Immuno-physiological adaptations confer wax moth Galleria mellonella resistance to Bacillus thuringiensis

The Greater wax moth, Galleria mellonella, is a pest of beehives and is gaining a reputation as an important organism for modelling host-pathogen interactions. A G. mellonella population was selected for enhanced resistance to Bacillus thuringiensis (Bt), which is a widely-used entomopathogenic biological pesticide. Resistance and defence mechanisms were investigated in this insect line, and compared with a non-selected (suspectible) line. We also investigated the possible cost of those survival strategies. In the uninfected state, resistant insects exhibited enhanced basal expression of genes related to regeneration and amelioration of Bt toxin activity in the midgut. In addition, these insects also exhibited elevated activity of genes linked to inflammation/stress management, and fat body immune defences. Following oral infection with Bt, several of these genes wwere more highly expressed in resistant insect than in the susceptible line. Gene expression analysis reveals a pattern of resistance mechanisms targeted to anatomical sites predominantly attacked by Bt. The resistant insect concentrates its response towards tissue repair. Unlike the susceptible insects, Bt infection significantly reduced the diversity and richness (abundance) of the gut microbiota in the resistant insects. These observations suggest that the resistant line not only has a more intact midgut but is secreting antimicrobial factors into the gut lumen which not only mitigate Bt activity but also affect the viability of other gut bacteria. Remarkably the resistant line employs these multifactorial adaptations for resistance to Bt without any detectable negative trade-off, since the insects also exhibited higher fecundity

DNA metabarcoding of benthic algae and associated eukaryotes from Lake Baikal in the face of rapid environmental changes

Here we report new data describing the biodiversity of phytobenthic communities based on DNA-metabarcoding using the 18S rDNA marker and the Illumina MiSeq system. The study was initiated due to the blooming of filamentous algae (mainly of the genus Spirogyra) and cyanobacteria in the coastal zone of Lake Baikal under climate change and anthropogenic impact. The composition and taxonomic diversity of algae and other organisms associated with them on different sites of Lake Baikal (near Bolshoi Ushkaniy Island, in Listvennichny Bay) and in the Kaya (within the city of Irkutsk, located in the same drainage basin as Lake Baikal) were determined using DNAmetabarcoding. About 15 thousand reads of the 18S rRNA marker were obtained by applying NGS (next-generation sequencing). The species of algae dominating in the number of reads, as well as the difficult-to-identify taxa (Stramenopiles, Alveolata, Euglenozoa, Chromista, Rhizaria, Amoebozoa, etc.), which play an important role in the functioning and formation of the structure of algal communities, were revealed. The Shannon index of the communities studied ranges from 1.56 to 2.72. The advantages and weaknesses of using DNA-metabarcoding based on the 18S rRNA gene fragment for studying the structure of algal communities are shown. The advantage of this method is the possibility to more fully determine the diversity of eukaryotes taxa, which are difficult to identify by morphology, without involving a large number of specialists, while the disadvantage of the method is the distortion that may occur during the PCR. Here, ways of solving this problem are proposed. The results of the study show that the analysis of the minor component of the eukaryotic community in samples (organisms with low biomass) consisting of a mixture of multicellular and unicellular organisms requires a read-depths of at least 100,000 sequences per sample. In general, the DNA-metabarcoding method is recommended for studying the structure of algal communities and eukaryotes associated with them

Phylogenetic analysis of the microbial mat in the hot spring Garga (Baikal rift zone) and the diversity of natural peptidases

Hydrolytic bacteria (in particular, proteolytics) are the primary destructors in hot springs. The proteolytic bacteria are able to secrete enzymes that are active in wide ranges of pH and temperature. The aim of this work was to study the taxonomic composition, the structure of the bacterial microbial mat, and to study the distribution of peptidases in the thermophilic microbial Garga community. For the study, we sampled the microbial mat at a water temperature of 54.2 °C and a pH of 8.3. Hydrochemical analysis of water showed a high content of sulfates, 390 mg/dm3. The microelement composition of water showed that the Garga water had increased concentrations of B, Rb, Li, Ba, Sr. We analyzed the taxonomic diversity of the microbial community in the hot spring Garga at a temperature zone of 54 °C. The structure of the microbial mat is represented by various phylogenetic groups of mesophilic and thermophilic bacteria, with various metabolic and ecological functions. The dominant group in this community was the phylum Firmicutes (64 %). The analysis of the collected metagenomic sequences of the microbial community allowed the detected peptidases in the microbial community in the hot spring Garga to be for the first time systematized and characterized. Comparisons of metagenomic sequences of representative data showed a dominance of serine peptidase class enzymes. Natural peptidases in the investigated microbial community ensure the hydrolysis of biopolymers at the first stages of the destruction of organic matter and may have biotechnological relevance

Chromosomal-level assembly of the Asian Seabass genome using long sequence reads and multi-layered scaffolding

We report here the ~670 Mb genome assembly of the Asian seabass (Lates calcarifer), a tropical marine teleost. We used long-read sequencing augmented by transcriptomics, optical and genetic mapping along with shared synteny from closely related fish species to derive a chromosome-level assembly with a contig N50 size over 1 Mb and scaffold N50 size over 25 Mb that span ~90% of the genome. The population structure of L. calcarifer species complex was analyzed by re-sequencing 61 individuals representing various regions across the species' native range. SNP analyses identified high levels of genetic diversity and confirmed earlier indications of a population stratification comprising three clades with signs of admixture apparent in the South-East Asian population. The quality of the Asian seabass genome assembly far exceeds that of any other fish species, and will serve as a new standard for fish genomics

Сравнение профиля генной экспрессии колониеформирующих эндотелиальных клеток из периферической крови человека и эндотелиальных клеток коронарной артерии

Aim. To compare gene expression profiles of CFEC to human coronary artery endothelial cells, based on the results of whole transcriptome analysis.Methods. CFEC were isolated from peripheral blood of patients during percutaneous coronary intervention. Human coronary artery endothelial cells were purchased from Cell Applications (300K-05a, USA). Cells were lysed with TRIzol with the following total RNA isolation and DNAse treatment. Then rRNA depletion was performed, followed by DNA library preparation. DNA libraries were then quantified by qPCR (CFX96 Touch, Bio-Rad, USA), pooled in equimolar amounts and sequenced (HiSeq 2000, Illumina) using 2 * 125 bp chemistry.Results. RNA-seq demonstrated that CFEC were generally similar to human coronary artery endothelial cells, regarding their global gene expression profile. However, CFEC overexpressed specific markers of all endothelial lineages (NRP2, NOTCH4, LYVE1), in particular, lymphatic EC (LYVE1) and had upregulated extracellular matrix and basement membrane genes (COLlAl, COL1A2, COL4A1, COL4A2).Conclusion. Baseline gene expression in CFEC is close to that of human coronary artery endothelial cells, testifying about their utility for the seeding of tubular scaffolds before the implantation to improve their short- and long-term performance.Цель. Сравнительный анализ профиля генной экспрессии колониеформирующих эндотелиальных клеток и эндотелиальных клеток коронарной артерии человека на основе результатов полнотранскриптомного секвенирования.Материалы и методы. Культура колониеформирующих эндотелиальных клеток получена из периферической крови пациентов, перенесших чрескожное коронарное вмешательство. Первичные эндотелиальные клетки коронарной артерии были приобретены у CellApplications (300K-05a, США). Клетки лизированы тризолом с последующим выделением тотальной РНК и сопутствующей обработкой ДНКазой. Проводилась деплеция рРНК с дальнейшим конструированием ДНК-би-блиотек. Концентрация ДНК-библиотек определялась с помощью количественной полимеразной цепной реакции с детекцией результата в реальном времени на амплификаторе CFX96 Touch (Bio-Rad, США). Далее ДНК-библиотеки смешивались эквимолярно и секвенировались на платформе HiSeq 2000 (Illumina, США) с длиной парно-концевых прочтений 2 я 125 нуклеотидов.Результаты. Полнотранскриптомное секвенирование продемонстрировало, что колониеформирующие эндотелиальные клетки были схожи с первичными эндотелиальными клетками коронарной артерии в отношении их профиля генной экспрессии, при этом гиперэкспрессировали специфичные маркеры всех направлений эндотелиальной дифференцировки (NRP2, NOTCH4, LYVE1), в особенности лимфатической (LYVE1), и обладали повышенной экспрессией генов компонентов внеклеточного матрикса и базальной мембраны (COL1A1, COL1A2, COL4A1, COL4A2).Заключение. Базовый профиль генной экспрессии колониеформирующих эндотелиальных клеток близок к таковому у эндотелиальных клеток коронарной артерии, что свидетельствует о применимости колониеформирующих эндотелиальных клеток для заселения трубчатых полимерных каркасов перед имплантацией для улучшения их кратко- и долгосрочной проходимости

Development and characterization of novel 2′-F-RNA aptamers specific to human total and glycated hemoglobins

Aptamers are short DNA and RNA fragments which bind their molecular targets with affinity and specificity comparable to those of antibodies. Here, we describe the selection of 2'-F-RNA aptamers against total human hemoglobin or its glycated form HbA1c. After SELEX and high-throughput sequencing of the enriched libraries, affinities and specificities of candidate aptamers and their truncated variants were examined by the solid-phase bioluminescent assay. As a result, we identified aptamers specific to both hemoglobins or only glycated HbA1c. The developed 2'-F-RNA aptamers have shown their applicability for detection of total and glycated hemoglobin in one sample using the solid-phase sandwich assay

P124 Targeted bisulfite sequencing of potential prostate cancer cell-free DNA markers: novel approach to reveal diagnostically significant molecules

Cancer cells display altered methylation signatures distinguishing them from normal cells. Originating from all tissues and cells of the body cell-free DNA (cfDNA) including aberrantly methylated DNA reflect epigenetic aberrations occurs not only in tumor cells but also in tumor microenvironment. Actually, aberrantly methylated cfDNA has proved to be a promising biomarker for noninvasive detection of cancer with several clinically-certified tests (Epi-pro Colon®, Epi-pro Lung®, Cologuard®). However only one test (Epi-pro Colon®) uses blood plasma – the most convenient source of cfDNA. Input of tissue and age specific methylation along with unidentified reasons lead to the presence of molecules with every conceivable cytosine-methylation patterns which decrease probability of tumor DNA identification. Among numerous variants of cfDNA methylation patterns only few reflect cancer related changes. To identify those tumor-specific profiles single nucleotide resolution of methylated cytosine locations in the individual circulating DNA molecules is obviously required.We performed target bisulfite sequencing of potential prostate cancer (PC) cfDNA markers (GSTP1; RNF219) isolated from blood plasma of 18 healthy donors (HD), 17 benign hyperplasia (BPH) and 20 PC patients using MiSeq platform (Illumina). RNF219 gene was shown to have high diagnostic potential in our previous comparative study of cfDNA from HD, PC and BHP patients using HCGI12k microarrays (Cortese, et al., 2012). GSTP1 is a common pathological DNA methylation event in PC and is most widely studied and promising methylation marker in the cfDNA of PC patients (Wu, et al., 2011).Selected loci were amplified after bisulfite conversion (Zymo Research) of cfDNA with methyl-independent barcoded primers and sequenced with coverage ranging from 23509 to 143953. Identification of CpG methylation status in DNA fragments was performed with the BiQ Analyzer HT Software. All statistical analysis was performed using R Statistical Software (version 3.1.1) To reveal diagnostically significant differences, several approaches to data analysis were used. Conventional approach is a prediction of patient’s diagnosis based on differences in methylation level of CpG-sites. Another approach used in this study relies on discrimination of cancer-related correlation between methylation statuses of CpG-sites within individual molecules of cfDNA – Intramolecular Correlation of Methylation Statuses (ICoMS).Study population was randomly subsampled into training and test cohorts. The logit regression model based on methylation level of CpG-sites achieved an area under the ROC curve (AUC) exceeding 0.94 in both cohorts for GSTP1 gene and 0.81 – for RNF219 gene. A novel approach to identify diagnostic significance of cfDNA methylation was based on comparison of correlation matrices (pairwise phi coefficient between methylation statuses of CpG sites) for HD, BHP and PC groups. Binomial regression models with LASSO penalization were used to predict patient’s diagnosis. ROC curves for fitted models show AUC, specificity and sensitivity as 0.99, 100% and 92% for GSTP1 gene and 0.99, 100% and 90% for RNF219 (test cohort).The ICoMS approach estimates diagnostic value of target genes and reveals cancer-related cytosine methylation. That could potentially input in gene expression, identify the features of tumor physiology and on a par with conventional approaches provide helpful information for rational design of noninvasive, methylation-specific cfDNA-based diagnostics for PC.Moreover, the planned further evaluation of relationship between the revealed correlations associated with cancer and tumor biology could increase the basic knowledge of carcinogenesis

<i>Bacillus thuringiensis</i> Spores and Cry3A Toxins Act Synergistically to Expedite Colorado Potato Beetle Mortality

The insect integument (exoskeleton) is an effective physiochemical barrier that limits disease-causing agents to a few portals of entry, including the gastrointestinal and reproductive tracts. The bacterial biopesticide Bacillus thuringiensis (Bt) enters the insect host via the mouth and must thwart gut-based defences to make its way into the body cavity (haemocoel) and establish infection. We sought to uncover the main antibacterial defences of the midgut and the pathophysiological features of Bt in a notable insect pest, the Colorado potato beetle Leptinotarsa decemlineata (CPB). Exposing the beetles to both Bt spores and their Cry3A toxins (crystalline δ-endotoxins) via oral inoculation led to higher mortality levels when compared to either spores or Cry3A toxins alone. Within 12 h post-exposure, Cry3A toxins caused a 1.5-fold increase in the levels of reactive oxygen species (ROS) and malondialdehyde (lipid peroxidation) within the midgut – key indicators of tissue damage. When Cry3A toxins are combined with spores, gross redox imbalance and ‘oxidation stress’ is apparent in beetle larvae. The insect detoxification system is activated when Bt spores and Cry3A toxins are administered alone or in combination to mitigate toxicosis, in addition to elevated mRNA levels of candidate defence genes (pattern-recognition receptor, stress-regulation, serine proteases, and prosaposin-like protein). The presence of bacterial spores and/or Cry3A toxins coincides with subtle changes in microbial community composition of the midgut, such as decreased Pseudomonas abundance at 48 h post inoculation. Both Bt spores and Cry3A toxins have negative impacts on larval health, and when combined, likely cause metabolic derangement, due to multiple tissue targets being compromised