can be classified into ten distinct serotypes differing in the chemical composition of their capsular polysaccharide. However, non-typeable strains that do not react with anti-capsular sera are frequently isolated from colonized and infected humans and cattle. To gain a comprehensive insight into the molecular basis for the loss of capsule expression in GBS, a collection of well-characterized non-typeable strains was investigated by genome sequencing. Genome based phylogenetic analysis extended to a wide population of sequenced strains confirmed the recently observed high clonality among GBS lineages mainly containing human strains, and revealed a much higher degree of diversity in the bovine population. Remarkably, non-typeable strains were equally distributed in all lineages. A number of distinct mutations in the cps operon were identified that were apparently responsible for inactivation of capsule synthesis. The most frequent genetic alterations were point mutations leading to stop codons in the cps genes, and the main target was found to be cpsE encoding the portal glycosyl trasferase of capsule biosynthesis. Complementation of strains carrying missense mutations in cpsE with a wild-type gene restored capsule expression allowing the identification of amino acid residues essential for enzyme activity. PMID:25946017

  19. Genomic analysis reveals the molecular basis for capsule loss in the group B Streptococcus population.

    PubMed

    Rosini, Roberto; Campisi, Edmondo; De Chiara, Matteo; Tettelin, Hervé; Rinaudo, Daniela; Toniolo, Chiara; Metruccio, Matteo; Guidotti, Silvia; Sørensen, Uffe B Skov; Kilian, Mogens; Ramirez, Mario; Janulczyk, Robert; Donati, Claudio; Grandi, Guido; Margarit, Immaculada

    2015-01-01

    The human and bovine bacterial pathogen Streptococcus agalactiae (Group B Streptococcus, GBS) expresses a thick polysaccharide capsule that constitutes a major virulence factor and vaccine target. GBS can be classified into ten distinct serotypes differing in the chemical composition of their capsular polysaccharide. However, non-typeable strains that do not react with anti-capsular sera are frequently isolated from colonized and infected humans and cattle. To gain a comprehensive insight into the molecular basis for the loss of capsule expression in GBS, a collection of well-characterized non-typeable strains was investigated by genome sequencing. Genome based phylogenetic analysis extended to a wide population of sequenced strains confirmed the recently observed high clonality among GBS lineages mainly containing human strains, and revealed a much higher degree of diversity in the bovine population. Remarkably, non-typeable strains were equally distributed in all lineages. A number of distinct mutations in the cps operon were identified that were apparently responsible for inactivation of capsule synthesis. The most frequent genetic alterations were point mutations leading to stop codons in the cps genes, and the main target was found to be cpsE encoding the portal glycosyl transferase of capsule biosynthesis. Complementation of strains carrying missense mutations in cpsE with a wild-type gene restored capsule expression allowing the identification of amino acid residues essential for enzyme activity. PMID:25946017

  20. Comparative genomic analysis of Lactobacillus plantarum ZJ316 reveals its genetic adaptation and potential probiotic profiles* #

    PubMed Central

    Li, Ping; Li, Xuan; Gu, Qing; Lou, Xiu-yu; Zhang, Xiao-mei; Song, Da-feng; Zhang, Chen

    2016-01-01

    Objective: In previous studies, Lactobacillus plantarum ZJ316 showed probiotic properties, such as antimicrobial activity against various pathogens and the capacity to significantly improve pig growth and pork quality. The purpose of this study was to reveal the genes potentially related to its genetic adaptation and probiotic profiles based on comparative genomic analysis. Methods: The genome sequence of L. plantarum ZJ316 was compared with those of eight L. plantarum strains deposited in GenBank. BLASTN, Mauve, and MUMmer programs were used for genome alignment and comparison. CRISPRFinder was applied for searching the clustered regularly interspaced short palindromic repeats (CRISPRs). Results: We identified genes that encode proteins related to genetic adaptation and probiotic profiles, including carbohydrate transport and metabolism, proteolytic enzyme systems and amino acid biosynthesis, CRISPR adaptive immunity, stress responses, bile salt resistance, ability to adhere to the host intestinal wall, exopolysaccharide (EPS) biosynthesis, and bacteriocin biosynthesis. Conclusions: Comparative characterization of the L. plantarum ZJ316 genome provided the genetic basis for further elucidating the functional mechanisms of its probiotic properties. ZJ316 could be considered a potential probiotic candidate. PMID:27487802

  1. Comparative genome analysis reveals the molecular basis of nicotine degradation and survival capacities of Arthrobacter

    PubMed Central

    Yao, Yuxiang; Tang, Hongzhi; Su, Fei; Xu, Ping

    2015-01-01

    Arthrobacter is one of the most prevalent genera of nicotine-degrading bacteria; however, studies of nicotine degradation in Arthrobacter species remain at the plasmid level (plasmid pAO1). Here, we report the bioinformatic analysis of a nicotine-degrading Arthrobacter aurescens M2012083, and show that the moeB and mogA genes that are essential for nicotine degradation in Arthrobacter are absent from plasmid pAO1. Homologues of all the nicotine degradation-related genes of plasmid pAO1 were found to be located on a 68,622-bp DNA segment (nic segment-1) in the M2012083 genome, showing 98.1% nucleotide acid sequence identity to the 69,252-bp nic segment of plasmid pAO1. However, the rest sequence of plasmid pAO1 other than the nic segment shows no significant similarity to the genome sequence of strain M2012083. Taken together, our data suggest that the nicotine degradation-related genes of strain M2012083 are located on the chromosome or a plasmid other than pAO1. Based on the genomic sequence comparison of strain M2012083 and six other Arthrobacter strains, we have identified 17 σ70 transcription factors reported to be involved in stress responses and 109 genes involved in environmental adaptability of strain M2012083. These results reveal the molecular basis of nicotine degradation and survival capacities of Arthrobacter species. PMID:25721465

  2. In silico analysis of missense mutations in LPAR6 reveals abnormal phospholipid signaling pathway leading to hypotrichosis.

    PubMed

    Raza, Syed Irfan; Muhammad, Dost; Jan, Abid; Ali, Raja Hussain; Hassan, Mubashir; Ahmad, Wasim; Rashid, Sajid

    2014-01-01

    Autosomal recessive hypotrichosis is a rare genetic irreversible hair loss disorder characterized by sparse scalp hair, sparse to absent eyebrows and eyelashes, and sparse axillary and body hair. The study, presented here, established genetic linkage in four families showing similar phenotypes to lysophosphatidic acid receptor 6 (LPAR6) gene on chromosome 13q14.11-q21.32. Subsequently, sequence analysis of the gene revealed two previously reported missense mutations including p.D63V in affected members of one and p.I188F in three other families. Molecular modeling and docking analysis was performed to investigate binding of a ligand oleoyl-L-alpha-lysophosphatidic acid (LPA) to modeled protein structures of normal and mutated (D63V, G146R, I188F, N248Y, S3T, L277P) LPAR6 receptors. The mutant receptors showed a complete shift in orientation of LPA at the binding site. In addition, hydropathy analysis revealed a significant change in the membrane spanning topology of LPAR6 helical segments. The present study further substantiated involvement of LPAR6-LPA signaling in the pathogenesis of hypotrichosis/woolly hair and provided additional insight into the molecular mechanism of hair development. PMID:25119526

  3. In Silico Analysis of Missense Mutations in LPAR6 Reveals Abnormal Phospholipid Signaling Pathway Leading to Hypotrichosis

    PubMed Central

    Raza, Syed Irfan; Muhammad, Dost; Jan, Abid; Ali, Raja Hussain; Hassan, Mubashir; Ahmad, Wasim; Rashid, Sajid

    2014-01-01

    Autosomal recessive hypotrichosis is a rare genetic irreversible hair loss disorder characterized by sparse scalp hair, sparse to absent eyebrows and eyelashes, and sparse axillary and body hair. The study, presented here, established genetic linkage in four families showing similar phenotypes to lysophosphatidic acid receptor 6 (LPAR6) gene on chromosome 13q14.11-q21.32. Subsequently, sequence analysis of the gene revealed two previously reported missense mutations including p.D63V in affected members of one and p.I188F in three other families. Molecular modeling and docking analysis was performed to investigate binding of a ligand oleoyl-L-alpha-lysophosphatidic acid (LPA) to modeled protein structures of normal and mutated (D63V, G146R, I188F, N248Y, S3T, L277P) LPAR6 receptors. The mutant receptors showed a complete shift in orientation of LPA at the binding site. In addition, hydropathy analysis revealed a significant change in the membrane spanning topology of LPAR6 helical segments. The present study further substantiated involvement of LPAR6-LPA signaling in the pathogenesis of hypotrichosis/woolly hair and provided additional insight into the molecular mechanism of hair development. PMID:25119526

  4. Protons and Psalmotoxin-1 reveal nonproton ligand stimulatory sites in chicken acid-sensing ion channel

    PubMed Central

    Smith, Rachel N; Gonzales, Eric B

    2014-01-01

    Acid-sensing ion channels (ASICs) are proton-sensitive, sodium-selective channels expressed in the nervous system that sense changes in extracellular pH. These ion channels are sensitive to an increasing number of nonproton ligands that include natural venom peptides and guanidine compounds. In the case of chicken ASIC1, the spider toxin Psalmotoxin-1 (PcTx1) activates the channel, resulting in an inward current. Furthermore, a growing class of ligands containing a guanidine group has been identified that stimulate peripheral ASICs (ASIC3), but exert subtle influence on other ASIC subtypes. The effects of the guanidine compounds on cASIC1 have not been the focus of previous study. Here, we investigated the interaction of the guanidine compound 2-guanidine-4-methylquinazoline (GMQ) on cASIC1 proton activation and PcTx1 stimulation. Exposure of expressed cASIC1 to PcTx1 resulted in biphasic currents consisting of a transient peak followed by an irreversible cASIC1 PcTx1 persistent current. This cASIC1 PcTx1 persistent current may be the result of locking the cASIC1 protein into a desensitized transition state. The guanidine compound GMQ increased the apparent affinity of protons on cASIC1 and decreased the half-maximal constant of the cASIC1 steady-state desensitization profile. Furthermore, GMQ stimulated the cASIC1 PcTx1 persistent current in a concentration-dependent manner, which resulted in a non-desensitizing inward current. Our data suggests that GMQ may have multiple sites within cASIC1 and may act as a “molecular wedge” that forces the PcTx1-desensitized ASIC into an open state. Our findings indicate that guanidine compounds, such as GMQ, may alter acid-sensing ion channel activity in combination with other stimuli, and that additional ASIC subtypes (along with ASIC3) may serve to sense and mediate signals from multiple stimuli. PMID:24262969

  5. Characterization of VuMATE1 Expression in Response to Iron Nutrition and Aluminum Stress Reveals Adaptation of Rice Bean (Vigna umbellata) to Acid Soils through Cis Regulation

    PubMed Central

    Liu, Meiya; Xu, Jiameng; Lou, Heqiang; Fan, Wei; Yang, Jianli; Zheng, Shaojian

    2016-01-01

    Rice bean (Vigna umbellata) VuMATE1 appears to be constitutively expressed at vascular system but root apex, and Al stress extends its expression to root apex. Whether VuMATE1 participates in both Al tolerance and Fe nutrition, and how VuMATE1 expression is regulated is of great interest. In this study, the role of VuMATE1 in Fe nutrition was characterized through in planta complementation assays. The transcriptional regulation of VuMATE1 was investigated through promoter analysis and promoter-GUS reporter assays. The results showed that the expression of VuMATE1 was regulated by Al stress but not Fe status. Complementation of frd3-1 with VuMATE1 under VuMATE1 promoter could not restore phenotype, but restored with 35SCaMV promoter. Immunostaining of VuMATE1 revealed abnormal localization of VuMATE1 in vasculature. In planta GUS reporter assay identified Al-responsive cis-acting elements resided between -1228 and -574 bp. Promoter analysis revealed several cis-acting elements, but transcription is not simply regulated by one of these elements. We demonstrated that cis regulation of VuMATE1 expression is involved in Al tolerance mechanism, while not involved in Fe nutrition. These results reveal the evolution of VuMATE1 expression for better adaptation of rice bean to acid soils where Al stress imposed but Fe deficiency pressure released. PMID:27148333

  6. Characterization of VuMATE1 Expression in Response to Iron Nutrition and Aluminum Stress Reveals Adaptation of Rice Bean (Vigna umbellata) to Acid Soils through Cis Regulation.

    PubMed

    Liu, Meiya; Xu, Jiameng; Lou, Heqiang; Fan, Wei; Yang, Jianli; Zheng, Shaojian

    2016-01-01

    Rice bean (Vigna umbellata) VuMATE1 appears to be constitutively expressed at vascular system but root apex, and Al stress extends its expression to root apex. Whether VuMATE1 participates in both Al tolerance and Fe nutrition, and how VuMATE1 expression is regulated is of great interest. In this study, the role of VuMATE1 in Fe nutrition was characterized through in planta complementation assays. The transcriptional regulation of VuMATE1 was investigated through promoter analysis and promoter-GUS reporter assays. The results showed that the expression of VuMATE1 was regulated by Al stress but not Fe status. Complementation of frd3-1 with VuMATE1 under VuMATE1 promoter could not restore phenotype, but restored with 35SCaMV promoter. Immunostaining of VuMATE1 revealed abnormal localization of VuMATE1 in vasculature. In planta GUS reporter assay identified Al-responsive cis-acting elements resided between -1228 and -574 bp. Promoter analysis revealed several cis-acting elements, but transcription is not simply regulated by one of these elements. We demonstrated that cis regulation of VuMATE1 expression is involved in Al tolerance mechanism, while not involved in Fe nutrition. These results reveal the evolution of VuMATE1 expression for better adaptation of rice bean to acid soils where Al stress imposed but Fe deficiency pressure released. PMID:27148333

  7. A lysophosphatidic acid analogue is revealed as a potent inhibitor of phosphatidylcholine synthesis, inducing apoptosis.

    PubMed Central

    Gueguen, Geneviéve; Granci, Virginie; Rogalle, Pierre; Briand-Mésange, Fabienne; Wilson, Michéle; Klaébé, Alain; Tercé, François; Chap, Hugues; Salles, Jean-Pierre; Simon, Marie-Françoise; Gaits, Frédérique

    2002-01-01

    A previous study demonstrated that cross-desensitization experiments performed with the lysophosphatidic acid (LPA) analogues (R)- and (S)-N-palmitoyl-norleucinol 1-phosphate (PNPAs) inhibited LPA-induced platelet aggregation without any stereospecificity. Here we report opposite biological effects of the two enantiomers on mitogenesis of IMR-90 fibroblasts in relation to their respective metabolism. (R)PNPA was proliferative, while (S)PNPA induced apoptosis by specifically inhibiting phosphatidylcholine biosynthesis at the last step of the CDP-choline pathway controlled by cholinephosphotransferase. This effect was not direct but required dephosphorylation of PNPAs by ecto-lipid phosphate phosphatase before cellular uptake of the generated N-palmitoyl-norleucinols (PNOHs). Inhibition of cholinephosphotransferase by the derivative (S)PNOH was confirmed by an in vitro assay. (S)PNPA proapoptotic effects led us to clarify the mechanism linking cholinephosphotransferase inhibition to apoptosis. Three proapoptotic responses were observed: the activation of caspase-3, the production of ceramides from newly synthesized pools (as demonstrated by the inhibitor Fumonisin B1) and finally the activation of stress-activated protein kinase, p38 and c-Jun N-terminal kinases 1/2, as a result of ceramide increase. Thus our data demonstrate that synthetic analogues of LPA might display stereospecific effects leading to apoptosis independently of classical LPA-activated pathways. PMID:12197836

  8. Competing mechanisms for perfluoroalkyl acid accumulation in plants revealed using an Arabidopsis model system.

    PubMed

    Müller, Claudia E; LeFevre, Gregory H; Timofte, Anca E; Hussain, Fatima A; Sattely, Elizabeth S; Luthy, Richard G

    2016-05-01

    Perfluoroalkyl acids (PFAAs) bioaccumulate in plants, presenting a human exposure route if present in irrigation water. Curiously, accumulation of PFAAs in plant tissues is greatest for both the short-chain and long-chain PFAAs, generating a U-shaped relationship with chain length. In the present study, the authors decouple competing mechanisms of PFAA accumulation using a hydroponic model plant system (Arabidopsis thaliana) exposed to a suite of 10 PFAAs to determine uptake, depuration, and translocation kinetics. Rapid saturation of root concentrations occurred for all PFAAs except perfluorobutanoate, the least-sorptive (shortest-chain) PFAA. Shoot concentrations increased continuously, indicating that PFAAs are efficiently transported and accumulate in shoots. Tissue concentrations of PFAAs during depuration rapidly declined in roots but remained constant in shoots, demonstrating irreversibility of the translocation process. Root and shoot concentration factors followed the U-shaped trend with perfluoroalkyl chain length; however, when normalized to dead-tissue sorption, this relationship linearized. The authors therefore introduce a novel term, the "sorption normalized concentration factor," to describe PFAA accumulation in plants; because of their hydrophobicity, sorption is the determining factor for long-chain PFAAs, whereas the shortest-chain PFAAs are most effectively transported in the plant. The present study provides a mechanistic explanation for previously unexplained PFAA accumulation trends in plants and suggests that shorter-chained PFAAs may bioaccumulate more readily in edible portions. Environ Toxicol Chem 2016;35:1138-1147. © 2015 SETAC. PMID:26383989

  9. Quantitative Analysis of Global Proteome and Lysine Acetylome Reveal the Differential Impacts of VPA and SAHA on HL60 Cells.

    PubMed

    Zhu, Xiaoyu; Liu, Xin; Cheng, Zhongyi; Zhu, Jun; Xu, Lei; Wang, Fengsong; Qi, Wulin; Yan, Jiawei; Liu, Ning; Sun, Zimin; Liu, Huilan; Peng, Xiaojun; Hao, Yingchan; Zheng, Nan; Wu, Quan

    2016-01-01

    Valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) are both HDAC inhibitors (HDACi). Previous studies indicated that both inhibitors show therapeutic effects on acute myeloid leukaemia (AML), while the differential impacts of the two different HDACi on AML treatment still remains elusive. In this study, using 3-plex SILAC based quantitative proteomics technique, anti-acetyllysine antibody based affinity enrichment, high resolution LC-MS/MS and intensive bioinformatic analysis, the quantitative proteome and acetylome in SAHA and VPA treated AML HL60 cells were extensively studied. In total, 5,775 proteins and 1,124 lysine acetylation sites were successfully obtained in response to VAP and SAHA treatment. It is found that VPA and SAHA treatment differently induced proteome and acetylome profiling in AML HL60 cells. This study revealed the differential impacts of VPA and SAHA on proteome/acetylome in AML cells, deepening our understanding of HDAC inhibitor mediated AML therapeutics. PMID:26822725

  10. Quantitative Analysis of Global Proteome and Lysine Acetylome Reveal the Differential Impacts of VPA and SAHA on HL60 Cells

    PubMed Central

    Zhu, Xiaoyu; Liu, Xin; Cheng, Zhongyi; Zhu, Jun; Xu, Lei; Wang, Fengsong; Qi, Wulin; Yan, Jiawei; Liu, Ning; Sun, Zimin; Liu, Huilan; Peng, Xiaojun; Hao, Yingchan; Zheng, Nan; Wu, Quan

    2016-01-01

    Valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) are both HDAC inhibitors (HDACi). Previous studies indicated that both inhibitors show therapeutic effects on acute myeloid leukaemia (AML), while the differential impacts of the two different HDACi on AML treatment still remains elusive. In this study, using 3-plex SILAC based quantitative proteomics technique, anti-acetyllysine antibody based affinity enrichment, high resolution LC-MS/MS and intensive bioinformatic analysis, the quantitative proteome and acetylome in SAHA and VPA treated AML HL60 cells were extensively studied. In total, 5,775 proteins and 1,124 lysine acetylation sites were successfully obtained in response to VAP and SAHA treatment. It is found that VPA and SAHA treatment differently induced proteome and acetylome profiling in AML HL60 cells. This study revealed the differential impacts of VPA and SAHA on proteome/acetylome in AML cells, deepening our understanding of HDAC inhibitor mediated AML therapeutics. PMID:26822725

  11. Exploration of Sulfur Assimilation of Aspergillus fumigatus Reveals Biosynthesis of Sulfur-Containing Amino Acids as a Virulence Determinant.

    PubMed

    Amich, Jorge; Dümig, Michaela; O'Keeffe, Gráinne; Binder, Jasmin; Doyle, Sean; Beilhack, Andreas; Krappmann, Sven

    2016-04-01

    Fungal infections are of major relevance due to the increased numbers of immunocompromised patients, frequently delayed diagnosis, and limited therapeutics. To date, the growth and nutritional requirements of fungi during infection, which are relevant for invasion of the host, are poorly understood. This is particularly true for invasive pulmonary aspergillosis, as so far, sources of (macro)elements that are exploited during infection have been identified to only a limited extent. Here, we have investigated sulfur (S) utilization by the human-pathogenic mold Aspergillus fumigatus during invasive growth. Our data reveal that inorganic S compounds or taurine is unlikely to serve as an S source during invasive pulmonary aspergillosis since a sulfate transporter mutant strain and a sulfite reductase mutant strain are fully virulent. In contrast, the S-containing amino acid cysteine is limiting for fungal growth, as proven by the reduced virulence of a cysteine auxotroph. Moreover, phenotypic characterization of this strain further revealed the robustness of the subordinate glutathione redox system. Interestingly, we demonstrate that methionine synthase is essential for A. fumigatus virulence, defining the biosynthetic route of this proteinogenic amino acid as a potential antifungal target. In conclusion, we provide novel insights into the nutritional requirements ofA. fumigatus during pathogenesis, a prerequisite to understanding and fighting infection. PMID:26787716

  12. A High-Throughput Fatty Acid Profiling Screen Reveals Novel Variations in Fatty Acid Biosynthesis in Chlamydomonas reinhardtii and Related Algae

    PubMed Central

    Pflaster, Erin L.; Schwabe, Michael J.; Becker, Joyanne; Wilkinson, Melissa S.; Parmer, Ashley; Clemente, Thomas E.; Cahoon, Edgar B.

    2014-01-01

    Analysis of fatty acid methyl esters (FAMEs) by gas chromatography (GC) is a common technique for the quantitative and qualitative analysis of acyl lipids. Methods for FAME preparation are typically time-consuming and labor-intensive and require multiple transfers of reagents and products between reaction tubes and autosampler vials. In order to increase throughput and lower the time and materials costs required for FAME preparation prior to GC analysis, we have developed a method in which 10-to-20-mg samples of microbial biomass are transferred to standard GC autosampler vials, transesterified using an emulsion of methanolic trimethylsulfonium hydroxide and hexane, and analyzed directly by GC without further sample handling. This method gives results that are essentially identical to those obtained by the more labor- and material-intensive FAME preparation methods, such as transmethylation with methanolic HCl. We applied this method to the screening of laboratory and environmental isolates of the green alga Chlamydomonas for variations in fatty acid composition. This screening method facilitated two novel discoveries. First, we identified a common laboratory strain of C. reinhardtii, CC-620, completely lacking all ω-3 fatty acids normally found in this organism and showed that this strain contains an inactivating mutation in the CrFAD7 gene, encoding the sole ω-3 desaturase activity in this organism. Second, we showed that some species of Chlamydomonas make Δ6-unsaturated polyunsaturated fatty acids (PUFA) rather than the Δ5 species normally made by the previously characterized laboratory strains of Chlamydomonas, suggesting that there is species-specific variation in the regiospecificity and substrate selectivity of front-end desaturases in this algal genus. PMID:25239975

  13. Proteomic analysis of chromoplasts from six crop species reveals insights into chromoplast function and development.

    PubMed

    Wang, Yong-Qiang; Yang, Yong; Fei, Zhangjun; Yuan, Hui; Fish, Tara; Thannhauser, Theodore W; Mazourek, Michael; Kochian, Leon V; Wang, Xiaowu; Li, Li

    2013-02-01

    Chromoplasts are unique plastids that accumulate massive amounts of carotenoids. To gain a general and comparative characterization of chromoplast proteins, this study performed proteomic analysis of chromoplasts from six carotenoid-rich crops: watermelon, tomato, carrot, orange cauliflower, red papaya, and red bell pepper. Stromal and membrane proteins of chromoplasts were separated by 1D gel electrophoresis and analysed using nLC-MS/MS. A total of 953-2262 proteins from chromoplasts of different crop species were identified. Approximately 60% of the identified proteins were predicted to be plastid localized. Functional classification using MapMan bins revealed large numbers of proteins involved in protein metabolism, transport, amino acid metabolism, lipid metabolism, and redox in chromoplasts from all six species. Seventeen core carotenoid metabolic enzymes were identified. Phytoene synthase, phytoene desaturase, ζ-carotene desaturase, 9-cis-epoxycarotenoid dioxygenase, and carotenoid cleavage dioxygenase 1 were found in almost all crops, suggesting relative abundance of them among the carotenoid pathway enzymes. Chromoplasts from different crops contained abundant amounts of ATP synthase and adenine nucleotide translocator, which indicates an important role of ATP production and transport in chromoplast development. Distinctive abundant proteins were observed in chromoplast from different crops, including capsanthin/capsorubin synthase and fibrillins in pepper, superoxide dismutase in watermelon, carrot, and cauliflower, and glutathione-S-transferease in papaya. The comparative analysis of chromoplast proteins among six crop species offers new insights into the general metabolism and function of chromoplasts as well as the uniqueness of chromoplasts in specific crop species. This work provides reference datasets for future experimental study of chromoplast biogenesis, development, and regulation in plants. PMID:23314817

  14. Proteomic Analysis of Ketogulonicigenium vulgare under Glutathione Reveals High Demand for Thiamin Transport and Antioxidant Protection

    PubMed Central

    Ma, Qian; Zhang, Weiwen; Zhang, Lu; Qiao, Bin; Pan, Chensong; Yi, Hong; Wang, Lili; Yuan, Ying-jin

    2012-01-01

    Ketogulonicigenium vulgare, though grows poorly when mono-cultured, has been widely used in the industrial production of the precursor of vitamin C with the coculture of Bacillus megaterium. Various efforts have been made to clarify the synergic pattern of this artificial microbial community and to improve the growth and production ability of K. vulgare, but there is still no sound explanation. In previous research, we found that the addition of reduced glutathione into K. vulgare monoculture could significantly improve its growth and productivity. By performing SEM and TEM, we observed that after adding GSH into K. vulgare monoculture, cells became about 4–6 folds elongated, and formed intracytoplasmic membranes (ICM). To explore the molecular mechanism and provide insights into the investigation of the synergic pattern of the co-culture system, we conducted a comparative iTRAQ-2-D-LC-MS/MS-based proteomic analysis of K. vulgare grown under reduced glutathione. Principal component analysis of proteomic data showed that after the addition of glutathione, proteins for thiamin/thiamin pyrophosphate (TPP) transport, glutathione transport and the maintenance of membrane integrity, together with several membrane-bound dehydrogenases had significant up-regulation. Besides, several proteins participating in the pentose phosphate pathway and tricarboxylic acid cycle were also up-regulated. Additionally, proteins combating intracellular reactive oxygen species were also up-regulated, which similarly occurred in K. vulgare when the co-cultured B. megaterium cells lysed from our former research results. This study reveals the demand for transmembrane transport of substrates, especially thiamin, and the demand for antioxidant protection of K. vulgare. PMID:22384164

  15. Intramolecular allosteric communication in dopamine D2 receptor revealed by evolutionary amino acid covariation

    PubMed Central

    Sung, Yun-Min; Wilkins, Angela D.; Rodriguez, Gustavo J.; Wensel, Theodore G.; Lichtarge, Olivier

    2016-01-01

    The structural basis of allosteric signaling in G protein-coupled receptors (GPCRs) is important in guiding design of therapeutics and understanding phenotypic consequences of genetic variation. The Evolutionary Trace (ET) algorithm previously proved effective in redesigning receptors to mimic the ligand specificities of functionally distinct homologs. We now expand ET to consider mutual information, with validation in GPCR structure and dopamine D2 receptor (D2R) function. The new algorithm, called ET-MIp, identifies evolutionarily relevant patterns of amino acid covariations. The improved predictions of structural proximity and D2R mutagenesis demonstrate that ET-MIp predicts functional interactions between residue pairs, particularly potency and efficacy of activation by dopamine. Remarkably, although most of the residue pairs chosen for mutagenesis are neither in the binding pocket nor in contact with each other, many exhibited functional interactions, implying at-a-distance coupling. The functional interaction between the coupled pairs correlated best with the evolutionary coupling potential derived from dopamine receptor sequences rather than with broader sets of GPCR sequences. These data suggest that the allosteric communication responsible for dopamine responses is resolved by ET-MIp and best discerned within a short evolutionary distance. Most double mutants restored dopamine response to wild-type levels, also suggesting that tight regulation of the response to dopamine drove the coevolution and intramolecular communications between coupled residues. Our approach provides a general tool to identify evolutionary covariation patterns in small sets of close sequence homologs and to translate them into functional linkages between residues. PMID:26979958

  16. Intramolecular allosteric communication in dopamine D2 receptor revealed by evolutionary amino acid covariation.

    PubMed

    Sung, Yun-Min; Wilkins, Angela D; Rodriguez, Gustavo J; Wensel, Theodore G; Lichtarge, Olivier

    2016-03-29

    The structural basis of allosteric signaling in G protein-coupled receptors (GPCRs) is important in guiding design of therapeutics and understanding phenotypic consequences of genetic variation. The Evolutionary Trace (ET) algorithm previously proved effective in redesigning receptors to mimic the ligand specificities of functionally distinct homologs. We now expand ET to consider mutual information, with validation in GPCR structure and dopamine D2 receptor (D2R) function. The new algorithm, called ET-MIp, identifies evolutionarily relevant patterns of amino acid covariations. The improved predictions of structural proximity and D2R mutagenesis demonstrate that ET-MIp predicts functional interactions between residue pairs, particularly potency and efficacy of activation by dopamine. Remarkably, although most of the residue pairs chosen for mutagenesis are neither in the binding pocket nor in contact with each other, many exhibited functional interactions, implying at-a-distance coupling. The functional interaction between the coupled pairs correlated best with the evolutionary coupling potential derived from dopamine receptor sequences rather than with broader sets of GPCR sequences. These data suggest that the allosteric communication responsible for dopamine responses is resolved by ET-MIp and best discerned within a short evolutionary distance. Most double mutants restored dopamine response to wild-type levels, also suggesting that tight regulation of the response to dopamine drove the coevolution and intramolecular communications between coupled residues. Our approach provides a general tool to identify evolutionary covariation patterns in small sets of close sequence homologs and to translate them into functional linkages between residues. PMID:26979958

  17. Ion Chromatography Analysis of Dibutyl Phosphoric Acid

    SciTech Connect

    Ray, R.J.

    1998-12-04

    Analysis of dibutyl phosphate (DBP), a degradation product of tributyl phosphate (TBP), has long been a problem analysis by Ion Chromatography at the Savannah River Site. Due to the presence of UO{sub 2}{sup +2} and high NO{sub 3}{sup {minus}1} concentrations, inadequate recovery and separation of DBP on the chromatographic column had rendered the analysis undependable and very inconsistent, thus causing high uncertainties in the data. The method presented here by the Savannah River Technology Center (SRTC)/Analytical Development Section (ADS) addresses the sample preparation problems encountered when analyzing for DBP in the presence of uranium and nitrate. The data presented reflects the improvements made to decrease data uncertainty and increase data accuracy and precision.

  18. SEM ANALYSIS OF THE ACID-ETCHED ENAMEL PATTERNS PROMOTED BY ACIDIC MONOMERS AND PHOSPHORIC ACIDS

    PubMed Central

    Shinohara, Mirela Sanae; de Oliveira, Marcelo Tavares; Hipólito, Vinícius Di; Giannin, Marcelo; de Goes, Mario Fernando

    2006-01-01

    Objective: Although self-etching bonding systems (SES) are indicated to prepare dental enamel for bonding, concerns have been expressed regarding their effectiveness. The aim of this study was to analyze the etching pattern (EP) of nine SES in comparison with 35% and 34% phosphoric acid etchants (FA) on intact (IN) and ground (GR) enamel surface. Materials and Methods: Twenty-two human third molars were sectioned in mesial-distal and buccal-lingual directions, and four dental fragments were obtained from each tooth. Half of the fragments were ground using 600-grit SiC paper and the other half remained intact. The fragments were randomly assigned into 22 groups, according to the texture of enamel surface (IN and GR) and the technique to etch the enamel (34% FA, 35% FA, AdheSE primer; Brush & Bond; Clearfil Protect Bond primer; iBond; One-up Bond F; OptiBond Solo Plus primer; Tyrian SPE primer; Unifil Bond primer and Xeno III). Conditioners were applied to IN and GR enamel surfaces, according to the manufacturer's instructions. Specimens etched with phosphoric acids were washed with water, while the surfaces treated with SES were submitted to alternate rinsing with alcohol and acetone. The specimens were dried, sputter-coated and examined under a scanning electron microscope. Results: For both IN and GR enamel surfaces, the EP of 34 and 35% FA was deeper and more homogeneous in comparison to EP of SES, except for Tyrian SPE. The acidic monomer action of self-etching systems was more effective on GR enamel. Conclusion: Most of the SES are less aggressive than phosphoric acid etchants and their etching effects were reduced on intact enamel surfaces. Uniterms: Dental acid etching; Dental enamel; Electron microscopy. PMID:19089243

  19. Typing of Histoplasma capsulatum strains by fatty acid profile analysis

    PubMed Central

    Zarnowski, Robert; Miyazaki, Makoto; Dobrzyn, Agnieszka; Ntambi, James M.; Woods, Jon P.

    2009-01-01

    The performance of fatty acid profiling for strain differentiation of Histoplasma capsulatum was assessed. Total fatty acids were isolated from the yeast-phase cells of seven stock and two previously unreported clinical strains of H. capsulatum var. capsulatum, as well as from one unreported clinical strain and one stock strain of H. capsulatum var. duboisii, and one strain of each of three other dimorphic zoopathogenic fungal species, Blastomyces dermatitidis, Paracoccidioides brasiliensis and Sporothrix schenckii. Different colony morphology and pigmentation types of the H. capsulatum strains were also included. The most frequently occurring fatty acids were oleic, palmitic, stearic and linoleic acids. There were variations in the relative percentage fatty acid contents of H. capsulatum strains that could be used for strain identification and discrimination. Differentiation between H. capsulatum strains was achieved by the comparison of detected fatty acids accompanied by principal component analysis using calculated Varimax-rotated principal component loadings. Statistical analysis yielded three major principal components that explained over 94% of total variance in the data. All the strains of H. capsulatum var. capsulatum RFLP classes II and III were grouped into two distinct clusters: the heterogenic RFLP class I formed a large, but also well-defined group, whereas the outgroup strains of H. capsulatum var. duboisii, B. dermatitidis, P. brasiliensis and S. schenckii were shifted away. These data suggest that fatty acid profiling can be used in H. capsulatum strain classification and epidemiological studies that require strain differentiation at the intraspecies level. PMID:17510264

  20. Function of Several Critical Amino Acids in Human Pyruvate Dehydrogenase Revealed by Its Structure

    NASA Technical Reports Server (NTRS)

    Korotchkina, Lioubov G.; Ciszak, E.; Patel, M.

    2004-01-01

    Pyruvate dehydrogenase (E1), an alpha 2 beta 2 tetramer, catalyzes the oxidative decarboxylation of pyruvate and reductive acetylation of lipoyl moieties of the dihydrolipoamide acetyltransferase. The roles of beta W135, alpha P188, alpha M181, alpha H15 and alpha R349 of E1 determined by kinetic analysis were reassessed by analyzing the three-dimensional structure of human E1. The residues identified above are found to play a structural role rather than being directly involved in catalysis: beta W135 is the center residue in the hydrophobic interaction between beta and beta' subunits; alpha P188 and alpha M181 are critical for the conformation of the TPP-binding motif and interaction between alpha and beta subunits; alpha H15, is necessary for the organization of the N-terminus of alpha and alpha'; subunits and alpha R349 supports the interaction of the C-terminus of the alpha subunits with the beta subunits. Analysis of several critical E1 residues confirms the importance of residues distant from the active site for subunit interactions and enzyme function.

  1. Proteomic Dissection of Endosperm Starch Granule Associated Proteins Reveals a Network Coordinating Starch Biosynthesis and Amino Acid Metabolism and Glycolysis in Rice Endosperms.

    PubMed

    Yu, Huatao; Wang, Tai

    2016-01-01

    Starch biosynthesis and starch granule packaging in cereal endosperms involve a coordinated action of starch biosynthesis enzymes and coordination with other metabolisms. Because directly binding to starch granules, starch granule-associated proteins (SGAPs) are essential to understand the underlying mechanisms, however the information on SGAPs remains largely unknown. Here, we dissected developmentally changed SGAPs from developing rice endosperms from 10 to 20 days after flowering (DAF). Starch granule packaging was not completed at 10 DAF, and was finished in the central endosperm at 15 DAF and in the whole endosperm at 20 DAF. Proteomic analysis with two-dimensional differential in-gel electrophoresis and mass spectrometry revealed 115 developmentally changed SGAPs, representing 37 unique proteins. 65% of the unique proteins had isoforms. 39% of the identified SGAPs were involved in starch biosynthesis with main functions in polyglucan elongation and granule structure trimming. Almost all proteins involved in starch biosynthesis, amino acid biosynthesis, glycolysis, protein folding, and PPDK pathways increased abundance as the endosperm developed, and were predicted in an interaction network. The network represents an important mechanism to orchestrate carbon partitioning among starch biosynthesis, amino acid biosynthesis and glycolysis for efficient starch and protein storage. These results provide novel insights into mechanisms of starch biosynthesis and its coordination with amino acid metabolisms and glycolysis in cereal endosperms. PMID:27252723

  2. Proteomic Dissection of Endosperm Starch Granule Associated Proteins Reveals a Network Coordinating Starch Biosynthesis and Amino Acid Metabolism and Glycolysis in Rice Endosperms

    PubMed Central

    Yu, Huatao; Wang, Tai

    2016-01-01

    Starch biosynthesis and starch granule packaging in cereal endosperms involve a coordinated action of starch biosynthesis enzymes and coordination with other metabolisms. Because directly binding to starch granules, starch granule-associated proteins (SGAPs) are essential to understand the underlying mechanisms, however the information on SGAPs remains largely unknown. Here, we dissected developmentally changed SGAPs from developing rice endosperms from 10 to 20 days after flowering (DAF). Starch granule packaging was not completed at 10 DAF, and was finished in the central endosperm at 15 DAF and in the whole endosperm at 20 DAF. Proteomic analysis with two-dimensional differential in-gel electrophoresis and mass spectrometry revealed 115 developmentally changed SGAPs, representing 37 unique proteins. 65% of the unique proteins had isoforms. 39% of the identified SGAPs were involved in starch biosynthesis with main functions in polyglucan elongation and granule structure trimming. Almost all proteins involved in starch biosynthesis, amino acid biosynthesis, glycolysis, protein folding, and PPDK pathways increased abundance as the endosperm developed, and were predicted in an interaction network. The network represents an important mechanism to orchestrate carbon partitioning among starch biosynthesis, amino acid biosynthesis and glycolysis for efficient starch and protein storage. These results provide novel insights into mechanisms of starch biosynthesis and its coordination with amino acid metabolisms and glycolysis in cereal endosperms. PMID:27252723

  3. Down-regulation of the caffeic acid O-methyltransferase gene in switchgrass reveals a novel monolignol analog

    PubMed Central

    2012-01-01

    Background Down-regulation of the caffeic acid 3-O-methyltransferase EC 2.1.1.68 (COMT) gene in the lignin biosynthetic pathway of switchgrass (Panicum virgatum) resulted in cell walls of transgenic plants releasing more constituent sugars after pretreatment by dilute acid and treatment with glycosyl hydrolases from an added enzyme preparation and from Clostridium thermocellum. Fermentation of both wild-type and transgenic switchgrass after milder hot water pretreatment with no water washing showed that only the transgenic switchgrass inhibited C. thermocellum. Gas chromatography–mass spectrometry (GCMS)-based metabolomics were undertaken on cell wall aqueous extracts to determine the nature of the microbial inhibitors. Results GCMS confirmed the increased concentration of a number of phenolic acids and aldehydes that are known inhibitors of microbial fermentation. Metabolomic analyses of the transgenic biomass additionally revealed the presence of a novel monolignol-like metabolite, identified as trans-3, 4-dimethoxy-5-hydroxycinnamyl alcohol (iso-sinapyl alcohol) in both non-pretreated, as well as hot water pretreated samples. iso-Sinapyl alcohol and its glucoside were subsequently generated by organic synthesis and the identity of natural and synthetic materials were confirmed by mass spectrometric and NMR analyses. The additional novel presence of iso-sinapic acid, iso-sinapyl aldehyde, and iso-syringin suggest the increased activity of a para-methyltransferase, concomitant with the reduced COMT activity, a strict meta-methyltransferase. Quantum chemical calculations were used to predict the most likely homodimeric lignans generated from dehydration reactions, but these products were not evident in plant samples. Conclusions Down-regulation of COMT activity in switchgrass resulted in the accumulation of previously undetected metabolites resembling sinapyl alcohol and its related metabolites, but that are derived from para-methylation of 5-hydroxyconiferyl

  4. Analysis and Annotation of Nucleic Acid Sequence

    SciTech Connect

    States, David J.

    2004-07-28

    The aims of this project were to develop improved methods for computational genome annotation and to apply these methods to improve the annotation of genomic sequence data with a specific focus on human genome sequencing. The project resulted in a substantial body of published work. Notable contributions of this project were the identification of basecalling and lane tracking as error processes in genome sequencing and contributions to improved methods for these steps in genome sequencing. This technology improved the accuracy and throughput of genome sequence analysis. Probabilistic methods for physical map construction were developed. Improved methods for sequence alignment, alternative splicing analysis, promoter identification and NF kappa B response gene prediction were also developed.

  5. Analysis and Annotation of Nucleic Acid Sequence

    SciTech Connect

    David J. States

    1998-08-01

    The aims of this project were to develop improved methods for computational genome annotation and to apply these methods to improve the annotation of genomic sequence data with a specific focus on human genome sequencing. The project resulted in a substantial body of published work. Notable contributions of this project were the identification of basecalling and lane tracking as error processes in genome sequencing and contributions to improved methods for these steps in genome sequencing. This technology improved the accuracy and throughput of genome sequence analysis. Probabilistic methods for physical map construction were developed. Improved methods for sequence alignment, alternative splicing analysis, promoter identification and NF kappa B response gene prediction were also developed.

  6. Analysis of TALE superclass homeobox genes (MEIS, PBC, KNOX, Iroquois, TGIF) reveals a novel domain conserved between plants and animals.

    PubMed Central

    Bürglin, T R

    1997-01-01

    A new Caenorhabditis elegans homeobox gene, ceh-25, is described that belongs to the TALE superclass of atypical homeodomains, which are characterized by three extra residues between helix 1 and helix 2. ORF and PCR analysis revealed a novel type of alternative splicing within the homeobox. The alternative splicing occurs such that two different homeodomains can be generated, which differ in their first 25 amino acids. ceh-25 is an orthologue of the vertebrate Meis genes and it shares a new conserved domain of 130 amino acids with them. A thorough analysis of all TALE homeobox genes was performed and a new classification is presented. Four TALE classes are identified in animals: PBC, MEIS, TGIF and IRO (Iroquois); two types in fungi: the mating type genes (M-ATYP) and the CUP genes; and two types in plants: KNOX and BEL. The IRO class has a new conserved motif downstream of the homeodomain. For the KNOX class, a conserved domain, the KNOX domain, was defined upstream of the homeodomain. Comparison of the KNOX domain and the MEIS domain shows significant sequence similarity revealing the existence of an archetypal group of homeobox genes that encode two associated conserved domains. Thus TALE homeobox genes were already present in the common ancestor of plants, fungi and animals and represent a branch distinct from the typical homeobox genes. PMID:9336443

  7. Fatty acid oxidation: systems analysis and applications.

    PubMed

    Cintolesi, Angela; Rodríguez-Moyá, María; Gonzalez, Ramon

    2013-01-01

    Fatty acids (FAs) are essential components of cellular structure and energy-generating routes in living organisms. They exist in a variety of chemical configurations and functionalities and are catabolized by different oxidative routes, according to their structure. α- and ω-Oxidation are minor routes that occur only in eukaryotes, while β-oxidation is the major degradation route in eukaroytes and prokaryotes. These pathways have been characterized and engineered from different perspectives for industrial and biomedical applications. The severity of FA oxidation disorders in humans initially guided the study of FA metabolism at a molecular-level. On the other hand, recent advances in metabolic engineering and systems biology have powered the study of FA biosynthetic and catabolic routes in microorganisms at a systems-level. Several studies have proposed these pathways as platforms for the production of fuels and chemicals from biorenewable sources. The lower complexity of microbial systems has allowed a more comprehensive study of FA metabolism and has opened opportunities for a wider range of applications. Still, there is a need for techniques that facilitate the translation of high-throughput data from microorganisms to more complex eukaryotic systems in order to aid the development of diagnostic and treatment strategies for FA oxidation disorders. In addition, further systems biology analyses on human systems could also provide valuable insights on oxidation disorders. This article presents a comparison of the three main FA oxidative routes, systems biology analyses that have been used to study FA metabolism, and engineering efforts performed on microbial systems. PMID:23661533

  8. Digital PCR analysis of circulating nucleic acids.

    PubMed

    Hudecova, Irena

    2015-10-01

    Detection of plasma circulating nucleic acids (CNAs) requires the use of extremely sensitive and precise methods. The commonly used quantitative real-time polymerase chain reaction (PCR) poses certain technical limitations in relation to the precise measurement of CNAs whereas the costs of massively parallel sequencing are still relatively high. Digital PCR (dPCR) now represents an affordable and powerful single molecule counting strategy to detect minute amounts of genetic material with performance surpassing many quantitative methods. Microfluidic (chip) and emulsion (droplet)-based technologies have already been integrated into platforms offering hundreds to millions of nanoliter- or even picoliter-scale reaction partitions. The compelling observations reported in the field of cancer research, prenatal testing, transplantation medicine and virology support translation of this technology into routine use. Extremely sensitive plasma detection of rare mutations originating from tumor or placental cells among a large background of homologous sequences facilitates unraveling of the early stages of cancer or the detection of fetal mutations. Digital measurement of quantitative changes in plasma CNAs associated with cancer or graft rejection provides valuable information on the monitoring of disease burden or the recipient's immune response and subsequent therapy treatment. Furthermore, careful quantitative assessment of the viral load offers great value for effective monitoring of antiviral therapy for immunosuppressed or transplant patients. The present review describes the inherent features of dPCR that make it exceptionally robust in precise and sensitive quantification of CNAs. Moreover, I provide an insight into the types of potential clinical applications that have been developed by researchers to date. PMID:25828047

  9. Analysis of the generating action of the acid from PAG using acid sensitive dyes

    NASA Astrophysics Data System (ADS)

    Sekiguchi, Atsushi; Matsumoto, Yoko; Konishi, Hiroko; Moriyasu, Kengo; Morimoto, Yukihiro

    2011-04-01

    The use of acid sensitive dyes to determine the quantity of acid generated from PAG and in the analysis of acid-generating reaction is currently being studied. The method would allow an easy understanding of the PAG acid-generating reaction simply by adding an acid sensitive dye to the resist. In the conventional method, a resist containing a chromogenic substance is applied to a quartz substrate, which is then exposed. Following the exposure, the absorbance of chromogenic component near 530 nm is measured and evaluated with a spectroscope. The rate constant for acid generation (Dill's C parameter) during the exposure is determined based on the relationship between transmittance at 530 nm and the exposure dose. However, the chromogenic substance used in this method degrades over time (fading reaction) after the exposure, resulting in variations in transmittance measurements due to the effects of time between the completion of the exposure and the measurement of transmittance. We devised a prototype instrument capable of in situ measurements of absorbance at 530 nm while irradiating a 193-nm light beam. Using this instrument, we obtained rate constants for acid generation (Dill's C parameter) and examined the differing results obtained with ArF resist polymers of differing PAG concentrations and structures as well as dependence on the quantity of the chromogenic substance.

  10. Reaction Mechanism of N-Acetylneuraminic Acid Lyase Revealed by a Combination of Crystallography, QM/MM Simulation, and Mutagenesis

    PubMed Central

    2014-01-01

    N-Acetylneuraminic acid lyase (NAL) is a Class I aldolase that catalyzes the reversible condensation of pyruvate with N-acetyl-d-mannosamine (ManNAc) to yield the sialic acid N-acetylneuraminic acid (Neu5Ac). Aldolases are finding increasing use as biocatalysts for the stereospecific synthesis of complex molecules. Incomplete understanding of the mechanism of catalysis in aldolases, however, can hamper development of new enzyme activities and specificities, including control over newly generated stereocenters. In the case of NAL, it is clear that the enzyme catalyzes a Bi-Uni ordered condensation reaction in which pyruvate binds first to the enzyme to form a catalytically important Schiff base. The identity of the residues required for catalysis of the condensation step and the nature of the transition state for this reaction, however, have been a matter of conjecture. In order to address, this we crystallized a Y137A variant of the E. coli NAL in the presence of Neu5Ac. The three-dimensional structure shows a full length sialic acid bound in the active site of subunits A, B, and D, while in subunit C, discontinuous electron density reveals the positions of enzyme-bound pyruvate and ManNAc. These ‘snapshot’ structures, representative of intermediates in the enzyme catalytic cycle, provided an ideal starting point for QM/MM modeling of the enzymic reaction of carbon–carbon bond formation. This revealed that Tyr137 acts as the proton donor to the aldehyde oxygen of ManNAc during the reaction, the activation barrier is dominated by carbon–carbon bond formation, and proton transfer from Tyr137 is required to obtain a stable Neu5Ac-Lys165 Schiff base complex. The results also suggested that a triad of residues, Tyr137, Ser47, and Tyr110 from a neighboring subunit, are required to correctly position Tyr137 for its function, and this was confirmed by site-directed mutagenesis. This understanding of the mechanism and geometry of the transition states along the C

  11. Expression and characterization of manganese lipoxygenase of the rice blast fungus reveals prominent sequential lipoxygenation of α-linolenic acid.

    PubMed

    Wennman, Anneli; Jernerén, Fredrik; Magnuson, Ann; Oliw, Ernst H

    2015-10-01

    Magnaporthe oryzae causes rice blast disease and has become a model organism of fungal infections. M. oryzae can oxygenate fatty acids by 7,8-linoleate diol synthase, 10R-dioxygenase-epoxy alcohol synthase, and by a putative manganese lipoxygenase (Mo-MnLOX). The latter two are transcribed during infection. The open reading frame of Mo-MnLOX was deduced from genome and cDNA analysis. Recombinant Mo-MnLOX was expressed in Pichia pastoris and purified to homogeneity. The enzyme contained protein-bound Mn and oxidized 18:2n-6 and 18:3n-3 to 9S-, 11-, and 13R-hydroperoxy metabolites by suprafacial hydrogen abstraction and oxygenation. The 11-hydroperoxides were subject to β-fragmentation with formation of 9S- and 13R-hydroperoxy fatty acids. Oxygen consumption indicated apparent kcat values of 2.8 s(-1) (18:2n-6) and 3.9 s(-1) (18:3n-3), and UV analysis yielded apparent Km values of 8 and 12 μM, respectively, for biosynthesis of cis-trans conjugated metabolites. 9S-Hydroperoxy-10E,12Z,15Z-octadecatrienoic acid was rapidly further oxidized to a triene, 9S,16S-dihydroperoxy-10E,12Z,14E-octadecatrienoic acid. In conclusion, we have expressed, purified and characterized a new MnLOX from M. oryzae. The pathogen likely secretes Mo-MnLOX and phospholipases to generate oxylipins and to oxidize lipid membranes of rice cells and the cuticle. PMID:26264916

  12. Comparative Genome Analysis of Megasphaera sp. Reveals Niche Specialization and Its Potential Role in the Human Gut

    PubMed Central

    Lanjekar, Vikram; Ranade, Dilip; Shouche, Yogesh S.

    2013-01-01

    With increasing number of novel bacteria being isolated from the human gut ecosystem, there is a greater need to study their role in the gut ecosystem and their effect on the host health. In the present study, we carried out in silico genome-wide analysis of two novel Megasphaera sp. isolates NM10 (DSM25563) and BL7 (DSM25562), isolated from feces of two healthy individuals and validated the key features by in vitro studies. The analysis revealed the general metabolic potential, adaptive features and the potential effects of these isolates on the host. The comparative genome analysis of the two human gut isolates NM10 and BL7 with ruminal isolate Megasphaera elsdenii (DSM20460) highlighted the differential adaptive features for their survival in human gut. The key findings include features like bile resistance, presence of various sensory and regulatory systems, stress response systems, membrane transporters and resistance to antibiotics. Comparison of the “glycobiome” based on the genomes of the ruminal isolate with the human gut isolates NM10 and BL revealed the presence of diverse and unique sets of Carbohydrate-Active enzymes (CAZymes) amongst these isolates, with a higher collection of CAZymes in the human gut isolates. This could be attributed to the difference in host diet and thereby the environment, consequently suggesting host specific adaptation in these isolates. In silico analysis of metabolic potential predicted the ability of these isolates to produce important metabolites like short chain fatty acids (butyrate, acetate, formate, and caproate), vitamins and essential amino acids, which was further validated by in vitro experiments. The ability of these isolates to produce important metabolites advocates for a potential healthy influence on the host. Further in vivo studies including transcriptomic and proteomic analysis will be required for better understanding the role and impact of these Megasphaera sp. isolates NM10 and BL7 on the human host

  13. Comparative metabolomics analysis of docosahexaenoic acid fermentation processes by Schizochytrium sp. under different oxygen availability conditions.

    PubMed

    Li, Juan; Ren, Lu-Jing; Sun, Guan-Nan; Qu, Liang; Huang, He

    2013-05-01

    The intracellular metabolic profile characterization of Schizochytrium sp. throughout docosahexaenoic acid fermentation was investigated using gas chromatography-mass spectrometry (GC-MS). Metabolite profiles originating from Schizochytrium sp. under normal and limited oxygen supply conditions were distinctive and distinguished by principal components analysis (PCA). A total of more than 60 intracellular metabolites were detected and quantified with the levels of some metabolites involved in central carbon metabolism varying throughout both processes. Both fermentation processes were differentiated into three main phases by principal components analysis. Potential biomarkers responsible for distinguishing the different fermentation phases were identified as glutamic acid, proline, glycine, alanine, and glucose. In addition, alanine, glutamic acid, glucose, inositol, ornithine, and galactose were found to make great contribution for dry cell weight and fatty acid composition during normal and limited oxygen supply fermentations. Furthermore, significantly higher levels of succinate and several amino acids in cells of limited oxygen supply fermentation revealed that they might play important roles in resisting oxygen deficiency and increasing DHA synthesis during the lipid accumulation. These findings provide novel insights into the metabolomic characteristics during docosahexaenoic acid fermentation processes by Schizochytrium sp. PMID:23586678

  14. Bile acids: analysis in biological fluids and tissues

    PubMed Central

    Griffiths, William J.; Sjövall, Jan

    2010-01-01

    The formation of bile acids/bile alcohols is of major importance for the maintenance of cholesterol homeostasis. Besides their functions in lipid absorption, bile acids/bile alcohols are regulatory molecules for a number of metabolic processes. Their effects are structure-dependent, and numerous metabolic conversions result in a complex mixture of biologically active and inactive forms. Advanced methods are required to characterize and quantify individual bile acids in these mixtures. A combination of such analyses with analyses of the proteome will be required for a better understanding of mechanisms of action and nature of endogenous ligands. Mass spectrometry is the basic detection technique for effluents from chromatographic columns. Capillary liquid chromatography-mass spectrometry with electrospray ionization provides the highest sensitivity in metabolome analysis. Classical gas chromatography-mass spectrometry is less sensitive but offers extensive structure-dependent fragmentation increasing the specificity in analyses of isobaric isomers of unconjugated bile acids. Depending on the nature of the bile acid/bile alcohol mixture and the range of concentration of individuals, different sample preparation sequences, from simple extractions to group separations and derivatizations, are applicable. We review the methods currently available for the analysis of bile acids in biological fluids and tissues, with emphasis on the combination of liquid and gas phase chromatography with mass spectrometry. PMID:20008121

  15. Nanopore sensors for nucleic acid analysis

    NASA Astrophysics Data System (ADS)

    Venkatesan, Bala Murali; Bashir, Rashid

    2011-10-01

    Nanopore analysis is an emerging technique that involves using a voltage to drive molecules through a nanoscale pore in a membrane between two electrolytes, and monitoring how the ionic current through the nanopore changes as single molecules pass through it. This approach allows charged polymers (including single-stranded DNA, double-stranded DNA and RNA) to be analysed with subnanometre resolution and without the need for labels or amplification. Recent advances suggest that nanopore-based sensors could be competitive with other third-generation DNA sequencing technologies, and may be able to rapidly and reliably sequence the human genome for under $1,000. In this article we review the use of nanopore technology in DNA sequencing, genetics and medical diagnostics.

  16. Computerized image analysis for acetic acid induced intraepithelial lesions

    NASA Astrophysics Data System (ADS)

    Li, Wenjing; Ferris, Daron G.; Lieberman, Rich W.

    2008-03-01

    Cervical Intraepithelial Neoplasia (CIN) exhibits certain morphologic features that can be identified during a visual inspection exam. Immature and dysphasic cervical squamous epithelium turns white after application of acetic acid during the exam. The whitening process occurs visually over several minutes and subjectively discriminates between dysphasic and normal tissue. Digital imaging technologies allow us to assist the physician analyzing the acetic acid induced lesions (acetowhite region) in a fully automatic way. This paper reports a study designed to measure multiple parameters of the acetowhitening process from two images captured with a digital colposcope. One image is captured before the acetic acid application, and the other is captured after the acetic acid application. The spatial change of the acetowhitening is extracted using color and texture information in the post acetic acid image; the temporal change is extracted from the intensity and color changes between the post acetic acid and pre acetic acid images with an automatic alignment. The imaging and data analysis system has been evaluated with a total of 99 human subjects and demonstrate its potential to screening underserved women where access to skilled colposcopists is limited.

  17. Comparative Genome Analysis Reveals Metabolic Versatility and Environmental Adaptations of Sulfobacillus thermosulfidooxidans Strain ST

    PubMed Central

    Guo, Xue; Yin, Huaqun; Liang, Yili; Hu, Qi; Zhou, Xishu; Xiao, Yunhua; Ma, Liyuan; Zhang, Xian; Qiu, Guanzhou; Liu, Xueduan

    2014-01-01

    The genus Sulfobacillus is a cohort of mildly thermophilic or thermotolerant acidophiles within the phylum Firmicutes and requires extremely acidic environments and hypersalinity for optimal growth. However, our understanding of them is still preliminary partly because few genome sequences are available. Here, the draft genome of Sulfobacillus thermosulfidooxidans strain ST was deciphered to obtain a comprehensive insight into the genetic content and to understand the cellular mechanisms necessary for its survival. Furthermore, the expressions of key genes related with iron and sulfur oxidation were verified by semi-quantitative RT-PCR analysis. The draft genome sequence of Sulfobacillus thermosulfidooxidans strain ST, which encodes 3225 predicted coding genes on a total length of 3,333,554 bp and a 48.35% G+C, revealed the high degree of heterogeneity with other Sulfobacillus species. The presence of numerous transposases, genomic islands and complete CRISPR/Cas defence systems testifies to its dynamic evolution consistent with the genome heterogeneity. As expected, S. thermosulfidooxidans encodes a suit of conserved enzymes required for the oxidation of inorganic sulfur compounds (ISCs). The model of sulfur oxidation in S. thermosulfidooxidans was proposed, which showed some different characteristics from the sulfur oxidation of Gram-negative A. ferrooxidans. Sulfur oxygenase reductase and heterodisulfide reductase were suggested to play important roles in the sulfur oxidation. Although the iron oxidation ability was observed, some key proteins cannot be identified in S. thermosulfidooxidans. Unexpectedly, a predicted sulfocyanin is proposed to transfer electrons in the iron oxidation. Furthermore, its carbon metabolism is rather flexible, can perform the transformation of pentose through the oxidative and non-oxidative pentose phosphate pathways and has the ability to take up small organic compounds. It encodes a multitude of heavy metal resistance systems to

  18. Molecular Determinants of Juvenile Hormone Action as Revealed by 3D QSAR Analysis in Drosophila

    PubMed Central

    Beňo, Milan; Farkaš, Robert

    2009-01-01

    Background Postembryonic development, including metamorphosis, of many animals is under control of hormones. In Drosophila and other insects these developmental transitions are regulated by the coordinate action of two principal hormones, the steroid ecdysone and the sesquiterpenoid juvenile hormone (JH). While the mode of ecdysone action is relatively well understood, the molecular mode of JH action remains elusive. Methodology/Principal Findings To gain more insights into the molecular mechanism of JH action, we have tested the biological activity of 86 structurally diverse JH agonists in Drosophila melanogaster. The results were evaluated using 3D QSAR analyses involving CoMFA and CoMSIA procedures. Using this approach we have generated both computer-aided and species-specific pharmacophore fingerprints of JH and its agonists, which revealed that the most active compounds must possess an electronegative atom (oxygen or nitrogen) at both ends of the molecule. When either of these electronegative atoms are replaced by carbon or the distance between them is shorter than 11.5 Å or longer than 13.5 Å, their biological activity is dramatically decreased. The presence of an electron-deficient moiety in the middle of the JH agonist is also essential for high activity. Conclusions/Significance The information from 3D QSAR provides guidelines and mechanistic scope for identification of steric and electrostatic properties as well as donor and acceptor hydrogen-bonding that are important features of the ligand-binding cavity of a JH target protein. In order to refine the pharmacophore analysis and evaluate the outcomes of the CoMFA and CoMSIA study we used pseudoreceptor modeling software PrGen to generate a putative binding site surrogate that is composed of eight amino acid residues corresponding to the defined molecular interactions. PMID:19547707

  19. A novel mechanism of gall midge resistance in the rice variety Kavya revealed by microarray analysis.

    PubMed

    Rawat, Nidhi; Chiruvuri Naga, Neeraja; Raman Meenakshi, Sundaram; Nair, Suresh; Bentur, Jagadish S

    2012-06-01

    The Asian rice gall midge [Orseolia oryzae (Wood-Mason)] is an important rice pest causing an annual average yield loss of about US $80 million in India. Rice varieties possess several discrete resistance (R) genes conferring resistance against the pest in two distinct ways, i.e., with (HR+ type) or without (HR- type) the expression of hypersensitive reaction (HR). The aim of the present work is to understand the molecular basis of compatible and incompatible (HR- type) rice gall midge interactions between the rice variety Kavya and the two gall midge biotypes: the virulent GMB4M and the avirulent GMB1 using transcriptional microarray gene expression analysis. A large number of differentially expressed genes (602genes in incompatible interaction and 1,330 genes in compatible interaction with at least twofold changes, p value <0.05) was obtained from the microarray analysis that could be grouped into six clusters based on their induction during both or either of the interactions. MapMan software was used for functional characterization of these genes into 13 categories (BINs). Real-time polymerase chain reaction validation of 26 genes selected through the analysis revealed four genes viz. NADPH oxidase, AtrbohF, cinnamoyl-CoA reductase, and von Willebrand factor type A domain containing protein coding genes to be significantly upregulated during the incompatible interaction. But most of the signature genes related to HR+ type resistance like salicylic acid pathway-related genes and disease resistance protein coding genes were downregulated. On the other hand, during the compatible interaction, genes related to primary metabolism and nutrient transport were upregulated and genes for defense and signaling were downregulated. We propose a hypothesis that HR- type of resistance in the rice variety Kavya against gall midge could be due to the constitutive expression of an R gene and a case of extreme resistance which is devoid of cell death. Compatible interaction

  20. Integrated metabolomic and proteomic analysis reveals systemic responses of Rubrivivax benzoatilyticus JA2 to aniline stress.

    PubMed

    Mujahid, Md; Prasuna, M Lakshmi; Sasikala, Ch; Ramana, Ch Venkata

    2015-02-01

    Aromatic amines are widely distributed in the environment and are major environmental pollutants. Although degradation of aromatic amines is well studied in bacteria, physiological adaptations and stress response to these toxic compounds is not yet fully understood. In the present study, systemic responses of Rubrivivax benzoatilyticus JA2 to aniline stress were deciphered using metabolite and iTRAQ-labeled protein profiling. Strain JA2 tolerated high concentrations of aniline (30 mM) with trace amounts of aniline being transformed to acetanilide. GC-MS metabolite profiling revealed aniline stress phenotype wherein amino acid, carbohydrate, fatty acid, nitrogen metabolisms, and TCA (tricarboxylic acid cycle) were modulated. Strain JA2 responded to aniline by remodeling the proteome, and cellular functions, such as signaling, transcription, translation, stress tolerance, transport and carbohydrate metabolism, were highly modulated. Key adaptive responses, such as transcription/translational changes, molecular chaperones to control protein folding, and efflux pumps implicated in solvent extrusion, were induced in response to aniline stress. Proteo-metabolomics indicated extensive rewiring of metabolism to aniline. TCA cycle and amino acid catabolism were down-regulated while gluconeogenesis and pentose phosphate pathways were up-regulated, leading to the synthesis of extracellular polymeric substances. Furthermore, increased saturated fatty acid ratios in membranes due to aniline stress suggest membrane adaptation. The present study thus indicates that strain JA2 employs multilayered responses: stress response, toxic compound tolerance, energy conservation, and metabolic rearrangements to aniline. PMID:25388363

  1. Design and Exploration of Novel Boronic Acid Inhibitors Reveals Important Interactions with a Clavulanic Acid-Resistant Sulfhydryl-Variable (SHV) β-Lactamase

    PubMed Central

    Winkler, Marisa L.; Rodkey, Elizabeth A.; Taracila, Magdalena A.; Drawz, Sarah M.; Bethel, Christopher R.; Papp-Wallace, Krisztina M.; Smith, Kerri M.; Xu, Yan; Dwulit-Smith, Jeffrey R.; Romagnoli, Chiara; Caselli, Emilia; Prati, Fabio; van den Akker, Focco; Bonomo, Robert A.

    2014-01-01

    Inhibitor resistant (IR) class A β-lactamases pose a significant threat to many current antibiotic combinations. The K234R substitution in the SHV β-lactamase, from Klebsiella pneumoniae, results in resistance to ampicillin/clavulanate. After site-saturation mutagenesis of Lys-234 in SHV, microbiological and biochemical characterization of the resulting β-lactamases revealed that only –Arg conferred resistance to ampicillin/clavulanate. X-ray crystallography revealed two conformations of Arg-234 and Ser-130 in SHV K234R. The movement of Ser-130 is the principal cause of the observed clavulanate resistance. A panel of boronic acid inhibitors was designed and tested against SHV-1 and SHV K234R. A chiral ampicillin analogue was discovered to have a 2.4 ± 0.2 nM Ki for SHV K234R; the chiral ampicillin analogue formed a more complex hydrogen-bonding network in SHV K234R vs SHV-1. Consideration of the spatial position of Ser-130 and Lys-234 and this hydrogen-bonding network will be important in the design of novel antibiotics targeting IR β-lactamases. PMID:23252553

  2. Activation of acid-sensing ion channels by localized proton transient reveals their role in proton signaling

    PubMed Central

    Zeng, Wei-Zheng; Liu, Di-Shi; Liu, Lu; She, Liang; Wu, Long-Jun; Xu, Tian-Le

    2015-01-01

    Extracellular transients of pH alterations likely mediate signal transduction in the nervous system. Neuronal acid-sensing ion channels (ASICs) act as sensors for extracellular protons, but the mechanism underlying ASIC activation remains largely unknown. Here, we show that, following activation of a light-activated proton pump, Archaerhodopsin-3 (Arch), proton transients induced ASIC currents in both neurons and HEK293T cells co-expressing ASIC1a channels. Using chimera proteins that bridge Arch and ASIC1a by a glycine/serine linker, we found that successful coupling occurred within 15 nm distance. Furthermore, two-cell sniffer patch recording revealed that regulated release of protons through either Arch or voltage-gated proton channel Hv1 activated neighbouring cells expressing ASIC1a channels. Finally, computational modelling predicted the peak proton concentration at the intercellular interface to be at pH 6.7, which is acidic enough to activate ASICs in vivo. Our results highlight the pathophysiological role of proton signalling in the nervous system. PMID:26370138

  3. Genetic dissection of the polyoxin building block-carbamoylpolyoxamic acid biosynthesis revealing the “pathway redundancy” in metabolic networks

    PubMed Central

    2013-01-01

    Background Polyoxin, a peptidyl nucleoside antibiotic, consists of three building blocks including a nucleoside skeleton, polyoximic acid (POIA), and carbamoylpolyoxamic acid (CPOAA), however, little is known about the “pathway redundancy” of the metabolic networks directing the CPOAA biosynthesis in the cell factories of the polyoxin producer. Results Here we report the genetic characterization of CPOAA biosynthesis with revealing a “pathway redundancy” in metabolic networks. Independent mutation of the four genes (polL-N and polP) directly resulted in the accumulation of polyoxin I, suggesting their positive roles for CPOAA biosynthesis. Moreover, the individual mutant of polN and polP also partially retains polyoxin production, suggesting the existence of the alternative homologs substituting their functional roles. Conclusions It is unveiled that argA and argB in L-arginine biosynthetic pathway contributed to the “pathway redundancy”, more interestingly, argB in S. cacaoi is indispensible for both polyoxin production and L-arginine biosynthesis. These data should provide an example for the research on the “pathway redundancy” in metabolic networks, and lay a solid foundation for targeted enhancement of polyoxin production with synthetic biology strategies. PMID:24314013

  4. Super-resolution Microscopy of Clickable Amino Acids Reveals the Effects of Fluorescent Protein Tagging on Protein Assemblies.

    PubMed

    Vreja, Ingrid C; Nikić, Ivana; Göttfert, Fabian; Bates, Mark; Kröhnert, Katharina; Outeiro, Tiago F; Hell, Stefan W; Lemke, Edward A; Rizzoli, Silvio O

    2015-11-24

    The advent of super-resolution microscopy (nanoscopy) has set high standards for fluorescence tagging. Fluorescent proteins (FPs) are convenient tags in conventional imaging, but their use in nanoscopy has been questioned due to their relatively large size and propensity to form multimers. Here, we compared the nanoscale organization of proteins with or without FP tags by introducing the unnatural amino acid propargyl-L-lysine (PRK) in 26 proteins known to form multimolecular arrangements and into their FP-tagged variants. We revealed the proteins by coupling synthetic fluorophores to PRK via click chemistry and visualized them using ground-state depletion microscopy followed by individual molecule return, as well as stimulated emission depletion microscopy. The arrangements formed by the FP-tagged and nontagged proteins were similar. Mild, but statistically significant differences were observed for only six proteins (23% of all proteins tested). This suggests that FP-based nanoscopy is generally reliable. Unnatural amino acids should be a reliable alternative for the few proteins that are sensitive to FP tagging. PMID:26498474

  5. Fluorescence spectroscopy reveals accompanying occurrence of ammonium with fulvic acid-like organic matter in a fluvio-lacustrine aquifer of Jianhan Plain.

    PubMed

    Huang, Shuangbing; Wang, Yanxin; Ma, Teng; Wang, Yanyan; Zhao, Long

    2016-05-01

    This study is the first to investigate the simultaneous presence of NH4 (+) and fluorescent organic matter components (FOCs) from a fluvio-lacustrine aquifer in Central Jianghan Plain. Sediment, groundwater, and surface water samples were collected for the sediment organic matter extraction, 3D fluorescence spectroscopy characterization, and/or hydrochemical analysis. NH4 (+) and dissolved organic carbon was ubiquitous in the groundwater. The fluorescence spectroscopy revealed good relationships between NH4 (+) and fulvic acid-like components (FALCs) in the groundwater and sediment-extracted organic matter (SEOM) solutions. NH4 (+) also exhibited significant positive correlation with protein-like component (PLC) (p < 0.001), with the stronger in the SEOM solutions than that in groundwater. Comparisons of spectroscopic indices [e.g., humification index (HIX), biological index (BIX), spectra slope (S275-295), and specific UV absorbance (SUVA254)] between the groundwater and SEOM solutions revealed more labile properties of SEOM. This result indicates that the decreasing NH4 (+)-FOCs correlations of groundwater relative to sediments may be attributed to microbial degradation. Factor analysis identifies important factors that cause NH4 (+) occurrence in the groundwater. The accompanying increase of FALC (C1) and NH4-N with the mole concentration of the normalized HCO3 (-)/(Ca(2+)+Mg(2+)) and [H(+)] suggests that couple effects of various biodegradations simultaneously occur in the aquifer, promoting the occurrence of NH4-DOMs. PMID:26791026

  6. Low-coverage exome sequencing screen in formalin-fixed paraffin-embedded tumors reveals evidence of exposure to carcinogenic aristolochic acid

    PubMed Central

    Castells, Xavier; Karanović, Sandra; Ardin, Maude; Tomić, Karla; Xylinas, Evanguelos; Durand, Geoffroy; Villar, Stephanie; Forey, Nathalie; Le Calvez-Kelm, Florence; Voegele, Catherine; Karlović, Krešimir; Mišić, Maja; Dittrich, Damir; Dolgalev, Igor; McKay, James; Shariat, Shahrokh F.; Sidorenko, Viktoria S.; Fernandes, Andrea; Heguy, Adriana; Dickman, Kathleen G.; Olivier, Magali; Grollman, Arthur P.; Jelaković, Bojan; Zavadil, Jiri

    2015-01-01

    Background Dietary exposure to cytotoxic and carcinogenic aristolochic acid (AA) causes severe nephropathy typically associated with urological cancers. Monitoring of AA exposure uses biomarkers such as aristolactam-DNA adducts, detected by mass spectrometry in the kidney cortex, or the somatic A>T transversion pattern characteristic of exposure to AA, as revealed by previous DNA sequencing studies using fresh frozen tumors. Methods Here we report a low-coverage whole-exome sequencing method (LC-WES) optimized for multi-sample detection of the AA mutational signature, and demonstrate its utility in 17 formalin-fixed paraffin-embedded urothelial tumors obtained from 15 patients with endemic nephropathy, an environmental form of aristolochic acid nephropathy. Results LC-WES identified the AA signature, alongside signatures of age and APOBEC enzyme activity, in 15 samples sequenced at the average per-base coverage of ~10x. Analysis at 3–9x coverage revealed the signature in 91% of the positive samples. The exome-wide distribution of the predominant A>T transversions exhibited a stochastic pattern whereas 83 cancer driver genes were enriched for recurrent non-synonymous A>T mutations. In two patients, pairs of tumors from different parts of the urinary tract, including the bladder, harbored overlapping mutation patterns, suggesting tumor dissemination via cell seeding. Conclusion LC-WES analysis of archived tumor tissues is a reliable method applicable to investigations of both the exposure to AA and its biologic effects in human carcinomas. Impact By detecting cancers associated with AA exposure in high-risk populations, LC-WES can support future molecular epidemiology studies and provide evidence-base for relevant preventive measures. PMID:26383547

  7. Quality Analysis of Chlorogenic Acid and Hyperoside in Crataegi fructus

    PubMed Central

    Weon, Jin Bae; Jung, Youn Sik; Ma, Choong Je

    2016-01-01

    Background: Crataegi fructus is a herbal medicine for strong stomach, sterilization, and alcohol detoxification. Chlorogenic acid and hyperoside are the major compounds in Crataegi fructus. Objective: In this study, we established novel high-performance liquid chromatography (HPLC)-diode array detection analysis method of chlorogenic acid and hyperoside for quality control of Crataegi fructus. Materials and Methods: HPLC analysis was achieved on a reverse-phase C18 column (5 μm, 4.6 mm × 250 mm) using water and acetonitrile as mobile phase with gradient system. The method was validated for linearity, precision, and accuracy. About 31 batches of Crataegi fructus samples collected from Korea and China were analyzed by using HPLC fingerprint of developed HPLC method. Then, the contents of chlorogenic acid and hyperoside were compared for quality evaluation of Crataegi fructus. Results: The results have shown that the average contents (w/w %) of chlorogenic acid and hyperoside in Crataegi fructus collected from Korea were 0.0438% and 0.0416%, respectively, and the average contents (w/w %) of 0.0399% and 0.0325%, respectively. Conclusion: In conclusion, established HPLC analysis method was stable and could provide efficient quality evaluation for monitoring of commercial Crataegi fructus. SUMMARY Quantitative analysis method of chlorogenic acid and hyperoside in Crataegi fructus is developed by high.performance liquid chromatography.(HPLC).diode array detectionEstablished HPLC analysis method is validated with linearity, precision, and accuracyThe developed method was successfully applied for quantitative analysis of Crataegi fructus sample collected from Korea and China. Abbreviations used: HPLC: High-performance liquid chromatography, GC: Gas chromatography, MS: Mass spectrometer, LOD: Limits of detection, LOQ: Limits of quantification, RSD: Relative standard deviation, RRT: Relative retention time, RPA: Relation peak area. PMID:27076744

  8. Genome wide transcriptome analysis reveals ABA mediated response in Arabidopsis during gold (AuCl(-) 4) treatment.

    PubMed

    Shukla, Devesh; Krishnamurthy, Sneha; Sahi, Shivendra V

    2014-01-01

    The unique physico-chemical properties of gold nanoparticles (AuNPs) find manifold applications in diagnostics, medicine and catalysis. Chemical synthesis produces reactive AuNPs and generates hazardous by-products. Alternatively, plants can be utilized to produce AuNPs in an eco-friendly manner. To better control the biosynthesis of AuNPs, we need to first understand the detailed molecular response induced by AuCl(-) 4 In this study, we carried out global transcriptome analysis in root tissue of Arabidopsis grown for 12- h in presence of gold solution (HAuCl4) using the novel unbiased Affymetrix exon array. Transcriptomics analysis revealed differential regulation of a total of 704 genes and 4900 exons. Of these, 492 and 212 genes were up- and downregulated, respectively. The validation of the expressed key genes, such as glutathione-S-transferases, auxin responsive genes, cytochrome P450 82C2, methyl transferases, transducin (G protein beta subunit), ERF transcription factor, ABC, and MATE transporters, was carried out through quantitative RT-PCR. These key genes demonstrated specific induction under AuCl4(-) treatment relative to other heavy metals, suggesting a unique plant-gold interaction. GO enrichment analysis reveals the upregulation of processes like oxidative stress, glutathione binding, metal binding, transport, and plant hormonal responses. Changes predicted in biochemical pathways indicated major modulation in glutathione mediated detoxification, flavones and derivatives, and plant hormone biosynthesis. Motif search analysis identified a highly significant enriched motif, ACGT, which is an abscisic acid responsive core element (ABRE), suggesting the possibility of ABA- mediated signaling. Identification of abscisic acid response element (ABRE) points to the operation of a predominant signaling mechanism in response to AuCl(-) 4 exposure. Overall, this study presents a useful picture of plant-gold interaction with an identification of candidate genes

  9. Genome wide transcriptome analysis reveals ABA mediated response in Arabidopsis during gold (AuCl−4) treatment

    PubMed Central

    Shukla, Devesh; Krishnamurthy, Sneha; Sahi, Shivendra V.

    2014-01-01

    The unique physico-chemical properties of gold nanoparticles (AuNPs) find manifold applications in diagnostics, medicine and catalysis. Chemical synthesis produces reactive AuNPs and generates hazardous by-products. Alternatively, plants can be utilized to produce AuNPs in an eco-friendly manner. To better control the biosynthesis of AuNPs, we need to first understand the detailed molecular response induced by AuCl−4 In this study, we carried out global transcriptome analysis in root tissue of Arabidopsis grown for 12- h in presence of gold solution (HAuCl4) using the novel unbiased Affymetrix exon array. Transcriptomics analysis revealed differential regulation of a total of 704 genes and 4900 exons. Of these, 492 and 212 genes were up- and downregulated, respectively. The validation of the expressed key genes, such as glutathione-S-transferases, auxin responsive genes, cytochrome P450 82C2, methyl transferases, transducin (G protein beta subunit), ERF transcription factor, ABC, and MATE transporters, was carried out through quantitative RT-PCR. These key genes demonstrated specific induction under AuCl4− treatment relative to other heavy metals, suggesting a unique plant-gold interaction. GO enrichment analysis reveals the upregulation of processes like oxidative stress, glutathione binding, metal binding, transport, and plant hormonal responses. Changes predicted in biochemical pathways indicated major modulation in glutathione mediated detoxification, flavones and derivatives, and plant hormone biosynthesis. Motif search analysis identified a highly significant enriched motif, ACGT, which is an abscisic acid responsive core element (ABRE), suggesting the possibility of ABA- mediated signaling. Identification of abscisic acid response element (ABRE) points to the operation of a predominant signaling mechanism in response to AuCl−4 exposure. Overall, this study presents a useful picture of plant-gold interaction with an identification of candidate genes

  10. Revealing biogenic sulfuric acid corrosion in sludge digesters: detection of sulfur-oxidizing bacteria within full-scale digesters.

    PubMed

    Huber, B; Drewes, J E; Lin, K C; König, R; Müller, E

    2014-01-01

    Biogenic sulfuric acid corrosion (BSA) is a costly problem affecting both sewerage infrastructure and sludge handling facilities such as digesters. The aim of this study was to verify BSA in full-scale digesters by identifying the microorganisms involved in the concrete corrosion process, that is, sulfate-reducing (SRB) and sulfur-oxidizing bacteria (SOB). To investigate the SRB and SOB communities, digester sludge and biofilm samples were collected. SRB diversity within digester sludge was studied by applying polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) targeting the dsrB-gene (dissimilatory sulfite reductase beta subunit). To reveal SOB diversity, cultivation dependent and independent techniques were applied. The SRB diversity studies revealed different uncultured SRB, confirming SRB activity and H2S production. Comparable DGGE profiles were obtained from the different sludges, demonstrating the presence of similar SRB species. By cultivation, three pure SOB strains from the digester headspace were obtained including Acidithiobacillus thiooxidans, Thiomonas intermedia and Thiomonas perometabolis. These organisms were also detected with PCR-DGGE in addition to two new SOB: Thiobacillus thioparus and Paracoccus solventivorans. The SRB and SOB responsible for BSA were identified within five different digesters, demonstrating that BSA is a problem occurring not only in sewer systems but also in sludge digesters. In addition, the presence of different SOB species was successfully associated with the progression of microbial corrosion. PMID:25353947

  11. GENOMIC ANALYSIS OF THE TESTICULAR TOXICITY OF HALOACETIC ACIDS

    EPA Science Inventory

    Genomic analysis of the testicular toxicity of haloacetic acids

    David J. Dix and John C. Rockett
    Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, R...

  12. Transcriptome Analysis Reveals Regulation of Gene Expression for Lipid Catabolism in Young Broilers by Butyrate Glycerides

    PubMed Central

    Yin, Fugui; Yu, Hai; Lepp, Dion; Shi, Xuejiang; Yang, Xiaojian; Hu, Jielun; Leeson, Steve; Yang, Chengbo; Nie, Shaoping; Hou, Yongqing; Gong, Joshua

    2016-01-01

    indicated that dietary BG intervention induced 79 and 205 characterized DEGs in the jejunum and liver, respectively. In addition, 255 and 165 TSEGs were detected in the liver and jejunum of BG-fed group, while 162 and 211 TSEGs genes were observed in the liver and jejunum of BD-fed birds, respectively. Bioinformatic analysis with both IPA and DAVID-BR further revealed a significant enrichment of DEGs and TSEGs in the biological processes for reducing the synthesis, storage, transportation and secretion of lipids in the jejunum, while those in the liver were for enhancing the oxidation of ingested lipids and fatty acids. In particular, transcriptional regulators of THRSP and EGR-1 as well as several DEGs involved in the PPAR-α signaling pathway were significantly induced by dietary BG intervention for lipid catabolism. Conclusions Our results demonstrate that BG reduces body fat deposition via regulation of gene expression, which is involved in the biological events relating to the reduction of synthesis, storage, transportation and secretion, and improvement of oxidation of lipids and fatty acids. PMID:27508934

  13. Genome-Wide Analysis of Branched-Chain Amino Acid Levels in Arabidopsis Seeds[W

    PubMed Central

    Angelovici, Ruthie; Lipka, Alexander E.; Deason, Nicholas; Gonzalez-Jorge, Sabrina; Lin, Haining; Cepela, Jason; Buell, Robin; Gore, Michael A.; DellaPenna, Dean

    2013-01-01

    Branched-chain amino acids (BCAAs) are three of the nine essential amino acids in human and animal diets and are important for numerous processes in development and growth. However, seed BCAA levels in major crops are insufficient to meet dietary requirements, making genetic improvement for increased and balanced seed BCAAs an important nutritional target. Addressing this issue requires a better understanding of the genetics underlying seed BCAA content and composition. Here, a genome-wide association study and haplotype analysis for seed BCAA traits in Arabidopsis thaliana revealed a strong association with a chromosomal interval containing two BRANCHED-CHAIN AMINO ACID TRANSFERASES, BCAT1 and BCAT2. Linkage analysis, reverse genetic approaches, and molecular complementation analysis demonstrated that allelic variation at BCAT2 is responsible for the natural variation of seed BCAAs in this interval. Complementation analysis of a bcat2 null mutant with two significantly different alleles from accessions Bayreuth-0 and Shahdara is consistent with BCAT2 contributing to natural variation in BCAA levels, glutamate recycling, and free amino acid homeostasis in seeds in an allele-dependent manner. The seed-specific phenotype of bcat2 null alleles, its strong transcription induction during late seed development, and its subcellular localization to the mitochondria are consistent with a unique, catabolic role for BCAT2 in BCAA metabolism in seeds. PMID:24368787

  14. Functional genomic analysis reveals overlapping and distinct features of chronologically long-lived yeast populations.

    PubMed

    Wierman, Margaret B; Matecic, Mirela; Valsakumar, Veena; Li, Mingguang; Smith, Daniel L; Bekiranov, Stefan; Smith, Jeffrey S

    2015-03-01

    Yeast chronological lifespan (CLS) is extended by multiple genetic and environmental manipulations, including caloric restriction (CR). Understanding the common changes in molecular pathways induced by such manipulations could potentially reveal conserved longevity mechanisms. We therefore performed gene expression profiling on several long-lived yeast populations, including anade4∆mutant defective in de novo purine (AMP) biosynthesis, and a calorie restricted WT strain. CLS was also extended by isonicotinamide (INAM) or expired media derived from CR cultures. Comparisons between these diverse long-lived conditions revealed a common set of differentially regulated genes, several of which were potential longevity biomarkers. There was also enrichment for genes that function in CLS regulation, including a long-lived adenosine kinase mutant (ado1∆) that links CLS regulation to the methyl cycle and AMP. Genes co-regulated between the CR and ade4∆ conditions were dominated by GO terms related to metabolism of alternative carbon sources, consistent with chronological longevity requiring efficient acetate/acetic acid utilization. Alternatively, treating cells with isonicotinamide (INAM) or the expired CR media resulted in GO terms predominantly related to cell wall remodeling, consistent with improved stress resistance and protection against external insults like acetic acid. Acetic acid therefore has both beneficial and detrimental effects on CLS. PMID:25769345

  15. Transcriptome Analysis Revealed the Embryo-Induced Gene Expression Patterns in the Endometrium from Meishan and Yorkshire Pigs.

    PubMed

    Huang, Jiangnan; Liu, Ruize; Su, Lijie; Xiao, Qian; Yu, Mei

    2015-01-01

    The expression patterns in Meishan- and Yorkshire-derived endometrium during early (gestational day 15) and mid-gestation (gestational days 26 and 50) were investigated, respectively. Totally, 689 and 1649 annotated genes were identified to be differentially expressed in Meishan and Yorkshire endometrium during the three gestational stages, respectively. Hierarchical clustering analysis identified that, of the annotated differentially expressed genes (DEGs), 73 DEGs were unique to Meishan endometrium, 536 DEGs were unique to Yorkshire endometrium, and 228 DEGs were common in Meishan and Yorkshire endometriums. Subsequently, DEGs in each of the three types of expression patterns were grouped into four distinct categories according to the similarities in their temporal expression patterns. The expression patterns identified from the microarray analysis were validated by quantitative RT-PCR. The functional enrichment analysis revealed that the common DEGs were enriched in pathways of steroid metabolic process and regulation of retinoic acid receptor signaling. These unique DEGs in Meishan endometrium were involved in cell cycle and adherens junction. The DEGs unique to Yorkshire endometrium were associated with regulation of Rho protein signal transduction, maternal placenta development and cell proliferation. This study revealed the different gene expression patterns or pathways related to the endometrium remodeling in Meishan and Yorkshire pigs, respectively. These unique DEGs in either Meishan or Yorkshire endometriums may contribute to the divergence of the endometrium environment in the two pig breeds. PMID:26393584

  16. Structural analysis reveals the substrate-binding mechanism for the expanded substrate specificity of mutant meso-diaminopimelate dehydrogenase.

    PubMed

    Liu, Weidong; Guo, Rey-Ting; Chen, Xi; Li, Zhe; Gao, Xiuzhen; Huang, Chun-Hsiang; Wu, Qiaqing; Feng, Jinhui; Zhu, Dunming

    2015-04-13

    A meso-diaminopimelate dehydrogenase (DAPDH) from Clostridium tetani E88 (CtDAPDH) was found to have low activity toward the D-amino acids other than its native substrate. Site-directed mutagenesis similar to that carried out on the residues mutated by Vedha-Peters et al. resulted in a mutant enzyme with highly improved catalytic ability for the synthesis of D-amino acids. The crystal structures of the CtDAPDH mutant in apo form and in complex with meso-diaminopimelate (meso-DAP), D-leucine (D-leu), and 4-methyl-2-oxopentanoic acid (MOPA) were solved. meso-DAP was found in an area outside the catalytic cavity; this suggested a possible two-step substrate-binding mechanism for meso-DAP. D-leu and MOPA each bound both to Leu154 and to Gly155 in the open form of CtDAPDH, and structural analysis revealed the molecular basis for the expanded substrate specificity of the mutant meso-diaminopimelate dehydrogenases. PMID:25754803

  17. The manipulation of auxin in the abscission zone cells of Arabidopsis flowers reveals that indoleacetic acid signaling is a prerequisite for organ shedding.

    PubMed

    Basu, Manojit M; González-Carranza, Zinnia H; Azam-Ali, Sayed; Tang, Shouya; Shahid, Ahmad Ali; Roberts, Jeremy A

    2013-05-01

    A number of novel strategies were employed to examine the role of indoleacetic acid (IAA) in regulating floral organ abscission in Arabidopsis (Arabidopsis thaliana). Analysis of auxin influx facilitator expression in β-glucuronidase reporter plants revealed that AUXIN RESISTANT1, LIKE AUX1, and LAX3 were specifically up-regulated at the site of floral organ shedding. Flowers from mutants where individual family members were down-regulated exhibited a reduction in the force necessary to bring about petal separation; however, the effect was not additive in double or quadruple mutants. Using the promoter of a polygalacturonase (At2g41850), active primarily in cells undergoing separation, to drive expression of the bacterial genes iaaL and iaaM, we have shown that it is possible to manipulate auxin activity specifically within the floral organ abscission zone (AZ). Analysis of petal breakstrength reveals that if IAA AZ levels are reduced, shedding takes place prematurely, while if they are enhanced, organ loss is delayed. The At2g41850 promoter was also used to transactivate the gain-of-function AXR3-1 gene in order to disrupt auxin signaling specifically within the floral organ AZ cells. Flowers from transactivated lines failed to shed their sepals, petals, and anthers during pod expansion and maturity, and these organs frequently remained attached to the plant even after silique desiccation and dehiscence had taken place. These observations support a key role for IAA in the regulation of abscission in planta and reveal, to our knowledge for the first time, a requirement for a functional IAA signaling pathway in AZ cells for organ shedding to take place. PMID:23509178

  18. Systematic Analysis of Compositional Order of Proteins Reveals New Characteristics of Biological Functions and a Universal Correlate of Macroevolution

    PubMed Central

    Persi, Erez; Horn, David

    2013-01-01

    We present a novel analysis of compositional order (CO) based on the occurrence of Frequent amino-acid Triplets (FTs) that appear much more than random in protein sequences. The method captures all types of proteomic compositional order including single amino-acid runs, tandem repeats, periodic structure of motifs and otherwise low complexity amino-acid regions. We introduce new order measures, distinguishing between ‘regularity’, ‘periodicity’ and ‘vocabulary’, to quantify these phenomena and to facilitate the identification of evolutionary effects. Detailed analysis of representative species across the tree-of-life demonstrates that CO proteins exhibit numerous functional enrichments, including a wide repertoire of particular patterns of dependencies on regularity and periodicity. Comparison between human and mouse proteomes further reveals the interplay of CO with evolutionary trends, such as faster substitution rate in mouse leading to decrease of periodicity, while innovation along the human lineage leads to larger regularity. Large-scale analysis of 94 proteomes leads to systematic ordering of all major taxonomic groups according to FT-vocabulary size. This is measured by the count of Different Frequent Triplets (DFT) in proteomes. The latter provides a clear hierarchical delineation of vertebrates, invertebrates, plants, fungi and prokaryotes, with thermophiles showing the lowest level of FT-vocabulary. Among eukaryotes, this ordering correlates with phylogenetic proximity. Interestingly, in all kingdoms CO accumulation in the proteome has universal characteristics. We suggest that CO is a genomic-information correlate of both macroevolution and various protein functions. The results indicate a mechanism of genomic ‘innovation’ at the peptide level, involved in protein elongation, shaped in a universal manner by mutational and selective forces. PMID:24278003

  19. Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

    PubMed Central

    Smith, Steven D.; Bridou, Romain; Johs, Alexander; Parks, Jerry M.; Elias, Dwayne A.; Hurt, Richard A.; Brown, Steven D.; Podar, Mircea

    2015-01-01

    Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential for mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. This study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin. PMID:25724962

  20. Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

    SciTech Connect

    Smith, Steven D.; Bridou, Romain; Johs, Alexander; Parks, Jerry M.; Elias, Dwayne A.; Hurt, Richard A.; Brown, Steven D.; Podar, Mircea; Wall, Judy D.

    2015-02-27

    Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential for mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. Ultimately, this study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.

  1. Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

    DOE PAGESBeta

    Smith, Steven D.; Bridou, Romain; Johs, Alexander; Parks, Jerry M.; Elias, Dwayne A.; Hurt, Richard A.; Brown, Steven D.; Podar, Mircea; Wall, Judy D.

    2015-02-27

    Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential formore » mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative “cap helix” region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. Ultimately, this study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin.« less

  2. OTU Deubiquitinases Reveal Mechanisms of Linkage Specificity and Enable Ubiquitin Chain Restriction Analysis

    PubMed Central

    Mevissen, Tycho E.T.; Hospenthal, Manuela K.; Geurink, Paul P.; Elliott, Paul R.; Akutsu, Masato; Arnaudo, Nadia; Ekkebus, Reggy; Kulathu, Yogesh; Wauer, Tobias; El Oualid, Farid; Freund, Stefan M.V.; Ovaa, Huib; Komander, David

    2013-01-01

    Summary Sixteen ovarian tumor (OTU) family deubiquitinases (DUBs) exist in humans, and most members regulate cell-signaling cascades. Several OTU DUBs were reported to be ubiquitin (Ub) chain linkage specific, but comprehensive analyses are missing, and the underlying mechanisms of linkage specificity are unclear. Using Ub chains of all eight linkage types, we reveal that most human OTU enzymes are linkage specific, preferring one, two, or a defined subset of linkage types, including unstudied atypical Ub chains. Biochemical analysis and five crystal structures of OTU DUBs with or without Ub substrates reveal four mechanisms of linkage specificity. Additional Ub-binding domains, the ubiquitinated sequence in the substrate, and defined S1’ and S2 Ub-binding sites on the OTU domain enable OTU DUBs to distinguish linkage types. We introduce Ub chain restriction analysis, in which OTU DUBs are used as restriction enzymes to reveal linkage type and the relative abundance of Ub chains on substrates. PMID:23827681

  3. Factor analysis for isolation of the Raman spectra of aqueous sulfuric acid components

    SciTech Connect

    Malinowski, E.R.; Cox, R.A.; Haldna, U.L.

    1984-04-01

    The Raman spectra of 16 sulfuric acid/water mixtures over the entire mole fraction range were studied by various factor analysis techniques. Abstract factor analysis showed that three factors account for 98.69% of the variation in the data with a real error of 13%. Key-set factor analysis, was used to identify three spectral wavenumbers unique to each component. Spectral-isolation factor analysis, based on the key wavenumbers, revealed the spectra of each unknown component. Target factor analysis, based on the isolated spectra, yielded the relative amounts of the three spectral components. The concentration profiles obtained from the factor loadings, as well as the isolated spectra, were used to identify the chemical species.

  4. Analysis of chlorophenoxy acids and other acidic contaminants in food crops.

    PubMed

    Hajslová, J; Tahtah, W H; Jehlicková, Z; Kocourek, V; Cuhra, P

    1993-04-29

    Several chlorophenoxy acids and chlorinated phenols were determined by means of gas chromatography in contaminated samples of cereals. Extraction of plant matrix with acetone/water mixture followed by alkaline hydrolysis was proved to be suitable for isolation of both free and conjugated residues. The use of pentafluorobenzyl bromide for volatilization of analytes, despite of enhanced ECD response, cannot be recommended for routine analysis. Methylation with either methanol/sulphuric acid or methanol/BF3 reagent can substitute diazomethane-based esterification procedure. Mass fragmentography provided the highest selectivity of detection, moreover good sensitivity--5 ppb--was achieved in this way. Even methyl derivatives of monochlorinated analytes could be, contrary to GC/ECD analysis, quantitated at this level. PMID:8475369

  5. Comparative Proteomics Analysis Reveals L-Arginine Activates Ethanol Degradation Pathways in HepG2 Cells

    PubMed Central

    Yan, Guokai; Lestari, Retno; Long, Baisheng; Fan, Qiwen; Wang, Zhichang; Guo, Xiaozhen; Yu, Jie; Hu, Jun; Yang, Xingya; Chen, Changqing; Liu, Lu; Li, Xiuzhi; Purnomoadi, Agung; Achmadi, Joelal; Yan, Xianghua

    2016-01-01

    L-Arginine (Arg) is a versatile amino acid that plays crucial roles in a wide range of physiological and pathological processes. In this study, to investigate the alteration induced by Arg supplementation in proteome scale, isobaric tags for relative and absolute quantification (iTRAQ) based proteomic approach was employed to comparatively characterize the differentially expressed proteins between Arg deprivation (Ctrl) and Arg supplementation (+Arg) treated human liver hepatocellular carcinoma (HepG2) cells. A total of 21 proteins were identified as differentially expressed proteins and these 21 proteins were all up-regulated by Arg supplementation. Six amino acid metabolism-related proteins, mostly metabolic enzymes, showed differential expressions. Intriguingly, Ingenuity Pathway Analysis (IPA) based pathway analysis suggested that the three ethanol degradation pathways were significantly altered between Ctrl and +Arg. Western blotting and enzymatic activity assays validated that the key enzymes ADH1C, ALDH1A1, and ALDH2, which are mainly involved in ethanol degradation pathways, were highly differentially expressed, and activated between Ctrl and +Arg in HepG2 cells. Furthermore, 10 mM Arg significantly attenuated the cytotoxicity induced by 100 mM ethanol treatment (P < 0.0001). This study is the first time to reveal that Arg activates ethanol degradation pathways in HepG2 cells. PMID:26983598

  6. Molecular Cloning and Functional Characterization of Xenopus tropicalis Frog Transient Receptor Potential Vanilloid 1 Reveal Its Functional Evolution for Heat, Acid, and Capsaicin Sensitivities in Terrestrial Vertebrates*

    PubMed Central

    Ohkita, Masashi; Saito, Shigeru; Imagawa, Toshiaki; Takahashi, Kenji; Tominaga, Makoto; Ohta, Toshio

    2012-01-01

    The functional difference of thermosensitive transient receptor potential (TRP) channels in the evolutionary context has attracted attention, but thus far little information is available on the TRP vanilloid 1 (TRPV1) function of amphibians, which diverged earliest from terrestrial vertebrate lineages. In this study we cloned Xenopus tropicalis frog TRPV1 (xtTRPV1), and functional characterization was performed using HeLa cells heterologously expressing xtTRPV1 (xtTRPV1-HeLa) and dorsal root ganglion neurons isolated from X. tropicalis (xtDRG neurons) by measuring changes in the intracellular calcium concentration ([Ca2+]i). The channel activity was also observed in xtTRPV1-expressing Xenopus oocytes. Furthermore, we tested capsaicin- and heat-induced nocifensive behaviors of the frog X. tropicalis in vivo. At the amino acid level, xtTRPV1 displays ∼60% sequence identity to other terrestrial vertebrate TRPV1 orthologues. Capsaicin induced [Ca2+]i increases in xtTRPV1-HeLa and xtDRG neurons and evoked nocifensive behavior in X. tropicalis. However, its sensitivity was extremely low compared with mammalian orthologues. Low extracellular pH and heat activated xtTRPV1-HeLa and xtDRG neurons. Heat also evoked nocifensive behavior. In oocytes expressing xtTRPV1, inward currents were elicited by heat and low extracellular pH. Mutagenesis analysis revealed that two amino acids (tyrosine 523 and alanine 561) were responsible for the low sensitivity to capsaicin. Taken together, our results indicate that xtTRPV1 functions as a polymodal receptor similar to its mammalian orthologues. The present study demonstrates that TRPV1 functions as a heat- and acid-sensitive channel in the ancestor of terrestrial vertebrates. Because it is possible to examine vanilloid and heat sensitivities in vitro and in vivo, X. tropicalis could be the ideal experimental lower vertebrate animal for the study of TRPV1 function. PMID:22130664

  7. Structural analysis of DNA interaction with retinol and retinoic acid.

    PubMed

    Mandeville, J S; N'soukpoé-Kossi, C N; Neault, J F; Tajmir-Riahi, H A

    2010-06-01

    Dietary constituents of fresh fruits and vegetables may play a relevant role in DNA adduct formation by inhibiting enzymatic activities. Studies have shown the important role of antioxidant vitamins A, C, and E in the protection against cancer and cardiovascular diseases. The antioxidant activity of vitamin A and beta-carotene may consist of scavenging oxygen radicals and preventing DNA damage. This study was designed to examine the interaction of calf-thymus DNA with retinol and retinoic acid in aqueous solution at physiological conditions using a constant DNA concentration and various retinoid contents. Fourier transform infrared (FTIR), circular dichroism (CD), and fluorescence spectroscopic methods were used to determine retinoid binding mode, the binding constant, and the effects of retinol and retinoic acid complexation on DNA conformation and aggregation. Structural analysis showed that retinol and retinoic acid bind DNA via G-C and A-T base pairs and the backbone phosphate groups with overall binding constants of Kret = 3.0 (+/-0.50) x 10(3) (mol.L(-1))(-1) and Kretac = 1.0 (+/-0.20) x 10(4) (mol.L(-1))(-1). The number of bound retinoids per DNA were 0.84 for retinol and 1.3 for retinoic acid. Hydrophobic interactions were also observed at high retinol and retinoic acid contents. At a high retinoid concentration, major DNA aggregation occurred, while DNA remained in the B-family structure. PMID:20555389

  8. Structural analysis of Bacillus pumilus phenolic acid decarboxylase, a lipocalin-fold enzyme.

    PubMed

    Matte, Allan; Grosse, Stephan; Bergeron, Hélène; Abokitse, Kofi; Lau, Peter C K

    2010-11-01

    The decarboxylation of phenolic acids, including ferulic and p-coumaric acids, to their corresponding vinyl derivatives is of importance in the flavouring and polymer industries. Here, the crystal structure of phenolic acid decarboxylase (PAD) from Bacillus pumilus strain UI-670 is reported. The enzyme is a 161-residue polypeptide that forms dimers both in the crystal and in solution. The structure of PAD as determined by X-ray crystallography revealed a β-barrel structure and two α-helices, with a cleft formed at one edge of the barrel. The PAD structure resembles those of the lipocalin-fold proteins, which often bind hydrophobic ligands. Superposition of structurally related proteins bound to their cognate ligands shows that they and PAD bind their ligands in a conserved location within the β-barrel. Analysis of the residue-conservation pattern for PAD-related sequences mapped onto the PAD structure reveals that the conservation mainly includes residues found within the hydrophobic core of the protein, defining a common lipocalin-like fold for this enzyme family. A narrow cleft containing several conserved amino acids was observed as a structural feature and a potential ligand-binding site. PMID:21045284

  9. Structural analysis of Bacillus pumilus phenolic acid decarboxylase, a lipocalin-fold enzyme

    SciTech Connect

    Matte, Allan; Grosse, Stephan; Bergeron, Hélène; Abokitse, Kofi; Lau, Peter C.K.

    2012-04-30

    The decarboxylation of phenolic acids, including ferulic and p-coumaric acids, to their corresponding vinyl derivatives is of importance in the flavoring and polymer industries. Here, the crystal structure of phenolic acid decarboxylase (PAD) from Bacillus pumilus strain UI-670 is reported. The enzyme is a 161-residue polypeptide that forms dimers both in the crystal and in solution. The structure of PAD as determined by X-ray crystallography revealed a -barrel structure and two -helices, with a cleft formed at one edge of the barrel. The PAD structure resembles those of the lipocalin-fold proteins, which often bind hydrophobic ligands. Superposition of structurally related proteins bound to their cognate ligands shows that they and PAD bind their ligands in a conserved location within the -barrel. Analysis of the residue-conservation pattern for PAD-related sequences mapped onto the PAD structure reveals that the conservation mainly includes residues found within the hydrophobic core of the protein, defining a common lipocalin-like fold for this enzyme family. A narrow cleft containing several conserved amino acids was observed as a structural feature and a potential ligand-binding site.

  10. Two-hybrid analysis reveals multiple direct interactions for thrombospondin 1.

    PubMed

    Aho, S; Uitto, J

    1998-10-01

    The yeast two-hybrid system was used to reveal the interactions between proteins residing within the cutaneous basement membrane zone and other gene products expressed in cultured human keratinocytes. The proteins of interest included type VII collagen, the predominant component of anchoring fibrils, and laminin 5, a component of anchoring filaments. Although the two-hybrid system was not able to verify a direct interaction between the type VII collagen NC1 domain and the short arm of Lam(beta)3, the type VII collagen NC1 domain (tVII/NC1) and the laminin 5 beta3 chain globular domain VI (lam5/beta3) cDNAs, when used as baits, detected four overlapping cDNA clones encoding thrombospondin 1 (TSP1). The overlapping region of these cDNAs encodes amino acids 400-459, a segment included within a 70 kDa chymotryptic fragment known to bind type V collagen, laminin-1 and other matrix components. The type VII collagen NC1/TSP1 interaction was confirmed by exchanging the vectors, and the interacting domain was mapped by testing a set of both 5' and 3' deletion constructs. The central region of TSP1, when used as a bait in two-hybrid system, showed strong binding to the fibronectin (FN) type III-like repeats 4-7 of type VII collagen NC1 domain. The TSP1 bait also interacted with laminin 5 beta3 chain domain V/III, and the TSP1/laminin 5 beta3 chain interaction was verified by a GST-fusion protein interaction assay. The transcripts encoding TSP1, TSP2, Lam(beta)3 and type VII collagen were abundant in cultured foreskin keratinocytes, and the expression of TSP1 and TSP2 in a wide variety of adult and fetal tissues was confirmed by PCR analysis of multiple tissue cDNA panels. Furthermore, TSP1 type I repeats showed self interaction, and recognized a clone for extracellular matrix protein fibrillin-2. In addition, clones encoding angiogenesis related protein Jagged1 and a platelet enzyme phospholipase scramblase were identified. Thus, the results indicate several previously

  11. Phospho-selective mechanisms of arrestin conformations and functions revealed by unnatural amino acid incorporation and 19F-NMR

    PubMed Central

    Yang, Fan; Yu, Xiao; Liu, Chuan; Qu, Chang-Xiu; Gong, Zheng; Liu, Hong-Da; Li, Fa-Hui; Wang, Hong-Mei; He, Dong-Fang; Yi, Fan; Song, Chen; Tian, Chang-Lin; Xiao, Kun-Hong; Wang, Jiang-Yun; Sun, Jin-Peng

    2015-01-01

    Specific arrestin conformations are coupled to distinct downstream effectors, which underlie the functions of many G-protein-coupled receptors (GPCRs). Here, using unnatural amino acid incorporation and fluorine-19 nuclear magnetic resonance (19F-NMR) spectroscopy, we demonstrate that distinct receptor phospho-barcodes are translated to specific β-arrestin-1 conformations and direct selective signalling. With its phosphate-binding concave surface, β-arrestin-1 ‘reads' the message in the receptor phospho-C-tails and distinct phospho-interaction patterns are revealed by 19F-NMR. Whereas all functional phosphopeptides interact with a common phosphate binding site and induce the movements of finger and middle loops, different phospho-interaction patterns induce distinct structural states of β-arrestin-1 that are coupled to distinct arrestin functions. Only clathrin recognizes and stabilizes GRK2-specific β-arrestin-1 conformations. The identified receptor-phospho-selective mechanism for arrestin conformation and the spacing of the multiple phosphate-binding sites in the arrestin enable arrestin to recognize plethora phosphorylation states of numerous GPCRs, contributing to the functional diversity of receptors. PMID:26347956

  12. In Vivo Screening Using Transgenic Zebrafish Embryos Reveals New Effects of HDAC Inhibitors Trichostatin A and Valproic Acid on Organogenesis.

    PubMed

    Li, Ling; Bonneton, François; Tohme, Marie; Bernard, Laure; Chen, Xiao Yong; Laudet, Vincent

    2016-01-01

    The effects of endocrine disrupting chemicals (EDCs) on reproduction are well known, whereas their developmental effects are much less characterized. However, exposure to endocrine disruptors during organogenesis may lead to deleterious and permanent problems later in life. Zebrafish (Danio rerio) transgenic lines expressing the green fluorescent protein (GFP) in specific organs and tissues are powerful tools to uncover developmental defects elicited by EDCs. Here, we used seven transgenic lines to visualize in vivo whether a series of EDCs and other pharmaceutical compounds can alter organogenesis in zebrafish. We used transgenic lines expressing GFP in pancreas, liver, blood vessels, inner ear, nervous system, pharyngeal tooth and pectoral fins. This screen revealed that four of the tested chemicals have detectable effects on different organs, which shows that the range of effects elicited by EDCs is wider than anticipated. The endocrine disruptor tetrabromobisphenol-A (TBBPA), as well as the three drugs diclofenac, trichostatin A (TSA) and valproic acid (VPA) induced abnormalities in the embryonic vascular system of zebrafish. Moreover, TSA and VPA induced specific alterations during the development of pancreas, an observation that was confirmed by in situ hybridization with specific markers. Developmental delays were also induced by TSA and VPA in the liver and in pharyngeal teeth, resulting in smaller organ size. Our results show that EDCs can induce a large range of developmental alterations during embryogenesis of zebrafish and establish GFP transgenic lines as powerful tools to screen for EDCs effects in vivo. PMID:26900852

  13. In Vivo Screening Using Transgenic Zebrafish Embryos Reveals New Effects of HDAC Inhibitors Trichostatin A and Valproic Acid on Organogenesis

    PubMed Central

    Li, Ling; Bonneton, François; Tohme, Marie; Bernard, Laure; Chen, Xiao Yong; Laudet, Vincent

    2016-01-01

    The effects of endocrine disrupting chemicals (EDCs) on reproduction are well known, whereas their developmental effects are much less characterized. However, exposure to endocrine disruptors during organogenesis may lead to deleterious and permanent problems later in life. Zebrafish (Danio rerio) transgenic lines expressing the green fluorescent protein (GFP) in specific organs and tissues are powerful tools to uncover developmental defects elicited by EDCs. Here, we used seven transgenic lines to visualize in vivo whether a series of EDCs and other pharmaceutical compounds can alter organogenesis in zebrafish. We used transgenic lines expressing GFP in pancreas, liver, blood vessels, inner ear, nervous system, pharyngeal tooth and pectoral fins. This screen revealed that four of the tested chemicals have detectable effects on different organs, which shows that the range of effects elicited by EDCs is wider than anticipated. The endocrine disruptor tetrabromobisphenol-A (TBBPA), as well as the three drugs diclofenac, trichostatin A (TSA) and valproic acid (VPA) induced abnormalities in the embryonic vascular system of zebrafish. Moreover, TSA and VPA induced specific alterations during the development of pancreas, an observation that was confirmed by in situ hybridization with specific markers. Developmental delays were also induced by TSA and VPA in the liver and in pharyngeal teeth, resulting in smaller organ size. Our results show that EDCs can induce a large range of developmental alterations during embryogenesis of zebrafish and establish GFP transgenic lines as powerful tools to screen for EDCs effects in vivo. PMID:26900852

  14. Proteome Analysis of Streptococcus thermophilus Grown in Milk Reveals Pyruvate Formate-Lyase as the Major Upregulated Protein

    PubMed Central

    Derzelle, Sylviane; Bolotin, Alexander; Mistou, Michel-Yves; Rul, Françoise

    2005-01-01

    We investigated the adaptation to milk of Streptococcus thermophilus LMG18311 using a proteomic approach. Two-dimensional electrophoresis of cytosolic proteins were performed after growth in M17 medium or in milk. A major modification of the proteome concerned proteins involved in the supply of amino acids, like the peptidase PepX, and several enzymes involved in amino acid biosynthesis. In parallel, we observed the upregulation of the synthesis of seven enzymes directly involved in the synthesis of purines, as well as formyl-tetrahydrofolate (THF) synthetase and serine hydroxy-methyl transferase, two enzymes responsible for the synthesis of compounds (THF and glycine, respectively) feeding the purine biosynthetic pathway. The analysis also revealed a massive increase in the synthesis of pyruvate formate-lyase (PFL), the enzyme which converts pyruvate into acetyl coenzyme A and formate. PFL has been essentially studied for its role in mixed-acid product formation in lactic acid bacteria during anaerobic fermentation. However, formate is an important methyl group donor for anabolic pathway through the formation of folate derivates. We hypothesized that PFL was involved in purine biosynthesis during growth in milk. We showed that PFL expression was regulated at the transcriptional level and that pfl transcription occurred during the exponential growth phase in milk. The complementation of milk with formate or purine bases was shown to reduce pfl expression, to suppress PFL synthesis, and to stimulate growth of S. thermophilus. These results show a novel regulatory mechanism controlling the synthesis of PFL and suggest an unrecognized physiological role for PFL as a formate supplier for anabolic purposes. PMID:16332852

  15. Metabolomics analysis reveals 6‐benzylaminopurine as a stimulator for improving lipid and DHA accumulation of Aurantiochytriumsp.

    PubMed Central

    Yu, Xin‐Jun; Sun, Jie; Zheng, Jian‐Yong; Sun, Ya‐Qi

    2016-01-01

    Abstract BACKGROUND Phytohormones are chemical messengers that have a positive effect on biodiesel production of microalgae at low concentrations. However, the effect of phytohormone 6‐benzylaminopurine on lipid and docosahexaenoic acid (DHA) production in marine DHA‐producer Aurantiochytrium has never been reported. In this study, a GC‐MS‐based metabolomics method combined with a multivariate analysis is applied to reveal the metabolic mechanism of 6‐benzylaminopurine enhancing production of lipid and DHA in Aurantiochytrium sp.YLH70. RESULTS In total, 71 metabolites were identified by GC‐MS. The PCA model revealed that 76.9% of metabolite variation was related to 6‐benzylaminopurine treatment, and overall metabolomics profiles between the 6‐benzylaminopurine and control groups were clearly discriminated. Forty‐six metabolites identified by the PLS‐DA model were responsible for responding to 6‐benzylaminopurine. Metabolic analysis showed that 6‐benzylaminopurine could accelerate the rate of utilization of glucose in Aurantiochytrium sp. YLH70, and the metabolic flux from glycolysis, TCA cycle and mevalonate pathway to fatty acids biosynthesis was promoted. Moreover, the anti‐stress mechanism in Aurantiochytrium sp.YLH70 might be induced by 6‐benzylaminopurine. CONCLUSION Metabolomics is a suitable tool to discover the metabolic mechanism for improving lipid and DHA accumulation in a microorganism. 6‐benzylaminopurine has the potential to stimulate lipid and DHA production of Aurantiochytrium sp.YLH70 for industrial purposes. © 2015 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. PMID:27065509

  16. An isobolographic analysis of the antinociceptive effect of xylopic acid in combination with morphine or diclofenac

    PubMed Central

    Woode, Eric; Ameyaw, Elvis Ofori; Abotsi, Wonder Kofi Mensah; Boakye-Gyasi, Eric

    2015-01-01

    Background: A common practice of managing pain globally is the combination of analgesics and this is aimed at facilitating patient compliance, simplifying prescription, and improving efficacy without increasing adverse effects. Fruit extracts of Xylopia aethiopica are used traditionally in the management of pain disorders and xylopic acid (XA) present in the fruit extract have been shown to possess analgesic properties in animals. There is the likelihood of concomitant use of XA and the commonly used analgesics in traditional settings. This study, therefore, evaluated the pharmacologic interaction between XA/morphine and xylopic/diclofenac combinations. Methods: The formalin test and acetic acid writhing test were used to study the antinociceptive activity of XA, morphine, and diclofenac. The isobolographic analysis was used to study the antinociceptive interactions between XA co-administered with morphine or diclofenac. Results: Results obtained revealed that XA (10–100 mg/kg), morphine (1–10 mg/kg), and diclofenac (1–10 mg/kg) produced dose-related antinociception with different potencies in the formalin and acetic acid writhing tests. Isobolographic analysis of XA/morphine and XA/diclofenac combinations revealed potentiation of their antinociceptive effects. The degree of potentiation calculated as interaction index showed synergism for both combinations in all the nociceptive tests. Conclusion: In conclusion, the present study demonstrated synergism for the co-administration of XA with morphine or diclofenac. PMID:26692735

  17. [Principal component analysis of mineral elements and fatty acids composition in flaxseed from ten different regions].

    PubMed

    Xing, Li; Zhao, Feng-Min; Cao, You-Fu; Wang, Mei; Mei, Shuai; Li, Shao-Ping; Cai, Zhi-Yong

    2014-09-01

    Flaxseed is a kind of biomass with high edible and medical value. It is rich in many kinds of nutrients and mineral elements. China is one of the important producing places of flaxseed. In order to explore the main characteristic constituents of mineral elements and fatty acids in flaxseed, the study of analyzing the mineral elements and fatty acid composition from 10 different regions was carried out. The contents of seventeen kinds of mineral elements in flaxseed were determined by inductively coupled plasma mass spectrometry (ICP-MS). The contents of fatty acids of the flaxseed oil obtained under the same conditions were determined by gas chromatography-mass spectrometer (GC-MS). The principal component analysis (PCA) method was applied to the study of analyzing the mineral elements and fatty acid compositions in flaxseeds. The difference in mineral elements and fatty acids of flaxseed from different regions were discussed. The main characteristic constituents of mineral elements and fatty acids were analyzed. The results showed that K, Sr, Mg, Ni, Co, Cr, Cd, Se, Zn and Cu were the main characteristic constituents of the mineral elements. At the same time, C16:0, C18:0, C18: 2, C18:3, C20:0 and C20:1 were the main characteristic constituents of the fatty acids. The combination of ICP-MS, GS-MS and PCA can reveal the characteristics and difference of mineral elements and fatty acids from different regions. The results would provide important theoretical basis for the reasonable and effective utilization of flaxseed. PMID:25532360

  18. Stable isotope studies reveal pathways for the incorporation of non-essential amino acids in Acyrthosiphon pisum (pea aphids).

    PubMed

    Haribal, Meena; Jander, Georg

    2015-12-01

    Plant roots incorporate inorganic nitrogen into the amino acids glutamine, glutamic acid, asparagine and aspartic acid, which together serve as the primary metabolites of nitrogen transport to other tissues. Given the preponderance of these four amino acids, phloem sap is a nutritionally unbalanced diet for phloem-feeding insects. Therefore, aphids and other phloem feeders typically rely on microbial symbionts for the synthesis of essential amino acids. To investigate the metabolism of the four main transport amino acids by the pea aphid (Acyrthosiphon pisum), and its Buchnera aphidicola endosymbionts, aphids were fed defined diets with stable isotope-labeled glutamine, glutamic acid, asparagine or aspartic acid (U-(13)C, U-(15)N; U-(15)N; α-(15)N; or γ-(15)N). The metabolic fate of the dietary (15)N and (13)C was traced using gas chromatography-mass spectrometry (GC-MS). Nitrogen was the major contributor to the observed amino acid isotopomers with one additional unit mass (M+1). However, there was differential incorporation, with the amine nitrogen of asparagine being incorporated into other amino acids more efficiently than the amide nitrogen. Higher isotopomers (M+2, M+3 and M+4) indicated the incorporation of varying numbers of (13)C atoms into essential amino acids. GC-MS assays also showed that, even with an excess of dietary labeled glutamine, glutamic acid, asparagine or aspartic acid, the overall content of these amino acids in aphid bodies was mostly the product of catabolism of dietary amino acids and subsequent re-synthesis within the aphids. Thus, these predominant dietary amino acids are not passed directly to Buchnera endosymbionts for synthesis of essential amino acids, but are rather are produced de novo, most likely by endogenous aphid enzymes. PMID:26632455

  19. In silico Analysis for Predicting Fatty Acids of Black Cumin Oil as Inhibitors of P-Glycoprotein

    PubMed Central

    Ali, Babar; Jamal, Qazi Mohd. Sajid; Mir, Showkat R.; Shams, Saiba; Al-Wabel, Naser A.; Kamal, Mohammad A.

    2015-01-01

    glycoprotein as a receptor.Rat P-gp structure quality shows 88.5% residues in favored region obtained by Ramchandran plot analysis.Docking analysis revealed that Some amino acids common for all compounds like Ser221, Pro222, Ile224, Gly225, Ser228, Ala229, Lys233, Tyr302, Tyr309, Ile337, Leu338 and Thr341 in the P-gp and ligands binding patterns.Eicosadeinoic acid has highest binding affinity with P-gp as the amount of energy needed to bind with P-gp was lowest (-11.92 kcal/mol). Abbreviations used: P-gp: P-glycoprotein PMID:27013802

  20. Transcriptome analysis of Rhizopus oryzae in response to xylose during fumaric acid production.

    PubMed

    Xu, Qing; Liu, Ying; Li, Shuang; Jiang, Ling; Huang, He; Wen, Jianping

    2016-08-01

    Xylose is one of the most abundant lignocellulosic components, but it cannot be used by R. oryzae for fumaric acid production. Here, we applied high-throughput RNA sequencing to generate two transcriptional maps of R. oryzae following fermentation in glucose or xylose. The differential expression analysis showed that, genes involved in amino acid metabolism, fatty acid metabolism, and gluconeogenesis, were up-regulated in response to xylose. Moreover, we discovered the potential presence of oxidative stress in R. oryzae during xylose fermentation. To adapt to this unfavorable condition, R. oryzae displayed reduced growth and induce of a number of antioxidant enzymes, including genes involved in glutathione, trehalose synthesis, and the proteasomal pathway. These responses might divert the flow of carbon required for the accumulation of fumaric acid. Furthermore, using high-throughput RNA sequencing, we identified a large number of novel transcripts and a substantial number of genes that underwent alternative splicing. Our analysis provides remarkable insight into the mechanisms underlying xylose fermentation by R. oryzae. These results may reveal potential target genes or strategies to improve xylose fermentation. PMID:27170374

  1. A Δ-9 Fatty Acid Desaturase Gene in the Microalga Myrmecia incisa Reisigl: Cloning and Functional Analysis.

    PubMed

    Xue, Wen-Bin; Liu, Fan; Sun, Zheng; Zhou, Zhi-Gang

    2016-01-01

    The green alga Myrmecia incisa is one of the richest natural sources of arachidonic acid (ArA). To better understand the regulation of ArA biosynthesis in M. incisa, a novel gene putatively encoding the Δ9 fatty acid desaturase (FAD) was cloned and characterized for the first time. Rapid-amplification of cDNA ends (RACE) was employed to yield a full length cDNA designated as MiΔ9FAD, which is 2442 bp long in sequence. Comparing cDNA open reading frame (ORF) sequence to genomic sequence indicated that there are 8 introns interrupting the coding region. The deduced MiΔ9FAD protein is composed of 432 amino acids. It is soluble and localized in the chloroplast, as evidenced by the absence of transmembrane domains as well as the presence of a 61-amino acid chloroplast transit peptide. Multiple sequence alignment of amino acids revealed two conserved histidine-rich motifs, typical for Δ9 acyl-acyl carrier protein (ACP) desaturases. To determine the function of MiΔ9FAD, the gene was heterologously expressed in a Saccharomyces cerevisiae mutant strain with impaired desaturase activity. Results of GC-MS analysis indicated that MiΔ9FAD was able to restore the synthesis of monounsaturated fatty acids, generating palmitoleic acid and oleic acid through the addition of a double bond in the Δ9 position of palmitic acid and stearic acid, respectively. PMID:27438826

  2. A Δ-9 Fatty Acid Desaturase Gene in the Microalga Myrmecia incisa Reisigl: Cloning and Functional Analysis

    PubMed Central

    Xue, Wen-Bin; Liu, Fan; Sun, Zheng; Zhou, Zhi-Gang

    2016-01-01

    The green alga Myrmecia incisa is one of the richest natural sources of arachidonic acid (ArA). To better understand the regulation of ArA biosynthesis in M. incisa, a novel gene putatively encoding the Δ9 fatty acid desaturase (FAD) was cloned and characterized for the first time. Rapid-amplification of cDNA ends (RACE) was employed to yield a full length cDNA designated as MiΔ9FAD, which is 2442 bp long in sequence. Comparing cDNA open reading frame (ORF) sequence to genomic sequence indicated that there are 8 introns interrupting the coding region. The deduced MiΔ9FAD protein is composed of 432 amino acids. It is soluble and localized in the chloroplast, as evidenced by the absence of transmembrane domains as well as the presence of a 61-amino acid chloroplast transit peptide. Multiple sequence alignment of amino acids revealed two conserved histidine-rich motifs, typical for Δ9 acyl-acyl carrier protein (ACP) desaturases. To determine the function of MiΔ9FAD, the gene was heterologously expressed in a Saccharomyces cerevisiae mutant strain with impaired desaturase activity. Results of GC-MS analysis indicated that MiΔ9FAD was able to restore the synthesis of monounsaturated fatty acids, generating palmitoleic acid and oleic acid through the addition of a double bond in the Δ9 position of palmitic acid and stearic acid, respectively. PMID:27438826

  3. Analysis of amino acids and carbohydrates in green coffee.

    PubMed

    Murkovic, Michael; Derler, Karin

    2006-11-30

    The analysis of carbohydrates and amino acids in green coffee is of the utmost importance since these two classes of compounds act as precursors of the Maillard reaction during which the colour and aroma are formed. During the course of the Maillard reaction potentially harmful substances like acrylamide or 5-hydroxymethyl-furfural accrue as well. The carbohydrates were analysed by anion-exchange chromatography with pulsed amperometric detection and the amino acids by reversed phase chromatography after derivatization with 6-amino-quinolyl-N-hydroxysuccinimidyl carbamate and fluorescence detection. Both methods had to be optimized to obtain a sufficient resolution of the analytes for identification and quantification. Sucrose is the dominant carbohydrate in green coffee with a concentration of up to 90 mg/g (mean = 73 mg/g) in arabica beans and significantly lower amounts in robusta beans (mean=45 mg/g). Alanine is the amino acid with the highest concentration (mean = 1200 microg/g) followed by asparagine (mean = 680 microg/g) in robusta and 800 microg/g and 360 microg/g in arabica respectively. In general, the concentration of amino acids is higher in robusta than in arabica. PMID:16563515

  4. Thermodynamic analysis of acetic acid steam reforming for hydrogen production

    NASA Astrophysics Data System (ADS)

    Goicoechea, Saioa; Ehrich, Heike; Arias, Pedro L.; Kockmann, Norbert

    2015-04-01

    A thermodynamic analysis of hydrogen generation by acetic acid steam reforming has been carried out with respect to applications in solid oxide fuel cells. The effect of operating parameters on equilibrium composition has been examined focusing especially on hydrogen and carbon monoxide production, which are the fuels in this type of fuel cell. The temperature, steam to acetic acid ratio, and to a lesser extent pressure affect significantly the equilibrium product distribution due to their influence on steam reforming, thermal decomposition and water-gas shift reaction. The study shows that steam reforming of acetic acid with a steam to acetic acid ratio of 2 to 1 is thermodynamically feasible with hydrogen, carbon monoxide and water as the main products at the equilibrium at temperatures higher than 700 °C, and achieving CO/CO2 ratios higher than 1. Thus, it can be concluded that within the operation temperature range of solid oxide fuel cells - between 700 °C and 1000 °C - the production of a gas rich in hydrogen and carbon monoxide is promoted.

  5. Proteomic analysis of an unculturable bacterial endosymbiont (Blochmannia) reveals high abundance of chaperonins and biosynthetic enzymes

    PubMed Central

    Fan, Yongliang; Thompson, J. Will; Dubois, Laura G.; Moseley, M. Arthur; Wernegreen, Jennifer J.

    2013-01-01

    Many insect groups have coevolved with bacterial endosymbionts that live within specialized host cells. As a salient example, ants in the tribe Camponotini rely on Blochmannia, an intracellular bacterial mutualist that synthesizes amino acids and recycles nitrogen for the host. We performed a shotgun, label-free, LC/MS/MS quantitative proteomic analysis to investigate the proteome of Blochmannia associated with Camponotus chromaiodes. We identified more than 330 Blochmannia proteins, or 54% coverage of the predicted proteome, as well as 244 Camponotus proteins. Using the average intensity of the top 3 “best flier” peptides along with spiking of a surrogate standard at a known concentration, we estimated the concentration (fmol/μg) of those proteins with confident identification. The estimated dynamic range of Blochmannia protein abundance spanned three orders of magnitude and covered diverse functional categories, with particularly high representation of metabolism, information transfer, and chaperones. GroEL, the most abundant protein, totaled 6% of Blochmannia protein abundance. Biosynthesis of essential amino acids, fatty acids, and nucleotides, and sulfate assimilation had disproportionately high coverage in the proteome, further supporting a nutritional role of the symbiosis. This first quantitative proteomic analysis of an ant endosymbiont illustrates a promising approach to study the functional basis of intimate symbioses. PMID:23205679

  6. Genome-wide mutational spectra analysis reveals significant cancer-specific heterogeneity

    PubMed Central

    Tan, Hua; Bao, Jiguang; Zhou, Xiaobo

    2015-01-01

    Cancer is widely recognized as a genetic disease in which somatic mutations are sequentially accumulated to drive tumor progression. Although genomic landscape studies are informative for individual cancer types, a comprehensive comparative study of tumorigenic mutations across cancer types based on integrative data sources is still a pressing need. We systematically analyzed ~106 non-synonymous mutations extracted from COSMIC, involving ~8000 genome-wide screened samples across 23 major human cancers at both the amino acid and gene levels. Our analysis identified cancer-specific heterogeneity that traditional nucleotide variation analysis alone usually overlooked. Particularly, the amino acid arginine (R) turns out to be the most favorable target of amino acid alteration in most cancer types studied (P < 10−9, binomial test), reflecting its important role in cellular physiology. The tumor suppressor gene TP53 is mutated exclusively with the HYDIN, KRAS, and PTEN genes in large intestine, lung, and endometrial cancers respectively, indicating that TP53 takes part in different signaling pathways in different cancers. While some of our analyses corroborated previous observations, others indicated relevant candidates with high priority for further experimental validation. Our findings have many ramifications in understanding the etiology of cancer and the underlying molecular mechanisms in particular cancers. PMID:26212640

  7. Comparative genomics in acid mine drainage biofilm communities reveals metabolic and structural differentiation of co-occurring archaea

    PubMed Central

    2013-01-01

    Background Metal sulfide mineral dissolution during bioleaching and acid mine drainage (AMD) formation creates an environment that is inhospitable to most life. Despite dominance by a small number of bacteria, AMD microbial biofilm communities contain a notable variety of coexisting and closely related Euryarchaea, most of which have defied cultivation efforts. For this reason, we used metagenomics to analyze variation in gene content that may contribute to niche differentiation among co-occurring AMD archaea. Our analyses targeted members of the Thermoplasmatales and related archaea. These results greatly expand genomic information available for this archaeal order. Results We reconstructed near-complete genomes for uncultivated, relatively low abundance organisms A-, E-, and Gplasma, members of Thermoplasmatales order, and for a novel organism, Iplasma. Genomic analyses of these organisms, as well as Ferroplasma type I and II, reveal that all are facultative aerobic heterotrophs with the ability to use many of the same carbon substrates, including methanol. Most of the genomes share genes for toxic metal resistance and surface-layer production. Only Aplasma and Eplasma have a full suite of flagellar genes whereas all but the Ferroplasma spp. have genes for pili production. Cryogenic-electron microscopy (cryo-EM) and tomography (cryo-ET) strengthen these metagenomics-based ultrastructural predictions. Notably, only Aplasma, Gplasma and the Ferroplasma spp. have predicted iron oxidation genes and Eplasma and Iplasma lack most genes for cobalamin, valine, (iso)leucine and histidine synthesis. Conclusion The Thermoplasmatales AMD archaea share a large number of metabolic capabilities. All of the uncultivated organisms studied here (A-, E-, G-, and Iplasma) are metabolically very similar to characterized Ferroplasma spp., differentiating themselves mainly in their genetic capabilities for biosynthesis, motility, and possibly iron oxidation. These results indicate that

  8. Cytochrome P450 genes in coronary artery diseases: Codon usage analysis reveals genomic GC adaptation.

    PubMed

    Malakar, Arup Kumar; Halder, Binata; Paul, Prosenjit; Chakraborty, Supriyo

    2016-09-15

    Establishing codon usage biases are imperative for understanding the etiology of coronary artery diseases (CAD) as well as the genetic factors associated with these diseases. The aim of this study was to evaluate the contribution of 18 responsible cytochrome P450 (CYP) genes for the risk of CAD. Effective number of codon (Nc) showed a negative correlation with both GC3 and synonymous codon usage order (SCUO) suggesting an antagonistic relationship between codon usage and Nc of genes. The dinucleotide analysis revealed that CG and TA dinucleotides have the lowest odds ratio in these genes. Principal component analysis showed that GC composition has a profound effect in separating the genes along the first major axis. Our findings revealed that mutational pressure and natural selection could possibly be the major factors responsible for codon bias in these genes. The study not only offers an insight into the mechanisms of genomic GC adaptation, but also illustrates the complexity of CYP genes in CAD. PMID:27275533

  9. Autotriploid origin of Carassius auratus as revealed by chromosomal locus analysis.

    PubMed

    Qin, Qinbo; Wang, Juan; Hu, Min; Huang, Shengnan; Liu, Shaojun

    2016-06-01

    In the Dongting water system, the Carassius auratus (Crucian carp) complex is characterized by the coexistence of diploid forms (2n=100, 2nCC) and polyploid forms. Chromosomal and karyotypic analyses have suggested that the polyploid C. auratus has a triploid (3n=150, 3nCC) and a tetraploid origin (4n=200), respectively. However, there is a lack of direct genetic evidence to support this conclusion. In this paper, analysis of the 5S rDNA chromosomal locus revealed that the 3nCC is of triploid origin. Analysis of the species-specific chromosomal centromere locus revealed that 3nCC individuals possess three sets of C. auratus-derived chromosomes. Our results provide direct cytogenetic evidence suggesting that individuals with 150 chromosomes are of autotriploid origin within the C. auratus complex. It marks an important contribution to the study of polyploidization and the evolution of vertebrates. PMID:27084707

  10. Gas-Phase Fragmentation Analysis of Nitro-Fatty Acids

    NASA Astrophysics Data System (ADS)

    Bonacci, Gustavo; Asciutto, Eliana K.; Woodcock, Steven R.; Salvatore, Sonia R.; Freeman, Bruce A.; Schopfer, Francisco J.

    2011-09-01

    Nitro-fatty acids are electrophilic signaling mediators formed in increased amounts during inflammation by nitric oxide and nitrite-dependent redox reactions. A more rigorous characterization of endogenously-generated species requires additional understanding of their gas-phase induced fragmentation. Thus, collision induced dissociation (CID) of nitroalkane and nitroalkene groups in fatty acids were studied in the negative ion mode to provide mass spectrometric tools for their structural characterization. Fragmentation of nitroalkanes occurred mainly through loss of the NO{2/-} anion or neutral loss of HNO2. The CID of nitroalkenes proceeds via a more complex cyclization, followed by fragmentation to nitrile and aldehyde products. Gas-phase fragmentation of nitroalkene functional groups with additional γ or δ unsaturation occurred through a multiple step cyclization reaction process, leading to 5 and 6 member ring heterocyclic products and carbon chain fragmentation. Cyclization products were not obtained during nitroalkane fragmentation, highlighting the role of double bond π electrons during NO{2/-} rearrangements, stabilization and heterocycle formation. The proposed structures, mechanisms and products of fragmentation are supported by analysis of 13C and 15N labeled parent molecules, 6 different nitroalkene positional isomers, 6 nitroalkane positional isomers, accurate mass determinations at high resolution and quantum mechanics calculations. Multiple key diagnostic ion fragments were obtained through this analysis, allowing for the precise placement of double bonds and sites of fatty acid nitration, thus supporting an ability to predict nitro positions in biological samples.

  11. Binding and Inactivation Mechanism of a Humanized Fatty Acid Amide Hydrolase by [alpha]-Ketoheterocycle Inhibitors Revealed from Cocrystal Structures

    SciTech Connect

    Mileni, Mauro; Garfunkle, Joie; DeMartino, Jessica K.; Cravatt, Benjamin F.; Boger, Dale L.; Stevens, Raymond C.

    2010-08-17

    The cocrystal X-ray structures of two isomeric {alpha}-ketooxazole inhibitors (1 (OL-135) and 2) bound to fatty acid amide hydrolase (FAAH), a key enzymatic regulator of endocannabinoid signaling, are disclosed. The active site catalytic Ser241 is covalently bound to the inhibitors electrophilic carbonyl groups, providing the first structures of FAAH bound to an inhibitor as a deprotonated hemiketal mimicking the enzymatic tetrahedral intermediate. The work also offers a detailed view of the oxyanion hole and an exceptional 'in-action' depiction of the unusual Ser-Ser-Lys catalytic triad. These structures capture the first picture of inhibitors that span the active site into the cytosolic port providing new insights that help to explain FAAH's interaction with substrate leaving groups and their role in modulating inhibitor potency and selectivity. The role for the activating central heterocycle is clearly defined and distinguished from that observed in prior applications with serine proteases, reconciling the large electronic effect of attached substituents found unique to this class of inhibitors with FAAH. Additional striking active site flexibility is seen upon binding of the inhibitors, providing insights into the existence of a now well-defined membrane access channel with the disappearance of a spatially independent portion of the acyl chain-binding pocket. Finally, comparison of the structures of OL-135 (1) and its isomer 2 indicates that they bind identically to FAAH, albeit with reversed orientations of the central activating heterocycle, revealing that the terminal 2-pyridyl substituent and the acyl chain phenyl group provide key anchoring interactions and confirming the distinguishing role of the activating oxazole.

  12. Whole Genome Expression Analysis Reveals Differential Effects of TiO2 Nanotubes on Vascular Cells

    PubMed Central

    Peng, Lily; Barczak, Andrea J.; Barbeau, Rebecca A.; Xiao, Yuanyuan; LaTempa, Thomas J.; Grimes, Craig A.; Desai, Tejal A.

    2010-01-01

    The response of primary human endothelial (ECs) and vascular smooth muscle cells (VSMCs) to TiO2 nanotube arrays is studied through gene expression analysis. Microarrays revealed that nanotubes enhanced EC proliferation and motility, decreased VSMC proliferation, and decreased expression of molecules involved in inflammation and coagulation in both cell types. Networks generated from significantly affected genes suggest that cells may be sensing nanotopographical cues via pathways previously implicated in sensing shear stress. PMID:20030358

  13. Comparative analysis of fungal genomes reveals different plant cell wall degrading capacity in fungi

    PubMed Central

    2013-01-01

    Background Fungi produce a variety of carbohydrate activity enzymes (CAZymes) for the degradation of plant polysaccharide materials to facilitate infection and/or gain nutrition. Identifying and comparing CAZymes from fungi with different nutritional modes or infection mechanisms may provide information for better understanding of their life styles and infection models. To date, over hundreds of fungal genomes are publicly available. However, a systematic comparative analysis of fungal CAZymes across the entire fungal kingdom has not been reported. Results In this study, we systemically identified glycoside hydrolases (GHs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), and glycosyltransferases (GTs) as well as carbohydrate-binding modules (CBMs) in the predicted proteomes of 103 representative fungi from Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota. Comparative analysis of these CAZymes that play major roles in plant polysaccharide degradation revealed that fungi exhibit tremendous diversity in the number and variety of CAZymes. Among them, some families of GHs and CEs are the most prevalent CAZymes that are distributed in all of the fungi analyzed. Importantly, cellulases of some GH families are present in fungi that are not known to have cellulose-degrading ability. In addition, our results also showed that in general, plant pathogenic fungi have the highest number of CAZymes. Biotrophic fungi tend to have fewer CAZymes than necrotrophic and hemibiotrophic fungi. Pathogens of dicots often contain more pectinases than fungi infecting monocots. Interestingly, besides yeasts, many saprophytic fungi that are highly active in degrading plant biomass contain fewer CAZymes than plant pathogenic fungi. Furthermore, analysis of the gene expression profile of the wheat scab fungus Fusarium graminearum revealed that most of the CAZyme genes related to cell wall degradation were up-regulated during plant infection. Phylogenetic analysis also

  14. Proteomics Analysis Reveals a Potential Antibiotic Cocktail Therapy Strategy for Aeromonas hydrophila Infection in Biofilm.

    PubMed

    Li, Wanxin; Yao, Zujie; Sun, Lina; Hu, Wenjie; Cao, Jijuan; Lin, Wenxiong; Lin, Xiangmin

    2016-06-01

    Antibiotic fitness and acquired resistance are the two critical factors when bacteria respond to antibiotics, and the correlations and mechanisms between these two factors remain largely unknown. In this study, a TMT-labeling-based quantitative proteomics method was used to compare the differential expression of proteins between the fitness and acquired resistance to chlortetracycline in Aeromonas hydrophila biofilm. Bioinformatics analysis showed that translation-related ribosomal proteins, such as 30s ribosome subunits, increased in both factors; fatty acid biosynthesis related proteins, such as FabB, FabD, FabG, AccA, and AccD, increased in biofilm fitness, and some pathways (including propanoate-metabolism-related protein, such as PrpB, AtoB, PflB, AcsA, PrpD, and GabT) displayed decreased abundance in acquired resistance biofilm. The varieties of selected proteins involved in fatty acid biosynthesis and propanoate metabolism were further validated by q-PCR assay or Western blotting. Furthermore, the antibiotic-resistance-function assays showed that fatty-acid biosynthesis should be a protective antibiotics-resistance mechanism and a cocktail of chlortetracycline and triclosan, a fatty-acid-biosynthesis inhibitor, exhibited more efficient antimicrobial capability than did each antibiotic individually on biofilm, specifically on chlortetracycline-sensitive biofilm. We therefore demonstrate that the up-regulation of fatty acid biosynthesis may play an important role in antibiotic resistance and suggest that a cocktail of chlortetracycline and triclosan may be a potential cocktail therapy for pathogenic infections in biofilm. PMID:27110028

  15. Transcriptomic Analysis Reveals Possible Influences of ABA on Secondary Metabolism of Pigments, Flavonoids and Antioxidants in Tomato Fruit during Ripening

    PubMed Central

    Mou, Wangshu; Li, Dongdong; Luo, Zisheng; Mao, Linchun; Ying, Tiejin

    2015-01-01

    Abscisic acid (ABA) has been proven to be involved in the regulation of climacteric fruit ripening, but a comprehensive investigation of its influence on ripening related processes is still lacking. By applying the next generation sequencing technology, we conducted a comparative analysis of the effects of exogenous ABA and NDGA (Nordihydroguaiaretic acid, an inhibitor of ABA biosynthesis) on tomato fruit ripening. The high throughput sequencing results showed that out of the 25728 genes expressed across all three samples, 10388 were identified as significantly differently expressed genes. Exogenous ABA was found to enhance the transcription of genes involved in pigments metabolism, including carotenoids biosynthesis and chlorophyll degradation, whereas NDGA treatment inhibited these processes. The results also revealed the crucial role of ABA in flavonoids synthesis and regulation of antioxidant system. Intriguingly, we also found that an inhibition of endogenous ABA significantly enhanced the transcriptional abundance of genes involved in photosynthesis. Our results highlighted the significance of ABA in regulating tomato ripening, which provided insight into the regulatory mechanism of fruit maturation and senescence process. PMID:26053166

  16. Reticulate evolutionary history and extensive introgression in mosquito species revealed by phylogenetic network analysis.

    PubMed

    Wen, Dingqiao; Yu, Yun; Hahn, Matthew W; Nakhleh, Luay

    2016-06-01

    The role of hybridization and subsequent introgression has been demonstrated in an increasing number of species. Recently, Fontaine et al. (Science, 347, 2015, 1258524) conducted a phylogenomic analysis of six members of the Anopheles gambiae species complex. Their analysis revealed a reticulate evolutionary history and pointed to extensive introgression on all four autosomal arms. The study further highlighted the complex evolutionary signals that the co-occurrence of incomplete lineage sorting (ILS) and introgression can give rise to in phylogenomic analyses. While tree-based methodologies were used in the study, phylogenetic networks provide a more natural model to capture reticulate evolutionary histories. In this work, we reanalyse the Anopheles data using a recently devised framework that combines the multispecies coalescent with phylogenetic networks. This framework allows us to capture ILS and introgression simultaneously, and forms the basis for statistical methods for inferring reticulate evolutionary histories. The new analysis reveals a phylogenetic network with multiple hybridization events, some of which differ from those reported in the original study. To elucidate the extent and patterns of introgression across the genome, we devise a new method that quantifies the use of reticulation branches in the phylogenetic network by each genomic region. Applying the method to the mosquito data set reveals the evolutionary history of all the chromosomes. This study highlights the utility of 'network thinking' and the new insights it can uncover, in particular in phylogenomic analyses of large data sets with extensive gene tree incongruence. PMID:26808290

  17. Influences of acidic reaction and hydrolytic conditions on monosaccharide composition analysis of acidic, neutral and basic polysaccharides.

    PubMed

    Wang, Qing-Chi; Zhao, Xia; Pu, Jiang-Hua; Luan, Xiao-Hong

    2016-06-01

    Monosaccharide composition analysis is important for structural characterization of polysaccharides. To investigate the influences of acidic reaction and hydrolytic conditions on monosaccharide composition analysis of polysaccharides, we chose alginate, starch, chitosan and chondroitin sulfate as representative of acidic, neutral, basic and complex polysaccharides to compare the release degree of monosaccharides under different hydrolytic conditions. The hydrolysis stability of 10 monosaccharide standards was also explored. Results showed that the basic sugars were hard to release but stable, the acidic sugars (uronic acids) were easy to release but unstable, and the release and stability of neutral sugars were in between acidic and basic sugars. In addition, the hydrolysis process was applied to monosaccharide composition analysis of Hippocampus trimaculatus polysaccharide and the appropriate hydrolytic condition was accorded with that of the above four polysaccharides. Thus, different hydrolytic conditions should be used for the monosaccharide composition analysis of polysaccharides based on their structural characteristics. PMID:27083372

  18. Transcriptome, carbohydrate, and phytohormone analysis of Petunia hybrida reveals a complex disturbance of plant functional integrity under mild chilling stress

    PubMed Central

    Bauerfeind, Martin Andreas; Winkelmann, Traud; Franken, Philipp; Druege, Uwe

    2015-01-01

    Cultivation of chilling-tolerant ornamental crops at lower temperature could reduce the energy demands of heated greenhouses. To provide a better understanding of how sub-optimal temperatures (12°C vs. 16°C) affect growth of the sensitive Petunia hybrida cultivar ‘SweetSunshine Williams’, the transcriptome, carbohydrate metabolism, and phytohormone homeostasis were monitored in aerial plant parts over 4 weeks by use of a microarray, enzymatic assays and GC-MS/MS. The data revealed three consecutive phases of chilling response. The first days were marked by a strong accumulation of sugars, particularly in source leaves, preferential up-regulation of genes in the same tissue and down-regulation of several genes in the shoot apex, especially those involved in the abiotic stress response. The midterm phase featured a partial normalization of carbohydrate levels and gene expression. After 3 weeks of chilling exposure, a new stabilized balance was established. Reduced hexose levels in the shoot apex, reduced ratios of sugar levels between the apex and source leaves and a higher apical sucrose/hexose ratio, associated with decreased activity and expression of cell wall invertase, indicate that prolonged chilling induced sugar accumulation in source leaves at the expense of reduced sugar transport to and reduced sucrose utilization in the shoot. This was associated with reduced levels of indole-3-acetic acid and abscisic acid in the apex and high numbers of differentially, particularly up-regulated genes, especially in the source leaves, including those regulating histones, ethylene action, transcription factors, and a jasmonate-ZIM-domain protein. Transcripts of one Jumonji C domain containing protein and one expansin accumulated in source leaves throughout the chilling period. The results reveal a dynamic and complex disturbance of plant function in response to mild chilling, opening new perspectives for the comparative analysis of differently tolerant cultivars

  19. Nucleotide sequence analysis of a cloned DNA fragment from human cells reveals homology to retrotransposons.

    PubMed Central

    Flügel, R M; Maurer, B; Bannert, H; Rethwilm, A; Schnitzler, P; Darai, G

    1987-01-01

    During molecular cloning of proviral DNA of human spumaretrovirus, various recombinant clones were established and analyzed. Blot hybridization revealed that one of the recombinant plasmids had the characteristic features of a member of the long interspersed repetitive sequences family. The DNA element was analyzed by restriction mapping and nucleotide sequencing. It showed a high degree of amino acid sequence homology of 54.3% when compared with the 5'-terminal part of the pol gene product of the murine retrotransposon LIMd. The 3' region of the cloned DNA element encodes proteins with an even higher degree of homology of 67.4% in comparison to the corresponding parts of a member of the primate KpnI sequence family. Images PMID:3031462

  20. A structural homologue of colipase in black mamba venom revealed by NMR floating disulphide bridge analysis.

    PubMed

    Boisbouvier, J; Albrand, J P; Blackledge, M; Jaquinod, M; Schweitz, H; Lazdunski, M; Marion, D

    1998-01-01

    The solution structure of mamba intestinal toxin 1 (MIT1), isolated from Dendroaspis polylepis polylepis venom, has been determined. This molecule is a cysteine-rich polypeptide exhibiting no recognised family membership. Resistance to MIT1 to classical specific endoproteases produced contradictory NMR and biochemical information concerning disulphide-bridge topology. We have used distance restraints allowing ambiguous partners between S atoms in combination with NMR-derived structural information, to correctly determine the disulphide-bridge topology. The resultant solution structure of MIT1, determined to a resolution of 0.5 A, reveals an unexpectedly similar global fold with respect to colipase, a protein involved in fatty acid digestion. Colipase exhibits an analogous resistance to endoprotease activity, indicating for the first time the possible topological origins of this biochemical property. The biochemical and structural homology permitted us to propose a mechanically related digestive function for MIT1 and provides novel information concerning snake venom protein evolution. PMID:9761684

  1. Period-Amplitude Analysis Reveals Wake-Dependent Changes in the Electroencephalogram during Sleep Deprivation

    PubMed Central

    Ehlen, J. Christopher; Jefferson, Felicia; Brager, Allison J.; Benveniste, Morris; Paul, Ketema N.

    2013-01-01

    Study Objectives: Electroencephalographic slow wave activity (SWA) during non-rapid eye movement (NREM) sleep results from the synchronous oscillation of cortical neurons and is the standard measurement of sleep homeostasis. SWA is not a direct measure of sleep pressure accumulation, but rather a measure of the NREM-sleep response to accumulated sleep pressure. Currently, no practical standard for the direct measurement of sleep pressure accumulation exists. Recently, it was demonstrated that rat cortical neurons undergo oscillations during wake that are similar to the cortical oscillations responsible for SWA. Furthermore, these oscillations increase in number as time awake increases. Here we hypothesize that period-amplitude analysis of the electroencephalogram (EEG), which treats the EEG as a series of discrete waves, can measure these cortical oscillations, and thus, is a measure of sleep-pressure accumulation during extended wake. Design: Mice were sleep deprived for 24 h by confinement to a slowly rotating wheel in order to assess wake-dependent changes in EEG wave incidence. Measurements and Results: Continuous period-amplitude analysis of the waking EEG across 24 h of sleep deprivation revealed that the incidence of 2 to 6 Hz waves increased exponentially over the deprivation period. This increase in wave incidence appeared to occur in two phases with exponential time constants of approximately 0.12 h and 3 h. Further analysis revealed that the changes in wave incidence were significantly correlated with two established markers of sleep pressure, SWA and NREM sleep latency. Conclusions: The data suggest that wave incidence is an effective method of measuring sleep homeostasis in the waking EEG that provides better temporal resolution than spectral power analysis. Citation: Ehlen JC; Jefferson F; Brager AJ; Benveniste M; Paul KN. Period-amplitude analysis reveals wake-dependent changes in the electroencephalogram during sleep deprivation. SLEEP 2013

  2. Transcriptome Profiling and Physiological Studies Reveal a Major Role for Aromatic Amino Acids in Mercury Stress Tolerance in Rice Seedlings

    PubMed Central

    Trinh, Ngoc Nam; Huang, Li-Yao; Chen, Ying-Chih; Cheng, Kai-Teng; Huang, Tsai-Lien; Lin, Chung-Yi; Huang, Hao-Jen

    2014-01-01

    Mercury (Hg) is a serious environmental pollution threat to the planet. The accumulation of Hg in plants disrupts many cellular-level functions and inhibits growth and development, but the mechanism is not fully understood. To gain more insight into the cellular response to Hg, we performed a large-scale analysis of the rice transcriptome during Hg stress. Genes induced with short-term exposure represented functional categories of cell-wall formation, chemical detoxification, secondary metabolism, signal transduction and abiotic stress response. Moreover, Hg stress upregulated several genes involved in aromatic amino acids (Phe and Trp) and increased the level of free Phe and Trp content. Exogenous application of Phe and Trp to rice roots enhanced tolerance to Hg and effectively reduced Hg-induced production of reactive oxygen species. Hg induced calcium accumulation and activated mitogen-activated protein kinase. Further characterization of the Hg-responsive genes we identified may be helpful for better understanding the mechanisms of Hg in plants. PMID:24840062

  3. Transcriptome profiling and physiological studies reveal a major role for aromatic amino acids in mercury stress tolerance in rice seedlings.

    PubMed

    Chen, Yun-An; Chi, Wen-Chang; Trinh, Ngoc Nam; Huang, Li-Yao; Chen, Ying-Chih; Cheng, Kai-Teng; Huang, Tsai-Lien; Lin, Chung-Yi; Huang, Hao-Jen

    2014-01-01

    Mercury (Hg) is a serious environmental pollution threat to the planet. The accumulation of Hg in plants disrupts many cellular-level functions and inhibits growth and development, but the mechanism is not fully understood. To gain more insight into the cellular response to Hg, we performed a large-scale analysis of the rice transcriptome during Hg stress. Genes induced with short-term exposure represented functional categories of cell-wall formation, chemical detoxification, secondary metabolism, signal transduction and abiotic stress response. Moreover, Hg stress upregulated several genes involved in aromatic amino acids (Phe and Trp) and increased the level of free Phe and Trp content. Exogenous application of Phe and Trp to rice roots enhanced tolerance to Hg and effectively reduced Hg-induced production of reactive oxygen species. Hg induced calcium accumulation and activated mitogen-activated protein kinase. Further characterization of the Hg-responsive genes we identified may be helpful for better understanding the mechanisms of Hg in plants. PMID:24840062

  4. Quantitative Proteomic Analysis Reveals Populus cathayana Females Are More Sensitive and Respond More Sophisticatedly to Iron Deficiency than Males.

    PubMed

    Zhang, Sheng; Zhang, Yunxiang; Cao, Yanchun; Lei, Yanbao; Jiang, Hao

    2016-03-01

    Previous studies have shown that there are significant sexual differences in the morphological and physiological responses of Populus cathayana Rehder to nitrogen and phosphorus deficiencies, but little is known about the sex-specific differences in responses to iron deficiency. In this study, the effects of iron deficiency on the morphology, physiology, and proteome of P. cathayana males and females were investigated. The results showed that iron deficiency (25 days) significantly decreased height growth, photosynthetic rate, chlorophyll content, and tissue iron concentration in both sexes. A comparison between the sexes indicated that iron-deficient males had less height inhibition and photosynthesis system II or chloroplast ultrastructural damage than iron-deficient females. iTRAQ-based quantitative proteomic analysis revealed that 144 and 68 proteins were decreased in abundance (e.g., proteins involved in photosynthesis, carbohydrate and energy metabolism, and gene expression regulation) and 78 and 39 proteins were increased in abundance (e.g., proteins involved in amino acid metabolism and stress response) according to the criterion of ratio ≥1.5 in females and males, respectively. A comparison between the sexes indicated that iron-deficient females exhibited a greater change in the proteins involved in photosynthesis, carbon and energy metabolism, the redox system, and stress responsive proteins. This study reveals females are more sensitive and have a more sophisticated response to iron deficiency compared with males and provides new insights into differential sexual responses to nutrient deficiency. PMID:26842668

  5. Proteomics Analysis with a Nano Random Forest Approach Reveals Novel Functional Interactions Regulated by SMC Complexes on Mitotic Chromosomes*

    PubMed Central

    Ohta, Shinya; Montaño-Gutierrez, Luis F.; de Lima Alves, Flavia; Ogawa, Hiromi; Toramoto, Iyo; Sato, Nobuko; Morrison, Ciaran G.; Takeda, Shunichi; Hudson, Damien F.; Earnshaw, William C.

    2016-01-01

    Packaging of DNA into condensed chromosomes during mitosis is essential for the faithful segregation of the genome into daughter nuclei. Although the structure and composition of mitotic chromosomes have been studied for over 30 years, these aspects are yet to be fully elucidated. Here, we used stable isotope labeling with amino acids in cell culture to compare the proteomes of mitotic chromosomes isolated from cell lines harboring conditional knockouts of members of the condensin (SMC2, CAP-H, CAP-D3), cohesin (Scc1/Rad21), and SMC5/6 (SMC5) complexes. Our analysis revealed that these complexes associate with chromosomes independently of each other, with the SMC5/6 complex showing no significant dependence on any other chromosomal proteins during mitosis. To identify subtle relationships between chromosomal proteins, we employed a nano Random Forest (nanoRF) approach to detect protein complexes and the relationships between them. Our nanoRF results suggested that as few as 113 of 5058 detected chromosomal proteins are functionally linked to chromosome structure and segregation. Furthermore, nanoRF data revealed 23 proteins that were not previously suspected to have functional interactions with complexes playing important roles in mitosis. Subsequent small-interfering-RNA-based validation and localization tracking by green fluorescent protein-tagging highlighted novel candidates that might play significant roles in mitotic progression. PMID:27231315

  6. PhyloChip microarray analysis reveals altered gastrointestinal microbial communities in a rat model of colonic hypersensitivity

    SciTech Connect

    Nelson, T.A.; Holmes, S.; Alekseyenko, A.V.; Shenoy, M.; DeSantis, T.; Wu, C.H.; Andersen, G.L.; Winston, J.; Sonnenburg, J.; Pasricha, P.J.; Spormann, A.

    2010-12-01

    Irritable bowel syndrome (IBS) is a chronic, episodic gastrointestinal disorder that is prevalent in a significant fraction of western human populations; and changes in the microbiota of the large bowel have been implicated in the pathology of the disease. Using a novel comprehensive, high-density DNA microarray (PhyloChip) we performed a phylogenetic analysis of the microbial community of the large bowel in a rat model in which intracolonic acetic acid in neonates was used to induce long lasting colonic hypersensitivity and decreased stool water content and frequency, representing the equivalent of human constipation-predominant IBS. Our results revealed a significantly increased compositional difference in the microbial communities in rats with neonatal irritation as compared with controls. Even more striking was the dramatic change in the ratio of Firmicutes relative to Bacteroidetes, where neonatally irritated rats were enriched more with Bacteroidetes and also contained a different composition of species within this phylum. Our study also revealed differences at the level of bacterial families and species. The PhyloChip is a useful and convenient method to study enteric microflora. Further, this rat model system may be a useful experimental platform to study the causes and consequences of changes in microbial community composition associated with IBS.

  7. Proteomics Analysis with a Nano Random Forest Approach Reveals Novel Functional Interactions Regulated by SMC Complexes on Mitotic Chromosomes.

    PubMed

    Ohta, Shinya; Montaño-Gutierrez, Luis F; de Lima Alves, Flavia; Ogawa, Hiromi; Toramoto, Iyo; Sato, Nobuko; Morrison, Ciaran G; Takeda, Shunichi; Hudson, Damien F; Rappsilber, Juri; Earnshaw, William C

    2016-08-01

    Packaging of DNA into condensed chromosomes during mitosis is essential for the faithful segregation of the genome into daughter nuclei. Although the structure and composition of mitotic chromosomes have been studied for over 30 years, these aspects are yet to be fully elucidated. Here, we used stable isotope labeling with amino acids in cell culture to compare the proteomes of mitotic chromosomes isolated from cell lines harboring conditional knockouts of members of the condensin (SMC2, CAP-H, CAP-D3), cohesin (Scc1/Rad21), and SMC5/6 (SMC5) complexes. Our analysis revealed that these complexes associate with chromosomes independently of each other, with the SMC5/6 complex showing no significant dependence on any other chromosomal proteins during mitosis. To identify subtle relationships between chromosomal proteins, we employed a nano Random Forest (nanoRF) approach to detect protein complexes and the relationships between them. Our nanoRF results suggested that as few as 113 of 5058 detected chromosomal proteins are functionally linked to chromosome structure and segregation. Furthermore, nanoRF data revealed 23 proteins that were not previously suspected to have functional interactions with complexes playing important roles in mitosis. Subsequent small-interfering-RNA-based validation and localization tracking by green fluorescent protein-tagging highlighted novel candidates that might play significant roles in mitotic progression. PMID:27231315

  8. Behavioral Analysis of Dopaminergic Activation in Zebrafish and Rats Reveals Similar Phenotypes.

    PubMed

    Ek, Fredrik; Malo, Marcus; Åberg Andersson, Madelene; Wedding, Christoffer; Kronborg, Joel; Svensson, Peder; Waters, Susanna; Petersson, Per; Olsson, Roger

    2016-05-18

    Zebrafish is emerging as a complement to mammals in behavioral studies; however, there is a lack of comparative studies with rodents and humans to establish the zebrafish as a predictive translational model. Here we present a detailed phenotype evaluation of zebrafish larvae, measuring 300-3000 variables and analyzing them using multivariate analysis to identify the most important ones for further evaluations. The dopamine agonist apomorphine has previously been shown to have a complex U-shaped dose-response relationship in the variable distance traveled. In this study, we focused on breaking down distance traveled into more detailed behavioral phenotypes for both zebrafish and rats and identified in the multivariate analysis low and high dose phenotypes with characteristic behavioral features. Further analysis of single parameters also identified an increased activity at the lowest concentration indicative of a U-shaped dose-response. Apomorphine increased the distance of each swim movement (bout) at both high and low doses, but the underlying behavior of this increase is different; at high dose, both bout duration and frequency increased whereas bout max speed was higher at low dose. Larvae also displayed differences in place preference. The low dose phenotype spent more time in the center, indicative of an anxiolytic effect, while the high-dose phenotype had a wall preference. These dose-dependent effects corroborated findings in a parallel rat study and previous observations in humans. The translational value of pharmacological zebrafish studies was further evaluated by comparing the amino acid sequence of the dopamine receptors (D1-D4), between zebrafish, rats and humans. Humans and zebrafish share 100% of the amino acids in the binding site for D1 and D3 whereas D2 and D4 receptors share 85-95%. Molecular modeling of dopamine D2 and D4 receptors indicated that nonconserved amino acids have limited influence on important ligand-receptor interactions. PMID

  9. Crystal Structure of the Mp1p Ligand Binding Domain 2 Reveals Its Function as a Fatty Acid-binding Protein*

    PubMed Central

    Liao, Shuang; Tung, Edward T. K.; Zheng, Wei; Chong, Ken; Xu, Yuanyuan; Dai, Peng; Guo, Yingying; Bartlam, Mark; Yuen, Kwok-Yung; Rao, Zihe

    2010-01-01

    Penicillium marneffei is a dimorphic, pathogenic fungus in Southeast Asia that mostly afflicts immunocompromised individuals. As the only dimorphic member of the genus, it goes through a phase transition from a mold to yeast form, which is believed to be a requisite for its pathogenicity. Mp1p, a cell wall antigenic mannoprotein existing widely in yeast, hyphae, and conidia of the fungus, plays a vital role in host immune response during infection. To understand the function of Mp1p, we have determined the x-ray crystal structure of its ligand binding domain 2 (LBD2) to 1.3 Å. The structure reveals a dimer between the two molecules. The dimer interface forms a ligand binding cavity, in which electron density was observed for a palmitic acid molecule interacting with LBD2 indirectly through hydrogen bonding networks via two structural water molecules. Isothermal titration calorimetry experiments measured the ligand binding affinity (Kd) of Mp1p at the micromolar level. Mutations of ligand-binding residues, namely S313A and S332A, resulted in a 9-fold suppression of ligand binding affinity. Analytical ultracentrifugation assays demonstrated that both LBD2 and Mp1p are mostly monomeric in vitro, no matter with or without ligand, and our dimeric crystal structure of LBD2 might be the result of crystal packing. Based on the conformation of the ligand-binding pocket in the dimer structure, a model for the closed, monomeric form of LBD2 is proposed. Further structural analysis indicated the biological importance of fatty acid binding of Mp1p for the survival and pathogenicity of the conditional pathogen. PMID:20053994

  10. Chromatographic analysis of amino and organic acids in physiological fluids to detect inborn errors of metabolism.

    PubMed

    Woontner, Michael; Goodman, Stephen I

    2006-11-01

    This unit describes methods for the preparation of samples for analysis of physiological amino acids and organic acids. Amino acids are analyzed by ion-exchange chromatography using an automated system. Organic acids are analyzed by gas-chromatography/mass spectrometry (GC-MS). Analysis of amino and organic acids is necessary to detect and monitor the treatment of many inborn errors of metabolism. PMID:18428392

  11. Genomic structural analysis of porcine fatty acid desaturase cluster on chromosome 2.

    PubMed

    Taniguchi, Masaaki; Arakawa, Aisaku; Motoyama, Michiyo; Nakajima, Ikuyo; Nii, Masahiro; Mikawa, Satoshi

    2015-04-01

    Fatty acid composition is an economically important trait in meat-producing livestock. To gain insight into the molecular genetics of fatty acid desaturase (FADS) genes in pigs, we investigated the genomic structure of the porcine FADS gene family on chromosome 2. We also examined the tissue distribution of FADS gene expression. The genomic structure of FADS family in mammals consists of three isoforms FADS1, FADS2 and FADS3. However, porcine FADS cluster in the latest pig genome assembly (Sscrofa 10.2) containing some gaps is distinct from that in other mammals. We therefore sought to determine the genomic structure, including the FADS cluster in a 200-kbp range by sequencing gap regions. The structure we obtained was similar to that in other mammals. We then investigated the porcine FADS1 transcription start site and identified a novel isoform named FADS1b. Phylogenetic analysis revealed that the three members of the FADS cluster were orthologous among mammals, whereas the various FADS1 isoforms identified in pigs, mice and cattle might be attributable to species-specific transcriptional regulation with alternative promoters. Porcine FADS1b and FADS3 isoforms were predominantly expressed in the inner layer of the subcutaneous adipose tissue. Additional analyses will reveal the effects of these functionally unknown isoforms on fatty acid composition in pig fat tissues. PMID:25409917

  12. Genome-wide analysis of longevity in nutrient-deprived Saccharomyces cerevisiae reveals importance of recycling in maintaining cell viability.

    PubMed

    Davey, Hazel M; Cross, Emma J M; Davey, Christopher L; Gkargkas, Konstantinos; Delneri, Daniela; Hoyle, David C; Oliver, Stephen G; Kell, Douglas B; Griffith, Gareth W

    2012-05-01

    Although typically cosseted in the laboratory with constant temperatures and plentiful nutrients, microbes are frequently exposed to much more stressful conditions in their natural environments where survival and competitive fitness depend upon both growth rate when conditions are favourable and on persistence in a viable and recoverable state when they are not. In order to determine the role of genetic heterogeneity in environmental fitness we present a novel approach that combines the power of fluorescence-activated cell sorting with barcode microarray analysis and apply this to determining the importance of every gene in the Saccharomyces cerevisiae genome in a high-throughput, genome-wide fitness screen. We have grown > 6000 heterozygous mutants together and exposed them to a starvation stress before using fluorescence-activated cell sorting to identify and isolate those individual cells that have not survived the stress applied. Barcode array analysis of the sorted and total populations reveals the importance of cellular recycling mechanisms (autophagy, pexophagy and ribosome breakdown) in maintaining cell viability during starvation and provides compelling evidence for an important role for fatty acid degradation in maintaining viability. In addition, we have developed a semi-batch fermentor system that is a more realistic model of environmental fitness than either batch or chemostat culture. Barcode array analysis revealed that arginine biosynthesis was important for fitness in semi-batch culture and modelling of this regime showed that rapid emergence from lag phase led to greatly increased fitness. One hundred and twenty-five strains with deletions in unclassified proteins were identified as being over-represented in the sorted fraction, while 27 unclassified proteins caused a haploinsufficient phenotype in semi-batch culture. These methods thus provide a screen to identifying other genes and pathways that have a role in maintaining cell viability. PMID

  13. Dynamic transcriptome analysis reveals AP2/ERF transcription factors responsible for cold stress in rapeseed (Brassica napus L.).

    PubMed

    Du, Chunfang; Hu, Kaining; Xian, Shuanshi; Liu, Chunqing; Fan, Jianchun; Tu, Jinxing; Fu, Tingdong

    2016-06-01

    The APETALA2/ethylene response factor (AP2/ERF) transcription factor (TF) superfamily plays an important regulatory role in signal transduction of the plant responses to various stresses including low temperature. Significant progress has been made in understanding the mechanism of cold resistance in Brassica napus, an important oilseed crop. However, comprehensive studies on the induction and activity of these TFs under low temperature have been lacking. In this study, 132 AP2/ERF genes were identified by transcriptome sequencing of rapeseed leaves exposed to 0, 2, 6, 12, and 24 h of low (4 °C) temperature stress. The genes were classified into 4 subfamilies (AP2, DREB, ERF, and RAV) and 13 subgroups, among which the DREB subfamily and ERF subfamily contained 114 genes, no genes were assigned to soloist or DREB A3 subgroups. One hundred and eighteen genes were located on chromosomes A1 to C9. GO functional analysis and promoter sequence analysis revealed that these genes are involved in many molecular pathways that may enhance cold resistance in plants, such as the low-temperature responsiveness, methyl jasmonate, abscisic acid, and ethylene-responsiveness pathways. Their expression patterns revealed dynamic control at different times following initiation of cold stress; the RAV and DREB subfamilies were expressed at the early stage of cold stress, whereas the AP2 subfamily was expressed later. Quantitative PCR analyses of 13 cold-induced AP2/ERF TFs confirmed the accuracy of above results. This study is the first dynamic analysis of the AP2/ERF TFs responsible for cold stress in rapeseed. These findings will serve as a reference for future functional research on transcription in rapeseed. PMID:26728151

  14. Quantitative proteomic analysis of amniocytes reveals potentially dysregulated molecular networks in Down syndrome

    PubMed Central

    2013-01-01

    Background Down syndrome (DS), caused by an extra copy of chromosome 21, affects 1 in 750 live births and is characterized by cognitive impairment and a constellation of congenital defects. Currently, little is known about the molecular pathogenesis and no direct genotype-phenotype relationship has yet been confirmed. Since DS amniocytes are expected to have a distinct biological behaviour compared to normal amniocytes, we hypothesize that relative quantification of proteins produced from trisomy and euploid (chromosomally normal) amniocytes will reveal dysregulated molecular pathways. Results Chromosomally normal- and Trisomy 21-amniocytes were quantitatively analyzed by using Stable Isotope Labeling of Amino acids in Cell culture and tandem mass spectrometry. A total of 4919 unique proteins were identified from the supernatant and cell lysate proteome. More specifically, 4548 unique proteins were identified from the lysate, and 91% of these proteins were quantified based on MS/MS spectra ratios of peptides containing isotope-labeled amino acids. A total of 904 proteins showed significant differential expression and were involved in 25 molecular pathways, each containing a minimum of 16 proteins. Sixty of these proteins consistently showed aberrant expression from trisomy 21 affected amniocytes, indicating their potential role in DS pathogenesis. Nine proteins were analyzed with a multiplex selected reaction monitoring assay in an independent set of Trisomy 21-amniocyte samples and two of them (SOD1 and NES) showed a consistent differential expression. Conclusions The most extensive proteome of amniocytes and amniotic fluid has been generated and differentially expressed proteins from amniocytes with Trisomy 21 revealed molecular pathways that seem to be most significantly affected by the presence of an extra copy of chromosome 21. PMID:23394617

  15. Analysis of the mineral acid-base components of acid-neutralizing capacity in Adirondack Lakes

    NASA Astrophysics Data System (ADS)

    Munson, R. K.; Gherini, S. A.

    1993-04-01

    Mineral acids and bases influence pH largely through their effects on acid-neutralizing capacity (ANC). This influence becomes particularly significant as ANC approaches zero. Analysis of data collected by the Adirondack Lakes Survey Corporation (ALSC) from 1469 lakes throughout the Adirondack region indicates that variations in ANC in these lakes correlate well with base cation concentrations (CB), but not with the sum of mineral acid anion concentrations (CA). This is because (CA) is relatively constant across the Adirondacks, whereas CB varies widely. Processes that supply base cations to solution are ion-specific. Sodium and silica concentrations are well correlated, indicating a common source, mineral weathering. Calcium and magnesium also covary but do not correlate well with silica. This indicates that ion exchange is a significant source of these cations in the absence of carbonate minerals. Iron and manganese concentrations are elevated in the lower waters of some lakes due to reducing conditions. This leads to an ephemeral increase in CB and ANC. When the lakes mix and oxic conditions are restored, these ions largely precipitate from solution. Sulfate is the dominant mineral acid anion in ALSC lakes. Sulfate concentrations are lowest in seepage lakes, commonly about 40 μeq/L less than in drainage lakes. This is due in part to the longer hydraulic detention time in seepage lakes, which allows slow sulfate reduction reactions more time to decrease lake sulfate concentration. Nitrate typically influences ANC during events such as snowmelt. Chloride concentrations are generally low, except in lakes impacted by road salt.

  16. Cloning and transcriptional analysis of Crepis alpina fatty acid desaturases affecting the biosynthesis of crepenynic acid.

    PubMed

    Nam, Jeong-Won; Kappock, T Joseph

    2007-01-01

    Crepis alpina acetylenase is a variant FAD2 desaturase that catalyses the insertion of a triple bond at the Delta12 position of linoleic acid, forming crepenynic acid in developing seeds. Seeds contain a high level of crepenynic acid but other tissues contain none. Using reverse transcriptase-coupled PCR (RT-PCR), acetylenase transcripts were identified in non-seed C. alpina tissues, which were highest in flower heads. To understand why functional expression of the acetylenase is limited to seeds, genes that affect acetylenase activity by providing substrate (FAD2) or electrons (cytochrome b5), or that compete for substrate (FAD3), were cloned. RT-PCR analysis indicated that the availability of a preferred cytochrome b5 isoform is not a limiting factor. Developing seeds co-express acetylenase and FAD2 isoform 2 (FAD2-2) at high levels. Flower heads co-express FAD2-3 and FAD3 at high levels, and FAD2-2 and acetylenase at moderate levels. FAD2-3 was not expressed in developing seed. Real-time RT-PCR absolute transcript quantitation showed 10(4)-fold higher acetylenase expression in developing seeds than in flower heads. Collectively, the results show that both the acetylenase expression level and the co-expression of other desaturases may contribute to the tissue specificity of crepenynate production. Helianthus annuus contains a Delta12 acetylenase in a polyacetylene biosynthetic pathway, so does not accumulate crepenynate. Real-time RT-PCR analysis showed relatively strong acetylenase expression in young sunflowers. Acetylenase transcription is observed in both species without accumulation of the enzymatic product, crepenynate. Functional expression of acetylenase appears to be affected by competition and collaboration with other enzymes. PMID:17329262

  17. Structure and function analysis of protein–nucleic acid complexes

    NASA Astrophysics Data System (ADS)

    Kuznetsova, S. A.; Oretskaya, T. S.

    2016-05-01

    The review summarizes published data on the results and achievements in the field of structure and function analysis of protein–nucleic acid complexes by means of main physical and biochemical methods, including X-ray diffraction, nuclear magnetic resonance spectroscopy, electron and atomic force microscopy, small-angle X-ray and neutron scattering, footprinting and cross-linking. Special attention is given to combined approaches. The advantages and limitations of each method are considered, and the prospects of their application for wide-scale structural studies in vivo are discussed. The bibliography includes 145 references.

  18. Isolation and characterization of a low phytic acid rice mutant reveals a mutation in the rice orthologue of maize mik.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using a forward genetics approach, we isolated two independent low phytic acid (lpa) rice mutants, N15-186 and N15-375. Both mutants are caused by single gene, recessive non-lethal mutations which result in approximately 75% (N15-186) and 43% (N15-375) reductions in seed phytic acid (inositol hexaki...

  19. Newly identified essential amino acid residues affecting ^8-sphingolipid desaturase activity revealed by site-directed mutagenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In order to identify amino acid residues crucial for the enzymatic activity of ^8-sphingolipid desaturases, a sequence comparison was performed among ^8-sphingolipid desaturases and ^6-fatty acid desaturase from various plants. In addition to the known conserved cytb5 (cytochrome b5) HPGG motif and...

  20. Analysis of Amino Acid Isotopomers using FT-ICR MS

    SciTech Connect

    Pingitore, Francesco; Tang, Yinjie; Kruppa, Gary H.; Keasling,Jay D.

    2006-10-08

    Fluxes through known metabolic pathways and the presence ofnovel metabolic reactions are often determined by feedingisotopically-labeled substrate to an organism and then determining theisotopomer distribution in amino acids in proteins. However, commonlyused techniques to measure the isotopomer distributions requirederivatization prior to analysis (gas chromatography-mass spectrometry(GC-MS)) or large sample sizes (nuclear magnetic resonance (NMR)spectroscopy). Here, we demonstrate the use of Fourier Transform-IonCyclotron Resonance Mass Spectrometry (FT-ICR MS) with direct infusionvia electrospray ionization to rapidly measure the amino acid isotopomerdistribution in a biomass hydrolysate of the soil bacterium Desulfovibriovulgaris Hildenborough. By applying high front-end resolution for theprecursor ion selection followed by sustained off-resonance irradiation -collision-induced dissociation (SORI-CID), it was possible to determineexactly and unambiguously the specific locations of the labeled atoms inthe amino acids, which usually requires a combination of 2-D 13C NMRspectroscopy and GC-MS. This method should be generally applicable toallbiomass samples and will allow more accurate determination of metabolicfluxes with less work and less sample.

  1. Specific polyunsaturated fatty acids modulate lipid delivery and oocyte development in C. elegans revealed by molecular-selective label-free imaging

    PubMed Central

    Chen, Wei-Wen; Yi, Yung-Hsiang; Chien, Cheng-Hao; Hsiung, Kuei-Ching; Ma, Tian-Hsiang; Lin, Yi-Chun; Lo, Szecheng J.; Chang, Ta-Chau

    2016-01-01

    Polyunsaturated fatty acids (PUFAs) exhibit critical functions in biological systems and their importance during animal oocyte maturation has been increasingly recognized. However, the detailed mechanism of lipid transportation for oocyte development remains largely unknown. In this study, the transportation of yolk lipoprotein (lipid carrier) and the rate of lipid delivery into oocytes in live C. elegans were examined for the first time by using coherent anti-Stokes Raman scattering (CARS) microscopy. The accumulation of secreted yolk lipoprotein in the pseudocoelom of live C. elegans can be detected by CARS microscopy at both protein (~1665 cm−1) and lipid (~2845 cm−1) Raman bands. In addition, an image analysis protocol was established to quantitatively measure the levels of secreted yolk lipoprotein aberrantly accumulated in PUFA-deficient fat mutants (fat-1, fat-2, fat-3, fat-4) and PUFA-supplemented fat-2 worms (the PUFA add-back experiments). Our results revealed that the omega-6 PUFAs, not omega-3 PUFAs, play a critical role in modulating lipid/yolk level in the oocytes and regulating reproductive efficiency of C. elegans. This work demonstrates the value of using CARS microscopy as a molecular-selective label-free imaging technique for the study of PUFA regulation and oocyte development in C. elegans. PMID:27535493

  2. Suberoylanilide Hydroxamic Acid Treatment Reveals Crosstalks among Proteome, Ubiquitylome and Acetylome in Non-Small Cell Lung Cancer A549 Cell Line

    PubMed Central

    Wu, Quan; Cheng, Zhongyi; Zhu, Jun; Xu, Weiqing; Peng, Xiaojun; Chen, Chuangbin; Li, Wenting; Wang, Fengsong; Cao, Lejie; Yi, Xingling; Wu, Zhiwei; Li, Jing; Fan, Pingsheng

    2015-01-01

    Suberoylanilide hydroxamic acid (SAHA) is a well-known histone deacetylase (HDAC) inhibitor and has been used as practical therapy for breast cancer and non-small cell lung cancer (NSCLC). It is previously demonstrated that SAHA treatment could extensively change the profile of acetylome and proteome in cancer cells. However, little is known about the impact of SAHA on other protein modifications and the crosstalks among different modifications and proteome, hindering the deep understanding of SAHA-mediated cancer therapy. In this work, by using SILAC technique, antibody-based affinity enrichment and high-resolution LC-MS/MS analysis, we investigated quantitative proteome, acetylome and ubiquitylome as well as crosstalks among the three datasets in A549 cells toward SAHA treatment. In total, 2968 proteins, 1099 acetylation sites and 1012 ubiquitination sites were quantified in response to SAHA treatment, respectively. With the aid of intensive bioinformatics, we revealed that the proteome and ubiquitylome were negatively related upon SAHA treatment. Moreover, the impact of SAHA on acetylome resulted in 258 up-regulated and 99 down-regulated acetylation sites at the threshold of 1.5 folds. Finally, we identified 55 common sites with both acetylation and ubiquitination, among which ubiquitination level in 43 sites (78.2%) was positive related to acetylation level. PMID:25825284

  3. Interaction of Di-2-pyridylketone 2-pyridine Carboxylic Acid Hydrazone and Its Copper Complex with BSA: Effect on Antitumor Activity as Revealed by Spectroscopic Studies.

    PubMed

    Li, Cuiping; Huang, Tengfei; Fu, Yun; Liu, Youxun; Zhou, Sufeng; Qi, Zhangyang; Li, Changzheng

    2016-01-01

    The drug, di-2-pyridylketone-2-pyridine carboxylic acid hydrazone (DPPCAH) and its copper complex (DPPCAH-Cu) exhibit significant antitumor activity. However, the mechanism of their pharmacological interaction with the biological molecule bovine serum albumin (BSA) remains poorly understood. The present study elucidates the interactions between the drug and BSA through MTT assays, spectroscopic methods and molecular docking analysis. Our results indicate that BSA could attenuate effect on the cytotoxicity of DPPCAH, but not DPPCAH-Cu. Data from fluorescence quenching measurements demonstrated that both DPPCAH and DPPCAH-Cu could bind to BSA, with a reversed effect on the environment of tryptophan residues in polarity. CD spectra revealed that the DPPCAH-Cu exerted a slightly stronger effect on the secondary structure of BSA than DPPCAH. The association constant of DPPCAH with BSA was greater than that of DPPCAH-Cu. Docking studies indicated that the binding of DPPCAH to BSA involved a greater number of hydrogen bonds compared to DPPCAH-Cu. The calculated distances between bound ligands and tryptophans in BSA were in agreement with fluorescence resonance energy transfer results. Thus, the binding affinity of the drug (DPPCAH or DPPCAH-Cu) with BSA partially contributes to its antitumor activity; the greater the drug affinity is to BSA, the less is its antitumor activity. PMID:27136517

  4. Specific polyunsaturated fatty acids modulate lipid delivery and oocyte development in C. elegans revealed by molecular-selective label-free imaging.

    PubMed

    Chen, Wei-Wen; Yi, Yung-Hsiang; Chien, Cheng-Hao; Hsiung, Kuei-Ching; Ma, Tian-Hsiang; Lin, Yi-Chun; Lo, Szecheng J; Chang, Ta-Chau

    2016-01-01

    Polyunsaturated fatty acids (PUFAs) exhibit critical functions in biological systems and their importance during animal oocyte maturation has been increasingly recognized. However, the detailed mechanism of lipid transportation for oocyte development remains largely unknown. In this study, the transportation of yolk lipoprotein (lipid carrier) and the rate of lipid delivery into oocytes in live C. elegans were examined for the first time by using coherent anti-Stokes Raman scattering (CARS) microscopy. The accumulation of secreted yolk lipoprotein in the pseudocoelom of live C. elegans can be detected by CARS microscopy at both protein (~1665 cm(-1)) and lipid (~2845 cm(-1)) Raman bands. In addition, an image analysis protocol was established to quantitatively measure the levels of secreted yolk lipoprotein aberrantly accumulated in PUFA-deficient fat mutants (fat-1, fat-2, fat-3, fat-4) and PUFA-supplemented fat-2 worms (the PUFA add-back experiments). Our results revealed that the omega-6 PUFAs, not omega-3 PUFAs, play a critical role in modulating lipid/yolk level in the oocytes and regulating reproductive efficiency of C. elegans. This work demonstrates the value of using CARS microscopy as a molecular-selective label-free imaging technique for the study of PUFA regulation and oocyte development in C. elegans. PMID:27535493

  5. Virus-induced gene silencing reveals control of reactive oxygen species accumulation and salt tolerance in tomato by γ-aminobutyric acid metabolic pathway.

    PubMed

    Bao, Hexigeduleng; Chen, Xianyang; Lv, Sulian; Jiang, Ping; Feng, Juanjuan; Fan, Pengxiang; Nie, Lingling; Li, Yinxin

    2015-03-01

    γ-Aminobutyric acid (GABA) accumulates in many plant species in response to environmental stress. However, the physiological function of GABA or its metabolic pathway (GABA shunt) in plants remains largely unclear. Here, the genes, including glutamate decarboxylases (SlGADs), GABA transaminases (SlGABA-Ts) and succinic semialdehyde dehydrogenase (SlSSADH), controlling three steps of the metabolic pathway of GABA, were studied through virus-induced gene silencing approach in tomato. Silencing of SlGADs (GABA biosynthetic genes) and SlGABA-Ts (GABA catabolic genes) led to increased accumulation of reactive oxygen species (ROS) as well as salt sensitivity under 200 mm NaCl treatment. Targeted quantitative analysis of metabolites revealed that GABA decreased and increased in the SlGADs- and SlGABA-Ts-silenced plants, respectively, whereas succinate (the final product of GABA metabolism) decreased in both silenced plants. Contrarily, SlSSADH-silenced plants, also defective in GABA degradation process, showed dwarf phenotype, curled leaves and enhanced accumulation of ROS in normal conditions, suggesting the involvement of a bypath for succinic semialdehyde catabolism to γ-hydroxybutyrate as reported previously in Arabidopsis, were less sensitive to salt stress. These results suggest that GABA shunt is involved in salt tolerance of tomato, probably by affecting the homeostasis of metabolites such as succinate and γ-hydroxybutyrate and subsequent ROS accumulation under salt stress. PMID:25074245

  6. Quantitative Proteomics by SWATH-MS Reveals Altered Expression of Nucleic Acid Binding and Regulatory Proteins in HIV-1-Infected Macrophages

    PubMed Central

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) infection remains a worldwide epidemic, and innovative therapies to combat the virus are needed. Developing a host-oriented antiviral strategy capable of targeting the biomolecules that are directly or indirectly required for viral replication may provide advantages over traditional virus-centric approaches. We used quantitative proteomics by SWATH-MS in conjunction with bioinformatic analyses to identify host proteins, with an emphasis on nucleic acid binding and regulatory proteins, which could serve as candidates in the development of host-oriented antiretroviral strategies. Using SWATH-MS, we identified and quantified the expression of 3608 proteins in uninfected and HIV-1-infected monocyte-derived macrophages. Of these 3608 proteins, 420 were significantly altered upon HIV-1 infection. Bioinformatic analyses revealed functional enrichment for RNA binding and processing as well as transcription regulation. Our findings highlight a novel subset of proteins and processes that are involved in the host response to HIV-1 infection. In addition, we provide an original and transparent methodology for the analysis of label-free quantitative proteomics data generated by SWATH-MS that can be readily adapted to other biological systems. PMID:24564501

  7. Suberoylanilide hydroxamic acid treatment reveals crosstalks among proteome, ubiquitylome and acetylome in non-small cell lung cancer A549 cell line.

    PubMed

    Wu, Quan; Cheng, Zhongyi; Zhu, Jun; Xu, Weiqing; Peng, Xiaojun; Chen, Chuangbin; Li, Wenting; Wang, Fengsong; Cao, Lejie; Yi, Xingling; Wu, Zhiwei; Li, Jing; Fan, Pingsheng

    2015-01-01

    Suberoylanilide hydroxamic acid (SAHA) is a well-known histone deacetylase (HDAC) inhibitor and has been used as practical therapy for breast cancer and non-small cell lung cancer (NSCLC). It is previously demonstrated that SAHA treatment could extensively change the profile of acetylome and proteome in cancer cells. However, little is known about the impact of SAHA on other protein modifications and the crosstalks among different modifications and proteome, hindering the deep understanding of SAHA-mediated cancer therapy. In this work, by using SILAC technique, antibody-based affinity enrichment and high-resolution LC-MS/MS analysis, we investigated quantitative proteome, acetylome and ubiquitylome as well as crosstalks among the three datasets in A549 cells toward SAHA treatment. In total, 2968 proteins, 1099 acetylation sites and 1012 ubiquitination sites were quantified in response to SAHA treatment, respectively. With the aid of intensive bioinformatics, we revealed that the proteome and ubiquitylome were negatively related upon SAHA treatment. Moreover, the impact of SAHA on acetylome resulted in 258 up-regulated and 99 down-regulated acetylation sites at the threshold of 1.5 folds. Finally, we identified 55 common sites with both acetylation and ubiquitination, among which ubiquitination level in 43 sites (78.2%) was positive related to acetylation level. PMID:25825284

  8. Local coexistence of VO2 phases revealed by deep data analysis

    NASA Astrophysics Data System (ADS)

    Strelcov, Evgheni; Ievlev, Anton; Belianinov, Alex; Tselev, Alexander; Kolmakov, Andrei; Kalinin, Sergei V.

    2016-07-01

    We report a synergistic approach of micro-Raman spectroscopic mapping and deep data analysis to study the distribution of crystallographic phases and ferroelastic domains in a defected Al-doped VO2 microcrystal. Bayesian linear unmixing revealed an uneven distribution of the T phase, which is stabilized by the surface defects and uneven local doping that went undetectable by other classical analysis techniques such as PCA and SIMPLISMA. This work demonstrates the impact of information recovery via statistical analysis and full mapping in spectroscopic studies of vanadium dioxide systems, which is commonly substituted by averaging or single point-probing approaches, both of which suffer from information misinterpretation due to low resolving power.

  9. A versatile multivariate image analysis pipeline reveals features of Xenopus extract spindles.

    PubMed

    Grenfell, Andrew W; Strzelecka, Magdalena; Crowder, Marina E; Helmke, Kara J; Schlaitz, Anne-Lore; Heald, Rebecca

    2016-04-11

    Imaging datasets are rich in quantitative information. However, few cell biologists possess the tools necessary to analyze them. Here, we present a large dataset ofXenopusextract spindle images together with an analysis pipeline designed to assess spindle morphology across a range of experimental conditions. Our analysis of different spindle types illustrates how kinetochore microtubules amplify spindle microtubule density. Extract mixing experiments reveal that some spindle features titrate, while others undergo switch-like transitions, and multivariate analysis shows the pleiotropic morphological effects of modulating the levels of TPX2, a key spindle assembly factor. We also apply our pipeline to analyze nuclear morphology in human cell culture, showing the general utility of the segmentation approach. Our analyses provide new insight into the diversity of spindle types and suggest areas for future study. The approaches outlined can be applied by other researchers studying spindle morphology and adapted with minimal modification to other experimental systems. PMID:27044897

  10. Local coexistence of VO2 phases revealed by deep data analysis

    PubMed Central

    Strelcov, Evgheni; Ievlev, Anton; Belianinov, Alex; Tselev, Alexander; Kolmakov, Andrei; Kalinin, Sergei V.

    2016-01-01

    We report a synergistic approach of micro-Raman spectroscopic mapping and deep data analysis to study the distribution of crystallographic phases and ferroelastic domains in a defected Al-doped VO2 microcrystal. Bayesian linear unmixing revealed an uneven distribution of the T phase, which is stabilized by the surface defects and uneven local doping that went undetectable by other classical analysis techniques such as PCA and SIMPLISMA. This work demonstrates the impact of information recovery via statistical analysis and full mapping in spectroscopic studies of vanadium dioxide systems, which is commonly substituted by averaging or single point-probing approaches, both of which suffer from information misinterpretation due to low resolving power. PMID:27384473

  11. Local coexistence of VO2 phases revealed by deep data analysis.

    PubMed

    Strelcov, Evgheni; Ievlev, Anton; Belianinov, Alex; Tselev, Alexander; Kolmakov, Andrei; Kalinin, Sergei V

    2016-01-01

    We report a synergistic approach of micro-Raman spectroscopic mapping and deep data analysis to study the distribution of crystallographic phases and ferroelastic domains in a defected Al-doped VO2 microcrystal. Bayesian linear unmixing revealed an uneven distribution of the T phase, which is stabilized by the surface defects and uneven local doping that went undetectable by other classical analysis techniques such as PCA and SIMPLISMA. This work demonstrates the impact of information recovery via statistical analysis and full mapping in spectroscopic studies of vanadium dioxide systems, which is commonly substituted by averaging or single point-probing approaches, both of which suffer from information misinterpretation due to low resolving power. PMID:27384473

  12. Local coexistence of VO2 phases revealed by deep data analysis

    DOE PAGESBeta

    Strelcov, Evgheni; Ievlev, Anton; Tselev, Alexander; Kolmakov, Andrei; Kalinin, Sergei V.

    2016-07-07

    We report a synergistic approach of micro-Raman spectroscopic mapping and deep data analysis to study the distribution of crystallographic phases and ferroelastic domains in a defected Al-doped VO2 microcrystal. Bayesian linear unmixing revealed an uneven distribution of the T phase, which is stabilized by the surface defects and uneven local doping that went undetectable by other classical analysis techniques such as PCA and SIMPLISMA. This work demonstrates the impact of information recovery via statistical analysis and full mapping in spectroscopic studies of vanadium dioxide systems, which is commonly substituted by averaging or single point-probing approaches, both of which suffer frommore » information misinterpretation due to low resolving power.« less

  13. Single-Amino Acid Modifications Reveal Additional Controls on the Proton Pathway of [FeFe]-Hydrogenase.

    PubMed

    Cornish, Adam J; Ginovska, Bojana; Thelen, Adam; da Silva, Julio C S; Soares, Thereza A; Raugei, Simone; Dupuis, Michel; Shaw, Wendy J; Hegg, Eric L

    2016-06-01

    The proton pathway of [FeFe]-hydrogenase is essential for enzymatic H2 production and oxidation and is composed of four residues and a water molecule. A computational analysis of this pathway in the [FeFe]-hydrogenase from Clostridium pasteurianum revealed that the solvent-exposed residue of the pathway (Glu282) forms hydrogen bonds to two residues outside of the pathway (Arg286 and Ser320), implying that these residues could function in regulating proton transfer. In this study, we show that substituting Arg286 with leucine eliminates hydrogen bonding with Glu282 and results in an ∼3-fold enhancement of H2 production activity when methyl viologen is used as an electron donor, suggesting that Arg286 may help control the rate of proton delivery. In contrast, substitution of Ser320 with alanine reduces the rate ∼5-fold, implying that it either acts as a member of the pathway or influences Glu282 to permit proton transfer. Interestingly, quantum mechanics/molecular mechanics and molecular dynamics calculations indicate that Ser320 does not play a structural role or indirectly influence the barrier for proton movement at the entrance of the channel. Rather, it may act as an additional proton acceptor for the pathway or serve in a regulatory role. While further studies are needed to elucidate the role of Ser320, collectively these data provide insights into the complex proton transport process. PMID:27186945

  14. Genome-Based Metabolic Mapping and 13C Flux Analysis Reveal Systematic Properties of an Oleaginous Microalga Chlorella protothecoides

    SciTech Connect

    Wu, Chao; Xiong, Wei; Dai, Junbiao; Wu, Qingyu

    2014-12-15

    We report that integrated and genome-based flux balance analysis, metabolomics, and 13C-label profiling of phototrophic and heterotrophic metabolism in Chlorella protothecoides, an oleaginous green alga for biofuel. The green alga Chlorella protothecoides, capable of autotrophic and heterotrophic growth with rapid lipid synthesis, is a promising candidate for biofuel production. Based on the newly available genome knowledge of the alga, we reconstructed the compartmentalized metabolic network consisting of 272 metabolic reactions, 270 enzymes, and 461 encoding genes and simulated the growth in different cultivation conditions with flux balance analysis. Phenotype-phase plane analysis shows conditions achieving theoretical maximum of the biomass and corresponding fatty acid-producing rate for phototrophic cells (the ratio of photon uptake rate to CO2 uptake rate equals 8.4) and heterotrophic ones (the glucose uptake rate to O2 consumption rate reaches 2.4), respectively. Isotope-assisted liquid chromatography-mass spectrometry/mass spectrometry reveals higher metabolite concentrations in the glycolytic pathway and the tricarboxylic acid cycle in heterotrophic cells compared with autotrophic cells. We also observed enhanced levels of ATP, nicotinamide adenine dinucleotide (phosphate), reduced, acetyl-Coenzyme A, and malonyl-Coenzyme A in heterotrophic cells consistently, consistent with a strong activity of lipid synthesis. To profile the flux map in experimental conditions, we applied nonstationary 13C metabolic flux analysis as a complementing strategy to flux balance analysis. We found that the result reveals negligible photorespiratory fluxes and a metabolically low active tricarboxylic acid cycle in phototrophic C. protothecoides. In comparison, high throughput of amphibolic reactions and the tricarboxylic acid cycle with no glyoxylate shunt activities were measured for heterotrophic cells. Lastly, taken together, the

  15. Genome-Based Metabolic Mapping and 13C Flux Analysis Reveal Systematic Properties of an Oleaginous Microalga Chlorella protothecoides1[OPEN

    PubMed Central

    Wu, Chao; Xiong, Wei; Dai, Junbiao; Wu, Qingyu

    2015-01-01

    Integrated and genome-based flux balance analysis, metabolomics, and 13C-label profiling of phototrophic and heterotrophic metabolism in Chlorella protothecoides, an oleaginous green alga for biofuel. The green alga Chlorella protothecoides, capable of autotrophic and heterotrophic growth with rapid lipid synthesis, is a promising candidate for biofuel production. Based on the newly available genome knowledge of the alga, we reconstructed the compartmentalized metabolic network consisting of 272 metabolic reactions, 270 enzymes, and 461 encoding genes and simulated the growth in different cultivation conditions with flux balance analysis. Phenotype-phase plane analysis shows conditions achieving theoretical maximum of the biomass and corresponding fatty acid-producing rate for phototrophic cells (the ratio of photon uptake rate to CO2 uptake rate equals 8.4) and heterotrophic ones (the glucose uptake rate to O2 consumption rate reaches 2.4), respectively. Isotope-assisted liquid chromatography-mass spectrometry/mass spectrometry reveals higher metabolite concentrations in the glycolytic pathway and the tricarboxylic acid cycle in heterotrophic cells compared with autotrophic cells. We also observed enhanced levels of ATP, nicotinamide adenine dinucleotide (phosphate), reduced, acetyl-Coenzyme A, and malonyl-Coenzyme A in heterotrophic cells consistently, consistent with a strong activity of lipid synthesis. To profile the flux map in experimental conditions, we applied nonstationary 13C metabolic flux analysis as a complementing strategy to flux balance analysis. The result reveals negligible photorespiratory fluxes and a metabolically low active tricarboxylic acid cycle in phototrophic C. protothecoides. In comparison, high throughput of amphibolic reactions and the tricarboxylic acid cycle with no glyoxylate shunt activities were measured for heterotrophic cells. Taken together, the metabolic network modeling assisted by experimental metabolomics and 13C labeling

  16. Comparative metabolomic analysis reveals a reactive oxygen species-dominated dynamic model underlying chilling environment adaptation and tolerance in rice.

    PubMed

    Zhang, Jingyu; Luo, Wei; Zhao, Yuan; Xu, Yunyuan; Song, Shuhui; Chong, Kang

    2016-09-01

    Cold, a major environmental stress for plants, has been studied intensively for decades. Its response system has been revealed, especially at the transcriptional level. The mechanisms underlying recovery growth and environmental adaptation, however, remain unknown. Taking advantage of a naturally existing system, two subspecies of Asian cultivated rice (Oryza sativa) with significant divergence in chilling tolerance, we analyzed representative japonica and indica varieties, Nipponbare and 93-11, using comparative metabolomic analysis at six time points covering chilling treatment and recovery. In total, 223 known metabolites were detected. During chilling treatment, significant biochemical changes were centered on antioxidation. During recovery, a wide-ranging chilling response was observed. Large-scale amino acid accumulation occurred, consistent with the appearance of chilling injury. At the mid-treatment stage, the accumulation of antioxidation-related compounds appeared earlier in Nipponbare than in 93-11, consistent with the higher reactive oxygen species (ROS) levels in japonica vs indica varieties. A significant contribution of ROS-mediated gene regulation, rather than the C-repeat binding factor/dehydration-responsive-element binding factor (CBF/DREB) regulon, to the more vigorous transcriptional stress response in Nipponbare was revealed by RNA-seq. Accordingly, during recovery, the induction of stress-tolerant-related metabolites was more active in the chilling-tolerant variety Nipponbare. Senescence-related compounds accumulated only in the chilling-sensitive variety 93-11. Our study uncovers the dynamic metabolic models underlying chilling response and recovery, and reveals a ROS-dominated rice adaptation mechanism to low-temperature environments. PMID:27198693

  17. In vitro reconstitution and steady-state analysis of the fatty acid synthase from Escherichia coli

    PubMed Central

    Yu, Xingye; Liu, Tiangang; Zhu, Fayin; Khosla, Chaitan

    2011-01-01

    Microbial fatty acid derivatives are emerging as promising alternatives to fossil fuel derived transportation fuels. Among bacterial fatty acid synthases (FAS), the Escherichia coli FAS is perhaps the most well studied, but little is known about its steady-state kinetic behavior. Here we describe the reconstitution of E. coli FAS using purified protein components and report detailed kinetic analysis of this reconstituted system. When all ketosynthases are present at 1 μM, the maximum rate of free fatty acid synthesis of the FAS exceeded 100 μM/ min. The steady-state turnover frequency was not significantly inhibited at high concentrations of any substrate or cofactor. FAS activity was saturated with respect to most individual protein components when their concentrations exceeded 1 μM. The exceptions were FabI and FabZ, which increased FAS activity up to concentrations of 10 μM; FabH and FabF, which decreased FAS activity at concentrations higher than 1 μM; and holo-ACP and TesA, which gave maximum FAS activity at 30 μM concentrations. Analysis of the S36T mutant of the ACP revealed that the unusual dependence of FAS activity on holo-ACP concentration was due, at least in part, to the acyl-phosphopantetheine moiety. MALDI-TOF mass spectrometry analysis of the reaction mixture further revealed medium and long chain fatty acyl-ACP intermediates as predominant ACP species. We speculate that one or more of such intermediates are key allosteric regulators of FAS turnover. Our findings provide a new basis for assessing the scope and limitations of using E. coli as a biocatalyst for the production of diesel-like fuels. PMID:22042840

  18. Metagenomic analysis of a Mexican ripened cheese reveals a unique complex microbiota.

    PubMed

    Escobar-Zepeda, Alejandra; Sanchez-Flores, Alejandro; Quirasco Baruch, Maricarmen

    2016-08-01

    Cotija cheese is a Mexican handcrafted product made from raw cow milk whose ripening process occurs spontaneously and, presumably, it is influenced by environmental conditions. Its sensory characteristics and safety are probably the result of the balance between microbial populations and their metabolic capacity. In this work, we studied the dominance and richness of the bacteria in the Cotija cheese microbiome, as well as their metabolic potential by high-throughput sequencing. By the analysis of 16S ribosomal sequences, it was found that this metagenome is composed mainly of three dominant genera: Lactobacillus, Leuconostoc and Weissella, and more than 500 of non-dominant genera grouped in 31 phyla of both bacteria and archaea. The analysis of single-copy marker genes reported a similar result for dominant genera, although with greater resolution that reached the species level. Pathogenic bacteria such as Salmonella, Listeria monocytogenes, Brucella or Mycobacterium were not found. The Cotija cheese microbiome has the metabolic capacity for the synthesis of a wide range of flavor compounds, mainly involved with the metabolism of branched chain amino acids and free fatty acids. Genes associated with bacteriocin production and immunity were also found. Arguably, this is one of the most diverse metagenomes among the microbial communities related to fermented products. PMID:27052710

  19. Gas chromatographic organic acid profiling analysis of brandies and whiskeys for pattern recognition analysis.

    PubMed

    Park, Y J; Kim, K R; Kim, J H

    1999-06-01

    An efficient gas chromatographic profiling and pattern recognition method is described for brandy and whiskey samples according to their organic acid contents. It involves solid-phase extraction of organic acids using Chromosorb P with subsequent conversion to stable tert-butyldimethylsilyl derivatives for the direct analysis by capillary column gas chromatography and gas chromatography-mass spectrometry. A total of 12 organic acids were reproducibly identified in liquor samples (1 mL). When the GC profiles were simplified to their retention index spectra, characteristic patterns were obtained for each liquor sample as well as for each group average. Stepwise discriminant analysis provided star symbols characteristic for each liquor sample and group average. As expected, canonical discriminant analysis correctly classified 23 liquor samples studied into two groups of either brandy or whiskey. PMID:10794629

  20. Crystal structure of cardiac troponin C regulatory domain in complex with cadmium and deoxycholic acid reveals novel conformation

    PubMed Central

    Borek, Dominika; Otwinowski, Zbyszek; Tibbits, Glen F.; Paetzel, Mark

    2014-01-01

    Summary The amino-terminal regulatory domain of cardiac troponin C (cNTnC) plays an important role as the calcium sensor for the troponin complex. Calcium binding to cNTnC results in conformational changes that trigger a cascade of events that leads to cardiac muscle contraction. Cardiac NTnC consists of two EF-hand calcium binding motifs, one of which is dysfunctional in binding calcium. Nevertheless, the defunct EF-hand still maintains a role in cNTnC function. For its structural analysis by X-ray crystallography, human cNTnC with wild-type primary sequence was crystallized in a novel crystallization condition. The crystal structure was solved from single wavelength anomalous dispersion method and refined to 2.2 Å resolution. The structure displays several novel features. Firstly, both EF-hand motifs coordinate cadmium ions derived from the crystallization milieu. Secondly, the ion coordination in the defunct EF-hand motif accompanies unusual changes in the protein conformation. Thirdly, deoxycholic acid, also derived from the crystallization milieu, is bound in the central hydrophobic cavity. This is reminiscent of the interactions observed for cardiac calcium sensitizer drugs that bind to the same core region and maintain the ‘open’ conformational state of calcium bound cNTnC. The cadmium ion coordination in the defunct EF-hand indicates that this vestigial calcium binding site retains the structural and functional elements that allow it to coordinate a cadmium ion. However, it is a result of, or concomitant with, large and unusual structural changes in cNTnC. PMID:21920370

  1. Crystal structure of cardiac troponin C regulatory domain in complex with cadmium and deoxycholic acid reveals novel conformation.

    PubMed

    Li, Alison Yueh; Lee, Jaeyong; Borek, Dominika; Otwinowski, Zbyszek; Tibbits, Glen F; Paetzel, Mark

    2011-10-28

    The amino-terminal regulatory domain of cardiac troponin C (cNTnC) plays an important role as the calcium sensor for the troponin complex. Calcium binding to cNTnC results in conformational changes that trigger a cascade of events that lead to cardiac muscle contraction. The cardiac N-terminal domain of TnC consists of two EF-hand calcium binding motifs, one of which is dysfunctional in binding calcium. Nevertheless, the defunct EF-hand still maintains a role in cNTnC function. For its structural analysis by X-ray crystallography, human cNTnC with the wild-type primary sequence was crystallized under a novel crystallization condition. The crystal structure was solved by the single-wavelength anomalous dispersion method and refined to 2.2 Å resolution. The structure displays several novel features. Firstly, both EF-hand motifs coordinate cadmium ions derived from the crystallization milieu. Secondly, the ion coordination in the defunct EF-hand motif accompanies unusual changes in the protein conformation. Thirdly, deoxycholic acid, also derived from the crystallization milieu, is bound in the central hydrophobic cavity. This is reminiscent of the interactions observed for cardiac calcium sensitizer drugs that bind to the same core region and maintain the "open" conformational state of calcium-bound cNTnC. The cadmium ion coordination in the defunct EF-hand indicates that this vestigial calcium binding site retains the structural and functional elements that allow it to coordinate a cadmium ion. However, it is a result of, or concomitant with, large and unusual structural changes in cNTnC. PMID:21920370

  2. Multilocus sequence analysis of nectar pseudomonads reveals high genetic diversity and contrasting recombination patterns.

    PubMed

    Alvarez-Pérez, Sergio; de Vega, Clara; Herrera, Carlos M

    2013-01-01

    The genetic and evolutionary relationships among floral nectar-dwelling Pseudomonas 'sensu stricto' isolates associated to South African and Mediterranean plants were investigated by multilocus sequence analysis (MLSA) of four core housekeeping genes (rrs, gyrB, rpoB and rpoD). A total of 35 different sequence types were found for the 38 nectar bacterial isolates characterised. Phylogenetic analyses resulted in the identification of three main clades [nectar groups (NGs) 1, 2 and 3] of nectar pseudomonads, which were closely related to five intrageneric groups: Pseudomonas oryzihabitans (NG 1); P. fluorescens, P. lutea and P. syringae (NG 2); and P. rhizosphaerae (NG 3). Linkage disequilibrium analysis pointed to a mostly clonal population structure, even when the analysis was restricted to isolates from the same floristic region or belonging to the same NG. Nevertheless, signatures of recombination were observed for NG 3, which exclusively included isolates retrieved from the floral nectar of insect-pollinated Mediterranean plants. In contrast, the other two NGs comprised both South African and Mediterranean isolates. Analyses relating diversification to floristic region and pollinator type revealed that there has been more unique evolution of the nectar pseudomonads within the Mediterranean region than would be expected by chance. This is the first work analysing the sequence of multiple loci to reveal geno- and ecotypes of nectar bacteria. PMID:24116076

  3. Multilocus Sequence Analysis of Nectar Pseudomonads Reveals High Genetic Diversity and Contrasting Recombination Patterns

    PubMed Central

    Álvarez-Pérez, Sergio; de Vega, Clara; Herrera, Carlos M.

    2013-01-01

    The genetic and evolutionary relationships among floral nectar-dwelling Pseudomonas ‘sensu stricto’ isolates associated to South African and Mediterranean plants were investigated by multilocus sequence analysis (MLSA) of four core housekeeping genes (rrs, gyrB, rpoB and rpoD). A total of 35 different sequence types were found for the 38 nectar bacterial isolates characterised. Phylogenetic analyses resulted in the identification of three main clades [nectar groups (NGs) 1, 2 and 3] of nectar pseudomonads, which were closely related to five intrageneric groups: Pseudomonas oryzihabitans (NG 1); P. fluorescens, P. lutea and P. syringae (NG 2); and P. rhizosphaerae (NG 3). Linkage disequilibrium analysis pointed to a mostly clonal population structure, even when the analysis was restricted to isolates from the same floristic region or belonging to the same NG. Nevertheless, signatures of recombination were observed for NG 3, which exclusively included isolates retrieved from the floral nectar of insect-pollinated Mediterranean plants. In contrast, the other two NGs comprised both South African and Mediterranean isolates. Analyses relating diversification to floristic region and pollinator type revealed that there has been more unique evolution of the nectar pseudomonads within the Mediterranean region than would be expected by chance. This is the first work analysing the sequence of multiple loci to reveal geno- and ecotypes of nectar bacteria. PMID:24116076

  4. Simultaneous transcriptome analysis of Colletotrichum gloeosporioides and tomato fruit pathosystem reveals novel fungal pathogenicity and fruit defense strategies.

    PubMed

    Alkan, Noam; Friedlander, Gilgi; Ment, Dana; Prusky, Dov; Fluhr, Robert

    2015-01-01

    The fungus Colletotrichum gloeosporioides breaches the fruit cuticle but remains quiescent until fruit ripening signals a switch to necrotrophy, culminating in devastating anthracnose disease. There is a need to understand the distinct fungal arms strategy and the simultaneous fruit response. Transcriptome analysis of fungal-fruit interactions was carried out concurrently in the appressoria, quiescent and necrotrophic stages. Conidia germinating on unripe fruit cuticle showed stage-specific transcription that was accompanied by massive fruit defense responses. The subsequent quiescent stage showed the development of dendritic-like structures and swollen hyphae within the fruit epidermis. The quiescent fungal transcriptome was characterized by activation of chromatin remodeling genes and unsuspected environmental alkalization. Fruit response was portrayed by continued highly integrated massive up-regulation of defense genes. During cuticle infection of green or ripe fruit, fungi recapitulate the same developmental stages but with differing quiescent time spans. The necrotrophic stage showed a dramatic shift in fungal metabolism and up-regulation of pathogenicity factors. Fruit response to necrotrophy showed activation of the salicylic acid pathway, climaxing in cell death. Transcriptome analysis of C. gloeosporioides infection of fruit reveals its distinct stage-specific lifestyle and the concurrent changing fruit response, deepening our perception of the unfolding fungal-fruit arms and defenses race. PMID:25377514

  5. Amino acid sequence analysis and characterization of a ribonuclease from starfish Asterias amurensis.

    PubMed

    Motoyoshi, Naomi; Kobayashi, Hiroko; Itagaki, Tadashi; Inokuchi, Norio

    2016-09-01

    The aim of this study was to phylogenetically characterize the location of the RNase T2 enzyme in the starfish (Asterias amurensis). We isolated an RNase T2 ribonuclease (RNase Aa) from the ovaries of starfish and determined its amino acid sequence by protein chemistry and cloning cDNA encoding RNase Aa. The isolated protein had 231 amino acid residues, a predicted molecular mass of 25,906 Da, and an optimal pH of 5.0. RNase Aa preferentially released guanylic acid from the RNA. The catalytic sites of the RNase T2 family are conserved in RNase Aa; furthermore, the distribution of the cysteine residues in RNase Aa is similar to that in other animal and plant T2 RNases. RNase Aa is cleaved at two points: 21 residues from the N-terminus and 29 residues from the C-terminus; however, both fragments may remain attached to the protein via disulfide bridges, leading to the maintenance of its conformation, as suggested by circular dichroism spectrum analysis. The phylogenetic analysis revealed that starfish RNase Aa is evolutionarily an intermediate between protozoan and oyster RNases. PMID:26920046

  6. The mosaicism of plasmids revealed by atypical genes detection and analysis

    PubMed Central

    2011-01-01

    Background From an evolutionary viewpoint, prokaryotic genomes are extremely plastic and dynamic, since large amounts of genetic material are continuously added and/or lost through promiscuous gene exchange. In this picture, plasmids play a key role, since they can be transferred between different cells and, through genetic rearrangement(s), undergo gene(s) load, leading, in turn, to the appearance of important metabolic innovations that might be relevant for cell life. Despite their central position in bacterial evolution, a massive analysis of newly acquired functional blocks [likely the result of horizontal gene transfer (HGT) events] residing on plasmids is still missing. Results We have developed a computational, composition-based, pipeline to scan almost 2000 plasmids for genes that differ significantly from their hosting molecule. Plasmids atypical genes (PAGs) were about 6% of the total plasmids ORFs and, on average, each plasmid possessed 4.4 atypical genes. Nevertheless, conjugative plasmids were shown to possess an amount of atypical genes than that found in not mobilizable plasmids, providing strong support for the central role suggested for conjugative plasmids in the context of HGT. Part of the retrieved PAGs are organized into (mainly short) clusters and are involved in important biological processes (detoxification, antibiotic resistance, virulence), revealing the importance of HGT in the spreading of metabolic pathways within the whole microbial community. Lastly, our analysis revealed that PAGs mainly derive from other plasmid (rather than coming from phages and/or chromosomes), suggesting that plasmid-plasmid DNA exchange might be the primary source of metabolic innovations in this class of mobile genetic elements. Conclusions In this work we have performed the first large scale analysis of atypical genes that reside on plasmid molecules to date. Our findings on PAGs function, organization, distribution and spreading reveal the importance of

  7. Field enhancement sample stacking for analysis of organic acids in traditional Chinese medicine by capillary electrophoresis.

    PubMed

    Zhu, Qianqian; Xu, Xueqin; Huang, Yuanyuan; Xu, Liangjun; Chen, Guonan

    2012-07-13

    A technique known as field enhancement sample stacking (FESS) and capillary electrophoresis (CE) separation has been developed to analyze and detect organic acids in the three traditional Chinese medicines (such as Portulaca oleracea L., Crataegus pinnatifida and Aloe vera L.). In FESS, a reverse electrode polarity-stacking mode (REPSM) was applied as on-line preconcentration strategy. Under the optimized condition, the baseline separation of eight organic acids (linolenic acid, lauric acid, p-coumaric acid, ascorbic acid, benzoic acid, caffeic acid, succinic acid and fumaric acid) could be achieved within 20 min. Validation parameters of this method (such as detection limits, linearity and precision) were also evaluated. The detection limits ranged from 0.4 to 60 ng/mL. The results indicated that the proposed method was effective for the separation of mixtures of organic acids. Satisfactory recoveries were also obtained in the analysis of these organic acids in the above traditional Chinese medicine samples. PMID:22381886

  8. Cluster analysis reveals risk factors for repeated suicide attempts in a multi-ethnic Asian population.

    PubMed

    Choo, Carol; Diederich, Joachim; Song, Insu; Ho, Roger

    2014-04-01

    This study explores underlying patterns in suicide risk factors using data mining techniques. Medical records of suicide attempters who were admitted to a teaching hospital in January 2004 - December 2006 were studied. Cluster analysis revealed hidden patterns for repeated and single attempters (n=418). Repeated attempters had a more complex clinical picture. Symptoms of psychotic illness, borderline personality disorder, and psychosomatic complaints of insomnia and headaches, reports of adverse life events such as unemployment, divorce and quarrels, experience of negative feelings, and usage of alcohol were associated with risk of repeated overdoses with benzodiazepines and paracetamol. The findings have implications for suicide assessments and interventions. PMID:24655624

  9. Correction: Comparative analysis of fungal genomes reveals different plant cell wall degrading capacity in fungi

    PubMed Central

    2014-01-01

    Abstract The version of this article published in BMC Genomics 2013, 14: 274, contains 9 unpublished genomes (Botryobasidium botryosum, Gymnopus luxurians, Hypholoma sublateritium, Jaapia argillacea, Hebeloma cylindrosporum, Conidiobolus coronatus, Laccaria amethystina, Paxillus involutus, and P. rubicundulus) downloaded from JGI website. In this correction, we removed these genomes after discussion with editors and data producers whom we should have contacted before downloading these genomes. Removing these data did not alter the principle results and conclusions of our original work. The relevant Figures 1, 2, 3, 4 and 6; and Table 1 have been revised. Additional files 1, 3, 4, and 5 were also revised. We would like to apologize for any confusion or inconvenience this may have caused. Background Fungi produce a variety of carbohydrate activity enzymes (CAZymes) for the degradation of plant polysaccharide materials to facilitate infection and/or gain nutrition. Identifying and comparing CAZymes from fungi with different nutritional modes or infection mechanisms may provide information for better understanding of their life styles and infection models. To date, over hundreds of fungal genomes are publicly available. However, a systematic comparative analysis of fungal CAZymes across the entire fungal kingdom has not been reported. Results In this study, we systemically identified glycoside hydrolases (GHs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), and glycosyltransferases (GTs) as well as carbohydrate-binding modules (CBMs) in the predicted proteomes of 94 representative fungi from Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota. Comparative analysis of these CAZymes that play major roles in plant polysaccharide degradation revealed that fungi exhibit tremendous diversity in the number and variety of CAZymes. Among them, some families of GHs and CEs are the most prevalent CAZymes that are distributed in all of the fungi analyzed

  10. Single-cell analysis reveals a novel uncultivated magnetotactic bacterium within the candidate division OP3.

    PubMed

    Kolinko, Sebastian; Jogler, Christian; Katzmann, Emanuel; Wanner, Gerhard; Peplies, Jörg; Schüler, Dirk

    2012-07-01

    Magnetotactic bacteria (MTB) are a diverse group of prokaryotes that orient along magnetic fields using membrane-coated magnetic nanocrystals of magnetite (Fe(3) O(4) ) or greigite (Fe(3) S(4) ), the magnetosomes. Previous phylogenetic analysis of MTB has been limited to few cultivated species and most abundant members of natural populations, which were assigned to Proteobacteria and the Nitrospirae phyla. Here, we describe a single cell-based approach that allowed the targeted phylogenetic and ultrastructural analysis of the magnetotactic bacterium SKK-01, which was low abundant in sediments of Lake Chiemsee. Morphologically conspicuous single cells of SKK-01 were micromanipulated from magnetically collected multi-species MTB populations, which was followed by whole genome amplification and ultrastructural analysis of sorted cells. Besides intracellular sulphur inclusions, the large ovoid cells of SKK-01 harbour ∼175 bullet-shaped magnetosomes arranged in multiple chains that consist of magnetite as revealed by TEM and EDX analysis. Sequence analysis of 16 and 23S rRNA genes from amplified genomic DNA as well as fluorescence in situ hybridization assigned SKK-01 to the candidate division OP3, which so far lacks any cultivated representatives. SKK-01 represents the first morphotype that can be assigned to the OP3 group as well as the first magnetotactic member of the PVC superphylum. PMID:22003954

  11. NMR studies reveal the role of biomembranes in modulating ligand binding and release by intracellular bile acid binding proteins.

    PubMed

    Pedò, Massimo; Löhr, Frank; D'Onofrio, Mariapina; Assfalg, Michael; Dötsch, Volker; Molinari, Henriette

    2009-12-18

    Bile acid molecules are transferred vectorially between basolateral and apical membranes of hepatocytes and enterocytes in the context of the enterohepatic circulation, a process regulating whole body lipid homeostasis. This work addresses the role of the cytosolic lipid binding proteins in the intracellular transfer of bile acids between different membrane compartments. We present nuclear magnetic resonance (NMR) data describing the ternary system composed of the bile acid binding protein, bile acids, and membrane mimetic systems, such as anionic liposomes. This work provides evidence that the investigated liver bile acid binding protein undergoes association with the anionic membrane and binding-induced partial unfolding. The addition of the physiological ligand to the protein-liposome mixture is capable of modulating this interaction, shifting the equilibrium towards the free folded holo protein. An ensemble of NMR titration experiments, based on nitrogen-15 protein and ligand observation, confirm that the membrane and the ligand establish competing binding equilibria, modulating the cytoplasmic permeability of bile acids. These results support a mechanism of ligand binding and release controlled by the onset of a bile salt concentration gradient within the polarized cell. The location of a specific protein region interacting with liposomes is highlighted. PMID:19836400

  12. Transcriptome Analysis in Haematococcus pluvialis: Astaxanthin Induction by Salicylic Acid (SA) and Jasmonic Acid (JA)

    PubMed Central

    Wu, Guanxun; Li, Guoqiang; Sun, Haifeng; Deng, Suzhen; Shen, Yicheng; Chen, Guoqiang; Zhang, Ruihao; Meng, Chunxiao; Zhang, Xiaowen

    2015-01-01

    Haematococcus pluvialis is an astaxanthin-rich microalga that can increase its astaxanthin production by salicylic acid (SA) or jasmonic acid (JA) induction. The genetic transcriptome details of astaxanthin biosynthesis were analyzed by exposing the algal cells to 25 mg/L of SA and JA for 1, 6 and 24 hours, plus to the control (no stress). Based on the RNA-seq analysis, 56,077 unigenes (51.7%) were identified with functions in response to the hormone stress. The top five identified subcategories were cell, cellular process, intracellular, catalytic activity and cytoplasm, which possessed 5600 (~9.99%), 5302 (~9.45%), 5242 (~9.35%), 4407 (~7.86%) and 4195 (~7.48%) unigenes, respectively. Furthermore, 59 unigenes were identified and assigned to 26 putative transcription factors (TFs), including 12 plant-specific TFs. They were likely associated with astaxanthin biosynthesis in Haematococcus upon SA and JA stress. In comparison, the up-regulation of differential expressed genes occurred much earlier, with higher transcript levels in the JA treatment (about 6 h later) than in the SA treatment (beyond 24 h). These results provide valuable information for directing metabolic engineering efforts to improve astaxanthin biosynthesis in H. pluvialis. PMID:26484871

  13. Transcriptome Analysis in Haematococcus pluvialis: Astaxanthin Induction by Salicylic Acid (SA) and Jasmonic Acid (JA).

    PubMed

    Gao, Zhengquan; Li, Yan; Wu, Guanxun; Li, Guoqiang; Sun, Haifeng; Deng, Suzhen; Shen, Yicheng; Chen, Guoqiang; Zhang, Ruihao; Meng, Chunxiao; Zhang, Xiaowen

    2015-01-01

    Haematococcus pluvialis is an astaxanthin-rich microalga that can increase its astaxanthin production by salicylic acid (SA) or jasmonic acid (JA) induction. The genetic transcriptome details of astaxanthin biosynthesis were analyzed by exposing the algal cells to 25 mg/L of SA and JA for 1, 6 and 24 hours, plus to the control (no stress). Based on the RNA-seq analysis, 56,077 unigenes (51.7%) were identified with functions in response to the hormone stress. The top five identified subcategories were cell, cellular process, intracellular, catalytic activity and cytoplasm, which possessed 5600 (~9.99%), 5302 (~9.45%), 5242 (~9.35%), 4407 (~7.86%) and 4195 (~7.48%) unigenes, respectively. Furthermore, 59 unigenes were identified and assigned to 26 putative transcription factors (TFs), including 12 plant-specific TFs. They were likely associated with astaxanthin biosynthesis in Haematococcus upon SA and JA stress. In comparison, the up-regulation of differential expressed genes occurred much earlier, with higher transcript levels in the JA treatment (about 6 h later) than in the SA treatment (beyond 24 h). These results provide valuable information for directing metabolic engineering efforts to improve astaxanthin biosynthesis in H. pluvialis. PMID:26484871

  14. Human retroviruses and AIDS 1996. A compilation and analysis of nucleic acid and amino acid sequences

    SciTech Connect

    Myers, G.; Foley, B.; Korber, B.; Mellors, J.W.; Jeang, K.T.; Wain-Hobson, S.

    1997-04-01

    This compendium and the accompanying floppy diskettes are the result of an effort to compile and rapidly publish all relevant molecular data concerning the human immunodeficiency viruses (HIV) and related retroviruses. The scope of the compendium and database is best summarized by the five parts that it comprises: (1) Nuclear Acid Alignments and Sequences; (2) Amino Acid Alignments; (3) Analysis; (4) Related Sequences; and (5) Database Communications. Information within all the parts is updated throughout the year on the Web site, http://hiv-web.lanl.gov. While this publication could take the form of a review or sequence monograph, it is not so conceived. Instead, the literature from which the database is derived has simply been summarized and some elementary computational analyses have been performed upon the data. Interpretation and commentary have been avoided insofar as possible so that the reader can form his or her own judgments concerning the complex information. In addition to the general descriptions of the parts of the compendium, the user should read the individual introductions for each part.

  15. Integrative analysis of RNA, translation, and protein levels reveals distinct regulatory variation across humans

    PubMed Central

    Cenik, Can; Cenik, Elif Sarinay; Byeon, Gun W.; Grubert, Fabian; Candille, Sophie I.; Spacek, Damek; Alsallakh, Bilal; Tilgner, Hagen; Araya, Carlos L.; Tang, Hua; Ricci, Emiliano; Snyder, Michael P.

    2015-01-01

    Elucidating the consequences of genetic differences between humans is essential for understanding phenotypic diversity and personalized medicine. Although variation in RNA levels, transcription factor binding, and chromatin have been explored, little is known about global variation in translation and its genetic determinants. We used ribosome profiling, RNA sequencing, and mass spectrometry to perform an integrated analysis in lymphoblastoid cell lines from a diverse group of individuals. We find significant differences in RNA, translation, and protein levels suggesting diverse mechanisms of personalized gene expression control. Combined analysis of RNA expression and ribosome occupancy improves the identification of individual protein level differences. Finally, we identify genetic differences that specifically modulate ribosome occupancy—many of these differences lie close to start codons and upstream ORFs. Our results reveal a new level of gene expression variation among humans and indicate that genetic variants can cause changes in protein levels through effects on translation. PMID:26297486

  16. Comprehensive genome-wide analysis reveals different classes of enigmatic old yellow enzyme in fungi

    PubMed Central

    Nizam, Shadab; Verma, Sandhya; Borah, Nilam Nayan; Gazara, Rajesh Kumar; Verma, Praveen Kumar

    2014-01-01

    In this study, we systematically identify Old Yellow Enzymes (OYEs) from a diverse range of economically important fungi representing different ecology and lifestyle. Using active site residues and sequence alignments, we present a classification for these proteins into three distinct classes including a novel class (Class III) and assign names to sequences. Our in-depth phylogenetic analysis suggests a complex history of lineage-specific expansion and contraction for the OYE gene family in fungi. Comparative analyses reveal remarkable diversity in the number and classes of OYE among fungi. Quantitative real-time PCR (qRT-PCR) of Ascochyta rabiei OYEs indicates differential expression of OYE genes during oxidative stress and plant infection. This study shows relationship of OYE with fungal ecology and lifestyle, and provides a foundation for future functional analysis and characterization of OYE gene family. PMID:24500274

  17. Novel aspects of COP9 signalosome functions revealed through analysis of hypomorphic csn mutants

    PubMed Central

    Parker, Jane E

    2009-01-01

    The COP9 signalosome (CSN) is a conserved eukaryotic protein complex implicated in the regulation of cullin-RING type E3 ubiquitin ligases by cleaving the small peptide RUB/Nedd8 from cullins. However, detailed analysis of CSN physiological functions in Arabidopsis has been hampered by the early seedling-lethality of csn null mutants. We and others have now identified a number of viable hypomorphic csn mutants which start to reveal novel CSN-dependent activities in adult Arabidopsis plants.1 Here, we present a detailed comparative analysis of the csn5a-1 and csn2-5 mutants as a mean to improve understanding of CSN functions in plant cells. Our observations point to CSN-independent activities of CSN5 and suggest a role of the CSN in cytoskeleton assembly/organization. PMID:19847120

  18. Analysis of the Tyrosine Kinome in Melanoma Reveals Recurrent Mutations in ERBB4

    PubMed Central

    Prickett, Todd D.; Agrawal, Neena S.; Wei, Xiaomu; Yates, Kristin E.; Lin, Jimmy C.; Wunderlich, John; Cronin, Julia C.; Cruz, Pedro; Rosenberg, Steven A.; Samuels, Yardena

    2010-01-01

    Tyrosine phosphorylation is important in signaling pathways underlying tumorigenesis. A mutational analysis of the Protein Tyrosine Kinase (PTK) gene family in cutaneous metastatic melanoma identified 30 somatic mutations in the kinase domain of 19 PTKs. The whole of the coding region of these 19 PTKs was further evaluated for somatic mutations in a total of 79 melanoma samples. This analysis revealed novel ERBB4 mutations in 19% of melanoma patients and that an additional two kinases (FLT1 and PTK2B) are mutated in 10% of melanomas. Seven missense mutations in the most commonly altered PTK (ERBB4) were examined and found to increase kinase activity and transformation ability. Melanoma cells expressing mutant ERBB4 had reduced cell growth after shRNA–mediated knockdown of ERBB4 or treatment with the ERBB inhibitor lapatinib. These studies might lead to personalized therapeutics specifically targeting the kinases that are mutationally altered in individual melanomas. PMID:19718025

  19. Global analysis of gene expression in pulmonary fibrosis reveals distinct programs regulating lung inflammation and fibrosis

    NASA Astrophysics Data System (ADS)

    Kaminski, Naftali; Allard, John D.; Pittet, Jean F.; Zuo, Fengrong; Griffiths, Mark J. D.; Morris, David; Huang, Xiaozhu; Sheppard, Dean; Heller, Renu A.

    2000-02-01

    The molecular mechanisms of pulmonary fibrosis are poorly understood. We have used oligonucleotide arrays to analyze the gene expression programs that underlie pulmonary fibrosis in response to bleomycin, a drug that causes lung inflammation and fibrosis, in two strains of susceptible mice (129 and C57BL/6). We then compared the gene expression patterns in these mice with 129 mice carrying a null mutation in the epithelial-restricted integrin 6 subunit (6/-), which develop inflammation but are protected from pulmonary fibrosis. Cluster analysis identified two distinct groups of genes involved in the inflammatory and fibrotic responses. Analysis of gene expression at multiple time points after bleomycin administration revealed sequential induction of subsets of genes that characterize each response. The availability of this comprehensive data set should accelerate the development of more effective strategies for intervention at the various stages in the development of fibrotic diseases of the lungs and other organs.

  20. In Silico Phylogenetic Analysis and Molecular Modelling Study of 2-Haloalkanoic Acid Dehalogenase Enzymes from Bacterial and Fungal Origin

    PubMed Central

    Satpathy, Raghunath; Konkimalla, V. B.; Ratha, Jagnyeswar

    2016-01-01

    2-Haloalkanoic acid dehalogenase enzymes have broad range of applications, starting from bioremediation to chemical synthesis of useful compounds that are widely distributed in fungi and bacteria. In the present study, a total of 81 full-length protein sequences of 2-haloalkanoic acid dehalogenase from bacteria and fungi were retrieved from NCBI database. Sequence analysis such as multiple sequence alignment (MSA), conserved motif identification, computation of amino acid composition, and phylogenetic tree construction were performed on these primary sequences. From MSA analysis, it was observed that the sequences share conserved lysine (K) and aspartate (D) residues in them. Also, phylogenetic tree indicated a subcluster comprised of both fungal and bacterial species. Due to nonavailability of experimental 3D structure for fungal 2-haloalkanoic acid dehalogenase in the PDB, molecular modelling study was performed for both fungal and bacterial sources of enzymes present in the subcluster. Further structural analysis revealed a common evolutionary topology shared between both fungal and bacterial enzymes. Studies on the buried amino acids showed highly conserved Leu and Ser in the core, despite variation in their amino acid percentage. Additionally, a surface exposed tryptophan was conserved in all of these selected models. PMID:26880911

  1. Analysis of acid rain effects on vegetation in eco-regions in China based on AVHRR/NDVI

    NASA Astrophysics Data System (ADS)

    Jin, Jiaxin; Jiang, Hong; Zhang, Xiuying; Xu, Xiaobin

    2010-09-01

    The vegetation, as the main component of the ecosystems, is the main receptor of acid rain pollution. Because of the discrepancy of the vegetation characteristics and environment, the responses of the different types of vegetation to acid rain in different regions are different. In this paper, we chose 9 eco-regions in southern China as study area, based on the acid rain and NOAA/NDVI data from 1992 to 2006, and revealed the impact of acid rain on the vegetation by using spatial interpolation, cluster analysis and curve fitting. The result shows that the most tropical and subtropical moist broadleaf forests were positively correlated with the acidity of precipitation and the growth was inhibited obviously. On the contrary, the growth of temperate coniferous forests was promoted by acid rain to some extent. In generally, the vegetation in the condition of the weak acid rain grew better, especially the Qin Ling Mountains deciduous forests and the Changjiang Plain evergreen forests. For South China-Vietnam subtropical evergreen forest, Yunnan Plateau subtropical evergreen forests and Qionglai-Minshan conifer forests, the significant difference of NDVI between the different gradients of acid rain lasted almost the whole year, while that of the other eco-regions only appeared most obviously in Winter.

  2. Co-occurrence correlations of heavy metals in sediments revealed using network analysis.

    PubMed

    Liu, Lili; Wang, Zhiping; Ju, Feng; Zhang, Tong

    2015-01-01

    In this study, the correlation-based study was used to identify the co-occurrence correlations among metals in marine sediment of Hong Kong, based on the long-term (from 1991 to 2011) temporal and spatial monitoring data. 14 stations out of the total 45 marine sediment monitoring stations were selected from three representative areas, including Deep Bay, Victoria Harbour and Mirs Bay. Firstly, Spearman's rank correlation-based network analysis was conducted as the first step to identify the co-occurrence correlations of metals from raw metadata, and then for further analysis using the normalized metadata. The correlations patterns obtained by network were consistent with those obtained by the other statistic normalization methods, including annual ratios, R-squared coefficient and Pearson correlation coefficient. Both Deep Bay and Victoria Harbour have been polluted by heavy metals, especially for Pb and Cu, which showed strong co-occurrence with other heavy metals (e.g. Cr, Ni, Zn and etc.) and little correlations with the reference parameters (Fe or Al). For Mirs Bay, which has better marine sediment quality compared with Deep Bay and Victoria Harbour, the co-occurrence patterns revealed by network analysis indicated that the metals in sediment dominantly followed the natural geography process. Besides the wide applications in biology, sociology and informatics, it is the first time to apply network analysis in the researches of environment pollutions. This study demonstrated its powerful application for revealing the co-occurrence correlations among heavy metals in marine sediments, which could be further applied for other pollutants in various environment systems. PMID:24559934

  3. Archetypal analysis of diverse Pseudomonas aeruginosa transcriptomes reveals adaptation in cystic fibrosis airways

    PubMed Central

    2013-01-01

    Background Analysis of global gene expression by DNA microarrays is widely used in experimental molecular biology. However, the complexity of such high-dimensional data sets makes it difficult to fully understand the underlying biological features present in the data. The aim of this study is to introduce a method for DNA microarray analysis that provides an intuitive interpretation of data through dimension reduction and pattern recognition. We present the first “Archetypal Analysis” of global gene expression. The analysis is based on microarray data from five integrated studies of Pseudomonas aeruginosa isolated from the airways of cystic fibrosis patients. Results Our analysis clustered samples into distinct groups with comprehensible characteristics since the archetypes representing the individual groups are closely related to samples present in the data set. Significant changes in gene expression between different groups identified adaptive changes of the bacteria residing in the cystic fibrosis lung. The analysis suggests a similar gene expression pattern between isolates with a high mutation rate (hypermutators) despite accumulation of different mutations for these isolates. This suggests positive selection in the cystic fibrosis lung environment, and changes in gene expression for these isolates are therefore most likely related to adaptation of the bacteria. Conclusions Archetypal analysis succeeded in identifying adaptive changes of P. aeruginosa. The combination of clustering and matrix factorization made it possible to reveal minor similarities among different groups of data, which other analytical methods failed to identify. We suggest that this analysis could be used to supplement current methods used to analyze DNA microarray data. PMID:24059747

  4. Serum Metabolomics Reveals Higher Levels of Polyunsaturated Fatty Acids in Lepromatous Leprosy: Potential Markers for Susceptibility and Pathogenesis

    PubMed Central

    Al-Mubarak, Reem; Vander Heiden, Jason; Broeckling, Corey D.; Balagon, Marivic; Brennan, Patrick J.; Vissa, Varalakshmi D.

    2011-01-01

    Background Leprosy is a disease of the skin and peripheral nervous system caused by the obligate intracellular bacterium Mycobacterium leprae. The clinical presentations of leprosy are spectral, with the severity of disease determined by the balance between the cellular and humoral immune response of the host. The exact mechanisms that facilitate disease susceptibility, onset and progression to certain clinical phenotypes are presently unclear. Various studies have examined lipid metabolism in leprosy, but there has been limited work using whole metabolite profiles to distinguish the clinical forms of leprosy. Methodology and Principal Findings In this study we adopted a metabolomics approach using high mass accuracy ultrahigh pressure liquid chromatography mass spectrometry (UPLC-MS) to investigate the circulatory biomarkers in newly diagnosed untreated leprosy patients. Sera from patients having bacterial indices (BI) below 1 or above 4 were selected, subjected to UPLC-MS, and then analyzed for biomarkers which distinguish the polar presentations of leprosy. We found significant increases in the abundance of certain polyunsaturated fatty acids (PUFAs) and phospholipids in the high-BI patients, when contrasted with the levels in the low-BI patients. In particular, the median values of arachidonic acid (2-fold increase), eicosapentaenoic acid (2.6-fold increase) and docosahexaenoic acid (1.6-fold increase) were found to be greater in the high-BI patients. Significance