Molecular dynamics study of tropical calcific pancreatitis (TCP) associated calcium-sensing receptor single nucleotide variation.

Tropical Calcific Pancreatitis (TCP) is a chronic non-alcoholic pancreatitis characterised by extensive calcification. The disease usually appears at a younger age and is more common in tropical regions. This disease's progression can lead to pancreatic diabetes, which can subsequently lead to pancreatic cancer. The CASR gene encodes a calcium-sensing receptor (CaSR), which is a GPCR protein of class C. It is expressed in the islets of Langerhans, the parathyroid gland, and other tissues. It primarily detects small gradients in circulating calcium concentrations and couples this information to intracellular signalling, which helps to regulate PTH (parathyroid hormone) secretion and mineral ion homeostasis. From co-leading insulin release, CaSR modulates ductal HCO3- secretion, Ca2+ concentration, cell-cell communication, ß-cell proliferation, and intracellular Ca2+ release. In pancreatic cancer, the CaSR limits cell proliferation. TCP-related four novel missense mutations P163R, I427S, D433H and V477A, found in CaSR extracellular domain (ECD) protein, which were reported in the mutTCPdb Database (https://lms.snu.edu.in/mutTCPDB/index.php). P163R mutation occurs in ligand-binding domain 1 (LBD-1) of the CaSR ECD. To investigate the influence of these variations on protein function and structural activity multiple in-silico prediction techniques such as SIFT, PolyPhen, CADD scores, and other methods have been utilized. A 500 ns molecular dynamic simulation was performed on the CaSR ECD crystal structure and the corresponding mutated models. Furthermore, Principal Component Analysis (PCA) and Essential Dynamics analysis were used to forecast collective motions, thermodynamic stabilities, and the critical subspace crucial to CaSR functions. The results of molecular dynamic simulations showed that the mutations P163R, I427S, D433H, and V477A caused conformational changes and decreased the stability of protein structures. This study also demonstrates the significance of TCP associated mutations. As a result of our findings, we hypothesised that the investigated mutations may have an effect on the protein's structure and ability to interact with other molecules, which may be related to the protein's functional impairment.

Imidazolium-based ionic liquids cause mammalian cell death due to modulated structures and dynamics of cellular membrane.

Here, we report the toxic effects of various imidazolium-based ionic liquids (ILs) with varying hydrocarbon chain lengths, on different human cell lines. Multiple biological assays have shown that the ILs with long hydrocarbon chains have stronger adverse effect especially on human liver cancer cells (Huh-7.5 cells). Further, our study has confirmed that the ILs induce necrosis dependent cell death and that it is related to cell membrane damage. To understand the molecular mechanism of such an effect, the cellular membranes were mimicked as lipid monolayers formed at the air-water interface and then as lipid bilayer vesicles. The pressure area-isotherms measured from the monolayer have shown that the interaction of ILs with the lipid layer is energetically favourable. The addition of these ILs reduces the in-plane elasticity of the self-assembled molecular layer. Quasielastic neutron scattering data clearly indicate that ILs in liver lipid vesicles significantly affects the dynamics of the lipid, in particular, the lateral motion of the lipids. It has been concluded that the mammalian cell death induced by these ILs is due to the modulated structure and altered physical properties of the cellular membrane.

Structural, functional and molecular dynamics analysis of cathepsin B gene SNPs associated with tropical calcific pancreatitis, a rare disease of tropics.

Tropical Calcific Pancreatitis (TCP) is a neglected juvenile form of chronic non-alcoholic pancreatitis. Cathepsin B (CTSB), a lysosomal protease involved in the cellular degradation process, has recently been studied as a potential candidate gene in the pathogenesis of TCP. According to the Cathepsin B hypothesis, mutated CTSB can lead to premature intracellular activation of trypsinogen, a key regulatory mechanism in pancreatitis. So far, CTSB mutations have been studied in pancreatitis and neurodegenerative disorders, but little is known about the structural and functional effect of variants in CTSB. In this study, we investigated the effect of single nucleotide variants (SNVs) specifically associated with TCP, using molecular dynamics and simulation algorithms. There were two non-synonymous variants (L26V and S53G) of CTSB, located in the propeptide region. We tried to predict the effect of these variants on structure and function using multiple algorithms: SIFT, Polyphen2, PANTHER, SDM sever, i-Mutant2.0 suite, mCSM algorithm, and Vadar. Further, using databases like miRdbSNP, PolymiRTS, and miRNASNP, two SNPs in the 3'UTR region were predicted to affect the miRNA binding sites. Structural mutated models of nsSNP mutants (L26V and S53G) were prepared by MODELLER v9.15 and evaluated using TM-Align, Verify 3D, ProSA and Ramachandran plot. The 3D mutated structures were simulated using GROMACS 5.0 to predict the impact of these SNPs on protein stability. The results from in silico analysis and molecular dynamics simulations suggested that these variants in the propeptide region of Cathepsin B could lead to structural and functional changes in the protein and thus could be pathogenic. Hence, the structural and functional analysis results have given interim conclusions that these variants can have a deleterious effect in TCP pathogenesis, either uniquely or in combination with other mutations. Thus, it could be extrapolated that Cathepsin B gene can be screened in samples from all TCP patients in future, to decipher the distribution of variants in patients.

A plant based protective antigen [PA(dIV)] vaccine expressed in chloroplasts demonstrates protective immunity in mice against anthrax.

The currently available anthrax vaccines are limited by being incompletely characterized, potentially reactogenic and have an expanded dosage schedule. Plant based vaccines offer safe alternative for vaccine production. In the present study, we expressed domain IV of Bacillus anthracis protective antigen gene [PA(dIV)] in planta (by nuclear agrobacterium and chloroplast transformation) and E. coli [rPA(dIV)]. The presence of transgene and the expression of PA(dIV) in planta was confirmed by molecular analysis. Expression levels up to 5.3% of total soluble protein (TSP) were obtained with AT rich (71.8% AT content) PA(dIV) gene in transplastomic plants while 0.8% of TSP was obtained in nuclear transformants. Further, we investigated the protective response of plant and E. coli derived PA(dIV) in mice by intraperitoneal (i.p.) and oral immunizations with or without adjuvant. Antibody titers of >10(4) were induced upon i.p. and oral immunizations with plant derived PA(dIV) and oral immunization with E. coli derived PA(dIV). Intraperitoneal injections with adjuvanted E. coli derived PA(dIV), generated highest antibody titers of >10(5). All the immunized groups demonstrated predominant IgG1 titers over IgG2a indicating a polarized Th2 type response. We also evaluated the mucosal antibody response in orally immunized groups. When fecal extracts were analyzed, low sIgA titer was demonstrated in adjuvanted plant and E. coli derived PA(dIV) groups. Further, PA(dIV) antisera enhanced B. anthracis spore uptake by macrophages in vitro and also demonstrated an anti-germinating effect suggesting a potent role at mucosal surfaces. The antibodies from various groups were efficient in neutralizing the lethal toxin in vitro. When mice were challenged with B. anthracis, mice immunized with adjuvanted plant PA(dIV) imparted 60% and 40% protection while E. coli derived PA(dIV) conferred 100% and 80% protection upon i.p. and oral immunizations. Thus, our study is the first attempt in highlighting the efficacy of plant expressed PA(dIV) by oral immunization in murine model.