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SARS-CoV-2 entry into host cells is a crucial step for virus tropism, transmission, and pathogenesis. Angiotensin-converting enzyme 2 (ACE2) has been identified as the primary entry receptor for SARS-CoV-2; however, the possible involvement of other cellular components in the viral entry has not yet been fully elucidated. Here we describe the identification of vimentin (VIM), an intermediate filament protein widely expressed in cells of mesenchymal origin, as an important attachment factor for SARS-CoV-2 on human endothelial cells. Using liquid chromatography–tandem mass spectrometry, we identified VIM as a protein that binds to the SARS-CoV-2 spike (S) protein. We showed that the S-protein receptor binding domain (RBD) is sufficient for S-protein interaction with VIM. Further analysis revealed that extracellular VIM binds to SARS-CoV-2 S-protein and facilitates SARS-CoV-2 infection, as determined by entry assays performed with pseudotyped viruses expressing S and with infectious SARS-CoV-2. Coexpression of VIM with ACE2 increased SARS-CoV-2 entry in HEK-293 cells, and shRNA-mediated knockdown of VIM significantly reduced SARS-CoV-2 infection of human endothelial cells. Moreover, incubation of A549 cells expressing ACE2 with purified VIM increased pseudotyped SARS-CoV-2-S entry. CR3022 antibody, which recognizes a distinct epitope on SARS-CoV-2-S-RBD without interfering with the binding of the spike with ACE2, inhibited the binding of VIM with CoV-2 S-RBD, and neutralized viral entry in human endothelial cells, suggesting a key role for VIM in SARS-CoV-2 infection of endothelial cells. This work provides insight into the pathogenesis of COVID-19 linked to the vascular system, with implications for the development of therapeutics and vaccines.

As the COVID-19 pandemic continues to spread, investigating the processes underlying the interactions between SARS-CoV-2 and its hosts is of high importance. Here, we report the identification of CD209L/L-SIGN and the related protein CD209/DC-SIGN as receptors capable of mediating SARS-CoV-2 entry into human cells. Immunofluorescence staining of human tissues revealed prominent expression of CD209L in the lung and kidney epithelium and endothelium. Multiple biochemical assays using a purified recombinant SARS-CoV-2 spike receptor binding domain (S-RBD) or S1 encompassing both NTB and RBD and ectopically expressed CD209L and CD209 revealed that CD209L and CD209 interact with S-RBD. CD209L contains two N-glycosylation sequons, at sites N92 and N361, but we determined that only site N92 is occupied. Removal of the N-glycosylation at this site enhances the binding of S-RBD with CD209L. CD209L also interacts with ACE2, suggesting a role for heterodimerization of CD209L and ACE2 in SARS-CoV-2 entry and infection in cell types where both are present. Furthermore, we demonstrate that human endothelial cells are permissive to SARS-CoV-2 infection and interference with CD209L activity by knockdown strategy or with soluble CD209L inhibits virus entry. Our observations demonstrate that CD209L and CD209 serve as alternative receptors for SARS-CoV-2 in disease-relevant cell types, including the vascular system. This property is particularly important in tissues where ACE2 has low expression or is absent, and may have implications for antiviral drug development.As the COVID-19 pandemic continues to spread, investigating the processes underlying the interactions between SARS-CoV-2 and its hosts is of high importance. Here, we report the identification of CD209L/L-SIGN and the related protein CD209/DC-SIGN as receptors capable of mediating SARS-CoV-2 entry into human cells. Immunofluorescence staining of human tissues revealed prominent expression of CD209L in the lung and kidney epithelium and endothelium. Multiple biochemical assays using a purified recombinant SARS-CoV-2 spike receptor binding domain (S-RBD) or S1 encompassing both NTB and RBD and ectopically expressed CD209L and CD209 revealed that CD209L and CD209 interact with S-RBD. CD209L contains two N-glycosylation sequons, at sites N92 and N361, but we determined that only site N92 is occupied. Removal of the N-glycosylation at this site enhances the binding of S-RBD with CD209L. CD209L also interacts with ACE2, suggesting a role for heterodimerization of CD209L and ACE2 in SARS-CoV-2 entry and infection in cell types where both are present. Furthermore, we demonstrate that human endothelial cells are permissive to SARS-CoV-2 infection and interference with CD209L activity by knockdown strategy or with soluble CD209L inhibits virus entry. Our observations demonstrate that CD209L and CD209 serve as alternative receptors for SARS-CoV-2 in disease-relevant cell types, including the vascular system. This property is particularly important in tissues where ACE2 has low expression or is absent, and may have implications for antiviral drug development.

Abstract As the COVID-19 pandemic continues to spread, investigating the processes underlying the interactions between SARS-CoV-2 and its hosts is of high importance. Here, we report the identification of CD209L/L-SIGN and the related protein CD209/DC-SIGN as receptors capable of mediating SARS-CoV-2 entry into human cells. Immunofluorescence staining of human tissues revealed prominent expression of CD209L in the lung and kidney epithelium and endothelium. Multiple biochemical assays using a purified recombinant SARS-CoV-2 spike receptor binding domain (S-RBD) and ectopically expressed CD209L and CD209 revealed that CD209L and CD209 interact with S-RBD. CD209L contains two N-glycosylation sequons, at sites N92 and N361, but only site N92 is occupied. Removal of the N-glycosylation at this site enhances the binding of S-RBD with CD209L. CD209L also interacts with ACE2, suggesting a role for heterodimerization of CD209L and ACE2 in SARS-CoV-2 entry and infection in cell types where both are present. Furthermore, we demonstrate that human endothelial cells are permissive to SARS-CoV-2 infection and interference with CD209L activity by knockdown strategy or with soluble CD209L inhibits virus entry. Our observations demonstrate that CD209L and CD209 serve as alternative receptors for SARS-CoV-2 in disease-relevant cell types, including the vascular system. This property is particularly important in tissues where ACE2 has low expression or is absent, and may have implications for antiviral drug development. Significance Understanding the interactions between SARS-CoV-2 with host cells is of high importance. ACE2 is recognized as a major entry receptor, but SARS-CoV-2 may also employ alternative receptors for cell entry and these may hold the key to infection in tissues, where ACE2 has a low expression level or is absent. We identify CD209L/L-SIGN and CD209/DC-SIGN as receptors for SARS-CoV-2. We show that CD209L is N-glycosylated and this modification modulates the binding of CD209L with spike protein. CD209L interacts with ACE2, suggesting that CD209L and ACE2 could function as co-receptors for SARS-CoV-2 entry and infection. Human endothelial cells are permissive to SARS-CoV-2 infection. We show that interfering with CD209L activity in endothelial cells by knockdown or with introduction of soluble CD209L inhibits virus entry, suggesting a novel target for development of antiviral drugs. Competing Interest Statement The authors have declared no competing interest. Footnotes * This is a revised manuscript with more data and information.

is an ubiquitous apicomplexan parasite which causes toxoplasmosis in humans and animals. Felids especially cats are definitive hosts and almost all warm-blooded mammals, including livestock and human can serve as intermediate hosts. Food animals can be reservoirs for and act as one of the sources for parasite transmission to humans. The objective of this study is to collect serological data on the prevalence of anti- antibody, and risk factors for certain food animals from Africa to provide a quantitative estimate of infection among these species from different African countries. Four databases were used to search seroepidemiological data on the prevalence of anti- antibody in food animals between 1969 and 2016 from African countries. The search focused on data obtained by serologic test in food animals and meta-analyses were performed per species. A total of 30,742 individual samples from 24 countries, described in 68 articles were studied. The overall estimated prevalence for toxoplasmosis in chicken, camel, cattle, sheep, goat, pig were 37.4% (29.2-46.0%), 36% (18-56%), 12% (8-17%), 26.1% (17.0-37.0%), 22.9% (12.3-36.0%), and 26.0% (20-32.0%), respectively. Moreover, major risk factor of infection was age, farming system, and farm location. A significant variation in the seroepidemiological data was observed within each species and country. The results can aid in an updated epidemiological analysis but also can be used as an important input in quantitative microbial risk assessment models. Further studies are required for a better and continual evaluation of the occurrence of this zoonotic infection.

Chronic kidney disease (CKD) imposes a strong and independent risk for peripheral artery disease (PAD). While solutes retained in CKD patients (uremic solutes) inflict vascular damage, their role in PAD remains elusive. Here, we show that the dietary tryptophan-derived uremic solutes including indoxyl sulfate (IS) and kynurenine (Kyn) at concentrations corresponding to those in CKD patients suppress β-catenin in several cell types, including microvascular endothelial cells (ECs), inhibiting Wnt activity and proangiogenic Wnt targets in ECs. Mechanistic probing revealed that these uremic solutes downregulated β-catenin in a manner dependent on serine 33 in its degron motif and through the aryl hydrocarbon receptor (AHR). Hindlimb ischemia in adenine-induced CKD and IS solute-specific mouse models showed diminished β-catenin and VEGF-A in the capillaries and reduced capillary density, which correlated inversely with blood levels of IS and Kyn and AHR activity in ECs. An AHR inhibitor treatment normalized postischemic angiogenic response in CKD mice to a non-CKD level. In a prospective cohort of PAD patients, plasma levels of tryptophan metabolites and plasma's AHR-inducing activity in ECs significantly increased the risk of future adverse limb events. This work uncovers the tryptophan metabolite/AHR/β-catenin axis as a mediator of microvascular rarefaction in CKD patients and demonstrates its targetability for PAD in CKD models.

Background and aims The differential diagnosis of solitary pancreatic cystic lesions is sometimes difficult. Needle-based confocal laser endomicroscopy (nCLE) performed during endoscopic ultrasound–fine-needle aspiration (EUS-FNA) enables real-time imaging of the internal structure of such cysts. Criteria have already been described for serous cystadenoma and intraductal papillary mucinous neoplasm (IPMN). The aims of the study were to determine new nCLE criteria for the diagnosis of pancreatic cystic lesions, to propose a comprehensive nCLE classification for the characterization of those lesions, and to carry out a first external retrospective validation . Methods Thirty-three patients with a lone pancreatic cystic lesion were included (CONTACT 1 study). EUS-FNA was combined with nCLE. Diagnosis was based on either pathology result (Group 1, n  = 20) or an adjudication committee consensus (Group 2, n  = 13). Six investigators, unblinded, studied cases from Group 1 and identified nCLE criteria for mucinous cystic neoplasm (MCN), pseudocyst (PC), and cystic neuroendocrine neoplasm (NEN). Four external reviewers assessed, blinded, the yield and interobserver agreement for the newly identified (MCN, PC) and previously described (IPMN, SC) criteria in a subset of 31 cases. Results New nCLE criteria were described for MCN (thick gray line), PC (field of bright particles), and cystic NEN (black neoplastic cells clusters with white fibrous areas). These criteria correlated with the histological features of the corresponding lesions. In the retrospective validation, a conclusive nCLE result was obtained for 74 % of the cases (87 % “true” and 13 % “false” with respect to the final diagnosis). On this limited case series, the nCLE criteria showed a trend for high diagnostic specificity (>90 % for mucinous cysts, 100 % for non-mucinous cysts). Conclusions Based on this newly completed atlas of interpretation criteria, nCLE could facilitate the diagnosis of pancreatic cystic lesion types.

Cleidocranial dysplasia (CCD) is an autosomal dominant human disorder characterized by abnormal bone development that is mainly due to defective intramembranous bone formation by osteoblasts. Here, we describe a mouse strain lacking the E3 ubiquitin ligase RNF146 that shows phenotypic similarities to CCD. Loss of RNF146 stabilized its substrate AXIN1, leading to impairment of WNT3a-induced β-catenin activation and reduced Fgf18 expression in osteoblasts. We show that FGF18 induces transcriptional coactivator with PDZ-binding motif (TAZ) expression, which is required for osteoblast proliferation and differentiation through transcriptional enhancer associate domain (TEAD) and runt-related transcription factor 2 (RUNX2) transcription factors, respectively. Finally, we demonstrate that adipogenesis is enhanced in Rnf146-/- mouse embryonic fibroblasts. Moreover, mice with loss of RNF146 within the osteoblast lineage had increased fat stores and were glucose intolerant with severe osteopenia because of defective osteoblastogenesis and subsequent impaired osteocalcin production. These findings indicate that RNF146 is required to coordinate β-catenin signaling within the osteoblast lineage during embryonic and postnatal bone development.

Casitas B lymphoma (c-Cbl) is an E3 ubiquitin ligase and a negative regulator of colorectal cancer (CRC). Despite its high expression in immune cells, the effect of c-Cbl on the tumor microenvironment remains poorly understood. Here we demonstrate that c-Cbl alters the tumor microenvironment and suppresses Programmed cell death-1 (PD-1) protein, an immune checkpoint receptor. Using syngeneic CRC xenografts, we observed significantly higher growth of xenografts and infiltrating immune cells in c-Cbl +/− compared to c-Cbl +/+ mice. Tumor-associated CD8+ T-lymphocytes and macrophages of c-Cbl +/− mice showed 2–3-fold higher levels of PD-1. Functionally, macrophages from c-Cbl +/− mice showed a 4–5-fold reduction in tumor phagocytosis, which was restored with an anti-PD-1 neutralizing antibody suggesting regulation of PD-1 by c-Cbl. Further mechanistic probing revealed that C-terminus of c-Cbl interacted with the cytoplasmic tail of PD-1. c-Cbl destabilized PD-1 through ubiquitination- proteasomal degradation depending on c-Cbl’s RING finger function. This data demonstrates c-Cbl as an E3 ligase of PD-1 and a regulator of tumor microenvironment, both of which were unrecognized components of its tumor suppressive activity. Advancing immune checkpoint and c-Cbl biology, our study prompts for probing of PD-1 regulation by c-Cbl in conditions driven by immune checkpoint abnormalities such as cancers and autoimmune diseases.

ObjectiveAmpullary neoplastic lesions can be resected by endoscopic papillectomy (EP) or transduodenal surgical ampullectomy (TSA) while pancreaticoduodenectomy is reserved for more advanced lesions. We present the largest retrospective comparative study analysing EP and TSA.DesignOf all patients in the database, lesions with prior interventions, benign histology advanced malignancy (T2 and more), patients with hereditary syndromes and those undergoing pancreatoduodenectomy were excluded. All remaining cases as well as a subgroup of them, after propensity-score matching (nearest-neighbour-method) based on age, gender, anthropometrics, comorbidities, size and histological subtype, were analysed. The median follow-up was 21 months (IQR 10–47) after the primary intervention. Primary outcomes were rates of complete resection (R0) and complications. Groups were compared by Fisher’s exact or χ2 test, Mann-Whitney-U-test and log-rank test for survival.ResultsOf 1673 patients in the database, 1422 underwent EP and 251 TSA. Of them, 23.2% were excluded for missing or inconclusive data and 19.8% of patients for prior interventions or hereditary syndromes. Final histology showed in 24.2% of EP and 14.8% of TSA patients a histology other than adenoma or adenocarcinoma while advanced cancers were recorded in 10.9% of EP and 36.6% of TSA patients. Finally, 569 EP and 63 TSA were included in the overall analysis, with a higher rate of more advanced cases and higher R0 resection rates in the TSA groups (90.5% vs 73.1%; p<0.01), with additional ablation in the EP group in 14.4%. Severe adverse event rates were 3.2% (TSA) vs 1.9% (EP). Recurrence after histological R0 resection was 16% (EP) vs 3.2% (TSA; p=0.01), and additional therapy for R1 resection was applied in 67% of the 159 cases. Propensity-score-based matching identified 62 pairs of EP/TSA patients with comparable baseline patient and lesion characteristics. The initial R0-rate was 72.6% (EP) compared with 90.3% (TSA, p=0.02) with recurrences found in 8% (EP) vs 3.2% (TSA; p=0.07); reinterventions were more frequent in the EP group. Overall survival was comparable.ConclusionsThe rate of patients with poor indications due to non-neoplastic disease or advanced cancer is still high for both EP and TSA; multiple retreatments were necessary for EP. Although EP can be considered an appropriate primary therapy for certain ampullary adenomas, case selection for both therapies (especially with regard to the best step-up approach) should be studied further.

Toxoplasma gondii is a parasitic protozoan, the etiological agent of toxoplasmosis, a worldwide zoonosis responsible for abortion and congenital malformation in animal and human. The present study reports, for the first time, the occurrence of T. gondii infection among sheep and goats from Benin. A total of 368 small ruminants: 215 serum samples from sheep raised in Sahelian area of North Benin and 153 serum samples from goats raised in a family farm from South-Benin, were collected and screened for anti- T. gondii IgG antibodies by the ELISA-indirect method. The results show the presence of anti- T. gondii IgG in 53% (83/153) of goats and 1.4% of sheep (3/215). Age, sex and breed did not seem to affect the frequency of this infection. Among goats, T. gondii infection was higher in animals reared in the coastal zone (Cotonou municipality) than those raised on the island (Allada municipality) [odds ratio (OR) = 4, 95% confidence interval (CI) = 1.07–15.002, p  = 0.032, (χ 2 ) test]. Humidity would be the determining factor in the disparity of recorded infection rates among sheep and goat. The high prevalence of caprine toxoplasmosis observed in southern Benin shows strong environmental contamination. Sensitization campaigns should therefore be undertaken by the public health authorities to inform the inhabitants of this area about risks and preventive measures of this zoonose.