Structural and accessibility studies highlight the differential binding of clemizole to TRPC5 and TRPC6.

Transient Receptor Potential Canonical 5 (T RP C5) and T RP C6 channels play critical physiological roles in various cell types. Their involvement in numerous disease progression mechanisms has led to extensive searches for their inhibitors. Although several potent T RP C inhibitors have been developed and the structure of their binding sites were mapped using cryo electron microscopy, a comprehensive understanding of the molecular interactions within the inhibitor binding site of T RP Cs remains elusive. This study aimed to decipher the structural determinants and molecular mechanisms contributing to the differential binding of clemizole to T RP C5 and T RP C6, with a particular focus on the accessibility of binding site residues. This information can help better understand what molecular features allow for selective binding, which is a key characteristic of clinically effective pharmacological agents. Using computational methodologies, we conducted an in-depth molecular docking analysis of clemizole with T RP C5 and T RP C6 channels. The protein structures were retrieved from publicly accessible protein databases. Discovery Studio 2020 Client Visualizer and Chimera software facilitated our in-silico mutation experiments and enabled us to identify the critical structural elements influencing clemizole binding. Our study reveals key molecular determinants at the clemizole binding site, specifically outlining the role of residues' Accessible Surface Area (ASA) and Relative Accessible Surface Area (RASA) in differential binding. We found that lower accessibility of T RP C6 binding site residues, compared to those in T RP C5, could account for the lower affinity binding of clemizole to T RP C6. This work illuminates the pivotal role of binding site residue accessibility in determining the affinity of clemizole to T RP C5 and T RP C6. A nuanced understanding of the distinct binding properties between these homologous proteins may pave the way for the development of more selective inhibitors, promising improved therapeutic efficacy and fewer off-target effects. By demystifying the structural and molecular subtleties of T RP C inhibitors, this research could significantly accelerate the drug discovery process, offering hope to patients afflicted with T RP C-related diseases.

Mannose-binding lectin gene 2 variant DD (rs 5030737) is associated with susceptibility to COVID-19 infection in the urban population of Patna City (India).

The study aimed to measure plasma levels of Mannose-Binding Lectin (MBL) and MBL-associated serine protease-2 (MASP-2) and their polymorphisms in COVID-19 patients and controls to detect association. As MBL is a protein of immunological importance, it may contribute to the first-line host defence against SARS-CoV-2. MBL initiates the lectin pathway of complement activation with help of MASP-1 and MASP-2. Hence, appropriate serum levels of MBL and MASPs are crucial in getting protection from the disease. The polymorphisms of MBL and MASP genes affect their plasma levels, impacting their protective function and thus may manifest susceptibility, extreme variability in the clinical symptoms and progression of COVID-19 disease. The present study was conducted to find plasma levels and genetic variations in MBL and MASP-2 in COVID-19 patients and controls using PCR-RFLP and ELISA, respectively.The present study was conducted to find plasma levels and genetic variations in MBL and MASP-2 in COVID-19 patients and controls using PCR-RFLP and ELISA, respectively. Our results indicate that median serum levels of MBL and MASP-2 were significantly low in diseased cases but attained normal levels on recovery. Only genotype DD was found to be associated with COVID-19 cases in the urban population of Patna city.

Transient Receptor Potential Canonical 6 (TRPC6) Channel in the Pathogenesis of Diseases: A Jack of Many Trades.

The mammalian Transient Receptor Potential Canonical (TRPC) subfamily comprises seven transmembrane proteins (TRPC1-7) forming cation channels in the plasma membrane of mammalian cells. TRPC channels mediate Ca2+ and Na+ influx into the cells. Amongst TRPCs, TRPC6 deficiency or increased activity due to gain-of-function mutations has been associated with a multitude of diseases, such as kidney disease, pulmonary disease, and neurological disease. Indeed, the TRPC6 protein is expressed in various organs and is involved in diverse signalling pathways. The last decade saw a surge in the investigative studies concerning the physiological roles of TRPC6 and describing the development of new pharmacological tools modulating TRPC6 activity. The current review summarizes the progress achieved in those investigations.

Evaluation of C4b as an adjunct marker in symptomatic RT-PCR negative Covid-19 cases.

Introduction: Detecting low viral load has been a challenge in this pandemic, which has led to its escalated transmission. Complement activation has been implicated in pathogenesis of Covid-19 infection. Thus, evaluation of complement activation in suspected Covid-19 infection may help to detect infection and limit false negative cases thus limiting transmission of infection. We speculate that measuring C4b, produced from an activated complement system due to the presence of Covid-19 may help in its detection, even when the viral titers are low. Methods: Plasma C4b levels of symptomatic RT-PCR positive patients (cases, n = 40); symptomatic RT-PCR negative patients (n = 35) and asymptomatic RT-PCR negative controls (n = 40) were evaluated. Plasma C5b-9, IL-6, D-dimer and C1-Inhibitor (C1-INH) were also measured in cases and controls. ELISA kits were used for all measurements. Statistical analyses were carried out using Stata, version 12 (Stata Corp., Texas, USA). Results: C4b levels were found to be significantly increased in RT-PCR positive patients as compared to asymptomatic RT-PCR negative controls. RT-PCR negative but symptomatic patients still showed increased C4b levels. The significantly higher levels of C4b in cases with a cut-off value of ≥ 116 ng/ml with optimum sensitivity and specificity of 80% and 52% respectively is indicative of its possible use as an adjunct marker. Increased levels of D-dimer, IL6, along with decreased levels of C1-INH were found in cases compared to controls. Whereas, C5b-9 levels were not significantly raised in cases. Conclusions: The results of our study suggests that plasma C4b may help to detect infection in false negative cases of RT-PCR that escape detection owing to low viral load. However, to confirm it a large-scale study is needed. Supplementary Information: The online version contains supplementary material available at 10.1007/s12291-022-01033-z.

Identification of substrates of MBL Associated Serine Protease-1 (MASP-1) from human plasma using N-terminomics strategy.

MBL Associated Serine Protease-1 (MASP-1) is an abundant enzyme of the lectin complement pathway. MASP-1 cleaves numerous substrates like MASP-2, MASP-3, C2, C3i, fibrinogen, FXIII and prothrombin. It has thrombin-like specificity and can cleave thrombin substrates. Owing to its high concentration and relaxed substrate specificity, MASP-1 has substrates outside the complement system and can influence other proteolytic cascades and physiological processes. The unidentified substrates may assist us to ascertain the role(s) of MASP-1. In this study, we used a high-throughput N-terminomics method to identify substrates of MASP-1 from human plasma. We have identified 35 putative substrates of MASP-1. Among the identified proteins, alpha 2-antiplasmin, alpha-1-acid glycoprotein, antithrombin III, and siglec-6 were demonstrated to be cleaved by MASP-1. We have discussed the physiological relevance of cleavage of these substrates by MASP-1. The expression of Siglec-6 and MASP-1 has been reported in the B cells. Alpha-1-acid glycoprotein cleavage by MASP-1 may occur in the acute phase as it is known to be an inhibitor of platelet aggregation, whereas MASP-1 triggers platelet aggregation. The cleavage alpha2 antiplasmin by MASP-1 implies that MASP-1 may be promoting plasmin-mediated fibrinolysis. Our study supports that MASP-1 may be implicated in thrombosis as well as thrombolysis.

Mobile technology: A tool for healthcare and a boon in pandemic.

Healthcare systems deal with disease prevention, early detection, diagnosis, investigation, and timely, affordable, and safe treatment. For the delivery of services in the health sector, communication is the key to linking the service provider and the patients. Mobile technology in the recent past has rendered various platforms of communications for the healthcare system. Thus, in health, mobile technology has greatly contributed to time management and cost reduction for healthcare at every level including hospital visits to individual appointments with doctors, hence the convenience. With advancements in mobile technologies and the growing number of mobile users, newer opportunities have opened up for the use of mobiles for patient care. Emerging information and communication technologies with the help of the Internet of Things (IoT) have been instrumental in integrating different domains of the health sector with mobile technology. Thus, the technology may have the potential to become powerful medical tools to support the health sector at all levels of care. In this review, the concept, applications, and advantages of mobile technology for health and the present pandemic have been discussed. It also discusses mobile health technology, as a support system for convenient and safer healthcare for public health, and the opportunities to improve its applications for unseen future health crises.

Mannose-binding lectin-associated serine protease-1 cleaves plasminogen and plasma fibronectin: prefers plasminogen over known fibrinogen substrate.

Mannose-binding lectin-associated serine protease-1 (MASP-1) is known to interact with complement and coagulation pathways. Recently it was reported that MASP-1 interacts with the fibrinolytic system but details remain unclear. The objective of the study is to find MASP-1 substrates that participate in the fibrinolytic system. Commercially available fibrinogen might contain some impurities. Fibrinogen was treated with MASP-1 followed by analysis on SDS-PAGE and the obtained cleaved fragments were identified by matrix-assisted laser desorption/ionization-time of flight/time of flight. Functional analysis of identified substrate was confirmed by fluorogenic and turbidimetric assay. Statistical analysis was done by using the Student t test. This study reports that plasminogen and plasma fibronectin are two hitherto unknown substrates of MASP-1. Conversion of plasminogen to plasmin like molecule by MASP-1 was confirmed by cleavage of plasmin specific substrate and digestion of fibrin clot. The role of MASP-1 in clot dissolution was confirmed by turbidity assay. Our study shows that MASP-1 selects plasminogen over fibrinogen to be a preferable substrate. MASP-1 promotes the fibrinolytic activity by the generation of plasmin like molecule from plasminogen and further destabilizes the clot by digestion of plasma fibronectin.

Mannose-binding lectin and associate serine protease complex modulates neutrophil respiratory burst and gene expression in Capra hircus.

Neutrophils are an essential cellular component of the innate immune system, responsible for multiple effector mechanisms and aspects of inflammation. Neutrophil priming results in a rapid elevation in antimicrobial activities and can be measured by reactive oxygen species production, bacterial endocytosis, and de-novo synthesis of components such as interleukins. Mannose binding lectin (MBL), a C-type lectin pathogen recognition receptor is associated with immune functions including complement activation, opsonization and modulating immune responses. Whether MBL opsonization of pathogen can induce neutrophil priming has not been studied so far. Hence, studies were performed using MBL and neutrophils of Capra hircus (domestic goat) to evaluate the effects of MBL + MASPs interactions on neutrophil functions. It was found that MBL + MASPs opsonization of zymosan stimulates neutrophil functions including increased oxidative burst, enhanced endocytosis and modulates the expression level of NCF4, XBP1, CCL2, and CR1 genes. The results suggest that MBL-MASP complex can regulate neutrophil functioning.

Identification of unexplored substrates of the serine protease, thrombin, using N-terminomics strategy.

The function and regulation of thrombin is a complex as well as an intriguing aspect of evolution and has captured the interest of many investigators over the years. The reported substrates of thrombin are coagulation factors V, VIII, XI, XIII, protein C and fibrinogen. However, these may not be all the substrate of thrombin and therefore its functional role(s), may not have been completely comprehended. The purpose of our study was to identify hitherto unreported substrates of thrombin from human plasma using a N-terminomics protease substrate identification method. We identified 54 putative substrates of thrombin of which 12 are already known and 42 are being reported for the first time. Amongst the proteins identified, recombinant siglec-6 and purified serum alpha-1-acid glycoprotein were validated by cleavage with thrombin. We have discussed the probable relevance of siglec-6 cleavage by thrombin in human placenta mostly because an upregulation in the expression of siglec-6 and thrombin has been reported in the placenta of preeclampsia patients. We also speculate the role of alpha-1-acid glycoprotein cleavage by thrombin in the acute phase as alpha-1-acid glycoprotein is known to be an inhibitor of platelet aggregation whereas thrombin is known to trigger platelet aggregation.

Proteolysis to Identify Protease Substrates: Cleave to Decipher.

Proteolysis is an irreversible post-translational modification process, characterized by highly precise yet stable cleavage of proteins. Downstream events in signaling processes are reliant on proteolysis triggered by the protease activity. Studies indicate that abnormal proteolytic activity may lead to the manifestation of diseased conditions. Therefore, characterization of proteases may provide clues to understand their role in fundamental cellular processes like cellular growth, differentiation, apoptosis, and survival. The relevance of proteases and their substrates as clinical targets are being studied. Understanding the mechanism of proteolytic activity, the identity, and the role of repertoire of its substrates in a physiological pathway has opened avenues for novel drug designing. However, only a limited knowledge of protease substrates is currently available. In this review, the authors recapitulate the library screening, proteomics, and bioinformatics based approaches that have been employed for the identification of protease substrates.

Allelic loss at PTEN locus leads to progression of colorectal carcinoma among North Indian patients.

We evaluated the loss of heterozygosity (LOH) at 10q23.3 locus of microsatellite markers; D10S198, D10S192, and D10S541 of PTEN gene in 223 North Indian colorectal cancer (CRC) specimens. DNA was isolated and microsatellite-specific markers polymerase chain reaction was performed. Out of total 223 cases 102 showed LOH for at least one of the locus. In addition, thereto a significant association was found with the clinicopathologic features like grade of differentiation, clinical stage, invasion, lymph node invasion, and the clinical outcome (p < 0.05). These data argue that the given markers to check the possible LOH of PTEN gene at locus 10q23.3 could be considered as one of the diagnostic markers in CRC.

Divalent metal ions binding properties of goat serum mannose binding lectin.

Mannose binding lectin (MBL) is a collectin with C-terminus Carbohydrate Recognition Domain (CRD) which binds with pathogen and arbitrate functions like activation of complement pathway, opsonization etc. The CRD required Ca(2+) ions to recognize the sugar moieties. In the present study the binding properties of CRD with divalent ions were characterized by Electron Paramagnetic Resonance (EPR) spectroscopy. The results revealed that the metal binding site of CRD is of approximately 1 Å diameter and ions greater than the size are not able to enter.

Mutational and expressional analyses of PTEN gene in colorectal cancer from Northern India.

The PTEN is a tumor-suppressor gene located on chromosome 10q23.3 and established to play key role in the varied types of cancer. To elucidate the possible effect of mutations and inactivation of PTEN gene on the occurrence and development of colorectal cancer (CRC), 223 cancer specimens were selected to probe PTEN gene mutations through the micro dissection of the genome. Polymerase chain reaction single-strand conformation polymorphism and DNA sequencing methods were applied for mutations while protein expression was evaluated by immunohistochemistry. Mutations in exons 7 and 8 of PTEN were observed in 12.5% and PTEN loss of expression was identified in 48% in CRC. In exon 7, we found the insertion of "G" resulted into the change at codon 218 from TGC to GTC leading to change in the reading frame starting downward from Cystein to Valine. In addition, the insertion of "A" in the same exon at codon 213 resulted into the change of codon CCT to CCA, which cause silent mutation. In exon 8, however, "A" is replaced by C at codon 282, but both encodes for Glycine. Statistically significant loss of PTEN expression was observed in cancerous tissue when compared with the adjacent control (P < 0.05). Furthermore, weak PTEN expression in CRC tissues were significantly associated with tumor size, depth of invasion, lymphatic invasion, lymph node metastasis, grade of differentiation, and TNM stage (P < 0.05). Our results suggested that PTEN gene mutation and loss of PTEN expression may provide valuable prognostic information to aid treatment strategies for CRC patients.

Cleavage of kininogen and subsequent bradykinin release by the complement component: mannose-binding lectin-associated serine protease (MASP)-1.

Bradykinin (BK), generated from high-molecular-weight kininogen (HK) is the major mediator of swelling attacks in hereditary angioedema (HAE), a disease associated with C1-inhibitor deficiency. Plasma kallikrein, activated by factor XIIa, is responsible for most of HK cleavage. However other proteases, which activate during episodes of angioedema, might also contribute to BK production. The lectin pathway of the complement system activates after infection and oxidative stress on endothelial cells generating active serine proteases: MASP-1 and MASP-2. Our aim was to study whether activated MASPs are able to digest HK to release BK. Initially we were trying to find potential new substrates of MASP-1 in human plasma by differential gel electrophoresis, and we identified kininogen cleavage products by this proteomic approach. As a control, MASP-2 was included in the study in addition to MASP-1 and kallikrein. The proteolytic cleavage of HK by MASPs was followed by SDS-PAGE, and BK release was detected by HPLC. We showed that MASP-1 was able to cleave HK resulting in BK production. MASP-2 could also cleave HK but could not release BK. The cleavage pattern of MASPs is similar but not strictly identical to that of kallikrein. The catalytic efficiency of HK cleavage by a recombinant version of MASP-1 and MASP-2 was about 4.0×10(2) and 2.7×10(2) M(-1) s(-1), respectively. C1-inhibitor, the major inhibitor of factor XIIa and kallikrein, also prevented the cleavage of HK by MASPs. In all, a new factor XII- and kallikrein-independent mechanism of bradykinin production by MASP-1 was demonstrated, which may contribute to the pro-inflammatory effect of the lectin pathway of complement and to the elevated bradykinin levels in HAE patients.

The ORF3 protein of hepatitis E virus delays degradation of activated growth factor receptors by interacting with CIN85 and blocking formation of the Cbl-CIN85 complex.

Hepatitis E virus (HEV) causes an acute self-limiting disease that is endemic in developing countries. Previous studies suggested that the ORF3 protein (pORF3) of HEV is required for infection in vivo and is likely to modulate the host response. Our previous work showed that pORF3 localizes to early and recycling endosomes and causes a delay in the postinternalization trafficking of epidermal growth factor receptor (EGFR) to late endosomes/lysosomes. Here we report that pORF3 also delays the trafficking and degradation of activated hepatocyte growth factor receptor (c-Met) and delineate the mechanistic details of these effects. A mutant ORF3 protein, which does not localize to endosomes, also showed similar effects on growth factor receptor trafficking, making this effect independent of the endosomal localization of pORF3. The ORF3 protein was found to interact with CIN85, a multidomain adaptor protein implicated in the Cbl-mediated downregulation of receptor tyrosine kinases. This interaction competed with the formation of the growth factor receptor-Cbl-CIN85 complex, resulting in the reduced ubiquitination of CIN85 and trafficking of the growth factor receptor complex toward late endosomes/lysosomes. We propose that through its effects on growth factor receptor trafficking, pORF3 prolongs endomembrane growth factor signaling and promotes cell survival to contribute positively to viral replication and pathogenesis.

Activation of mannan-binding lectin-associated serine proteases leads to generation of a fibrin clot.

The lectin pathway of complement is activated upon binding of mannan-binding lectin (MBL) or ficolins (FCNs) to their targets. Upon recognition of targets, the MBL-and FCN-associated serine proteases (MASPs) are activated, allowing them to generate the C3 convertase C4b2a. Recent findings indicate that the MASPs also activate components of the coagulation system. We have previously shown that MASP-1 has thrombin-like activity whereby it cleaves and activates fibrinogen and factor XIII. MASP-2 has factor Xa-like activity and activates prothrombin through cleavage to form thrombin. We now report that purified L-FCN-MASPs complexes, bound from serum to N-acetylcysteine-Sepharose, or MBL-MASPs complexes, bound to mannan-agarose, generate clots when incubated with calcified plasma or purified fibrinogen and factor XIII. Plasmin digestion of the clot and analysis using anti-D-dimer antibodies revealed that the clot was made up of fibrin and was similar to that generated by thrombin in normal human plasma. Fibrinopeptides A and B (FPA and FPB, respectively) were released after fibrinogen cleavage by L-FCN-MASPs complexes captured on N-acetylcysteine-Sepharose. Studies of inhibition of fibrinopeptide release indicated that the dominant pathway for clotting catalysed by the MASPs is via MASP-2 and prothrombin activation, as hirudin, a thrombin inhibitor that does not inhibit MASP-1 and MASP-2, substantially inhibits fibrinopeptide release. In the light of their potent chemoattractant effects on neutrophil and fibroblast recruitment, the MASP-mediated release of FPA and FPB may play a role in early immune activation. Additionally, MASP-catalysed deposition and polymerization of fibrin on the surface of micro-organisms may be protective by limiting the dissemination of infection.

Functional estimation of mannose binding lectin associated serine protease (MBL-MASPs) in human serum.

BACKGROUND & OBJECTIVE: Mannose binding lectin (MBL), a C-type or Ca(2+) dependent lectin, plays a major role in lectin pathway of complement activation. MBL deficiency/insufficiency is associated with susceptibility to many infections. It is important to know the association of functional lectin levels with disease condition. Therefore, we carried out this study to develop a simple assay to estimate the functional MBL-associated serine proteases (MBL-MASPs) levels in human serum samples. METHODS: A novel method was developed based on direct haemolysis of mannan coated human erythrocytes in autologous human serum for functional estimation of MBL and associated serine proteases (MBLMASPs complex). Functional MBL-MASPs serum levels in 75 healthy individuals was estimated. Results were compared with those obtained by ELISA based assay. RESULTS: Lysis of mannan coated human RBC in autologous serum was highly specific and mediated by MBL-MASPs lectin complement pathway. Concentration of MBL-MASPs in serum of normal healthy individuals (n=75) was found to be 1.579 mug/ml (median= 1.149 mug/ml) by the haemolytic assay which was comparable to the values obtained by ELISA method. INTERPRETATION & CONCLUSION: Our findings showed that the method developed for the estimation of functional MBL-MASPs levels in human serum is simple, cost-effective and comparable with existing ELISA method.

Vaccination with 73kDa recombinant heavy chain myosin generates high level of protection against Brugia malayi challenge in jird and mastomys models.

We have earlier reported identification, expression and purification of a 2.0kb cDNA clone coding for Brugia malayi heavy chain myosin which exhibited strong immuno-reactivity with bancroftian sera from endemic normal (EN) human subjects which are considered to be putatively immune. In the present study, immunoprophylactic characterization of B. malayi recombinant myosin was carried out in rodent models and the protective efficacy was evaluated by assessing the microfilarial burden and adult worm counts in vaccinated host after an infective larval challenge. Data indicates that immunization resulted in to a significant reduction in microfilarial burden (approximately 76%) and adult worm establishment (54-58%), accompanied with embryostatic effect (70-75%) in both the animal models. The findings suggest that immune-protection by recombinant myosin was conferred through both humoral and cellular arms of immunity as indicated by an increased antibody titer with predominance of IgG2a and IgG2b isotypes along with elevated level of IgG1 apart from significant proliferation of lymphocytes, increased nitric oxide production and profound adherence of splenocytes causing cytotoxicity to microfilariae and infective larvae. The present study indicates that the recombinant B. malayi myosin is a promising vaccine candidate against human lymphatic filarial infection.

Disease associations of mannose-binding lectin & potential of replacement therapy.

Mannose-binding lectin (MBL) is an important component of the immune defence able to bind to repeating mannose based structural patterns typical of microbial surface (bacteria, viruses, fungi, parasites) leading to opsonization and phagocytosis, and activation of the complement pathway resulting in lysis of the pathogen. MBL thus plays a very important role in the first line of host immune response. MBL deficiency has been implicated in susceptibility and modulating the severity in viral, bacterial, fungal, and protozoan infections. High MBL levels, on the contrary might be helpful to intracellular organisms, which take the advantage of C3 opsonization and C3 receptor on monocytes/macrophages to enter their host. MBL replacement therapy to help patients with MBL deficiency has undergone phase I clinical trials. Phase II and III trials and production of recombinant MBL for replacement therapy are currently underway.