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Abstract 137Cs is a long-lived man-made radionuclide introduced in the environment worldwide at the early beginning of the nuclear Era during atmospheric nuclear testing’s followed by the civil use of nuclear energy. Atmospheric fallout deposition of this major artificial radionuclide was reconstructed at the scale of French large river basins since 1945, and trajectories in French nuclearized rivers were established using sediment coring. Our results show that 137Cs contents in sediments of the studied rivers display a large spatial and temporal variability in response to the various anthropogenic pressures exerted on their catchment. The Loire, Rhone, and Rhine rivers were the most affected by atmospheric fallout from the global deposition from nuclear tests. Rhine and Rhone also received significant fallout from the Chernobyl accident in 1986 and recorded significant 137Cs concentrations in their sediments over the 1970–1985 period due to the regulatory releases from the nuclear industries. The Meuse River was notably impacted in the early 1970s by industrial releases. In contrast, the Seine River display the lowest 137Cs concentrations regardless of the period. All the rivers responded similarly over time to atmospheric fallout on their catchment, underlying a rather homogeneous resilience capacity of these river systems to this source of contamination.

Crotalphine is an analgesic peptide identified from the venom of the South American rattlesnake Crotalus durissus terrificus. Although its antinociceptive effect is well documented, its direct mechanisms of action are still unclear. The aim of the present work was to study the action of the crotalid peptide on the NaV1.7 channel subtype, a genetically validated pain target. To this purpose, the effects of crotalphine were evaluated on the NaV1.7 component of the tetrodotoxin-sensitive Na+ current in the dorsal root ganglion neurons of adult mice, using the whole-cell patch-clamp configuration, and on cell viability, using propidium iodide fluorescence and trypan blue assays. The results show that 18.7 µM of peptide inhibited 50% of the Na+ current. The blocking effect occurred without any marked change in the current activation and inactivation kinetics, but it was more important as the membrane potential was more positive. In addition, crotalphine induced an increase in the leakage current amplitude of approximately 150% and led to a maximal 31% decrease in cell viability at a high 50 µM concentration. Taken together, these results point out, for the first time, the effectiveness of crotalphine in acting on the NaV1.7 channel subtype, which may be an additional target contributing to the peptide analgesic properties and, also, although less efficiently, on a second cell plasma membrane component, leading to cell loss.

Abstract Background The excessive inflammatory responses provoked by SARS-CoV-2 infection are critical factors affecting the severity and mortality of COVID-19. Previous work found that two adjacent co-occurring mutations R203K and G204R (KR) on the nucleocapsid (N) protein correlate with increased disease severity in COVID-19 patients. However, links with the host immune response remain unclear. Methods Here, we grouped nasopharyngeal swab samples of COVID-19 patients into two cohorts based on the presence and absence of SARS-CoV-2 nucleocapsid KR mutations. We performed nasopharyngeal transcriptome analysis of age, gender, and ethnicity-matched COVID-19 patients infected with either SARS-CoV-2 with KR mutations in the N protein (KR patients n = 39) or with the wild-type N protein (RG patients n = 39) and compared to healthy controls (n = 34). The impact of KR mutation on immune response was further characterized experimentally by transcriptomic and proteomic profiling of virus-like-particle (VLP) incubated cells. Results We observed markedly elevated expression of proinflammatory cytokines, chemokines, and interferon-stimulated (ISGs) genes in the KR patients compared to RG patients. Using nasopharyngeal transcriptome data, we found significantly higher levels of neutrophils and neutrophil-to-lymphocyte (NLR) ratio in KR patients than in the RG patients. Furthermore, transcriptomic and proteomic profiling of VLP incubated cells confirmed a similar hyper-inflammatory response mediated by the KR variant. Conclusions Our data demonstrate an unforeseen connection between nucleocapsid KR mutations and augmented inflammatory immune response in severe COVID-19 patients. These findings provide insights into how mutations in SARS-CoV-2 modulate host immune output and pathogenesis and may contribute to more efficient therapeutics and vaccine development.

Abstract A perennial criticism of provisioning ecotourism is that it alters the natural behavior and ecology of the target species by providing an artificial food source. Here we evaluate its impact on the long-term site fidelity patterns of tiger sharks in French Polynesia. We hypothesized that a significant impact of provisioning would lead to (1) increases in individual site fidelity over time, and (2) an increase in the number of resident individuals over time. Of 53 individuals photo-identified and monitored during > 500 dives over five years, 10 individuals accounted for > 75% of all sightings, whereas 35 sharks were sighted very infrequently. Even the most frequently observed tiger sharks exhibited overall low fidelity at the site and showed no increase in site fidelity over time. Furthermore, the number of tiger sharks sighted during each dive did not increase. The observed patterns of tiger shark sightings were best explained by natural movements, including general roaming within home ranges along the coastline and seasonal migrations. Despite the apparent lack of impact of provisioning ecotourism on tiger shark ecology in Tahitian waters, it would be prudent to implement a strict code of conduct during any future provisioning activities to maximize the safety of participants and animals involved.

Coralsnakes (Micrurus spp.) are the only elapids found throughout the Americas. They are recognized for their highly neurotoxic venom, which is comprised of a wide variety of toxins, including the stable, low-mass toxins known as three-finger toxins (3FTx). Due to difficulties in venom extraction and availability, research on coralsnake venoms is still very limited when compared to that of other Elapidae snakes like cobras, kraits, and mambas. In this study, two previously described 3FTx from the venom of M. corallinus, NXH1 (3SOC1_MICCO), and NXH8 (3NO48_MICCO) were characterized. Using in silico, in vitro, and ex vivo experiments, the biological activities of these toxins were predicted and evaluated. The results showed that only NXH8 was capable of binding to skeletal muscle cells and modulating the activity of nAChRs in nerve–diaphragm preparations. These effects were antagonized by anti-rNXH8 or antielapidic sera. Sequence analysis revealed that the NXH1 toxin possesses eight cysteine residues and four disulfide bonds, while the NXH8 toxin has a primary structure similar to that of non-conventional 3FTx, with an additional disulfide bond on the first loop. These findings add more information related to the structural diversity present within the 3FTx class, while expanding our understanding of the mechanisms of the toxicity of this coralsnake venom and opening new perspectives for developing more effective therapeutic interventions.

Abstract A fungal metabolite, FR235222, specifically inhibits a histone deacetylase of the apicomplexan parasite Toxoplasma gondii and TgHDAC3 has emerged as a key factor regulating developmental stage transition in this species. Here, we exploited FR235222 to ask if changes in histone acetylation regulate developmental stage transition of Theileria annulata, another apicomplexan species. We found that FR235222 treatment of T. annulata-infected transformed leukocytes induced a proliferation arrest. The blockade in proliferation was due to drug-induced conversion of intracellular schizonts to merozoites that lack the ability to maintain host leukocyte cell division. Induction of merogony by FR235222 leads to an increase in expression of merozoite-marker (rhoptry) proteins. RNA-seq of FR235222-treated T. annulata-infected B cells identified deregulated expression of 468 parasite genes including a number encoding parasite ApiAP2 transcription factors. Thus, similar to T. gondii, FR235222 inhibits T. annulata HDAC (TaHDAC1) activity and places parasite histone acetylation as a major regulatory event of the transition from schizonts to merozoites.

Observers of the Polynesian Shark Observatory (ORP), a citizen science network organized mainly through the Polynesian dive centers, collected an unprecedented amount of data from more than 13,916 dives spanning 43% of the islands of French Polynesia between July 8, 2011, and April 11, 2018. The objective for this type of data collection, which is not accessible within the standard research context, was to provide a unique dataset, and the opportunity to explore the specific diversity, distribution, seasonality and abundance of many elasmobranch species spread out throughout the territory of French Polynesia. Since the data are based on random citizen observations, the spatial distribution was biased toward the most frequented sites and islands where scuba diving is most developed. Overall, the increase in observed abundance of rays and sharks observed in French Polynesia, and the three most sampled islands as well as the high specific diversity recorded for the region, provide first evidence on the effectiveness of the French Polynesia’s Shark Sanctuary, established in 2006. These data, collected randomly by the volunteers, also provide insights into potential movement patterns and site fidelity of some of the more commonly observed species. While no final conclusions can be drawn, it is clear that the network of volunteers that regularly contributes information to the Polynesian Shark Observatory plays a very important role in the delivery of much needed data for conservation and management action, as well as providing perspectives for new directions in research on sharks and rays in French Polynesia.

Cell cycle transitions are generally triggered by variation in the activity of cyclin-dependent kinases (CDKs) bound to cyclins. Malaria-causing parasites have a life cycle with unique cell-division cycles, and a repertoire of divergent CDKs and cyclins of poorly understood function and interdependency. We show that Plasmodium berghei CDK-related kinase 5 (CRK5), is a critical regulator of atypical mitosis in the gametogony and is required for mosquito transmission. It phosphorylates canonical CDK motifs of components in the pre-replicative complex and is essential for DNA replication. During a replicative cycle, CRK5 stably interacts with a single Plasmodium-specific cyclin (SOC2), although we obtained no evidence of SOC2 cycling by transcription, translation or degradation. Our results provide evidence that during Plasmodium male gametogony, this divergent cyclin/CDK pair fills the functional space of other eukaryotic cell-cycle kinases controlling DNA replication.

Abstract Mamba venoms contain a multiplicity of three-finger fold aminergic toxins known to interact with various α-adrenergic, muscarinic and dopaminergic receptors with different pharmacological profiles. In order to generate novel functions on this structural scaffold and to avoid the daunting task of producing and screening an overwhelming number of variants generated by a classical protein engineering strategy, we accepted the challenge of resurrecting ancestral proteins, likely to have possessed functional properties. This innovative approach that exploits molecular evolution models to efficiently guide protein engineering, has allowed us to generate a small library of six ancestral toxin (AncTx) variants and associate their pharmacological profiles to key functional substitutions. Among these variants, we identified AncTx1 as the most α1A-adrenoceptor selective peptide known to date and AncTx5 as the most potent inhibitor of the three α2 adrenoceptor subtypes. Three positions in the ρ-Da1a evolutionary pathway, positions 28, 38 and 43 have been identified as key modulators of the affinities for the α1 and α2C adrenoceptor subtypes. Here, we present a first attempt at rational engineering of the aminergic toxins, revealing an epistasis phenomenon.

A novel human protoparvovirus related to human bufavirus and preliminarily named cutavirus has been discovered. We detected cutavirus in a sample of cutaneous malignant melanoma by using viral enrichment and high-throughput sequencing. The role of cutaviruses in cutaneous cancers remains to be investigated.

Dysfunction of the oxidative phosphorylation (OXPHOS) system is a major cause of human disease and the cellular consequences are highly complex. Here, we present comparative analyses of mitochondrial proteomes, cellular transcriptomes and targeted metabolomics of five knockout mouse strains deficient in essential factors required for mitochondrial DNA gene expression, leading to OXPHOS dysfunction. Moreover, we describe sequential protein changes during post-natal development and progressive OXPHOS dysfunction in time course analyses in control mice and a middle lifespan knockout, respectively. Very unexpectedly, we identify a new response pathway to OXPHOS dysfunction in which the intra-mitochondrial synthesis of coenzyme Q (ubiquinone, Q) and Q levels are profoundly decreased, pointing towards novel possibilities for therapy. Our extensive omics analyses provide a high-quality resource of altered gene expression patterns under severe OXPHOS deficiency comparing several mouse models, that will deepen our understanding, open avenues for research and provide an important reference for diagnosis and treatment.

Ageing is a progressive decline of intrinsic physiological functions. We examined the impact of ageing on the ultrastructure and function of mitochondria in mouse and fruit flies (Drosophila melanogaster) by electron cryo-tomography and respirometry. We discovered distinct age-related changes in both model organisms. Mitochondrial function and ultrastructure are maintained in mouse heart, whereas subpopulations of mitochondria from mouse liver show age-related changes in membrane morphology. Subpopulations of mitochondria from young and old mouse kidney resemble those described for apoptosis. In aged flies, respiratory activity is compromised and the production of peroxide radicals is increased. In about 50% of mitochondria from old flies, the inner membrane organization breaks down. This establishes a clear link between inner membrane architecture and functional decline. Mitochondria were affected by ageing to very different extents, depending on the organism and possibly on the degree to which tissues within the same organism are protected against mitochondrial damage.

BACKGROUND:Physical activity at moderate-high intensity is recommended to prevent lifestyle diseases. Patients with atrial fibrillation are at risk of a sedentary lifestyle due to fear of exercise-induced episodes of atrial fibrillation. The burden of arrhythmia can be reduced by physical exercise. The effect of exercise intensity on burden of atrial fibrillation needs to be studied further. METHODS AND RESULTS:In a 12-week randomized controlled trial, 76 patients with paroxysmal/persistent atrial fibrillation were allocated to perform exercise at either low intensity or high intensity (50% and 80% of maximal perceived exertion, respectively). Primary outcome was burden of AF measured by daily electrocardiography-reporting during 12 weeks. Secondarily, change in maximal oxygen uptake (peak VO2) and 1-year hospitalization was compared between low and high intensity exercise. Sixty-three patients completed the follow-up. In the intention-to-treat analysis, we found no statistical difference in burden of atrial fibrillation between low and high intensity exercise (incidence rate ratio 0.742, 95% CI 0.29-1.91, P = 0.538). No serious adverse events were reported and there was no difference in hospitalization between the two exercise groups. Both exercise groups improved significantly in peak VO2 (low intensity: 3.62 mL O2/kg/min, SD 3.77; high intensity: 2.87 mL O2/kg/min, SD 4.98), with no statistical difference between-groups (mean difference: 0.76 mL O2/kg/min, 95% CI -3.22-1.7). CONCLUSIONS:High intensity physical exercise was not superior to low intensity physical exercise in reducing burden of atrial fibrillation. HI exercise was well tolerated; no evidence of an increased risk was found for HI compared to LI exercise. Larger studies are required to further prove our findings. TRIAL REGISTRATION:ClinicalTrials.gov NCT01817998.

Over the two last decades, venom toxins have been explored as alternatives to opioids to treat chronic debilitating pain. At present, approximately 20 potential analgesic toxins, mainly from spider venoms, are known to inhibit with high affinity the NaV1.7 subtype of voltage-gated sodium (NaV) channels, the most promising genetically validated antinociceptive target identified so far. The present study aimed to consolidate the development of phlotoxin 1 (PhlTx1), a 34-amino acid and 3-disulfide bridge peptide of a Phlogiellus genus spider, as an antinociceptive agent by improving its affinity and selectivity for the human (h) NaV1.7 subtype. The synthetic homologue of PhlTx1 was generated and equilibrated between two conformers on reverse-phase liquid chromatography and exhibited potent analgesic effects in a mouse model of NaV1.7-mediated pain. The effects of PhlTx1 and 8 successfully synthetized alanine-substituted variants were studied (by automated whole-cell patch-clamp electrophysiology) on cell lines stably overexpressing hNaV subtypes, as well as two cardiac targets, the hCaV1.2 and hKV11.1 subtypes of voltage-gated calcium (CaV) and potassium (KV) channels, respectively. PhlTx1 and D7A-PhlTx1 were shown to inhibit hNaV1.1−1.3 and 1.5−1.7 subtypes at hundred nanomolar concentrations, while their affinities for hNaV1.4 and 1.8, hCaV1.2 and hKV11.1 subtypes were over micromolar concentrations. Despite similar analgesic effects in the mouse model of NaV1.7-mediated pain and selectivity profiles, the affinity of D7A-PhlTx1 for the NaV1.7 subtype was at least five times higher than that of the wild-type peptide. Computational modelling was performed to deduce the 3D-structure of PhlTx1 and to suggest the amino acids involved in the efficiency of the molecule. In conclusion, the present structure−activity relationship study of PhlTx1 results in a low improved affinity of the molecule for the NaV1.7 subtype, but without any marked change in the molecule selectivity against the other studied ion channel subtypes. Further experiments are therefore necessary before considering the development of PhlTx1 or synthetic variants as antinociceptive drug candidates.

The eukaryotic phylum Apicomplexa encompasses thousands of obligate intracellular parasites of humans and animals with immense socio-economic and health impacts. We sequenced nuclear genomes of Chromera velia and Vitrella brassicaformis, free-living non-parasitic photosynthetic algae closely related to apicomplexans. Proteins from key metabolic pathways and from the endomembrane trafficking systems associated with a free-living lifestyle have been progressively and non-randomly lost during adaptation to parasitism. The free-living ancestor contained a broad repertoire of genes many of which were repurposed for parasitic processes, such as extracellular proteins, components of a motility apparatus, and DNA- and RNA-binding protein families. Based on transcriptome analyses across 36 environmental conditions, Chromera orthologs of apicomplexan invasion-related motility genes were co-regulated with genes encoding the flagellar apparatus, supporting the functional contribution of flagella to the evolution of invasion machinery. This study provides insights into how obligate parasites with diverse life strategies arose from a once free-living phototrophic marine alga.

The conserved MICOS complex functions as a primary determinant of mitochondrial inner membrane structure. We address the organization and functional roles of MICOS and identify two independent MICOS subcomplexes: Mic27/Mic10/Mic12, whose assembly is dependent on respiratory complexes and the mitochondrial lipid cardiolipin, and Mic60/Mic19, which assembles independent of these factors. Our data suggest that MICOS subcomplexes independently localize to cristae junctions and are connected via Mic19, which functions to regulate subcomplex distribution, and thus, potentially also cristae junction copy number. MICOS subunits have non-redundant functions as the absence of both MICOS subcomplexes results in more severe morphological and respiratory growth defects than deletion of single MICOS subunits or subcomplexes. Mitochondrial defects resulting from MICOS loss are caused by misdistribution of respiratory complexes in the inner membrane. Together, our data are consistent with a model where MICOS, mitochondrial lipids and respiratory complexes coordinately build a functional and correctly shaped mitochondrial inner membrane.

Prematurity affects 11% of the births and is the main cause of infant mortality. On the opposite case, the failure of induction of parturition in the case of delayed spontaneous birth is associated with fetal suffering. Both conditions are associated with precocious and/or delayed cervical ripening. Quantitative and objective information about the temporal evolution of the cervical ripening may provide a complementary method to identify cases at risk of preterm delivery and to assess the likelihood of successful induction of labour. In this study, the cervical stiffness was measured in vivo in pregnant sheep by using Shear Wave Elastography (SWE). This technique assesses the stiffness of tissue through the measurement of shear waves speed (SWS). In the present study, 9 pregnant ewes were used. Cervical ripening was induced at 127 days of pregnancy (term: 145 days) by dexamethasone injection in 5 animals, while 4 animals were used as control. Elastographic images of the cervix were obtained by two independent operators every 4 hours during 24 hours after injection to monitor the cervical maturation induced by the dexamethasone. Based on the measurements of SWS during vaginal ultrasound examination, the stiffness in the second ring of the cervix was quantified over a circular region of interest of 5 mm diameter. SWS was found to decrease significantly in the first 4-8 hours after dexamethasone compared to controls, which was associated with cervical ripening induced by dexamethasone (from 1.779 m/s ± 0.548 m/s, p < 0.0005, to 1.291 m/s ± 0.516 m/s, p < 0.000). Consequently a drop in the cervical elasticity was quantified too (from 9.5 kPa ± 0.9 kPa, p < 0.0005, to 5.0 kPa ± 0.8 kPa, p < 0.000). Moreover, SWE measurements were highly reproducible between both operators at all times. Cervical ripening induced by dexamethasone was confirmed by the significant increase in maternal plasma Prostaglandin E2 (PGE2), as evidenced by the assay of its metabolite PGEM. Histological analyses and two-photon excitation microscopy, combining both Second Harmonic Generation (SHG) and Two-photon Fluorescence microscopy (2PF) contrasts, were used to investigate, at the microscopic scale, the structure of cervical tissue. Results show that both collagen and 2PF-active fibrillar structures could be closely related to the mechanical properties of cervical tissue that are perceptible in elastography. In conclusion, SWE may be a valuable method to objectively quantify the cervical stiffness and as a complementary diagnostic tool for preterm birth and for labour induction success.

Viral infections cause many different diseases stemming both from well-characterized viral pathogens but also from emerging viruses, and the search for novel viruses continues to be of great importance. High-throughput sequencing is an important technology for this purpose. However, viral nucleic acids often constitute a minute proportion of the total genetic material in a sample from infected tissue. Techniques to enrich viral targets in high-throughput sequencing have been reported, but the sensitivity of such methods is not well established. This study compares different library preparation techniques targeting both DNA and RNA with and without virion enrichment. By optimizing the selection of intact virus particles, both by physical and enzymatic approaches, we assessed the effectiveness of the specific enrichment of viral sequences as compared to non-enriched sample preparations by selectively looking for and counting read sequences obtained from shotgun sequencing. Using shotgun sequencing of total DNA or RNA, viral targets were detected at concentrations corresponding to the predicted level, providing a foundation for estimating the effectiveness of virion enrichment. Virion enrichment typically produced a 1000-fold increase in the proportion of DNA virus sequences. For RNA virions the gain was less pronounced with a maximum 13-fold increase. This enrichment varied between the different sample concentrations, with no clear trend. Despite that less sequencing was required to identify target sequences, it was not evident from our data that a lower detection level was achieved by virion enrichment compared to shotgun sequencing.

Toxoplasma gondii is an important protozoan parasite that infects all warm-blooded animals and causes opportunistic infections in immuno-compromised humans. Its closest relative, Neospora caninum, is an important veterinary pathogen that causes spontaneous abortion in livestock. Comparative genomics of these two closely related coccidians has been of particular interest to identify genes that contribute to varied host cell specificity and disease. Here, we describe a manual evaluation of these genomes based on strand-specific RNA sequencing and shotgun proteomics from the invasive tachyzoite stages of these two parasites. We have corrected predicted structures of over one third of the previously annotated gene models and have annotated untranslated regions (UTRs) in over half of the predicted protein-coding genes. We observe distinctly long UTRs in both the organisms, almost four times longer than other model eukaryotes. We have also identified a putative set of cis-natural antisense transcripts (cis-NATs) and long intergenic non-coding RNAs (lincRNAs). We have significantly improved the annotation quality in these genomes that would serve as a manually curated dataset for Toxoplasma and Neospora research communities.

Sarafotoxin-m (24 amino acids) from the venom of Atractaspis microlepidota microlepidota was the first long-sarafotoxin to be identified, while sarafotoxin-b (21 aa) is a short-sarafotoxin from Atractaspis engaddensis. Despite the presence of three additional C-terminus residues in sarafotoxin-m, these two peptides display a high sequence homology and share similar three-dimensional structures. However, unlike sarafotoxin-b, sarafotoxin-m shows a very low in vitro affinity for endothelin receptors, but still has a very high in vivo toxicity in mammals, similar to that of sarafotoxin-b. We have previously demonstrated, in vitro, the crucial role of the C-terminus extension in terms of pharmacological profiles and receptor affinities of long- versus short-sarafotoxins. One possible hypothesis to explain the high in vivo toxicity of sarafotoxin-m could be that its C-terminus extension is processed in vivo, resulting in short-like sarafotoxin. To address this possibility, we investigated, in the present study, the in vivo cardiovascular effects of sarafotoxin-b, sarafotoxin-m and sarafotoxin-m-Cter (sarafotoxin-m without the C -terminus extension). Male Wistar rats were anaesthetised and mechanically ventilated. Invasive haemodynamic measurements and echocardiographic measurements of left and right ventricular function were performed. The rats were divided into four groups that respectively received intravenous injections of: saline, sarafotoxin-b (one LD50), sarafotoxin-m (one LD50) or sarafotoxin-m-Cter (one LD50). All measurements were performed at baseline, at 1 minute (+1) and at 6 minutes (+6) after injection.Sarafotoxin-b and sarafotoxin-m-Cter decreased cardiac output and impaired left ventricle systolic and diastolic function, whilst sarafotoxin-m decreased cardiac output, increased airway pressures and led to acute right ventricular dilatation associated with a decreased tricuspid annulus peak systolic velocity. Sarafotoxin-b and sarafotoxin-m-Cter appear to exert toxic effects via impairment of left ventricular function, whilst sarafotoxin-m increases airway pressures and impairs right ventricular function. These results do not support the hypothesis of an in vivo processing of long sarafotoxins.

In 2010, a chimpanzee died at Copenhagen Zoo following an outbreak of respiratory disease among chimpanzees in the zoo. Identification of coxsackie B3 virus, a common human pathogen, as the causative agent, and its severe manifestation, raise questions about pathogenicity and transmissibility among humans and other primates.

The present study aimed to determine the effects of breed and sex on growth patterns and metabolic features of advanced-pregnancy foetuses exposed to the same environmental conditions. Thus, at Day 62 of pregnancy, swine foetuses from an obese breed with leptin resistance (Iberian breed) were compared to lean crossbred foetuses (25% Large White ×25% Landrace ×50% Pietrain). There were differential developmental patterns in foetuses with leptin resistance, mainly a higher relative weight of the brain resembling "brain-sparing effect". Prioritization of brain growth may be protective for the adequate growth and postnatal survival of the Iberian individuals, an ancient breed reared in extensive semi-feral conditions for centuries. There were also clear sex-related differences in foetal development and metabolism in the Iberian breed. Female Iberian foetuses were similar in size and weight to male littermates but had a significantly higher relative liver to body weight ratio resembling "liver-sparing effect" and a trend for a higher relative intestine to body ratio. Moreover, the availability of triglycerides, cholesterol and IL-6 in female Iberian foetuses was similar to that of lean crossbred foetuses. Overall, these features may favour a better postnatal survival and development of females, the sex more critical for the species survival. These findings set the basis for future translational studies aimed at increasing the knowledge on the interaction between genetic and environmental factors in the early programming of the adult phenotype.

The population dynamics of shark species are generally poorly described because highly mobile marine life is challenging to investigate. Here we investigate the genetic population structure of the blacktip reef shark (Carcharhinus melanopterus) in French Polynesia. Five demes were sampled from five islands with different inter-island distances (50-1500 km). Whether dispersal occurs between islands frequently enough to prevent moderate genetic structure is unknown. We used 11 microsatellites loci from 165 individuals and a strong genetic structure was found among demes with both F-statistics and Bayesian approaches. This differentiation is correlated with the geographic distance between islands. It is likely that the genetic structure seen is the result of all or some combination of the following: low gene flow, time since divergence, small effective population sizes, and the standard issues with the extent to which mutation models actually fit reality. We suggest low levels of gene flow as at least a partial explanation of the level of genetic structure seen among the sampled blacktip demes. This explanation is consistent with the ecological traits of blacktip reef sharks, and that the suitable habitat for blacktips in French Polynesia is highly fragmented. Evidence for spatial genetic structure of the blacktip demes we studied highlights that similar species may have populations with as yet undetected or underestimated structure. Shark biology and the market for their fins make them highly vulnerable and many species are in rapid decline. Our results add weight to the case that total bans on shark fishing are a better conservation approach for sharks than marine protected area networks.

Human pressures have put many top predator populations at risk of extinction. Recent years have seen alarming declines in sharks worldwide, while their resilience remains poorly understood. Studying the ecology of small populations of marine predators is a priority to better understand their ability to withstand anthropogenic and environmental stressors. In the present study, we monitored a naturally small island population of 40 adult sicklefin lemon sharks in Moorea, French Polynesia over 5 years. We reconstructed the genetic relationships among individuals and determined the population's mating system. The genetic network illustrates that all individuals, except one, are interconnected at least through one first order genetic relationship. While this species developed a clear inbreeding avoidance strategy involving dispersal and migration, the small population size, low number of breeders, and the fragmented environment characterizing these tropical islands, limits its complete effectiveness.

Many Caenorhabditis elegans mutants with dysfunctional mitochondrial electron transport chain are surprisingly long lived. Both short-lived (gas-1(fc21)) and long-lived (nuo-6(qm200)) mutants of mitochondrial complex I have been identified. However, it is not clear what are the pathways determining the difference in longevity. We show that even in a short-lived gas-1(fc21) mutant, many longevity assurance pathways, shown to be important for lifespan prolongation in long-lived mutants, are active. Beside similar dependence on alternative metabolic pathways, short-lived gas-1(fc21) mutants and long-lived nuo-6(qm200) mutants also activate hypoxia-inducible factor -1α (HIF-1α) stress pathway and mitochondrial unfolded protein response (UPR(mt)). The major difference that we detected between mutants of different longevity, is in the massive loss of complex I accompanied by upregulation of complex II levels, only in short-lived, gas-1(fc21) mutant. We show that high levels of complex II negatively regulate longevity in gas-1(fc21) mutant by decreasing the stability of complex I. Furthermore, our results demonstrate that increase in complex I stability, improves mitochondrial function and decreases mitochondrial stress, putting it inside a "window" of mitochondrial dysfunction that allows lifespan prolongation.

Sarafotoxins (SRTX) are endothelin-like peptides extracted from the venom of snakes belonging to the Atractaspididae family. A recent in vivo study on anesthetized and ventilated animals showed that sarafotoxin-b (SRTX-b), extracted from the venom of Atractaspis engaddensis, decreases cardiac output by inducing left ventricular dysfunction while sarafotoxin-m (SRTX-m), extracted from the venom of Atractaspis microlepidota microlepidota, induces right ventricular dysfunction with increased airway pressure. The aim of the present experimental study was to compare the respiratory effects of SRTX-m and SRTX-b. Male Wistar rats were anesthetized, tracheotomized and mechanically ventilated. They received either a 1 LD50 IV bolus of SRTX-b (n = 5) or 1 LD50 of SRTX-m (n = 5). The low-frequency forced oscillation technique was used to measure respiratory impedance. Airway resistance (Raw), parenchymal damping (G) and elastance (H) were determined from impedance data, before and 5 min after SRTX injection. SRTX-m and SRTX-b injections induced acute hypoxia and metabolic acidosis with an increased anion gap. Both toxins markedly increased Raw, G and H, but with a much greater effect of SRTX-b on H, which may have been due to pulmonary edema in addition to bronchoconstriction. Therefore, despite their structural analogy, these two toxins exert different effects on respiratory function. These results emphasize the role of the C-terminal extension in the in vivo effect of these toxins.

Protein engineering approaches are often a combination of rational design and directed evolution using display technologies. Here, we test "loop grafting," a rational design method, on three-finger fold proteins. These small reticulated proteins have exceptional affinity and specificity for their diverse molecular targets, display protease-resistance, and are highly stable and poorly immunogenic. The wealth of structural knowledge makes them good candidates for protein engineering of new functionality. Our goal is to enhance the efficacy of these mini-proteins by modifying their pharmacological properties in order to extend their use in imaging, diagnostics and therapeutic applications. Using the interaction of three-finger fold toxins with muscarinic and adrenergic receptors as a model, chimeric toxins have been engineered by substituting loops on toxin MT7 by those from toxin MT1. The pharmacological impact of these grafts was examined using binding experiments on muscarinic receptors M1 and M4 and on the α(1A)-adrenoceptor. Some of the designed chimeric proteins have impressive gain of function on certain receptor subtypes achieving an original selectivity profile with high affinity for muscarinic receptor M1 and α(1A)-adrenoceptor. Structure-function analysis supported by crystallographic data for MT1 and two chimeras permits a molecular based interpretation of these gains and details the merits of this protein engineering technique. The results obtained shed light on how loop permutation can be used to design new three-finger proteins with original pharmacological profiles.

The majority of metabolic principles are evolutionarily conserved from nematodes to humans. Caenorhabditis elegans has widely accelerated the discovery of new genes important to maintain organismic metabolic homeostasis. Various methods exist to assess the metabolic state in worms, yet they often require large animal numbers and tend to be performed as bulk analyses of whole worm homogenates, thereby largely precluding a detailed studies of metabolic changes in specific worm tissues. Here, we have adapted well-established histochemical methods for the use on C. elegans fresh frozen sections and demonstrate their validity for analyses of morphological and metabolic changes on tissue level in wild type and various mutant strains. We show how the worm presents on hematoxylin and eosin (H&E) stained sections and demonstrate their usefulness in monitoring and the identification of morphological abnormalities. In addition, we demonstrate how Oil-Red-O staining on frozen worm cross-sections permits quantification of lipid storage, avoiding the artifact-prone fixation and permeabilization procedures of traditional whole-mount protocols. We also adjusted standard enzymatic stains for respiratory chain subunits (NADH, SDH, and COX) to monitor metabolic states of various C. elegans tissues. In summary, the protocols presented here provide technical guidance to obtain robust, reproducible and quantifiable tissue-specific data on worm morphology as well as carbohydrate, lipid and mitochondrial energy metabolism that cannot be obtained through traditional biochemical bulk analyses of worm homogenates. Furthermore, analysis of worm cross-sections overcomes the common problem with quantification in three-dimensional whole-mount specimens.

IL-2 has been reported to be critical for peripheral T(reg) survival in mouse models. Here, we examined T(reg) maintenance in a series of paediatric liver transplant recipients who received basiliximab, a therapeutic anti-CD25 monoclonal antibody.FoxP3+ CD4 T cells were analyzed by flow cytometry before liver grafting and more than 9 months later. We found that in vivo CD25 blockade did not lead to T(reg) depletion: the proportion of FoxP3+ cells among CD4 T cells and the level of FoxP3 expression were both unchanged. IL-2Rbeta expression was enhanced in FoxP3+ cells both before and after basiliximab treatment, while the level of IL-2Rgamma expression was similar in T(regs) and non-T(regs). No significant change in the weak or absent expression of IL-7Ralpha and IL-15Ralpha expression on FoxP3+ cells was observed. Although the proportion of FoxP3+ cells among CD4 T cells did not vary, food allergies occurred more rapidly after liver grafting in patients who received basiliximab, raising questions as to T(reg) functionality in vivo in the absence of functional CD25.CD25 appears non essential for human T(reg) peripheral maintenance in vivo. However, our results raise questions as to T(reg) functionality after therapeutic CD25 targeting.

BACKGROUND: Eukaryotic genomes are scattered with retroelements that proliferate through retrotransposition. Although retroelements make up around 40 percent of the human genome, large regions are found to be completely devoid of retroelements. This has been hypothesised to be a result of genomic regions being intolerant to insertions of retroelements. The inadvertent transcriptional activity of retroelements may affect neighbouring genes, which in turn could be detrimental to an organism. We speculate that such retroelement transcription, or transcriptional interference, is a contributing factor in generating and maintaining retroelement-free regions in the human genome. METHODOLOGY/PRINCIPAL FINDINGS: Based on the known transcriptional properties of retroelements, we expect long interspersed elements (LINEs) to be able to display a high degree of transcriptional interference. In contrast, we expect short interspersed elements (SINEs) to display very low levels of transcriptional interference. We find that genomic regions devoid of long interspersed elements (LINEs) are enriched for protein-coding genes, but that this is not the case for regions devoid of short interspersed elements (SINEs). This is expected if genes are subject to selection against transcriptional interference. We do not find microRNAs to be associated with genomic regions devoid of either SINEs or LINEs. We further observe an increased relative activity of genes overlapping LINE-free regions during early embryogenesis, where activity of LINEs has been identified previously. CONCLUSIONS/SIGNIFICANCE: Our observations are consistent with the notion that selection against transcriptional interference has contributed to the maintenance and/or generation of retroelement-free regions in the human genome.