Integration of lipidomics and metabolomics for in-depth understanding of cellular mechanism and disease progression

Mass spectrometry(MS)-based omics technologies are now widely used to profile small molecules in multiple matrices to confer comprehensive snapshots of cellular metabolic phenotypes.The metabolomes of cells,tissues,and organisms comprise a variety of molecules including lipids,amino acids,sugars,organic acids,and so on.Metabolomics mainly focus on the hydrophilic classes,while lipidomics has emerged as an independent omics owing to the complexities of the organismal lipidomes.The potential roles of lipids and small metabolites in disease pathogenesis have been widely investigated in various human diseases,but system-level understanding is largely lacking,which could be partly attributed to the insufficiency in terms of metabolite coverage and quantitation accuracy in current analytical technologies.While scientists are continuously striving to develop high-coverage omics approaches,integration of metabolomics and lipidomics is becoming an emerging approach to mechanistic investigation.Integration of metabolome and lipidome offers a complete atlas of the metabolic landscape,enabling comprehensive network analysis to identify critical metabolic drivers in disease pathology,facilitating the study of interconnection between lipids and other metabolites in disease progression.In this review,we summarize omics-based findings on the roles of lipids and metabolites in the pathogenesis of selected major diseases threatening public health.We also discuss the advantages of integrating lipidomics and metabolomics for in-depth understanding of molecular mechanism in disease pathogenesis.     

Investigation of Combining Plant Genotypic Values and Molecular Marker Information for Constructing Core Subsets

In the present study, a strategy was proposed for constructing plant core subsets by clusters based on the combination of continuous data for genotypic values and discrete data for molecular marker InformaUon. A mixed linear model approach was used to predict genotyplc values for eliminating the environment effect. The ＂mixed genetic distance＂ was designed to solve the difficult problem of combining continuous and discrete data to construct a core subset by cluster. Four commonly used genetic distances for continuous data （Euclidean distance, standardized Euclidean distance, city block distance, and Mahalanobls distance） were used to assess the validity of the conUnuous data part of the mixed genetic distance; three commonly used genetic distances for discrete data （cosine distance, correlaUon distance, and Jaccard distance） were used to assess the validity of the discrete data part of the mixed genetic distance, A rice germplasm group with eight quantitative traits and information for 60 molecular markers was used to evaluate the validity of the new strategy. The results suggest that the validity of both parts of the mixed geneUc distance are equal to or higher than the common geneUc distance. The core subset constructed on the basis of a combination of data for genotyplc values and molecular marker information was more representative than that constructed on the basis of data from genotypic values or molecular marker informaUon alone. Moreover, the strategy of using combined data was able to treat dominant marker informaUon and could combine any other continuous data and discrete data together to perform cluster to construct a plant core subset.     

Ray Wu, Cornell’s acclaimed pioneer of genetic engineering and developer of insect-resistant rice

ITHACA, N.Y. -- Ray J. Wu, Cornell University professor of molecular biology and genetics, who was widely recog-nized as one of the fathers of genetic engineering and who developed and sought to feed the world with a higher yield-ing rice that resists insects and drought, died of cardiac arrest in Ithaca, Feb. 10.     

Characterization and Use of Male Sterility in Hybrid Rice Breeding

The hybrid rice （Oryza sativa L.） breeding that was Initiated In China in the 1970s led to a great improvement in rice productivity. In general, It increases the grain yield by over 20% to the inbred rice varieties, and now hybrid rice has been widely introduced into Africa, Southern Asia and America. These hybrid varieties are generated through either three-line hybrid and two-line hybrid systems; the former is derived from cytoplasmic male sterility （CMS） and the latter derived from genlc male sterility （GMS）. There are three major types of CMS （HL, BT and WA） and two types of GMS （photoperlod-sensitlve （PGMS） and temperature-sensitive （TGMS））. The BT- and HL-type CMS genes are characterized as orf79 and orfH79, which are chimeric toxic genes derived from mltochondrial rearrangement. Rf3 for CMS-WA Is located on chromosome 1, while Rf1, Rf4, Rf5 and Rf6 correspond to CMS-BT, CMSoWA and CMS- HL, located on chromosome 10. The Rfl gene for BT-CMS has been cloned recently, and encodes a mltochondriatargeted PPR protein. PGMS Is thought to be controlled by two recessive loci on chromosomes 7 and 12, whereas nine recessive alleles have been identified for TGMS and mapped on different chromosomes. Attention Is still urgently needed to resolve the molecular complexity of male sterility to assist rice breeding.     

A High-throughput Genomic Tool： Diversity Array Technology Complementary for Rice Genotyping

Diversity array technology （DART^TM） was a genotyping tool characterized gel-independent and high throughput. The main purpose of present study is to validate DArT for rice （Oryza sativa L.）genotyping in a high throughput manner. Technically, the main objective was to generate a rice general purpose gene pool, and optimize this genomic tool in order to evaluate rice germplasm genetic diversity. To achieve this, firstly, a generalpurpose DArT array was developed. Ten representatives from 24 varieties were hybridized with the general-purpose array to determine the informativeness of the clones printed on the array. The informative 1 152 clones were re-arrayed on a slide and used to fingerprint 17 of 24 germplasms. Hybridizing targets prepared from the germplasm to be assayed to the DNA array gave DNA fingerprints of germplasms. Raw data were normalized and transformed into binary data, which were then analyzed by using NTSYSpc （Numerical taxonomy system for cluster and ordination analysis, v. 2.02j） software package. The graphically displayed dendrogram derived from the array experimental data was matched with simple sequence repeats genotyping outline and varieties＇ pedigree deviation of the different varieties. Considering DArT is a sequence-independent genotyping approach, it will be applied in studies of the genetic diversity and the gene mapping of diverse of organisms, especially for those crops with less-developed molecular markers.     

Identification of optimal hyperspectral bands for estimation of rice biophysical parameters

The present study aims to identify the narrow spectral bands that are most suitable for characterizing rice biophysical parameters. The data used for this study come from ground-level hyperspectral reflectance measurements for five rice species at three levels of nitrogen fertilization during the growing period. Reflectance was measured in discrete narrow bands between 350 and 2 500 nm. Observed rice biophysical parameters included leaf area index （LAI）, wet biomass and dry biomass. The stepwise regression method was applied to identify the optimal bands for rice biophysical parameter estimation. This research indicated that combinations of four narrow bands in stepwise regression models explained 69% to 83% variability for LAI, 56% to 73% for aboveground wet biomass and 70% to 83% for leaf wet biomass. An overwhelming proportion of rice information was in a particular portion of near infrared （NIR） （1 100-1 150 nm）, red-edge （700-750 nm）, and a longer portion of green （550-600 nm）. These were followed by the moisture-sensitive NIR （950-1 000 nm）, the intermediate portion of shortwave infrared （SWlR） （1 650-1 700 nm）, and another portion of NIR （1 000-1 050 nm）.     

Cloning of genomic DNA of rice 5-enolpyruvylshikimate 3-phosphate synthase gene and chromosomal localization of the gene

The shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPs) is the target of nonselective herbicide glyphosate. A partial rice epsps cDNA was generated by RT-PCR with primers designed according to EST sequence in GenBank and used as probe for rice genomic library screening. In a screen of approximately 8.0×104 clones from the rice genomic library, sixteen positive clones were obtained, which strongly hybridized to the probe. One clone, E11, was selected for further analysis and the full-length 3661 bp rice epsps genomic sequence was obtained. Sequence analysis and homologous comparison revealed that epsps gene is composed of 8 exons and 7 introns. Analysis by restriction fragment length polymorphism with the probe of rice epsps cDNA fragment confirmed that rice epsps is located on chromosome 6 with an indica-japonica (ZYQ8-JX17) double-haploid (DH) population. This is the first report on the EPSP synthase from monocotyledons.     

A Lipidomic Study of the Effects of N-methyl-N＇-nitro-N-nitrosoguanidine on Sphingomyelin Metabolism

Systems biology is a new and rapidly developing research area in which, by quantitatively describing the interaction among all the individual components of a cell, a systems-level understanding of a biological response can be achieved. Therefore, it requires high-throughput measurement technologies for biological molecules, such as genomic and proteomic approaches for DNA/RNA and protein, respectively.Recently, a new concept, lipidomics, which utilizes the mass spectrometry （MS） method for lipid analysis,has been proposed. Using this lipidomic approach, the effects of N-methyl-N＇-nitro-N-nitrosoguanidine （MNNG） on sphingomyelin metabolism, a major class of sphingolipids, were evaluated. Sphingomyelin molecules were extracted from cells and analyzed by matrix-assisted laser desorption ionization-time of flight MS. It was found that MNNG induced profound changes in sphingomyelin metabolism, including the appearance of some new sphingomyelin species and the disappearance of some others, and the concentrations of several sphingomyelin species also changed. This was accompanied by the redistribution of acid sphingomyelinase （ASM）, a key player in sphingomyelin metabolism. On the other hand, imipramine, an inhibitor of ASM,caused the accumulation of sphingomyelin. It also prevented some of the effects of MNNG, as well as the redistribution of ASM. Taken together, these data suggested that the lipidomic approach is highly effective for the systematic analysis of cellular lipids metabolism.     

Comparative Analysis of the Endosperm Proteins Separated by 2-D Electrophoresis for Two Cultivars of Hybrid Rice （Oryza sativa L,）

Liangyoupeijiu is a two-parental-line, and Shanyou63 is a three-parental-line hybrid rice （Oryza sativa L.）. Although both belong to the indica subspecies, they have obvious differences with respect to morphology, physiology and grain quality. Variations in endosperm protein compositions were studied by comparing the 2-D electrophoresis （2-DE） maps for these two cultivars of hybrid rice. After matrix-assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF/MS） analysis, a 21-kDa precursor of 19- kDa globulin was identified as the major storage protein for both cultivars. Some isoforms of peroxiredoxin and seed maturation protein were found to only exist in Shanyou63, whereas aldose reductase and starch granule-bound starch synthase were only detected in Liangyoupeijiu. These data might provide a foundation for further comparative studies of these two cultivars of hybrid rice.     

Construction and characterization of a bacterial artificial chromosome library of thermo-sensitive genie male-sterile rice 5460S

In order to develop a detailed physical map of the thermo-sensitive genie male-sterile (TGMS) gene-encompassing region and finally clone the TGMS gene, a high-quality rice bacterial artificial chromosome (BAC) library from TGMS rice 5460S was constructed. The method of constructing BAC library was examined and optimized. The 5460S library consists of 19 584 BAC clones with an average insert size of 110 kb, which represents about 5 times rice haploid genome equivalents. Rice inserts of up to 140 kb and 250 kb were isolated and appeared stable after 100 generations of serial growth. Hybridization of BAC clones with mitochondrial and chloroplastic genes as probes demonstrated that this library has no organellar contamination. The 5460S library was screened with 3 molecular markers linked to tmsl gene as probes and at least 1 BAC clone was identified with each probe. The insert ends of positive clones were successfully isolated using thermal asymmetric interlaced PCR (TAIL-PCR) technique.     

Construction of a DNA library from chromosome 4 of rice （Oryza sativa） by microdissection

A simple method to create a chromosome-specific DNA librqary of rice,including microdissection,amplification,charterization and cloning,is described.Rice chromosome 4 from a metaphase cell has been isolated and amplified by the Linker Adapter PCR (LA-PCR).The PCR products were labeled as probes with DIG-11-dUTP using the random priming method.Southern blot analysis with rice genomic DNA and specific RFLP markers demonstrated that the PCR products were derived from rice chromosome 4.A large library comprising over 100,000 recombinant plasmid microclones from rice chromosome 4 was constructed.Colony hybridization showed that 58% of the clones contained single or low-copy sequences and 42% contained repetitive sequences.The size of inserts generated by PCR ranged from 140bp to 500bp.This method will facilitate cloning of the specific chromosome DNA markers and important genes of rice.     

Plant Cell Wall Proteomics： Mass Spectrometry Data,a Trove for Research on Protein Structure/Function Relationships

Proteomics allows the large-scale study of protein expression either in whole organisms or in purified organelles. In particular, mass spectrometry （MS） analysis of gel-separated proteins produces data not only for protein identification, but for protein structure, location, and processing as well. An in-depth analysis was performed on MS data from etiolated hypocotyl cell wall proteomics ofArabidopsis thaliana. These analyses show that highly homologous members of multigene families can be differentiated. Two lectins presenting 93% amino acid identity were identified using peptide mass fingerprinting. Although the identification of structural proteins such as extensins or hydroxyproline/proline-rich proteins （H/PRPs） is arduous, different types of MS spectra were exploited to identify and characterize an H/PRP. Maturation events in a couple of cell wall proteins （CWPs） were analyzed using site mapping. N-glycosylation of CWPs as well as the hydroxylation or oxidation of amino acids were also explored, adding information to improve our understanding of CWP structure/function relationships. A bioinformatic tool was developed to locate by means of MS the N-terminus of mature secreted proteins and N-glycosylation.     

Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene （cbh1） promoter optimization

To improve heterologous gene expression in Trichoderma reesei, a set of optimal artificial cellobiohydrolase I gene （cbhl） promoters was obtained. The region from -677 to -724 with three potential glucose repressor binding sites was deleted. Then the region from -620 to -820 of the modified cbhl promoter, including the CCAAT box and the Ace2 binding site, was repeatedly inserted into the modified cbhl promoter, obtaining promoters with copy numbers 2, 4, and 6. The results showed that the glucose repression effects were abolished and the expression level of the glucuronidase （gus） reporter gene regulated by these multi-copy promoters was markedly enhanced as the copy number increased simultaneously. The data showed the great promise of using the promoter artificial modification strategy to increase heterologous gene expression in filamentous fungi and provided a set of optional high-expression vectors for gene function investigation and strain modification.     

Genetic mapping of a new semi-dwarf gene, sd-t(t), in indica rice and estimating of the physical distance of the mapping region

Application and functional study of dwarf and semi-dwarf genes are of great importance to both crop breeding and molecular biology. A new semi-dwarf gene, sd-t(t), non-allelic to sd-1, had been identified in an indica rice variety, Aitaiyin 2. In this study the gene was genetically mapped by using an F2 population, which consisted of 474 individuals developed from a cross between Aitaiyin 2 and B30. The sd-t(t) gene was located between the RFLP markers R514 and R1408B with a distance of 1.1 cM to R514, and 4.5 cM to R1408B on chromosome 4. A physical contig covering the sd-t(t) mapping region was further constructed by screening a BAG library with R514 and R1408B as probes, and the physical distance between R514 and R1408B was estimated at approximately 147 kb. This result will facilitate map-based cloning of the sd-t(t) gene.     

Expression and localization of GhH6L,a putative classical arabinogalactan protein in cotton （Gossypium hirsutum）

Arabinogalactan proteins （AGPs） are a large family of highly glycosylated of hydroxyproline-rich glycoproteins that play important roles in plant growth, development, and signal transduction. A cDNA encoding a putative classical AGP named GhH6L was isolated from cotton fiber cDNA libraries, and the deduced protein contains 17 copies of repetitive motif of X-Y-proline-proline-proline （where X is serine or alanine and Y is threonine or serine）. Northern blotting analysis and quantitative RT-PCR results showed that it was preferentially expressed in 10 days post-anthesis （dpa） fibers and was also developmentally regulated. A promoter fragment was isolated from cotton （Gossypium hirsutum） by genome walking PCR. Expression of β-glucuronidase （GUS） gene under the GhH6L promoter was examined in the transgenic Arabidopsis plants; only petiole and pedicel were stained, no staining was detected in other tissues. Subcellular localization indicated that GhH6L was localized to the plasma membrane and in the cytoplasm. These data further our understanding of GhH6L as well as shed light on functional insight to GhH6L in cotton.     

Development and Characterization of 15 Polymorphic Micro satellite Markers for a Highly Adaptive and Wide-range Frog （Microhyla fissipes）

In order to analyze population genetic structure at multiple spatial scales, microsatellite loci were developed for the ornamented pygmy flog （Microhylafissipes）, and 15 polymorphic microsatellite loci were successfully screened from 105 individuals, of which 82 from four populations distributed in the Sichuan Basin and 23 from the Sangzhi population in western Hunan. Five loci were found to deviate significantly from Hardy-Weinberg equilibrium in one to three popu lations, probably due to small sample size or null alleles. The average number of alleles in all loci was 8.5, ranging from 4 to 13, and the observed and expected heterozygosity ranged from 0.26 to 0.90 and 0.63 to 0.90, respectively. The Sangzhi population and the remaining four populations can be clearly separated using Bayesian clustering methods, showing that the genetic structure of M. fissipes was probably affected by the topography, especially mountain barriers. These polymorphic microsatellite loci could be used for further study on the landscape genetics of this highly adaptive and widely distributed species.     

Metabolic responses of Eucalyptus species to different temperature regimes

Species and hybrids of Eucalyptus are the world's most widely planted hardwood trees. They are cultivated across a wide range of latitudes and therefore environmental conditions. In this context, comprehensive metabolomics approaches have been used to assess how different temperature regimes may affect the metabolism of three species of Eucalyptus, E. dunnii, E. grandis and E. pellita.Young plants were grown for 53 d in the greenhouse and then transferred to growth chambers at 10°C, 20°C or30°C for another 7 d. In all three species the leaf chlorophyll content was positively correlated to temperature, and in E.pellita the highest temperature also resulted in a significant increase in stem biomass. Comprehensive metabolomics was performed using untargeted gas chromatography mass spectrometry(GC-MS) and liquid chromatography(LC)-MS.This approach enabled the comparison of the relativeabundance of 88 polar primary metabolites from GC-MS and625 semi-polar secondary metabolites from LC-MS. Using principal components analysis, a major effect of temperature was observed in each species which was larger than that resulting from the genetic background. Compounds mostly affected by temperature treatment were subsequently selected using partial least squares discriminant analysis and were further identified. These putative annotations indicated that soluble sugars and several polyphenols, including tannins, triterpenes and alkaloids were mostly influenced.     

Expression and functional analysis of the rice plasma-membrane intrinsic protein gene family

Plasma membrane intrinsic proteins （PIPs） are a subfamily ofaquaporins that enable fast and controlled translocation of water across the membrane. In this study, we systematically identified and cloned ten PIP genes from rice. Based on the similarity of the amino acid sequences they encoded, these rice PIP genes were classified into two groups and designated as OsPIP1-1 to OsPIP1-3 and OsPIP2-1 to OsPIP2-7 following the nomenclature of PIP genes in maize. Quantitative RT-PCR analysis identified three root-specific and one leaf-specific OsPIP genes. Furthermore, the expression profile of each OsPIP gene in response to salt, drought and ABA treatment was examined in detail. Analysis on transgenic plants over-expressing of either OsPIP1 （OsPIP1-1） or OsPIP2 （OsPIP2-2） in wild-type Arabidopsis, showed enhanced tolerance to salt （100 mM of NaCl） and drought （200 mM ofmannitol）, but not to salt treatment of higher concentration （150 mM of NaCl）. Taken together, these data suggest a distinct role of each OsPIP gene in response to different stresses, and should add a new layer to the understanding of the physiological function of rice PIP genes.