Towards an Inerian DNA barcode reference library of freshwater macroinvertebrates and fishes

Freshwater macroinvertebrates and fishes are focal groups in major ecosystem biomonitoring programs in the Iberian Peninsula. Yet, their use as bioindicators is sometimes constrained by the time and cost needed for sorting macroinvertebrates specimens and their challenging taxonomic identification, and the huge sampling procedures for capturing rare or incipient fish species, respec- tively. Given the increasing cost-effectiveness of metazoan identification based on metabarcoding [i.e., high-throughput sequenc- ing (HTS) of DNA barcodes] and reliability of species-level identification and the high number of samples that can be processed, its use in biomonitoring of freshwater communities can provide an alternative to morphology-based approaches. However, the accuracy of species assignment in metabarcoding approaches relies on the availability of a comprehensive DNA barcode reference library. Because of the high level of endemicity in the Iberian Peninsula, current public repositories for DNA barcodes may not be informative enough to identify the Iberian fauna to species level. Here, we compiled the Iberian freshwater macroinvertebrates and fishes taxonomic list (including indigenous and non-indigenous species) and the available molecular data for the cytochrome oxidase I DNA barcode (cox1, COI-5P) in public repositories to assess the extent of DNA barcode coverage. The DNA barcode coverage was reported for DNA fragments within the Folmer region (658 bp). Given that HTS platforms provide DNA sequence in the range of 50-400 bp in length, we also reported the second half of the DNA barcode (313 bp, Leray region) and the first part of the Leray region (285 bp, Leray-285), which are short DNA barcodes useful to assign metabarcoding cox1 data. For macroin- vertebrates, the final taxonomic checklist comprises 3348 species including Mollusca (65 species), Crustacea (101 species) and Insecta (3182 species). We present an initial DNA barcode reference library, with an overall coverage of ~ 35 % of the Iberian taxa. Exploring this data, we find a strong taxonomic bias. Based on Leray-285, Odonata (43 of 79 species barcoded, 54.43 %) and Hemiptera (44 of 81 species barcoded, 54.32 %) were the best represented lineages. In contrast, Diptera (393 of 1693 species barcoded, 23.21 %), and Plecoptera (42 of 135 species barcoded, 31.11 %) were underrepresented. For fishes, the available DNA barcode data covered 98.11 % of the indigenous (76) and non-indigenous (30) species. By revealing and quantifying current gaps on the available data (~ 65 %), we aim to improve efficiency in designing the next steps towards the ambitious yet necessary goal of compiling a complete DNA barcode reference library for Iberian macroinvertebrates and fishes

Liver Resection for Primary Hepatic Neoplasms.

Subtotal hepatic resection was performed in 356 patients; 87 had primary hepatic malignancies, 108 had metastatic tumors, and 161 had benign lesions including 8 traumatic injuries. The global mortality was 4.2%. The experience has elucidated the role of subtotal hepatic resection both for benign and malignant neoplasms

HMG1A and PPARG are differently expressed in the liver of fat and lean broilers

The expression of nine functional candidates for QT abdominal fat weight and relative abdominal fat content was investigated by real-time polymerase chain reaction (PCR) in the liver, adipose tissue, colon, muscle, pituitary gland and brain of broilers. The high mobility group AT-hook 1 (HMG1A) gene was up-regulated in liver with a ratio of means of 2.90 (P ≤ 0.01) in the «fatty» group (relative abdominal fat content 3.5 ± 0.18%, abdominal fat weight 35.4 ± 6.09 g) relative to the «lean» group (relative abdominal fat content 1.9 ± 0.56%, abdominal fat weight 19.2 ± 5.06 g). Expression of this gene was highly correlated with the relative abdominal fat content (0.70, P ≤ 0.01) and abdominal fat weight (0.70, P ≤ 0.01). The peroxisome proliferator-activated receptor gamma (PPARG) gene was also up-regulated in the liver with a ratio of means of 3.34 (P ≤ 0.01) in the «fatty» group relative to the «lean» group. Correlation of its expression was significant with both the relative abdominal fat content (0.55, P ≤ 0.05) and the abdominal fat weight (0.57, P ≤ 0.01). These data suggest that the HMG1A and PPARG genes were candidate genes for abdominal fat deposition in chickens. Searching of rSNPs in regulatory regions of the HMG1A and PPARG genes could provide a tool for gene-assisted selection

Non‐specific amplification compromises environmental DNA metabarcoding with COI

1. Metabarcoding extra-organismal DNA from environmental samples is now a key technique in aquatic biomonitoring and ecosystem health assessment. However, choice of genetic marker and primer set is a critical consideration when designing experiments, especially so when developing community standards and legislative frameworks. Mitochondrial cytochrome c oxidase subunit I (COI), the standard DNA barcode marker for animals, with its extensive reference library, taxonomic discriminatory power, and predictable sequence variation, is the natural choice for many metabarcoding applications such as the bulk sequencing of invertebrates. However, the overall utility of COI for environmental sequencing of targeted taxonomic groups has yet to be fully scrutinised. 2. Here, by using a case study of marine and freshwater fishes from the British Isles, we quantify the in silico performance of twelve mitochondrial primer pairs from COI, cytochrome b, 12S and 16S, in terms of reference library coverage, taxonomic discriminatory power, and primer universality. We subsequently test in vitro three COI primer pairs and one 12S pair for their specificity, reproducibility, and congruence with independent datasets derived from traditional survey methods at five estuarine and coastal sites in the English Channel and North Sea coast. 3. Our results show that for aqueous extra-organismal DNA at low template concentrations, both metazoan and fish-targeted COI primers perform poorly in comparison to 12S, exhibiting low levels of reproducibility due to non-specific amplification of prokaryotic and non-target eukaryotic DNAs. 4. An ideal metabarcode would have an extensive reference library for which custom primer sets can be designed for either broad assessments of biodiversity or taxon specific surveys, but unfortunately, low primer specificity hinders the use of COI, while the paucity of reference sequences is problematic for 12S. The latter, however, can be mitigated by expanding the concept of DNA barcodes to include whole mitochondrial genomes generated by genome-skimming existing tissue collections

Human malarial disease: a consequence of inflammatory cytokine release

Malaria causes an acute systemic human disease that bears many similarities, both clinically and mechanistically, to those caused by bacteria, rickettsia, and viruses. Over the past few decades, a literature has emerged that argues for most of the pathology seen in all of these infectious diseases being explained by activation of the inflammatory system, with the balance between the pro and anti-inflammatory cytokines being tipped towards the onset of systemic inflammation. Although not often expressed in energy terms, there is, when reduced to biochemical essentials, wide agreement that infection with falciparum malaria is often fatal because mitochondria are unable to generate enough ATP to maintain normal cellular function. Most, however, would contend that this largely occurs because sequestered parasitized red cells prevent sufficient oxygen getting to where it is needed. This review considers the evidence that an equally or more important way ATP deficency arises in malaria, as well as these other infectious diseases, is an inability of mitochondria, through the effects of inflammatory cytokines on their function, to utilise available oxygen. This activity of these cytokines, plus their capacity to control the pathways through which oxygen supply to mitochondria are restricted (particularly through directing sequestration and driving anaemia), combine to make falciparum malaria primarily an inflammatory cytokine-driven disease