Development and characterization of microsatellite markers for the French endemic Angelica heterocarpa (Apiaceae) and congeneric sympatric species

Objective Angelica heterocarpa (Apiaceae) is a wild endemic French species with special conservation interest in the European Union. It belongs to Angelica complex genus which is widespread throughout the north temperate zone, and is sympatric with other congeneric species. The objective of this work is to develop and characterize microsatellite markers as a new tool for understanding the ecology and evolution of Angelica species complex. Results We identified simple sequence repeat (SSR) regions in a microsatellite‐enriched library from A. heterocarpa and A. sylvestris. All 16 selected SSR regions were found to amplify in these species and were highly polymorphic. Marker transferability was validated in A. razulii and A. archangelica. These markers will help us to better understand the evolutionary dynamic between rare endemics and widespread sister species, and be useful for conservation of the endemic species. Moreover, they can provide new tools for studying the numerous traditional medicinal herbs of the Angelica genus.

Phenotypic Screening of Molecular Docking Enriched Chemical Libraries from Targets Identified in Ischemic Stroke Genome Data by Network-Based Method

Cerebral ischemia (IS) is one of the main cardiovascular diseases threatening life and disability. Like most cardiovascular events, the disease progression of is affects a variety of signaling pathways and changes multiple overexpressed genes in the body. The use of new therapeutic agents to interfere with the disease progression of cardiovascular diseases (such as is) can be achieved by selectively regulating small molecules of the target set of different signal pathways, also known as selective multipharmacology. Phenotypic screening can be an effective method to solve this problem, but the lack of targeted methods for ischemic stroke limits its impact. Here, we aim to identify IS-specific targets by RNA sequencing data with a network-based approach. Molecular docking approach was applied to screen over 210,000 molecules from SPECS compound library. Screening of this enriched library resulted in 605 candidates that led to several potent active hits. The novelty analysis suggested that the structure scaffolds of the compounds were dissimilar to existing IKKB inhibitors, and further biological test result confirmed two identified compounds represented novel IKKB inhibitors. Further, docking exploration with IKKB (PDB id: 4KIK) showed that the three selective compounds were stable inside the binding pocket of IKKB which shared a homology of compound-protein interactions in comparison with the bioactive inhibitor of CHEMBL1762621. Our screening method is expected to produce selective multidrug lead compounds for the development of treatments for complex diseases, such as ischemic stroke.

Cell Culture Isolation and Whole Genome Characterization of Hepatitis E Virus Strains from Wild Boars in Germany

Infection with hepatitis E virus (HEV) can cause acute and chronic hepatitis in humans. The HEV genotype 3 can be zoonotically transmitted from animals to humans, with wild boars representing an important reservoir species. Cell culture isolation of HEV is generally difficult and mainly described for human isolates so far. Here, five sera and five liver samples from HEV-RNA-positive wild boar samples were inoculated onto PLC/PRF/5 cells, incubated for 3 months and thereafter passaged for additional 6 weeks. As demonstrated by RT-qPCR, immunofluorescence and immune electron microscopy, virus was successfully isolated from two liver samples, which originally contained high HEV genome copy numbers. Both isolates showed slower growth than the culture-adapted HEV strain 47832c. In contrast to this strain, the isolated strains had no insertions in their hypervariable genome region. Next generation sequencing using an HEV sequence-enriched library enabled full genome sequencing. Strain Wb108/17 belongs to subtype 3f and strain Wb257/17 to a tentative novel subtype recently described in Italian wild boars. The results indicate that HEV can be successfully isolated in cell culture from wild boar samples containing high HEV genome copy numbers. The isolates may be used further to study the zoonotic potential of wild boar-derived HEV subtypes.

Microsatellites for the Neotropical Ant, Odontomachus chelifer (Hymenoptera: Formicidae)

Odontomachus chelifer (Latreille) (Ponerinae) is a ground-dwelling, predominantly carnivorous ant whose colonies may contain multiple egg-laying queens and are potentially susceptible to border effects in the Brazilian savanna known as Cerrado. The ecology and natural history of O. chelifer is well studied, but very little is known about the genetic diversity of O. chelifer colonies. In this study, we developed microsatellite markers for the study of genetic variation in O. chelifer. We created a microsatellite-enriched library that resulted in the development and characterization of 22 markers, of which 18 were found to be polymorphic in the population studied. The mean expected heterozygosity was 0.59, whereas the mean rarified allelic richness was determined as 4.27 alleles per locus. The polymorphism level detected was similar to genetic diversity estimates found in other poneromorph ant species. The microsatellites developed here are likely to be useful for the investigation of colony structure, functional polygyny, breeding system, and population genetics in O. chelifer. Moreover, the description of O. chelifer’s genetic diversity is crucial for its conservation and maintenance of its ecological role in the Cerrado savanna.

Interrogating the recognition landscape of a conserved HIV-specific TCR reveals distinct bacterial peptide cross-reactivity

T cell cross-reactivity ensures that diverse pathogen-derived epitopes encountered during a lifetime are recognized by the available TCR repertoire. A feature of cross-reactivity where previous exposure to one microbe can alter immunity to subsequent, non-related pathogens has been mainly explored for viruses. Yet cross-reactivity to additional microbes is important to consider, especially in HIV infection where gut-intestinal barrier dysfunction could facilitate T cell exposure to commensal/pathogenic microbes. Here we evaluated the cross-reactivity of a ‘public’, HIV-specific, CD8 T cell-derived TCR (AGA1 TCR) using MHC class I yeast display technology. Via screening of MHC-restricted libraries comprising ~2×108 sequence-diverse peptides, AGA1 TCR specificity was mapped to a central peptide di-motif. Using the top TCR-enriched library peptides to probe the non-redundant protein database, bacterial peptides that elicited functional responses by AGA1-expressing T cells were identified. The possibility that in context-specific settings, MHC class I proteins presenting microbial peptides influence virus-specific T cell populations in vivo is discussed.

TM3’seq: A Tagmentation-Mediated 3’ Sequencing Approach for Improving Scalability of RNAseq Experiments

RNA-seq has become the standard tool for collecting genome-wide expression data in diverse fields, from quantitative genetics and medical genomics to ecology and developmental biology. However, RNA-seq library preparation is still prohibitive for many laboratories. Recently, the field of single-cell transcriptomics has reduced costs and increased throughput by adopting early barcoding and pooling of individual samples —producing a single final library containing all samples. In contrast, RNA-seq protocols where each sample is processed individually are significantly more expensive and lower throughput than single-cell approaches. Yet, many projects depend on individual library generation to preserve important samples or for follow-up re-sequencing experiments. Improving on currently available RNA-seq methods we have developed TM3′seq, a 3′-enriched library preparation protocol that uses Tn5 transposase and preserves sample identity at each step. TM3′seq is designed for high-throughput processing of individual samples (96 samples in 6h, with only 3h hands-on time) at a fraction of the cost of commercial kits ($1.5 per sample). The protocol was tested in a range of human and Drosophila melanogaster RNA samples, recovering transcriptomes of the same quality and reliability than the commercial NEBNext kit. We expect that the cost- and time-efficient features of TM3′seq make large-scale RNA-seq experiments more permissive for the entire scientific community.

TM3’seq: a tagmentation-mediated 3’ sequencing approach for improving scalability of RNA-seq experiments

RNA-seq has become the standard tool for collecting genome-wide expression data in diverse fields, from quantitative genetics and medical genomics to ecology and developmental biology. However, RNA-seq library preparation is still prohibitive for many laboratories. Recently, the field of single-cell transcriptomics has reduced costs and increased throughput by adopting early barcoding and pooling of individual samples —producing a single final library containing all samples. In contrast, RNA-seq protocols where each sample is processed individually are significantly more expensive and lower throughput than single-cell approaches. Yet, many projects depend on individual library generation to preserve important samples or for follow-up re-sequencing experiments. Improving on currently available RNA-seq methods we have developed TM3’seq, a 3’-enriched library preparation protocol that uses Tn5 transposase and preserves sample identity at each step. TM3’seq is designed for high-throughput processing of individual samples (96 samples in 6h, with only 3h hands-on time) at a fraction of the cost of commercial kits ($1.5 per sample). The protocol was tested in a range of human andDrosophila melanogasterRNA samples, recovering transcriptomes of the same quality and reliability than the commercial NEBNext®kit. We expect that the cost-and time-efficient features of TM3’seq make large-scale RNA-seq experiments more permissive for the entire scientific community.

An Evaluation of Students’ and Teachers’ Opinions About Enriched Libraries (Z-Libraries)

The purpose of this study is to evaluate Enriched libraries, established in schools in Turkey in the recent years. In particular, the study intended to investigate how relevant, effective, useful and functional Enriched libraries have been in real life, regarding students’ and teachers’ opinions. The research was conducted in line with the mixed model approach. In other words, both the quantitative and qualitative research methods were used in the research. Quantitative data was gathered from 469 students from one primary school, two secondary schools and two high schools with an enriched library in Giresun/Turkey in 2017-2018 academic year and the qualitative data was obtained from 18 teachers. Two types of data collection tools were used to collect quantitative and qualitative data in the study. Quantitative data of the research was collected from the students through the Z-Library Student Assessment Questionnaire developed by the researcher. The qualitative data of the study were collected from the teachers by the semi-structured interview form prepared by the researcher. Quantitative data of the study were analyzed using the SPSS statistical package program. In the analysis of the qualitative data descriptive analysis techniques were used to interpret the data. The results obtained from the analysis of quantitative and qualitative data were evaluated and interpreted together. According to the results of the survey, students and teachers think that they are appropriate, effective, functional and useful in compliance with the purpose of establishing Z-Library; they meet the expectations.

Selection and Identification of Novel Aptamers Specific for Clenbuterol Based on ssDNA Library Immobilized SELEX and Gold Nanoparticles Biosensor

We describe a multiple combined strategy to discover novel aptamers specific for clenbuterol (CBL). An immobilized ssDNA library was used for the selection of specific aptamers using the systematic evolution of ligands by exponential enrichment (SELEX). Progress was monitored using real-time quantitative PCR (Q-PCR), and the enriched library was sequenced by high-throughput sequencing. Candidate aptamers were picked and preliminarily identified using a gold nanoparticles (AuNPs) biosensor. Bioactive aptamers were characterized for affinity, circular dichroism (CD), specificity and sensitivity. The Q-PCR amplification curve increased and the retention rate was about 1% at the eighth round. Use of the AuNPs biosensor and CD analyses determined that six aptamers had binding activity. Affinity analysis showed that aptamer 47 had the highest affinity (Kd = 42.17 ± 8.98 nM) with no cross reactivity to CBL analogs. Indirect competitive enzyme linked aptamer assay (IC-ELAA) based on a 5′-biotin aptamer 47 indicated the limit of detection (LOD) was 0.18 ± 0.02 ng/L (n = 3), and it was used to detect pork samples with a mean recovery of 83.33–97.03%. This is the first report of a universal strategy including library fixation, Q-PCR monitoring, high-throughput sequencing, and AuNPs biosensor identification to select aptamers specific for small molecules.