Adaptation and convergence in circadian-related genes in Iberian freshwater fish.

BACKGROUND: The circadian clock is a biological timing system that improves the ability of organisms to deal with environmental fluctuations. At the molecular level it consists of a network of transcription-translation feedback loops, involving genes that activate (bmal and clock - positive loop) and repress expression (cryptochrome (cry) and period (per) - negative loop). This is regulated by daily alternations of light but can also be affected by temperature. Fish, as ectothermic, depend on the environmental temperature and thus are good models to study its integration within the circadian system. Here, we studied the molecular evolution of circadian genes in four Squalius freshwater fish species, distributed across Western Iberian rivers affected by two climatic types with different environmental conditions (e.g., light and temperature). S. carolitertii and S. pyrenaicus inhabit the colder northern region under Atlantic climate type, while S. torgalensis, S. aradensis and some populations of S. pyrenaicus inhabit the warmer southern region affected by summer droughts, under Mediterranean climate type. RESULTS: We identified 16 circadian-core genes in the Squalius species using a comparative transcriptomics approach. We detected evidence of positive selection in 12 of these genes using methods based on dN/dS. Positive selection was mainly found in cry and per genes of the negative loop, with 55 putatively adaptive substitutions, 16 located on protein domains. Evidence for positive selection is predominant in southern populations affected by the Mediterranean climate type. By predicting protein features we found that changes at sites under positive selection can impact protein thermostability by changing their aliphatic index and isoelectric point. Additionally, in nine genes, the phylogenetic clustering of species that belong to different clades but inhabit southern basins with similar environmental conditions indicated evolutionary convergence. We found evidence for increased nonsynonymous substitution rate in convergent lineages, likely due to positive selection at 27 sites, mostly in cry genes. CONCLUSIONS: Our results support that temperature may be a selective pressure driving the evolution of genes involved in the circadian system. By integrating sequence-based functional protein prediction with dN/dS-based methods to detect selection we uncovered adaptive convergence in the southern populations, probably related to their similar thermal conditions.

The Personal Genome Project-UK, an open access resource of human multi-omics data.

Integrative analysis of multi-omics data is a powerful approach for gaining functional insights into biological and medical processes. Conducting these multifaceted analyses on human samples is often complicated by the fact that the raw sequencing output is rarely available under open access. The Personal Genome Project UK (PGP-UK) is one of few resources that recruits its participants under open consent and makes the resulting multi-omics data freely and openly available. As part of this resource, we describe the PGP-UK multi-omics reference panel consisting of ten genomic, methylomic and transcriptomic data. Specifically, we outline the data processing, quality control and validation procedures which were implemented to ensure data integrity and exclude sample mix-ups. In addition, we provide a REST API to facilitate the download of the entire PGP-UK dataset. The data are also available from two cloud-based environments, providing platforms for free integrated analysis. In conclusion, the genotype-validated PGP-UK multi-omics human reference panel described here provides a valuable new open access resource for integrated analyses in support of personal and medical genomics.

Author Correction: Critical steps in clinical shotgun metagenomics for the concomitant detection and typing of microbial pathogens.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Plasmid ATLAS: plasmid visual analytics and identification in high-throughput sequencing data.

Plasmid ATLAS (pATLAS, http://www.patlas.site) provides an easy-to-use web accessible database with visual analytics tools to explore the relationships of plasmids available in NCBI's RefSeq database. pATLAS has two main goals: (i) to provide an easy way to search for plasmids deposited in NCBI RefSeq and their associated metadata; (ii) to visualize the relationships of plasmids in a graph, allowing the exploration of plasmid evolution. pATLAS allows searching by plasmid name, bacterial host taxa, antibiotic resistance and virulence genes, plasmid families, and by sequence length and similarity. pATLAS is also able to represent in the plasmid network, plasmid sets identified by external pipelines using mapping, mash screen or assembly from high-throughput sequencing data. By representing the identified hits within the network of relationships between plasmids, allowing the possibility of removing redundant results, and by taking advantage of the browsing capabilities of pATLAS, users can more easily interpret the pipelines' results. All these analyses can be saved to a JSON file for sharing and future re-evaluation. Furthermore, by offering a REST-API, the pATLAS database and network display are easily accessible by other interfaces or pipelines.

Critical steps in clinical shotgun metagenomics for the concomitant detection and typing of microbial pathogens.

High throughput sequencing has been proposed as a one-stop solution for diagnostics and molecular typing directly from patient samples, allowing timely and appropriate implementation of measures for treatment, infection prevention and control. However, it is unclear how the variety of available methods impacts the end results. We applied shotgun metagenomics on diverse types of patient samples using three different methods to deplete human DNA prior to DNA extraction. Libraries were prepared and sequenced with Illumina chemistry. Data was analyzed using methods likely to be available in clinical microbiology laboratories using genomics. The results of microbial identification were compared to standard culture-based microbiological methods. On average, 75% of the reads corresponded to human DNA, being a major determinant in the analysis outcome. None of the kits was clearly superior suggesting that the initial ratio between host and microbial DNA or other sample characteristics were the major determinants of the proportion of microbial reads. Most pathogens identified by culture were also identified through metagenomics, but substantial differences were noted between the taxonomic classification tools. In two cases the high number of human reads resulted in insufficient sequencing depth of bacterial DNA for identification. In three samples, we could infer the probable multilocus sequence type of the most abundant species. The tools and databases used for taxonomic classification and antimicrobial resistance identification had a key impact on the results, recommending that efforts need to be aimed at standardization of the analysis methods if metagenomics is to be used routinely in clinical microbiology.

Different ecophysiological responses of freshwater fish to warming and acidification.

Future climate change scenarios predict threatening outcomes to biodiversity. Available empirical data concerning biological response of freshwater fish to climate change remains scarce. In this study, we investigated the physiological and biochemical responses of two Iberian freshwater fish species (Squalius carolitertii and the endangered S. torgalensis), inhabiting different climatic conditions, to projected future scenarios of warming (+3°C) and acidification (ΔpH=-0.4). Herein, metabolic enzyme activities of glycolytic (citrate synthase - CS, lactate dehydrogenase - LDH) and antioxidant (glutathione S-transferase, catalase and superoxide dismutase) pathways, as well as the heat shock response (HSR) and lipid peroxidation were determined. Our results show that, under current water pH, warming causes differential interspecific changes on LDH activity, increasing and decreasing its activity in S. carolitertii and in S. torgalensis, respectively. Furthermore, the synergistic effect of warming and acidification caused an increase in LDH activity of S. torgalensis, comparing with the warming condition. As for CS activity, acidification significantly decreased its activity in S. carolitertii whereas in S. torgalensis no significant effect was observed. These results suggest that S. carolitertii is more vulnerable to climate change, possibly as the result of its evolutionary acclimatization to milder climatic condition, while S. torgalensis evolved in the warmer Mediterranean climate. However, significant changes in HSR were observed under the combined warming and acidification (S. carolitertii) or under acidification (S. torgalensis). Our results underlie the importance of conducting experimental studies and address species endpoint responses under projected climate change scenarios to improve conservation strategies, and to safeguard endangered freshwater fish.

Protein analysis and gene expression indicate differential vulnerability of Iberian fish species under a climate change scenario.

Current knowledge on the biological responses of freshwater fish under projected scenarios of climate change remains limited. Here, we examine differences in the protein configuration of two endemic Iberian freshwater fish species, Squalius carolitertii and the critically endangered S. torgalensis that inhabit in the Atlantic-type northern and in the Mediterranean-type southwestern regions, respectively. We performed protein structure modeling of fourteen genes linked to protein folding, energy metabolism, circadian rhythms and immune responses. Structural differences in proteins between the two species were found for HSC70, FKBP52, HIF1α and GPB1. For S. torgalensis, besides structural differences, we found higher thermostability for two proteins (HSP90 and GBP1), which can be advantageous in a warmer environment. Additionally, we investigated how these species might respond to projected scenarios of 3° climate change warming, acidification (ΔpH = -0.4), and their combined effects. Significant changes in gene expression were observed in response to all treatments, particularly under the combined warming and acidification. While S. carolitertii presented changes in gene expression for multiple proteins related to folding (hsp90aa1, hsc70, fkbp4 and stip1), only one such gene was altered in S. torgalensis (stip1). However, S. torgalensis showed a greater capacity for energy production under both the acidification and combined scenarios by increasing cs gene expression and maintaining ldha gene expression in muscle. Overall, these findings suggest that S. torgalensis is better prepared to cope with projected climate change. Worryingly, under the simulated scenarios, disturbances to circadian rhythm and immune system genes (cry1aa, per1a and gbp1) raise concerns for the persistence of both species, highlighting the need to consider multi-stressor effects when evaluating climate change impacts upon fish. This work also highlights that assessments of the potential of endangered freshwater species to cope with environmental change are crucial to help decision-makers adopt future conservation strategies.

Different levels of hsp70 and hsc70 mRNA expression in Iberian fish exposed to distinct river conditions.

Comprehension of the mechanisms by which ectotherms, such as fish, respond to thermal stress is paramount for understanding the threats that environmental changes may pose to wild populations. Heat shock proteins are molecular chaperones with an important role in several stress conditions such as high temperatures. In the Iberian Peninsula, particularly in Portugal, freshwater fish of the genus Squalius are subject to daily and seasonal temperature variations. To examine the extent to which different thermal regimes influence the expression patterns of hsp70 and hsc70 transcripts we exposed two species of Squalius (S. torgalensis and S. carolitertii) to different temperatures (20, 25, 30 and 35 °C). At 35 °C, there was a significant increase in the expression of hsp70 and hsc70 in the southern species, S. torgalensis, while the northern species, S. carolitertii, showed no increase in the expression of these genes; however, some individuals of the latter species died when exposed to 35 °C. These results suggest that S. torgalensis may cope better with harsher temperatures that are characteristic of this species natural environment; S. carolitertii, on the other hand, may be unable to deal with the extreme temperatures faced by the southern species.