A Novel Natural Siderophore Antibiotic Conjugate Reveals a Chemical Approach to Macromolecule Coupling.

Inspired by natural sideromycins, the conjugation of antibiotics to siderophores is an attractive strategy to facilitate "Trojan horse" delivery of antibiotics into bacteria. Genome analysis of a soil bacterium, Dactylosporangium fulvum, found a "hybrid" biosynthetic gene cluster responsible for the production of both an antibiotic, pyridomycin, and a novel chlorocatechol-containing siderophore named chlorodactyloferrin. While both of these natural products were synthesized independently, analysis of the culture supernatant also identified a conjugate of both molecules. We then found that the addition of ferric iron to purified chlorodactyloferrin and pyridomycin instigated their conjugation, leading to the formation of a covalent bond between the siderophore-catechol and the pyridomycin-pyridine groups. Using model reactants, this iron-based reaction was found to proceed through a Michael-type addition reaction, where ferric iron oxidizes the siderophore-catechol group into its quinone form, which is then attacked by the antibiotic pyridyl-nitrogen to form the catechol-pyridinium linkage. These findings prompted us to explore if other "cargo" molecules could be attached to chlorodactyloferrin in a similar manner, and this was indeed confirmed with a pyridine-substituted TAMRA fluorophore as well as with pyridine-substituted penicillin, rifampicin, and norfloxacin antibiotic analogues. The resultant biomimetic conjugates were demonstrated to effectively enter a number of bacteria, with TAMRA-chlorodactyloferrin conjugates causing fluorescent labeling of the bacteria, and with penicillin and rifampicin conjugates eliciting antibiotic activity. These findings open up new opportunities for the design and facile synthesis of a novel class of biomimetic siderophore conjugates with antibiotic activity.

Low dose dietary contamination with deoxynivalenol mycotoxin exacerbates enteritis and colorectal cancer in mice.

BACKGROUND: The mycotoxin deoxynivalenol (DON) is a frequent contaminant of grain and cereal products worldwide. Exposure to DON can cause gastrointestinal inflammation, disturb gut barrier function, and induce gut dysbiosis in vivo under basal conditions, but little is known about the effects of DON ingestion in individuals with pre-existing gastrointestinal disease. OBJECTIVES: Mice were orally exposed to 10 and 100 µg/kg bw/day of DON, corresponding to 10 to 100-fold human tolerable daily intake concentrations, and to the translation in mice of current human daily intake. The effects of DON exposure were explored under steady-state conditions, and in murine models of enteritis and colorectal cancer (CRC). RESULTS: After 8 days of DON exposure, an increase of histomorphological and molecular parameters of epithelial proliferation were observed in normal mice, from the duodenum to the colon. The same exposure in a murine model of indomethacin-induced enteritis led to exacerbation of lesion development and induction of ileal cytokines. DON exposure also worsened the development of colitis-associated CRC in mice as shown by increases in endoscopic and histological colitis scores, tumor grades, and histological hyperplasia. In colon of DON-exposed mice, upstream and downstream ERK signaling genes were upregulated including Mapk1, Mapk3, Map 2k1, Map2k2 core ERK pathway effectors, and Bcl2 and Bcl2l1 antiapoptotic genes. The effects observed in the CRC model were associated with alterations in cecal microbiota taxonomic composition and metabolism of bacterial fucose and rhamnose. Strong Spearman's correlations were revealed between the relative abundance of the changed bacterial genera and CRC-related variables. DISCUSSION: Ingestion of DON mycotoxin at concentrations representative of human real-world exposure worsened the development of indomethacin-induced enteritis and colitis-associated CRC in mice. Our results suggest that even at low doses, which are currently tolerated in the human diet, DON could promote the development of intestinal inflammatory diseases and CRC.

A Protective Role of NOD2 on Oxazolone-induced Intestinal Inflammation Through IL-1ß-mediated Signalling Pathway.

BACKGROUND AND AIMS: NOD2 has emerged as a critical player in the induction of both Th1 and Th2 responses for potentiation and polarisation of antigen-dependent immunity. Loss-of-function mutations in the NOD2-encoding gene and deregulation of its downstream signalling pathway have been linked to Crohn's disease. Although it is well documented that NOD2 is capable of sensing bacterial muramyl dipeptide, it remains counter-intuitive to link development of overt intestinal inflammation to a loss of bacterial-induced inflammatory response. We hypothesised that a T helper bias could also contribute to an autoimmune-like colitis different from inflammation that is fully fledged by Th1 type cells. METHODS: An oedematous bowel wall with a mixed Th1/Th2 response was induced in mice by intrarectal instillation of the haptenating agent oxazolone. Survival and clinical scoring were evaluated. At several time points after instillation, colonic damage was assessed by macroscopic and microscopic observations. To evaluate the involvement of NOD2 in immunochemical phenomena, quantitative polymerase chain reaction [PCR] and flow cytometry analysis were performed. Bone marrow chimera experimentation allowed us to evaluate the role of haematopoietic/non-hematopoietic NOD2-expressing cells. RESULTS: Herein, we identified a key regulatory circuit whereby NOD2-mediated sensing of a muramyl dipeptide [MDP] by radio-resistant cells improves colitis with a mixed Th1/Th2 response that is induced by oxazolone. Genetic ablation of either Nod2 or Ripk2 precipitated oxazolone colitis that is predominantly linked to a lack of interferon-gamma. Bone marrow chimera experiments revealed that inactivation of Nod2 signalling in non-haematopoietic cells is causing a biased M1-M2 polarisation of macrophages and a decreased frequency of splenic regulatory T cells that correlates with an impaired activation of CD4 + T cells within mesenteric lymph nodes. Mechanistically, mice were protected from oxazolone-induced colitis upon administration of MDP in an interleukin-1- and interleukin-23-dependent manner. CONCLUSIONS: These findings indicate that Nod2 signalling may prevent pathological conversion of T helper cells for maintenance of tissue homeostasis.

An Arginine-Rich Motif in the ORF2 capsid protein regulates the hepatitis E virus lifecycle and interactions with the host cell.

Hepatitis E virus (HEV) infection is the most common cause of acute viral hepatitis worldwide. Hepatitis E is usually asymptomatic and self-limiting but it can become chronic in immunocompromised patients and is associated with increased fulminant hepatic failure and mortality rates in pregnant women. HEV genome encodes three proteins including the ORF2 protein that is the viral capsid protein. Interestingly, HEV produces 3 isoforms of the ORF2 capsid protein which are partitioned in different subcellular compartments and perform distinct functions in the HEV lifecycle. Notably, the infectious ORF2 (ORF2i) protein is the structural component of virions, whereas the genome-free secreted and glycosylated ORF2 proteins likely act as a humoral immune decoy. Here, by using a series of ORF2 capsid protein mutants expressed in the infectious genotype 3 p6 HEV strain as well as chimeras between ORF2 and the CD4 glycoprotein, we demonstrated how an Arginine-Rich Motif (ARM) located in the ORF2 N-terminal region controls the fate and functions of ORF2 isoforms. We showed that the ARM controls ORF2 nuclear translocation likely to promote regulation of host antiviral responses. This motif also regulates the dual topology and functionality of ORF2 signal peptide, leading to the production of either cytosolic infectious ORF2i or reticular non-infectious glycosylated ORF2 forms. It serves as maturation site of glycosylated ORF2 by furin, and promotes ORF2-host cell membrane interactions. The identification of ORF2 ARM as a unique central regulator of the HEV lifecycle uncovers how viruses settle strategies to condense their genetic information and hijack cellular processes.

Murine in utero exposure to simulated complex urban air pollution disturbs offspring gut maturation and microbiota during intestinal suckling-to-weaning transition in a sex-dependent manner.

BACKGROUND: Emerging data indicate that prenatal exposure to air pollution may lead to higher susceptibility to several non-communicable diseases. Limited research has been conducted due to difficulties in modelling realistic air pollution exposure. In this study, pregnant mice were exposed from gestational day 10-17 to an atmosphere representative of a 2017 pollution event in Beijing, China. Intestinal homeostasis and microbiota were assessed in both male and female offspring during the suckling-to-weaning transition. RESULTS: Sex-specific differences were observed in progeny of gestationally-exposed mice. In utero exposed males exhibited decreased villus and crypt length, vacuolation abnormalities, and lower levels of tight junction protein ZO-1 in ileum. They showed an upregulation of absorptive cell markers and a downregulation of neonatal markers in colon. Cecum of in utero exposed male mice also presented a deeply unbalanced inflammatory pattern. By contrast, in utero exposed female mice displayed less severe intestinal alterations, but included dysregulated expression of Lgr5 in colon, Tjp1 in cecum, and Epcam, Car2 and Sis in ileum. Moreover, exposed female mice showed dysbiosis characterized by a decreased weighted UniFrac ß-diversity index, a higher abundance of Bacteroidales and Coriobacteriales orders, and a reduced Firmicutes/Bacteroidetes ratio. CONCLUSION: Prenatal realistic modelling of an urban air pollution event induced sex-specific precocious alterations of structural and immune intestinal development in mice.

Exposure to atmospheric Ag, TiO2, Ti and SiO2 engineered nanoparticles modulates gut inflammatory response and microbiota in mice.

The development of nanotechnologies is leading to greater abundance of engineered nanoparticles (EN) in the environment, including in the atmospheric air. To date, it has been shown that the most prevalent EN found in the air are silver (Ag), titanium dioxide (TiO2), titanium (Ti), and silicon dioxide (SiO2). As the intestinal tract is increasingly recognized as a target for adverse effects induced by inhalation of air particles, the aim of this study was to assess the impact of these 4 atmospheric EN on intestinal inflammation and microbiota. We assessed the combined toxicity effects of Ag, Ti, TiO2, and SiO2 following a 28-day inhalation protocol in male and female mice. In distal and proximal colon, and in jejunum, EN mixture inhalation did not induce overt histological damage, but led to a significant modulation of inflammatory cytokine transcript abundance, including downregulation of Tnfα, Ifnγ, Il1ß, Il17a, Il22, IL10, and Cxcl1 mRNA levels in male jejunum. A dysbiosis was observed in cecal microbiota of male and female mice exposed to the EN mixture, characterized by sex-dependent modulations of specific bacterial taxa, as well as sex-independent decreased abundance of the Eggerthellaceae family. Under dextran sodium sulfate-induced inflammatory conditions, exposure to the EN mixture increased the development of colitis in both male and female mice. Moreover, the direct dose-response effects of individual and mixed EN on gut organoids was studied and Ag, TiO2, Ti, SiO2, and EN mixture were found to generate specific inflammatory responses in the intestinal epithelium. These results indicate that the 4 most prevalent atmospheric EN could have the ability to disturb intestinal homeostasis through direct modulation of cytokine expression in gut epithelium, and by altering the inflammatory response and microbiota composition following inhalation.

Oral exposure to polyethylene microplastics alters gut morphology, immune response, and microbiota composition in mice.

The ubiquitous and growing presence of microplastics (MPs) in all compartments of the environment raises concerns about their possible harmful effects on human health. Human exposure to MPs occurs largely through ingestion. Polyethylene (PE) is widely employed for reusable bags and food packaging and found to be present in drinking water and food. It is also one of the major polymers detected in human stool. The aim of this study was to characterize the effects of intestinal exposure to PE MPs on gut homeostasis. Mice were orally exposed for 6 weeks to PE microbeads of 2 different sizes, 36 and 116 µm, that correspond to those found in human stool. They were administrated either individually or as a mixture at a dose of 100 µg/g of food. Both PE microbead sizes were detected in mouse stool. Different parameters related to major intestinal functions were compared between control mice, mice exposed to each type of microbead, or co-exposed to the 2 types of microbeads. Intestinal disturbances were observed after individual exposure to each size of PE microbead, and the most marked deleterious effects were found in co-exposed mice. At the histomorphological level, crypt depth was increased throughout the intestinal tissues. Significant variations of gene expression related to epithelial, permeability, and inflammatory biomarkers were quantified. Defective recruitment of some intestinal immune cells was observed from the proximal portion of the small intestine to the colon. Several bacterial taxa at the order level were found to be affected by exposure to the MPs by metagenomic analysis of cecal microbiota. These results show that ingestion of PE microbeads induces significant alterations of crucial intestinal markers in mice and underscores the need to further study the health impact of MP exposure in humans.

Persistent Cryptosporidium parvum Infection Leads to the Development of the Tumor Microenvironment in an Experimental Mouse Model: Results of a Microarray Approach.

Cryptosporidium spp. are enteric protozoa parasites that infect a variety of vertebrate hosts. These parasites are capable of inducing life-threatening gastrointestinal disease in immunocompromised individuals. With the rising epidemiological evidence of the occurrence of Cryptosporidium infections in humans with digestive cancer, the tumorigenic potential of the parasite has been speculated. In this regard, Cryptosporidium parvum has been reported to induce digestive adenocarcinoma in a rodent model of chronic cryptosporidiosis. However, the processes by which the parasite could induce this carcinogenesis are still unknown. Therefore, the transcriptomes of C. parvum infected ileo-cecal regions of mice developing tumors were analyzed in the current study. For the first time, downregulation of the expression of α-defensin, an anti-microbial target of the parasite in response to C. parvum infection was observed in the transformed tissues. This phenomenon has been speculated to be the result of resistance of C. parvum to the host defense through the upregulated expression of interferon γ-stimulated genes. The inflammatory response generated as result of attenuated expression of anti-microbial peptides highlights the role of immune evasion in the C. parvum-induced tumorigenesis. The study has also succeeded in the characterization of the tumor microenvironment (TME) which is characterized by the presence of cancer associated fibroblasts, myeloid-derived suppressor cells, tumor-associated macrophages and extracellular matrix components. Identification of immune suppressor cells and accumulation of pro-inflammatory mediators speculates that chronic inflammation induced by persistent C. parvum infection assists in development of an immunosuppressive tumor microenvironment.

A Unique Reverse Adaptation Mechanism Assists Bordetella pertussis in Resistance to Both Scarcity and Toxicity of Manganese.

The ability of bacterial pathogens to acquire essential micronutrients is critical for their survival in the host environment. Manganese plays a complex role in the virulence of a variety of pathogens due to its function as an antioxidant and enzymatic cofactor. Therefore, host cells deprive pathogens of manganese to prevent or attenuate infection. Here, we show that evolution of the human-restricted pathogen Bordetella pertussis has selected for an inhibitory duplication within a manganese exporter of the calcium:cation antiporter superfamily. Intriguingly, upon exposure to toxic levels of manganese, the nonfunctional exporter becomes operative in resister cells due to a unique reverse adaptation mechanism. However, compared with wild-type (wt) cells, the resisters carrying a functional copy of the exporter displayed strongly reduced intracellular levels of manganese and impaired growth under oxidative stress. Apparently, inactivation of the manganese exporter and the resulting accumulation of manganese in the cytosol benefited the pathogen by improving its survival under stress conditions. The inhibitory duplication within the exporter gene is highly conserved among B. pertussis strains, absent from all other Bordetella species and from a vast majority of organisms across all kingdoms of life. Therefore, we conclude that inactivation of the exporter gene represents an exceptional example of a flexible genome decay strategy employed by a human pathogen to adapt to its exclusive host. IMPORTANCE Bordetella pertussis, a respiratory pathogen restricted to humans, continuously adapts its genome to its exclusive host. We show that speciation of this reemerging pathogen was accompanied by loss of function of the manganese exporter. Intriguingly, the functionality of the exporter can be restored in the presence of toxic levels of manganese by a unique genetic modification. However, compared with the wt strain, the strain carrying the functional exporter failed to resist the oxidative stress in vitro. Thus, our data demonstrate that inactivation of the exporter resulting in manganese accumulation assists B. pertussis in adaptation to oxidative stress. We conclude that this sophisticated process of reverse adaptation enables B. pertussis to adjust to rapidly changing environments by facilitating its resistance to both manganese toxicity and manganese scarcity.

Erratum: Rémy et al. Modelling the Impact of Chronic Cigarette Smoke Exposure in Obese Mice: Metabolic, Pulmonary, Intestinal, and Cardiac Issues. Nutrients 2020, 12, 827.

The authors have requested that the following changes be made to their paper [...].

Rubrolone production by Dactylosporangium vinaceum: biosynthesis, modulation and possible biological function.

Rare actinomycetes are likely treasure troves for bioactive natural products, and it is therefore important that we enrich our understanding of biosynthetic potential of these relatively understudied bacteria. Dactylosporangium are a genus of such rare Actinobacteria that are known to produce a number of important antibacterial compounds, but for which there are still no fully assembled reference genomes, and where the extent of encoded biosynthetic capacity is not defined. Dactylosporangium vinaceum (NRRL B-16297) is known to readily produce a deep wine red-coloured diffusible pigment of unknown origin, and it was decided to define the chemical identity of this natural product pigment, and in parallel use whole genome sequencing and transcriptional analysis to lay a foundation for understanding the biosynthetic capacity of these bacteria. Results show that the produced pigment is made of various rubrolone conjugates, the spontaneous product of the reactive pre-rubrolone, produced by the bacterium. Genome and transcriptome analysis identified the highly expressed biosynthetic gene cluster (BGC) for pre-rubrolone. Further analysis of the fully assembled genome found it to carry 24 additional BGCs, of which the majority were poorly transcribed, confirming the encoded capacity of this bacterium to produce natural products but also illustrating the main bottleneck to exploiting this capacity. Finally, analysis of the potential environmental role of pre-rubrolone found it to react with a number of amine containing antibiotics, antimicrobial peptides and siderophores pointing to its potential role as a "minesweeper" of xenobiotic molecules in the bacterial environment. KEY POINTS: • D. vinaceum encodes many BGC, but the majority are transcriptionally silent. • Chemical screening identifies molecules that modulate rubrolone production. • Pre-rubrolone is efficient at binding and inactivating many natural antibiotics.

Streamlined copper defenses make Bordetella pertussis reliant on custom-made operon.

Copper is both essential and toxic to living beings, which tightly controls its intracellular concentration. At the host-pathogen interface, copper is used by phagocytic cells to kill invading microorganisms. We investigated copper homeostasis in Bordetella pertussis, which lives in the human respiratory mucosa and has no environmental reservoir. B. pertussis has considerably streamlined copper homeostasis mechanisms relative to other Gram-negative bacteria. Its single remaining defense line consists of a metallochaperone diverted for copper passivation, CopZ, and two peroxide detoxification enzymes, PrxGrx and GorB, which together fight stresses encountered in phagocytic cells. Those proteins are encoded by an original, composite operon assembled in an environmental ancestor, which is under sensitive control by copper. This system appears to contribute to persistent infection in the nasal cavity of B. pertussis-infected mice. Combining responses to co-occurring stresses in a tailored operon reveals a strategy adopted by a host-restricted pathogen to optimize survival at minimal energy expenditure.

Association between schizophrenia and Toxoplasma gondii infection in Algeria.

Toxoplasmosis has been previously associated with an increased risk of having schizophrenia in several epidemiological studies. The aim of this prospective study was to examine for the first time a possible association between positive serology to Toxoplasma gondii (T. gondii) and schizophrenia in the Algerian population. Seventy patients affected by schizophrenia according to DSM-5 criteria and seventy healthy controls were enrolled in the study. We found a significant association between schizophrenia and the infection status with a seroprevalence of 70% in patients with schizophrenia compared to 52.9% in controls and a calculated odds ratio of 2.081. In addition, while T. gondii seroprevalence increases significantly with age in controls, this association was not observed in patients with schizophrenia, which display a high percentage of seropositive subjects under 38 years of age, suggesting that T. gondii infection may promote the onset of schizophrenia. Moreover, our analysis also revealed that patients with schizophrenia had significantly lower levels of serum immunoglobulins G (IgG) to T. gondii compared to controls. Thus, this study adds to previous research questioning the asymptomatic aspect of chronic toxoplasmosis and the etiology of schizophrenia.

Combined RNAseq and ChIPseq Analyses of the BvgA Virulence Regulator of Bordetella pertussis.

Bordetella pertussis regulates the production of its virulence factors by the two-component system BvgAS. In the virulence phase, BvgS phosphorylates BvgA, which then activates the transcription of virulence-activated genes (vags). In the avirulence phase, such as during growth in the presence of MgSO4, BvgA is not phosphorylated and the vags are not expressed. Instead, a set of virulence-repressed genes (vrgs) is expressed. Here, we performed transcriptome sequencing (RNAseq) analyses on B. pertussis cultivated with or without MgSO4 and on a BvgA-deficient Tohama I derivative. We observed that 146 genes were less expressed under modulating conditions or in the BvgA-deficient strain than under the nonmodulating condition, while 130 genes were more expressed. Some of the genes code for proteins with regulatory functions, suggesting a BvgA/S regulation cascade. To determine which genes are directly regulated by BvgA, we performed chromatin immunoprecipitation sequencing (ChIPseq) analyses. We identified 148 BvgA-binding sites, 91 within putative promoter regions, 52 within open reading frames, and 5 in noncoding regions. Among the former, 32 are in BvgA-regulated putative promoter regions. Some vags, such as dnt and fhaL, contain no BvgA-binding site, suggesting indirect BvgA regulation. Unexpectedly, BvgA also bound to some vrg putative promoter regions. Together, these observations indicate an unrecognized complexity of BvgA/S biology.IMPORTANCE Bordetella pertussis, the etiological agent of whooping cough, remains a major global health problem. Despite the global usage of whole-cell vaccines since the 1950s and of acellular vaccines in the 1990s, it still is one of the most prevalent vaccine-preventable diseases in industrialized countries. Virulence of B. pertussis is controlled by BvgA/S, a two-component system responsible for upregulation of virulence-activated genes (vags) and downregulation of virulence-repressed genes (vrgs). By transcriptome sequencing (RNAseq) analyses, we identified more than 270 vags or vrgs, and chromatin immunoprecipitation sequencing (ChIPseq) analyses revealed 148 BvgA-binding sites, 91 within putative promoter regions, 52 within open reading frames, and 5 in noncoding regions. Some vags, such as dnt and fhaL, do not contain a BvgA-binding site, suggesting indirect regulation. In contrast, several vrgs and some genes not identified by RNAseq analyses under laboratory conditions contain strong BvgA-binding sites, indicating previously unappreciated complexities of BvgA/S biology.

Influenza infection rewires energy metabolism and induces browning features in adipose cells and tissues.

Like all obligate intracellular pathogens, influenza A virus (IAV) reprograms host cell's glucose and lipid metabolism to promote its own replication. However, the impact of influenza infection on white adipose tissue (WAT), a key tissue in the control of systemic energy homeostasis, has not been yet characterized. Here, we show that influenza infection induces alterations in whole-body glucose metabolism that persist long after the virus has been cleared. We report depot-specific changes in the WAT of IAV-infected mice, notably characterized by the appearance of thermogenic brown-like adipocytes within the subcutaneous fat depot. Importantly, viral RNA- and viral antigen-harboring cells are detected in the WAT of infected mice. Using in vitro approaches, we find that IAV infection enhances the expression of brown-adipogenesis-related genes in preadipocytes. Overall, our findings shed light on the role that the white adipose tissue, which lies at the crossroads of nutrition, metabolism and immunity, may play in influenza infection.

Modelling the Impact of Chronic Cigarette Smoke Exposure in Obese Mice: Metabolic, Pulmonary, Intestinal, and Cardiac Issues.

Unhealthy lifestyle choices, such as bad eating behaviors and cigarette smoking, have major detrimental impacts on health. However, the inter-relations between obesity and smoking are still not fully understood. We thus developed an experimental model of high-fat diet-fed obese C57BL/6 male mice chronically exposed to cigarette smoke. Our study evaluated for the first time the resulting effects of the combined exposure to unhealthy diet and cigarette smoke on several metabolic, pulmonary, intestinal, and cardiac parameters. We showed that the chronic exposure to cigarette smoke modified the pattern of body fat distribution in favor of the visceral depots in obese mice, impaired the respiratory function, triggered pulmonary inflammation and emphysema, and was associated with gut microbiota dysbiosis, cardiac hypertrophy and myocardial fibrosis.

Use of whole-genome sequencing in the molecular investigation of care-associated HCoV-OC43 infections in a hematopoietic stem cell transplant unit.

BACKGROUND: While respiratory viral infections are recognized as a frequent cause of illness in hematopoietic stem cell transplantation (HSCT) recipients, HCoV-OC43 infections have rarely been investigated as healthcare-associated infections in this population. OBJECTIVES: In this report, HCoV-OC43 isolates collected from HSCT patients were retrospectively characterized to identify potential clusters of infection that may stand for a hospital transmission. STUDY DESIGN: Whole-genome and S gene sequences were obtained from nasal swabs using next-generation sequencing and phylogenetic trees were constructed. Similar identity matrix and determination of the most common ancestor were used to compare clusters of patient's sequences. Amino acids substitutions were analysed. RESULTS: Genotypes B, E, F and G were identified. Two clusters of patients were defined from chronological data and phylogenetic trees. Analyses of amino acids substitutions of the S protein sequences identified substitutions specific for genotype F strains circulating among European people. CONCLUSIONS: HCoV-OC43 may be implicated in healthcare-associated infections.

Let-7f: A New Potential Circulating Biomarker Identified by miRNA Profiling of Cells Isolated from Human Abdominal Aortic Aneurysm.

Abdominal aortic aneurysm (AAA) is a progressive vascular disease responsible for 1-4% of the deaths in elderly men. This study aimed to characterize specific microRNA (miRNA) expression in aneurysmal smooth muscle cells (SMCs) and macrophages in order to identify circulating miRNAs associated with AAA. We screened 850 miRNAs in aneurysmal SMCs, M1 and M2 macrophages, and in control SMCs isolated by micro-dissection from aortic biopsies using microarray analysis. In all, 92 miRNAs were detected and 10 miRNAs were selected for validation by qRT-PCR in isolated cells (n = 5), whole control and aneurysmal aorta biopsies (n = 13), and plasma from patients (n = 24) undergoing AAA (over 50 mm) repair matched to patients (n = 18) with peripheral arterial disease (PAD) with atherosclerosis but not AAA. Seven miRNAs were modulated similarly in all aneurysmal cells. The Let-7f was downregulated in aneurysmal cells compared to control SMCs with a significant lower expression in M1 compared to M2 macrophages (0.1 fold, p = 0.03), correlated with a significant downregulation in whole aneurysmal aorta compared to control aorta (0.2 fold, p = 0.03). Significant levels of circulating let-7f (p = 0.048) were found in AAA patients compared to PAD patients with no significant correlation with aortic diameter (R2 = 0.03). Our study underlines the utility of profiling isolated aneurysmal cells to identify other miRNAs for which the modulation of expression might be masked when the whole aorta is used. The results highlight let-7f as a new potential biomarker for AAA.

A complete protocol for whole-genome sequencing of virus from clinical samples: Application to coronavirus OC43.

Genome sequencing of virus has become a useful tool for better understanding of virus pathogenicity and epidemiological surveillance. Obtaining virus genome sequence directly from clinical samples is still a challenging task due to the low load of virus genetic material compared to the host DNA, and to the difficulty to get an accurate genome assembly. Here we introduce a complete sequencing and analyzing protocol called V-ASAP for Virus Amplicon Sequencing Assembly Pipeline. Our protocol is able to generate the viral dominant genome sequence starting from clinical samples. It is based on a multiplex PCR amplicon sequencing coupled with a reference-free analytical pipeline. This protocol was applied to 11 clinical samples infected with coronavirus OC43 (HcoV-OC43), and led to seven complete and two nearly complete genome assemblies. The protocol introduced here is shown to be robust, to produce a reliable sequence, and could be applied to other virus.

Proteasomal degradation of NOD2 by NLRP12 in monocytes promotes bacterial tolerance and colonization by enteropathogens.

Mutations in the nucleotide-binding oligomerization domain protein 12 (NLRP12) cause recurrent episodes of serosal inflammation. Here we show that NLRP12 efficiently sequesters HSP90 and promotes K48-linked ubiquitination and degradation of NOD2 in response to bacterial muramyl dipeptide (MDP). This interaction is mediated by the linker-region proximal to the nucleotide-binding domain of NLRP12. Consequently, the disease-causing NLRP12 R284X mutation fails to repress MDP-induced NF-κB and subsequent activity of the JAK/STAT signaling pathway. While NLRP12 deficiency renders septic mice highly susceptible towards MDP, a sustained sensing of MDP through NOD2 is observed among monocytes lacking NLRP12. This loss of tolerance in monocytes results in greater colonization resistance towards Citrobacter rodentium. Our data show that this is a consequence of NOD2-dependent accumulation of inflammatory mononuclear cells that correlates with induction of interferon-stimulated genes. Our study unveils a relevant process of tolerance towards the gut microbiota that is exploited by an attaching/effacing enteric pathogen.