In silico and empirical evaluation of twelve metabarcoding primer sets for insectivorous diet analyses

During the most recent decade, environmental DNA metabarcoding approaches have been both developed and improved to minimize the biological and technical biases in these protocols. However, challenges remain, notably those relating to primer design. In the current study, we comprehensively assessed the performance of ten COI and two 16S primer pairs for eDNA metabarcoding, including novel and previously published primers. We used a combined approach of in silico, in vivo‐mock community (33 arthropod taxa from 16 orders), and guano‐based analyses to identify primer sets that would maximize arthropod detection and taxonomic identification, successfully identify the predator (bat) species, and minimize the time and financial costs of the experiment. We focused on two insectivorous bat species that live together in mixed colonies: the greater horseshoe bat (Rhinolophus ferrumequinum) and Geoffroy's bat (Myotis emarginatus). We found that primer degeneracy is the main factor that influences arthropod detection in silico and mock community analyses, while amplicon length is critical for the detection of arthropods from degraded DNA samples. Our guano‐based results highlight the importance of detecting and identifying both predator and prey, as guano samples can be contaminated by other insectivorous species. Moreover, we demonstrate that amplifying bat DNA does not reduce the primers' capacity to detect arthropods. We therefore recommend the simultaneous identification of predator and prey. Finally, our results suggest that up to one‐third of prey occurrences may be unreliable and are probably not of primary interest in diet studies, which may decrease the relevance of combining several primer sets instead of using a single efficient one. In conclusion, this study provides a pragmatic framework for eDNA primer selection with respect to scientific and methodological constraints.     

Relationship between Obesity and Massive Transfusion Needs in Trauma Patients,and Validation of TASH Score in Obese Population: A Retrospective Study on 910 Trauma Patients

Background

Prediction of massive transfusion (MT) is challenging in management of trauma patients. However, MT and its prediction were poorly studied in obese patients. The main objective was to assess the relationship between obesity and MT needs in trauma patients. The secondary objectives were to validate the Trauma Associated Severe Hemorrhage (TASH) score in predicting MT in obese patients and to use a grey zone approach to optimize its ability to predict MT.

Methods and Findings

An observational retrospective study was conducted in a Level I Regional Trauma Center Trauma in obese and non-obese patients. MT was defined as ≥10U of packed red blood cells in the first 24h and obesity as a BMI≥30kg/m². Between January 2008 and December 2012, 119 obese and 791 non-obese trauma patients were included. The rate of MT was 10% (94/910) in the whole population. The MT rate tended to be higher in obese patients than in non-obese patients: 15% (18/119, 95%CI 9‒23%) versus 10% (76/791, 95%CI 8‒12%), OR, 1.68 [95%CI 0.97‒2.92], p = 0.07. After adjusting for Injury Severity Score (ISS), obesity was significantly associated with MT rate (OR, 1.79[95%CI 1.00‒3.21], p = 0.049). The TASH score was higher in the obese group than in the non-obese group: 7(4–11) versus 5(2–10)(p<0.001). The area under the ROC curves of the TASH score in predicting MT was very high and comparable between the obese and non-obese groups: 0.93 (95%CI, 0.89‒0.98) and 0.94 (95%CI, 0.92‒0.96), respectively (p = 0.80). The grey zone ranged respectively from 10 to 13 and from 9 to 12 in obese and non obese patients, and allowed separating patients at low, intermediate or high risk of MT using the TASH score.

Conclusions

Obesity was associated with a higher rate of MT in trauma patients. The predictive performance of the TASH score and the grey zones were robust and comparable between obese and non-obese patients.     

Differential expression levels of aroma-related genes during ripening of apricot (Prunus armeniaca L.)

Fruit aroma is a complex trait, particularly in terms of the number of different biosynthetic pathways involved, the complexity of the final metabolites, and their regulation. In order to understand the underlying biochemical processes involved in apricot aroma, four cDNAs (Pa-aat, EU784138; Pa-adh EU395433; Pa-pdc EU395434; and Pa-lox EU439430) encoding an alcohol acyl transferase (AAT), alcohol dehydrogenase (ADH), pyruvate decarboxylase (PDC), and lipoxygenase (LOX), respectively, were isolated and characterized at four stages of maturity in Prunus armeniaca L. cv. Modesto. We observed a reduction in aldehyde and alcohol production between early-harvested fruit and late-harvest fruit, concomitant with an increase in ester production. qPCR analyses showed that the expression levels of the adh gene and the lox gene stayed constant at all stages. Interestingly, aat levels showed a sharp increase in the late-harvest stages concurrent with the changes observed in ester levels. The significance of these changes in relation to aroma production in apricot is discussed.     

Metabarcoding for the parallel identification of several hundred predators and their prey: Application to bat species diet analysis

Assessing diet variability is of main importance to better understand the biology of bats and design conservation strategies. Although the advent of metabarcoding has facilitated such analyses, this approach does not come without challenges. Biases may occur throughout the whole experiment, from fieldwork to biostatistics, resulting in the detection of false negatives, false positives or low taxonomic resolution. We detail a rigorous metabarcoding approach based on a short COI minibarcode and two‐step PCR protocol enabling the “all at once” taxonomic identification of bats and their arthropod prey for several hundreds of samples. Our study includes faecal pellets collected in France from 357 bats representing 16 species, as well as insect mock communities that mimic bat meals of known composition, negative and positive controls. All samples were analysed using three replicates. We compare the efficiency of DNA extraction methods, and we evaluate the effectiveness of our protocol using identification success, taxonomic resolution, sensitivity and amplification biases. Our parallel identification strategy of predators and prey reduces the risk of mis‐assigning prey to wrong predators and decreases the number of molecular steps. Controls and replicates enable to filter the data and limit the risk of false positives, hence guaranteeing high confidence results for both prey occurrence and bat species identification. We validate 551 COI variants from arthropod including 18 orders, 117 family, 282 genus and 290 species. Our method therefore provides a rapid, resolutive and cost‐effective screening tool for addressing evolutionary ecological issues or developing “chirosurveillance” and conservation strategies.     

Monocyte/macrophage dysfunctions do not impair the promotion of myelofibrosis by high levels of thrombopoietin

Several lines of evidence indicate that the megakaryocyte/platelet lineage is crucial in myelofibrosis induction. The demonstration that NOD/SCID mice with functionally deficient monocytes do not develop fibrotic changes when exposed to thrombopoietin (TPO) also suggests an important role for monocyte/macrophages. However, in this animal model, the development of myelofibrosis is dependent on the level of TPO. This study was conducted to investigate whether NOD/SCID mice exposed to high TPO levels mediated by a retroviral vector would be refractory to the development of bone marrow fibrosis. We show that TPO and TGF-beta1 in plasma from NOD/SCID and SCID mice engrafted with TPO-overexpressing hemopoietic cells reach levels similar to the ones reached in immunocompetent mice, and all animals develop a myeloproliferative disease associated with a dense myelofibrosis at 8 wk posttransplantation. Monocytes in NOD/SCID mice are functionally deficient to secrete cytokines such as IL-1alpha in response to stimuli, even under TPO expression. Surprisingly, the plasma of these mice displays high levels of IL-alpha, which was demonstrated to originate from platelets. Together, these data suggest that completely functional monocytes are not required to develop myelofibrosis and that platelets are able, under TPO stimulation, to synthesize inflammatory cytokines, which may be involved in the pathogenesis of myelofibrosis and osteosclerosis.     

Combination of Linkage Mapping and Microarray-Expression Analysis Identifies NF-κB Signaling Defect as a Cause of Autosomal-Recessive Mental Retardation

Autosomal-recessive inheritance accounts for nearly 25% of nonsyndromic mental retardation (MR), but the extreme heterogeneity of such conditions markedly hampers gene identification. Combining autozygosity mapping and RNA expression profiling in a consanguineous Tunisian family of three MR children with mild microcephaly and white-matter abnormalities identified the TRAPPC9 gene, which encodes a NF-κB-inducing kinase (NIK) and IκB kinase complex β (IKK-β) binding protein, as a likely candidate. Sequencing analysis revealed a nonsense variant (c.1708C>T [p.R570X]) within exon 9 of this gene that is responsible for an undetectable level of TRAPPC9 protein in patient skin fibroblasts. Moreover, TNF-α stimulation assays showed a defect in IkBα degradation, suggesting impaired NF-κB signaling in patient cells. This study provides evidence of an NF-κB signaling defect in isolated MR.