Molecular Dynamics Simulation of Kir6.2 Variants Reveals Potential Association with Diabetes Mellitus.

Diabetes mellitus (DM) represents a problem for the healthcare system worldwide. DM has very serious complications such as blindness, kidney failure, and cardiovascular disease. In addition to the very bad socioeconomic impacts, it influences patients and their families and communities. The global costs of DM and its complications are huge and expected to rise by the year 2030. DM is caused by genetic and environmental risk factors. Genetic testing will aid in early diagnosis and identification of susceptible individuals or populations using ATP-sensitive potassium (KATP) channels present in different tissues such as the pancreas, myocardium, myocytes, and nervous tissues. The channels respond to different concentrations of blood sugar, stimulation by hormones, or ischemic conditions. In pancreatic cells, they regulate the secretion of insulin and glucagon. Mutations in the KCNJ11 gene that encodes the Kir6.2 protein (a major constituent of KATP channels) were reported to be associated with Type 2 DM, neonatal diabetes mellitus (NDM), and maturity-onset diabetes of the young (MODY). Kir6.2 harbors binding sites for ATP and phosphatidylinositol 4,5-diphosphate (PIP2). The ATP inhibits the KATP channel, while the (PIP2) activates it. A Kir6.2 mutation at tyrosine330 (Y330) was demonstrated to reduce ATP inhibition and predisposes to NDM. In this study, we examined the effect of mutations on the Kir6.2 structure using bioinformatics tools and molecular dynamic simulations (SIFT, PolyPhen, SNAP2, PANTHER, PhD&SNP, SNP&Go, I-Mutant, MuPro, MutPred, ConSurf, HOPE, and GROMACS). Our results indicated that M199R, R201H, R206H, and Y330H mutations influence Kir6.2 structure and function and therefore may cause DM. We conclude that MD simulations are useful techniques to predict the effects of mutations on protein structure. In addition, the M199R, R201H, R206H, and Y330H variant in the Kir6.2 protein may be associated with DM. These results require further verification in protein-protein interactions, Kir6.2 function, and case-control studies.

Protein Misfolding and Aggregation in Proteinopathies: Causes, Mechanism and Cellular Response.

Proteins are central to life functions. Alterations in the structure of proteins are reflected in their function. Misfolded proteins and their aggregates present a significant risk to the cell. Cells have a diverse but integrated network of protection mechanisms. Streams of misfolded proteins that cells are continuously exposed to must be continually monitored by an elaborated network of molecular chaperones and protein degradation factors to control and contain protein misfolding problems. Aggregation inhibition properties of small molecules such as polyphenols are important as they possess other beneficial properties such as antioxidative, anti-inflammatory, and pro-autophagic properties and help neuroprotection. A candidate with such desired features is important for any possible treatment development for protein aggregation diseases. There is a need to study the protein misfolding phenomenon so that we can treat some of the worst kinds of human ailments related to protein misfolding and aggregation.

Phytochemical Analysis and Binding Interaction of Cotton Seed Cake Derived Compounds with Target Protein of Meloidogyne incognita for Nematicidal Evaluation.

The root-knot nematode Meloidogyne incognita is one of the most damaging plant-parasitic nematodes and is responsible for significant crop losses worldwide. Rising human health and environmental concerns have led to the withdrawal of commonly used chemical nematicides. There has been a tremendous demand for eco-friendly bio-nematicides with beneficial properties to the nematode hosting plants, which encourages the need for alternative nematode management practices. The current study was undertaken to determine the nematicidal potential of cotton seed cake (CSC) against second-stage juvenile (J2) hatching, J2 mortality, and J2 penetration of M. incognita in tomato plants in vitro. J2s and egg masses of M. incognita were exposed to four concentrations (250, 500, 750, and 1000 mg/L) of CSC extracts. The higher J2 mortality and inhibition of J2 hatching were found at 1000 mg/L, while the least effective result was observed at 250 mg/L of the CSC extract. The CSC extract applied with the concentrations mentioned above also showed inhibition of J2 penetration in tomato roots; 1000 mg/L showed the highest inhibition of penetration, while 250 mg/L displayed the least inhibition. Using gas chromatography-mass spectroscopy, we identified 11 compounds, out of which 9,12-Octadecadienoic acid, Hexadecanoic acid, and Tetradecanoic acid were found as major compounds. Subsequently, in silico molecular docking was conducted to confirm the nematicidal behavior of CSC based on binding interactions of the above three major compounds with the targeted protein acetylcholine esterase (AChE) of M. incognita. The values of binding free energy are -5.3, -4.5, and -4.9 kcal/mol, observed for 9,12-Octadecadienoic acid, n-Hexadecanoic acid, and Tetradecanoic acid, respectively, suggesting that 9,12-Octadecadienoic acid binds with the receptor AChE more efficiently than the other two ligands. This study indicates that CSC has nematicidal potential that can be used to control M. incognita for sustainable agriculture.

Protective Effect of Catharanthus roseus Extract on Cadmium-Induced Toxicity in Albino Rats: A Putative Mechanism of Detoxification.

Globally, people are highly affected by Cadmium (Cd), the most hazardous heavy metal. It has been implicated in various pathogeneses. Oxidative stress may be one the main reasons for Cd-induced disorders in the body. This article investigates the protective ability of Catharanthus roseus (CR) extract on oxidative stress in the kidney and liver of rats exposed to Cd. After 21 days, a significant increase in MDA concentration (6.81 ± 0.05), (6.64 ± 0.03) was observed in Cd-treated groups compared to the control (5.54 ± 0.02), (5.39 ± 0.04) for the kidney and liver, respectively, while significant changes were observed in the haematological parameters. Antioxidant enzymes, GPx, CAT, and SOD showed a significant decrease in their activity. We established that increasing the concentration of Cd in the presence of H2O2 was able to cause stand scission in pBR322 plasmid DNA, which may be due to the mediation of ROS generated in the process. The antioxidant ability of CR extract was tested in DPPH and H2O2 scavenging assay, depicted by the increase in the percentage inhibition. Upon treatment of CR extract to rats, MDA concentration was decreased for the kidney and liver compared to the Cd-treated groups. This was again confirmed by comet assay of both tissues, where the degree of cellular DNA breakage caused by Cd toxicity decreased significantly upon treatment with CR extract. Overall, the results suggest that Cd plays a major role as an effector metal ion, causing a decrease in the concentration and activity of AO enzymes and enhanced lipid peroxidation. ROS production resulted in oxidative DNA damage within the cell, whereas CR extract showed potential antioxidant activity against ROS-mediated DNA damage induced by Cd poisoning.

Moxifloxacin Disrupts and Attenuates Aß42 Fibril and Oligomer Formation: Plausibly Repositioning an Antibiotic as Therapeutic against Alzheimer's Disease.

The aggregation of Aß42 is established as a key factor in the development of Alzheimer's disease (AD). Consequently, molecules that inhibit aggregation of peptide may lead to therapies to prevent or control AD. Several studies suggest that oligomeric intermediates present during aggregation may be more cytotoxic than fibrils themselves. In this work, we examine the inhibitory activity of an antibiotic MXF on aggregation (fibrils and oligomers) and disaggregation of Aß42 using various biophysical and microscopic studies. Computational analysis was done to offer mechanistic insight. The amyloid formation of Aß42 is suppressed by MXF, as demonstrated by the decrease in both the corresponding ThT fluorescence intensity and other biophysical techniques. The lag phase of amyloid formation doubled from 4.53 to 9.66 h in the presence of MXF. The addition of MXF at the completion of the fibrillation reaction, as monitored by ThT, led to a rapid, concentration dependent, exponential decrease in fluorescence signal that was consistent with loss of fibrils. We used TEM to directly demonstrate that MXF caused fibrils to disassemble. Our docking results show that MXF binds to both monomeric and fibrillar forms of Aß42 with significant affinities. We also observed breaking of fibrils in the presence of MXF through molecular dynamics simulation. These findings suggest that antibiotic MXF could be a promising lead compound with dual role as fibril/oligomer inhibitor and disaggregase for further development as potential repurposed therapeutic against AD.

Luteolin Causes 5'CpG Demethylation of the Promoters of TSGs and Modulates the Aberrant Histone Modifications, Restoring the Expression of TSGs in Human Cancer Cells.

Cancer progression is linked to abnormal epigenetic alterations such as DNA methylation and histone modifications. Since epigenetic alterations, unlike genetic changes, are heritable and reversible, they have been considered as interesting targets for cancer prevention and therapy by dietary compounds such as luteolin. In this study, epigenetic modulatory behaviour of luteolin was analysed on HeLa cells. Various assays including colony forming and migration assays, followed by biochemical assays of epigenetic enzymes including DNA methyltransferase, histone methyl transferase, histone acetyl transferase, and histone deacetylases assays were performed. Furthermore, global DNA methylation and methylation-specific PCR for examining the methylation status of CpG promoters of various tumour suppressor genes (TSGs) and the expression of these TSGs at transcript and protein level were performed. It was observed that luteolin inhibited migration and colony formation in HeLa cells. It also modulated DNA methylation at promoters of TSGs and the enzymatic activity of DNMT, HDAC, HMT, and HAT and reduced the global DNA methylation. Decrease in methylation resulted in the reactivation of silenced tumour suppressor genes including FHIT, DAPK1, PTEN, CDH1, SOCS1, TIMPS, VHL, TP53, TP73, etc. Hence, luteolin-targeted epigenetic alterations provide a promising approach for cancer prevention and intervention.

On the binding reaction of loratadine with human serum acute phase protein alpha 1-acid glycoprotein.

Loratadine is an important anti-allergic drug. It is a second generation antihistamine drug used to treat allergic rhinitis, hay fever and urticaria. Human serum alpha 1-acid glycoprotein (AG) is an important acute phase protein and its serum concentration is found to increase in inflammation and acute response.The binding interaction between loratadine and AG is studied using spectroscopy and molecular docking techniques. The results obtained from fluorescence quenching experiments demonstrated that the fluorescence intensity of AG is quenched by loratadine. Loratadine was found to bind AG with the binding constant of ≈104 at 298 K. The Gibb's free energy change was found to be negative for the interaction of loratadine with AG indicating the binding process is spontaneous. Binding of loratadine with AG induced ordered structures in the protein. Hydrogen bonding and hydrophobic interactions were the main bonding forces between AG-loratadine as revealed by molecular docking results. This study suggests the importance of binding of anti-allergic drug to AG spatially in the diseases where the plasma concentration of AG increases many folds and interaction with this protein becomes significant. This study will help in design of drug dosage and adjustment accordingly to achieve optimal treatment outcome. Communicated by Ramaswamy H. Sarma.

Mechanistic In Vitro Dissection of the Inhibition of Amyloid Fibrillation by n-Acetylneuraminic Acid: Plausible Implication in Therapeutics for Neurodegenerative Disorders.

A variety of neurodegenerative disorders including Parkinson's disease are due to fibrillation in amyloidogenic proteins. The development of therapeutics for these disorders is a topic of extensive research as effective treatments are still unavailable. The present study establishes that n-acetylneuraminic acid (Neu5ac) inhibits the amyloid fibrillation of hen egg-white lysozyme (HEWL) and α-synuclein (SYN), as observed using various biophysical techniques and cellular assays. Neu5ac inhibits the amyloid formation in both proteins, as suggested from the reduction in the ThT fluorescence and remnant structures in transmission electron microscopy micrographs observed in its presence. In HEWL fibrillation, Neu5ac decreases the hydrophobicity and resists the transition of the α-helix to a ß-sheet, as observed by an ANS binding assay, circular dichroism (CD) spectra, and Fourier transform infrared measurements, respectively. Neu5ac stabilizes the states that facilitate the amyloid formation in HEWL and SYN, as demonstrated by an enhanced intrinsic fluorescence in its presence, which is further confirmed by an increase in Tm obtained from differential scanning calorimetry thermograms and an increase in the near-UV CD signal for HEWL with Neu5ac. However, the increase in stability is not a manifestation of Neu5ac binding to amyloid facilitating (partially folded or native) states of both proteins, as verified by isothermal titration calorimetry and fluorescence binding measurements. Besides, Neu5ac also attenuates the cytotoxicity of amyloid fibrils, as evaluated by a cell toxicity assay. These findings provide mechanistic insights into the Neu5ac action against amyloid fibrillation and may establish it as a plausible inhibitor molecule against neurodegenerative disorders.

Correction: The Impact of COVID-19 Management Policies Tailored to Airborne SARS-CoV-2 Transmission: Policy Analysis.

[This corrects the article DOI: 10.2196/20699.].

The Impact of COVID-19 Management Policies Tailored to Airborne SARS-CoV-2 Transmission: Policy Analysis.

BACKGROUND: Daily new COVID-19 cases from January to April 2020 demonstrate varying patterns of SARS-CoV-2 transmission across different geographical regions. Constant infection rates were observed in some countries, whereas China and South Korea had a very low number of daily new cases. In fact, China and South Korea successfully and quickly flattened their COVID-19 curve. To understand why this was the case, this paper investigated possible aerosol-forming patterns in the atmosphere and their relationship to the policy measures adopted by select countries. OBJECTIVE: The main research objective was to compare the outcomes of policies adopted by countries between January and April 2020. Policies included physical distancing measures that in some cases were associated with mask use and city disinfection. We investigated whether the type of social distancing framework adopted by some countries (ie, without mask use and city disinfection) led to the continual dissemination of SARS-CoV-2 (daily new cases) in the community during the study period. METHODS: We examined the policies used as a preventive framework for virus community transmission in some countries and compared them to the policies adopted by China and South Korea. Countries that used a policy of social distancing by 1-2 m were divided into two groups. The first group consisted of countries that implemented social distancing (1-2 m) only, and the second comprised China and South Korea, which implemented distancing with additional transmission/isolation measures using masks and city disinfection. Global daily case maps from Johns Hopkins University were used to provide time-series data for the analysis. RESULTS: The results showed that virus transmission was reduced due to policies affecting SARS-CoV-2 propagation over time. Remarkably, China and South Korea obtained substantially better results than other countries at the beginning of the epidemic due to their adoption of social distancing (1-2 m) with the additional use of masks and sanitization (city disinfection). These measures proved to be effective due to the atmosphere carrier potential of SARS-CoV-2 transmission. CONCLUSIONS: Our findings confirm that social distancing by 1-2 m with mask use and city disinfection yields positive outcomes. These strategies should be incorporated into prevention and control policies and be adopted both globally and by individuals as a method to fight the COVID-19 pandemic.

Biophysical insight into the interaction of levocabastine with human serum albumin: spectroscopy and molecular docking approach.

Interaction of levocabastine with human serum albumin (HSA) is investigated by applying fluorescence spectroscopy, circular dichroism spectroscopy and molecular docking methods. Levocabastine is an important drug in treatment of allergy and currently a target drug for drug repurposing to treat other diseases like vernal keratoconjuctivitis. Fluorescence quenching data revealed that levocabastine bind weakly to protein with binding constant in the order of 103 M-1. Förster resonance energy transfer results indicated the binding distance of 2.28 nm for levocabastine. Synchronous fluorescence result suggest slight blue shift for tryptophan upon levocabastine binding, binding of levocabastine impelled rise in α-helical structure in protein, while there are minimal changes in tertiary structure in protein. Moreover, docking results indicate levocabastine binds to pocket near to the drug site-I in HSA via hydrogen bonding and hydrophobic interactions. Understanding the interaction of levocabastine with HSA is significant for the advancement of therapeutic and diagnostic strategies for optimal treatment results.Communicated by Ramaswamy H. Sarma.

Biophysical Insight into the Interaction of Human Lysozyme with Anticancer Drug Anastrozole: A Multitechnique Approach.

In the present study, we employ fluorescence spectroscopy, dynamic light scattering, and molecular docking methods. Binding of anticancer drug anastrozole with human lysozyme (HL) is studied. Binding of anastrozole to HL is moderate but spontaneous. There is anastrozole persuaded hydrodynamic change in HL, leading to molecular compaction. Binding of anastrozole to HL also decreased in vitro lytic activity of HL. Molecular docking results suggest the electrostatic interactions and van der Waals forces played key role in binding interaction of anastrozole near the catalytic site. Binding interaction of anastrozole to proteins other than major transport proteins in blood can significantly affect pharmacokinetics of this molecule. Hence, rationalizing drug dosage is important. This study also points to unrelated effects that small molecules bring in the body that are considerable and need thorough investigation.

Biophysical insights into the interaction of clofazimine with human alpha 1-acid glycoprotein: a multitechnique approach.

Alpha1-acid glycoprotein (AAG) is a major acute phase protein of human plasma. Binding of clofazimine to AAG is investigated using optical spectroscopy and molecular docking tools. We found significant quenching of intrinsic fluorescence of AAG upon the binding of clofazimine, binding mode is static with binding constant of 3.52 × 104at 298 K. The Gibbs free energy change is found to be negative for the interaction of clofazimine with AAG indicating spontaneity of the binding process. Binding of clofazimine induced ordered structure in protein and lead to molecular compaction. Molecular docking results indicate the binding site is located in the central beta barrel, hydrogen bonding and hydrophobic interactions are main bonding forces between AAG-clofazimine.

Amino group of salicylic acid exhibits enhanced inhibitory potential against insulin amyloid fibrillation with protective aptitude toward amyloid induced cytotoxicity.

Protein misfolding and aggregation lead to amyloid generation that in turn may induce cell membrane disruption and leads to cell apoptosis. In an effort to prevent or treat amyloidogenesis, large number of studies has been paying attention on breakthrough of amyloid inhibitors. In the present work, we aim to access the effect of two drugs, that is, acetylsalicylic acid and 5-amino salicylic acid on insulin amyloids by using various biophysical, imaging, cell viability assay, and computational approaches. We established that both drugs reduce the turbidity, light scattering and fluorescence intensity of amyloid indicator dye thioflavin T. Premixing of drugs with insulin inhibited the nucleation phase and inhibitory potential was boosted by increasing the concentration of the drug. Moreover, addition of drugs at the studied concentrations attenuated the insulin fibril induced cytotoxicity in breast cancer cell line MDA-MB-231. Our results highlight the amino group of salicylic acid exhibited enhanced inhibitory effects on insulin fibrillation in comparison to acetyl group. It may be due to presence of amino group that helps it to prolong the nucleation phase with strong binding as well as disruption of aromatic and hydrophobic stacking that plays a key role in amyloid progression.

Investigating the site selective binding of busulfan to human serum albumin: Biophysical and molecular docking approaches.

We have studied the binding of busulfan (BN) to human serum albumin (HSA) at physiological pH 7.4 by using fluorescence, UV-vis and circular dichroism (CD) spectroscopic tools, as well as dynamic light scattering (DLS) measurements and molecular simulation approaches. HSA fluorescence quenching experiments showed that BN reduces the HSA native fluorescence intensity through the static mechanism. In addition, a single binding site on the HSA is occupied by BN with a binding constant at 298K of 1.84×103M-1. The enthalpy change (ΔH) and entropy change (ΔS) of BN-HSA interaction were calculated as -1.40kcalmol-1 and +10.14calmol-1K-1 respectively, which suggest the possible interaction mode as hydrophobic and hydrogen bonding. Moreover, the secondary structure alteration of HSA following its complexation with BN was studied and showed that α-helical content of HSA gets increased on interacting with BN. Ligand binding site to HSA was further investigated by site-specific markers in fluorescence measurements as well molecular modeling approach which indicated that BN bind to the nearby sudlow site II of HSA through hydrophobic as well as hydrogen bonding interaction. The present study will be helpful for understanding the binding mechanism of BN to human serum albumin.

Interaction of catecholamine precursor l-Dopa with lysozyme: A biophysical insight.

The current study comprises of an inclusive biophysical study, enlightening the binding of L-3, 4-dihydroxyphenylalanine (l-Dopa) with human lysozyme (HL) and hen egg white lysozyme (HEWL). Spectroscopic and molecular docking tools have been utilized to study the interaction of l-Dopa with both HL and HEWL. Spectrofluorimetric measurements exhibited that l-Dopa quenched the HL and HEWL intrinsic fluorescence. A binding constant (Kb) of ∼104M-1 for both HL and HEWL was obtained, asserting a significant binding. Negative value of ΔG affirmed that the reaction between proteins and l-Dopa was spontaneous. Far-UV CD spectra revealed a boost to the proteins helical content in the presence of l-Dopa. Furthermore, DLS measurements displayed the decrease in hydrodynamic radii (Rh) of HL and HEWL in the presence of l-Dopa. Molecular docking studies established that l-Dopa formed complexes with both the proteins through hydrogen bonding and hydrophobic interaction. The present study characterizing the l-Dopa interaction with lysozyme could be noteworthy in realizing both pharmaco-dynamics and/or -kinetics of drugs used in various diseases.

Biophysical insight into the interaction mechanism of plant derived polyphenolic compound tannic acid with homologous mammalian serum albumins.

Numerous phenolic compounds have been reported in the last decade that have a good antioxidant property and interaction affinity towards mammalian serum albumins. In the present study, we have utilized mammalian serum albumins as a model protein to examine their comparative interaction property with polyphenolic compound tannic acid (TA) by using various spectroscopic and calorimetric methods We have also monitored the esterase and antioxidant activity of mammalian serum albumins in the absence and presence of TA. The obtain results recommended that the TA have a good binding affinity (∼104 to 106M-1) towards mammalian serum albumins and shows double sequential binding sites, which depends on the concentration of TA that induced the conformational alteration which responsible for the thermal stability of proteins. Binding affinity, structural transition and thermodynamic parameters were calculated from spectroscopic and calorimetric method reveals that non-covalent interaction causes partial conformational alteration in the secondary structure of protein ie.; increase in α-helical content with decrease in ß-sheet, random coil and other structure. Meanwhile, we have found that esterase activities of serum albumins were also stabilized against hydrolysis and shows higher antioxidant activity in the presence of TA because albumins its self have an immense antioxidant activity beside TA.

Cysteine as a potential anti-amyloidogenic agent with protective ability against amyloid induced cytotoxicity.

Protein aggregation and misfolding have been allied with numerous human disorders and thus inhibition of such occurrence has been center for intense research efforts against these diseases. Here, we investigated anti-fibrillation activity of cysteine and its effect on kinetics of stem bromelain amyloid fibril formation. We established the anti-fibrillation and anti aggregation activities of cysteine by using multiple approaches like turbidity measurements, dye binding assays (ThT and ANS) and structural changes were monitored by circular dichroism (CD) followed by electron microscopy. Our experimental study inferred that cysteine inhibits temperature induced fibrillation of protein in a concentration dependent way. In addition, MDA-MB-231 cell viability of pre-formed amyloid was increased in presence of cysteine as compared to the fibrils alone. Furthermore, dynamic light scattering studies of native, aggregated as well as incubated (amyloids in presence of cysteine) samples indicates that cysteine restores native like structures of stem bromelain. Isothermal titration calorimetric results revealed that hydrogen bonding between cysteine and stem bromelain plays a significant role during inhibition of stem bromelain aggregation. However, thiophilic interaction between thiol group of cysteine and aromatic amino acid residue of stem bromelain may also have noteworthy role in inhibition of amyloid formation.

Probing the interaction of cephalosporin antibiotic-ceftazidime with human serum albumin: A biophysical investigation.

The fate of drug administered to a living organism depends on drug's pharmacokinetics as well as pharmacological behavior. Serum albumins (proteins in blood plasma of human) act as a carrier molecule to deliver the drug at specific site. In the present study, we have explored the mechanism of interaction between cephalosporin antibiotic-ceftazidime (CFD) and human serum albumin (HSA) by spectroscopic and molecular docking studies. Quenching of HSA fluorescence by CFD inferred that it binds to HSA through static quenching mechanism; with binding affinity in order of 104M-1. Fluorescence resonance energy transfer (FRET) results shows that donor and acceptor molecule are at 2.08nm apart and also reflects the high probability of energy transfer between HSA and CFD. Change in secondary structure as well as microenvironment around both tryptophan and tyrosine residue, were monitored by Circular Dichroism (CD) and Synchronous fluorescence spectroscopy respectively; confirms that CFD increases the alpha helical secondary structure as well as altered the environment around tryptophan and tyrosine. The specific binding site of CFD on HSA was determined by site-specific markers and molecular docking methods. CFD preferably bind to subdomain IIIA (Sudlow site II) on HSA.