Objective: To isolate and characterize a novel phytocystatin from walnut and investigate it for biopesticide development. Methods: A battery of methodology was employed. Initially, phytocystatin was extracted and purified from walnut using ammonium sulfate saturation (60-80%), followed by gel filtration chromatography on the Sephacryl S-100 HR column. Further characterization studies including pH and temperature stability, molecular weight, secondary structure, protease inhibitory assay and antimicrobial activity were carried using various techniques viz: spectroscopy, electrophoresis, and circular dichroism (CD) techniques. Results: Thiol protease inhibitor from walnut (WCPI) was isolated and purified with high (71.4%) yield and 184-fold purification. The molecular weight of the purified inhibitor was found to be around 11.2 kDa. Kinetic tests revealed that the inhibitor competitively inhibited papain and other cysteine proteases such as ficin and bromelain. It also exhibited significant antimicrobial activity against bacterial species. Conclusion: Walnut cysteine protease inhibitor (WCPI) from Juglans regia (Kashmiri walnut) was isolated to homogeneity and had features of other members of the phytocystatin family. It demonstrated potential antimicrobial activity and may serve as an initial step towards developing enhanced pest control methods based on natural molecules. (C) 2022 The Authors. Published by Elsevier B.V. on behalf of King Saud University.

Coronaviruses are enveloped non-segmented positive-sense RNA viruses belonging to the family Coronaviridae. The human coronavirus infections are mild; the epidemics of the two β-coronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) have caused more than ten thousand cumulative cases in the past twodecades. There is a new public health crisis threatening the world with the emergence and spread of 2019 novel coronavirus (2019-nCoV). The virus originated in bats and was transmitted to humans through yet unknown intermediary animals in Wuhan, Hubei province in China during the month of December 2019. Till date around 7,823,289 reported cases of coronavirus disease 2019 (COVID-2019) and 431,541 reported deaths till date. The disease is transmitted by inhalation or contact with infected droplets with incubation period of 2–14 days. The symptoms are usually fever, sore throat, dry cough, breathlessness, fatigue while many people are asymptomatic. Coronavirus (2019-nCoV) may progress to pneumonia, acute respiratory distress syndrome (ARDS) and can cause multi-organ dysfunction. Currently diagnosis is done by demonstration of the virus in respiratory secretions by special molecular tests like real-time reverse-transcription–polymerase-chain-reaction (RT-PCR), Radiological examinations (chest CT). Common laboratory tests like white cell counts and C-reactive protein (CRP) and measure symptoms can be used as preliminary screening at large scale after lock down the area or country. Treatment is essentially supportive; role of antiviral agents is yet to be established. It is paramount to implement infection control practices by infection source controlling, transmission route blocking, and susceptible population protection. Early preventive measures can be home isolation of suspected cases and those with mild illnesses and strict infection control measures at hospitals that include contact and droplet precautions. The worldwide impact of this Coronavirus new epidemic is yet uncertain.

BACKGROUND:Under certain circumstances, the path for protein folding deviates and attains an alternative path forming misfolded states, which are the key precursors for protein aggregation. Protein aggregation is associated with variety of diseases and leads to the cytotoxicity. These protein aggregate related diseases have been untreated so far. However, extensive attempts have been applied to develop anti-aggregating agents as possible approaches to overcome protein aggregation. Different types of substances have been reported to halt or decrease the formation of ordered protein aggregates both in vitro and in vivo, such as polyphenols and metal ions.OBJECTIVE:In the present study the in vitro aggregation of human serum albumin (HSA) by using a reactive dicarbonyl glyoxal has been investigated, simultaneously an attempt has been done to inhibit the glyoxal (GO) induced aggregation of (HSA) by caffeic acid (CA).METHODS:Different methods have been employed to investigate the process, fluorescence spectroscopy, circular dichroism, cango red binding assay, thioflavin T dye binding, turbidimetric analysis, docking study and transmission electron microscopy.RESULTS:Results have shown that elevated concentration of GO forms aggregates of HSA, and the activity of CA suggested the possibility of inhibiting the HSA aggregation at higher concentrations, and this compound was found to have an anti-aggregation property.CONCLUSION:The present study explained that micro molar concentrations of CA inhibits the aggregation of HSA and showed pronounced anti-aggregation effect at increasing concentrations in the presence of GO which is elevated in diabetic and hyperglycaemia conditions.

Tuberculosis (TB) is a dangerous infection affecting about one third of the world population, despite the availability of affordable and effective chemotherapy, remaining one of the major causes of death from a single infectious agent worldwide.

Hyperglycaemic conditions facilitate the glycation of serum proteins which may have predisposition to aggregation and thus lead to complications. The current study investigates the glycation induced structural and functional modifications of chickpea cystatin (CPC) as well as biological toxicity of the modified protein forms, using CPC-glucose as a model system. Several structural intermediates were formed during the incubation of CPC with glucose (day 4, 8, 12, & 16) as revealed by circular dichroism (CD), altered intrinsic fluorescence, and high ANS binding. Further incubation of CPC with glucose (day 21) formed abundant β structures as revealed by Fourier transform infrared spectroscopy and CD analysis which may be due to the aggregation of protein. High thioflavin T fluorescence intensity and increased Congo red absorbance together with enhanced turbidity and Rayleigh scattering by this modified form confirmed the aggregation. Electron microscopy finally provided the valid physical authentication about the presence of aggregate structures. Functional inactivation of glucose incubated CPC was also observed with time. Single cell electrophoresis of lymphocytes and plasmid nicking assays in the presence of modified CPC showed the DNA damage which confirmed its biological toxicity. Hence, our study suggests that glycation of CPC not only leads to structural and functional alterations in proteins but also to biotoxic AGEs and aggregates.

Reactive dicarbonyl species such as methylglyoxal (MGO) and glyoxal (GO) have recently received extensive attention due to their high reactivity and ability to modify biological substances such as proteins, phospholipids, and DNA. In case of proteins these reactive species mainly react with lysine and arginine residues to form AGEs, oxidative products, and aggregates. Chickpea cystatin (CPC) was incubated with varying concentrations of glyoxal and methylglyoxal which caused, along with altered secondary and tertiary structures, glycation, functional inactivation, altered redox state, cross-linking and high-molecular-mass aggregation. All these processes were examined and characterized by UV-Vis, fluorescence, and CD spectroscopies. Further characterization of CPC modified by reactive dicarbonyls was done by polyacrylamide gel electrophoresis which also showed alterations in the CPC molecules. Thus, in addition to describing the effects of GO and MGO on structure, conformation and function of CPC, this study also shows the relatively superior modifying effect of methylglyoxal for CPC in terms of glycation, oxidation and aggregation. This model system could shed some more light on the role of the reactive dicarbonyls in the specific alterations of proteins with different biological consequences having implications to ageing and disease such as diabetes.

Several mammalian proteins form pathological deposits under nonphysiological conditions that are associated with many degenerative diseases. Protein aggregation is associated with aging, as well as a variety of diseases, including cystic fibrosis, amyotrophic lateral sclerosis (ALS), and hypertrophic cardiomyopathy. There is a lack of any potential anti-amyloidogenic agents and therapeutics till date. Polyphenols have been accredited with myriad biological effects. An analysis of the effects of natural agents like baicalin (BC) and gallocatechin (GC) on aggregation process can open new avenues for the treatment of protein misfolding diseases. Thus, investigation of the effects of these flavonoids on Buffalo Heart Cystatin (BHC) aggregation induced by a reactive metabolic dialdehyde, glyoxal (GO), was taken up. Results have shown that elevated concentration of GO forms aggregates of BHC, which was characterized by an increase in the ANS fluorescence intensity, an increase in ThT fluorescence intensity, red shift in Congo red absorbance, negative ellipticity peak at 217 nm in the far-UVCD and BHC aggregates displaying by TEM. Using fluorescence spectroscopic analysis with Thioflavin T, CD and electron microscopic studies, anti-aggregation effects of polyphenols, BC and GC were analyzed. The study showed that BC and GC produced concentration-dependent anti-aggregation effects with GC producing a more pronounced effect than BC. The study proposed a mechanistic approach assuming structural constraints and specific aromatic interactions of polyphenols with sheets of BHC aggregates.

Herein, we have examined the interaction of oxy-diester novel twin tailed (gemini) surfactant, 2,2'-[(oxybis(ethane-1,2-diyl))bis(oxy)]bis(N-hexadecyl-N,Ndimethyl-2-oxoethanaminium) dichloride (C16-E2O-C16) with hen egg white lysozyme (HEWL), utilizing a spectroscopic and molecular docking techniques. Steady-state fluorescence infers ground state C16-E2O-C16-HEWL complex formation. Other spectroscopic results validated the conformational, structural and micro-environmental changes in HEWL upon interaction with C16-E2O-C16. Molecular modeling has shown that C16-E2O-C16 binds in the proximity of hydrophobic moieties (Trp-62/108). We believe the results of the current study will assist in designing the surfactant-enzyme systems for their end use as ingredients in pharmaceutical, cosmetic, drug delivery and industrial compilations. In terms of scientific literature standpoint, it will also enrich and widen the scope of biomacromolecule-surfactant interactions.

Metals such as Cu-2+, Fe-3+,Fe- and Zn2+ are major contributors to the biology of a brain in stages of health, aging, and disease because of their unique effects on both protein structures (misfolding) and oxidative stress. The relationship between metal ions and neurodegenerative diseases is very complicated. Our study highlights how metal ions influence amyloid formation at low pH and on preformed amyloid fibrils. By using thioflavin T assay, ANS fluorescence, Congo red assay, circular dichroism, and microscopy to elucidate the effects of Cu-2+,Cu- Fe3+, and Zn2+ on goat brain cystatin (GBC) aggregation at low pH. Results showed that Cu2+ and Fe3+ inhibit fibril formation of GBC by promoting amorphous aggregates. However, Zn2+ exclusively promotes fibril formation at low pH, leading to the formation of more ordered aggregates. Furthermore, the combined results of these complementary methods also suggested that Cu2+ and Fe3+ destabilize the beta-sheet secondary structure of preformed amyloid fibrils of GBC.

Cystatins, known for their ubiquitous presence in mammalian system are thiol protease inhibitors serving important physiological functions. Here, we present a variant of cystatin isolated from brain of Capra hircus (goat) which is glycosylated but lacks disulphide bonds. Caprine brain cystatin (CBC) was isolated using alkaline treatment, ammonium sulphate fractionation (40–60%) and gel filtration chromatography on Sephacryl S-100HR column with an overall yield of 26.29% and 322-fold purification. The inhibitor gave a molecular mass of ~44 kDa as determined by SDS-PAGE and gel filtration behaviour. The Stokes radius and diffusion coefficient of CBC were 27.14 Å and 8.18 × 10−7 cm2 s−1, respectively. Kinetic data revealed that CBC inhibited thiol proteases reversibly and competitively, with the highest inhibition towards papain (Ki = 4.10 nM) followed by ficin and bromelain. CBC possessed 34.7% α-helical content as observed by CD spectroscopy. UV, fluorescence, CD and FTIR spectroscopy revealed significant conformational change upon CBC-papain complex formation. Isothermal titration calorimetry (ITC) was used to measure the thermodynamic parameters – ΔH, ΔS, ΔG along with N (binding stoichiometry) for CBC-papain complex formation. Binding stoichiometry (N = .97 ± .07 sites) for the CBC-papain complex indicates that cystatin is surrounded by nearly one papain molecule. Negative ΔH (−5.78 kcal mol−1) and positive ΔS (11.01 cal mol−1 deg−1) values suggest that the interaction between CBC and papain is enthalpically as well as entropically favoured process. The overall negative ΔG (−9.19 kcal mol−1) value implies a spontaneous CBC-papain interaction.

The binding study of pesticides with proteins is of great importance in ecotoxicology. (a) Chlorpyrifos (CPF), an insecticide, (b) Mancozeb (MCZ), a fungicide, and (c) Glyphosate (GPS), a herbicide.

Many protein misfolding diseases in mammalian system are characterised by the accumulation of protein aggregates in amyloid fibrillar forms. Several therapeutic approaches include reduction in the production of the amyloidogenic form of proteins, increase in the clearance rate of misfolded or aggregated proteins, and direct inhibition of the self-assembly process have been explained. One of the possible remedial treatments for such disorders may be to identify molecules which are capable of either preventing formation of fibrils or disintegrating the formed fibrils. In this work, we have studied the effect of conventional surfactants; sodium dodecylsulphate (SDS), cetyl trimethylammonium bromide (CTAB) and dicationic gemini (16-4-16) surfactant on the disintegration of the goat brain cystatin (GBC) fibrils above their critical micelle concentrations (CMC) using ThT fluorescence, CD, TEM, Congo red and turbidity approaches. The results obtained are significant and showing the best disintegrating potency on GBC fibrils with gemini surfactant. The outcome from this work will aid in the development and/or design of potential inhibitory agents against amyloid deposits associated with amyloid diseases.

Herein we report the interaction of a biodegradable gemini surfactant, ethane-1,2-diyl bis(N,N-dimethyl-N-hexadecylammoniumacetoxy) dichloride (16-E2-16) with bovine milk xanthine oxidase (XO), employing tensiometry, fluorescence spectroscopy, UV spectroscopy, far-UV circular dichroism spectroscopy (CD), Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and computational molecular modeling. Surface tension results depict substantial changes in the micellar as well as interfacial parameters (CMC, ΠCMC, γCMC, Γmax, Amin, ΔGmic° and ΔGads°) of 16-E2-16 gemini surfactant upon XO combination, deciphering the interaction of XO with the gemini surfactant. Fluorescence measurements reveal that 16-E2-16 gemini surfactant causes quenching in the xanthine oxidase (XO) fluorescence spectra via static procedure and the values of various evaluated binding parameters (KSV, Kb, kq, ΔGb° and n) describe that 16-E2-16 effectively binds to XO. Three dimensional fluorescence, 8-anilino-1-naphthalene sulfonic acid (ANS) binding, F1F3 ratio, UV, CD, FTIR, SEM and TEM results delineate changes in the secondary structure of xanthine oxidase. Molecular docking results provide complement to the steady-state fluorescence findings and support the view that quenching occurs due to non-polar environment experienced by aromatic residues of the enzyme. The results of this study can help scientists to tune the conformation of an enzyme (XO) with biocompatible amphiphilic microstructures, which will help to unfold further understanding in the treatment modes of various diseases like gout, hyperuricemia, liver and brain necrosis.

More than 20 human diseases involve the fibrillation of a specific protein/peptide which forms pathological deposits at various sites. Hereditary lysozyme amyloidosis is a systemic disorder which mostly affects liver, spleen and kidney. This conformational disorder is featured by lysozyme fibril formation. In vivo lysozyme fibrillation was simulated under in vitro conditions using a strong denaturant GdHCl at 3 M concentration. Sharp decline in the ANS fluorescence intensity compared to the partially unfolded states, almost 20-fold increase in ThT fluorescence intensity, increase in absorbance at 450 nm suggesting turbidity, negative ellipticity peak in the far-UVCD at 217 nm, red shift of 50 nm compared to the native state in Congo red assay and appearance of a network of long rope-like fibrils in transmission electron microscope (TEM) analysis suggested HEWL fibrillation. Anti-fibrillation potency of baicalein against the preformed fibrils of HEWL was investigated following ThT assay in which there was a dose-dependent decrease in ThT fluorescence intensity compared to the fibrillar state of HEWL with the maximum effect observed at 150-μM baicalein concentration, loss of negative ellipticity peak in the far-UVCD region, dip in the Rayleigh scattering intensity and absorbance at 350 and 450 nm, respectively, together with a reduction in the density of fibrillar structure in TEM imaging. Thus, it could be suggested that baicalein could prove to be a positive therapeutics for hereditary human lysozyme amyloidosis. Graphical abstract

Thiol protease inhibitors (cystatins) are implicated in various disease states from cancer to neurodegenerative conditions and immune responses. Cystatins have high amyloidogenic propensity and they are prone to form fibrillar aggregates leading to amyloidosis. Particularly challenging examples of such disorders occur in type 2 diabetes, Alzheimer’s and Parkinson’s diseases. The aim of the present study is to find an interaction between the compound methylglyoxal (MG) which is particularly elevated in type 2 diabetes with caprine brain cystatin (CBC). Results have shown that elevated concentration of MG forms amyloid aggregates of CBC. This was achieved by allowing slow growth in a solution containing moderate to high concentrations of MG. When analysed with microscopy, the protein aggregate present in the sample after incubation consisted of extended filaments with ordered structures. This fibrillar material possesses extensive β-sheet structure as revealed by far-UV CD and IR spectroscopy. Furthermore, the fibrils exhibit increased Thioflavin T fluorescence.

Germplasm of Psoralea corylifolia L.,one of the most important endangered medicinal plants of Chinese and Indian Pharmacopeia were treated with ethyl methane sulphonate (0, 0.1, 0.25, 0.50, 0.75 and 1 %) and sampled for observations in the form of growth, non enzymatic systems and stress proteins at pre-and post-flowering stages of development. Higher concentrations of EMS showed significant (P < 0.001) inhibition in the germination percentage from 3.88 to 35.46 % at pre-flowering stage and from 4.90 to 35.97 % at post-flowering stage when compared to control. Other growth related parameters viz shoot growth and root growth also declined within increase in the dose as compared to control plants. Root growth was severely affected as compared to shoot growth. Roots showed a significant correlation with tolerance index as increasing concentration of EMS progressively inhibited the root growth. Plants developed from treated germplasm at higher doses exhibit higher activity of ascorbate peroxidase (APX), up to flowering and after that declined. Contrast to that, total soluble protein (TSP) content decreased in age and dose wise manner. The EMS dose applied and oxidative stress was correlated and derived from the increased antioxidant activities and decrease in total glutathione content from 122.75 % (GSH) and 103.29 % (GSSG) in plants raised from treated germplasm towards the post-flowering stage. Pronounced accumulation of psoralen from 10.74 to 93.24 % was observed following higher doses of EMS at post-flowering stage, where EMS induced oxidative stress-enhanced proline accumulation and lipid peroxidation. In addition, the prevalence of maximum antioxidant activity, psoralen accumulation was an efficient process in acclimatizing P. corylifolia to stress. 2-Dimensional gel electrophoresis (2D-GE) of the protein expression profile and MALDI TOF–MS revealed an expression of 20 different proteins which are associated with such functions as cellular detoxification, stress defense, psoralen production, photosynthesis, signal transduction, and cell cycle. The increase in the psoralen pool after EMS treatment proved to be a best protective antioxidant to counterbalance EMS-induced ROS detoxification and can be used as a biomarker and a scavenger in stress physiology.

Several mammalian proteins fold abnormally under non physiological conditions, to form pathological deposits that are associated with many degenerative diseases. In vitro variation of solvent conditions and pH can lead to partial unfolding and subsequent fibril formation. In the present study, we examined the effects of low pH on goat brain cystatin (GBC) with a focus on amyloid fibril formation. The results demonstrate that GBC can form amyloid like fibrils at pH 3.0. Moreover this study is aimed at exploring the inhibitory activity of polyphenols, Kaempferol (KM) and Catechin (CA) against the fibrillation of GBC. Using fluorescence spectroscopic analysis with Thioflavin T, CD and electron microscopic studies, anti-fibrillation effects of polyphenols, KM and CA were analyzed. The study also revealed that KM and CA produced a concentration dependent anti-fibrillogenic effects with KM producing more pronounced effect compared to CA. The study proposed a mechanistic approach assuming structural constraints and specific aromatic interactions of polyphenols with β sheets of GBC fibrils.

Physiological conditions corresponding to oxidative stress deplete the level of enzyme glyoxalase, facilitating a hike in the serum concentration of glyoxal. Simulating an elevated in vivo level of glyoxal, we tested (50%, v/v) concentration of glyoxal to interact with HEWL. Initially, docking study revealed that glyoxal binds in the hydrophobic core of the enzyme. The interaction between the dialdehyde (glyoxal) and the enzyme (HEWL) followed a three step transition involving pre-molten and molten globule states formed on days 7 and 15 of incubation respectively, which were characterised by an increase in the ANS fluorescence intensity compared to the native state. These molten globule states upon further incubation on day 20 resulted in the formation of aggregates which were characterised by an increase in ThT fluorescence intensity, red shift in Congo red absorbance, negative ellipticity peak at 217nm in the far-UV CD and the loss of signals at 284, 290 and 294nm in the near-UV CD spectra. Finally, TEM confirmed the authenticity of lysozyme fibril formation by displaying rod like fibrillar structure.