BACKGROUND:Histiocytic sarcoma (HS) is a rare but highly aggressive neoplasm in humans, with limited knowledge on indicators markers and no standardized treatment. Dogs naturally develop HS, offering a powerful comparative model. Activating mutations in PTPN11, affecting two recurrent hotspots (E76 and G503), are the most frequent drivers of canine HS and are strongly associated with the disseminated form (DHS). While DHS is typically considered metastatic, we hypothesize that multiple independent tumor clones may arise within the same individual. METHODS:We investigated clonal heterogeneity in 38 dogs affected with DHS presenting tumors in both abdominal and thoracic organs. PTPN11 hotspot mutations were analyzed by high-sensitivity droplet digital PCR (ddPCR) to detect low-abundance variants. The frequency of divergent mutations between tumor sites was used to infer the proportion of cases arising from independent clonal origins. RESULTS:Divergent PTPN11 mutations were directly observed in 3 of 28 dogs with PTPN11-mutated tumor pairs. After correcting for the limited detectability inherent to hotspot-based analysis, we estimated that, in this cohort, up to 24-45% of DHS cases may harbor clonally independent PTPN11 mutations in distinct tumors. In addition, analysis of somatic mutations and copy number variants by Next Generation Sequencing (NGS) in two cell lines derived from the same patient confirmed the independent origin of the clones. CONCLUSIONS:Our findings suggest that a significant numbers of dogs with DHS develop several genetically distinct HS lesions either simultaneously or within a short time interval. This need to be confirmed in larger cohorts but this clonal diversity challenges the assumption that DHS is predominantly metastatic and has major implications for prognosis, therapeutic strategies, and the use of canine HS as a comparative model for human disease. Further exploration of clonal evolution in HS is warranted to improve treatment approaches in both species.

SprA1 and SprA2 are small hydrophobic peptides that belong to the type I toxin-antitoxin systems expressed by Staphylococcus aureus. Both peptides induce S. aureus death when overexpressed. Although they share 71% of amino acids sequence similarity, SprA2 exhibits stronger hemolytic activity than SprA1. In this study, we investigated the mode of action of these toxins on both prokaryotic-like and eukaryotic-like membranes. We first confirmed that SprA2, like SprA1, is an alpha-helical peptide located at the S. aureus membrane. By overexpressing each toxin, we demonstrated that SprA1 forms stable pores in the S. aureus membrane, evidenced by concomitant membrane depolarization, permeabilization and ATP release leading to growth arrest, whereas SprA2 forms transient pores, causing concomitant membrane depolarization, ATP release, and growth arrest. We showed that the unique cysteine residue present in SprA1 and SprA2 is required for toxicity through disulfide bond formation. Next, we found that both synthetic peptides induce slight leakage in anionic DOPC-DOPG lipid vesicles mimicking prokaryotic membranes, concomitant with lipid vesicles aggregation and/or fusion. Moreover, we observed that SprA1 permeabilizes S. aureus protoplasts, via its ability to form stable pores, whereas SprA2 permeabilizes and lyses them. However, no permeabilization of intact bacteria was detected after the addition of SprA1 and SprA2 in the extracellular medium. Finally, we confirmed that SprA2 has strong activity on zwitterionic DOPC lipid vesicles mimicking eukaryotic membranes, without inducing aggregation. This work highlights the strong selectivity of SprA2 for eukaryotic membranes, suggesting that this toxin may play a role in S. aureus virulence.

Bacterial regulatory RNAs (sRNAs) are important players to control gene expression. InStaphylococcus aureus, SprC is an antivirulenttrans-acting sRNA known to base-pair with the major autolysinatlmRNA, preventing its translation. Using MS2-affinity purification coupled with RNA sequencing, we looked for its sRNA-RNA interactome and identified 14 novel mRNA targets. In vitro biochemical investigations revealed that SprC binds two of them,czrBanddeoD,and uses a single accessible region to regulate its targets, including Atl translation. Unlike Atl regulation, the characterization of the SprC-czrBinteraction pinpointed a destabilization of theczrABcotranscript, leading to a decrease of the mRNA level that impaired CzrB zinc efflux pump expression. On a physiological standpoint, we showed that SprC expression is detrimental to combat against zinc toxicity. In addition, phagocyctosis assays revealed a significant, but moderate, increase ofczrBmRNA levels in asprC-deleted mutant, indicating a functional link between SprC andczrBupon internalization in macrophages, and suggesting a role in resistance to both oxidative and zinc bursts. Altogether, our data uncover a novel pathway in which SprC is implicated, highlighting the multiple strategies used byS. aureusto balance virulence using an RNA regulator.

Hereditary sensory and autonomic neuropathies (HSAN) represent a group of genetic diseases affecting the peripheral nervous system. In humans, at least 16 loci have been associated with the disorder but do not explain the disease origin of all patients. In dogs, similar conditions have been documented for decades in various breeds with a severe impact on life quality and are often referred to as acral mutilation syndrome (AMS). Causal variants in three genes have been identified to date, suggesting larger genetic heterogeneity in the dog population. Our aim was to explain the genetic etiology of an early-onset HSAN/AMS in a purebred German Spitz. The affected dog showed progressive loss of pain sensation in the distal extremities, which led to intense licking, biting, and self-mutilation of digits and paw pads. Whole-genome sequencing identified a single candidate causal variant on chromosome 4 in the RETREG1 gene (c.656C>T, p.Pro219Leu). This missense variant was previously recognized as deleterious in a mixed breed dog family with similar clinical signs. Haplotype analyses and targeted genotyping revealed a likely German Spitz ancestry of these mixed breed dogs. Further screening of an extensive cohort of ~900 000 dogs of various breeds hinted at the variant allele origin in the German Spitz breed. Disruption of RETREG1 inhibits endoplasmic reticulum turnover and leads to neuron degeneration. Our findings provide evidence that this variant underlies the recessive form of HSAN/AMS in the German Spitz and support the use of whole-genome sequencing-based veterinary precision medicine for early diagnosis and prevention via a genetic test.

Download This Paper Open PDF in Browser Add Paper to My Library Share: Permalink Using these links will ensure access to this page indefinitely Copy URL Copy DOI

Staphylococcus aureus RNAIII is a dual-function regulatory RNA that controls the expression of multiple virulence genes and especially the transition from adhesion to the production of exotoxins. However, its contribution to S. aureus central metabolism remains unclear. Using MS2-affinity purification coupled with RNA sequencing, we uncovered more than 50 novel RNAIII-mRNA interactions. Among them, we demonstrate that RNAIII is a major activator of the rpiRc gene, encoding a regulator of the pentose phosphate pathway (PPP). RNAIII binds the 5' UTR of rpiRc mRNA to favor ribosome loading, leading to an increased expression of RpiRc and, subsequently, of two PPP enzymes. Finally, we show that RNAIII and RpiRc are involved in S. aureus fitness in media supplemented with various carbohydrate sources related to PPP and glycolysis. Collectively, our data depict an unprecedented phenotype associated with the RNAIII regulon, especially the direct implication of RNAIII in central metabolic activity modulation. These findings show that the contribution of RNAIII in Staphylococcus aureus adaptation goes far beyond what was initially reported. IMPORTANCE:Staphylococcus aureus is a major human pathogen involved in acute and chronic infections. Highly recalcitrant to antibiotic treatment, persistent infections are mostly associated with the loss of RNAIII expression, a master RNA regulator responsible for the switch from colonization to infection. Here, we used the MS2 affinity purification coupled with RNA sequencing approach to identify novel mRNA targets of RNAIII and uncover novel functions. We demonstrate that RNAIII is an activator of the expression of genes involved in the pentose phosphate pathway and is implicated in the adjustment of bacterial fitness as a function of carbohydrate sources. Taken together, our results demonstrate an unprecedented role of RNAIII that goes beyond the knowledge gained so far and contributes to a better understanding of the role of RNAIII in bacterial adaptation expression and the coordination of a complex regulatory network.

T-cell-based adoptive immunotherapy is a new pillar of cancer care. Tumor-redirected B cells could also contribute to therapy if their manipulation to rewire immunoglobulin (Ig) genes is mastered. We designed a single-chain Ig-encoding cassette (“scFull-Ig”) that redirects antigen specificity when inserted at a single position of the IgH locus. This design, which places combined IgH and IgL variable genes downstream of a pVH promoter, nevertheless preserves all Ig functional domains and the intrinsic mechanisms that regulate expression from the IgM B cell receptor (BCR) expression to Ig secretion, somatic hypermutation and class switching. This single-locus editing provides an efficient and safe strategy to both disrupt endogenous Ig expression and encode a new Ig paratope. As a proof of concept, the functionality of scFull BCR and/or secreted Ig was validated against two different classical human tumor antigens, HER2 and hCD20. Once validated in cell lines, the strategy was extended to primary B cells, confirming the successful engineering of BCR and Ig expression and the ability of scFull-Ig to undergo further class switching. These results further pave the way for future B cell-based adoptive immunotherapy and strategies to express a therapeutic mAb with a variety of switched H-chains that provide complementary functions.

Sexual development is a complex process relying on numerous genes. Disruptions in some of these genes are known to cause differences of sexual development (DSDs). Advances in genome sequencing allowed the discovery of new genes implicated in sexual development, such as PBX1. We present here a fetus with a new PBX1 NM_002585.3: c.320G>A,p.(Arg107Gln) variant, presenting with severe DSD along with renal and lung malformations. Using CRISPR-Cas9 gene editing on HEK293T cells, we generated a KD cell line for PBX1. The KD cell line showed reduced proliferation and adhesion properties compared with HEK293T cells. HEK293T and KD cells were then transfected plasmids coding either PBX1 WT or PBX1-320G>A (mutant). WT or mutant PBX1 overexpression rescued cell proliferation in both cell lines. RNA-seq analyses showed less than 30 differentially expressed genes, in ectopic mutant-PBX1-expressing cells compared with WT-PBX1. Among them, U2AF1, encoding a splicing factor subunit, is an interesting candidate. Overall, mutant PBX1 seems to have modest effects compared with WT PBX1 in our model. However, the recurrence of PBX1 Arg107 substitution in patients with closely related phenotypes calls for its impact in human diseases. Further functional studies are needed to explore its effects on cellular metabolism.

Ganglioside-monosialic acid (GM1) gangliosidosis, a rare autosomal recessive disorder, is frequently caused by deleterious single nucleotide variants (SNVs) in GLB1 gene. These variants result in reduced β-galactosidase (β-gal) activity, leading to neurodegeneration associated with premature death. Currently, no effective therapy for GM1 gangliosidosis is available. Three ongoing clinical trials aim to deliver a functional copy of the GLB1 gene to stop disease progression. In this study, we show that 41% of GLB1 pathogenic SNVs can be replaced by adenine base editors (ABEs). Our results demonstrate that ABE efficiently corrects the pathogenic allele in patient-derived fibroblasts, restoring therapeutic levels of β-gal activity. Off-target DNA analysis did not detect off-target editing activity in treated patient's cells, except a bystander edit without consequences on β-gal activity based on 3D structure bioinformatics predictions. Altogether, our results suggest that gene editing might be an alternative strategy to cure GM1 gangliosidosis.

sprG1/SprF1 is a type I toxin-antitoxin system located on Staphylococcus aureus prophage. It has previously been shown that the two toxins, SprG131 and SprG144, encoded by the sprG1 gene, are two membrane-associated peptides structured in a single α-helix. Overexpression of these two peptides leads to growth inhibition and even S. aureus death. In this study, we investigated the involvement of each peptide in this toxicity, the sequence requirements necessary for SprG131 toxicity, and the mechanism of action of these two peptides. Our findings show that both peptides, when expressed individually, are able to stop growth, with higher toxicity observed for SprG131. The combination of a hydrophobic domain and a charged domain located only at the C-terminus is necessary for this toxicity, likely to retain the orientation of the transmembrane domain. A net cationic charge for SprG131 is not essential to induce a growth defect in S. aureus. Furthermore, we established a chronology of toxic events following overexpression to gain insights into the mode of action of SprG144 and SprG131. We demonstrated that mesosome-like structures are already formed when membrane is depolarized, about 20 min after peptides induction. This membrane depolarization occurs concomitantly with a depletion of intracellular ATP, leading to S. aureus growth arrest. Moreover, we hypothesized that SprG144 and SprG131 do not form large pores in the S. aureus membrane, as ATP is not excreted into the extracellular medium, and membrane permeabilization is delayed relative to membrane depolarization. The next challenge is to identify the conditions under which SprG144 and SprG131 are naturally expressed, and to uncover their potential roles during staphylococcal growth, colonization, and infection.

Population genetics of threatened species provides information about evolutionary pressures over those populations and thus may inform conservation management strategies. However, conservation genetics still has a low impact on conservation practices. This study’s aim is to integrate genetics in the conservation management of the only-known population of an extremely narrow-range endemic Corsican snail – Tyrrhenaria ceratina – , whose distribution area is restricted to the Ricantu site in Corsica. Using non-invasive DNA samples of 210 individuals, we amplified 13 microsatellites loci to assess the population viability, genetic structure and demographic history of the population, along with the estimation of the historical and contemporary gene flow between identified genetic clusters. We also estimated the dispersal ability of the species. Our results showed a surprisingly high genetic diversity, along with a pattern of isolation by distance (IBD) and a strongly spatialized genetic structure. Furthermore, we underlined a low functional connectivity, along with evidence of a recent decline in the population size, which are both likely due to a historical fragmentation between the sampled areas, caused by anthropization. Overall, this study allows to provide a first insight about the functioning of the population, to guide future conservation actions for the species.

Ganglioside-monosialic acid (GM1) gangliosidosis, a rare autosomal recessive disorder, is frequently caused by deleterious single nucleotide variants (SNVs) in GLB1 gene. These variants result in reduced β-galactosidase (β-gal) activity, leading to neurodegeneration associated with premature death. Currently, no effective therapy for GM1 gangliosidosis is available. Three ongoing clinical trials aim to deliver a functional copy of the GLB1 gene to stop disease progression. Here, we show that 41% of GLB1 pathogenic SNVs might be cured by adenine base editors (ABEs). Our results demonstrate that ABE efficiently corrects the pathogenic allele in patient-derived fibroblasts, restoring a therapeutic level of β-gal activity. Unbiased off-target DNA analysis did not detect off-target editing activity in treated patient’s cells except a bystander edit without consequences on β-gal activity. Altogether our results suggest that gene editing is an alternative strategy to cure GM1 gangliosidosis, by correcting the root cause of disease and avoiding repetitive adeno-associated virus injections.

In forest-specialist mammals, forest loss may induce resistance to animal movement and reduce gene flow between populations, and thereby increase genetic erosion and extinction risks for populations. Understanding how landscape features affect gene flow is of critical importance for conservation. Using landscape genetic tools at multiple spatial scales, we assessed the effects of landscape heterogeneity (in particular the presence of wide open or rural habitats) on gene flow in an endangered forest-specialist species - the Barbary macaque (Macaca sylvanus) -, in its major forest site in Morocco. We genotyped 248 individuals from 23 macaque groups using 11 microsatellite loci. We modelled different scenarios of isolation by landscape resistance. We further tested the relationships between genetic distance and isolation by resistance, after controlling for the effect of isolation by distance. Our results revealed a significant genetic structure and a disruption of gene flow even in geographic proximity. Whatever the spatial scale, remoteness from the forest edge beyond 1 km acted as a barrier to macaque movements. In addition, at a fine scale, human-dominated areas were also detected as a barrier. The detection of private alleles in each population suggests an ongoing process of isolation. The preservation of the Barbary macaque implies 1) strictly avoiding all silvicultural practices (in particular clear-cutting of holm oak forests) that could contribute to increase distances between forest patches, 2) restoring corridors between forests, 3) and preserving key small forest patches as potential stepping stones facilitating macaque dispersal.

Social networks are the result of interactions between individuals at different temporal scales. Thus, sporadic intergroup encounters and individual forays play a central role in defining the dynamics of populations in social species. We assessed the rate of intergroup encounters for three western lowland gorilla (Gorilla gorilla gorilla) groups with daily observations over 5 years, and non-invasively genotyped a larger population over four months. Both approaches revealed a social system much more dynamic than anticipated, with non-aggressive intergroup encounters that involved social play by immature individuals, exchanges of members between groups likely modulated by kinship, and absence of infanticide evidenced by infants not fathered by the silverback of the group where they were found. This resulted in a community composed of groups that interacted frequently and not-aggressively, contrasting with the more fragmented and aggressive mountain gorilla (G. beringei beringei) societies. Such extended sociality can promote the sharing of behavioural and cultural traits, but might also increase the susceptibility of western lowland gorillas to infectious diseases that have decimated their populations in recent times.

Abstract C. (Morphocarabus) rothi comptus Dejean 1831, is a endemic species of the Romania, appearing localized to some mountainous massifs of Romanian Banat and their piedmont areas (Poiana Rusca Mountains, Țarcu Mountains). Its presence in the Apuseni Mountains is doubtful, due to the lack of recent captures and to the confusion with C. (Morphocarabus) hampei diffinis Csiki 1905, morphologically like. In Banat the species occurs in two forms: szorenyensis Csiki 1908 at higher altitude (1300-2000 m) and ulrichhoffmanni Lie 1982 in hilly areas, which is distinguished by a larger size. Whatever the altitude and the ecological conditions, the period of activity is brief, spread over a month with a maximum of over two weeks. Molecular biology studies: confirm the belonging of comptus to rothi as a subspecies of her (28SRNA5 marker); suggest (concatenation COI I / cyt b) a low infra-specific variability between provenances of the szorenyensis (Rusca, Muntele Mic) and between the origins of the ulrichhoffmanni.

Interspecific hybridization represents an evolutionary force resulting in novel genotypes. The genomic changes that occur as a result of hybridization affect both genome structure and gene expression and consequently determine hybrid phenotypes and ecology. This study provides new data on the dynamics of hybrid invasions, integrating effects of the genetic, phenotypic, geographical and environmental scenarios with hybridization following invasion of a halophyte community by an exotic plant species. We analyzed the spatial genetic structure of sterile Spartina Fl hybrid populations established at the Gulf of Cadiz (Southwest Iberian Peninsula) and that of their parental species native S. maritima and invasive S. densiflora using nuclear DNA (Simple Sequence Repeats) and chloroplast DNA sequences. We also analyzed the relationships between the spatial genetic structure of the hybrids, their phenotypic variability and their marsh environment. The studied populations of Spartina hybrids were establishing hybrid zones with a spatial genetic structure inherited from both parental species. The hybrids were genetically more similar to the native than to the invasive species. The hybrid populations with greater genetic differentiation were those more spatially separated from each other and that were present in more contrasted sedimentary environments, revealing respective isolation processes by distance and by environment. The hybrids in the Guadiana Estuary were the most genetically differentiated and with the highest transgressive behavior in terms of tiller height.

Abstract Species can respond to environmental pressures through genetic and epigenetic changes and through phenotypic plasticity, but few studies have evaluated the relationships between genetic differentiation and phenotypic plasticity of plant species along changing environmental conditions throughout wide latitudinal ranges. We studied inter‐ and intrapopulation genetic diversity (using simple sequence repeats and chloroplast DNA sequencing) and inter‐ and intrapopulation phenotypic variability of 33 plant traits (using field and common‐garden measurements) for five populations of the invasive cordgrass Spartina densiflora Brongn. along the Pacific coast of North America from San Francisco Bay to Vancouver Island. Studied populations showed very low genetic diversity, high levels of phenotypic variability when growing in contrasted environments and high intrapopulation phenotypic variability for many plant traits. This intrapopulation phenotypic variability was especially high, irrespective of environmental conditions, for those traits showing also high phenotypic plasticity. Within‐population variation represented 84% of the total genetic variation coinciding with certain individual plants keeping consistent responses for three plant traits (chlorophyll b and carotenoid contents, and dead shoot biomass) in the field and in common‐garden conditions. These populations have most likely undergone genetic bottleneck since their introduction from South America; multiple introductions are unknown but possible as the population from Vancouver Island was the most recent and one of the most genetically diverse. S. densiflora appears as a species that would not be very affected itself by climate change and sea‐level rise as it can disperse, establish, and acclimate to contrasted environments along wide latitudinal ranges.

Bacterial small regulatory RNAs (sRNAs) play a major role in the regulation of various cellular functions. Most sRNAs interact with mRNA targets via an antisense mechanism, modifying their translation and/or degradation. Despite considerable progresses in discovering sRNAs in Gram-positive bacteria, their functions, for the most part, are unknown. This is mainly due to difficulties in identifying their targets. To aid in the identification of sRNA targets in Gram-positive bacteria, we set up an in vivo method for fast analysis of sRNA-mediated post-transcriptional control at the 5΄ regions of target mRNAs. The technology is based on the co-expression of an sRNA and a 5΄ sequence of an mRNA target fused to a green fluorescent protein (GFP) reporter. The system was challenged on Staphylococcus aureus, an opportunistic Gram-positive pathogen. We analyzed several established sRNA-mRNA interactions, and in addition, we identified the ecb mRNA as a novel target for SprX2 sRNA. Using our in vivo system in combination with in vitro experiments, we demonstrated that SprX2 uses an antisense mechanism to prevent ecb mRNA translation initiation. Furthermore, we used our reporter assay to validate sRNA regulations in other Gram-positive organisms, Bacillus subtilis and Listeria monocytogenes. Overall, our method is broadly applicable to challenge the predicted sRNA-mRNA interactions in Gram-positive bacteria.