Shotgun Approach Research Articles

Metagenomic Approach to Bacterial Diversity and Lipolytic Enzymes’ Genes from a Steam Soil of Los Humeros Geothermal Field (Puebla, México)

Los Humeros volcanic complex, located at the eastern end of the Trans Mexican Volcanic Belt, possess superficial thermal manifestations (fumaroles, heat steam-soils and alteration zones), with temperatures between 50 °C and 90 °C. This is the first work to explore the bacterial diversity of a steam soil of Los Humeros geothermal field using a metagenomic 16S RNA gene analysis, and the presence of lipolytic enzymes’ genes after an enrichment culture by a whole-genome shotgun approach. High diversity indexes were found in soil samples, with Firmicutes, Chloroflexi, Proteobacteria, Actinobacteria and Acidobacteria as the most prevalent phyla. Alicyclobacillus was the most prevalent genus at 15 cm and Anoxybacillus at 50 cm depth; anaerobic and obligate thermophilic and hyperthermophilic members were found at 50 cm depth. Firmicutes was favored by the oil-rich medium, and the dominant genera were Geobacillus and Caldibacillus. 165 genes related to lipolytic enzymes were found; carboxilesterases and phospholipases C were the most abundant, and 43% were assigned to uncharacterized lipolytic enzymes. The diversity as well as the high percentage of non-characterized lipolytic enzymes found after soil enrichment in an oil-rich medium suggest the presence of biotechnologically promising organisms and enzymes that deserve to be investigated.

Sequenced application of glutathione as an antioxidant with an organic biostimulant improves physiological and metabolic adaptation to salinity in wheat

Globally, salinity threatens the agricultural crops productivity by inhibiting plant growth and development through osmotic stress and ionic cytotoxicity. The polygenic nature of salinity offers several pragmatic shotgun approaches to improve salinity tolerance. The present study investigated the potential of glutathione (GSH; 1 mM) as an antioxidant and moringa leaf extract (MLE; 3%) as an organic biostimulant applied in sequence as seed priming and foliar spray on wheat growth, physiology and metabolic adaptation under saline conditions (9.16 dS m−1). Plants without any treatment and water spray (H2O) were considered controls. Salinity induced osmotic stress reduced the plant tissue water status and photosynthetic performance, and perturbed ionic (K+/Na+, Ca2+/Na+, K++Ca2+/Na+) and hormonal (IAA, GA3, zeatin, ABA) homeostasis, consequently affected growth and yield in wheat. Sequenced applied MLE and/or GSH improved osmotic stress tolerance by stabilizing membrane integrity and decreasing electrolyte leakage. These positive results were owed to enhanced endogenous GSH and ascorbate levels. Improved tissue water status was attributed to increased osmotic adjustment, better ionic and hormonal homeostasis contributed to improving photosynthetic efficiency and growth under salinity. Exogenously applied MLE and GSH sequences improved grain yield, which was attributed to the maintenance of green leaf area and delayed senescence associated with an increase in photosynthetic pigments and chlorophyll fluorescence traits. In crux, exogenous applied MLE and/or GSH can be the best physiological strategy to reduce the deleterious effects of salinity and improve physiological and metabolic adaptation in wheat under saline field conditions.

A low-cost pipeline for soil microbiome profiling.

Common bottlenecks in environmental and crop microbiome studies are the consumable and personnel costs necessary for genomic DNA extraction and sequencing library construction. This is harder for challenging environmental samples such as soil, which is rich in Polymerase Chain Reaction (PCR) inhibitors. To address this, we have established a low‐cost genomic DNA extraction method for soil samples. We also present an Illumina‐compatible 16S and ITS rRNA gene amplicon library preparation workflow that uses common laboratory equipment. We evaluated the performance of our genomic DNA extraction method against two leading commercial soil genomic DNA kits (MoBio PowerSoil® and MP Biomedicals™ FastDNA™ SPIN) and a recently published non‐commercial extraction method by Zou et al. (PLoS Biology, 15, e2003916, 2017). Our benchmarking experiment used four different soil types (coniferous, broad‐leafed, and mixed forest plus a standardized cereal crop compost mix) assessing the quality and quantity of the extracted genomic DNA by analyzing sequence variants of 16S V4 and ITS rRNA amplicons. We found that our genomic DNA extraction method compares well to both commercially available genomic DNA extraction kits in DNA quality and quantity. The MoBio PowerSoil® kit, which relies on silica column‐based DNA extraction with extensive washing, delivered the cleanest genomic DNA, for example, best A260:A280 and A260:A230 absorbance ratios. The MP Biomedicals™ FastDNA™ SPIN kit, which uses a large amount of binding material, yielded the most genomic DNA. Our method fits between the two commercial kits, producing both good yields and clean genomic DNA with fragment sizes of approximately 10 kb. Comparative analysis of detected amplicon sequence variants shows that our method correlates well with the two commercial kits. Here, we present a low‐cost genomic DNA extraction method for soil samples that can be coupled to an Illumina‐compatible simple two‐step amplicon library construction workflow for 16S V4 and ITS marker genes. Our method delivers high‐quality genomic DNA at a fraction of the cost of commercial kits and enables cost‐effective, large‐scale amplicon sequencing projects. Notably, our extracted gDNA molecules are long enough to be suitable for downstream techniques such as full gene sequencing or even metagenomics shotgun approaches using long reads (PacBio or Nanopore), 10x Genomics linked reads, and Dovetail genomics.

Parallel Factor Analysis Enables Quantification and Identification of Highly Convolved Data-Independent-Acquired Protein Spectra

SummaryHigh-throughput data-independent acquisition (DIA) is the method of choice for quantitative proteomics, combining the best practices of targeted and shotgun approaches. The resultant DIA spectra are, however, highly convolved and with no direct precursor-fragment correspondence, complicating biological sample analysis. Here, we present CANDIA (canonical decomposition of data-independent-acquired spectra), a GPU-powered unsupervised multiway factor analysis framework that deconvolves multispectral scans to individual analyte spectra, chromatographic profiles, and sample abundances, using parallel factor analysis. The deconvolved spectra can be annotated with traditional database search engines or used as high-quality input for de novo sequencing methods. We demonstrate that spectral libraries generated with CANDIA substantially reduce the false discovery rate underlying the validation of spectral quantification. CANDIA covers up to 33 times more total ion current than library-based approaches, which typically use less than 5% of total recorded ions, thus allowing quantification and identification of signals from unexplored DIA spectra.

The Microbial Composition in Circumneutral Thermal Springs from Chignahuapan, Puebla, Mexico Reveals the Presence of Particular Sulfur-Oxidizing Bacterial and Viral Communities.

Terrestrial thermal springs are widely distributed globally, and these springs harbor a broad diversity of organisms of biotechnological interest. In Mexico, few studies exploring this kind of environment have been described. In this work, we explore the microbial community in Chignahuapan hot springs, which provides clues to understand these ecosystems’ diversity. We assessed the diversity of the microorganism communities in a hot spring environment with a metagenomic shotgun approach. Besides identifying similarities and differences with other ecosystems, we achieved a systematic comparison against 11 metagenomic samples from diverse localities. The Chignahuapan hot springs show a particular prevalence of sulfur-oxidizing bacteria from the genera Rhodococcus, Thermomonas, Thiomonas, Acinetobacter, Sulfurovum, and Bacillus, highlighting those that are different from other recovered bacterial populations in circumneutral hot springs environments around the world. The co-occurrence analysis of the bacteria and viruses in these environments revealed that within the Rhodococcus, Thiomonas, Thermonas, and Bacillus genera, the Chignahuapan samples have specific species of bacteria with a particular abundance, such as Rhodococcus erytropholis. The viruses in the circumneutral hot springs present bacteriophages within the order Caudovirales (Siphoviridae, Myoviridae, and Podoviridae), but the family of Herelleviridae was the most abundant in Chignahuapan samples. Furthermore, viral auxiliary metabolic genes were identified, many of which contribute mainly to the metabolism of cofactors and vitamins as well as carbohydrate metabolism. Nevertheless, the viruses and bacteria present in the circumneutral environments contribute to the sulfur cycle. This work represents an exhaustive characterization of a community structure in samples collected from hot springs in Mexico and opens opportunities to identify organisms of biotechnological interest.

Genomic Adaptations and Evolutionary History of the Extinct Scimitar-Toothed Cat, Homotherium latidens

SummaryHomotherium was a genus of large-bodied scimitar-toothed cats, morphologically distinct from any extant felid species, that went extinct at the end of the Pleistocene [1, 2, 3, 4]. They possessed large, saber-form serrated canine teeth, powerful forelimbs, a sloping back, and an enlarged optic bulb, all of which were key characteristics for predation on Pleistocene megafauna [5]. Previous mitochondrial DNA phylogenies suggested that it was a highly divergent sister lineage to all extant cat species [6, 7, 8]. However, mitochondrial phylogenies can be misled by hybridization [9], incomplete lineage sorting (ILS), or sex-biased dispersal patterns [10], which might be especially relevant for Homotherium since widespread mito-nuclear discrepancies have been uncovered in modern cats [10]. To examine the evolutionary history of Homotherium, we generated a ∼7x nuclear genome and a ∼38x exome from H. latidens using shotgun and target-capture sequencing approaches. Phylogenetic analyses reveal Homotherium as highly divergent (∼22.5 Ma) from living cat species, with no detectable signs of gene flow. Comparative genomic analyses found signatures of positive selection in several genes, including those involved in vision, cognitive function, and energy consumption, putatively consistent with diurnal activity, well-developed social behavior, and cursorial hunting [5]. Finally, we uncover relatively high levels of genetic diversity, suggesting that Homotherium may have been more abundant than the limited fossil record suggests [3, 4, 11, 12, 13, 14]. Our findings complement and extend previous inferences from both the fossil record and initial molecular studies, enhancing our understanding of the evolution and ecology of this remarkable lineage.

Toxic trace element resistance genes and systems identified using the shotgun metagenomics approach in an Iranian mine soil.

This study aimed to identify the microbial communities, resistance genes, and resistance systems in an Iranian mine soil polluted with toxic trace elements (TTE). The polluted soil samples were collected from a mining area and compared against non-polluted (control) collected soils from the vicinity of the mine. The soil total DNA was extracted and sequenced, and bioinformatic analysis of the assembled metagenomes was conducted to identify soil microbial biodiversity, TTE resistance genes, and resistance systems. The results of the employed shotgun approach indicated that the relative abundance of Proteobacteria, Firmicutes, Bacteroidetes, and Deinococcus-Thermus was significantly higher in the TTE-polluted soils compared with those in the control soils, while the relative abundance of Actinobacteria and Acidobacteria was significantly lower in the polluted soils. The high concentration of TTE increased the ratio of archaea to bacteria and decreased the alpha diversity in the polluted soils compared with the control soils. Canonical correspondence analysis (CCA) demonstrated that heavy metal pollution was the major driving factor in shaping microbial communities compared with any other soil characteristics. In the identified heavy metal resistome (HV-resistome) of TTE-polluted soils, major functional pathways were carbohydrates metabolism, stress response, amino acid and derivative metabolism, clustering-based subsystems, iron acquisition and metabolism, cell wall synthesis and capsulation, and membrane transportation. Ten TTE resistance systems were identified in the HV-resistome of TTE-polluted soils, dominated by "P-type ATPases," "cation diffusion facilitators," and "heavy metal efflux-resistance nodulation cell division (HME-RND)." Most of the resistance genes (69%) involved in resistance systems are affiliated to cell wall, outer membrane, periplasm, and cytoplasmic membrane. The finding of this study provides insight into the microbial community in Iranian TTE-polluted soils and their resistance genes and systems.

Adaptive Acquisition Schemes for Photon-Limited Free-Space Optical Communications

Acquisition and tracking systems form an important component of free-space optical communications due to directional nature of the optical signal. Acquisition subsystems are needed in order to search and locate the receiver terminal in an uncertainty/search region with very narrow laser beams. In this paper, we have proposed and analyzed two adaptive search schemes for acquisition systems that perform better-for the low probability of detection-than the spiral scanning approach. The first of these schemes, the adaptive spiral search, provides a better acquisition time performance by dividing the search region into a number of smaller subregions, and prioritizing search in regions of higher probability mass. The second technique-the shotgun approach-searches the region in a random manner by sampling the search region according to a Gaussian distribution. The adaptive spiral scheme outperforms the shotgun approach in terms of acquisition time, especially if the number of search subregions is large enough. However, a higher pointing accuracy is required by the adaptive spiral search in order to search the region precisely. On the other hand, the shotgun scanning approach does not require such stringent pointing accuracy.

Shotgun metagenomic data of microbiomes on plastic fabrics exposed to harsh tropical environments.

The development of more affordable high-throughput DNA sequencing technologies and powerful bioinformatics is making of shotgun metagenomics a common tool for effective characterization of microbiomes and robust functional genomics. A shotgun metagenomic approach was applied in the characterization of microbial communities associated with plasticized fabric materials exposed to a harsh tropical environment for 14 months. High-throughput sequencing of TruSeq paired-end libraries was conducted using a whole-genome shotgun (WGS) approach on an Illumina HiSeq2000 platform generating 100 bp reads. A multifaceted bioinformatics pipeline was developed and applied to conduct quality control and trimming of raw reads, microbial classification, assembly of multi-microbial genomes, binning of assembled contigs to individual genomes, and prediction of microbial genes and proteins. The bioinformatic analysis of the large 161 Gb sequence dataset generated 3,314,688 contigs and 120 microbial genomes. The raw metagenomic data and the detailed description of the bioinformatics pipeline applied in data analysis provide an important resource for the genomic characterization of microbial communities associated with biodegraded plastic fabric materials. The raw shotgun metagenomics sequence data of microbial communities on plastic fabric materials have been deposited in MG-RAST (https://www.mg-rast.org/) under accession numbers: mgm4794685.3–mgm4794690.3. The datasets and raw data presented here were associated with the main research work “Metagenomic characterization of microbial communities on plasticized fabric materials exposed to harsh tropical environments” (Radwan et al., 2020).

Organic Farming Enhances the Diversity and Community Structure of Endophytic Archaea and Fungi in Maize Plant: a Shotgun Approach

This study compares, for the first time, the diversity and community structure of the endophytic archaea and fungi inhabiting the roots of maize plant cultivated using two different fertilizer regimes and the control field via shotgun metagenomics. This was used to verify our hypothesis that organic farming positively influences the diversity, beneficial effects, and abundance of plant endophytic communities as compared with conventional farming. Total DNA extraction was carried out using the roots of maize plants cultivated with organic fertilizer (FK), inorganic fertilizer (NK), and without any fertilizer (CK) at different farming sites in an experimental field and sequenced using shotgun metagenomics. In all, 3 archaea phyla, namely, Crenarchaeota, Euryarchaeota, and Thaumarchaeota, and 2 fungi phyla, namely, Ascomycota and Basidiomycota, were identified in the samples with higher abundance in samples from the FK site. Our diversity results revealed that the abundance of endophytic archaea and fungi species across the farming sites are in the order NK < CK < FK. The results indicate that organic farming enhances the abundance of endophytic archaea and fungi which might be a boost to sustainable agricultural practices. Furthermore, some new genera of endophytic archaea and fungi were identified from the maize roots. Future studies into how these novel endophytes can be cultured are important in a bid to explore their functions in sustainable agriculture as well as identify their biotechnological importance.

Profiling of rice Cd-tolerant genes through yeast-based cDNA library survival screening

The bioaccumulation of cadmium (Cd) in crop and the subsequent food chain has aroused extensive concerns. However, the underlying molecular mechanisms of plant Cd tolerance remain to be clarified from the viewpoint of novel candidate genes. Here we described a highly efficient approach for preliminary identifying rice Cd-tolerant genes through the yeast-based cDNA library survival screening combined with high-throughput sequencing strategy. About 690 gene isoforms were identified as being Cd-tolerant candidates using this shotgun approach. Among the Cd-tolerant genes identified, several categories of genes such as BAX inhibitor (BI), NAC transcription factors and Rapid ALkalinization Factors (RALFs) were of particular interest, and their function of Cd tolerance was further validated via heterologous expression, which suggested that SNAC1, RALF12, OsBI-1 can confer Cd tolerance in yeast and tobacco leaves. Regarding the genes involved in ion transport, the validated Cd-tolerant heavy metal-associated domain (HMAD) isoprenylated protein HIPP42 was particularly noteworthy. Further elucidation of these genes associated with Cd tolerance in rice will benefit agricultural activities.

Antimicrobial and Anti-Oxidative Activities of 12-Arylbenzoacridines

Searching biologically active compounds has been one of the major duties of organic chemists. We previously constructed a library of synthetic 12-arylbenzoacridines and found estrogenic and anti-estrogenic agents. With taking the shot-gun approach into consideration, further exploration for bioactive species in the above library was carried out. As a result, new antibacterial agents against S. aureus (MIC = 16 μM), E. coli (MIC = 7.8 μM), E. faecalis (MIC = 0.98 μM), and A. baumannii (MIC = 31 μM) were found. Moreover, we also unveiled that some of the tested compounds exhibit cell-based anti-oxidative activity. These results clearly show a good potential of 12-arylbenzoacridines as novel pharmacophores, which is useful to establish various medicines.

Critical evaluation of equations for serum osmolality: Proposals for effective clinical utility

BackgroundMany studies have assessed the predictive accuracy of serum osmolality equations. Different approaches for selecting a usable equation were compared using thirty published equations and patient data from a regional hospital laboratory. MethodsLaboratory records were extracted with same-sample results for measured serum osmolality, sodium, potassium, urea and glucose analysed in a regional hospital laboratory between 1/1/2017–31/12/2018. Differences were analysed using Passing-Bablok and difference (Bland-Altman) analysis. Three approaches were compared: the shotgun approach, adjusting for bias, and deriving a novel equation using multivariate analysis. The criteria for success included bias ≤0.7%, a 230 – 400 mOsm/kg range, and osmolal gap (OG) 95% reference limits within ±10 mOsm/kg. ResultsThe majority of equations produced proportionally negative-biased results. The shotgun approach identified two equations (EQ19, EQ6) with bias ≤0.7% but unworkable OG reference limits. The bias adjustment approach produced several equations with bias ≤ 0.7% and OG reference limits within or equivalent to ±10 mOsm/kg. A novel equation generated by us (1.89Na+ + 1.71 K+ + 1.08 Urea + 1.08 Glucose + 13.7) improved with the adjustment of bias and was not superior to the adjusted published equations. ConclusionFew published equations are immediately usable. Adjustment of bias derives several usable equations of which the best had OG ranges <20 mOsm/kg. We conclude that adjustment of bias can generate equations of equal or superior performance to that of novel equations.

The complete mitochondrial genome of Iphisa elegans (Reptilia: Squamata: Gymnophthalmidae)

The complete mitogenome of the lizard Iphisa elegans Gray, 1851 was sequenced using a shotgun approach on an Illumina HiSeq 3000 platform, providing the first mitogenome for Gymnophthalmidae. The genome was 18,622 bp long, with 13 protein-coding genes, two rRNA (12S and 16S), and 22 tRNA, as well as the control region. A maximum likelihood phylogenetic analysis including I. elegans and all other available mitogenomes of Squamata provided a tree in accordance with previous phylogenetic relationships inferred for Squamata.

Comparative characterization of Viperidae snake venoms from Perú reveals two compositional patterns of phospholipase A2 expression.

Snake species within the Bothrops complex (sensu lato) are of medical relevance in Latin America, but knowledge on their venom characteristics is limited, or even unavailable, for some taxa. Perú harbors 17 species of pit vipers, within the genera Bothrops, Bothriechis, Bothrocophias, Porthidium, Crotalus, and Lachesis. This study compared the venoms of twelve species, through chromatographic and electrophoretic profiles, as well as proteolytic and phospholipase A2 (PLA2) activities. Also, proteomic profiles were analyzed for nine of the venoms using a shotgun approach. Results unveiled conspicuous differences in the expression of venom PLA2s among species, six of them presenting scarce levels as judged by RP-HPLC profiles. Since most species within the bothropoid lineage possess venoms with high to intermediate abundances of this protein family, our findings suggest the existence of a phenotypic duality in the expression of venom PLA2s within the Bothrops (sensu lato) complex. Bothrops barnetti and Bothrocophias andianus venoms, very scarce in PLA2s, were shown to lack significant myotoxic activity, highlighting that the observed variability in PLA2 expression bears toxicological correlations with effects attributed to these proteins. Finally, an attempt to identify phylogenetic relationships of bothropoid species from Perú presenting low- or high-PLA2 venom phenotypes showed an interspersed pattern, thus precluding a simple phylogenetic interpretation of this venom compositional dichotomy.

Comparative Analysis of Porcine Follicular Fluid Proteomes of Small and Large Ovarian Follicles.

Ovarian follicular fluid is widely used for in vitro oocyte maturation, but its in-depth characterization to extract full beneficial effects remains unclear. Here, we performed both shotgun (nanoscale liquid chromatography coupled to tandem mass spectrometry or nanoLC-MS/MS) and gel-based (two dimension-differential in-gel electrophoresis or 2D-DIGE) proteomics, followed by functional bioinformatics to compare the proteomes of follicular fluids collected from small (<4 mm) and large (>6–12 mm) follicles of pig ovaries. A total of 2321 unique spots were detected with the 2D-DIGE across small and large follicles, while 2876 proteins with 88% successful annotations were detected with the shotgun approach. The shotgun and 2D-DIGE approaches revealed about 426 and 300 proteins that were respectively common across samples. Six proteins detected with both technical approaches were significantly differently expressed between small and large follicles. Pathways such as estrogen and PI3K-Akt signaling were significantly enriched in small follicles while the complement and coagulation cascades pathways were significantly represented in large follicles. Up-regulated proteins in small follicles were in favor of oocyte maturation, while those in large follicles were involved in the ovulatory process preparation. Few proteins with potential roles during sperm–oocyte interactions were especially detected in FF of large follicles and supporting the potential role of the ovarian FF on the intrafallopian sperm migration and interaction with the oocyte.

Quantitative Profiling of the Human Substantia Nigra Proteome from Laser-capture Microdissected FFPE Tissue

Laser-capture microdissection (LCM) allows the visualization and isolation of morphologically distinct subpopulations of cells from heterogeneous tissue specimens. In combination with formalin-fixed and paraffin-embedded (FFPE) tissue it provides a powerful tool for retrospective and clinically relevant studies of tissue proteins in a healthy and diseased context. We first optimized the protocol for efficient LCM analysis of FFPE tissue specimens. The use of SDS containing extraction buffer in combination with the single-pot solid-phase-enhanced sample preparation (SP3) digest method gave the best results regarding protein yield and protein/peptide identifications. Microdissected FFPE human substantia nigra tissue samples (∼3,000 cells) were then analyzed, using tandem mass tag (TMT) labeling and LC-MS/MS, resulting in the quantification of >5,600 protein groups. Nigral proteins were classified and analyzed by abundance, showing an enrichment of extracellular exosome and neuron-specific gene ontology (GO) terms among the higher abundance proteins. Comparison of microdissected samples with intact tissue sections, using a label-free shotgun approach, revealed an enrichment of neuronal cell type markers, such as tyrosine hydroxylase and alpha-synuclein, as well as proteins annotated with neuron-specific GO terms. Overall, this study provides a detailed protocol for laser-capture proteomics using FFPE tissue and demonstrates the efficiency of LCM analysis of distinct cell subpopulations for proteomic analysis using low sample amounts.

Lipidomic Analysis Demonstrates that 36:1 Phosphatidylcholine is Enriched in Cell Models of Drug Resistance Prostate Cancer

A lack of understanding exists between the association between circulating lipids and clinical outcomes of drug‐resistant castration‐resistant prostate cancer (CRPC). Although an increase in select circulating lipids correlates to decreased patient survival, neither the mechanisms mediating alterations in these lipids, nor the correlation to drug resistance are well characterized. This gap‐in‐knowledge was addressed using in vitro models of non‐cancerous, hormone‐sensitive, CRPC and drug‐resistant cell lines combined with quantitative HPLC‐ESI‐Orbitrap‐MS lipidomic analysis.Lipid extracts were obtained using Bligh‐Dyer (methanol/chloroform/water) extraction from non‐cancerous, hormone‐sensitive, CRPC and drug‐resistant prostate cell lines. Lipids present in both cells and media were initially analyzed using an untargeted shotgun approach (ESI‐MS), followed by a targeted‐based quantitative HPLC‐ESI‐Orbitrap‐MS approach. Shotgun experiments were performed using commercially available standards. For target‐based lipidomics, LC separation yielded specific lipid class separation prior to ESI/MS/MS enabling enhanced detection of lipids suppressed in the shotgun approach. Post data acquisition for both shotgun and HPLC‐ESI/MS/MS lipidomics were based on multiple methods including isotope, carbon number (to internal standards) and ionization efficiency‐based corrections. Pathway analysis was also performed on the various prostate cell lines. Online resources and software’s utilized included MetaboAnalyst, LIPIDMATCH LIPID MAPS, XCMS, and MZmine.Principle component analysis (PCA) generated scores plots of non‐cancerous, hormone‐sensitive, CRPC parent cell lines and drug‐resistant CRPC cell lines in the positive mode (ESI+) had a total variance of 66%. Additionally, a distinct separation between drug‐resistant and parent control cell lines was shown. Several discriminatory ion features were identified between prostate cell lines, especially between drug‐resistant and parent control neuroendocrine cell lines. Of these discriminatory features, 83 showed significant alterations. Two of the identified lipids had alterations matching those measured in plasma from prostate cancer patients.Distinct phospholipid classes were observed to have increased levels in prostate cancer cells, when compared to non‐cancer cells. Lipidomic analysis identified phosphatidylcholines (PC 36:1), whose levels were increased in Docetaxel resistant CRPC. These data also show that the lipidomic profiles of prostate cancer cell lines mirror that seen in the plasma of prostate cancer patients. These data may lead to new early detection strategies and more personalized treatment options for patients.Support or Funding InformationDepartment of Defense Prostate Cancer Research Program Idea Development Award (PC150431 GRANT11996600)Phospholipids in Prostate Cancer(A. Scores Plot) PCA scores plot comparing drug‐resistant CRPC cells to DU145 control parent cells in the positive mode (ESI+). ~80% of the total variance is explained by Component 1 and Component 2. (B. MTT Assay) MTT assay shows decrease in MTT staining in parent cell lines as compared to docetaxel‐resistant cell lines. This suggests DU145‐DR (DR) maintain resistance to Docetaxel as compared to DU145 parent cell line. (C. Heat Map) Each row represents a metabolite feature and each column represents a sample. Metabolite features whose levels vary significantly (p&lt; 0.01) are projected on the heat map and used for sample clustering displayed above. (D. Cloud Plot) Differential feature plot: Only features that are dysregulated in both DU145‐DR cells and DU145‐DR media (p‐value&lt;= 0.05 threshold, fold change &gt;= 1.5 threshold) are displayed above. Up‐regulated features (features that have a positive fold change) are graphed above the x‐axis while down‐regulated features (features that have a negative fold change) are graphed below the x‐axis.Figure 1PC 36‐1 in Prostate Cancer(A) Cloud plot showing 7460 discriminatory ion features (positive ion data) in human cell lines. Only dysregulated features are displayed above (p‐value &lt;= 0.05 threshold, fold change &gt;= 1.5 threshold). The radius of each bubble corresponds to the log fold change of that feature. Features with higher fold change (or higher intensity) will have larger radii. The shade of bubbles corresponds to the magnitude of the p‐value (the darker the color, the smaller the p‐value). The statistical significance of the fold change, as calculated by a Welch t‐test with unequal variances.Figure 6. (B) Box‐plot identified 788.6 feature in human cell lines. Of the lipids identified by the cloud plot, PC(36:1) was one of the few lipid species enriched in all drug resistant prostate cancer cell lines as compared to control cells. This was previously observed in castration‐resistant prostate cancer patient‐derived plasma samples.Figure 2

Metagenomic sequencing of clinical samples reveals a single widespread clone of Lawsonia intracellularis responsible for porcine proliferative enteropathy.

Lawsonia intracellularis is a Gram-negative obligate intracellular bacterium that is the aetiological agent of proliferative enteropathy (PE), a common intestinal disease of major economic importance in pigs and other animal species. To date, progress in understanding the biology of L. intracellularis for improved disease control has been hampered by the inability to culture the organism in vitro. In particular, our understanding of the genomic diversity and population structure of clinical L. intercellularis is very limited. Here, we utilized a metagenomic shotgun approach to directly sequence and assemble 21 L . intracellularis genomes from faecal and ileum samples of infected pigs and horses across three continents. Phylogenetic analysis revealed a genetically monomorphic clonal lineage responsible for infections in pigs, with distinct subtypes associated with infections in horses. The genome was highly conserved, with 94 % of genes shared by all isolates and a very small accessory genome made up of only 84 genes across all sequenced strains. In part, the accessory genome was represented by regions with a high density of SNPs, indicative of recombination events importing novel gene alleles. In summary, our analysis provides the first view of the population structure for L. intracellularis , revealing a single major lineage associated with disease of pigs. The limited diversity and broad geographical distribution suggest the recent emergence and clonal expansion of an important livestock pathogen.

Seminal plasma protein networks and enriched functions in varicocele: Effect of smoking.

To verify a possible synergistic effect of smoking and varicocele on the seminal plasma proteome and biological functions, a cross-sectional study was performed in 25 smokers and 24 nonsmokers. Samples were used for conventional semen analysis, functional analysis (DNA fragmentation, acrosome integrity and mitochondrial activity) and proteomics by a shotgun approach. Functional enrichment of biological pathways was performed in differentially expressed proteins. Smokers presented lower ejaculate volume (p=.027), percentage of progressively motile spermatozoa (p=.002), total sperm count (p=.039), morphology (p=.001) and higher percentage of immotile spermatozoa (p=.03), round cell (p=.045) and neutrophil count (p=.009). Smokers also presented lower mitochondrial activity and acrosome integrity and higher DNA fragmentation. We identified and quantified 421 proteins in seminal plasma, of which one was exclusive, 21 were overexpressed and 70 were underexpressed in the seminal plasma of smokers. The proteins neprilysin, beta-defensin 106A and histone H4A were capable of predicting the smoker group. Enriched functions were related to immune function and sperm machinery in testis/epididymis. Based on our findings, we can conclude that cigarette smoking leads to the establishment of inflammatory protein pathways in the testis/epididymis in the presence of varicocele that seems to act in synergy with the toxic components of the cigarette.