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According to official data, the Democratic Republic of the Congo (DRC) has a low prevalence of the coronavirus disease 19 (COVID-19) pandemic. The goal of this cross-sectional study was to determine the COVID-19 seroprevalence in people attending Goma’s two largest markets, Kituku and Virunga. This study was conducted between September and November 2021, overlapping by one month with another similar study carried out in a slum of Bukavu, and using the same methodology.COVID-19 unvaccinated participants (n = 796 including 454 vendors and 342 customers, 60% of whom were women) were surveyed. The median age of vendors and customers was 34.2 and 30.1 years, respectively.The crude and adjusted anti-SARS-CoV-2 antibody seroprevalence rates were 70.2% (95 % CI 66.9-73.4%) and 98.8% (95% CI 94.1-100%), respectively, with no difference between vendors and customers. COVID-19 symptoms were mild or absent in 58.9% and 41.1% of participants with anti-SARS-CoV-2 antibodies respectively. COVID-19 did not require hospitalisation for any of the seropositive participants.These findings are consistent with those reported in Bukavu. They confirm that SARS-CoV-2 spread without causing severe symptoms in densely populated settlements and markets, and suggest that many COVID-19 cases went unreported. Based on these results, relevance of an untargeted hypothetical vaccination programme in these communities should be questioned.

Multidrug-resistant (MDR) and Extended Spectrum β-Lactamase (ESBL)-producing extraintestinalK. pneumoniaeare associated with increased morbidity and mortality. This study aimed to characterize the resistance and virulence profiles of extra-intestinal MDR ESBL-producingK. pneumoniaeassociated with infections at a tertiary hospital in South-Kivu province, DRC. Whole-genome sequencing (WGS) was carried out on 37 K.pneumoniaeisolates displaying MDR, and ESBL-producing phenotype. The assembled genomes were analysed for phylogeny, virulence factors and antimicrobial resistance genes (ARG) determinants. These isolates were compared to sub-Saharan counterparts.K. pneumoniaeisolates displayed a high genetic variability with up to 16 sequence types (ST). AMR was widespread against β-lactamases (including third and fourth-generation cephalosporins, but not carbapenems), aminoglycosides, ciprofloxacin, tetracycline, erythromycin, nitrofurantoin, and cotrimoxazole. TheblaCTXM-15gene was the most common β-lactamase gene amongK. pneumoniaeisolates. No carbapenemase gene was found. ARG for aminoglycosides, quinolones, phenicols, tetracyclines, sulfonamides, nitrofurantoin were widely distributed among the isolates. Nine isolates had the colistin-resistant R256G substitution in thepmrBefflux pump gene without displaying reduced susceptibility to colistin. Despite carrying virulence genes, none had hypervirulence genes. Our results highlight the genetic diversity of MDR ESBL-producingK. pneumoniaeisolates, and underscore the importance of monitoring simultaneously the evolution of phenotypic and genotypic AMR in Bukavu and DRC, while calling for caution in administering colistin and carbapenem to patients.

Data on coronavirus disease 2019 (COVID-19) prevalence in the Democratic Republic of Congo are scarce. We conducted a cross-sectional study to determine the seroprevalence of antibodies against anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the slum of Kadutu, city of Bukavu, between June and September 2021. The survey participants were all unvaccinated against SARS-CoV-2. The crude seroprevalence rate was adjusted to the known characteristics of the assay. Participants aged 15–49 years old made up 80% of the population enrolled in the study (n = 507; 319 women and 188 men). The overall crude and adjusted seroprevalence rates of antibodies for COVID-19 were 59.7% (95% CI 55.4–63.9%) and 84.0% (95% CI 76.2–92.4%), respectively. This seroprevalence rate indicates widespread dissemination of SARS-CoV-2 in these communities. COVID-19 symptoms were either absent or mild in more than half of the participants with antibodies for COVID-19 and none of the participants with antibodies for COVID-19 required hospitalisation. These results suggest that SARS-CoV-2 spread did not appear to be associated with severe symptoms in the population of these settlements and that many cases went unreported in these densely populated locations. The relevance of vaccination in these communities should be thoroughly investigated.

Rodent studies on the effects of engineered nanomaterials (ENM) on the gut microbiome have revealed contradictory results. Our aim was to assess the effects of four well-investigated model ENM using a realistic exposure scenario. Two independent ad libitum feeding studies were performed. In study 1, female mice from the local breeding facility received feed pellets containing 1% CeO2 or 1% SiO2 for three weeks. In study 2, both female and male mice were purchased and exposed to 0.2% Ag-PVP or 1% TiO2 for four weeks. A next generation 16S rDNA sequencing-based approach was applied to assess impacts on the gut microbiome. None of the ENM had an effect on the α- or β-diversity. A decreased relative abundance of the phylum Actinobacteria was observed in SiO2 exposed mice. In female mice, the relative abundance of the genus Roseburia was increased with Ag exposure. Furthermore, in study 2, a sex-related difference in the β-diversity was observed. A difference in the β-diversity was also shown between the female control mice of the two studies. We did not find major effects on the gut microbiome. This contrast to other studies may be due to variations in the study design. Our investigation underlined the important role of the sex of test animals and their microbiome composition prior to ENM exposure initiation. Hence, standardization of microbiome studies is strongly required to increase comparability. The ENM-specific effects on Actinobacteria and Roseburia, two taxa pivotal for the human gut homeostasis, warrant further research on their relevance for health.

Dependence on multiple nutritional bacterial symbionts forming a metabolic unit has repeatedly evolved in many insect species that feed on nutritionally unbalanced diets such as plant sap. This is the case for aphids of the subfamilies Lachninae and Chaitophorinae, which have evolved di-symbiotic systems in which the ancient obligate nutritional symbiont Buchnera aphidicola is metabolically complemented by an additional nutritional symbiont acquired more recently. Deciphering how different symbionts integrate both metabolically and anatomically in such systems is crucial to understanding how complex nutritional symbiotic systems function and evolve. In this study, we sequenced and analyzed the genomes of the symbionts B. aphidicola and Serratia symbiotica associated with the Chaitophorinae aphids Sipha maydis and Periphyllus lyropictus. Our results show that, in these two species, B. aphidicola and S. symbiotica complement each other metabolically (and their hosts) for the biosynthesis of essential amino acids and vitamins but with distinct metabolic reactions supported by each symbiont depending on the host species. Furthermore, the S. symbiotica symbiont associated with S. maydis appears to be strictly compartmentalized into the specialized host cells housing symbionts in aphids, the bacteriocytes, whereas the S. symbiotica symbiont associated with P. lyropictus exhibits a highly invasive phenotype, presumably because it is capable of expressing a larger set of virulence factors, including a complete flagellum for bacterial motility. Such contrasting levels of metabolic and anatomical integration for two S. symbiotica symbionts that were recently acquired as nutritional co-obligate partners reflect distinct coevolutionary processes specific to each association.

Dependence on multiple nutritional bacterial symbionts forming a metabolic unit has repeatedly evolved in many insect species that feed on nutritionally unbalanced diets such as plant sap. This is the case for aphids of the subfamilies Lachninae and Chaitophorinae, which have evolved di-symbiotic systems in which the ancient obligate nutritional symbiont

PURPOSE: Housekeeping genes (HKGs), reference or endogenous control genes, are vital to normalize mRNA levels between different samples. Since using inappropriate HKGs can lead to unreliable results, selecting the proper ones is critical for gene expression studies. To this end, normal human ovaries, as well as those from patients diagnosed with ovarian endometrioid adenocarcinoma (OEA), ovarian mucinous adenocarcinoma (OMA), ovarian serous papillary carcinoma (OSPC), and polycystic ovary syndrome (PCOS), were used to identify the most suitable housekeeping genes. METHODS: RNA was isolated from 5 normal human ovaries (52–79 years of age), 9 cancerous ovaries (3 OEA, 3 OMA, 3 OSPC; 49–75 years of age), and 4 PCOS ovaries (18–35 years of age) in women undergoing hysterectomy. cDNA was synthesized using a whole transcriptome kit, and quantitative real-time PCR was performed using TaqMan array 96-well plates containing 32 human endogenous controls in triplicate. RESULTS: Among 32 HKGs studied, RPS17, RPL37A, PPIA, 18srRNA, B2M, RPLP0, RPLP30, HPRT1, POP4, CDKN1B, and ELF1 were selected as the best reference genes. CONCLUSIONS: This study confirms recent investigations demonstrating that conventional HKGs, such as GAPDH and beta-actin, are not suitable reference genes for specific pathological conditions, emphasizing the importance of determining the best HKGs on a case-by-case basis and according to tissue type. Our results have identified reliable HKGs for studies of normal human ovaries and those affected by OEA, OMA, OSPC, or PCOS, as well as combined studies of control subjects vs. each cancer or PCOS group. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-020-01901-8) contains supplementary material, which is available to authorized users.

PURPOSE: With the development of minimal invasive procedure, trans-oral robotic surgery (TORS) is expanding in the field of ENT. Most reviews focus on oropharyngeal and laryngeal (supra-glottic) localization. We report here the feasibility and outcomes of TORS hypopharyngectomy (TORSH) for selected patients with hypopharyngeal tumor. METHODS: Between September 2009 and July 2017, 22 patients, retrospectively included, underwent TORSH with curative intent. RESULTS: From 22 successful hypopharyngectomy, no conversion to open procedure was needed. Three patients (13%) presented a post-operative bleeding and were managed by surgical revision. No fistula was encountered. The 3-year overall survival and disease-specific survival rates were 54 and 92%, respectively. Patients started oral feeding after an average of 7 days. Naso-gastric feeding tubes were removed after a median period of 16 days. Two patients (9%) needed a transient gastrostomy (< 1 year). Three patients (13%) received a transient tracheostomy (< 2 months). Median hospitalization stay was 13 days. CONCLUSIONS: TORSH is a safe technique. Patients' outcomes are favorable and the post-operative morbidity is reduced compared to open neck approach. Hospitalization length and safe swallowing time are reduced.

Background Acetyl-CoA carboxylase (ACC), the first enzyme regulating lipid synthesis, promotes thrombus formation by increasing platelet phospholipid content and thromboxane A2 generation. Purpose Our study sought to evaluate whether ACC1 platelet-specific deletion may affect platelet functions by decreasing phospholipid content. Methods We generated a new Cre transgenic mouse strain that allows megakaryocyte/platelet specific ACC1 deletion (GpIbCre+/− x ACC1 flx/flx mouse). In vitro, platelet functions were assessed by aggregometry and flow cytometry. In vivo, hemostasis was assessed via the measurement of bleeding time. Lipidomics analysis was carried out on the commercial Lipidyzer platform. Thromboxane A2 secretion was evaluated by ELISA. Results As expected, ACC1 deletion was restricted to the megakaryocytic lineage. Hematological parameters in platelet-specific ACC1 knockout mice showed a decrease in platelet count by 30% and an increase in platelet volume by 31%, compared to ACC1 flx/flx platelets. In vitro, platelets from platelet-specific ACC1 knockout mice displayed a decrease in thrombin and CRP-induced platelet aggregation, associated with impaired dense granules secretion. In contrast, ADP-induced platelet aggregation was higher in the absence of ACC1. In vivo, platelet-specific ACC1 knockout mice showed a normal bleeding time. In agreement with our hypothesis, lipidomics analyses showed that ACC1 deletion in platelets was associated with a significant decrease in arachidonic acid-contaning phosphatidylethanolamine plasmalogen, and subsequently with a reduced production of thromboxane A2 upon thrombin or CRP stimulation. Conclusion Platelet-specific ACC1 deletion led to a decrease in phospholipid content which, in turn, decreased platelet thromboxane A2 generation, dense granules secretion and aggregation upon thrombin and CRP, but not ADP stimulation. Further studies are needed to elucidate the impact of ADP on platelet functions Funding Acknowledgement Type of funding sources: Other. Main funding source(s): Fonds pour la formation à la Recherche dans l'Industrie et l'Agriculture (FRIA)

Background While pulsed UV-C light sources such as excimer lasers are used in emerging non-thermal food decontamination method, they also have a high potential for a wide range of other microbial decontamination applications. The acaricidal effect of an experimental UV-C irradiation device was assessed using two-spotted spider mite female adults Tetranychus urticae and eggs as a model. Methods The UV-C light source was generated by a pulsed krypton fluoride (KrF) excimer laser operating at 248 nm emission wavelength. Pulse energy and pulse repetition rate were 5mJ, and up to 100Hz rate, respectively. Distance from light source to target was 150 mm with a target surface of 21.6 cm². The exposure time of the mite and fresh eggs varied from 1 to 4 minutes at a power of 300 mW. The post-irradiation acaricidal effects (mites’ mortality) were assessed immediately and measured 24 h post-irradiation. Effects on the hatchability of irradiated eggs were observed daily post-irradiation for up to 12 days. Results The mortality of mites observed with an energy amount of 5 and 40 mJ/mm² was 26% and 90%, respectively. The mortality at 80 mJ/mm² reached 99%. The effect of exposure duration on mortality was minimal. The effect of irradiation of eggs hatchability was even more important, with about 100% mortality with as little as 5 mJ/mm² for each exposure time. Conclusions A high mite mortality and lethal egg damage was already observed after less than 1 min exposure to UV-C pulsed irradiation. Pending further developments and feasibility studies, the current results and features of the UV-C generator (energy output modulation and applicability to varying spot sizes) open up a large field of germicidal applications requiring long-range radiation.

Rodent studies on the effects of engineered nanomaterials (ENM) on the gut microbiome have revealed contradictory results. Our aim was to assess the effects of four well-investigated model ENM using a realistic exposure scenario. Two independent

Macrophages are not only derived from circulating blood monocytes or embryonic precursors but also expand by proliferation. The origin determines macrophage fate and functions in steady state and pathological conditions. Macrophages predominantly infiltrate fibre-induced mesothelioma tumors and contribute to cancer development. Here, we revealed their ontogeny by comparing the response to needle-like mesotheliomagenic carbon nanotubes (CNT-7) with tangled-like non-mesotheliomagenic CNT-T. In a rat peritoneal cavity model of mesothelioma, both CNT induced a rapid macrophage disappearance reaction (MDR) of MHCIIlow resident macrophages generating an empty niche available for macrophage repopulation. Macrophage depletion after mesotheliomagenic CNT-7 was followed by a substantial inflammatory reaction, and macrophage replenishment completed after 7 days. Thirty days after non-mesotheliomagenic CNT-T, macrophage repopulation was still incomplete and accompanied by a limited inflammatory reaction. Cell depletion experiments, flow cytometry and RNA-seq analysis demonstrated that, after mesotheliomagenic CNT-7 exposure, resident macrophages were mainly replaced by an influx of monocytes, which differentiated locally into MHCIIhigh inflammatory macrophages. In contrast, the low inflammatory response induced by CNT-T was associated by the accumulation of self-renewing MHCIIlow macrophages that initially derive from monocytes. In conclusion, the mesotheliomagenic response to CNT specifically relies on macrophage niche recolonization by monocyte-derived inflammatory macrophages. In contrast, the apparent homeostasis after non-mesotheliomagenic CNT treatment involves a macrophage regeneration by proliferation. Macrophage depletion and repopulation are thus decisive events characterizing the carcinogenic activity of particles and fibres.

Background When a patient is seen with a newly diagnosed oropharyngeal squamous cell carcinoma, it remains unclear to the treating physicians how fast the tumor growth rate is. Methods From patients with oropharynx squamous cell carcinoma treated by radiotherapy, the investigators selected comparable diagnostic CT-scan (DiCT) and radiotherapy planning CT-scan (RtCT). Tumor and pathological lymph node volumes were measured in order to calculate tumor progression. Results From the selection of 19 patients, the mean absolute tumor progression rate was 0.23 ± 0.2 cm3 /d and mean relative progression rate was 1.84 ± 1.64%/d. Mean tumor doubling time is 286 days (range 7-1282 days), demonstrating a wide range of tumor growth pattern. Significant tumor progression (>20%) between DiCT and RtCT was shown in 73% of patients, and 53% of the patients were seen a tumor progression of >50% within a mean waiting time of 42.1 days. Kaplan-Meier curves showed a non-significative link between fast progression tumors (>1%/d) and higher risk of recurrence (HR: 2.2; P = .23). Conclusions Tumor progression can be assessed based on DiCT and RtCT. Treatment delay should be avoided at all cost. Different growth patterns were evidenced. For the fast-growing tumors subgroup, pejorative clinical outcomes were suggested. Prospective studies are needed to confirm a link between fast-growing tumors and higher risk for recurrence.

Dependence on multiple nutritional bacterial symbionts forming a metabolic unit has repeatedly evolved in many insect species that feed on nutritionally unbalanced diets such as plant sap. This is the case for aphids of the subfamilies Lachninae and Chaitophorinae, which have evolved di-symbiotic systems in which the ancient obligate nutritional symbiont Buchnera aphidicola is metabolically complemented by an additional nutritional symbiont acquired more recently. Deciphering how different symbionts integrate both metabolically and anatomically in such systems is crucial to understanding how complex nutritional symbiotic systems function and evolve. In this study, we sequenced and analyzed the genomes of the symbionts B. aphidicola and Serratia symbiotica associated with the Chaitophorinae aphids Sipha maydis and Periphyllus lyropictus. Our results show that, in these two species, B. aphidicola and S. symbiotica complement each other metabolically (and their hosts) for the biosynthesis of essential amino acids and vitamins, but with distinct metabolic reactions supported by each symbiont depending on the host species. Furthermore, the S. symbiotica symbiont associated with S. maydis appears to be strictly compartmentalized into the specialized host cells housing symbionts in aphids, the bacteriocytes, whereas the S. symbiotica symbiont associated with P. lyropictus exhibits a highly invasive phenotype, presumably because it is capable of expressing a larger set of virulence factors, including a complete flagellum for bacterial motility. Such contrasting levels of metabolic and anatomical integration for two S. symbiotica symbionts that were recently acquired as nutritional co-obligate partners reflect distinct coevolutionary processes specific to each association.

RESEARCH QUESTION: Could a modification in the ovarian tissue freezing protocol improve follicle survival after cryopreservation and xenotransplantation? DESIGN: Ovarian tissue was used from 13 adult patients, frozen either with our original protocol, or a modified version involving a higher concentration of dimethyl sulphoxide (DMSO), larger volume of cryopreservation solution and lower seeding temperature. After thawing, the ovarian fragments were xenotransplanted to six mice with severe combined immunodeficiency (SCID) for 3 weeks. RESULTS: The proportion of primordial follicles decreased, and the proportion of growing follicles increased significantly (all P < 0.01) after cryopreservation and xenografting compared with fresh controls for both protocols. Follicle density, development, ultrastructure and function were similar between treatments. CONCLUSIONS: This study showed that, although the higher DMSO concentration did not improve survival of preantral follicles, it did not seem to induce any major toxicity in the follicle population either.

BackgroundChronic obstructive pulmonary disease (COPD) is a devastating lung disease, representing the third cause of mortality worldwide. In COPD, the bronchial epithelium displays several structural and functional abnormalities affecting barrier integrity, cell polarity, and differentiation, as well as epithelial-to-mesenchymal transition (EMT) and inflammation. Although COPD is currently considered an irreversible disease, the (ir)reversible nature of epithelium changes ex vivo remains poorly known.MethodsThe persistence of COPD epithelial abnormalities was addressed in very long-term (10 weeks) primary cultures of air/liquid interface (ALI)-reconstituted airway epithelium from non-smoker controls, smoker controls, and COPD patients. The role of inflammation was also explored by stimulating ALI cultures with a cytokine mix consisting of TNF-α, IL-6 and IL-1β. Finally, the cellular niche holding epithelial memory was studied by exploiting a single cell RNA-sequencing database.ResultsAlmost all epithelial defects (barrier dysfunction, impaired polarity, lineage abnormalities) observed in smokers and COPD patients persisted in vitro up to week 10, except IL-8/CXCL-8 release and EMT which declined over time. Cytokine treatment induced COPD-like changes and reactivated EMT in COPD cells. Progenitor cells of large and small airways, namely basal and club cells, exhibited EMT-related signatures reminiscent of features observed in situ.ConclusionsThe airway epithelium from smokers and COPD patients displays a memory of its native state and previous injuries by cigarette smoking, which is multidimensional and sustained for years. This memory probably resides in progenitor cells of the airway epithelium.

Objectives Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli are an increasingly significant cause of hospital- and community-acquired infections worldwide. Whereas several reports have highlighted their increased prevalence also in North African countries, genomic data on isolates associated with these infections are still scarce. This study aimed to provide data on ESBL-producing E. coli isolates from patients with extraintestinal infections at the Military Teaching Hospital Mohamed V of Rabat, Morocco. Methods Whole-genome sequencing was carried out on 18 ESBL-producing extraintestinal pathogenic E. coli (ExPEC) isolates for analysis of phylogenomic evolution, virulence factors and antimicrobial resistance genes. Data were compared with ExPEC lineages from several surrounding countries using multilocus sequence typing (MLST) and single nucleotide polymorphism-based phylogenetic approaches. Results The majority of E. coli isolates were ST131 (n = 15), followed by ST617 (n = 2) and a novel sequence type (ST10703) that is closely related to the pandemic ST405 clone. All ST131 isolates belonged to the O25b-ST131 pandemic clone. They harboured more virulence genes than their non-ST131 counterparts. IncF plasmid replicons and the blaCTX-M-15 β-lactamase gene were identified in all isolates. No ESBL-producing E. coli isolates carried any known carbapenemase gene. Conclusion Our findings underscore the pre-eminence of ST131 as the major factor driving the expansion of ExPEC in the Rabat region while highlighting the potential links with isolates circulating in other neighbouring countries.

We have previously demonstrated that systemic AMP-activated protein kinase α1 (AMPKα1) invalidation enhanced adverse LV remodelling by increasing fibroblast proliferation, while myodifferentiation and scar maturation were impaired. We thus hypothesised that fibroblastic AMPKα1 was a key signalling element in regulating fibrosis in the infarcted myocardium and an attractive target for therapeutic intervention. The present study investigates the effects of myofibroblast (MF)-specific deletion of AMPKα1 on left ventricular (LV) adaptation following myocardial infarction (MI), and the underlying molecular mechanisms. MF-restricted AMPKα1 conditional knockout (cKO) mice were subjected to permanent ligation of the left anterior descending coronary artery. cKO hearts exhibit exacerbated post-MI adverse LV remodelling and are characterised by exaggerated fibrotic response, compared to wild-type (WT) hearts. Cardiac fibroblast proliferation and MF content significantly increase in cKO infarcted hearts, coincident with a significant reduction of connexin 43 (Cx43) expression in MFs. Mechanistically, AMPKα1 influences Cx43 expression by both a transcriptional and a post-transcriptional mechanism involving miR-125b-5p. Collectively, our data demonstrate that MF-AMPKα1 functions as a master regulator of cardiac fibrosis and remodelling and might constitute a novel potential target for pharmacological anti-fibrotic applications. Supplementary Information The online version contains supplementary material available at 10.1007/s00395-021-00846-y.

On Friday, 19 March 2021, WHO organized a virtual global workshop highlighting the need for a globally coordinated plan to increase SARS-CoV-2 genetic sequencing capacities to detect SARS-CoV-2 mutations and variants, and to monitor virus genomic evolution worldwide. One week later, in another virtual meeting, it focused on sero epidemiology for SARS-CoV-2 variants of concern and variants of interest. Efficient monitoring of the virus relies on the storage, handling and sharing of the genomic data and the associated metadata. In this manuscript, we demonstrate how the Bioconductor VariantExperiment class addresses these needs, offering a robust and efficient solution to the requirements laid out by the WHO.

Background Ambient air pollution by particulate matters, including diesel exhaust particles (DEP), is a major cause of cardiovascular and metabolic mortality worldwide. The mechanisms by which DEP cause these adverse outcomes are not completely understood. Because the gut microbiota controls cardiovascular and metabolic health, we hypothesized that the fraction of inhaled DEP which reach the gut after mucociliary clearance and swallowing might induce gut dysbiosis and, in turn, contribute to aggravate or induce cardiovascular and metabolic diseases. Results Female ApoE−/− mice fed a Western diet, and wild-type (C57Bl/6) mice fed standard diet were gavaged with DEP (SRM2975) doses corresponding to mucociliary clearance from inhalation exposure (200 or 1000 ng/day, 3 times a week for 3 months; and 40, 200 or 1000 ng/day, 3 times a week for 6 months, respectively). No mortality, overt systemic or digestive toxicity was observed. A dose-dependent alteration of the gut microbiota was recorded in both strains. In ApoE−/−, β-diversity was modified by DEP, but no significant modification of the relative abundance of the phyla, families or genera was identified. In C57BL/6 mice, DEP reduced α-diversity (Shannon and Simpson indices), and modified β-diversity, including a reduction of the Proteobacteria and Patescibacteria phyla, and an increase of the Campylobacterota phylum. In both mouse models, perturbation of the gut microbiota composition was associated with a dose-dependent reduction of bacterial short chain fatty acids (butyrate and propionate) in cecal content. However, DEP ingestion did not aggravate (ApoE−/−), or induce (C57BL/6 mice) atherosclerotic plaques, and no metabolic alteration (glucose tolerance, resistance to insulin, or lipidemia) was recorded. Conclusions We show here that oral exposure to DEP, at doses relevant for human health, changes the composition and function of the gut microbiota. These modifications were, however, not translated into ultimate atherosclerotic or metabolic outcomes.

We report here the complete genome sequence of a Bacillus cereus isolate identified in a soil sample from Namibia. This isolate is closely related to the B. anthracis clade. While the plasmids (500 and 12 kb) carry no detectable B. anthracis virulence gene, the large plasmid shares a 50-kb continuous region similar to plasmid pXO1.

Objectives We explored histological and transcriptomic profiles of paired synovial biopsies from rheumatoid arthritis (RA) patients, in order to assess homogeneity in synovial tissue at the individual level. Methods Synovial biopsies were performed simultaneously in one small and one large joint per patient using needle-arthroscopy for the knee and ultrasound-guided biopsy for the hand or wrist. Synovium from individuals with osteoarthritis was used as controls. Paraffin-embedded samples were stained for CD3, CD20, and CD68. Total RNA was hybridized on high-density microarrays. TCRB variable sequences were obtained from synovial and blood RNA samples. Results Twenty paired biopsies from 10 RA patients with active disease were analyzed. Semi-quantification of histological markers showed a positive correlation for synovial hyperplasia, inflammatory infiltrates and CD3-positive T cells between pairs. Pairwise comparison of transcriptomic profiles showed similar expression of RA-related molecular pathways (TCR signaling, T cell costimulation and response to TNFα). T cells clonotypes were enriched in all but one joints compared to blood, regardless of the magnitude of T cell infiltration. Enriched clonotypes were shared between pairs (23–100%), but this was less the case in pairs of joints displaying weaker T cell signatures and more pronounced germinal center-like transcriptomic profiles. Conclusion Cellular and molecular alterations in RA synovitis are similar between small and large joints from the same patient. Interindividual differences in magnitude of T cell infiltrates and distribution of enriched T cell clonotypes support the concept of distinct synovial pathotypes in RA that are associated with systemic versus local antigen-driven activation of T cells.

Background: Multigene panels are routinely used to assess for predisposing germline mutations in families at high breast cancer risk. The number of variants of unknown significance thereby identified increases with the number of sequenced genes. We aimed to determine whether tumor sequencing can help refine the analysis of germline variants based on second somatic genetic events in the same gene. Methods: Whole-exome sequencing (WES) was performed on whole blood DNA from 70 unrelated breast cancer patients referred for genetic testing and without a BRCA1, BRCA2, TP53, or CHEK2 mutation. Rare variants were retained in a list of 735 genes. WES was performed on matched tumor DNA to identify somatic second hits (copy number alterations (CNAs) or mutations) in the same genes. Distinct methods (among which immunohistochemistry, mutational signatures, homologous recombination deficiency, and tumor mutation burden analyses) were used to further study the role of the variants in tumor development, as appropriate. Results: Sixty-eight patients (97%) carried at least one germline variant (4.7 ± 2.0 variants per patient). Of the 329 variants, 55 (17%) presented a second hit in paired tumor tissue. Of these, 53 were CNAs, resulting in tumor enrichment (28 variants) or depletion (25 variants) of the germline variant. Eleven patients received variant disclosure, with clinical measures for five of them. Seven variants in breast cancer-predisposing genes were considered not implicated in oncogenesis. One patient presented significant tumor enrichment of a germline variant in the oncogene ERBB2, in vitro expression of which caused downstream signaling pathway activation. Conclusion: Tumor sequencing is a powerful approach to refine variant interpretation in cancer-predisposing genes in high-risk breast cancer patients. In this series, the strategy provided clinically relevant information for 11 out of 70 patients (16%), adapted to the considered gene and the familial clinical phenotype., SCOPUS: ar.j, info:eu-repo/semantics/published

RESEARCH QUESTION: Does ulipristal acetate (UPA) modify the expression of genes related to apoptosis or the extracellular matrix in uterine myomas and are any modifications associated with a clinical response? DESIGN: Targeted analysis of 176 apoptosis- or extracellular-matrix-related genes was conducted using polymerase chain reaction (PCR) arrays. Relevant results were validated by quantitative PCR. Four groups were established: responsive short-term (one course, n = 9), responsive long-term (two to four courses, n = 9), non-responsive (n = 9), and the control group who was not given any hormone therapy (n = 9). The clinical response was monitored by medical imagery and considered significant when volume reduction was greater than 25%. RESULTS: Compared with untreated myomas, significant changes in expression of four genes were found in UPA-treated myomas. Gene expression of integrin subunit beta 4 was repressed by UPA treatment (fold change [FC] = -12.50, P < 0.001, q < 0.001), tenascin-C expression was downregulated in UPA-responsive patients (FC = -2.50, P = 0.010, q = 0.090), survivin was repressed in short-term UPA-responsive tumours (FC = -7.69, P < 0.001, q = 0.010), and catenin delta 2 gene expression was upregulated in non-responsive myomas (FC = +7.36, P < 0.001, q = 0.010). CONCLUSION: This characterization provides the first molecular distinction between myomas responsive or non-responsive to UPA treatment.

Study question How does the formation of the blood-testis barrier (BTB), as reflected by the expression of connexin 43 and claudin 11 proteins during the pubertal transition period, take place in vitro compared to samples from a large cohort of pre/peripubertal boys? Summary answer The BTB connexin 43 and claudin 11 expression patterns appeared to be partially achieved in organotypic culture when compared to that in samples from 71 pre/peripubertal patients. What is known already Although alterations in the protein expression patterns of the BTB, whose main components are connexin 43 and claudin 11, are known to be associated with impaired spermatogenesis in mice and adult men, there is a lack of knowledge on its formation in pre-peripubertal human tissue both in vitro and in vivo. Moreover, despite Sertoli cell (SC) maturation during long-term organotypic culture of immature testicular tissue (ITT), initiation of spermatogenesis has not yet been achieved. Study design, size, duration Histological sections from 71 pre-peripubertal patients were evaluated for the formation of the BTB acting as in vivo controls according to age, SC maturation, clinical signs of puberty and germ cell differentiation. Testicular tissue fragments retrieved from three prepubertal boys were cultured in a long-term organotypic system to analyze the BTB formation and expression pattern in correlation with SC maturation. Participants/materials, setting, methods Testicular histological sections from 71 patients aged 0-16 years who underwent a biopsy between 2005 and 2014 to preserve their fertility before gonadotoxic treatment were examined. Immunohistochemistry (IHC) results for connexin 43 and claudin 11 as BTB markers, using a semi-quantitative score for their expression, and for Anti-Mullerian hormone (AMH), as SC maturation marker, were analyzed. Germ cell differentiation was evaluated on Hematoxylin-Eosin sections. Tanner stages at the time of biopsy were recorded from medical files. A longitudinal analysis of connexin 43, claudin 11 and AMH expressions on immunohistological sections of organotypic cultured testicular tissue from three prepubertal boys who underwent a biopsy for fertility preservation was performed. Immunostaining was evaluated at culture Days 0, 1, 3, 10, 16, 27, 32, 53, 64 and 139 for two different types of culture media. Main results and the role of chance Immunohistochemical control sections showed progressive maturation of SCs, as shown by the decrease in AMH expression, with increasing age (P ≤ 0.01) and the AMH expression was negatively correlated with the expression of connexin 43 and claudin 11 (P ≤ 0.01 for both proteins). Androgen receptor (AR) expression increased with age (P ≤ 0.01) and was significantly correlated with the expression of connexin 43 (P = 0.002) and claudin 11 (P = 0.03). A statistical correlation was also found between the reduction of AMH expression and both the advancement of Tanner stages (P ≤ 0.01) and the differentiation of germ cells (P ≤ 0.01). Furthermore, positive correlations between BTB formation (using connexin 43 and claudin 11 expression) and age (P ≤ 0.01 for both the proteins), higher Tanner stages (P ≤ 0.001 and P ≤ 0.01 for connexin 43 and claudin 11, respectively), and presence of more advanced germ cells (P ≤ 0.001 for both proteins) were observed. In the subanalysis on organotypic cultured ITT, where a significant decrease in AMH expression as a marker of SC maturation was already reported, we showed the onset of expression of connexin 43 at Day 16 (P ≤ 0.001) and a constant expression of claudin 11 from Days 0 to 139, for all three patients, without differences between the two types of culture media. Large scale data N/A. Limitations reasons for caution Accessibility of prepubertal human testicular tissue is a major limiting factor to the analysis of cultured tissue samples from a wide number of patients, as would be needed to assess the in vitro development of the BTB according to the age. The impossibility of performing longitudinal studies on in vivo BTB formation in the same patient prevents a comparison of the time needed to achieve effective BTB formation and protein expression patterns in vivo and in vitro. Wider implications of the findings To the best of our knowledge, this is the first report describing the expression of two BTB proteins in samples from a cohort of prepubertal and peripubertal boys, for the in vivo pattern, and in cultured ITT from a few prepubertal boys, for the in vitro evaluation. Since the formation of this barrier is essential for spermatogenesis and because little is known about its protein expression patterns and development in humans, a deeper understanding of the testicular microenvironment is essential to improve ITT in vitro culture conditions. The final aim is to restore fertility by acheiving in vitro differentiation of spermatogonial stem cells, using cryopreserved ITT collected before gonadotoxic therapies. Study funding and competing interest(s) Funding was received from Fonds National de la Recherche Scientifique de Belgique (Grant Televie Nos. 7.4554.14F and 7.6511.16) and Fondation Salus Sanguinis. No conflict of interest has to be disclosed.

International audience; Acetyl-CoA carboxylase (ACC) is the first enzyme regulating de novo lipid synthesis via the carboxylation of acetyl-CoA into malonyl-CoA. The inhibition of its activity decreases lipogenesis and, in parallel, increases the acetyl-CoA content, which serves as a substrate for protein acetylation. Several findings support a role for acetylation signaling in coordinating signaling systems that drive platelet cytoskeletal changes and aggregation. Therefore, we investigated the impact of ACC inhibition on tubulin acetylation and platelet functions. Human platelets were incubated 2 h with CP640.186, a pharmacological ACC inhibitor, prior to thrombin stimulation. We have herein demonstrated that CP640.186 treatment does not affect overall platelet lipid content, yet it is associated with increased tubulin acetylation levels, both at the basal state and after thrombin stimulation. This resulted in impaired platelet aggregation. Similar results were obtained using human platelets that were pretreated with tubacin, an inhibitor of tubulin deacetylase HDAC6. In addition, both ACC and HDAC6 inhibitions block key platelet cytoskeleton signaling events, including Rac1 GTPase activation and the phosphorylation of its downstream effector, p21-activated kinase 2 (PAK2). However, neither CP640.186 nor tubacin affects thrombin-induced actin cytoskeleton remodeling, while ACC inhibition results in decreased thrombin-induced reactive oxygen species (ROS) production and extracellular signal-regulated kinase (ERK) phosphorylation. We conclude that when using washed human platelets, ACC inhibition limits tubulin deacetylation upon thrombin stimulation, which in turn impairs platelet aggregation. The mechanism involves a downregulation of the Rac1/PAK2 pathway, being independent of actin cytoskeleton.

Introduction Acetyl-CoA carboxylase (ACC), the first enzyme regulating lipid synthesis, promotes thrombus formation by increasing platelet phospholipid content. Inhibition of its activity decreases lipogenesis and increases the content in acetyl-CoA which can serve as a substrate for protein acetylation. This posttranslational modification plays a key role in the regulation of platelet aggregation, via tubulin acetylation. Purpose To demonstrate that ACC inhibition may affect platelet functions via an alteration of lipid content and/or tubulin acetylation. Methods Platelets were treated 2 hours with CP640.186, a pharmacological ACC inhibitor, prior to thrombin stimulation. Platelet functions were assessed by aggregometry and flow cytometry. Lipogenesis was measured via 14C-acetate incorporation into lipids. Lipidomics analysis was carried out on the commercial Lipidyzer platform. Protein phosphorylation and acetylation were evaluated by western blot. Results Treatment with CP640.186 drastically decreased platelet lipogenesis. However, the quantitative lipidomics analyses showed that preincubation with the compound did not affect global platelet lipid content. Interestingly, this short-term ACC inhibition was sufficient to increase tubulin acetylation level, at basal state and after thrombin stimulation. It was associated with an impaired platelet aggregation, in response to low thrombin concentration, while granules secretion was not affected. Mechanistically, we highlighted a decrease in Rac1 activity, associated with a reduced phosphorylation of its downstream effector PAK2. Surprisingly, actin cytoskeleton was not impacted but we evidenced a significant decrease in ROS production which could result from a decreased NOX2 activity. Conclusion Pharmacological ACC inhibition decreases platelet aggregation upon thrombin stimulation. The mechanism depends on increased tubulin acetylation, with subsequent alteration of the Rac1/PAK2/NOX2 signaling pathway Funding Acknowledgement Type of funding sources: Other. Main funding source(s): Fonds pour la formation à la Recherche dans l'Industrie et l'Agriculture (FRIA)

Background Nanotechnologies provide new opportunities for improving the safety, quality, shelf life, flavor and appearance of foods. The most common nanoparticles (NPs) in human diet are silver metal, mainly present in food packaging and appliances, and silicon and titanium dioxides used as additives. The rapid development and commercialization of consumer products containing these engineered NPs is, however, not well supported by appropriate toxicological studies and risk assessment. Local and systemic toxicity and/or disruption of the gut microbiota (GM) have already been observed after oral administration of NPs in experimental animals, but results are not consistent and doses used were often much higher than the estimated human intakes. In view of the strong evidence linking alterations of the GM to cardiometabolic (CM) diseases, we hypothesized that dietary NPs might disturb this GM-CM axis. Materials and methods We exposed male C57BL/6JRj mice (n = 13 per dose group) to dietary NPs mixed in food pellets at doses relevant for human exposure: Ag (0, 4, 40 or 400 μg/kg pellet), SiO2 (0, 0.8, 8 and 80 mg/kg pellet) or TiO2 (0, 0.4, 4 or 40 mg/kg pellet). After 24 weeks of exposure, we assessed effects on the GM and CM health (n = 8 per dose group). The reversibility of the effects was examined after 8 additional weeks without NPs exposure (recovery period, n ≤ -5 per dose group). Results No overt toxicity was recorded. The GM β-diversity was dose-dependently disrupted by the three NPs, and the bacterial short chain fatty acids (SCFAs) were dose-dependently reduced after the administration of SiO2 and TiO2 NPs. These effects disappeared completely or partly after the recovery period, strengthening the association with dietary NPs. We did not observe atheromatous disease or glucose intolerance after NP exposure. Instead, dose-dependent decreases in the expression of IL-6 in the liver, circulating triglycerides (TG) and urea nitrogen (BUN) were recorded after administration of the NPs. Conclusion We found that long-term oral exposure to dietary NPs at doses relevant for estimated human intakes disrupts the GM composition and function. These modifications did not appear associated with atheromatous or deleterious metabolic outcomes.

The Aristolochic Acid (AA)-specific mutational pattern was recently characterized in urothelial carcinoma (UC) from Belgian AA Nephropathy (AAN) patients (n=5). Besides the A>T transversion hallmark, a specific AA-mutational pattern was found in the TP53 hotspot region in p53-positive immunohistochemistry (IHC) areas and consisted of poly- or multiclonal TP53 alterations and an unusual high prevalence of G>T transversion. In the current study, these data were complemented using the same validated methodology for assessing the complete coding sequence of the TP53 gene in tumor areas stratified according to the percentage of p53-stained cells (i.e., [+], ≥60%; [+/-], 1-59%, [-], no staining). Results were compared to existing data (i.e., other series of AA-associated UC and IARC TP53 database). Aside of the TP53 hotspot region (exons 5-8), multiple mutations were also found in exons 4 and 10. A unique TP53 mutational pattern was characterized by a large spectrum of mutations among which A>T and C>T have the highest prevalence. Most A>T mutations were characterized by the 5'Py-A-Pu sequence. Interestingly, the majority of p53-negative areas were dysplastic (low grade intra-urothelial neoplasia) and disclosed TP53 mutations among which a majority of A>T transversions. Beside nonsense p53-negative mutations, several missense mutations are also known to affect p53 DNA- or zinc-binding domains, hence probably impairing the antigen binding site and/or masking the antigenic epitope. These uncommon histologic, immunopathologic and genetic features in Belgian AAN patients probably result from the unique pattern of duration and extent of AA exposure which led to a cumulative toxic dose ingested over a short period of time. Consequenlty, current results bring additional and valuable evidence unravelling the molecular pathogenesis of AA-induced carcinogenesis and the origin of related high-grade UCs.

We presented here the first draft genome sequence of the trypanosomatid Herpetomonas muscarum ingenoplastis. This parasite was isolated repeatedly in the black blowfly, Phormia regina. This is the first draft genome of a flagellate from the phylogenetically distinct clade of Trypanosomatidae.

Rationale: Research into pathways that drive COPD pathophysiology are key to unravel novel therapeutic targets. In contrast with the alveolar epithelium, the role of Wnt in the COPD bronchial epithelium (BE) remains unclear. Methods: We performed a targeted RNA sequencing (RNA-seq) of 92 preselected canonical Wnt-related genes in air/liquid interface-reconstituted BE from 47 patients (9 non-smoker controls, 10 smoker controls, 9 mild COPD, 10 moderate COPD and 9 very severe COPD). Quantification of active and total β-catenin was performed in immunostained BE. In addition, a pilot in vitro modulation experiment was carried out using CHIR99021 and XAV939 to activate or inhibit, respectively, the canonical Wnt pathway, to assess its role in broncho-epithelial differentiation. Results: RNA-seq data of Wnt genes was consistent with a global activation of the canonical Wnt pathway in COPD, as evidenced by the upregulation of PORCN, FZD7, CTNNB1, CSNK2A1&2, SOX4, TCF7L2, and MMP2&7 amongst others. This activation was more pronounced in very severe disease. Increased levels of both active and total β-catenin were observed in BE. In the pilot modulation experiment, CHIR99021 strongly inhibited epithelial differentiation for both goblet (SPDEF, MUC5AC) and ciliated cells (FOXJ1, DNAI1&2), as well as epithelial polarization (pIgR), while XAV939 promoted broncho-epithelial polarization. Conclusions: Canonical Wnt signalling is activated in the COPD bronchial epithelium, and could contribute to the COPD airway epithelial phenotype which will be further assessed through modulation experiments and in situ BE laser microdissection.

Multiple-Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) is widely used by laboratory-based surveillance networks for subtyping pathogens causing foodborne and water-borne disease outbreaks. However, Whole Genome Sequencing (WGS) has recently emerged as the new more powerful reference for pathogen subtyping, making a data conversion method necessary which enables the users to compare the MLVA identified by either method. TheMLVATypeshiny application was designed to extract MLVA profiles from WGS data while ensuring backward compatibility with traditional MLVA typing methods.To test and validate theMLVATypealgorithm, WGS-derived MLVA profiles of nineteenVibrio choleraeisolates from Democratic Republic of the Congo (n=9) and Uganda (n=10) were compared to MLVA profiles generated by microchip electrophoresis (Bioanalyzer Agilent 2100), GeneScan analysis, and Sanger sequencing as the reference method. Unlike amplicon-size derived MLVA profiles, results obtained by Sanger sequencing and WGS were totally concordant. However, the latter were affected by censored estimations whose percentage was inversely proportional to the k-mer parameter used during genome assembly. With a k-mer of 127, less than 15% estimation ofV. choleraeVNTR was censored. Preventing censored estimation was only achievable when using a longer k-mer size (i.e. 175), which is not proposed in the SPAdes v.3.13.0 software.In silicoanalysis showed that this limitation does not apply to other microbial species (e.g. Mycobacterium, Streptococcus, Staphylococcus, andPseudomonas) characterized by smaller lengths of motif repeats. As NGS read lengths and qualities tend to increase with time, one may expect the increase of k-mer size in a near future. UsingMLVATypeapplication with a longer k-mer size will then efficiently retrieve MLVA profiles from WGS data while avoiding censored estimation irrespective of the microbial species.Author summaryNext Generation Sequencing (NGS) has emerged as a powerful high throughput genomic approach enabling the Whole Genome Sequence (WGS) of pathogens to be assembled in a relatively short time. A major advantage of WGS, compared to traditional genotypic identification and typing methods, is its ability to generate data that can be exploitedin silicofor multiple bacterial tests including accurate subtyping, determination of genetic relatedness, and characterization of virulence and antimicrobial resistance determinants. Accordingly, WGS is now rapidly replacing traditional methods like Multi-Locus Variable Number of Tandem Repeats Analysis (MLVA) that has long been used in the public health sector for laboratory-based surveillance of pathogens and outbreak response. While these missions require maintenance of data comparability within networks, the lack of backward compatibility between WGS-derived and traditional MLVA methods is a well-recognized issue. As illustrated here withVibrio choleraeisolates from DRC and Uganda, theMLVATypesoftware application analyzes WGS data to generate MLVA profiles that are identical to those determined with traditional typing. Interestingly, this tool has also the potential to extract MLVA profiles from any bacterial genome that are characterized by a small number of tandem repeats,e.g. Streptococcus, Staphylococcus, Pseudomonas, andMycobacteriumspecies. This restriction can be lifted if subsequences of length k, called k-mers, are longer than what is currently proposed by genome assembly algorithm like SPAdes v.3.13.0.

Many genetic risk factors have been identified for causing venous thromboembolism (VTE). Most of them affect the function of natural anticoagulant pathways, particularly the protein C system, although recent studies suggest a role of components of the hematopoietic pathway in the etiology of venous thrombosis. In this case–control study, we aimed to determine the frequency of prothrombin G20210A and factor V Leiden (FVL) G1691A polymorphisms and protein C, protein S, and antithrombin III deficiencies in the East Algerian population and to investigate whether these genetic factors are associated with VTE. On the other hand, our study tends to evaluate the status of JAK2V617F and calreticulin (CALR) mutations among these cases. The participants consisted of 121 cases with VTE and 146 healthy controls. Polymorphisms of FVL G1691A and prothrombin G20210A were genotyped by polymerase chain reaction (PCR) restriction fragment length polymorphism. JAK2-V617F and calreticulin mutations were analyzed by quantitative PCR and PCR followed by capillary electrophoresis sequencing, respectively. Protein C, protein S, and antithrombin levels were determined and then hereditary deficiencies were identified. Of all cases and controls, none was a carrier of the antithrombin III deficiency, prothrombin gene G20210A, and CALR mutations. Only 1 case reported having a positive JAK2 mutation (mutant allele burden was 15%). The FVL mutation (GA/AA) was found in 14 (11.6%) cases and 2 (1.4%) controls and it was significantly different between both the groups ( P = .001). Deficiencies of protein S and protein C were detected in 17 (18.8%) cases. The univariate analysis resulted in a significant impact of FVL (odds ratio [OR] = 9.4, 95% confidence interval [CI] = 2.1-42.3; P = .003) and of protein S deficiency (OR = 16.9, 95% CI =2.1-132.8, P = .007) on the VTE status. Both factors stayed significant after adjustment for sex and age. The OR of the protein C deficiency was slightly elevated (OR = 6.4, 95% CI = 0.7-55.5), but it did not reach the level of statistical significance ( P = .091), and it was therefore not considered as a risk factor. In conclusion, coagulant factor V gene G1691A mutation and protein S deficiency constitute important genetic risk factors in patients with VTE in Eastern Algeria. The somatic mutation of JAK2 V617F and CALR mutations are less frequent causes of VTE, thus routine testing for these mutations is not recommended.

Aim To determine predisposing factors of idiopathic allograft fibrosis among pediatric liver transplant recipients. Background Protocol biopsies (PB) from stable liver transplant (LT) recipient children frequently exhibit idiopathic fibrosis. The relation between allograft inflammation, humoral immune response and fibrosis is uncertain. Also the role of HLA-DRB1 genotype has not been evaluated, though it's associated with fibrosis in autoimmune hepatitis. Patients and Methods This observational study, included 89 stable LT recipient transplanted between 2004–2012 with mean follow-up of 4.3 years, 281 serial PBs (3.1 biopsy/child) and human leukocyte antigen (HLA) antibody data. PBs were taken 1–2, 2–3, 3–5, 5–7, and 7–10 years post-LT, and evaluated for inflammation and fibrosis using liver allograft fibrosis score (LAFSc). The evolution of fibrosis, inflammation and related predisposing factors were analysed. Findings HLA-DRB1*03/04 allele and Class II DSA were significantly associated with portal fibrosis (p = 0.03; p = 0.03, respectively). Portal inflammation was predisposed by Class II DSA (p = 0.02) and non-HLA antibody presence (p = 0.01). Non-portal fibrosis wasn't predisposed by inflammation. Lobular inflammation was associated with non-HLA antibodies. Interpretation We conclusively demonstrated that allograft inflammation results in fibrosis and is associated with post-LT Class II DSA and non-HLA antibodies. The HLA-DRB1*03/04 allele caused genetic predisposition for fibrosis. Funding None., Highlights • There is a link between genetic predisposition, alloimmune antibodies, allograft inflammation and fibrosis • Description of allograft natural history by assessing evolution of inflammation, fibrosis in different histological zones • Allograft inflammation results in allograft fibrosis and is associated with post-LT Class II DSA and non-HLA antibodies • The HLA-DRB1*03/04 allele causes genetic predisposition for fibrosis Among pediatric liver transplant recipients, the graft longevity is expected to parallel the life expectancy of the recipient. A major impediment in this is progressive allograft fibrosis, the cause of which is not well understood. If this pathogenesis could be found, then interventions to arrest the progressive fibrosis could be explored. In our 89 patients for whom we did periodic surveillance liver biopsies, inflammation was found to precede the fibrosis and associated with presence of specific antibodies. Certain genetic predisposition for higher fibrogenesis was also found, thus establishing a link between genetic predisposition, alloimmune response, inflammation, and allograft fibrosis.

Introduction Acetyl-CoA carboxylase (ACC) is the first committed enzyme regulating lipids synthesis. We have recently demonstrated that the overexpression of a constitutively active form of ACC in platelets leads to an increase in the phospholipid content which stimulates thrombus formation. Based on these data, we believe that ACC inhibition might be a counter-regulatory mechanism limiting endogenous lipogenesis and platelet reactivity in certain pathological circumstances. Aim Our study aims to assess the impact of pharmacological ACC1 inhibition on platelet functions, de novo lipogenesis and lipid content. Methods Platelets were treated with the long-chain fatty acid (FA) analog TOFA (30 μM) or the active site-directed CP640.186 (CP, 60 μM), two ACC inhibitors, for 2H before thrombin stimulation. Lipogenesis was measured via 14C-acetate incorporation into FA. Platelet functions were assessed by aggregometry. Lipidomics analysis was carried out on the commercial Lipidyzer platform. Results Both ACC inhibitors decreased lipogenesis and thrombin-induced platelet aggregation. TOFA, but not CP, drastically decreased mitochondrial oxygen consumption rate, an effect associated with an increased reactive oxygen species production and AMPK activation. Surprisingly, the quantitative lipidomics analyses showed that a preincubation with CP did not affect global platelet lipid content. However, the short-term ACC inhibition (and potential acetyl-CoA accumulation) was sufficient to modify the level of acetylated proteins such as tubulin, a key player in the regulation of platelet shape change and aggregation. Conclusion Given the effect of TOFA on mitochondrial respiration, oxidative stress and AMPK activation, CP seems more appropriate to investigate the impact of ACC inhibition in platelets. A short-term acute treatment with CP inhibits thrombin-induced aggregation by a mechanism which doesn’t depend on lipid content but might involve changes in protein acetylation.

The molecular landscape of head and neck squamous cell carcinoma (HNSCC) harbors potentially actionable genomic alterations. We aimed to study the utility of liquid biopsy to (i) characterize the mutational landscape of recurrent/metastatic HNSCC using a comprehensive gene panel and (ii) estimate the concordance between DNA mutations identified from circulating tumor DNA (ctDNA) and matched tumor tissues. Targeted next-generation sequencing (NGS) was performed on cell-free DNA (cfDNA) of 39 patients with locoregional recurrent (n = 19) and/or metastatic (n = 20) HNSCC. Tumor biopsy (n = 18) was sequenced using the same technique. ctDNA was detected in 51% of patients (20/39) with a higher probability of detection in metastatic than locoregional recurrent disease (70% versus 30%, p = 0.025). 81% and 58% of the tissue tumor variants were not detected in plasma when considering all patients and only metastatic patients with detectable ctDNA, respectively. In a multivariate analysis, the likelihood of detecting the tissue tumor variant in plasma was related to metastatic status (p = 0.012), tumor variant allele frequency (p

BACKGROUND Breast cancer (BC) is a complex disease. While Mendelian mutations in high-penetrance genes predispose to some familial forms, others are due to multiple common low-penetrance polymorphisms. However, it is unclear if the combination of some rare coding variants has an effect. As some Mendelian forms demonstrate genotype-phenotype correlations, we hypothesized co-segregation of rare variants in cancer-related genes would be more frequent in high-risk families with a uniform BC phenotype among relatives. METHODS Whole-exome sequencing was performed on germline DNA from unrelated BC patients referred for genetic testing but without a causative mutation and for which DNA was available from at least one second relative with BC. We retained rare (< 1.5% in ExaC) in silico possibly damaging variants (splice alteration, nonsense or frameshift mutation, or missense variant considered deleterious by at least 2 different algorithms) in a list of 735 cancer or DNA repair related genes. We assessed correlation between segregation of these variants in affected relatives (studied by Sanger sequencing) and tumor phenotypes. A familial uniform BC phenotype was denoted as sharing the same BC histology and the same clinical molecular classification, based on the expression of the hormone receptors and HER2. RESULTS Of the 54 families, 23 (43%) presented a uniform phenotype across the affected relatives. We identified 251 variants. The mean number of variants tested did not differ between families with a similar BC phenotype and families with different BC phenotypes (4.3 vs 4.9 respectively, two-sided T-test p=0.19). The proportion of disease co-segregating variants inside each family was however significantly higher in families with a uniform BC phenotype than in families with a heterogeneous BC phenotype (57.8% vs 30.2%, p Citation Format: Cedric Van Marcke, Raphaël Helaers, Céline A Schoonjans, Jérôme Ambroise, Martine Berlière, Jean-Luc Canon, Peter Vuylsteke, Jean-Pascal Machiels, Miikka Vikkula, Francois P Duhoux. Co-segregation of rare possibly-damaging variants in cancer-related genes correlates with phenotypic homogeneity in familial breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-03-04.

Adult-derived human liver stem/progenitor cells (ADHLSCs) are a promising alternative to orthotopic liver transplantation in the treatment of inborn errors of metabolism. However, as is the case with many mesenchymal stromal cells, ADHLSCs have shown a low level of engraftment, which could be explained by the fact that they lack expression of selectin ligand and LFA-1 and only slightly express VLA- 4, molecules that have been shown to be involved in cell adhesion to the endothelium. In this paper, we have investigated strategies to increase their rolling and adhesion during the homing process by (1) adding a selectin ligand (Sialyl Lewis X) to their surface using biotinyl- N-hydroxy-succinimide–streptavidin bridges, and (2) protecting the adhesion proteins from trypsinization-induced damage using a thermosensitive polymer for cell culture and a nonenzymatic cell dissociation solution (CDS) for harvest. Despite increasing adhesion of ADHLSCs to E-selectin during an adhesion test in vitro performed under shear stress, the addition of Sialyl Lewis X did not increase adhesion to endothelial cells under the same conditions. Cultivating cells on a thermosensitive polymer and harvesting them with CDS increased their adhesion to endothelial cells under noninflammatory conditions, compared to the use of trypsin. However, we were not able to demonstrate any improvement in cell adhesion to the endothelium following culture on polymer and harvest with CDS, suggesting that alternative methods of improving engraftment still need to be evaluated.

While in mice various studies have described the completion of spermatogenesis in vitro using either organotypic culture of prepubertal testicular tissue or 3D culture of isolated cells, in humans it has not been possible to achieve germ cell differentiation from immature testicular tissue (ITT). In our study, we evaluated the ability of human ITT to differentiate via a long-term organotypic culture of frozen–thawed 1 mm3 testicular fragments from five prepubertal boys in two different culture media. Tissue and supernatants were analyzed at regular intervals up to day 139. Sertoli cell (SC) viability and maturation was evaluated using immunohistochemistry (IHC) for SOX9, GDNF, anti-Mullerian hormone (AMH) and androgen receptor (AR), and AMH concentration in supernatants. Spermatogonia (SG) and proliferating cells were identified by MAGE-A4 (for SG) and Ki67 (for proliferating cells) via immunohistochemistry (IHC). Apoptotic cells were studied by active caspase 3. To evaluate Leydig cell (LC) functionality testosterone was measured in the supernatants and steroidogenic acute regulatory protein (STAR) IHC was performed. Germ cell differentiation was evaluated on Hematoxylin-Eosin histological sections, via IHC for synaptonemal complex 3 (SYCP3) for spermatocytes, Protein boule-like (BOLL) for spermatocytes and round spermatids, angiotensin-converting enzyme (ACE), protamine 2 and transition protein 1 (for elongated spermatids) and via chromogenic in situ hybridization (CISH). We reported the generation of meiotic and postmeiotic cells after 16 days of culture, as shown by the histological analyses, the presence of differentiation markers and the increase of haploid germ cells. We showed SC viability and maturation by a decrease of AMH secretion in the supernatants (p ≤ 0.001) while the number of SOX9 positive cells did not show any variation. A decrease of spermatogonia (p ≤ 0.001) was observed. The number of apoptotic cells did not vary. LC functionality was shown by the increase in STAR expression (p ≤ 0.007) and a peak in testosterone secretion, followed by a reduction (p ≤ 0.001) with stabilization. According to our knowledge, this is the first report of generation of haploid cells in human ITT. Differentiating germ cells have to be further evaluated for their ability to complete differentiation, their fecundability and epigenetic characteristics.

Four isothermal recombinase polymerase amplification (RPA) assays were developed for fast in-field identification ofBacillus anthracis. The RPA assays targeted three specific sequences (i.e., the BA_5345 chromosomal marker, the lethal factorlef[from pXO1], and the capsule-biosynthesis-relatedcapA[from pXO2]) and a conserved sequence in the adenylate cyclase gene (adk) for theBacillus cereusgroup.B. anthracis-specific RPA assays were tested first with purified genomic DNAs (n= 60), including 11 representatives ofB. anthracis, and then with soil (n= 8) and white powder (n= 8) samples spiked with inactivatedB. anthracisspores and/or other biological agents. The RPA assays were also tested in another laboratory facility, which blindly provided DNA and lysate samples (n= 30, including 20B. anthracisstrains). RPA assays displayed 100% specificity and sensitivity. The hands-off turnaround times at 42°C ranged from 5 to 6 min for 102genomic copies. The analytical sensitivity of each RPA assay was ∼10 molecules per reaction. In addition, the BA_5345 andadkRPA assays were assessed under field conditions with a series of surface swabs (n= 13, including 11 swabs contaminated withB. thuringiensisspores) that were blindly brought to the field laboratory by a chemical, biological, radiological, and nuclear (CBRN) sampling team. None of the 13 samples, except the control, tested positive forB. anthracis, and all samples that had been harvested from spore-contaminated surfaces tested positive with theadkRPA assay. All threeB. anthracis-specific RPA assays proved suitable for rapid and reliable identification ofB. anthracisand therefore could easily be used by first responders under field conditions to quickly discriminate between a deliberate release ofB. anthracisspores and a hoax attack involving white powder.IMPORTANCEIn recent decades, particularly following the 11 September 2001 and Amerithrax attacks, the world has experienced attempts to sow panic and chaos in society through thousands of white-powder copycats using household powders to mimic real bioterrorism attacks. In such circumstances, field-deployable detection methods are particularly needed to screen samples collected from the scene. The aim is to test the samples directly using a fast and reliable assay for detection of the presence ofB. anthracis. While this would not preclude further confirmatory tests from being performed in reference laboratories, it would bring useful, timely, and relevant information to local crisis managers and help them make appropriate decisions without having to wait for quantitative PCR results (with turnaround times of a few hours) or phenotypic identification and sequencing (with turnaround times of a few days). In the current investigation, we developed a set of isothermal RPA assays for the rapid screening and identification ofB. anthracisin powders and soil samples, with the purpose of discriminating a deliberate release ofB. anthracisspores from a hoax attack involving white powder; this would also apply to dispersion by spraying of aerosolized forms ofB. anthracis. Further work is now ongoing to confirm the first observations and validate the on-site use of these assays by first responders.

Background Circulating tumor DNA (ctDNA) is a minimally-invasive technique to profile the tumor genome in different cancers but is still underexplored in squamous cell carcinoma of the head and neck (SCCHN). Our aim was to study the relevance of mutational profiling through liquid biopsy in SCCHN. Methods We collected plasma cell-free DNA from 39 patients (pts) with locoregional recurrent (LR) (n = 19) and/or metastatic (M) (n = 20) SCCHN. Targeted next-generation sequencing (Illumina, HiSeq4000) was performed on a custom panel of 604 genes (median coverage 1000x). When available (n = 18), a tumor biopsy was sequenced to evaluate the concordance of somatic mutations. Results M SCCHN pts had a higher probability of carrying ctDNA than LR pts (70% vs 31%, p = 0.0256). When ctDNA was detected, the quantity (estimated using the median of the allele frequencies (AF) of all the somatic plasma variants) did not differ between the two groups (median 2.3% vs 2.6%, p = 0.97). The concordance between liquid and solid biopsy showed that 26% of the plasma variants (14/54) were not detected in the matched tumors, reflecting some tumor heterogeneity. Conversely, 81% of the variants (168/208) identified in the solid tumors were not detected in the plasma. In a logistic regression model, the detection of the variant in the plasma was related to the metastatic status (Odds Ratio (OR) 4.50, p = 0.012), the variant AF in the tumor (OR = 5.31, p Conclusions Detection of ctDNA using targeted NGS is feasible in pts with SCCHN, essentially for metastatic disease. Liquid biopsy alone can identify potentially actionable alterations but does not reflect the full landscape of the solid tumor. Presence of a solid tumor variant in the plasma is related to the variant AF, the metastatic status and the ctDNA quantity of the sample. Clinical trial identification NCT02139020. Legal entity responsible for the study Cliniques Universitaires Saint-Luc - Universite Catholique de Louvain, Brussels, Belgium. Funding Region Wallonne, Belgium - Fondation Louvain UCLouvain. Disclosure R. Galot: Advisory / Consultancy: Innate Pharma; Travel / Accommodation / Expenses: Astellas; Travel / Accommodation / Expenses: Amgen. J. Machiels: Honoraria (institution), Research grant / Funding (institution), Travel / Accommodation / Expenses: Pfizer; Advisory / Consultancy, Research grant / Funding (institution): Roche; Honoraria (institution), Research grant / Funding (institution): AstraZeneca; Advisory / Consultancy, Research grant / Funding (institution): Bayer; Advisory / Consultancy: Innate Pharma; Honoraria (institution), Research grant / Funding (institution): Merck Serono; Honoraria (institution), Advisory / Consultancy, Research grant / Funding (institution): Boehringer Ingelheim; Honoraria (institution), Research grant / Funding (institution), Travel / Accommodation / Expenses: BMS; Honoraria (institution), Research grant / Funding (institution): Novartis; Honoraria (institution), Research grant / Funding (institution): Janssen; Honoraria (institution), Research grant / Funding (institution): Incyte; Research grant / Funding (institution), Travel / Accommodation / Expenses: Amgen; Research grant / Funding (institution), Travel / Accommodation / Expenses, Uncompensated advisory role: MSD; Advisory / Consultancy, Data safety monitoring board with honoraria: Debio; Advisory / Consultancy, Data safety monitoring board with honoraria: Nanobiotix; Non-remunerated activity/ies, investigator and study coordinator: EORTC. All other authors have declared no conflicts of interest.

Objectives Activation of the adenosine monophosphate (AMP)-activated protein kinase (AMPK) leads to phosphorylation of acetyl-CoA carboxylase (ACC) on serine 79 (phosphoACC) and its subsequent inhibition. Given the role of AMPK-ACC signaling in platelet lipid metabolism and function, this pathway could be affected in the platelets of patients with coronary artery disease (CAD), where the atherogenic environment has an impact on platelet biology. We hypothesized that platelet AMPKACC signaling is activated by atherogenic lipids and could be considered as a metabolic signature in high-risk CAD patients. We also explored the consequences of platelet phosphoACC on platelet lipid metabolic profile. Approach and results Blood samples from 188 consecutive patients admitted for coronary angiography were processed. Lipid extracts from the platelets of 31 patients were subjected to lipidomic analysis. PhosphoACC level in circulating platelets of CAD patients were significantly increased and was linked to the severity of coronary artery calcification as well as acute coronary syndrome (OR: 6.71, 95% CI: 2.06–21.91; P = 0.002). The triglyceride/high-density lipoprotein cholesterol (TG/HDL-C) ratio, a well-known atherogenic marker, was strongly associated with increased phosphoACC in our CAD cohort. Hence, oxLDL activated AMPK-ACC signaling through a CD36-dependent pathway. Lipidomic analysis revealed that increased phosphoACC led to a down-regulation of intraplatelet TG, particularly of those containing C14:0 fatty acid chains. Conclusion Platelet phosphoACC is a marker for risk stratification in suspected CAD patients and reveals an interaction between platelets and lipids. Inhibitory phosphoACC impacts platelet lipid content by down-regulating TG, which in turn may affect platelet function (ACCTHEROMA, NCT03034148).

Purpose Heart failure (HF) is a complex syndrome. Its appropriate management should combine several health measurements. We assessed the relationship between the Kansas City Cardiomyopathy Questionnaire (KCCQ) and the Pocock’s clinical score. Methods We conducted a prospective registry of HF outpatients. The main outcome was occurrence of death or hospitalization during a 6-month follow-up. A multivariate logistic regression was performed, including the KCCQ overall summary score, the Pocock’s clinical score and their interaction in the model. Results From January 2008 to December 2010, 143 patients were involved. Mean age of patients was 68 years, and 74 % were men. KCCQ’s overall summary score and Pocock’s clinical score were inversely correlated (r = −0.24, p = 0.026). A total of 61 (42.7 %) events occurred. There was a high proportion of events (77.8 %) in patients with a Pocock’s clinical score >50 %, whatever the KCCQ score value. When the KCCQ score was ≤50 %, there was a low increase in risk as the Pocock’s clinical score increased (OR 2.0 [0.6; 6.6]). However, when the KCCQ score was between 50 and 75 or ≥75 %, there was a high increase in risk as the Pocock’s clinical score increased (OR 6.9 [1.2; 38.9] and OR 7.4 [0.8; 69.7], respectively). Conclusions Patients with a high Pocock’s clinical score are at a high risk of death or hospitalization. For patients with a low Pocock’s clinical score, the KCCQ score can identify those at risk of these events. Electronic supplementary material The online version of this article (doi:10.1007/s11136-015-1154-9) contains supplementary material, which is available to authorized users.

What is the best source of ovarian cells for the artificial ovary: medulla or cortex, cryopreserved or fresh?Ovarian cells from fresh medullary tissue, which can be isolated in larger numbers, show higher viability and are able to improve graft vascularization.In a previous study, addition of endothelial cells along with ovarian cells was found to be crucial for formation of a well-vascularized ovary-like structure. This study is the first to evaluate both the effect of cryopreservation and the source of ovarian tissue on isolated ovarian cells.Prospective experimental study in an academic research unit using ovarian tissue from seven patients undergoing surgery for benign gynecologic disease.Ovarian tissue was retrieved from seven patients, with one half processed as fresh (fresh group) and the other half frozen and thawed before processing (frozen group). In each group, ovarian cells from the cortex and medulla were isolated separately, and their viability was tested using a calcein AM/ethidium homodimer viability assay. Fifty thousand cells were then encapsulated in fibrin and grafted to peritoneal pockets in nude mice (14 in all). Grafts recovered after 7 days were analyzed by immunohistochemistry for the presence of ovarian cells (vimentin), proliferation (Ki67) and graft vascularization (double CD34). Cell apoptosis was analyzed by TUNEL assay.Cryopreservation decreased ovarian cell yield (-2804 cells/mg, P = 0.015) and viability (-9.72%, P = 0.052) before grafting and had a considerable (5-fold, P = 0.2) but non-significant negative impact on ovarian cell presence in grafts. The medulla yielded many more cells (+3841 cells/mg, P0.001) with higher viability (+18.23%, P0.001) than did the cortex. Moreover, grafts with cells from the medulla exhibited a statistically significant 6.44- and 2.47-fold increase in human and total vascular surface area, respectively. P-values were adjusted for multiple testing using the Benjamini-Hochberg method to achieve a 10% false discovery rate and adjusted P-values0.1 were therefore considered significant.Pilot study involving a limited number of experiments.Knowing that fresh medullary tissue is the best source of stromal cells is important for construction of the artificial ovary, as isolated follicles require structural support and a rich vascular network for their survival and development.This work was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (5/4/150/5 and 7.4518.12F), Fonds Spéciaux de Recherche, Fondation Saint Luc and Foundation Against Cancer, and donations from Mr Pietro Ferrero, Baron Frère and Viscount Philippe de Spoelberch. None of the authors have any conflicting interests to declare.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most common of all pediatric cancers. Aside from predisposing to ALL, polymorphisms could also be associated with poor outcome. Indeed, genetic variations involved in drug metabolism could, at least partially, be responsible for heterogeneous responses to standardized leukemia treatments, hence requiring more personalized therapy. AIMS: (a) to determine the prevalence of seven common genetic polymorphisms including those that affect the folate and/or thiopurine metabolic pathways, i.e., cyclin D1 (CCND1-G870A), ɣ-glutamyl hydrolase (GGH-C452T), methylenetetrahydrofolate reductase (MTHFR-C677T and MTHFR-A1298C), thymidylate synthase promoter (TYMS-TSER), thiopurine methyltransferase (TPMT*3A and TPMT*3C) and inosine triphosphate pyrophosphatase (ITPA-C94A), in Caucasian (N=94, age< 20) and Vietnamese (N=141, age< 16) childhood ALL; (b) to assess the impact of a multilocus genetic risk score (MGRS) on relapse-free survival (RFS) using a cox proportional-hazards regression model. RESULTS: The prevalence of MTHFR-677TT genotype was significantly higher in Caucasian (p = 0.008), in contrast to the prevalence of TYMS-TSER*3R/3R and ITPA-94AA/AC genotypes which were significantly higher in Vietnamese (p < 0.001 and p = 0.02, respectively). Compared to children with a low MGRS (≤3), those with a high MGRS (≥4) were 2.06 (95% CI = 1.01 - 4.22; p = 0.04) times more likely to relapse. Adding MGRS into a multivariate Cox regression model with race/ethnicity and four clinical variables improved the predictive accuracy of the model (AUC from 0.682 to 0.709 at 24 months). CONCLUSION: Including MGRS into a clinical model improved the predictive accuracy of short and medium-term prognosis, hence confirming the association between well determined pharmacogenotypes and outcome of pediatric ALL. Whether variants on other genes associated with folate metabolism can substantially improve the predictive value of current MGRS is not known but deserves further evaluation.