Shotgun Strategy Research Articles

The Postpharyngeal Gland: Specialized Organ for Lipid Nutrition in Leaf-Cutting Ants.

There are several hypotheses about the possible functions of the postpharyngeal gland (PPG) in ants. The proposed functions include roles as cephalic or gastric caeca and diverticulum of the digestive tract, mixing of hydrocarbons, nestmate recognition, feeding larvae, and the accumulation of lipids inside this gland, whose origin is contradictory. The current study aimed to investigate the functions of these glands by examining the protein expression profile of the PPGs of Atta sexdens rubropilosa (Hymenoptera, Formicidae). Mated females received lipid supplementation and their glands were extracted and analyzed using a proteomic approach. The protocol used combined two-dimensional electrophoresis and shotgun strategies, followed by mass spectrometry. We also detected lipid β-oxidation by immunofluorescent marking of acyl-CoA dehydrogenase. Supplying ants with lipids elicited responses in the glandular cells of the PPG; these included increased expression of proteins related to defense mechanisms and signal transduction and reorganization of the cytoskeleton due to cell expansion. In addition, some proteins in PPG were overexpressed, especially those involved in lipid and energy metabolism. Part of the lipids may be reduced, used for the synthesis of fatty alcohol, transported to the hemolymph, or may be used as substrate for the synthesis of acetyl-CoA, which is oxidized to form molecules that drive oxidative phosphorylation and produce energy for cellular metabolic processes. These findings suggest that this organ is specialized for lipid nutrition of adult leaf-cutting ants and characterized like a of diverticulum foregut, with the ability to absorb, store, metabolize, and mobilize lipids to the hemolymph. However, we do not rule out that the PPG may have other functions in other species of ants.

Draft Genome Sequence of Lactobacillus plantarum CRL1506, an Immunomodulatory Strain Isolated from Goat Milk.

This report describes a draft genome sequence of Lactobacillus plantarum CRL1506, a probiotic strain with immunomodulatory properties isolated from goat milk. The reads generated by a whole-genome shotgun (WGS) strategy on an Illumina MiSeq sequencer were assembled into contigs with a total size of 3,228,096 bp. The draft genome sequence of L. plantarum CRL1506 will be useful for further studies of specific genetic features of this strain and for understanding the mechanisms of its immunobiotic properties.

Using a proteometabolomic approach to investigate the role of Dufour's gland in pheromone biosynthesis in the social wasp Polybia paulista

Dufour's gland is associated with the venom apparatuses of social wasps and bees. This location and its evolutionary adaptations indicate that it could be involved in the production of alarm pheromones in the social wasp Polybia paulista. To investigate this hypothesis, the volatile composition of this gland was analyzed and compared to that in the venom. Eighteen compounds were identified as secreted by Dufour's gland, and 16 of these compounds were also identified in the venom, suggesting that the compounds produced by the gland are secreted and mixed with venom in the venom reservoir of this wasp. These compounds were subjected to a field bioassay to investigate their potential action as alarm pheromones. Alcohols and aldehydes elicited the alert behavior in workers, luring them outside the nest, whereas acids attracted the workers in the direction of the source; fatty acid methyl esters elicited aggression. These results suggest that Dufour's gland produces alarm pheromones. To corroborate this hypothesis the proteomic complement of this gland was assigned using a shot-gun strategy; 59 proteins were identified, and the results indicate specialization of Dufour's gland for the metabolism of fatty acids (elongation, esterification unsaturation, reduction, and decarboxylation) in the biosynthesis of alarm pheromones. Biological significanceThe present knowledge about the role of Dufour's gland among aculeate Hymenoptera insects suggests that it may have many different roles related to the biosynthesis and secretion of chemical markers for different biological functions, though none are related to the elicitation of alarm behaviors for coordinating a mass attack of the colony against intruders. The present study combined the analysis of secreted volatile compounds (as metabolites) with proteome assignments and a field bioassay with synthetic compounds to clearly demonstrate that Dufour's gland does in fact biosynthesize alarm pheromones in social wasps. This strategy may be reproduced in other investigations related to pheromone production in other insects.

Comparative Phosphoproteomics Reveals the Role of AmpC β-lactamase Phosphorylation in the Clinical Imipenem-resistant Strain Acinetobacter baumannii SK17

Nosocomial infectious outbreaks caused by multidrug-resistant Acinetobacter baumannii have emerged as a serious threat to human health. Phosphoproteomics of pathogenic bacteria has been used to identify the mechanisms of bacterial virulence and antimicrobial resistance. In this study, we used a shotgun strategy combined with high-accuracy mass spectrometry to analyze the phosphoproteomics of the imipenem-susceptible strain SK17-S and -resistant strain SK17-R. We identified 410 phosphosites on 248 unique phosphoproteins in SK17-S and 285 phosphosites on 211 unique phosphoproteins in SK17-R. The distributions of the Ser/Thr/Tyr/Asp/His phosphosites in SK17-S and SK17-R were 47.0%/27.6%/12.4%/8.0%/4.9% versus 41.4%/29.5%/17.5%/6.7%/4.9%, respectively. The Ser-90 phosphosite, located on the catalytic motif S(88)VS(90)K of the AmpC β-lactamase, was first identified in SK17-S. Based on site-directed mutagenesis, the nonphosphorylatable mutant S90A was found to be more resistant to imipenem, whereas the phosphorylation-simulated mutant S90D was sensitive to imipenem. Additionally, the S90A mutant protein exhibited higher β-lactamase activity and conferred greater bacterial protection against imipenem in SK17-S compared with the wild-type. In sum, our results revealed that in A. baumannii, Ser-90 phosphorylation of AmpC negatively regulates both β-lactamase activity and the ability to counteract the antibiotic effects of imipenem. These findings highlight the impact of phosphorylation-mediated regulation in antibiotic-resistant bacteria on future drug design and new therapies.

Lactobacillus wasatchensis sp. nov., a non-starter lactic acid bacteria isolated from aged Cheddar cheese.

A Gram-stain positive, rod-shaped, non-spore-forming strain (WDC04T), which may be associated with late gas production in cheese, was isolated from aged Cheddar cheese following incubation on MRS agar (pH 5.2) at 6 °C for 35 days. Strain WDC04T had 97 % 16S rRNA gene sequence similarity with Lactobacillus hokkaidonensis DSM 26202T, Lactobacillus oligofermentans 533, 'Lactobacillus danicus' 9M3, Lactobacillus suebicus CCUG 32233T and Lactobacillus vaccinostercus DSM 20634T. API 50 CH carbohydrate fermentation panels indicated strain WDC04T could only utilize one of the 50 substrates tested, ribose, although it does slowly utilize galactose. In the API ZYM system, strain WDC04T was positive for leucine arylamidase, valine arylamidase, cysteine arylamidase (weakly), naphthol-AS-BI-phosphohydrolase and β-galactosidase activities. Total genomic DNA was sequenced from strain WDC04T using a whole-genome shotgun strategy on a 454 GS Titanium pyrosequencer. The sequence was assembled into a 1.90 Mbp draft genome consisting of 105 contigs with preliminary genome annotation performed using the RAST algorithm (rast.nmpdr.org). Genome analysis confirmed the pentose phosphate pathway for ribose metabolism as well as galactose, N-acetylglucosamine, and glycerol fermentation pathways. Genomic analysis places strain WDC04T in the obligately heterofermentative group of lactobacilli and metabolic results confirm this conclusion. The result of genome sequencing, along with 16S rRNA gene sequence analysis, indicates WDC04T represents a novel species of the genus Lactobacillus, for which the name Lactobacillus wasatchensis sp. nov. is proposed. The type strain is WDC04T ( = DSM 29958T = LMG 28678T).

Genome sequencing and analysis of a granulovirus isolated from the Asiatic rice leafroller, Cnaphalocrocis medinalis.

The complete genome of Cnaphalocrocis medinalis granulovirus (CnmeGV) from a serious migratory rice pest, Cnaphalocrocis medinalis (Lepidoptera: Pyralidae), was sequenced using the Roche 454 Genome Sequencer FLX system (GS FLX) with shotgun strategy and assembled by Roche GS De Novo assembler software. Its circular double-stranded genome is 111,246 bp in size with a high A+T content of 64.8% and codes for 118 putative open reading frames (ORFs). It contains 37 conserved baculovirus core ORFs, 13 unique ORFs, 26 ORFs that were found in all Lepidoptera baculoviruses and 42 common ORFs. The analysis of nucleotide sequence repeats revealed that the CnmeGV genome differs from the rest of sequenced GVs by a 23 kb and a 17kb gene block inversions, and does not contain any typical homologous region (hr) except for a region of non-hr-like sequence. Chitinase and cathepsin genes, which are reported to have major roles in the liquefaction of the hosts, were not found in the CnmeGV genome, which explains why CnmeGV infected insects do not show the phenotype of typical liquefaction. Phylogenetic analysis, based on the 37 core baculovirus genes, indicates that CnmeGV is closely related to Adoxophyes orana granulovirus. The genome analysis would contribute to the functional research of CnmeGV, and would benefit to the utilization of CnmeGV as pest control reagent for rice production.

Two proving strategies of highly successful mathematics majors

We examined the proof-writing behaviors of six highly successful mathematics majors on novel proving tasks in calculus. We found two approaches that these students used to write proofs, which we termed the targeted strategy and the shotgun strategy. When using a targeted strategy students would develop a strong understanding of the statement they were proving, choose a plan based on this understanding, develop a graphical argument for why the statement is true, and formalize this graphical argument into a proof. When using a shotgun strategy, students would begin trying different proof plans immediately after reading the statement and would abandon a plan at the first sign of difficulty. The identification of these two strategies adds to the literature on proving by informing how elements of existing problem-solving models interrelate.

De novo assembly of a haplotype-resolved human genome.

The human genome is diploid, and knowledge of the variants on each chromosome is important for the interpretation of genomic information. Here we report the assembly of a haplotype-resolved diploid genome without using a reference genome. Our pipeline relies on fosmid pooling together with whole-genome shotgun strategies, based solely on next-generation sequencing and hierarchical assembly methods. We applied our sequencing method to the genome of an Asian individual and generated a 5.15-Gb assembled genome with a haplotype N50 of 484 kb. Our analysis identified previously undetected indels and 7.49 Mb of novel coding sequences that could not be aligned to the human reference genome, which include at least six predicted genes. This haplotype-resolved genome represents the most complete de novo human genome assembly to date. Application of our approach to identify individual haplotype differences should aid in translating genotypes to phenotypes for the development of personalized medicine.

Valuing museum specimens: high-throughput DNA sequencing on historical collections of New Guinea crowned pigeons (Goura)

Museum specimens are of particular importance for investigating systematics and the biogeography as well as other aspects of the evolution of biodiversity. They are also a depository of specimens accumulated over recent historical times and often the only way to study recently extinct or rare species. Unfortunately, most museum specimens yield low-quality DNA limiting their generalized use in phylogenetic and population genetic studies. Advances in sequencing technologies now offer opportunities to analyse such material even from very small tissue. Here, we applied the Illumina technology (HiSeq) on three specimens of crowned pigeon (Goura spp.) collected in New Guinea between 1879 and 1934. A shotgun strategy allowed us to quickly and reliably assemble complete mitochondrial genomes and fragments of three single-copy gene phylogenetic markers even with low double-stranded DNA quantity (9 to 69 ng). Phylogenetic assignments of these new sequences confirmed them as Gourasequences. Therefore, our study confirms the power of using next-generation sequencing methods to investigate the evolutionary history of species for which access to fresh samples is limited but museum collections are available. These approaches are considerably increasing the value of many natural history collections for genetic investigations, particularly from biodiversity hotspots that are presently difficult to prospect.

Choice of next-generation sequencing pipelines.

The next-generation sequencing (NGS) technologies are revolutionary tools which have made possible achieving remarkable advances in genetics since the beginning of the twenty-first century. Thanks to the possibility to produce large amount of sequence data, these tools are going to completely substitute other high-throughput technologies. Moreover, the large applications of NGS protocols are increasing the genetic decoding of biological systems through studies of genome anatomy and gene mapping, coupled to the transcriptome pictures. The application of NGS pipelines such as (1) de-novo genomic sequencing by mate-paired and whole-genome shotgun strategies; (2) specific gene sequencing on large bacterial communities; and (3) RNA-seq methods including whole transcriptome sequencing and Serial Analysis of Gene Expression (Sage-analysis) are fundamental in the genome-wide fields like metagenomics. Recently, the availability of these advanced protocols has allowed to overcome the usual sequencing technical issues related to the mapping specificity over standard shotgun library sequencing, the detection of large structural genomes variations and bridging sequencing gaps, as well as more precise gene annotation. In this chapter we will discuss how to manage a successful NGS pipeline from the planning of sequencing projects through the choice of the platforms up to the data analysis management.

Identification of protein network alterations upon retinal ischemia-reperfusion injury by quantitative proteomics using a Rattus norvegicus model.

Retinal ischemia is a common feature associated with several ocular diseases, including diabetic retinopathy. In this study, we investigated the effect of a retinal ischemia and reperfusion (I/R) injury on protein levels via a quantitative shotgun strategy using stable isotope dimethyl labeling combined with LC-MS/MS analysis. Based on the relative quantitation data of 1088 proteins, 234 proteins showed a greater than 1.5-fold change following I/R injury, 194 of which were up-regulated and 40 were down-regulated. Gene ontology analysis revealed that after I/R injury, there was an increase in the metabolic-process related proteins but a decline in cell communication, system process and transport-related proteins. A ribosome protein network and a secreted protein network consisting of many protease inhibitors were identified among the up-regulated proteins, despite a suppression of the mammalian target of rapamycin (mTOR) pathway following the I/R injury. A synaptic-related protein network was found to be significantly down-regulated, implicating a functional reduction of neurons following a retinal I/R injury. Our results provide new systems-biology clues for the study of retinal ischemia.

Comparative genomic data of the Avian Phylogenomics Project.

BackgroundThe evolutionary relationships of modern birds are among the most challenging to understand in systematic biology and have been debated for centuries. To address this challenge, we assembled or collected the genomes of 48 avian species spanning most orders of birds, including all Neognathae and two of the five Palaeognathae orders, and used the genomes to construct a genome-scale avian phylogenetic tree and perform comparative genomics analyses (Jarvis et al. in press; Zhang et al. in press). Here we release assemblies and datasets associated with the comparative genome analyses, which include 38 newly sequenced avian genomes plus previously released or simultaneously released genomes of Chicken, Zebra finch, Turkey, Pigeon, Peregrine falcon, Duck, Budgerigar, Adelie penguin, Emperor penguin and the Medium Ground Finch. We hope that this resource will serve future efforts in phylogenomics and comparative genomics.FindingsThe 38 bird genomes were sequenced using the Illumina HiSeq 2000 platform and assembled using a whole genome shotgun strategy. The 48 genomes were categorized into two groups according to the N50 scaffold size of the assemblies: a high depth group comprising 23 species sequenced at high coverage (>50X) with multiple insert size libraries resulting in N50 scaffold sizes greater than 1 Mb (except the White-throated Tinamou and Bald Eagle); and a low depth group comprising 25 species sequenced at a low coverage (~30X) with two insert size libraries resulting in an average N50 scaffold size of about 50 kb. Repetitive elements comprised 4%-22% of the bird genomes. The assembled scaffolds allowed the homology-based annotation of 13,000 ~ 17000 protein coding genes in each avian genome relative to chicken, zebra finch and human, as well as comparative and sequence conservation analyses.ConclusionsHere we release full genome assemblies of 38 newly sequenced avian species, link genome assembly downloads for the 7 of the remaining 10 species, and provide a guideline of genomic data that has been generated and used in our Avian Phylogenomics Project. To the best of our knowledge, the Avian Phylogenomics Project is the biggest vertebrate comparative genomics project to date. The genomic data presented here is expected to accelerate further analyses in many fields, including phylogenetics, comparative genomics, evolution, neurobiology, development biology, and other related areas.Electronic supplementary materialThe online version of this article (doi:10.1186/2047-217X-3-26) contains supplementary material, which is available to authorized users.

Patterns of genomic variation in the poplar rust fungus Melampsora larici-populina identify pathogenesis-related factors.

Melampsora larici-populina is a fungal pathogen responsible for foliar rust disease on poplar trees, which causes damage to forest plantations worldwide, particularly in Northern Europe. The reference genome of the isolate 98AG31 was previously sequenced using a whole genome shotgun strategy, revealing a large genome of 101 megabases containing 16,399 predicted genes, which included secreted protein genes representing poplar rust candidate effectors. In the present study, the genomes of 15 isolates collected over the past 20 years throughout the French territory, representing distinct virulence profiles, were characterized by massively parallel sequencing to assess genetic variation in the poplar rust fungus. Comparison to the reference genome revealed striking structural variations. Analysis of coverage and sequencing depth identified large missing regions between isolates related to the mating type loci. More than 611,824 single-nucleotide polymorphism (SNP) positions were uncovered overall, indicating a remarkable level of polymorphism. Based on the accumulation of non-synonymous substitutions in coding sequences and the relative frequencies of synonymous and non-synonymous polymorphisms (i.e., PN/PS), we identify candidate genes that may be involved in fungal pathogenesis. Correlation between non-synonymous SNPs in genes encoding secreted proteins (SPs) and pathotypes of the studied isolates revealed candidate genes potentially related to virulences 1, 6, and 8 of the poplar rust fungus.

Shotgun proteome analysis of honeybee venom using targeted enrichment strategies

The aim of this study was to explore the honeybee venom proteome applying a shotgun proteomics approach using different enrichment strategies (combinatorial peptide ligand libraries and solid phase extraction). The studies were conducted using nano-LC/MALDI-TOF/TOF-MS system. The MS analysis of peptide profiles (in the range of 900–4500 Da) and virtual gel-image of proteins from Lab-on-Chip assay (in the range of 10–250 kDa) confirm that use of targeted enrichment strategies increase detection of honeybee venom components. The gel-free shotgun strategy and sophisticated instrumentation led to a significant increase of the sensitivity and higher number of identified peptides in honeybee venom samples, comparing with the current literature. Moreover, 11 of 12 known honeybee venom allergens were acknowledged and 4 new, so far uncharacterized proteins were identified. In addition, similarity searches were performed in order to investigate biological relations and homology between newly identified proteins sequences from Apis mellifera and other Hymenoptera.

The common marmoset genome provides insight into primate biology and evolution.

A first analysis of the genome sequence of the common marmoset (Callithrix jacchus), assembled using traditional Sanger methods and Ensembl annotation, has permitted genomic comparison with apes and that old world monkeys and the identification of specific molecular features a rapid reproductive capacity partly due to may contribute to the unique biology of diminutive The common marmoset has prevalence of this dizygotic primate. twins. Remarkably, these twins share placental circulation and exchange hematopoietic stem cells in utero, resulting in adults that are hematopoietic chimeras.We observed positive selection or non-synonymous substitutions for genes encoding growth hormone / insulin-like growth factor (growth pathways), respiratory complex I (metabolic pathways), immunobiology, and proteases (reproductive and immunity pathways). In addition, both protein-coding and microRNA genes related to reproduction exhibit rapid sequence evolution. This New World monkey genome sequence enables significantly increased power for comparative analyses among available primate genomes and facilitates biomedical research application.

Low Density Lipoprotein Receptor Class A Repeats Are O-Glycosylated in Linker Regions

The low density lipoprotein receptor (LDLR) is crucial for cholesterol homeostasis and deficiency in LDLR functions cause hypercholesterolemia. LDLR is a type I transmembrane protein that requires O-glycosylation for stable expression at the cell surface. It has previously been suggested that LDLR O-glycosylation is found N-terminal to the juxtamembrane region. Recently we identified O-glycosylation sites in the linker regions between the characteristic LDLR class A repeats in several LDLR-related receptors using the "SimpleCell" O-glycoproteome shotgun strategy. Herein, we have systematically characterized O-glycosylation sites on recombinant LDLR shed from HEK293 SimpleCells and CHO wild-type cells. We find that the short linker regions between LDLR class A repeats contain an evolutionarily conserved O-glycosylation site at position -1 of the first cysteine residue of most repeats, which in wild-type CHO cells is glycosylated with the typical sialylated core 1 structure. The glycosites in linker regions of LDLR class A repeats are conserved in LDLR from man to Xenopus and found in other homologous receptors. O-Glycosylation is controlled by a large family of polypeptide GalNAc transferases. Probing into which isoform(s) contributed to glycosylation of the linker regions of the LDLR class A repeats by in vitro enzyme assays suggested a major role of GalNAc-T11. This was supported by expression of LDLR in HEK293 cells, where knock-out of the GalNAc-T11 isoform resulted in the loss of glycosylation of three of four linker regions.

Comparison at the peptide level with post‐translational modification consideration reveals more differences between two unenriched samples (778.1)

In shotgun strategy, peptide sequences are first identified from MS/MS spectra, and the existence and abundance of the proteins are then inferred from the peptide information. However, the protein inference step can produce errors and a loss of information. To identify the information that is lost using the traditional approaches, this study compared the proteomic data of two leukemia cell lines (Jurkat and K562) at peptide level with consideration of post‐translational modifications (PTMs). After database search, the observed modification name was matched with the modification classification on the Unimod website. Only the peptides with ‘post‐translational’ modifications were compared between the two cell lines. As a result, a total of 44887 non‐redundant peptides were identified in both the Jurkat and K562 cell lines. Of these peptides, even without specific PTM enrichment, 11.43% of them (with at least two spectra in one cell line) existed in different PTM forms between the two cell lines, and 1.89% of the peptides were modified in both cell lines, but with different modifications or on different sites. The sensitivity of PTM identification will become greater and more differences will be found if PTMs are enriched or if the peptides can be compared in a quantitative manner. Therefore, comparing proteomic data at the peptide level with consideration of PTMs can reveal differences between two unenriched samples.Grant Funding Source: Supported by National Basic Research Program of China (2012CB517606, 2013CB530805, 2011CB964901).

Seasonally variable intestinal metagenomes of the red palm weevil (Rhynchophorus ferrugineus)

The intestinal microbes residing in the red palm weevil (RPW, Rhynchophorus ferrugineus) larva consume tender interior fibrous tissues of date palm trunks. The understanding of such microbiota at molecular level provides vital clues for the biological control of this devastating pest. Using pyrosequencing and shotgun strategy, we first study taxonomic profiles of the microbiota sampled at different months (March, July and November), and then confirm the impact of high-temperature stress on the microbial populations based on data from 16S rRNA amplicons using both field and laboratory samples. We further identify Klebsiella pneumoniae in November and Lactococcus lactis in July as the dominant species of the microbiota. We find that the RPW gut microbiota degrades polysaccharides and sucrose with hydrolases and that different active bacterial species in November and July are responsible for the symbiotic relationship between the microbiota and the host. Our results provide vital information for pest control and cellulolytic bacterial species characterization.

Proteomic analysis of pleural effusion from lung adenocarcinoma patients by shotgun strategy

To construct a protein catalogue of malignant pleural effusion from lung adenocarcinoma patients and to screen the potential candidates of biomarkers for diagnostic value in human lung adenocarcinoma. Five malignant pleural effusion samples of lung adenocarcinoma patients were collected from January 2009 to September. A composite sample was analyzed using shotgun strategy. Pleural effusion samples were separated by means of SDS-PAGE. Proteomic analysis was performed by 1D-LC-MS/MS, and then the proteins were identified using SEQUEST software and protein database search. Among 230 unique proteins, 123 proteins were identified with higher confidence levels (at least two unique peptide sequences matched). Most of these proteins have been reported in plasma. However, there are 7 proteins, including JUP protein, suprabasin, annexin A2, transforming growth factor-beta-induced protein ig-h3 (βig-h3), V-set and immunoglobulin domain-containing protein 4 precursor, ifapsoriasin 2 and actin, cytoplasmic 1 have not been reported in serum. Seven proteins may represent potential candidates of biomarkers. Annexin A2 is of special interest since it may play a role in the regulation of intercellular adhesion and cell proliferation.

GW24-e0404 Proteomics screen to reveal molecular changes mediated by C722G missense mutation in CHRM2 gene

ObjectivesPreviously, we reported that a missense mutation (C722G) in the M2-muscarinic acetylcholine receptor (CHRM2) geneis associated with familial dilated cardiomyopathy (DCM). However, the exact molecular mechanisms by the fundamental changes...