Comparing diversity levels in environmental samples: DNA sequence capture and metabarcoding approaches using 18S and COI genes

Environmental DNA studies targeting multiple taxa using metabarcoding provide remarkable insights into levels of species diversity in any habitat. The main drawbacks are the presence of primer bias and difficulty in identifying rare species. We tested a DNA sequence‐capture method in parallel with the metabarcoding approach to reveal possible advantages of one method over the other. Both approaches were performed using the same eDNA samples and the same 18S and COI regions, followed by high throughput sequencing. Metabarcoded eDNA libraries were PCR amplified with one primer pair from 18S and COI genes. DNA sequence‐capture libraries were enriched with 3,639 baits targeting the same gene regions. We tested amplicon sequence variants (ASVs) and operational taxonomic units (OTUs) in silico approaches for both markers and methods, using for this purpose the metabarcoding data set. ASVs methods uncovered more species for the COI gene, whereas the opposite occurred for the 18S gene, suggesting that clustering reads into OTUs could bias diversity richness especially using 18S with relaxed thresholds. Additionally, metabarcoding and DNA sequence‐capture recovered 80%–90% of the control sample species. DNA sequence‐capture was 8x more expensive, nonetheless it identified 1.5x more species for COI and 13x more genera for 18S than metabarcoding. Both approaches offer reliable results, sharing ca. 40% species and 72% families and retrieve more taxa when nuclear and mitochondrial markers are combined. eDNA metabarcoding is quite well established and low‐cost, whereas DNA‐sequence capture for biodiversity assessment is still in its infancy, is more time‐consuming but provides more taxonomic assignments.     

Development and validation of an experimental and theoretical method for the chiral discrimination of dinotefuran

Dinotefuran is a low-cost agrochemical considered a highly toxic product. In this sense, there is a need for its constant environmental, biological, and food control, aiming to ensure its use to humans as well as to preserve biodiversity and ecosystems. In the present work, we developed an experimental and theoretical method for dinotefuran chiral discrimination. According to the main results, the dinotefuran enantioselective separation was efficiently optimized by high-performance liquid chromatography evaluating the influence of different percentage compositions in the mobile phase to improve the resolution of the peaks in the chromatogram. The novelty of this work was the proposition of a reduced molecular model for the chiral selector amylose-Tris-(3,5-dimethylphenylcarbamate) polysaccharide that was able to adequately describe at the molecular level its interaction with the dinotefuran enantiomers. Besides, the thermodynamic and structural parameters obtained via density functional theory calculations pointed out the chiral discrimination as well as the enantiomeric elution order of the analyte studied, confirming the experimental data, thus validating our proposed method. Finally, hydrogen bonds and repulsive interactions played a key role in the discrimination between the diastereomeric complexes, and consequently, for the dinotefuran enantioselective separation.     

Role of B lymphocytes in the infarcted mass in patients with acute myocardial infarction

Despite early reperfusion, patients with ST segment elevation myocardial infarction (STEMI) may present large myocardial necrosis and significant impairment of ventricular function. The present study aimed to evaluate the role of subtypes of B lymphocytes and related cytokines in the infarcted mass and left ventricular ejection fraction obtained by cardiac magnetic resonance imaging performed after 30 days of STEMI. This prospective study included 120 subjects with STEMI submitted to pharmacoinvasive strategy. Blood samples were collected in subjects in the first (D1) and 30th (D30) days post STEMI. The amount of CD11b+ B1 lymphocytes (cells/ml) at D1 were related to the infarcted mass (rho = 0.43; P=0.033), measured by cardiac MRI at D30. These B1 cells were associated with CD4+ T lymphocytes at D1 and D30, while B2 classic lymphocytes at day 30 were related to left ventricular ejection fraction (LVEF). Higher titers of circulating IL-4 and IL-10 were observed at D30 versus D1 (P=0.013 and P<0.001, respectively). Titers of IL-6 at D1 were associated with infarcted mass (rho = 0.41, P<0.001) and inversely related to LVEF (rho = −0.38, P<0.001). After multiple linear regression analysis, high-sensitivity troponin T and IL-6 collected at day 1 were independent predictors of infarcted mass and, at day 30, only HDL-C. Regarding LVEF, high-sensitivity troponin T and high-sensitivity C-reactive protein were independent predictors at day 1, and B2 classic lymphocytes, at day 30. In subjects with STEMI, despite early reperfusion, the amount of infarcted mass and ventricular performance were related to inflammatory responses triggered by circulating B lymphocytes.     

Computer-Guided Trypanocidal Activity of Natural Lactones Produced by Endophytic Fungus of Euphorbia umbellata

Hundreds of millions of people worldwide are affected by Chagas’ disease caused by Trypanosoma cruzi. Since the current treatment lack efficacy, specificity, and suffers from several side-effects, novel therapeutics are mandatory. Natural products from endophytic fungi have been useful sources of lead compounds. In this study, three lactones isolated from an endophytic strain culture were in silico evaluated for rational guidance of their bioassay screening. All lactones displayed in vitro activity against T. cruzi epimastigote and trypomastigote forms. Notably, the IC₅₀ values of (+)-phomolactone were lower than benznidazole (0.86 vs. 30.78 μM against epimastigotes and 0.41 vs. 4.88 μM against trypomastigotes). Target-based studies suggested that lactones displayed their trypanocidal activities due to T. cruzi glyceraldehyde-3-phosphate dehydrogenase (TcGAPDH) inhibition, and the binding free energy for all three TcGAPDH-lactone complexes suggested that (+)-phomolactone has a lower score value (−3.38), corroborating with IC₅₀ assays. These results highlight the potential of these lactones for further anti-T. cruzi drug development.     

The Crown Pearl: a draft genome assembly of the European freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758)

Since historical times, the inherent human fascination with pearls turned the freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758) into a highly valuable cultural and economic resource. Although pearl harvesting in M. margaritifera is nowadays residual, other human threats have aggravated the species conservation status, especially in Europe. This mussel presents a myriad of rare biological features, e.g. high longevity coupled with low senescence and Doubly Uniparental Inheritance of mitochondrial DNA, for which the underlying molecular mechanisms are poorly known. Here, the first draft genome assembly of M. margaritifera was produced using a combination of Illumina Paired-end and Mate-pair approaches. The genome assembly was 2.4 Gb long, possessing 105,185 scaffolds and a scaffold N50 length of 288,726 bp. The ab initio gene prediction allowed the identification of 35,119 protein-coding genes. This genome represents an essential resource for studying this species’ unique biological and evolutionary features and ultimately will help to develop new tools to promote its conservation.     

The glyceraldehyde-3-phosphate dehydrogenase promoter of the food yeast Candida utilis strain NRRL Y-660 is functional in Agrobacterium tumefaciens

The glyceraldehyde-3-phosphate dehydrogenase promoter of the food yeast Candida utilis strain NRRL Y-660 was cloned to create a novel integrative vector for Agrobacterium tumefaciens-mediated transformation. The new binary vector harbors β-glucuronidase activity as reporter and kanamicin/geneticin resistance as selection marker. Recombinant clones of A. tumefaciens show kanamycin resistance and high β-glucuronidase activity under the control of the C. utilis promoter. This finding can be explained by the presence of a prokaryotic core in the yeast promoter, predicted by in silico analysis of the sequence. This is the first report about functionality of a yeast promoter in A. tumefaciens.     

Intermittent Explosive Disorder amongst Women in Conflict Affected Timor-Leste: Associations with Human Rights Trauma,Ongoing Violence,Poverty, and Injustice

Introduction

Women in conflict-affected countries are at risk of mental disorders such as posttraumatic stress disorder and depression. No studies have investigated the association between experiences of abuse and injustice and explosive anger amongst women in these settings, and the impact of anger on women''s health, family relationships and ability to participate in development.

Methods

A mixed methods study including an epidemiological survey (n = 1513, 92.6% response) and qualitative interviews (n = 77) was conducted in Timor-Leste. The indices measured included Intermittent Explosive Disorder, posttraumatic stress disorder; severe distress; days out of role (the number of days that the person was unable to undertake normal activities); gender-specific trauma; conflict/violence; poverty; and preoccupations with injustice.

Results

Women with Intermittent Explosive Disorder (n = 184, 12.2%) were more disabled than those without the disorder (for >5 days out of role, 40.8% versus 31.5%, X² ₍₂₎  = 12.93 p = 0.0016). Multivariable associations with Intermittent Explosive Disorder, controlling for the presence of PTSD, psychological distress and other predictors in the model, included the sense of being sick (OR 1.73; 95% CI 1.08–2.77); victimization as a result of helping the resistance movement (OR 2.33, 95% CI 1.48–3.68); war-related trauma specific to being a woman (OR 1.95, 95%, CI 1.09–3.50); ongoing family violence and community conflict (OR 1.88, 95% CI 1.27–2.77); extreme poverty (OR 1.23, 95%, CI 1.08–1.39); and distressing preoccupations with injustice (relating to 2/3 historical periods, OR 2.10, 95% CI 1.35–3.28). In the qualitative study, women elaborated on the determinants of anger and its impact on their health, family and community functioning, child-rearing, and capacity to engage in development. Women reflected on the strategies that might help them overcome their anger.

Conclusions

Intermittent Explosive Disorder is prevalent and disabling amongst women in conflict-affected Timor-Leste, impacting on their health, child-rearing and ability to participate fully in socio-economic development.