The IMEx coronavirus interactome: an evolving map of Coronaviridae-host molecular interactions.

The current coronavirus disease of 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus (SARS-CoV)-2, has spurred a wave of research of nearly unprecedented scale. Among the different strategies that are being used to understand the disease and develop effective treatments, the study of physical molecular interactions can provide fine-grained resolution of the mechanisms behind the virus biology and the human organism response. We present a curated dataset of physical molecular interactions focused on proteins from SARS-CoV-2, SARS-CoV-1 and other members of the Coronaviridae family that has been manually extracted by International Molecular Exchange (IMEx) Consortium curators. Currently, the dataset comprises over 4400 binarized interactions extracted from 151 publications. The dataset can be accessed in the standard formats recommended by the Proteomics Standards Initiative (HUPO-PSI) at the IntAct database website (https://www.ebi.ac.uk/intact) and will be continuously updated as research on COVID-19 progresses.

The IMEx Coronavirus interactome: an evolving map of Coronaviridae-Host molecular interactions.

The current Coronavirus Disease 2019 (COVID-19) pandemic, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has spurred a wave of research of nearly unprecedented scale. Among the different strategies that are being used to understand the disease and develop effective treatments, the study of physical molecular interactions enables studying fine-grained resolution of the mechanisms behind the virus biology and the human organism response. Here we present a curated dataset of physical molecular interactions, manually extracted by IMEx Consortium curators focused on proteins from SARS-CoV-2, SARS-CoV-1 and other members of the Coronaviridae family. Currently, the dataset comprises over 2,200 binarized interactions extracted from 86 publications. The dataset can be accessed in the standard formats recommended by the Proteomics Standards Initiative (HUPO-PSI) at the IntAct database website ( www.ebi.ac.uk/intact ), and will be continuously updated as research on COVID-19 progresses.

Encompassing new use cases - level 3.0 of the HUPO-PSI format for molecular interactions.

BACKGROUND: Systems biologists study interaction data to understand the behaviour of whole cell systems, and their environment, at a molecular level. In order to effectively achieve this goal, it is critical that researchers have high quality interaction datasets available to them, in a standard data format, and also a suite of tools with which to analyse such data and form experimentally testable hypotheses from them. The PSI-MI XML standard interchange format was initially published in 2004, and expanded in 2007 to enable the download and interchange of molecular interaction data. PSI-XML2.5 was designed to describe experimental data and to date has fulfilled this basic requirement. However, new use cases have arisen that the format cannot properly accommodate. These include data abstracted from more than one publication such as allosteric/cooperative interactions and protein complexes, dynamic interactions and the need to link kinetic and affinity data to specific mutational changes. RESULTS: The Molecular Interaction workgroup of the HUPO-PSI has extended the existing, well-used XML interchange format for molecular interaction data to meet new use cases and enable the capture of new data types, following extensive community consultation. PSI-MI XML3.0 expands the capabilities of the format beyond simple experimental data, with a concomitant update of the tool suite which serves this format. The format has been implemented by key data producers such as the International Molecular Exchange (IMEx) Consortium of protein interaction databases and the Complex Portal. CONCLUSIONS: PSI-MI XML3.0 has been developed by the data producers, data users, tool developers and database providers who constitute the PSI-MI workgroup. This group now actively supports PSI-MI XML2.5 as the main interchange format for experimental data, PSI-MI XML3.0 which additionally handles more complex data types, and the simpler, tab-delimited MITAB2.5, 2.6 and 2.7 for rapid parsing and download.

MatrixDB, the extracellular matrix interaction database: updated content, a new navigator and expanded functionalities.

MatrixDB (http://matrixdb.ibcp.fr) is a freely available database focused on interactions established by extracellular proteins and polysaccharides. It is an active member of the International Molecular Exchange (IMEx) consortium and has adopted the PSI-MI standards for annotating and exchanging interaction data, either at the MIMIx or IMEx level. MatrixDB content has been updated by curation and by importing extracellular interaction data from other IMEx databases. Other major changes include the creation of a new website and the development of a novel graphical navigator, iNavigator, to build and expand interaction networks. Filters may be applied to build sub-networks based on a list of biomolecules, a specified interaction detection method and/or an expression level by tissue, developmental stage, and health state (UniGene data). Any molecule of the network may be selected and its partners added to the network at any time. Networks may be exported under Cytoscape and tabular formats and as images, and may be saved for subsequent re-use.

Surface characterization and efficiency of a matrix-free and flat carboxylated gold sensor chip for surface plasmon resonance (SPR).

We report the preparation and characterization of a matrix-free carboxylated surface plasmon resonance (SPR) sensor chip with high sensing efficiency by functionalizing a bare gold thin film with a self-assembled monolayer of 16-mercaptohexadecanoic acid (SAM-MHDA chip). The self assembled monolayer surface coverage of the gold layer was carefully evaluated and the SAM was characterized by infrared reflection absorption spectroscopy, X-ray photoemission spectroscopy, atomic force microscopy, X-ray reflectivity-diffraction, and SPR experiments with bovine serum albumin. We compared the SPR signal obtained on this chip made of a dense monolayer of carboxylic acid groups with commercially available carboxylated sensor chips built on the same gold substrate, a matrix-free C1 chip, and a CM5 chip with a ~100 nm dextran hydrogel matrix (GE Healthcare). Two well-studied interaction types were tested, the binding of a biotinylated antibody (immunoglobulin G) to streptavidin and an antigen-antibody interaction. For both interactions, the well characterized densely functionalized SAM-MHDA chip gave a high signal-to-noise ratio and showed a gain in the availability of immobilized ligands for their partners injected in buffer flow. It thus compared favourably with commercially available sensor chips.

Interaction networks: from protein functions to drug discovery. A review.

Most genes, proteins and other components carry out their functions within a complex network of interactions and a single molecule can affect a wide range of other cell components. A global, integrative, approach has been developed for several years, including protein-protein interaction networks (interactomes). In this review, we describe the high-throughput methods used to identify new interactions and to build large interaction datasets. The minimum information required for reporting a molecular interaction experiment (MIMIx) has been defined as a standard for storing data in publicly available interaction databases. Several examples of interaction networks from molecular machines (proteasome) or organelles (phagosome, mitochondrion) to whole organisms (viruses, bacteria, yeast, fly, and worm) are given and attempts to cover the entire human interaction network are discussed. The methods used to perform the topological analysis of interaction networks and to extract biological information from them are presented. These investigations have provided clues on protein functions, signalling and metabolic pathways, and physiological processes, unraveled the molecular basis of some diseases (cancer, infectious diseases), and will be very useful to identify new therapeutic targets and for drug discovery. A major challenge is now to integrate data from different sources (interactome, transcriptome, phenome, localization) to switch from static to dynamic interaction networks. The merging of a viral interactome and the human interactome has been used to simulate viral infection, paving the way for future studies aiming at providing molecular basis of human diseases.

Characterization of osteoprotegerin binding to glycosaminoglycans by surface plasmon resonance: role in the interactions with receptor activator of nuclear factor kappaB ligand (RANKL) and RANK.

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of nuclear factor kappaB ligand (RANKL), a key inducer of osteoclastogenesis via its receptor RANK. We previously showed that RANK, RANKL, and OPG are able to form a tertiary complex and that OPG must be also considered as a direct effector of osteoclast functions. As OPG contains a heparin-binding domain, the present study investigated the interactions between OPG and glycosaminoglycans (GAGs) by surface plasmon resonance and their involvement in the OPG functions. Kinetic data demonstrated that OPG binds to heparin with a high-affinity (KD: 0.28 nM) and that the pre-incubation of OPG with heparin inhibits in a dose-dependent manner the OPG binding to the complex RANK-RANKL. GAGs from different structure/origin (heparan sulfate, dermatan sulfate, and chondroitin sulfate) exert similar activity on OPG binding. The contribution of the sulfation pattern and the size of the oligosaccharide were determined in this inhibitory mechanism. The results demonstrated that sulfation is essential in the OPG-blocking function of GAGs since a totally desulfated heparin loses its capacity to bind and to block OPG binding to RANKL. Moreover, a decasaccharide is the minimal structure that totally inhibits the OPG binding to the complex RANK-RANKL. Western blot analysis performed in 293 cells surexpressing RANKL revealed that the pre-incubation of OPG with these GAGs strongly inhibits the OPG-induced decrease of membrane RANKL half-life. These data support an essential function of the related glycosaminoglycans heparin and heparan sulfate in the activity of the triad RANK-RANKL-OPG.

Circulating fibrosis markers, eosinophil cationic protein and eosinophil protein X in patients with Wuchereria bancrofti infection: association with clinical status.

We measured the concentrations of several circulating fibrosis markers (type I collagen I, type III procollagen, hyaluronan) and eosinophil granule proteins (ECP and EPX) in lymphatic filariosis patients to investigate their relationship with clinical, parasitological and immunological data. This study was conducted in Polynesian patients with various stages of the disease (acute lymphangitis, chyluria, hydrocoele, elephantiasis), a closely related microbial lymphangitis and endemic controls. We observed modifications of the different markers in this pathology. Serum type I collagen and PIIINP were decreased. Serum hyaluronan, linked to perilymphatic granulomatous inflammation, was significantly increased in acute lymphangitis and elephantiasis patients. Serum ECP was also increased, at the limit of significance in our sample, in elephantiasis patients. These two last markers, already validated in another helminth disease, schistosomiasis, have potential interest in terms of follow-up of morbidity in these parasitic diseases.

Transglutaminase-mediated cross-linking is involved in the stabilization of extracellular matrix in human liver fibrosis.

BACKGROUND/AIMS: Lysyl oxidase-mediated cross-linking contributes to the stabilization of collagen in liver fibrosis. We have investigated transglutaminase-mediated cross-linking, to determine if it participates in the stabilization of extracellular matrix in human liver fibrosis. METHODS: Transglutaminase activity was assessed in vitro by incorporation of biotinylated amine into liver proteins. The product of the transglutaminase-catalyzed cross-linking reaction, Nepsilon(gamma-glutamyl)lysine, and the extracellular proteins cross-linked by it, were localized by immunohistochemistry in fibrotic livers. The cross-linked complexes were extracted from liver tissue, immunopurified and characterized by Western blot. RESULTS: Transglutaminase, detected by immunohistochemistry, Western blot and by enzymatic activity, was found in higher amounts in fibrotic than in normal liver. The Nepsilon(gamma-glutamyl)lysine cross-link, undetectable in normal liver, was present extracellularly in fibrotic liver, where it was co-distributed with osteonectin, mostly in inflammatory areas submitted to an intense remodeling. Cross-linking of osteonectin by transglutaminase was confirmed by Western blot. In parasitic fibrosis transglutaminase also originates from the parasite. CONCLUSIONS: Transglutaminase-mediated cross-linking occurs in liver extracellular matrix during the early, inflammatory, stage of liver fibrosis, whereas cross-linking by pyridinoline occurs mostly later in the fibrotic process. This could lead to the development of new anti-fibrotic treatments targeted to a specific stage of fibrosis.

Relationship between urinary pyridinium cross-links, disease activity and disease subsets of ankylosing spondylitis.

OBJECTIVE: In this study, we aimed to determine the urinary levels of pyridinium cross-links and urinary beta-isomerized fragments derived from the C-telopeptide of the alpha1 chain of type I collagen (beta-CTX) as markers of bone resorption in patients with ankylosing spondylitis (AS), and to study their relationship to markers of disease activity [erythrocyte sedimentation rate (ESR)] and to disease subsets of this condition. METHODS: The serum calcium, osteocalcin (OC), parathormone (PTH), 25 OHD3 levels, beta-CTX and the urinary combined free pyridinolines (f-Pyr + f-Dpyr), urinary free deoxypyridinoline (f-Dpyr) and urinary free pyridinoline (f-Pyr) were evaluated and compared in 32 AS patients and 25 controls. Bone mineral density (BMD) was evaluated at the lumbar spine and the femoral neck. RESULTS: The serum markers of bone metabolism (serum calcium, PTH, 25 OHD3 and OC) were in the normal range in the AS group. AS patients had a lowered lumbar spine BMD (P = 0.01) (corresponding T score: P = 0.03), but femoral neck BMD did not differ significantly between AS and controls (P = 0.08) (corresponding T score: P = 0.11). There was no difference in the urinary levels of pyridinium cross-links and beta-CTX between AS patients and controls. A positive correlation between ESR, (f-Pyr + f-Dpyr) (r = 0.42; P = 0.018) and f-Dpyr (r = 0.49; P = 0.005) was observed. In the different disease subsets of AS, we found that patients with peripheral involvement had higher (f-Pyr + f-Dpyr) (P = 0.04) and f-Dpyr levels (P = 0.04), patients with early disease had elevated (f-Pyr + f-Dpyr) (P = 0.01), f-Dpyr (P = 0.02) and f-Pyr (P = 0.01) levels, and that those with raised ESR had enhanced f-Dpyr (P = 0.009) excretion. Patients were then stratified according to disease duration, peripheral involvement and sex, and this allowed us to observe that only urinary f-Dpyr remained elevated in patients independently from these variables and that raised ESR is the more relevant parameter for explaining this high level of excretion. CONCLUSION: We conclude that there was no difference in the levels of urinary pyridinium cross-links and beta-CTX between AS and controls. However, urinary excretion of some of these collagen compounds was enhanced in subgroups of AS, mainly in patients with raised ESR. Thus, AS patients with laboratory evidence of active disease could have a higher risk of bone loss.

Relationships between several markers of extracellular matrix turn-over and ultrasonography in human Schistosomiasis mansoni.

We measured the concentrations of several serum and urinary fibrosis markers, which are metabolites of extracellular matrix, in schistosomiasis patients to investigate their relationship with the ultrasonographic scoring system and with parasitologic data. This study was conducted in patients with various stages of the disease evaluated by ultrasonography (intestinal disease with no organ involvement, with minor hepatosplenic involvement and with severe disease) and in endemic controls. The level of hyaluronan, which were increased in infected patients compared with controls (P < 0.01), was the only fibrosis marker that correlated with the ultrasonographic score (P = 0.003) and is thus a potential serum marker of schistosomiasis-associated morbidity. Urinary free pyridinoline levels were lower (P < 0.001) in infected patients with fibrosis (score > or = 1) than in nonfibrotic patients. A two-year follow-up of the patients treated with praziquantel showed that type I collagen and hyaluronan decreased during the first year post-treatment, whereas free pyridinolines peaked after 12 months and decreased thereafter.

The level of the collagen cross-link pyridinoline reflects the improvement of cutaneous lesions in one case of skin alveolar echinococcosis.

Cutaneous parasitic lesions, associated with a dense fibrous reaction, markedly improved under albendazole treatment in one case of supraumbilical skin localization of alveolar echinococcosis. Since collagen cross-linking increases during fibrogenesis and contributes to the stability of fibrotic lesions, we monitored the level of the cross-links pyridinoline and pentosidine in skin lesions from this patient to determine if they would reflect the changes occurring during treatment. We looked at the deposition of cross-linked type I collagen by immunohistochemistry and also measured the serum concentrations of pentosidine and of a fragment of type I collagen (ICTP), which contains a site of pyridinoline formation. Albendazole treatment did not affect either the collagen content of skin lesions or the serum concentrations of ICTP and pentosidine, but it led to a pronounced decrease in pyridinoline level concomitant with the disappearance, observed by immunohistochemistry, of extensively cross-linked fibrotic type I collagen. The follow-up of collagen cross-linking by pyridinoline in skin tissue thus appears to be useful in reflecting the improvement of fibrotic skin diseases during therapy.

Monitoring of extracellular matrix metabolism and cross-linking in tissue, serum and urine of patients with chromoblastomycosis, a chronic skin fibrosis.

BACKGROUND: Chromoblastomycosis is a fungal disease leading to a granulomatous reaction associated with dermal fibrosis. METHODS: In an attempt to elucidate the mechanisms leading to improvement in the cutaneous lesions after treatment with terbinafine, a new antifungal drug, we analysed collagen content and cross-linking before and at the end of the treatment. The turnover of extracellular matrix was monitored for 1 year by following up serum and urinary metabolites. RESULTS: The serum levels of type III collagen and its N-terminal propeptide were correlated with the lesion size (P < 0.035) after 4 and 12 months of treatment respectively. After 4 months of treatment, urinary pyridinoline was higher (P = 0.04) in patients whose lesion size was reduced by more than 50% and serum hyaluronan was lower in patients who had lesions active for less than 5 years (P < 0.05). The treatment increased pyridinoline and pentosidine cross-links in the lesions but significantly reduced the collagen content (P = 0.05). CONCLUSION: This is the first demonstration that, in addition to its fungicidal activity, terbinafine acts in vivo as an antifibrotic drug.

Echinococcus multilocularis infection in mice: in vivo treatment with a low dose of IFN-gamma decreases metacestode growth and liver fibrogenesis.

As no antiparasitic drug is definitively efficient in patients with alveolar echinococcosis, the effects of exogenous IFN-gamma on murine Echinococcus multilocularis infection were assessed with regards to the parasite burden, parasite-specific immune responses, and the urinary level of the collagen cross-link pyridinolines. They were analyzed after 3-week treatments with 1 or 5 micrograms of IFN-gamma per day twice a week. The treatment with 1 microgram transiently reduced the liver metacestode load, and the metastase weight as far as 6 weeks after the end of treatment. It slightly increased Th 1-type T cell responses and reduced the excretion of pyridinolines. These results should encourage further study to assess whether the decrease in liver fibrosis leads to an improvement of the efficacy of albendazole therapy. In contrast, the treatment with 5 micrograms increased the liver metacestode load and was less efficient than that with 1 microgram in decreasing pyridinoline excretion. These results incitate to follow up carefully patients with alveolar echinococcosis who are treated with IFN-gamma.

In situ reverse transcription-polymerase chain reaction. Applications for light and electron microscopy.

Since its discovery in 1986 by Mullis, the polymerase chain reaction (PCR) has been extensively developed by morphologists in order to overcome the main limitation of in situ hybridization, the lack of sensitivity. In situ PCR combines the extreme sensitivity of PCR with the cell-localizing ability of in situ hybridization. The amplification of DNA (PCR) or a cDNA (RT-PCR) in cell or tissue sections has been developed at light and electron microscopic levels. A successful PCR experiment requires the careful optimization of several parameters depending on the tissue (or of cell types), and a compromise must be found between the fixation time, pretreatments and a good preservation of the morphology. Other crucial factors (primer design, concentration in MgCl2, annealing and elongation temperatures during the amplification steps) and their influence on the specificity and sensitivity of in situ PCR or RT-PCR are discussed. The necessity to run appropriate controls, especially to assess the lack of diffusion of the amplified products, is stressed. Current applications and future trends are also presented.

Immunohistochemical study of type I collagen turn-over and of matrix metalloproteinases in chromoblastomycosis before and after treatment by terbinafine.

The distribution of type I collagen, the major component of human dermis, was characterized by immunohistochemistry in skin lesions of chromoblastomycosis, a chronic cutaneous mycosis, before and after a specific antifungal treatment with terbinafine to study the changes induced in the lesions by the treatment. Newly synthesized type I collagen was studied with an antibody directed against the aminoterminal propeptide of the molecule (PINP), whereas mature, cross-linked type I collagen was detected with an antibody against the carboxyterminal telopeptide of type I collagen (ICTP). The isopeptide N epsilon gamma-glutamyl lysine (N epsilon gamma GL), synthesized by transglutaminase and able to cross-link several components of the extracellular matrix, has also been investigated with two monoclonal antibodies to determine if it is involved in the stabilisation of the fibrotic cutaneous lesions. The degradative process involved in the remodelling has also been assessed by immunohistochemistry with anti-metalloproteinase (MMP-1 and MMP-9) and anti-tissue inhibitor (TIMP-1) antibodies. All tissue macrophages stained for CD68 and MMP-9, but not for MMP-1, while the polymorphonuclear neutrophils had an elastase and a weak MMP-9 phenotype. The fibroblasts of fibrotic areas stained constantly for N epsilon gamma GL and PINP. The immunostaining of extracellular matrix for ICTP and N epsilon gamma GL, and the number of PINP-positive fibroblasts, decreased significantly after one year of antifungal treatment. Terbinafine treatment decreases the synthesis of type I collagen and leads to a partial reversal of the cutaneous fibrotic lesions, independently of the cure of the fungal infection.

Urinary excretion of the collagen cross-link pyridinoline increases during liver fibrogenesis.

BACKGROUND/AIMS: Pyridinoline, a specific cross-link of mature collagen, increases in liver during fibrogenesis and its hepatic level is related to the degree of reversibility of the fibrotic process. Since pyridinoline is excreted in urine, we have investigated the relationship between its urinary level and liver fibrogenesis in a model of mild and reversible liver fibrosis, murine schistosomiasis. METHODS: Pyridinoline was measured by HPLC in urine and in liver of Schistosoma mansoni-infected mice during the acute and the chronic phases of the infection. Collagen deposition was measured colorimetrically. Both the isolated granulomas and the surrounding liver parenchyma were analyzed. RESULTS: In infected mice, pyridinoline increased mainly in the isolated granulomas, corresponding to the fibrotic lesions, and slightly in the surrounding parenchyma. The urinary excretion of pyridinoline increased during liver fibrogenesis and was correlated to the duration of infection (r=0.81) and to the collagen content of granulomas (r=0.81). The treatment of infected mice by praziquantel, an antiparasitic drug, did not lead to significant changes in liver collagen cross-linking by pyridinoline either in granulomas or in parenchyma. The major effect of the drug was targeted at the collagen content of parenchyma, which decreased by 50%, 18 weeks after treatment. The urinary level of pyridinoline of treated mice was negatively correlated to the length of the treatment follow-up (r=-0.76). CONCLUSIONS: The measurement of the urinary excretion of pyridinoline could be helpful to monitor the remodeling of liver extracellular matrix occurring in fibrogenesis and the effect of chemotherapy.

Mechanism of collagen network stabilization in human irreversible granulomatous liver fibrosis.

BACKGROUND & AIMS: Cross-linking participates in the increased stability of collagen towards proteolytic degradation. Liver collagen cross-linking by pyridinoline, from the lysyl oxidase pathway, and by pentosidine, issued from glycation, was investigated to determine their respective contribution to collagen stabilization in patients with an irreversible liver fibrosis caused by the parasitic granulomatous disease alveolar echinococcosis. METHODS: Liver pyridinoline and pentosidine were analyzed by high-performance liquid chromatography, and urinary pyridinoline was analyzed by immunoassay. Cross-linked type I collagen was localized by immunohistochemistry with an antibody against the C-terminal part of the molecule, involved in pyridinoline formation, that was measured in serum by radioimmunoassay. RESULTS: In contrast to pyridinoline, pentosidine decreased in fibrotic lesions. Cross-linked I collagen was located predominantly in collagen bundles in the periparasitic granuloma. Serum pentosidine and urinary pyridinoline levels did not differ significantly from controls, but the serum concentration of the C-terminal telopeptide of type I collagen increased significantly. CONCLUSIONS: Lysyl oxidase-mediated cross-linking is the major process contributing to the stabilization of collagen in granulomatous fibrosis, and glycation is not significantly involved in it. The changes induced by alveolar echinococcosis in liver collagen metabolism are associated with an increase in serum C-telopeptide of type I collagen.

The carboxy-terminal cross-linked telopeptide of type I collagen (ICTP) is a potential serum marker of ongoing liver fibrosis.

We report for the first time the measurement of the serum concentration of the carboxy-terminal cross-linked telopeptide of type I collagen in patients with various liver diseases. This breakdown product of type I collagen, which is the major collagen type found in fibrotic liver, was measured by a radioimmunoassay in the serum of 149 patients with various liver diseases and in 67 controls. Its concentration is significantly elevated (P < 0.05) above reference intervals in sera from patients with liver diseases, except in patients with chronic active hepatitis of unknown origin and in patients with acute hepatitis A. In the 143 patients with liver fibrosis, the serum level of the carboxy-terminal telopeptide of type I collagen is correlated with the extent of fibrosis, as assessed by a histological scoring system (r = 0.3899, P < 0.0001), but not with inflammation and necrosis.