Simple and cost-effective SNP genotyping method for discriminating subpopulations of the fish pathogen, Nocardia seriolae

Nocardia seriolae has caused significant fish losses in Asia and the Americas in recent decades, including in Vietnam, which has witnessed devastating economic and social impacts due to this bacterial pathogen. Surveillance strategies are urgently needed to mitigate N. seriolae dissemination in Vietnamese aquaculture and mariculture industries. Whole-genome sequencing (WGS) offers the highest level of resolution to discriminate closely related strains and to determine their putative origin and transmission routes. However, WGS is impractical for epidemiological investigations and pathogen surveillance due to its time-consuming and costly nature, putting this technology out-of-reach for many industry end-users. To overcome this issue, we targeted two previously characterised, phylogenetically informative single-nucleotide polymorphisms (SNPs) in N. seriolae that accurately distinguish: i) Vietnamese from non-Vietnamese strains, and ii) the two Vietnamese subclades. Using the mismatch amplification mutation assay (MAMA) format, we developed assays that genotype strains based on differences in amplicon melting temperature (melt-MAMA) and size (agarose-MAMA). Our MAMA assays accurately genotyped strains both from culture and fish tissues at low cost, using either real-time (~AUD$1/per sample) or conventional (~AUD$0.50/per sample) PCR instrumentation. Our novel assays provide a rapid, reproducible, and cost-effective tool for routine genotyping of this pathogen, allowing faster identification and treatment of nocardiosis-effected permit fish within Vietnamese aquaculture/mariculture facilities, an essential step in mitigating N. seriolae-associated losses.

Rediscovery and redescription of Niphargus enslini Karaman, 1932 (Amphipoda, Niphargidae) in southern Germany

Niphargus enslini Karaman, 1932 was collected only once in 1905 from the Falkensteiner Höhle (Baden-Württemberg, Germany). Two years after its description, the species was synonymized with Niphargus virei and not studied any more. During recent surveys on German niphargids, further samples collected in this cave did not yield N. enslini specimens, but this species was collected in the Blätterteighöhle and in the Schwarzer Brunnen, two caves located in Baden-Württemberg and intercepting the same karstic aquifer feeding Falkensteiner Höhle. In an integrative taxonomic approach, we carefully studied the morphology of the newly collected specimens and sequenced two molecular markers (fragments of the cytochrome c oxidase subunit I (COI) and of the nuclear 28S rRNA gene) to test for possible conspecificity of N. enslini with N. virei. Morphological analysis confirmed that N. enslini is distinct from the N. virei species complex. We provide a redescription of newly collected material, together with new drawings of a more than 100 years old topotypic female. We briefly discuss the putative origin of N. enslini and the age of its split from the N. virei species complex.

Detection and Complete Genome Analysis of Circoviruses and Cycloviruses in the Small Indian Mongoose (Urva auropunctata): Identification of Novel Species

Fecal samples from 76 of 83 apparently healthy small Indian mongooses (Urva auropunctata) were PCR positive with circovirus/cyclovirus pan-rep (replicase gene) primers. In this case, 30 samples yielded high quality partial rep sequences (~400 bp), of which 26 sequences shared maximum homology with cycloviruses from an arthropod, bats, humans or a sheep. Three sequences exhibited maximum identities with a bat circovirus, whilst a single sequence could not be assigned to either genus. Using inverse nested PCRs, the complete genomes of mongoose associated circoviruses (Mon-1, -29 and -66) and cycloviruses (Mon-20, -24, -32, -58, -60 and -62) were determined. Mon-1, -20, -24, -29, -32 and -66 shared <80% maximum genome-wide pairwise nucleotide sequence identities with circoviruses/cycloviruses from other animals/sources, and were assigned to novel circovirus, or cyclovirus species. Mon-58, -60 and -62 shared maximum pairwise identities of 79.90–80.20% with human and bat cycloviruses, which were borderline to the cut-off identity value for assigning novel cycloviral species. Despite high genetic diversity, the mongoose associated circoviruses/cycloviruses retained the various features that are conserved among members of the family Circoviridae, such as presence of the putative origin of replication (ori) in the 5′-intergenic region, conserved motifs in the putative replication-associated protein and an arginine rich region in the amino terminus of the putative capsid protein. Since only fecal samples were tested, and mongooses are polyphagous predators, we could not determine whether the mongoose associated circoviruses/cycloviruses were of dietary origin, or actually infected the host. To our knowledge, this is the first report on detection and complete genome analysis of circoviruses/cycloviruses in the small Indian mongoose, warranting further studies in other species of mongooses.

Prediction and characterization of diffuse large B-cell lymphoma cell-of-origin subtypes using targeted sequencing

The aim of the present study was to determine cell of origin (COO) from a platform using a DNA-based method, COO DNA classifier (COODC). A targeted exome-sequencing platform that applies the mutational profile of a sample was used to classify COO subtype. Two major mutational signatures associated with COO were identified: Catalogue of Somatic Mutations in Cancer (COSMIC) signature 23 enriched in activated B-cell (ABC) and COSMIC signature 3, which suggested increased frequency in germinal center B-cell (GCB). Differential mutation signatures linked oncogenesis to mutational processes during B-cell activation, confirming the putative origin of GCB and ABC subtypes. Integrating COO with comprehensive genomic profiling enabled identification of features associated with COO and demonstrated the feasibility of determining COO without RNA.

Comparative phenotypic, genotypic and genomic analyses of Bacillus thuringiensis associated with foodborne outbreaks in France

Bacillus thuringiensis (Bt) belongs to the Bacillus cereus (Bc) group, well known as an etiological agent of foodborne outbreaks (FBOs). Bt distinguishes itself from other Bc by its ability to synthesize insecticidal crystals. However, the search for these crystals is not routinely performed in food safety or clinical investigation, and the actual involvement of Bt in the occurrence of FBOs is not known. In the present study, we reveal that Bt was detected in the context of 49 FBOs declared in France between 2007 and 2017. In 19 of these FBOs, Bt was the only microorganism detected, making it the most likely causal agent. Searching for its putative origin of contamination, we noticed that more than 50% of Bt isolates were collected from dishes containing raw vegetables, in particular tomatoes (48%). Moreover, the genomic characterization of isolates showed that most FBO-associated Bt isolates exhibited a quantified genomic proximity to Bt strains, used as biopesticides, especially those from subspecies aizawai and kurstaki. Taken together, these results strengthen the hypothesis of an agricultural origin for the Bt contamination and call for further investigations on Bt pesticides.

Deficiency and absence of endogenous isoprene in adults, disqualified its putative origin

Background: Isoprene (C5H8) is a clinically important breath metabolite. Although, hundreds of studies have reported differential expressions in isoprene exhalation as breath biomarker for diverse diseases, the substance couldn’t enter to clinical practice as diagnostic marker. Moreover, many experimental/basic observations upon breath isoprene remained unrelated to the corresponding pathophysiological effects on its putative metabolic origin (i.e. mevalonate pathway). Here, we investigated the fundamental reason that hindered the rational interpretation and translation of this marker from basic to clinical science. Methods: Via high-resolution mass-spectrometry based breathomics in 1026 human subjects, we discovered adults with significant deficiency (order of magnitude lower than the normal) and complete absence of breath isoprene. We prospectively applied real-time breathomics, quantitative gene expression analysis of the mevalonate pathway enzymes, lipid-profiling and hemodynamic monitoring on those isoprene deficient subjects and controls. Additionally, the subject with absence of isoprene was followed up throughout different phases of her womanhood.Results: In contrast to convention, we witnessed that adults can live healthy without exhaling isoprene or with significant deficiency. This rare phenotype represents a recessive inheritance. Despite physio-metabolic changes during menstrual cycle (that is known to profoundly affect isoprene exhalation) and profoundly increased plasma cholesterol during pregnancy and after childbirth, isoprene remained absent. All genes of mevalonate pathway enzymes were normally expressed in all participants, without any down-regulation or compensatory up-regulation. Conclusions: Absence/deficiency of isoprene despite normal lipid profiles and no mevalonate pathway malfunction disqualifies the long-believed metabolic origin of isoprene from cholesterol biosynthesis. Thus, clinical translation of breath isoprene expressions should not be generally attributed to corresponding pathophysiological effects onto mevalonate/cholesterol pathway. Our finding has refined and optimized the clinical interpretation of isoprene as biomarker in volatile metabolomics and breathomics. Future studies will address the correct metabolic origin of isoprene to imply this important marker to routine practice.

Deficiency and absence of endogenous isoprene in adults, disqualified its putative origin

Background: Isoprene (C5H8) is a clinically important breath metabolite. Although, hundreds of studies have reported differential expressions in isoprene exhalation as breath biomarker for diverse diseases, the substance couldn’t enter to clinical practice as diagnostic marker. Moreover, many experimental/basic observations upon breath isoprene remained unrelated to the corresponding pathophysiological effects on its putative metabolic origin (i.e. mevalonate pathway). Here, we investigated the fundamental reason that hindered the rational interpretation and translation of this marker from basic to clinical science.Methods: Via high-resolution mass-spectrometry based breathomics in 1026 human subjects, we discovered adults with significant deficiency (order of magnitude lower than the normal) and complete absence of breath isoprene. We prospectively applied real-time breathomics, quantitative gene expression analysis of the mevalonate pathway enzymes, lipid-profiling and hemodynamic monitoring on those isoprene deficient subjects and controls. Additionally, the subject with absence of isoprene was followed up throughout different phases of her womanhood.Results: In contrast to convention, we witnessed that adults can live healthy without exhaling isoprene or with significant deficiency. This rare phenotype represents a recessive inheritance. Despite physio-metabolic changes during menstrual cycle (that is known to profoundly affect isoprene exhalation) and profoundly increased plasma cholesterol during pregnancy and after childbirth, isoprene remained absent. All genes of mevalonate pathway enzymes were normally expressed in all participants, without any down-regulation or compensatory up-regulation.Conclusions: Absence/deficiency of isoprene despite normal lipid profiles and no mevalonate pathway malfunction disqualifies the long-believed metabolic origin of isoprene from cholesterol biosynthesis. Thus, clinical translation of breath isoprene expressions should not be generally attributed to corresponding pathophysiological effects onto mevalonate/cholesterol pathway. Our finding has refined and optimized the clinical interpretation of isoprene as biomarker in volatile metabolomics and breathomics. Future studies will address the correct metabolic origin of isoprene to imply this important marker to routine practice.

A putative origin of the insect chemosensory receptor superfamily in the last common eukaryotic ancestor

The insect chemosensory repertoires of Odorant Receptors (ORs) and Gustatory Receptors (GRs) together represent one of the largest families of ligand-gated ion channels. Previous analyses have identified homologous ‘Gustatory Receptor-Like’ (GRL) proteins across Animalia, but the evolutionary origin of this novel class of ion channels is unknown. We describe a survey of unicellular eukaryotic genomes for GRLs, identifying several candidates in fungi, protists and algae that contain many structural features characteristic of animal GRLs. The existence of these proteins in unicellular eukaryotes, together with ab initio protein structure predictions, provide evidence for homology between GRLs and a family of uncharacterized plant proteins containing the DUF3537 domain. Together, our analyses suggest an origin of this protein superfamily in the last common eukaryotic ancestor.