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Abstract Background Neoadjuvant chemotherapy (NAC) is the standard of care for patients with early-stage triple negative breast cancers (TNBC). However, more than half of TNBC patients do not achieve a pathological complete response (pCR) after NAC, and residual cancer burden (RCB) is associated with dismal long-term prognosis. Understanding the mechanisms underlying differential treatment outcomes is therefore critical to limit RCB and improve NAC efficiency. Methods Human TNBC cell lines and patient-derived organoids were used in combination with real-time metabolic assays to evaluate the effect of NAC (paclitaxel and epirubicin) on tumor cell metabolism, in particular glycolysis. Diagnostic biopsies (pre-NAC) from patients with early TNBC were analyzed by bulk RNA-sequencing to evaluate the predictive value of a glycolysis-related gene signature. Results Paclitaxel induced a consistent metabolic switch to glycolysis, correlated with a reduced mitochondrial oxidative metabolism, in TNBC cells. In pre-NAC diagnostic biopsies from TNBC patients, glycolysis was found to be upregulated in non-responders. Furthermore, glycolysis inhibition greatly improved response to NAC in TNBC organoid models. Conclusions Our study pinpoints a metabolic adaptation to glycolysis as a mechanism driving resistance to NAC in TNBC. Our data pave the way for the use of glycolysis-related genes as predictive biomarkers for NAC response, as well as the development of inhibitors to overcome this glycolysis-driven resistance to NAC in human TNBC patients.

Between 2018 and 2024, we conducted systematic whole-genome sequencing and phylogenomic analysis on 263 V. cholerae O1 isolates from cholera patients across four provinces in the Democratic Republic of Congo (North-Kivu, South-Kivu, Tanganyika, and Kasai Oriental). These isolates were classified into the AFR10d and AFR10e sublineages of AFR10 lineage, originating from the third wave of the seventh El Tor cholera pandemic (7PET). Compared to the strains analysed between 2014 and 2017, both sublineages had few genetic changes in the core genome but recent isolates (2022-2024) had significant CTX prophage rearrangement. AFR10e spread across all four provinces, while AFR10d appeared to be extinct by the end of 2020. Since 2022, most V. cholerae O1 isolates exhibited significant CTX prophage rearrangements, including a tandem repeat of an environmental satellite phage RS1 downstream the ctxB toxin gene of the CTX-Φ-3 prophage on the large chromosome, as well as two or more arrayed copies of an environmental pre-CTX-Φ prophage precursor on the small chromosome. We used Illumina data for mapping and coverage estimation to identify isolates with unique CTX-Φ genomic features. Gene localization was then determined on MinION-derived assemblies, revealing an organization similar to that of non-O1 V. cholerae isolates found in Asia (O139 VC1374, and environmental O4 VCE232), but never described in V. cholerae O1 El Tor from the third wave. In conclusion, while the core genome of AFR10d and AFR10e showed minimal changes, significant alterations in the CTX-Φ and pre-CTX-Φ prophage content and organization were identified in AFR10e from 2022 onwards.

Background and aimsHuman islet preparations designated for research exhibit diverse insulin-secretory profiles. This study aims to assess the impact of donor- and isolation-related factors on in vitro islet secretory function.MethodsA retrospective analysis of 46 isolations from 23 pancreata discarded for clinical transplantation was conducted. In vitro islet secretory function tests were performed on Day 1 and Day 7 of culture. Linear mixed-effects models (LMMs) were employed to investigate the relationships between various predictors characterizing the patient and donor characteristics as well as the isolation effectiveness and two functional outcomes including the islet stimulation index (SI) and area under the insulin curve (AUC). Fixed effects were introduced to represent the main effects of each predictor, and backward elimination was utilized to select the most significant fixed effects for the final model. Interaction effects between the timepoint (Day 7 vs. Day 1) and the predictors were also evaluated to assess whether predictors were associated with the temporal evolution of SI and AUC. Fold-change (Fc) values associated with each predictor were obtained by exponentiating the corresponding coefficients of the models, which were built on log-transformed outcomes.ResultsAnalysis using LMMs revealed that donor body mass index (BMI) (Fc = 0.961, 95% CI = 0.927–0.996, p = 0.05), donor gender (female vs. male, Fc = 0.702, 95% CI = 0.524–0.942, p = 0.04), and donor hypertension (Fc = 0.623, 95% CI = 0.466–0.832, p=

Abstract Background Pulsed ultraviolet (UV)-C light sources, such as excimer lasers, are used in emerging non-thermal food-decontamination methods and also have high potential for use in a wide range of microbial decontamination applications. The acaricidal effect of an experimental UV-C irradiation device was assessed using female adults and eggs of a model organism, the two-spotted spider mite Tetranychus urticae. Methods UV-C light was generated by a pulsed krypton fluoride excimer laser operating at 248-nm emission wavelength. The pulse energy and pulse repetition rate were 5 mJ and up to 100 Hz, respectively. The distance from the light source to the target was 150 mm; the target surface area was 2.16 cm2. The exposure time for the mites and fresh eggs varied from 1 to 4 min at 5–300 mW, which corresponded to UV doses of 5–80 kJ/m2. Post-irradiation acaricidal effects (mite mortality) were assessed immediately and also measured at 24 h. The effects of UV-C irradiation on the hatchability of eggs were observed daily for up to 12 days post-irradiation. Results The mortality of mites at 5 and 40 kJ/m2 was 26% and 92%, respectively. Mite mortality reached 98% at 80 kJ/m2. The effect of exposure duration on mortality was minimal. The effect of irradiation on egg hatchability was even more significant than that on adult mite mortality, i.e. about 100% egg mortality at an accumulated dose of as little as 5 kJ/m2 for each exposure time. Conclusions A high rate of mite mortality and lethal egg damage were observed after less than 1 min of exposure to 5 mJ UV-C pulsed irradiation at 60 Hz. Pending further developments (such as beam steering, beam shaping and miniaturisation) and feasibility studies (such as testing with mites in real-life situations), the reported results and characteristics of the UV-C generator (modulation of energy output and adaptability to varying spot sizes) open up the use of this technology for a vast field of acaricidal applications that require long-range radiation. Graphical Abstract

ABSTRACT: 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.

Abstract 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.

Multidrug-resistant (MDR) and extended spectrum β-lactamase (ESBL)-producing extra-intestinal K. pneumoniae are associated with increased morbidity and mortality. This study aimed to characterize the resistance and virulence profiles of extra-intestinal MDR ESBL-producing K. pneumoniae associated with infections at a tertiary hospital in South-Kivu province, DRC. Whole-genome sequencing (WGS) was carried out on 37 K. pneumoniae isolates 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. pneumoniae isolates 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. The blaCTX-M-15 gene was the most common β-lactamase gene among K. pneumoniae isolates. 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 the pmrB efflux 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-producing K. pneumoniae isolates 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.

Abstract 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.

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.

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.

Mutualistic associations between insects and heritable bacterial symbionts are ubiquitous in nature. The aphid symbiont Serratia symbiotica is a valuable candidate for studying the evolution of bacterial symbiosis in insects because it includes a wide diversity of strains that reflect the diverse relationships in which bacteria can be engaged with insects, from pathogenic interactions to obligate intracellular mutualism. The recent discovery of culturable strains, which are hypothesized to resemble the ancestors of intracellular strains, provide an opportunity to study the mechanisms underlying bacterial symbiosis in its early stages. In this study, we analyzed the genomes of three of these culturable strains that are pathogenic to aphid hosts, and performed comparative genomic analyses including mutualistic host-dependent strains. All three genomes are larger than those of the host-restricted S. symbiotica strains described so far, and show significant enrichment in pseudogenes and mobile elements, suggesting that these three pathogenic strains are in the early stages of the adaptation to their host. Compared to their intracellular mutualistic relatives, the three strains harbor a greater diversity of genes coding for virulence factors and metabolic pathways, suggesting that they are likely adapted to infect new hosts and are a potential source of metabolic innovation for insects. The presence in their genomes of secondary metabolism gene clusters associated with the production of antimicrobial compounds and phytotoxins supports the hypothesis that S. symbiotia symbionts evolved from plant-associated strains and that plants may serve as intermediate hosts. Mutualistic associations between insects and bacteria are the result of independent transitions to endosymbiosis initiated by the acquisition of environmental progenitors. In this context, the genomes of free-living S. symbiotica strains provide a rare opportunity to study the inventory of genes held by bacterial associates of insects that are at the gateway to a host-dependent lifestyle.

Many insect species are associated with bacterial partners that can significantly influence their evolutionary ecology. Compared to other insect groups, aphids harbor a bacterial microbiota that has the reputation of being poorly diversified, generally limited to the presence of the obligate nutritional symbiont Buchnera aphidicola and some facultative symbionts. In this study, we analyzed the bacterial diversity associated with the dogwood-grass aphid Anoecia corni, an aphid species that spends much of its life cycle in a subterranean environment. Little is known about the bacterial diversity associated with aphids displaying such a lifestyle, and one hypothesis is that close contact with the vast microbial community of the rhizosphere could promote the acquisition of a richer bacterial diversity compared to other aphid species. Using 16S rRNA amplicon Illumina sequencing on specimens collected on wheat roots in Morocco, we identified 10 bacterial operational taxonomic units (OTUs) corresponding to five bacterial genera. In addition to the obligate symbiont Buchnera, we identified the facultative symbionts Serratia symbiotica and Wolbachia in certain aphid colonies. The detection of Wolbachia is unexpected as it is considered rare in aphids. Moreover, its biological significance remains unknown in these insects. Besides, we also detected Arsenophonus and Dactylopiibacterium carminicum. These results suggest that, despite its subterranean lifestyle, A. corni shelter a bacterial diversity mainly limited to bacterial endosymbionts.

ObjectivesWe 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.MethodsSynovial 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.ResultsTwenty 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.ConclusionCellular 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: Chronic obstructive pulmonary disease (COPD) is a devastating lung disease, mainly due to cigarette smoking, which represents the third cause of mortality worldwide. The mechanisms driving its epithelial salient features remain largely elusive. We aimed to evaluate the activation and the role of the canonical, β-catenin-dependant WNT pathway in the airway epithelium from COPD patients. Methods: The WNT/β-catenin pathway was first assessed by WNT-targeted RNA sequencing of the air/liquid interface-reconstituted bronchial epithelium from COPD and control patients. Airway expression of total and active β-catenin was assessed in lung sections, as well as WNT components in laser-microdissected airway epithelium. Finally, we evaluated the role of WNT at the bronchial epithelial level by modulating the pathway in the reconstituted COPD epithelium. Findings: We show that the WNT/β-catenin pathway is upregulated in the COPD airway epithelium as compared with that of non-smokers and control smokers, in targeted RNA-sequencing of in vitro reconstituted airway epithelium, and in situ in lung tissue and laser-microdissected epithelium. Extrinsic activation of this pathway in COPD-derived airway epithelium inhibited epithelial differentiation, polarity and barrier function, and induced TGF-β-related epithelial-to-mesenchymal transition (EMT). Conversely, canonical WNT inhibition increased ciliated cell numbers, epithelial polarity and barrier function, whilst inhibiting EMT, thus reversing COPD features. Interpretation: In conclusion, the aberrant reactivation of the canonical WNT pathway in the adult airway epithelium recapitulates the diseased phenotype observed in COPD patients, suggesting that this pathway or its downstream effectors could represent a future therapeutic target. Funding: This study was supported by the Fondation Mont-Godinne, the FNRS and the WELBIO.

Klinefelter syndrome (KS), with an incidence between 1/600 and 1/1,000, is the main genetic cause of male infertility. Due to the lack of an accurate study model, the detailed pathogenic mechanisms by which this X chromosome aneuploidy leads to KS features remain unknown. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from a patient with KS: 47XXY-iPSCs. In order to compare the potentials of both 47XXY-iPSCs and 46XY-iPSCs to differentiate into the germ cell lineage, we developed a directed differentiation protocol by testing different combinations of factors including bone morphogenetic protein 4 (BMP4), glial-derived neurotrophic factor (GDNF), retinoic acid (RA) and stem cell factor (SCF) for 42 days. Importantly, we found a reduced ability of 47XXY-iPSCs to differentiate into germ cells when compared to 46XY-iPSCs. In particular, upon germ cell differentiation of 47XXY-iPSCs, we found a reduced proportion of cells positive for BOLL, a protein required for germ cell development and spermatogenesis, as well as a reduced proportion of cells positive for MAGEA4, a spermatogonia marker. This reduced ability to generate germ cells was not associated with a decrease of proliferation of 47XXY-iPSC-derived cells but rather with an increase of cell death upon germ cell differentiation as revealed by an increase of LDH release and of capase-3 expression in 47XXY-iPSC-derived cells. Our study supports the idea that 47XXY-iPSCs provides an excellent in vitro model to unravel the pathophysiology and to design potential treatments for KS patients.

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.

BackgroundOver the past recent years, Vibrio cholerae has been associated with outbreaks in sub-Saharan Africa, notably in Democratic Republic of the Congo (DRC). This study aimed to determine the genetic relatedness of isolates responsible for cholera outbreaks in eastern DRC between 2014 and 2017, and their potential spread to bordering countries.Methods/principal findingsPhenotypic analysis and whole genome sequencing (WGS) were carried out on 78 clinical isolates of V. cholerae associated with cholera in eastern provinces of DRC between 2014 and 2017. SNP-based phylogenomic data show that most isolates (73/78) were V. cholerae O1 biotype El Tor with CTX-3 type prophage. They fell within the third transmission wave of the current seventh pandemic El Tor (7PET) lineage and were contained in the introduction event (T)10 in East Africa. These isolates clustered in two sub-clades corresponding to Multiple Locus Sequence Types (MLST) profiles ST69 and the newly assigned ST515, the latter displaying a higher genetic diversity. Both sub-clades showed a distinct geographic clustering, with ST69 isolates mostly restricted to Lake Tanganyika basin and phylogenetically related to V. cholerae isolates associated with cholera outbreaks in western Tanzania, whereas ST515 isolates were disseminated along the Albertine Rift and closely related to isolates in South Sudan, Uganda, Tanzania and Zambia. Other V. cholerae isolates (5/78) were non-O1/non-O139 without any CTX prophage and no phylogenetic relationship with already characterized non-O1/non-O139 isolates.Conclusions/significanceCurrent data confirm the association of both DRC O1 7PET (T)10 sub-clades ST69 and ST515 with recurrent outbreaks in eastern DRC and at regional level over the past 10 years. Interestingly, while ST69 is predominantly a locally endemic sequence type, ST515 became adaptable enough to expand across DRC neighboring countries.

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.

BACKGROUND: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. The MLVAType shiny application was designed to extract MLVA profiles of Vibrio cholerae isolates from WGS data while ensuring backward compatibility with traditional MLVA typing methods. METHODS:To test and validate the MLVAType algorithm, WGS-derived MLVA profiles of nineteen Vibrio cholerae isolates from Democratic Republic of the Congo (n = 9) and Uganda (n = 10) were compared to MLVA profiles generated by an in silico PCR approach and Sanger sequencing, the latter being used as the reference method. RESULTS:Results obtained by Sanger sequencing and MLVAType 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 of V. cholerae VNTR 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. CONCLUSION:As NGS read lengths and qualities tend to increase with time, one may expect the increase of k-mer size in a near future. Using MLVAType application with a longer k-mer size will then efficiently retrieve MLVA profiles from WGS data while avoiding censored estimation.

Background. Cell transplantation is in clinical development for the treatment of various ailments including acquired and inborn hepatic diseases. Detection and quantification of the donor cells after infusion remain difficult. Traditional methods (sex-based FISH, HLA mismatch, and Short Tandem Repeat PCR) can only achieve low levels of sensitivity (1%) and therefore are seldom used. The use of a droplet digital PCR (ddPCR) assay based on mismatch of null alleles is a promising alternative. Methods. We selected genes with a high frequency of null genotype in the general population (SRY, RHD, TRY6, LEC3C, GSTM1, and GSTT1) and investigated their expression by liver progenitor cell donors and liver cell therapy recipients, in order to identify genes of interest for each donor/recipient couple. We first validated the detection of microchimerism by ddPCR and then used these assays to detect and quantify microchimerism in pre- and postinfusion liver biopsies. Results. We validated the ddPCR detection of the selected genes based on linearity, precision, lack of inhibition, and accuracy, and we established limits of blank, limits of detection, and limits of quantification to ensure the reliability of the results. After genotyping donors and recipients, we were able to identify at least one gene of interest for each donor/recipient couple. We detected donor cells in the three patients posttransplantation. However, analysis of several biopsies taken at the same timepoint revealed a heterogeneous cell distribution. In addition, the values obtained remained below the limit of quantification. Therefore, the actual quantification of microchimerism may not be entirely accurate. Conclusions. Overall, our study demonstrates that the detection of microchimerism post-liver cell transplantation can be performed using ddPCR amplification of null allele genes expressed by the donor but absent from the recipient. However, this technique can be extended to other cell types and target organs in cell transplantation.

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.

Here, we present first draft genome sequence of the trypanosomatid Herpetomonas muscarum ingenoplastis. This parasite was isolated repeatedly in the black blowfly, Phormia regina, and it forms a phylogenetically distinct clade in the Trypanosomatidae family.

Introduction: Focal adhesion kinase (FAK) plays a crucial role in cancer development and progression. FAK is overexpressed and/or activated and associated with poor prognosis in various malignancies. However, in lung cancer, activated FAK expression and its prognostic value are unknown. Methods: FAK and activated FAK (phospho-FAK Y397) expressions were analyzed by multiplex immunofluorescence staining in formalin-fixed paraffin-embedded tissues from 95 non-small-cell lung cancer (NSCLC) and 105 small-cell lung cancer (SCLC) patients, and 37 healthy donors. The FAK staining score was defined as the percentage (%) of FAK-stained tumor area multiplied by (×) FAK mean intensity and phospho-FAK staining score as the (% of phospho-FAK-stained area of low intensity × 1) + (% of phospho-FAK-stained area of medium intensity × 2) + (% of the phospho-FAK-stained area of high intensity × 3). FAK and phospho-FAK staining scores were compared between normal, NSCLC, and SCLC tissues. They were also tested for correlations with patient characteristics and clinical outcomes. Results: The median follow-up time after the first treatment was 42.5 months and 6.4 months for NSCLC and SCLC patients, respectively. FAK and phospho-FAK staining scores were significantly higher in lung cancer than in normal lung and significantly higher in SCLC compared to NSCLC tissues (p < 0.01). Moreover, the ratio between phospho-FAK and FAK staining scores was significantly higher in SCLC than in NSCLC tissues (p < 0.01). However, FAK and activated FAK expression in lung cancer did not correlate with recurrence-free and overall survival in NSCLC and SCLC patients. Conclusions: Total FAK and activated FAK expressions are significantly higher in lung cancer than in normal lung, and significantly higher in SCLC compared to NSCLC, but are not prognostic biomarkers in this study.

Xenotransplantation of human cells in animal models is an essential tool for evaluation of safety and efficacy of cell-based products for therapeutic use. Sensitive and reproducible methods are needed to detect and quantify human cells engrafted into the host tissue either in the targeted organ or in undesired locations. We developed a robust quantitative polymerase chain reaction (qPCR) assay based on amplification of human AluYb8 repeats, to assess the number of human cells present in rat or mouse tissues after transplantation. Standard curves of mixed human/rodent DNA and mixed human/rodent cells have been performed to determine the limit of detection and linear range of the assay. Standard curves from DNA mixing differed significantly from standard curves from cell mixing. We show here that the AluYb8 qPCR assay is highly reproducible and is able to quantify human cells in a rodent cell matrix over a large linear range that extends from 50% to 0.01% human cells. Short-term in vivo studies showed that human cells could be quantified in mouse liver up to 7 days after intrasplenic transplantation and in rat liver 4 h after intrahepatic transplantation.

Early diagnosis of rheumatoid arthritis (RA) is an unmet medical need in the field of rheumatology. Previously, we performed high-density transcriptomic studies on synovial biopsies from patients with arthritis, and found that synovial gene expression profiles were significantly different according to the underlying disorder. Here, we wanted to further explore the consistency of the gene expression signals in synovial biopsies of patients with arthritis, using low-density platforms.Low-density assays (cDNA microarray and microfluidics qPCR) were designed, based on the results of the high-density microarray data. Knee synovial biopsies were obtained from patients with RA, spondyloarthropathies (SA) or osteoarthritis (OA) (n = 39), and also from patients with initial undifferentiated arthritis (UA) (n = 49).According to high-density microarray data, several molecular pathways are differentially expressed in patients with RA, SA and OA: T and B cell activation, chromatin remodelling, RAS GTPase activation and extracellular matrix regulation. Strikingly, disease activity (DAS28-CRP) has a significant influence on gene expression patterns in RA samples. Using the low-density assays, samples from patients with OA are easily discriminated from RA and SA samples. However, overlapping molecular patterns are found, in particular between RA and SA biopsies. Therefore, prediction of the clinical diagnosis based on gene expression data results in a diagnostic accuracy of 56.8%, which is increased up to 98.6% by the addition of specific clinical symptoms in the prediction algorithm. Similar observations are made in initial UA samples, in which overlapping molecular patterns also impact the accuracy of the diagnostic algorithm. When clinical symptoms are added, the diagnostic accuracy is strongly improved.Gene expression signatures are overall different in patients with OA, RA and SA, but overlapping molecular signatures are found in patients with these conditions. Therefore, an accurate diagnosis in patients with UA requires a combination of gene expression and clinical data.

Separating CBRN mixed samples that contain both chemical and biological warfare agents (CB mixed sample) in liquid and solid matrices remains a very challenging issue. Parameters were set up to assess the performance of a simple filtration-based method first optimized on separate C- and B-agents, and then assessed on a model of CB mixed sample. In this model, MS2 bacteriophage, Autographa californica nuclear polyhedrosis baculovirus (AcNPV), Bacillus atrophaeus and Bacillus subtilis spores were used as biological agent simulants whereas ethyl methylphosphonic acid (EMPA) and pinacolyl methylphophonic acid (PMPA) were used as VX and soman (GD) nerve agent surrogates, respectively. Nanoseparation centrifugal devices with various pore size cut-off (30 kD up to 0.45 µm) and three RNA extraction methods (Invisorb, EZ1 and Nuclisens) were compared. RNA (MS2) and DNA (AcNPV) quantification was carried out by means of specific and sensitive quantitative real-time PCRs (qPCR). Liquid chromatography coupled to time-of-flight mass spectrometry (LC/TOFMS) methods was used for quantifying EMPA and PMPA. Culture methods and qPCR demonstrated that membranes with a 30 kD cut-off retain more than 99.99% of biological agents (MS2, AcNPV, Bacillus Atrophaeus and Bacillus subtilis spores) tested separately. A rapid and reliable separation of CB mixed sample models (MS2/PEG-400 and MS2/EMPA/PMPA) contained in simple liquid or complex matrices such as sand and soil was also successfully achieved on a 30 kD filter with more than 99.99% retention of MS2 on the filter membrane, and up to 99% of PEG-400, EMPA and PMPA recovery in the filtrate. The whole separation process turnaround-time (TAT) was less than 10 minutes. The filtration method appears to be rapid, versatile and extremely efficient. The separation method developed in this work constitutes therefore a useful model for further evaluating and comparing additional separation alternative procedures for a safe handling and preparation of CB mixed samples.

In the Balkan and Taiwan, the relationship between exposure to aristolochic acid and risk of urothelial neoplasms was inferred from the A>T genetic hallmark in TP53 gene from malignant cells. This study aimed to characterize the TP53 mutational spectrum in urothelial cancers consecutive to Aristolochic Acid Nephropathy in Belgium. Serial frozen tumor sections from female patients (n=5) exposed to aristolochic acid during weight-loss regimen were alternatively used either for p53 immunostaining or laser microdissection. Tissue areas with at least 60% p53-positive nuclei were selected for microdissecting sections according to p53-positive matching areas. All areas appeared to be carcinoma in situ. After DNA extraction, mutations in the TP53 hot spot region (exons 5-8) were identified using nested-PCR and sequencing. False-negative controls consisted in microdissecting fresh-frozen tumor tissues both from a patient with a Li-Fraumeni syndrome who carried a p53 constitutional mutation, and from KRas mutated adenocarcinomas. To rule out false-positive results potentially generated by microdissection and nested-PCR, a phenacetin-associated urothelial carcinoma and normal fresh ureteral tissues (n=4) were processed with high laser power. No unexpected results being identified, molecular analysis was pursued on malignant tissues, showing at least one mutation in all (six different mutations in two) patients, with 13/16 exonic (nonsense, 2; missense, 11) and 3/16 intronic (one splice site) mutations. They were distributed as transitions (n=7) or transversions (n=9), with an equal prevalence of A>T and G>T (3/16 each). While current results are in line with A>T prevalence previously reported in Balkan and Taiwan studies, they also demonstrate that multiple mutations in the TP53 hot spot region and a high frequency of G>T transversion appear as a complementary signature reflecting the toxicity of a cumulative dose of aristolochic acid ingested over a short period of time.