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Upon infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), patients with critical coronavirus disease 2019 (COVID-19) present with life-threatening respiratory distress, pulmonary damage, and cytokine storm. One unexplored component in COVID-19 is the neuropeptide calcitonin gene-related peptide (CGRP), which is highly abundant in the airways and could converge in multiple aspects of COVID-19-related pulmonary pathophysiology. Whether CGRP affects SARS-CoV-2 infection directly remains elusive. We show that in critical COVID-19 patients, CGRP is increased in both plasma and lungs. Importantly, CGRP pulmonary levels are elevated in early SARS-CoV-2-positive patients and restored to baseline upon subsequent viral clearance in SARS-CoV-2-negative patients. We further show that CGRP and its stable analog SAX directly inhibit infection of bronchial Calu-3 epithelial cells with SARS-CoV-2 Omicron and Alpha variants in a dose-dependent manner. Both pre- and post-infection treatments with CGRP and/or SAX are enough to block SARS-CoV-2 productive infection of Calu-3 cells. CGRP-mediated inhibition occurs via activation of the CGRP receptor and involves down-regulation of both SARS-CoV-2 entry receptors at the surface of Calu-3 cells. Together, we propose that increased pulmonary CGRP mediates beneficial viral clearance in critical COVID-19 patients by directly inhibiting SARS-CoV-2 propagation. Hence, CGRP-based interventions could be harnessed for management of COVID-19.IMPORTANCEThe neuropeptide CGRP is highly abundant in the airways. Due to its immunomodulatory, vasodilatory, and anti-viral functions, CGRP could affect multiple aspects of COVID-19-related pulmonary pathophysiology. Yet, the interplay between CGRP and SARS-CoV-2 during COVID-19 remains elusive. Herein, we show that pulmonary levels of CGRP are increased in critical COVID-19 patients, at an early stage of their disease when patients are SARS-CoV-2-positive. Upon subsequent viral clearance, CGRP levels are restored to baseline in SARS-CoV-2-negative patients. We further show that pre- and post-infection treatments with CGRP directly inhibit infection of Calu-3 bronchial epithelial cells with SARS -CoV-2, via activation of the CGRP receptor leading to decreased expression of both SARS-CoV-2 entry receptors. Together, we propose that increased pulmonary CGRP is beneficial in COVID-19, as CGRP-mediated inhibition of SARS-CoV-2 infection could contribute to viral clearance in critical COVID-19 patients. Accordingly, CGRP-based formulations could be useful for COVID-19 management.
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Although the Hepatitis E virus (HEV) is an emerging global health burden, little is known about its interaction with the host cell. HEV genome encodes three proteins including the ORF2 capsid protein that is produced in different forms, the ORF2i protein which is the structural component of viral particles, and the ORF2g/c proteins which are massively secreted but are not associated with infectious material. We recently demonstrated that the endocytic recycling compartment (ERC) is hijacked by HEV to serve as a viral factory. However, host determinants involved in the subcellular shuttling of viral proteins to viral factories are unknown. Here, we demonstrate that the AP-1 adaptor complex plays a pivotal role in the targeting of ORF2i protein to viral factories. This complex belongs to the family of adaptor proteins that are involved in vesicular transport between the trans-Golgi network and early/recycling endosomes. An interplay between the AP-1 complex and viral protein(s) has been described for several viral lifecycles. In the present study, we demonstrated that the ORF2i protein colocalizes and interacts with the AP-1 adaptor complex in HEV-producing or infected cells. We showed that silencing or drug-inhibition of the AP-1 complex prevents ORF2i protein localization in viral factories and reduces viral production in hepatocytes. Modeling of the ORF2i/AP-1 complex also revealed that the S domain of ORF2i likely interacts with the σ1 subunit of AP-1 complex. Hence, our study identified for the first time a host factor involved in addressing HEV proteins (i.e. ORF2i protein) to viral factories.
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Complejo 1 de Proteína Adaptadora , Proteínas de la Cápside , Virus de la Hepatitis E , Virus de la Hepatitis E/metabolismo , Virus de la Hepatitis E/fisiología , Virus de la Hepatitis E/genética , Humanos , Complejo 1 de Proteína Adaptadora/metabolismo , Complejo 1 de Proteína Adaptadora/genética , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/genética , Transporte de Proteínas , Proteínas Virales/metabolismo , Proteínas Virales/genética , Ensamble de Virus , Hepatitis E/metabolismo , Hepatitis E/virologíaRESUMEN
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.
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Virus de la Hepatitis E , Hepatitis E , Secuencias de Aminoácidos , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Femenino , Glicosilación , Hepatitis E/genética , Hepatitis E/metabolismo , Virus de la Hepatitis E/crecimiento & desarrollo , Humanos , EmbarazoRESUMEN
The binding of the SARS-CoV-2 spike to angiotensin-converting enzyme 2 (ACE2) promotes virus entry into the cell. Targeting this interaction represents a promising strategy to generate antivirals. By screening a phage-display library of biosynthetic protein sequences build on a rigid alpha-helicoidal HEAT-like scaffold (named αReps), we selected candidates recognizing the spike receptor binding domain (RBD). Two of them (F9 and C2) bind the RBD with affinities in the nM range, displaying neutralisation activity in vitro and recognizing distinct sites, F9 overlapping the ACE2 binding motif. The F9-C2 fusion protein and a trivalent αRep form (C2-foldon) display 0.1 nM affinities and EC50 of 8-18 nM for neutralization of SARS-CoV-2. In hamsters, F9-C2 instillation in the nasal cavity before or during infections effectively reduced the replication of a SARS-CoV-2 strain harbouring the D614G mutation in the nasal epithelium. Furthermore, F9-C2 and/or C2-foldon effectively neutralized SARS-CoV-2 variants (including delta and omicron variants) with EC50 values ranging from 13 to 32 nM. With their high stability and their high potency against SARS-CoV-2 variants, αReps provide a promising tool for SARS-CoV-2 therapeutics to target the nasal cavity and mitigate virus dissemination in the proximal environment.
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Enzima Convertidora de Angiotensina 2 , Tratamiento Farmacológico de COVID-19 , Proteínas Recombinantes de Fusión , Glicoproteína de la Espiga del Coronavirus , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/metabolismo , Antivirales/química , Antivirales/farmacología , Humanos , Peptidil-Dipeptidasa A/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/uso terapéutico , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Drug repurposing has the advantage of shortening regulatory preclinical development steps. Here, we screened a library of drug compounds, already registered in one or several geographical areas, to identify those exhibiting antiviral activity against SARS-CoV-2 with relevant potency. Of the 1,942 compounds tested, 21 exhibited a substantial antiviral activity in Vero-81 cells. Among them, clofoctol, an antibacterial drug used for the treatment of bacterial respiratory tract infections, was further investigated due to its favorable safety profile and pharmacokinetic properties. Notably, the peak concentration of clofoctol that can be achieved in human lungs is more than 20 times higher than its IC50 measured against SARS-CoV-2 in human pulmonary cells. This compound inhibits SARS-CoV-2 at a post-entry step. Lastly, therapeutic treatment of human ACE2 receptor transgenic mice decreased viral load, reduced inflammatory gene expression and lowered pulmonary pathology. Altogether, these data strongly support clofoctol as a therapeutic candidate for the treatment of COVID-19 patients.
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Tratamiento Farmacológico de COVID-19 , SARS-CoV-2 , Animales , Antivirales/farmacología , Clorobencenos , Chlorocebus aethiops , Cresoles , Humanos , Pulmón , Ratones , Células VeroRESUMEN
INTRODUCTION AND HYPOTHESIS: Lateral suspension is an abdominal prosthetic surgical procedure used to correct apical prolapse. The procedure involves the placement of a T-shaped mesh on the anterior vaginal wall and on the isthmus or uterine cervix that is suspended laterally and posteriorly to the abdominal wall. Since its description in the late 90s, modifications of the technique have been described. So far, no consensus on the correct indications, safety, advantages, and disadvantages of this emerging procedure has been reached. METHODS: A modified Delphi process was used to build consensus within a group of 21 international surgeons who are experts in the performance of laparoscopic lateral suspension (LLS). The process was held with a first online round, where the experts expressed their level of agreement on 64 statements on indications, technical features, and other aspects of LLS. A subsequent re-discussion of statements where a threshold of agreement was not reached was held in presence. RESULTS: The Delphi process allowed the identification of several aspects of LLS that represented areas of agreement by the experts. The experts agreed that LLS is a safe and effective technique to correct apical and anterior prolapse. The experts highlighted several key technical aspects of the procedure, including clinical indications and surgical steps. CONCLUSIONS: This Delphi consensus provides valuable guidance and criteria for the use of LLS in the treatment of pelvic organ prolapse, based on expert opinion by large volume surgeons' experts in the performance of this innovative procedure.
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Técnica Delphi , Laparoscopía , Prolapso de Órgano Pélvico , Mallas Quirúrgicas , Humanos , Prolapso de Órgano Pélvico/cirugía , Femenino , Laparoscopía/métodos , Procedimientos Quirúrgicos Ginecológicos/métodos , ConsensoRESUMEN
The coronavirus' (CoV) membrane (M) protein is the driving force during assembly, but this process remains poorly characterized. Previously, we described two motifs in the C-tail of the Middle East respiratory syndrome CoV (MERS-CoV) M protein involved in its endoplasmic reticulum (ER) exit (211DxE213) and trans-Golgi network (TGN) retention (199KxGxYR204). Here, their function in virus assembly was investigated by two different virus-like particle (VLP) assays and by mutating both motifs in an infectious MERS-CoV cDNA clone. It was shown that the 199KxGxYR204 motif was essential for VLP and infectious virus assembly. Moreover, the mislocalization of the M protein induced by mutation of this motif prevented M-E interaction. Hampering the ER export of M by mutating its 211DxE213 motif still allowed the formation of nucleocapsid-empty VLPs, but prevented the formation of fully assembled VLPs and infectious particles. Taken together, these data show that the MERS-CoV assembly process highly depends on the correct intracellular trafficking of its M protein, and hence that not only specific protein-protein interacting motifs but also correct subcellular localization of the M protein in infected cells is essential for virus formation and should be taken into consideration when studying the assembly process.
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Proteínas de la Membrana , Coronavirus del Síndrome Respiratorio de Oriente Medio , Proteínas de la Membrana/metabolismo , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , Coronavirus del Síndrome Respiratorio de Oriente Medio/metabolismo , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/metabolismo , Ensamble de Virus/genéticaRESUMEN
Coronaviruses are positive-stranded RNA enveloped viruses. The helical nucleocapsid is surrounded by a lipid bilayer in which are anchored three viral proteins: the spike (S), membrane (M) and envelope (E) proteins. The M protein is the major component of the viral envelope and is believed to be its building block. The M protein of Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contains a short N-terminal domain with an N-glycosylation site. We investigated their N-glycosylation and show that polylactosamine chains are conjugated to SARS-CoV-2 and MERS-CoV M proteins in transfected and infected cells. Acidic residues present in the first transmembrane segments of the proteins are required for their glycosylation. No specific signal to specify polylactosamine conjugation could be identified and high mannose-conjugated protein was incorporated into virus-like particles.
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COVID-19 , Coronavirus del Síndrome Respiratorio de Oriente Medio , Humanos , Coronavirus del Síndrome Respiratorio de Oriente Medio/genética , SARS-CoV-2/metabolismo , Proteínas de la Matriz Viral/genética , Proteínas de la Membrana , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
SARS-CoV-2 spike (S) envelope glycoprotein constitutes the main determinant of virus entry and the target of host immune response, thus being of great interest for antiviral research. It is constituted of S1 and S2 subunits, which are involved in ACE2 receptor binding and fusion between the viral envelope and host cell membrane, respectively. Induction of the fusion process requires S cleavage at the S1-S2 junction and the S2' site located upstream of the fusion peptide. Interestingly, the SARS-CoV-2 spike harbors a 4-residue insertion at the S1-S2 junction that is absent in its closest relatives and constitutes a polybasic motif recognized by furin-like proteases. In addition, the S2' site is characterized by the presence of conserved basic residues. Here, we sought to determine the importance of the furin cleavage site (FCS) and the S2' basic residues for S-mediated entry functions. We determined the impact of mutations introduced at these sites on S processing, fusogenic activity, and its ability to mediate entry in different cellular backgrounds. Strikingly, mutation phenotypes were highly dependent on the host cell background. We confirmed that although the FCS was not absolutely required for virus entry, it contributed to extending the fusogenic potential of S. Cleavage site mutations, as well as inhibition of furin protease activity, affected the cell surface expression of S in a host cell-dependent manner. Finally, inhibition of furin activity differentially affected SARS-CoV-2 virus infectivity in the tested host cells, thereby confirming the host cell-dependent effect of spike processing for the viral life cycle. IMPORTANCE SARS-CoV-2 is responsible for the current global pandemic that has resulted in several million deaths. As the key determinant of virus entry into host cells and the main target of host immune response, the spike glycoprotein constitutes an attractive target for therapeutics development. Entry functions of spike rely on its processing at two sites by host cell proteases. While SARS-CoV-2 spike differs from its closest relatives by the insertion of a basic furin cleavage motif at the first site, it harbors conserved basic residues at the second cleavage site. Characterization of the importance of the basic sequences present at the two cleavage sites revealed that they were influencing spike processing, intracellular localization, induction of fusion, and entry in a host cell-dependent manner. Thus, our results revealed a high heterogeneity in spike sequence requirement for entry functions in the different host cells, in agreement with the high adaptability of the SARS-CoV-2 virus.
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COVID-19 , Furina , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Internalización del Virus , COVID-19/virología , Furina/metabolismo , Humanos , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Dispersals have been shown to be critical to the evolution of the long isolated but megadiverse flora of Madagascar and the surrounding islands of the western Indian Ocean, but we are just beginning to understand the directionality of these dispersals. With more than half of its species occurring in the western Indian Ocean region (WIOR), the paleotropical subfamily Dombeyoideae provides a particularly useful case study through which to better understand the biogeography of the WIOR, and yet its biogeography is poorly understood. Here we sampled six molecular markers from all 20 genera in the Dombeyoideae to reconstruct the most complete phylogeny to date for the subfamily. From this, divergence times, calibrated with three fossils (two dombeyoid, one malvoid), and ancestral range estimations were hypothesized. Biogeographic stochastic mapping (BSM) analyses on the maximum clade credibility tree were completed and compared to BSM analyses on 1,000 trees randomly sampled from the posterior distribution of trees resulting from the dating analysis. We found the Dombeyoideae crown node diverged ca. 53 million years ago out of a broad ancestral range involving all three major areas of its distribution: Madagascar, Africa, and Asia. The majority of diversification and dispersals in the subfamily occurred within the last ca. 10 million years, mostly from the Pliocene onwards. There were roughly five dispersals from Madagascar to Africa (and only one in reverse), at least six from Madagascar to surrounding islands of the WIOR (Mascarenes and Comoros), and one dispersal from Madagascar to Asia (and ca. 1 in reverse). Other long-distance dispersals included one from Africa to St. Helena and one from Africa to Australasia, both from within the most widespread clade, the Cheirolaena & allies clade, and one dispersal from Asia to Africa. Critically, the Dombeyoideae provide strong evidence for considering the island of Madagascar as a source for the colonization of continents, as well as the surrounding islands of the WIOR. Furthermore, narrow sympatry was a key process in the evolution of the subfamily, particularly in Madagascar and the Mascarenes.
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Malvaceae , Filogenia , Madagascar , Filogeografía , ÁfricaRESUMEN
BACKGROUND AND AIMS: Epiphytism has evolved repeatedly in plants and has resulted in a considerable number of species with original characteristics. Because water supply is generally erratic compared to that in soils, succulent forms in particular are widespread in epiphytic species. However, succulent organs also exist in terrestrial plants, and the question of the concomitant evolution of epiphytism and succulence has received little attention, not even in the epidendroid orchids, which account for 67.6 % of vascular epiphytes. METHODS: We built a new time-calibrated phylogenetic tree of Epidendroideae with 203 genera treated in genus Orchidacearum, from which we reconstructed the evolution of epiphytism as well as traits related to water scarcity (stem and leaf succulence and the number of velamen layers), while testing for the correlated evolution between the two. Furthermore, we estimated the ancestral geographical ranges to evaluate the palaeoclimatic context in which epiphytism evolved. KEY RESULTS: Epiphytism evolved at least three times: 39.0 million years ago (Mya) in the common ancestor of the Malaxideae and Cymbidieae that probably ranged from the Neotropics to Southeast Asia and Australia, 11.5 Mya in the Arethuseae in Southeast Asia and Australia, and 7.1 Mya in the neotropical Sobralieae, and it was notably lost in the Malaxidiinae, Collabieae, Calypsoeae, Bletiinae and Eulophiinae. Stem succulence is inferred to have evolved once, in a terrestrial ancestor at least 4.1 Mya before the emergence of epiphytic lineages. If lost, stem succulence was almost systematically replaced by leaf succulence in epiphytic lineages. CONCLUSIONS: Epiphytism may have evolved in seasonally dry forests during the Eocene climatic cooling, among stem-succulent terrestrial orchids. Our results suggest that the emergence of stem succulence in early epidendroids was a key innovation in the evolution of epiphytism, facilitating the colonization of epiphytic environments that later led to the greatest diversification of epiphytic orchids.
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Orchidaceae , Suelo , Filogenia , Fenotipo , BosquesRESUMEN
BACKGROUND: Robotic surgery has gained popularity for the reconstruction of pelvic floor defects. Nonetheless, there is no evidence that robot-assisted reconstructive surgery is either appropriate or superior to standard laparoscopy for the performance of pelvic floor reconstructive procedures or that it is sustainable. The aim of this project was to address the proper role of robotic pelvic floor reconstructive procedures using expert opinion. METHODS: We set up an international, multidisciplinary group of 26 experts to participate in a Delphi process on robotics as applied to pelvic floor reconstructive surgery. The group comprised urogynecologists, urologists, and colorectal surgeons with long-term experience in the performance of pelvic floor reconstructive procedures and with the use of the robot, who were identified primarily based on peer-reviewed publications. Two rounds of the Delphi process were conducted. The first included 63 statements pertaining to surgeons' characteristics, general questions, indications, surgical technique, and future-oriented questions. A second round including 20 statements was used to reassess those statements where borderline agreement was obtained during the first round. The final step consisted of a face-to-face meeting with all participants to present and discuss the results of the analysis. RESULTS: The 26 experts agreed that robotics is a suitable indication for pelvic floor reconstructive surgery because of the significant technical advantages that it confers relative to standard laparoscopy. Experts considered these advantages particularly important for the execution of complex reconstructive procedures, although the benefits can be found also during less challenging cases. The experts considered the robot safe and effective for pelvic floor reconstruction and generally thought that the additional costs are offset by the increased surgical efficacy. CONCLUSION: Robotics is a suitable choice for pelvic reconstruction, but this Delphi initiative calls for more research to objectively assess the specific settings where robotic surgery would provide the most benefit.
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Laparoscopía , Procedimientos Quirúrgicos Robotizados , Robótica , Cirugía Plástica , Humanos , Diafragma Pélvico/cirugía , Técnica Delphi , Procedimientos Quirúrgicos Robotizados/métodos , Laparoscopía/métodosRESUMEN
Although hepatitis E virus (HEV) is the major leading cause of enterically transmitted viral hepatitis worldwide, many gaps remain in the understanding of the HEV lifecycle. Notably, viral factories induced by HEV have not been documented yet, and it is currently unknown whether HEV infection leads to cellular membrane modeling as many positive-strand RNA viruses. HEV genome encodes the ORF1 replicase, the ORF2 capsid protein and the ORF3 protein involved in virion egress. Previously, we demonstrated that HEV produces different ORF2 isoforms including the virion-associated ORF2i form. Here, we generated monoclonal antibodies that specifically recognize the ORF2i form and antibodies that recognize the different ORF2 isoforms. One antibody, named P1H1 and targeting the ORF2i N-terminus, recognized delipidated HEV particles from cell culture and patient sera. Importantly, AlphaFold2 modeling demonstrated that the P1H1 epitope is exposed on HEV particles. Next, antibodies were used to probe viral factories in HEV-producing/infected cells. By confocal microscopy, we identified subcellular nugget-like structures enriched in ORF1, ORF2 and ORF3 proteins and viral RNA. Electron microscopy analyses revealed an unprecedented HEV-induced membrane network containing tubular and vesicular structures. We showed that these structures are dependent on ORF2i capsid protein assembly and ORF3 expression. An extensive colocalization study of viral proteins with subcellular markers, and silencing experiments demonstrated that these structures are derived from the endocytic recycling compartment (ERC) for which Rab11 is a central player. Hence, HEV hijacks the ERC and forms a membrane network of vesicular and tubular structures that might be the hallmark of HEV infection.
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Virus de la Hepatitis E , Humanos , Virus de la Hepatitis E/genética , Compartimentos de Replicación Viral , Proteínas de la Cápside , Transporte Biológico , Anticuerpos MonoclonalesRESUMEN
SARS-CoV-2, although not being a circulatory virus, spread from the respiratory tract resulting in multiorgan failures and thrombotic complications, the hallmarks of fatal COVID-19. A convergent contributor could be platelets that beyond hemostatic functions can carry infectious viruses. Here, we profiled 52 patients with severe COVID-19 and demonstrated that circulating platelets of 19 out 20 non-survivor patients contain SARS-CoV-2 in robust correlation with fatal outcome. Platelets containing SARS-CoV-2 might originate from bone marrow and lung megakaryocytes (MKs), the platelet precursors, which were found infected by SARS-CoV-2 in COVID-19 autopsies. Accordingly, MKs undergoing shortened differentiation and expressing anti-viral IFITM1 and IFITM3 RNA as a sign of viral sensing were enriched in the circulation of deadly COVID-19. Infected MKs reach the lung concomitant with a specific MK-related cytokine storm rich in VEGF, PDGF and inflammatory molecules, anticipating fatal outcome. Lung macrophages capture SARS-CoV-2-containing platelets in vivo. The virus contained by platelets is infectious as capture of platelets carrying SARS-CoV-2 propagates infection to macrophages in vitro, in a process blocked by an anti-GPIIbIIIa drug. Altogether, platelets containing infectious SARS-CoV-2 alter COVID-19 pathogenesis and provide a powerful fatality marker. Clinical targeting of platelets might prevent viral spread, thrombus formation and exacerbated inflammation at once and increase survival in COVID-19.
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COVID-19 , Trombosis , Plaquetas , Humanos , Pulmón , Megacariocitos , Proteínas de la Membrana , Proteínas de Unión al ARN , SARS-CoV-2RESUMEN
The limited availability of antiviral therapy for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spurred the search for novel antiviral drugs. Here, we investigated the potential antiviral properties of plants adapted to high-salt environments collected in the north of France. Twenty-five crude methanolic extracts obtained from twenty-two plant species were evaluated for their cytotoxicity and antiviral effectiveness against coronaviruses HCoV-229E and SARS-CoV-2. Then, a bioguided fractionation approach was employed. The most active crude methanolic extracts were partitioned into three different sub-extracts. Notably, the dichloromethane sub-extract of the whole plant Hippophae rhamnoides L. demonstrated the highest antiviral activity against both viruses. Its chemical composition was evaluated by ultra-high performance liquid chromatography (UHPLC) coupled with mass spectrometry (MS) and then it was fractionated by centrifugal partition chromatography (CPC). Six cinnamoyl triterpenoid compounds were isolated from the three most active fractions by preparative high-performance liquid chromatography (HPLC) and identified by high resolution MS (HR-MS) and mono- and bi-dimensional nuclear magnetic resonance (NMR). Specifically, these compounds were identified as 2-O-trans-p-coumaroyl-maslinic acid, 3ß-hydroxy-2α-trans-p-coumaryloxy-urs-12-en-28-oic acid, 3ß-hydroxy-2α-cis-p-coumaryloxy-urs-12-en-28-oic acid, 3-O-trans-caffeoyl oleanolic acid, a mixture of 3-O-trans-caffeoyl oleanolic acid/3-O-cis-caffeoyl oleanolic acid (70/30), and 3-O-trans-p-coumaroyl oleanolic acid. Infection tests demonstrated a dose-dependent inhibition of these triterpenes against HCoV-229E and SARS-CoV-2. Notably, cinnamoyl oleanolic acids displayed activity against both SARS-CoV-2 and HCoV-229E. Our findings suggest that Hippophae rhamnoides could represent a source of potential antiviral agents against coronaviruses.
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Coronavirus Humano 229E , Hippophae , Ácido Oleanólico , Triterpenos , Triterpenos/química , Hippophae/química , Plantas Tolerantes a la Sal , Mar del Norte , SARS-CoV-2 , Antivirales/farmacología , Antivirales/análisisRESUMEN
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak has highlighted the need for broad-spectrum antivirals against coronaviruses (CoVs). Here, pheophorbide a (Pba) was identified as a highly active antiviral molecule against human CoV-229E after bioguided fractionation of plant extracts. The antiviral activity of Pba was subsequently shown for SARS-CoV-2 and Middle East respiratory syndrome coronavirus (MERS-CoV), and its mechanism of action was further assessed, showing that Pba is an inhibitor of coronavirus entry by directly targeting the viral particle. Interestingly, the antiviral activity of Pba depends on light exposure, and Pba was shown to inhibit virus-cell fusion by stiffening the viral membrane, as demonstrated by cryoelectron microscopy. Moreover, Pba was shown to be broadly active against several other enveloped viruses and reduced SARS-CoV-2 and MERS-CoV replication in primary human bronchial epithelial cells. Pba is the first described natural antiviral against SARS-CoV-2 with direct photosensitive virucidal activity that holds potential for COVID-19 therapy or disinfection of SARS-CoV-2-contaminated surfaces.
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Productos Biológicos , COVID-19 , Coronavirus del Síndrome Respiratorio de Oriente Medio , Antivirales/farmacología , Productos Biológicos/farmacología , Microscopía por Crioelectrón , Humanos , SARS-CoV-2RESUMEN
One of the challenges of all synchrotron facilities is to offer the highest performance detectors for all their specific experiments, in particular for X-ray diffraction imaging and its high throughput data collection. In that context, the DiffAbs beamline, the Detectors and the Design and Engineering groups at Synchrotron SOLEIL, in collaboration with ImXPAD and Cegitek companies, have developed an original and unique detector with a circular shape. This detector is based on the hybrid pixel photon-counting technology and consists of the specific assembly of 20 hybrid pixel array detector (XPAD) modules. This article aims to demonstrate the main characteristics of the CirPAD (for Circular Pixel Array Detector) and its performance - i.e. excellent pixel quality, flat-field correction, high-count-rate performance, etc. Additionally, the powder X-ray diffraction pattern of an LaB6 reference sample is presented and refined. The obtained results demonstrate the high quality of the data recorded from the CirPAD, which allows the proposal of its use to all scientific communities interested in performing experiments at the DiffAbs beamline.
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INTRODUCTION AND HYPOTHESIS: To assess the long-term satisfaction, cure rate and safety of a new titanium-covered transobturator tape compared to polypropylene tape for the treatment of stress urinary incontinence (SUI). METHODS: A prospective study was conducted with 151 patients. Seventy patients underwent transobturator sling surgery with titanium tape from 2011 to 2019, and a historical control group (CG) of 81 patients was treated with a noncoated tape and underwent incontinence surgery from 1999 to 2009. We compared patient-reported outcome measures (PROMs) with the incontinence outcome questionnaire (IOQ). RESULTS: The median follow-up was 2½ years in both groups. Based on responses to the IOQ, a statistically significantly shorter time of recovery (IOQ 15: 21.3 ± 26.4 [TG], 40.2 ± 38.5 [CG], p = 0.02), improvement of sex life (IOQ 13: 34.1 ± 29.4 [TG] vs. 65.3 ± 35.6 [CG], p = 0.01) and less voiding dysfunction (IOQ 19: 30.9 ± 28.1 [CG], 9.3 ± 18.6 [TG], p = 0.01) were observed in the TG. Objectively, no postoperative urinary retention was observed in the TG, but four cases were described in the CG. Ten patients needed a reoperation for SUI in the CG compared to three in the TG (p = 0.03). CONCLUSION: The titanium-covered transobturator sling had superior recovery time, improved sexual function and reduced reoperation rate compared to a historical polypropylene group.
Asunto(s)
Cabestrillo Suburetral , Incontinencia Urinaria de Esfuerzo , Incontinencia Urinaria , Femenino , Humanos , Masculino , Polipropilenos , Estudios Prospectivos , Titanio , Resultado del Tratamiento , Incontinencia Urinaria/cirugía , Incontinencia Urinaria de Esfuerzo/cirugía , Procedimientos Quirúrgicos UrológicosRESUMEN
Many studies on SARS-CoV-2 have been performed over short-time scale, but few have focused on the ultrastructural characteristics of infected cells. We used TEM to perform kinetic analysis of the ultrastructure of SARS-CoV-2-infected cells. Early infection events were characterized by the presence of clusters of single-membrane vesicles and stacks of membrane containing nuclear pores called annulate lamellae (AL). A large network of host cell-derived organelles transformed into virus factories was subsequently observed in the cells. As previously described for other RNA viruses, these replication factories consisted of double-membrane vesicles (DMVs) located close to the nucleus. Viruses released at the cell surface by exocytosis harbored the typical crown of spike proteins, but viral particles without spikes were also observed in intracellular compartments, possibly reflecting incorrect assembly or a cell degradation process.
Asunto(s)
SARS-CoV-2/crecimiento & desarrollo , Compartimentos de Replicación Viral/ultraestructura , Liberación del Virus/fisiología , Replicación Viral/fisiología , Animales , COVID-19/patología , Línea Celular , Chlorocebus aethiops , Microscopía Electrónica de Transmisión , Glicoproteína de la Espiga del Coronavirus/metabolismo , Células Vero , Compartimentos de Replicación Viral/fisiologíaRESUMEN
STUDY OBJECTIVE: To demonstrate the feasibility of transvaginal natural orifice transluminal endoscopic surgery (vNOTES) meshless anterior repair for the treatment of pelvic organ prolapse (POP). DESIGN: Stepwise demonstration of the technique with narrated video footage. SETTING: The vNOTES approach is a recent and rapidly developing technique that allows safe endoscopic transvaginal treatment of benign uterine pathologies [1]. Its use in the treatment of POP is still in its early stages; however, the first results are encouraging in terms of anatomy, functionality, and safety [2]. INTERVENTIONS: We describe the vNOTES technique of meshless anterior POP repair using a vaginal plastron [3]. The "vaginal plastron" technique uses an autologous vaginal strip that is left attached to the bladder and suspended from the arcus tendineus. After the delimitation of a 6 cm square vaginal strip, a lateral dissection is performed on each side between the bladder and the vaginal fascia to enter the paravesical space. The vaginal strip is left attached to the bladder wall and will later be fixed laterally to the arcus tendineus with 6 nonabsorbable monofilament sutures, caliber 0 (3 on each side of the plastron). The fixation points on the arcus tendineus concern the internal obturator fascia ventrally and the iliococcygeus fascia dorsally. The sutures are placed under endoscopic view using a transvaginal access platform (GelPOINT V-Path, Applied Medical, Rancho Santa Margarita, CA). After deflation and removal of the platform, the sutures are attached to the vaginal strip. Once the vaginal plastron is secured, the anterior vaginal wall is closed. CONCLUSION: The vNOTES approach offers an endoscopic anatomic view of the paravesical space, thus reducing any blind surgical procedure. It provides an alternative route in the performance of meshless anterior POP repair.