Volatiles of rhizobacteria Serratia and Stenotrophomonas alter growth and metabolite composition of Arabidopsis thaliana.

The emission of volatiles is a common, but mostly neglected, ability of bacteria that is important for inter- and intraspecific interactions. Currently, limited information is available on how the bacterial volatile (mVOC) signal is integrated into a plant's life at the physiological, transcriptional and metabolic level. Previous results provided evidence for volatile-dependent regulation of WRKY18, a pathogen-responsive transcription factor of Arabidopsis thaliana in co-culture with two rhizobacteria, Serratia plymuthica HRO-C48 and Stenotrophomonas maltophilia R3089. Dual cultures of these bacteria and A. thaliana; application of the common mVOC 2-phenyl-ethanol; extraction of metabolites of A. thaliana after exposure to bacterial volatiles; and analysis of the metabolomes (GC-TOF/MS) were carried out. The prominent microbial aromatic compound 2-phenyl-ethanol, emitted by both bacteria, negatively affects growth of A. thaliana wild type, whereas WRKY18 T-DNA insertion mutants were significantly more tolerant than wild-type seedlings. This paper also demonstrates for the first time the impact of the rhizobacterial volatiles on the metabolome of A. thaliana. Upon mVOC exposure the plants rearrange their metabolism by accumulation of e.g. amino acids and TCA intermediates that potentially allow plants to cope with and survive this stress. Our findings illustrate the high degree of complexity of metabolic rearrangements underlying the interactions of bacterial volatile elicitors and resulting plant responses. Furthermore, the impact of the volatile 2-phenyl-ethanol as a signal in the WRKY18-dependent pathway highlights this compound as an important molecular player.

Multi-platform metabolomics analyses of a broad collection of fragrant and non-fragrant rice varieties reveals the high complexity of grain quality characteristics.

The quality of rice in terms not only of its nutritional value but also in terms of its aroma and flavour is becoming increasingly important in modern rice breeding where global targets are focused on both yield stability and grain quality. In the present paper we have exploited advanced, multi-platform metabolomics approaches to determine the biochemical differences in 31 rice varieties from a diverse range of genetic backgrounds and origin. All were grown under the specific local conditions for which they have been bred and all aspects of varietal identification and sample purity have been guaranteed by local experts from each country. Metabolomics analyses using 6 platforms have revealed the extent of biochemical differences (and similarities) between the chosen rice genotypes. Comparison of fragrant rice varieties showed a difference in the metabolic profiles of jasmine and basmati varieties. However with no consistent separation of the germplasm class. Storage of grains had a significant effect on the metabolome of both basmati and jasmine rice varieties but changes were different for the two rice types. This shows how metabolic changes may help prove a causal relationship with developing good quality in basmati rice or incurring quality loss in jasmine rice in aged grains. Such metabolomics approaches are leading to hypotheses on the potential links between grain quality attributes, biochemical composition and genotype in the context of breeding for improvement. With this knowledge we shall establish a stronger, evidence-based foundation upon which to build targeted strategies to support breeders in their quest for improved rice varieties.

HIV-1 genetic diversity in Argentina and early diagnosis of perinatal infection

HIV-1 diagnosis of perinatally exposed children is usually performed by molecular biology-based methods, allowing the direct detection of the virus. Thus, HIV-1 genomic variability within and across strains plays a major role in relation to the sensitivity of these tests, often leading to misdiagnosis. We describe the performance of an in-house multiplex nested PCR (nPCR) for early detection of HIV-1 infection in perinatally exposed children born in Argentina, where the percentage of diverse BF recombinants is as high as 80%. After evaluation of 1316 HIV-1 perinatally exposed children collected over a 7-year period, the specificity and sensitivity of the diagnostic nPCR was of 100% and 99.2% respectively, with only two false negative cases indicating a good performance of the diagnostic nPCR in the Argentine pediatric cohort. In search of unusual HIV-1 subtypes among 22 HIV-1 infected cases presenting partial or complete HIV-1 gene amplification failure, we performed phylogenetic and recombination analysis of a vpu-env fragment in addition to gag and env Heteroduplex Mobility Assay screening. The most unusual findings included two subtypes A and a novel BC recombinant, while the majority of the strains were a variety of different BF recombinants. These results indicate the presence of novel and heterogeneous genotypes in our country and the need of continuous viral surveillance not only for diagnostic test optimization but also for the eventual implementation of a successful vaccine.(AU)

HIV-1 genetic diversity in Argentina and early diagnosis of perinatal infection

HIV-1 diagnosis of perinatally exposed children is usually performed by molecular biology-based methods, allowing the direct detection of the virus. Thus, HIV-1 genomic variability within and across strains plays a major role in relation to the sensitivity of these tests, often leading to misdiagnosis. We describe the performance of an in-house multiplex nested PCR (nPCR) for early detection of HIV-1 infection in perinatally exposed children born in Argentina, where the percentage of diverse BF recombinants is as high as 80%. After evaluation of 1316 HIV-1 perinatally exposed children collected over a 7-year period, the specificity and sensitivity of the diagnostic nPCR was of 100% and 99.2% respectively, with only two false negative cases indicating a good performance of the diagnostic nPCR in the Argentine pediatric cohort. In search of unusual HIV-1 subtypes among 22 HIV-1 infected cases presenting partial or complete HIV-1 gene amplification failure, we performed phylogenetic and recombination analysis of a vpu-env fragment in addition to gag and env Heteroduplex Mobility Assay screening. The most unusual findings included two subtypes A and a novel BC recombinant, while the majority of the strains were a variety of different BF recombinants. These results indicate the presence of novel and heterogeneous genotypes in our country and the need of continuous viral surveillance not only for diagnostic test optimization but also for the eventual implementation of a successful vaccine.(AU)

HIV-1 genetic diversity in Argentina and early diagnosis of perinatal infection

HIV-1 diagnosis of perinatally exposed children is usually performed by molecular biology-based methods, allowing the direct detection of the virus. Thus, HIV-1 genomic variability within and across strains plays a major role in relation to the sensitivity of these tests, often leading to misdiagnosis. We describe the performance of an in-house multiplex nested PCR (nPCR) for early detection of HIV-1 infection in perinatally exposed children born in Argentina, where the percentage of diverse BF recombinants is as high as 80%. After evaluation of 1316 HIV-1 perinatally exposed children collected over a 7-year period, the specificity and sensitivity of the diagnostic nPCR was of 100% and 99.2% respectively, with only two false negative cases indicating a good performance of the diagnostic nPCR in the Argentine pediatric cohort. In search of unusual HIV-1 subtypes among 22 HIV-1 infected cases presenting partial or complete HIV-1 gene amplification failure, we performed phylogenetic and recombination analysis of a vpu-env fragment in addition to gag and env Heteroduplex Mobility Assay screening. The most unusual findings included two subtypes A and a novel BC recombinant, while the majority of the strains were a variety of different BF recombinants. These results indicate the presence of novel and heterogeneous genotypes in our country and the need of continuous viral surveillance not only for diagnostic test optimization but also for the eventual implementation of a successful vaccine.

Metabolite profiling for plant functional genomics.

Multiparallel analyses of mRNA and proteins are central to today's functional genomics initiatives. We describe here the use of metabolite profiling as a new tool for a comparative display of gene function. It has the potential not only to provide deeper insight into complex regulatory processes but also to determine phenotype directly. Using gas chromatography/mass spectrometry (GC/MS), we automatically quantified 326 distinct compounds from Arabidopsis thaliana leaf extracts. It was possible to assign a chemical structure to approximately half of these compounds. Comparison of four Arabidopsis genotypes (two homozygous ecotypes and a mutant of each ecotype) showed that each genotype possesses a distinct metabolic profile. Data mining tools such as principal component analysis enabled the assignment of "metabolic phenotypes" using these large data sets. The metabolic phenotypes of the two ecotypes were more divergent than were the metabolic phenotypes of the single-loci mutant and their parental ecotypes. These results demonstrate the use of metabolite profiling as a tool to significantly extend and enhance the power of existing functional genomics approaches.

Technical advance: simultaneous analysis of metabolites in potato tuber by gas chromatography-mass spectrometry.

A new method is presented in which gas chromatography coupled to mass spectrometry (GC-MS) allows the quantitative and qualitative detection of more than 150 compounds within a potato tuber, in a highly sensitive and specific manner. In contrast to other methods developed for metabolite analysis in plant systems, this method represents an unbiased and open approach that allows the detection of unexpected changes in metabolite levels. Although the method represents a compromise for a wide range of metabolites in terms of extraction, chemical modification and GC-MS analysis, for 25 metabolites analysed in detail the recoveries were found to be within the generally accepted range of 70-140%. Further, the reproducibility of the method was high: the error occurring in the analysis procedures was found to be less than 6% for 30 out of 33 compounds tested. Biological variability exceeded the systematic error of the analysis by a factor of up to 10. The method is also suited for upscaling, potentially allowing the simultaneous analysis of a large number of samples. As a first example this method has been applied to soil- and in vitro-grown tubers. Due to the simultaneous analysis of a wide range of metabolites it was immediately apparent that these systems differ significantly in their metabolism. Furthermore, the parallel insight into many pathways allows some conclusions to be drawn about the underlying physiological differences between both tuber systems. As a second example, transgenic lines modified in sucrose catabolism or starch synthesis were analysed. This example illustrates the power of an unbiased approach to detecting unexpected changes in transgenic lines.

Identification of uncommon plant metabolites based on calculation of elemental compositions using gas chromatography and quadrupole mass spectrometry.

Unknown compounds in polar fractions of Arabidopsis thaliana crude leaf extracts were identified on the basis of calculations of elemental compositions obtained from gas chromatography/low-resolution quadrupole mass spectrometric data. Plant metabolites were methoximated and silylated prior to analysis. All known peaks were used as internal references to construct polynomial recalibration curves of from raw mass spectrometric data. Mass accuracies of 0.005 +/- 0.003 amu and isotope ratio errors of 0.5 +/- 0.3% (A + 1/A), respectively, 0.3 +/- 0.2% (A + 2/A), could be achieved. Both masses and isotope ratios were combined when the elemental compositions of unknown peaks were calculated. After calculation, compound identities were elucidated by searching metabolic databases, interpreting spectra, and, finally, by comparison with reference compounds. Sum formulas of more than 70 peaks were determined throughout single GC/MS chromatograms. Exact masses were confirmed by high-resolution mass spectrometric data. More than 15 uncommon plant metabolites were identified, some of which are novel in Arabidopsis, such as tartronate semialdehyde, citramalic acid, allothreonine, or glycolic amide.

Protective effect of CCR2-64I and not of CCR5-delta32 and SDF1-3'A in pediatric HIV-1 infection.

The effects of chemokine and chemokine receptor genetic polymorphisms such as stromal derived factor 1 (SDF1-3'A), CCR2-64I, and CCR5-delta32 associated with HIV-1 transmission and/or rate of disease progression in infected study subjects remain highly controversial and have been analyzed primarily only in adults. We have investigated whether these polymorphisms may provide similar beneficial effects in children exposed to HIV-1 perinatally. The prevalence of CCR2-64I allele was significantly increased (p = .03) and the CCR2-64I genotype distribution was not in Hardy-Weinberg equilibrium, among HIV-1-exposed uninfected infants. Moreover, in the HIV-1-infected group, a delay to AIDS progression was observed among carriers of CCR2-64I allele. This is the first report that suggests a protective role of CCR2-64I allele in mother-to-infant HIV-1 transmission and documents a delay in disease progression, after the child has been infected with HIV-1. However, SDFI-3'A and CCR5-delta32 alleles did not modify the rate of HIV-1 transmission or disease progression in HIV-1-infected children.

Transgenic Arabidopsis plants can accumulate polyhydroxybutyrate to up to 4% of their fresh weight.

Transgenic Arabidopsis thaliana (L.) Heynh. plants expressing the three enzymes encoding the biosynthetic route to polyhydroxybutyrate (PHB) are described. These plants accumulated more than 4% of their fresh weight (approximately 40% of their dry weight) in the form of PHB in leaf chloroplasts. These very high producers were obtained and identified following a novel strategy consisting of a rapid GC-MS analysis of a large number of transgenic Arabidopsis plants generated using a triple construct, thus allowing the parallel transfer of all three genes necessary for PHB synthesis in a single transformation event. The level of PHB produced was 4-fold greater than previously published values, thus demonstrating the large potential of plants to produce this renewable resource. However, the high levels of the polymer produced had severe effects on both plant development and metabolism. Stunted growth and a loss of fertility were observed in the high-producing lines. Analysis of the metabolite composition of these lines using a GC-MS method that we have newly developed showed that the accumulation of high levels of PHB was not accompanied by an appreciable change in either the composition or the amount of fatty acids. Substantial changes were, however, observed in the levels of various organic acids, amino acids, sugars and sugar alcohols.

Molecular and enzymatic characterization of three phosphoinositide-specific phospholipase C isoforms from potato.

Many cellular responses to stimulation of cell-surface receptors by extracellular signals are transmitted across the plasma membrane by hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2), which is cleaved into diacylglycerol and inositol-1,4,5-trisphosphate by phosphoinositide-specific phospholipase C (PI-PLC). We present structural, biochemical, and RNA expression data for three distinct PI-PLC isoforms, StPLC1, StPLC2, and StPLC3, which were cloned from a guard cell-enriched tissue preparation of potato (Solanum tuberosum) leaves. All three enzymes contain the catalytic X and Y domains, as well as C2-like domains also present in all PI-PLCs. Analysis of the reaction products obtained from PIP2 hydrolysis unequivocally identified these enzymes as genuine PI-PLC isoforms. Recombinant StPLCs showed an optimal PIP2-hydrolyzing activity at 10 microM Ca2+ and were inhibited by Al3+ in equimolar amounts. In contrast to PI-PLC activity in plant plasma membranes, however, recombinant enzymes could not be activated by Mg2+. All three stplc genes are expressed in various tissues of potato, including leaves, flowers, tubers, and roots, and are affected by drought stress in a gene-specific manner.

Potato guard cells respond to drying soil by a complex change in the expression of genes related to carbon metabolism and turgor regulation.

Altering stomatal function by a guard cell-targeted transgenic approach with the aim of increased stress tolerance and crop yield requires knowledge of the natural fluctuations of stomatal gene expression under stress conditions. We developed a fast method for the isolation of RNA from epidermal fragments of potato leaves (Solanum tuberosum L. cv. Désirée), demonstrated that this RNA preparation is highly enriched in guard cell transcripts and used this method to investigate the response of gene expression in guard cells to mild drought stress. Drought was applied in planta by withholding water over a period of 2-4 days. In the following work responses observed under these conditions are called 'long-term' in contrast to immediate (short-term) stomatal opening and closing responses to environmental stress. We observed both gene-specific increases and decreases of steady-state transcript levels. In particular, the mRNA levels of sucrose synthase and sucrose-phosphate synthase were elevated 5.5-fold and 1.4-fold, respectively. In contrast, expression of an inwardly rectifying K+ channel from guard cells (kst1) and of a plasma membrane H(+)-ATPase (pha2) was reduced to 26% and 36%, respectively, of the expression in watered controls. In addition, expression of vacuolar invertase, UDP-glucose pyrophosphorylase, ADP-glucose pyrophosphorylase (large subunit), cytosolic glyceraldehyde-3-phosphate dehydrogenase, a sucrose/H+ cotransporter, and a novel isoform of phosphoenolpyruvate carboxylase were also reduced. Other genes exhibited unaltered expression. Compared with the response in whole leaves, the transcript levels of phosphoenolpyruvate carboxylase, vacuolar invertase, and cytosolic glyceraldehyde-3-phosphate dehydrogenase were regulated guard cell specifically. Most importantly, changes in steady-state transcript levels were complete before the onset of a decrease in leaf water potential, when drought-induced stomatal closure was already obvious. These data support the hypothesis that a systemic drought-stress signal acts not only on short-term stomatal movements but also on long-term gene expression in guard cells. Such long-term changes in gene expression might contribute to the fine-tuning of guard cell responses to environmental stimuli.

Complementary DNAs encoding eukaryotic-type cytidine-5'-diphosphate-diacylglycerol synthases of two plant species.

Cytidine diphosphate (CDP)-diacylglycerol synthase (cytidine triphosphate:phosphatidate cytihyltransferase, EC 2.7.7.41) catalyzes the formation of CDP-diacylglycerol, which is the precursor of phosphatidylinositol, phosphatidylglycerol, and cardiolipin. We report the first cloning, to our knowledge, of two plant cDNAs, StCDS1 and AtCDS1, coding for CDP-diacylglycerol synthase from potato (Solanum tuberosum) and Arabidopsis thaliana, respectively. The two proteins belong to the eukaryotic type of CDP-diacylglycerol synthase and contain eight predicted transmembrane-spanning domains. We analyzed gene expression in shoot and root tissues of potato plants and demonstrated enzyme activity by expression of N-terminally truncated, recombinant StCDS1 in Escherichia coli.

Analysis of in vivo levels of acyl-thioesters with gas chromatography/mass spectrometry of the butylamide derivative.

The analysis of the in vivo level and composition of acyl-thioester pools, such as acyl-ACP, has been accomplished by selective formation of acyl-butylamides and subsequent analysis by gas chromatography/mass spectrometry. The acyl-butylamide derivative was synthesized by direct aminolysis with n-butylamine. The reaction was specific for thioester-linked acyl groups and 90% conversion was achieved with acyl-ACP and acyl-CoA in aqueous solution. Electron ionization mass spectra exhibited two intense diagnostic ions m/z 115 and 128 common to butylamides of saturated and monounsaturated fatty acids. Mixtures of butylamides with fatty acid moieties ranging between C4 and C20 were analyzed by selective ion monitoring gas chromatography/mass spectrometry set to 115 and 128 amu. The limit for the quantitative analysis of the long-chain 18:0- and 18:1-butylamides was 1.5 pmol and the detection limit was less than 0.5 pmol. The utility of this method was demonstrated by analysis of two model systems: standard acyl-CoA mixtures and in vivo levels of spinach leaf acyl-ACP. A purification protocol based on DE 52 anion exchange chromatography was necessary in order to separate spinach acyl-ACP and acyl-CoA from tissue extracts. The acyl composition obtained from total spinach leaf acyl-ACP by selective ion monitoring gas chromatography/mass spectrometry of the butylamide derivatives matched with the direct analysis of the same sample by urea-polyacrylamide gel electrophoresis and subsequent scanning densitometry of the anti-spinach ACP immunoblot.

Incubator noise and vibration--possible iatrogenic influence on neonate.

Analysis of noise and vibration from infant incubators (models Medicor and CN2) revealed average noise levels (peak values) of 60 dB (A-weighted) and 73.5 dB (linear) and values for vibration where the infant's head normally rests were 0.065 m/sec2 (A-weighted) and 0.14 m/sec2 (linear), and at the infants' feet were 0.105 m/sec 2 (A-weighted) and 0.58 m/sec2 (linear). On the basis of present knowledge of the physiologic effects of noise and vibration, we discuss the influence of long-term noise and vibration exposures on the neonate; they provide a threat to carbohydrate, aliphatic compound and protein-enzymatic metabolic transformations and produce changes in cardiovascular, pituitary--adrenal axis and auditory systems.