The clonal relationships between pre-cancer and cancer revealed by ultra-deep sequencing

The study of the relationships between pre-cancer and cancer and identification of early driver mutations is becoming increasingly important as the value of molecular markers of early disease and personalised drug targets is recognized, especially now the extent of clonal heterogeneity in fully invasive disease is being realized. It has been assumed that pre-cancerous lesions exhibit a fairly passive progression to invasive disease; the degree to which they, too, are heterogeneous is unknown. We performed ultra-deep sequencing of thousands of selected mutations, together with copy number analysis, from multiple, matched pre-invasive lesions, primary tumours and metastases from five patients with oral cancer, some with multiple primary tumours presenting either synchronously or metachronously, totalling 75 samples. This allowed the clonal relationships between the samples to be observed for each patient. We expose for the first time the unexpected variety and complexity of the relationships between this group of oral dysplasias and their associated carcinomas and, ultimately, the diversity of processes by which tumours are initiated, spread and metastasize. Instead of a series of genomic precursors of their adjacent invasive disease, we have shown dysplasia to be a distinct dynamic entity, refuting the belief that pre-cancer and invasive tumours with a close spatial relationship always have linearly related genomes. We show that oral pre-cancer exhibits considerable subclonal heterogeneity in its own right, that mutational changes in pre-cancer do not predict the onset of invasion, and that the genomic pathway to invasion is neither unified nor predictable. Sequence data from this study have been deposited in the European Nucleotide Archive, Accession No. PRJEB6588

Wolbachia in the flesh: symbiont intensities in germ-line and somatic tissues challenge the conventional view of Wolbachia transmission routes

Symbionts can substantially affect the evolution and ecology of their hosts. The investigation of the tissue-specific distribution of symbionts (tissue tropism) can provide important insight into host-symbiont interactions. Among other things, it can help to discern the importance of specific transmission routes and potential phenotypic effects. The intracellular bacterial symbiont Wolbachia has been described as the greatest ever panzootic, due to the wide array of arthropods that it infects. Being primarily vertically transmitted, it is expected that the transmission of Wolbachia would be enhanced by focusing infection in the reproductive tissues. In social insect hosts, this tropism would logically extend to reproductive rather than sterile castes, since the latter constitute a dead-end for vertically transmission. Here, we show that Wolbachia are not focused on reproductive tissues of eusocial insects, and that non-reproductive tissues of queens and workers of the ant Acromyrmex echinatior, harbour substantial infections. In particular, the comparatively high intensities of Wolbachia in the haemolymph, fat body, and faeces, suggest potential for horizontal transmission via parasitoids and the faecal-oral route, or a role for Wolbachia modulating the immune response of this host. It may be that somatic tissues and castes are not the evolutionary dead-end for Wolbachia that is commonly thought

Heat Resistance Mediated by pLM58 Plasmid-Borne ClpL in Listeria monocytogenes

Listeria monocytogenes is one of the most heat-resistant non-sporeforming food-borne pathogens and poses a notable risk to food safety, particularly when mild heat treatments are used in food processing and preparation. While general heat stress properties and response mechanisms of L. monocytogenes have been described, accessory mechanisms providing particular L. monocytogenes strains with the advantage of enhanced heat resistance are unknown. Here, we report plasmidmediated heat resistance of L. monocytogenes for the first time. This resistance is mediated by the ATP-dependent protease ClpL. We tested the survival of two wildtype L. monocytogenes strains-both of serotype 1/2c, sequence type ST9, and high sequence identity-at high temperatures and compared their genome composition in order to identify genetic mechanisms involved in their heat survival phenotype. L. monocytogenes AT3E was more heat resistant (0.0 CFU/ml log(10) reduction) than strain AL4E (1.4 CFU/ml log(10) reduction) after heating at 55 degrees C for 40 min. A prominent difference in the genome compositions of the two strains was a 58-kb plasmid (pLM58) harbored by the heat-resistant AT3E strain, suggesting plasmid-mediated heat resistance. Indeed, plasmid curing resulted in significantly decreased heat resistance (1.1 CFU/ml log(10) reduction) at 55 degrees C. pLM58 harbored a 2,115-bp open reading frame annotated as an ATP-dependent protease (ClpL)-encoding clpL gene. Introducing the clpL gene into a natively heat-sensitive L. monocytogenes strain (1.2 CFU/ml log(10) reduction) significantly increased the heat resistance of the recipient strain (0.4 CFU/ml log(10) reduction) at 55 degrees C. Plasmid-borne ClpL is thus a potential predictor of elevated heat resistance in L. monocytogenes. IMPORTANCE Listeria monocytogenes is a dangerous food pathogen causing the severe illness listeriosis that has a high mortality rate in immunocompromised individuals. Although destroyed by pasteurization, L. monocytogenes is among the most heat-resistant non-spore-forming bacteria. This poses a risk to food safety, as listeriosis is commonly associated with ready-to-eat foods that are consumed without thorough heating. However, L. monocytogenes strains differ in their ability to survive high temperatures, and comprehensive understanding of the genetic mechanisms underlying these differences is still limited. Whole-genome-sequence analysis and phenotypic characterization allowed us to identify a novel plasmid, designated pLM58, and a plasmid-borne ATP-dependent protease (ClpL), which mediated heat resistance in L. monocytogenes. As the first report on plasmid-mediated heat resistance in L. monocytogenes, our study sheds light on the accessory genetic mechanisms rendering certain L. monocytogenes strains particularly capable of surviving high temperatures-with plasmid-borne ClpL being a potential predictor of elevated heat resistance.Peer reviewe

A multiplex marker set for microsatellite typing and sexing of sooty terns Onychoprion fuscatus

OBJECTIVES: Seabirds have suffered dramatic population declines in recent decades with one such species being the sooty tern Onychoprion fuscatus. An urgent call to re-assess their conservation status has been made given that some populations, such as the one on Ascension Island, South Atlantic, have declined by over 80% in three generations. Little is known about their population genetics, which would aid conservation management through understanding ecological processes and vulnerability to environmental change. We developed a multiplex microsatellite marker set for sooty terns including sex-typing markers to assist population genetics studies. RESULTS: Fifty microsatellite loci were isolated and tested in 23 individuals from Ascension Island. Thirty-one were polymorphic and displayed between 4 and 20 alleles. Three loci were Z-linked and two autosomal loci deviated from Hardy-Weinberg equilibrium. The remaining 26 autosomal loci together with three sex-typing makers were optimised in seven polymerase chain reaction plexes. These 26 highly polymorphic markers will be useful for understanding genetic structure of the Ascension Island population and the species as a whole. Combining these with recently developed microsatellite markers isolated from Indian Ocean birds will allow for assessment of global population structure and genetic diversity

Genetic diversity, linkage disequilibrium and power of a large grapevine (Vitis vinifera L) diversity panel newly designed for association studies

UMR-AGAP Equipe DAVV (Diversité, adaptation et amélioration de la vigne) ; équipe ID (Intégration de Données)International audienceAbstractBackgroundAs for many crops, new high-quality grapevine varieties requiring less pesticide and adapted to climate change are needed. In perennial species, breeding is a long process which can be speeded up by gaining knowledge about quantitative trait loci linked to agronomic traits variation. However, due to the long juvenile period of these species, establishing numerous highly recombinant populations for high resolution mapping is both costly and time-consuming. Genome wide association studies in germplasm panels is an alternative method of choice, since it allows identifying the main quantitative trait loci with high resolution by exploiting past recombination events between cultivars. Such studies require adequate panel design to represent most of the available genetic and phenotypic diversity. Assessing linkage disequilibrium extent and panel power is also needed to determine the marker density required for association studies.ResultsStarting from the largest grapevine collection worldwide maintained in Vassal (France), we designed a diversity panel of 279 cultivars with limited relatedness, reflecting the low structuration in three genetic pools resulting from different uses (table vs wine) and geographical origin (East vs West), and including the major founders of modern cultivars. With 20 simple sequence repeat markers and five quantitative traits, we showed that our panel adequately captured most of the genetic and phenotypic diversity existing within the entire Vassal collection. To assess linkage disequilibrium extent and panel power, we genotyped single nucleotide polymorphisms: 372 over four genomic regions and 129 distributed over the whole genome. Linkage disequilibrium, measured by correlation corrected for kinship, reached 0.2 for a physical distance between 9 and 458 Kb depending on genetic pool and genomic region, with varying size of linkage disequilibrium blocks. This panel achieved reasonable power to detect associations between traits with high broad-sense heritability (> 0.7) and causal loci with intermediate allelic frequency and strong effect (explaining > 10 % of total variance).ConclusionsOur association panel constitutes a new, highly valuable resource for genetic association studies in grapevine, and deserves dissemination to diverse field and greenhouse trials to gain more insight into the genetic control of many agronomic traits and their interaction with the environment

Characterization of microsatellite markers for Moricandia moricandioides (Brassicaceae) and related species

PREMISE OF THE STUDY: Polymorphic microsatellite markers were developed to study population structure and mating patterns of the monocarpic herb Moricandia moricandioides (Brassicaceae). METHODS AND RESULTS: Illumina MiSeq sequencing was used to develop a panel of 15 polymorphic microsatellite markers that were tested across 77 individuals from three populations on the Iberian Peninsula. All markers were polymorphic in at least two studied populations, and the number of alleles ranged from one to 11 per locus. The levels of observed and expected heterozygosity ranged from 0.000 to 1.000 and from 0.153 to 0.865, respectively. Nine and 11 loci were successfully amplified in the congeneric species M. arvensis and M. foetida, respectively. CONCLUSIONS: The 15 microsatellite markers will be useful for population genetic studies of the genus Moricandia. These markers will serve as a useful tool for exploring population structure and mating patterns of M. moricandioides.This work received funding from the European Union’s Horizon 2020 (Marie Skłodowska-Curie No. 655653), Fundación BBVA (PR17-ECO- 0021), and the Spanish Ministerio de Economia y Competitividad (CGL2017-86626- C2- 1- P)

Low oxygen affects photophysiology and the level of expression of two-carbon metabolism genes in the seagrass <i>Zostera muelleri</i>

© 2017, Springer Science+Business Media B.V. Seagrasses are a diverse group of angiosperms that evolved to live in shallow coastal waters, an environment regularly subjected to changes in oxygen, carbon dioxide and irradiance. Zostera muelleri is the dominant species in south-eastern Australia, and is critical for healthy coastal ecosystems. Despite its ecological importance, little is known about the pathways of carbon fixation in Z. muelleri and their regulation in response to environmental changes. In this study, the response of Z. muelleri exposed to control and very low oxygen conditions was investigated by using (i) oxygen microsensors combined with a custom-made flow chamber to measure changes in photosynthesis and respiration, and (ii) reverse transcription quantitative real-time PCR to measure changes in expression levels of key genes involved in C4 metabolism. We found that very low levels of oxygen (i) altered the photophysiology of Z. muelleri, a characteristic of C3 mechanism of carbon assimilation, and (ii) decreased the expression levels of phosphoenolpyruvate carboxylase and carbonic anhydrase. These molecular-physiological results suggest that regulation of the photophysiology of Z. muelleri might involve a close integration between the C3 and C4, or other CO2 concentrating mechanisms metabolic pathways. Overall, this study highlights that the photophysiological response of Z. muelleri to changing oxygen in water is capable of rapid acclimation and the dynamic modulation of pathways should be considered when assessing seagrass primary production

Automatic identification of species-specific repetitive DNA sequences and their utilization for detecting microbial organisms

Motivation: The concentration of pathogen DNA in biological samples is often very low. Therefore, the sensitivity of diagnostic tests is always a critical factor

Characterization of Species-Specific Repeats in 613 Prokaryotic Species

Prokaryotes are in general believed to possess small, compactly organized genomes, with repetitive sequences forming only a small part of them. Nonetheless, many prokaryotic genomes in fact contain species-specific repeats (>85 bp long genomic sequences with less than 60% identity to other species) as we have previously demonstrated. However, it is not known at present how frequent such species-specific repeats are and what their functional roles in bacterial genomes may be. Therefore, we have conducted a comprehensive survey of prokaryotic species-specific repeats and characterized them to examine as to whether there are functional classes among different repeats or not and how they are mutually related to each other. Of the 613 distinct prokaryotic species analyzed, 97% were found to contain at least one species-specific repeats. It seems interesting to note that the species-specific repeats thus identified appear to be functionally variable in different genomes: in some genomes, they are mostly associated with duplicated protein-coding genes, whereas in some other genomes with rRNA and tRNA genes. Contrary to what may be expected, only one-fourth of the species-specific repeats were found to be associated with mobile genetic elements

PrimerHunter: a primer design tool for PCR-based virus subtype identification

Rapid and reliable virus subtype identification is critical for accurate diagnosis of human infections, effective response to epidemic outbreaks and global-scale surveillance of highly pathogenic viral subtypes such as avian influenza H5N1. The polymerase chain reaction (PCR) has become the method of choice for virus subtype identification. However, designing subtype-specific PCR primer pairs is a very challenging task: on one hand, selected primer pairs must result in robust amplification in the presence of a significant degree of sequence heterogeneity within subtypes, on the other, they must discriminate between the subtype of interest and closely related subtypes. In this article, we present a new tool, called PrimerHunter, that can be used to select highly sensitive and specific primers for virus subtyping. Our tool takes as input sets of both target and nontarget sequences. Primers are selected such that they efficiently amplify any one of the target sequences, and none of the nontarget sequences. PrimerHunter ensures the desired amplification properties by using accurate estimates of melting temperature with mismatches, computed based on the nearest neighbor model via an efficient fractional programming algorithm. Validation experiments with three avian influenza HA subtypes confirm that primers selected by PrimerHunter have high sensitivity and specificity for target sequences