Search

ABSTRACT 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 t oxin 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.

Despite the implementation of personalized medicine, patients with metastatic CRC (mCRC) still have a dismal overall survival due to the frequent occurrence of acquired resistance mechanisms thereby leading to clinical relapse. Understanding molecular mechanisms that support acquired resistance to anti-EGFR targeted therapy in mCRC is therefore clinically relevant and key to improving patient outcomes. Here, we observe distinct metabolic changes between cetuximab-resistant CRC cell populations, with in particular an increased glycolytic activity in KRAS-mutant cetuximab-resistant CRC cells (LIM1215 and OXCO2) but not in KRAS-amplified resistant DiFi cells. We show that cetuximab-resistant LIM1215 and OXCO2 cells have the capacity to recycle glycolysis-derived lactate to sustain their growth capacity. This is associated with an upregulation of the lactate importer MCT1 at both transcript and protein levels. Pharmacological inhibition of MCT1, with AR-C155858, reduces the uptake and oxidation of lactate and impairs growth capacity in cetuximab-resistant LIM1215 cells both in vitro and in vivo. This study identifies MCT1-dependent lactate utilization as a clinically actionable, metabolic vulnerability to overcome KRAS-mutant-mediated acquired resistance to anti-EGFR therapy in CRC., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: S.A. reports personal fees from MSD Italia and a patent (international PCT patent application No. WO 2023/199255 and Italian patent application No. 102022000007535) outside the submitted work. A.Ba. reports receipt of grants/research support from Neophore, AstraZeneca and Boehringer Ingelheim and honoraria/consultation fees from Guardant Health and Inivata. A.Ba. is a stock shareholder of Neophore and Kither Biotech. A.Ba. is an advisory board member for Inivata, Neophore, Roche/Genentech. The other authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background and Aims: Human 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., Methods: A 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., Results: Analysis 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= <0.01) were significantly and independently associated with SI. Moreover, donor gender (Fc = 0.512, 95% CI = 0.302-0.864, p = 0.02), donor cause of death (cerebrovascular accident vs. cardiac arrest, Fc = 2.129, 95% CI = 0.915-4.946, p = 0.09; trauma vs. cardiac arrest, Fc = 2.129, 95% CI = 1.112-7.106, p = 0.04), pancreas weight (Fc = 1.01, 95% CI = 1.001-1.019, p = 0.03), and islet equivalent (IEQ)/mg (Fc = 1.277, 95% CI = 1.088-1.510, p ≤ 0.01) were significantly and independently associated with AUC. There was no predictor significantly associated with the temporal evolution between Day 1 and Day 7 for both SI and AUC outcomes., Conclusion: This study identified donor- and isolation-related factors influencing in vitro islet secretory function. Further investigations are essential to validate the applicability of these results in clinical practice., Competing Interests: AB received a grant from the “Fondation Saint Luc,” and the “Fondation Recherche Clinique”. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Buemi, Mourad, Ambroise, Hoton, Devresse, Darius, Kanaan, Gianello and Mourad.)

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., (© 2024. The Author(s).)

Chronic obstructive pulmonary disease (COPD), a devastating and irreversible lung disease, causes structural and functional defects in the bronchial epithelium, the (ir)reversibility of which remains unexplored in vitro. This study aimed to investigate the persistence of COPD-related epithelial defects in long-term airway epithelial cultures derived from non-smokers, smokers, and COPD patients. Barrier function, polarity, cell commitment, epithelial-to-mesenchymal transition, and inflammation were evaluated and compared with native epithelium characteristics. The role of inflammation was explored using cytokines. We show that barrier dysfunction, compromised polarity, and lineage abnormalities observed in smokers and COPD persisted for up to 10 wk. Goblet cell hyperplasia was associated with recent cigarette smoke exposure. Conversely, increased IL-8/CXCL-8 release and abnormal epithelial-to-mesenchymal transition diminished over time. These ex vivo observations matched surgical samples' abnormalities. Cytokine treatment induced COPD-like changes in control cultures and reactivated epithelial-to-mesenchymal transition in COPD cells. In conclusion, these findings suggest that the airway epithelium of smokers and COPD patients retains a multidimensional memory of its original state and previous cigarette smoke-induced injuries, maintaining these abnormalities for extended periods., (© 2023 Carlier et al.)

The Democratic Republic of the Congo (DRC) officially reports low coronavirus disease 19 (COVID-19) prevalence. This cross-sectional study, conducted between September and November 2021, assessed the COVID-19 seroprevalence in people attending Goma's two largest markets, Kituku and Virunga. A similar study in a slum of Bukavu overlapped for 1 month using identical methods. 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 reported by survey participants in the previous 6 months were mild or absent in 58.9% and 41.1% of participants with anti-SARS-CoV-2 antibodies, respectively. No COVID-19-seropositive participants reported hospitalisation in the last 6 months. 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, the relevance of an untargeted hypothetical vaccination programme in these communities should be questioned.

Pulmonary fibrosis (PF) and pulmonary hypertension (PH) are chronic diseases of the pulmonary parenchyma and circulation, respectively, which may coexist, but underlying mechanisms remain elusive. Mutations in the GCN2 (general control nonderepressible 2) gene ( EIF2AK4 [eukaryotic translation initiation factor 2 alpha kinase 4]) were recently associated with pulmonary veno-occlusive disease. The aim of this study is to explore the involvement of the GCN2/eIF2α (eukaryotic initiation factor 2α) pathway in the development of PH during PF, in both human disease and in a laboratory animal model. Lung tissue from patients with PF with or without PH was collected at the time of lung transplantation, and control tissue was obtained from tumor resection surgery. Experimental lung disease was induced in either male wild-type or EIF2AK4 -mutated Sprague-Dawley rats, randomly receiving a single intratracheal instillation of bleomycin or saline. Hemodynamic studies and organ collection were performed 3 weeks after instillation. Only significant results ( P  < 0.05) are presented. In PF lung tissue, GCN2 protein expression was decreased compared with control tissue. GCN2 expression was reduced in CD31 + endothelial cells. In line with human data, GCN2 protein expression was decreased in the lung of bleomycin rats compared with saline. EIF2AK4 -mutated rats treated with bleomycin showed increased parenchymal fibrosis (hydroxyproline concentrations) and vascular remodeling (media wall thickness) as well as increased right ventricular systolic pressure compared with wild-type animals. Our data show that GCN2 is dysregulated in both humans and in an animal model of combined PF and PH. The possibility of a causative implication of GCN2 dysregulation in PF and/or PH development should be further studied.

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 bla CTX-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.

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.

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.

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: 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 cm 2 . 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/m 2 . 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/m 2 was 26% and 92%, respectively. Mite mortality reached 98% at 80 kJ/m 2 . 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/m 2 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., (© 2021. The Author(s).)

Background: Small-for-size syndrome (SFSS) looms over patients needing liver resection or living-donor transplantation. Hypoxia has been shown to be crucial for the successful outcome of liver resection in the very early postoperative phase. While poorly acceptable as such in real-world clinical practice, hypoxia responses can still be simulated by pharmacologically raising levels of its transducers, the hypoxia-inducible factors (HIFs). We aimed to assess the potential role of a selective inhibitor of HIF degradation in 70% hepatectomy (70%Hx)., Methods: In a pilot study, we tested the required dose of roxadustat to stabilize liver HIF1α. We then performed 70%Hx in 8-week-old male Lewis rats and administered 25 mg/kg of roxadustat (RXD25) at the end of the procedure. Regeneration was assessed: ki67 and 5-ethynyl-2'-deoxyuridine (EdU) immunofluorescent labeling, and histological parameters. We also assessed liver function via a blood panel and functional gadoxetate-enhanced magnetic resonance imaging (MRI), up to 47 h after the procedure. Metabolic results were analyzed by means of RNA sequencing (RNAseq)., Results: Roxadustat effectively increased early HIF1α transactivity. Liver function did not appear to be improved nor liver regeneration to be accelerated by the experimental compound. However, treated livers showed a mitigation in hepatocellular steatosis and ballooning, known markers of cellular stress after liver resection. RNAseq confirmed that roxadustat unexpectedly increases lipid breakdown and cellular respiration., Conclusions: Selective HIF stabilization did not result in an enhanced liver function after standard liver resection, but it induced interesting metabolic changes that are worth studying for their possible role in extended liver resections and fatty liver diseases., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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% CeO 2 or 1% SiO 2 for three weeks. In study 2, both female and male mice were purchased and exposed to 0.2% Ag-PVP or 1% TiO 2 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 SiO 2 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.

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 bla CTX-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., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

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), SiO 2 (0, 0.8, 8 and 80 mg/kg pellet) or TiO 2 (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 SiO 2 and TiO 2 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., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Renoz, Foray, Ambroise, Baa-Puyoulet, Bearzatto, Mendez, Grigorescu, Mahillon, Mardulyn, Gala, Calevro and Hance.)

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 MHCII low 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 MHCII high inflammatory macrophages. In contrast, the low inflammatory response induced by CNT-T was associated by the accumulation of self-renewing MHCII low 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Orsi, Palmai-Pallag, Yakoub, Ibouraadaten, De Beukelaer, Bouzin, Bearzatto, Ambroise, Gala, Brusa, Lison and Huaux.)

Context: The addition of silver (Ag) to food items, and its migration from food packaging and appliances results in a dietary exposure in humans, estimated to 70-90 µg Ag/day. In view of the well-known bactericidal activity of Ag ions, concerns arise about a possible impact of dietary Ag on the gut microbiota (GM), which is a master determinant of human health and diseases. Repeated oral administration of Ag acetate (AgAc) can also cause systemic toxicity in rats with reported NOAELs of 4 mg AgAc/b.w./d for impaired fertility and 0.4 mg AgAc/b.w./d for developmental toxicity., Objective: The objective of this study was to investigate whether oral exposure to AgAc can induce GM alterations at doses causing reproductive toxicity in rats., Methods: Male and female Wistar rats were exposed during 10 weeks to AgAc incorporated into food (0, 0.4, 4 or 40 mg/kg b.w./d), and we analyzed the composition of the GM (α- and β-diversity). We documented bacterial function by measuring short-chain fatty acid (SCFA) production in cecal content. Ferroxidase activity, a biomarker of systemic Ag toxicity, was measured in serum., Results and Conclusions: From 4 mg/kg b.w./d onwards, we recorded systemic toxicity, as indicated by the reduction of serum ferroxidase activity, as well as serum Cu and Se concentrations. This systemic toxic response to AgAc might contribute to explain reprotoxic manifestations. We observed a dose-dependent modification of the GM composition in male rats exposed to AgAc. No impact of AgAc exposure on the production of bacterial SCFA was recorded. The limited GM changes recorded in this study do not appear related to a reprotoxicity outcome.

Coronavirus disease 2019 (COVID-19) is an acute infectious disease caused by the novel coronavirus (SARS-CoV-2) identified in 2019. The COVID-19 outbreak continues to have devastating consequences for human lives and the global economy. The B-LiFe mobile laboratory in Piedmont, Italy, was deployed for the surveillance of COVID-19 cases by large-scale testing of first responders. The objective was to assess the seroconversion among the regional civil protection (CP), police, health care professionals, and volunteers. The secondary objective was to detect asymptomatic individuals within this cohort in the light of age, sex, and residence. In this paper, we report the results of serological testing performed by the B-LiFe mobile laboratory deployed from 10 June to 23 July 2020. The tests included whole blood finger-prick and serum sampling for detection of SARS-CoV-2 spike receptor-binding domain (S-RBD) antibodies. The prevalence of SARS-CoV-2 antibodies was approximately 5% (294/6013). The results of the finger-prick tests and serum sample analyses showed moderate agreement (kappa = 0.77). Furthermore, the detection rates of serum antibodies to the SARS-CoV-2 nucleocapsid protein (NP) and S-RBD among the seroconverted individuals were positively correlated (kappa = 0.60), at least at the IgG level. Seroprevalence studies based on serological testing for the S-RBD protein or SARS-CoV-2 NP antibodies are not sufficient for diagnosis but might help in screening the population to be vaccinated and in determining the duration of seroconversion.

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 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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Triaille, Vansteenkiste, Constant, Ambroise, Méric de Bellefon, Nzeusseu Toukap, Sokolova, Galant, Coulie, Carrasco, Durez and Lauwerys.)

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., (Copyright © 2020 Irenge et al.)

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., Competing Interests: Declaration of Competing Interest The authors have nothing to disclose., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Zellweger spectrum disorders (ZSD) are inborn errors of metabolism caused by mutations in PEX genes that lead to peroxisomal biogenesis disorder (PBD). No validated treatment is able to modify the dismal progression of the disease. ZSD mouse models used to develop therapeutic approaches are limited by poor survival and breeding restrictions. To overcome these limitations, we backcrossed the hypomorphic Pex1 p.G844D allele to NMRI background. NMRI mouse breeding restored an autosomal recessive Mendelian inheritance pattern and delivered twice larger litters. Mice were longitudinally phenotyped up to 6 months of age to make this model suitable for therapeutic interventions. ZSD mice exhibited growth retardation and relative hepatomegaly associated to progressive hepatocyte hypertrophy. Biochemical studies associated with RNA sequencing deciphered ZSD liver glycogen metabolism alterations. Affected fibroblasts displayed classical immunofluorescence pattern and biochemical alterations associated with PBD. Plasma and liver showed very long-chain fatty acids, specific oxysterols and C 27 bile acids intermediates elevation in ZSD mice along with a specific urine organic acid profile. With ageing, C 26 fatty acid and phytanic acid levels tended to normalize in ZSD mice, as described in patients reaching adulthood. In conclusion, our mouse model recapitulates a mild ZSD phenotype and is suitable for liver-targeted therapies evaluation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (Copyright © 2020 Botman, Hibaoui, Giudice, Ambroise, Creppe, Feki and Wyns.)

We report here a complete genome sequence of a Vibrio cholerae O1 El Tor (Inaba; sequence type 515 [ST515]) strain isolated from a cholera patient in North Kivu Province, Democratic Republic of the Congo (DRC), which showed a complete deletion (∼80 kb) of the Vibrio pathogenicity island 1., (Copyright © 2020 Irenge et al.)

Objectives: 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., Materials and Methods: 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., Results: 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 < 0.001) and ctDNA quantity (p < 0.001). 26% of the variants were detected only in liquid and not in the solid biopsy. Three patients without an available tumor sample had plasma containing three different potentially actionable PIK3CA mutations., Conclusion: CtDNA detection and characterization using targeted NGS is feasible in metastatic HNSCC. Liquid biopsies do not reflect the complete mutation profile of the tumor but have the potential to identify actionable mutations when tumor biopsies are not available as well as variants not found in matched tumor tissue., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Over 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 Findings: Phenotypic 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/significance: Current 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., Competing Interests: The authors have declared that no competing interests exist.

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.

Serratia symbiotica , one of the most frequent symbiont species in aphids, includes strains that exhibit various lifestyles ranging from free-living to obligate intracellular mutualism. Here, we report the draft genome sequences of two strains, namely, 24.1 and Apa8A1, isolated from aphids of the genus Aphis , consisting of genome sizes of 3,089,091 bp and 3,232,107 bp, respectively. These genome sequences may provide new insights into how mutualistic interactions between bacteria and insects evolve and are shaped., (Copyright © 2020 Renoz et al.)

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., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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.

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., Competing Interests: The authors have declared that no competing interests exist.

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.

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 cm 3 /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., (© 2019 Wiley Periodicals, Inc.)

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.

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., (Copyright © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

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 mm 3 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.

AMP-activated protein kinase (AMPK) α1 is activated in platelets on thrombin or collagen stimulation, and as a consequence, phosphorylates and inhibits acetyl-CoA carboxylase (ACC). Because ACC is crucial for the synthesis of fatty acids, which are essential for platelet activation, we hypothesized that this enzyme plays a central regulatory role in platelet function. To investigate this, we used a double knock-in (DKI) mouse model in which the AMPK phosphorylation sites Ser79 on ACC1 and Ser212 on ACC2 were mutated to prevent AMPK signaling to ACC. Suppression of ACC phosphorylation promoted injury-induced arterial thrombosis in vivo and enhanced thrombus growth ex vivo on collagen-coated surfaces under flow. After collagen stimulation, loss of AMPK-ACC signaling was associated with amplified thromboxane generation and dense granule secretion. ACC DKI platelets had increased arachidonic acid-containing phosphatidylethanolamine plasmalogen lipids. In conclusion, AMPK-ACC signaling is coupled to the control of thrombosis by specifically modulating thromboxane and granule release in response to collagen. It appears to achieve this by increasing platelet phospholipid content required for the generation of arachidonic acid, a key mediator of platelet activation., (© 2018 by The American Society of Hematology.)

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., (Copyright © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Four isothermal recombinase polymerase amplification (RPA) assays were developed for fast in-field identification of Bacillus anthracis The RPA assays targeted three specific sequences (i.e., the BA&#95;5345 chromosomal marker, the lethal factor lef [from pXO1], and the capsule-biosynthesis-related capA [from pXO2]) and a conserved sequence in the adenylate cyclase gene ( adk ) for the Bacillus cereus group. B. anthracis -specific RPA assays were tested first with purified genomic DNAs ( n = 60), including 11 representatives of B. anthracis , and then with soil ( n = 8) and white powder ( n = 8) samples spiked with inactivated B. anthracis spores 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 20 B. anthracis strains). RPA assays displayed 100% specificity and sensitivity. The hands-off turnaround times at 42°C ranged from 5 to 6 min for 10 2 genomic copies. The analytical sensitivity of each RPA assay was ∼10 molecules per reaction. In addition, the BA&#95;5345 and adk RPA assays were assessed under field conditions with a series of surface swabs ( n = 13, including 11 swabs contaminated with B. thuringiensis spores) 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 for B. anthracis , and all samples that had been harvested from spore-contaminated surfaces tested positive with the adk RPA assay. All three B. anthracis -specific RPA assays proved suitable for rapid and reliable identification of B. anthracis and therefore could easily be used by first responders under field conditions to quickly discriminate between a deliberate release of B. anthracis spores and a hoax attack involving white powder. IMPORTANCE In 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 of B. 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 of B. anthracis in powders and soil samples, with the purpose of discriminating a deliberate release of B. anthracis spores from a hoax attack involving white powder; this would also apply to dispersion by spraying of aerosolized forms of B. anthracis Further work is now ongoing to confirm the first observations and validate the on-site use of these assays by first responders., (Copyright © 2018 Bentahir et al.)

Insects are frequently associated with bacteria that can have significant ecological and evolutionary impacts on their hosts. To date, few studies have examined the influence of environmental factors to microbiome composition of aphids. The current work assessed the diversity of bacterial communities of five cereal aphid species (Sitobion avenae, Rhopalosiphum padi, R. maidis, Sipha maydis and Diuraphis noxia) collected across Morocco, covering a wide range of environmental conditions. We aimed to test whether symbiont combinations are host or environment specific. Deep 16S rRNA sequencing enabled us to identify 17 bacterial operational taxonomic units (OTUs). The obligate symbiont Buchnera aphidicola was represented by five OTUs with multiple haplotypes in many single samples. Facultative endosymbionts were presented by a high prevalence of Regiella insecticola and Serratia symbiotica in S. avenae and Si. maydis, respectively. In addition to these symbiotic partners, Pseudomonas, Acinetobacter, Pantoea, Erwinia and Staphyloccocus were also identified in aphids, suggesting that the aphid microbiome is not limited to the presence of endosymbiotic bacteria. Beside a significant association between host species and bacterial communities, an inverse correlation was also found between altitude and α-diversity. Overall, our results support that symbiont combinations are mainly host specific., (© FEMS 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Humans are increasingly exposed via the diet to Ag nanoparticles (NP) used in the food industry. Because of their anti-bacterial activity, ingested Ag NP might disturb the gut microbiota that is essential for local and systemic homeostasis. We explored here the possible impact of dietary Ag NP on the gut microbiota in mice at doses relevant for currently estimated human intake., Methods: Mice were orally exposed to food (pellets) supplemented with increasing doses of Ag NP (0, 46, 460 or 4600 ppb) during 28 d. Body weight, systemic inflammation and gut integrity were investigated to determine overall toxicity, and feces DNA collected from the gut were analyzed by Next Generation Sequencing (NGS) to assess the effect of Ag NP on the bacterial population. Ag NP were characterized alone and in the supplemented pellets by scanning transmission electron microscopy (STEM) and energy dispersive X-ray analysis (EDX)., Results: No overall toxicity was recorded in mice exposed to Ag NP. Ag NP disturbed bacterial evenness (α-diversity) and populations (β-diversity) in a dose-dependent manner. Ag NP increased the ratio between Firmicutes (F) and Bacteroidetes (B) phyla. At the family level, Lachnospiraceae and the S24-7 family mainly accounted for the increase in Firmicutes and decrease in Bacteroidetes, respectively. Similar effects were not observed in mice identically exposed to the same batch of Ag NP-supplemented pellets aged during 4 or 8 months and the F/B ratio was less or not modified. Analysis of Ag NP-supplemented pellets showed that freshly prepared pellets released Ag ions faster than aged pellets. STEM-EDX analysis also showed that Ag sulfidation occurred in aged Ag NP-supplemented pellets., Conclusions: Our data indicate that oral exposure to human relevant doses of Ag NP can induce microbial alterations in the gut. The bacterial disturbances recorded after Ag NP are similar to those reported in metabolic and inflammatory diseases, such as obesity. It also highlights that Ag NP aging in food, and more specifically sulfidation, can reduce the effects of Ag NP on the microbiota by limiting the release of toxic Ag ions.

Aims: Slide digitalization has brought pathology to a new era, including powerful image analysis possibilities. However, while being a powerful prognostic tool, immunostaining automated analysis on digital images is still not implemented worldwide in routine clinical practice., Methods and Results: Digitalized biopsy sections from two independent cohorts of patients, immunostained for membrane or nuclear markers, were quantified with two automated methods. The first was based on stained cell counting through tissue segmentation, while the second relied upon stained area proportion within tissue sections. Different steps of image preparation, such as automated tissue detection, folds exclusion and scanning magnification, were also assessed and validated. Quantification of either stained cells or the stained area was found to be correlated highly for all tested markers. Both methods were also correlated with visual scoring performed by a pathologist. For an equivalent reliability, quantification of the stained area is, however, faster and easier to fine-tune and is therefore more compatible with time constraints for prognosis., Conclusions: This work provides an incentive for the implementation of automated immunostaining analysis with a stained area method in routine laboratory practice., (© 2015 John Wiley & Sons Ltd.)

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.

Rapid and specific detection of extended-spectrum β-lactamase-producing (ESBL) bacteria is crucial both for timely antibiotic therapy when treating infected patients as well as for appropriate infection control measures aimed at curbing the spread of ESBL-producing isolates. Whereas a variety of phenotypic methods are currently available for ESBL detection, they remain time consuming and sometimes difficult to interpret while being also affected by a lack of sensitivity and specificity. Considering the longer turnaround time (TAT) of susceptibility testing and culture results, DNA-based ESBL identification would be a valuable surrogate for phenotypic-based methods. Putative ESBL-positive Enterobacteriaceae isolates (n = 330) from clinical specimen were prospectively collected in Bulgaria, Romania and Democratic Republic of Congo and tested in this study. All isolates were assessed for ESBL-production by the E-test method and those giving undetermined ESBL status were re-tested using the combination disk test. A genotypic assay successively combining qPCR detection of blaCTX-M, blaTEM and blaSHV genes with a multiplex pyrosequencing of blaTEM and blaSHV genes was developed in order to detect the most common ESBL-associated TEM and SHV single nucleotides polymorphisms, irrespective of their plasmid and/or chromosomal location. This assay was applied on all Enterobacteriaceae isolates (n = 330). Phenotypic and genotypic results matched in 324/330 (98.2%). Accordingly, real-time PCR combined with multiplex pyrosequencing appears to be a reliable and easy-to-perform assay with high-throughput identification and fast TAT (~5 h).