Sulfonamide based pyrimidine derivatives combating Plasmodium parasite by inhibiting falcipains-2 and falcipains-3 as antimalarial agents.

In this report, we present the design and synthesis of a novel series of pyrimidine-tethered spirochromane-based sulfonamide derivatives aimed at combating drug resistance in malaria. The antimalarial effectiveness of these compounds was assessed in vitro. Structural validation of the synthesized compounds was conducted using mass spectrometry and NMR spectroscopy. Strong antimalarial activity against CQ-sensitive (3D7) and CQ-resistant (W2) strains of Plasmodium falciparum was demonstrated by the majority of the compounds. Notably, compounds SZ14 and SZ9 demonstrated particularly potent effects, with compound SZ14 showing IC50 values of 2.84 µM and SZ9 3.22 µM, indicating single-digit micromolar activity. The compounds exhibiting strong antimalarial activity were assessed through enzymatic tests against the cysteine protease enzymes of P. falciparum, falcipain-2 and falcipain-3. The results indicated that SZ14 and SZ9 inhibited PfFP-2 (IC50 values: 4.1 and 5.4 µM, respectively), and PfFP-3 (IC50 values: 4.9 and 6.3 µM, respectively). To confirm the compounds' specificity towards the parasite, we investigated their cytotoxicity against Vero cell lines, revealing strong selectivity indices and no significant cytotoxic effects. Additionally, in vitro hemolysis testing showed these compounds to be non-toxic to normal human blood cells. Moreover, predicted in silico ADME parameters and physiochemical characteristics demonstrated the drug-likeness of the synthetic compounds. These collective findings suggest that sulfonamide derivatives based on pyrimidine-tethered oxospirochromane could serve as templates for the future development of potential antimalarial drugs.

Reinvestigation of diphenylmethylpiperazine analogues of pyrazine as new class of Plasmodial cysteine protease inhibitors for the treatment of malaria.

Malaria eradication is still a global challenge due to the lack of a broadly effective vaccine and the emergence of drug resistance to most of the currently available drugs as part of the mainline artemisinin-based combination therapy. A variety of experimental approaches are quite successful in identifying and synthesizing new promising pharmacophore hybrids with distinct mechanisms of action. Based on our recent findings, the current study demonstrates the reinvestigation of a series of diphenylmethylpiperazine and pyrazine-derived molecular hybrids. Pyrazine-derived molecular hybrids were screened to investigate the antiplasmodial activity on drug-susceptible Pf3D7 and drug-resistant PfW2 strains. The selected compounds were shown to be potent dual inhibitors of cysteine protease PfFP2 and PfFP3. Time-course parasitic development study demonstrated that compounds were able to arrest the growth of the parasite at the early trophozoite stage. The compounds did not show hemolysis of red blood cells and showed selectivity to the parasite compared with the mammalian Vero and A5489 cell lines. The study underlined HR5 and HR15 as a new class of Plasmodial falcipain inhibitors with an IC50 of 6.2 µM and 5.9 µM for PfFP2 and 6.8 µM and 6.4 µM for PfFP3, respectively. Both compounds have antimalarial efficacy with IC50 values of 3.05 µM and 2.80 µM for the Pf3D7 strain, and 4.35 µM and 3.39 µM for the PfW2 strain, respectively. Further structural optimization may turn them into potential Plasmodial falcipain inhibitors for malaria therapeutics.

Clay-Polymer Nanocomposites Mediated Inhibition of Protein Aggregation: Possible Role in the Prevention of Proteinopathies.

BACKGROUND: The transformation of proteins from their native conformation into highly ordered fibrillar structures due to their misfolding and aggregation under particular conditions are described as beta-sheet enriched amyloid fibrils. The accumulation of these fibrils in different body parts is the major cause of several neurological and non-neurological conditions (proteinopathies). OBJECTIVES: To prevent these proteinopathies, inhibition of protein aggregation is considered a promising strategy. Therefore, in this study, we synthesized montmorillonite (MMT) based poly- orthophenylenediamine (PoPD) nanocomposites (NCs) and characterized their size and morphology due to their remarkable biological properties. Further, the effect of these nanocomposites on inhibition of fibril formation was assessed. METHODS: These nanocomposites were evaluated for their anti-amyloidogenic potential on two model proteins of amyloidopathies, i.e., human lysozyme and human serum albumin (HL & HSA), by using several biophysical methods, such as Thioflavin T (ThT) and 1-anilino-8-naphthalene sulfonate (ANS) fluorescence, congo red dye binding assay (CR). Secondary structural content was evaluated by Circular dichroism (CD) spectroscopy. RESULTS: Results demonstrated that synthesized nanocomposites significantly inhibited fibril formation in dose-dependent manner that corresponds to their ability to arrest fibrillation. It is suggested that they may adsorb proteins to protect them against aggregation when they are subjected to aggregating conditions. CONCLUSION: This study offers an opportunity to understand the mechanism of inhibition of fibril formation by nanocomposites, showing that they inhibit amyloid formation and amyloid diseases. Thus, the study concludes that these nanocomposites are promising candidates as therapeutic molecules for proteinopathies and are envisaged to enrich the area of personalized medicine, augmenting the human healthcare system.

Anionic surfactant causes dual conformational changes in insulin.

Amyloid fibrillation is a process by which proteins aggregate and form insoluble fibrils that are implicated in several neurodegenerative diseases. In n this study, we aimed to investigate the impact of the negatively charged detergent sodium dodecyl sulfate (SDS) on insulin amyloid fibrillation at pH 7.4 and 2.0, as SDS has been linked to the acceleration of amyloid fibrillation in vitro, but the underlying molecular mechanism is not fully understood. Our findings show that insulin forms amyloid-like aggregates in the presence of SDS at concentrations ranging from 0.05 to 1.8 mM at pH 2.0, while no aggregates were observed at SDS concentrations greater than 1.8 mM, and insulin remained soluble. However, at pH 7.4, insulin remained soluble regardless of the concentration of SDS. Interestingly, the aggregated insulin had a cross-ß sheet secondary structure, and when incubated with higher SDS concentrations, it gained more alpha-helix. The electrostatics and hydrophobic interaction of SDS and insulin may contribute to amyloid induction. Moreover, the SDS-induced aggregation was not affected by the presence of salts. Furthermore, as the concentration of SDS increased, the preformed insulin amyloid induced by SDS began to disintegrate. Overall, our study sheds light on the mechanism of surfactant-induced amyloid fibrillation in insulin protein.

Design, synthesis, and biological evaluation of morpholinopyrimidine derivatives as anti-inflammatory agents.

Here, we outline the synthesis of a few 2-methoxy-6-((4-(6-morpholinopyrimidin-4-yl)piperazin-1-yl)(phenyl)methyl)phenol derivatives and assess their anti-inflammatory activity in macrophage cells that have been stimulated by LPS. Among these newly synthesized morpholinopyrimidine derivatives, 2-methoxy-6-((4-methoxyphenyl)(4-(6-morpholinopyrimidin-4-yl)piperazin-1-yl)methyl)phenol (V4) and 2-((4-fluorophenyl)(4-(6-morpholinopyrimidin-4-yl)piperazin-1-yl)methyl)-6-methoxyphenol (V8) are two of the most active compounds which can inhibit the production of NO at non-cytotoxic concentrations. Our findings also showed that compounds V4 and V8 dramatically reduced iNOS and cyclooxygenase mRNA expression (COX-2) in LPS-stimulated RAW 264.7 macrophage cells; western blot analysis showed that the test compounds decreased the amount of iNOS and COX-2 protein expression, hence inhibiting the inflammatory response. We find through molecular docking studies that the chemicals had a strong affinity for the iNOS and COX-2 active sites and formed hydrophobic interactions with them. Therefore, use of these compounds could be suggested as a novel therapeutic strategy for inflammation-associated disorders.

Development of diphenylmethylpiperazine hybrids of chloroquinoline and triazolopyrimidine using Petasis reaction as new cysteine proteases inhibitors for malaria therapeutics.

Malaria is a widespread infectious disease, causing nearly 247 million cases in 2021. The absence of a broadly effective vaccine and rapidly decreasing effectiveness of most of the currently used antimalarials are the major challenges to malaria eradication efforts. To design and develop novel antimalarials, we synthesized a series of 4,7-dichloroquinoline and methyltriazolopyrimidine analogues using a multi-component Petasis reaction. The synthesized molecules (11-31) were screened for in-vitro antimalarial activity against drug-sensitive and drug-resistant strains of Plasmodium falciparum with an IC50 value of 0.53 µM. The selected compounds were screened to evaluate in-vitro and in-silico enzyme inhibition efficacy against two cysteine proteases, PfFP2 and PfFP3. The compounds 15 and 17 inhibited PfFP2 with an IC50 = 3.5 and 4.8 µM, respectively and PfFP3 with an IC50 = 4.9 and 4.7 µM, respectively. Compounds 15 and 17 were found equipotent against the Pf3D7 strain with an IC50 value of 0.74 µM, whereas both were displayed IC50 values of 1.05 µM and 1.24 µM for the PfW2 strain, respectively. Investigation of effect of compounds on parasite development demonstrated that compounds were able to arrest the growth of the parasites at trophozoite stage. The selected compounds were screened for in-vitro cytotoxicity against mammalian lines and human red-blood-cell (RBC), which demonstrated no significant cytotoxicity associated with the molecules. In addition, in silico ADME prediction and physiochemical properties supported the drug-likeness of the synthesized molecules. Thus, the results highlighted the diphenylmethylpiperazine group cast on 4,7-dichloroquinoline and methyltriazolopyrimidine using Petasis reaction may serve as models for the development of new antimalarial agents.

Physico-Chemical Properties and Deposition Potential of PM2.5 during Severe Smog Event in Delhi, India.

The present work studies a severe smog event that occurred in Delhi (India) in 2017, targeting the characterization of PM2.5 and its deposition potential in human respiratory tract of different population groups in which the PM2.5 levels raised from 124.0 µg/m3 (pre-smog period) to 717.2 µg/m3 (during smog period). Higher concentration of elements such as C, N, O, Na, Mg, Al, Si, S, Fe, Cl, Ca, Ti, Cr, Pb, Fe, K, Cu, Cl, P, and F were observed during the smog along with dominant organic functional groups (aldehyde, ketones, alkyl halides (R-F; R-Br; R-Cl), ether, etc.), which supported potential contribution from transboundary biomass-burning activities along with local pollution sources and favorable meteorological conditions. The morphology of individual particles were found mostly as non-spherical, including carbon fractals, aggregates, sharp-edged, rod-shaped, and flaky structures. A multiple path particle dosimetry (MPPD) model showed significant deposition potential of PM2.5 in terms of deposition fraction, mass rate, and mass flux during smog conditions in all age groups. The highest PM2.5 deposition fraction and mass rate were found for the head region followed by the alveolar region of the human respiratory tract. The highest mass flux was reported for 21-month-old (4.7 × 102 µg/min/m2), followed by 3-month-old (49.2 µg/min/m2) children, whereas it was lowest for 21-year-old adults (6.8 µg/min/m2), indicating babies and children were more vulnerable to PM2.5 pollution than adults during smog. Deposition doses of toxic elements such as Cr, Fe, Zn, Pb, Cu, Mn, and Ni were also found to be higher (up to 1 × 10-7 µg/kg/day) for children than adults.

The adverse impact of excessive smartphone screen-time on sleep quality among young adults: A prospective cohort.

INTRODUCTION: Problematic over usage of smartphones has led to various deteriorating effects including poor sleep quality. Screen exposure, especially near bedtime, directly leads to poor sleep quality. We aimed to measure smartphone screen-time (ST) statistics of the participants directly using a smartphone application. Furthermore, we aimed to assess sleep quality using the Pittsburgh sleep quality index (PSQI), and to investigate the association between ST & PSQI. MATERIAL AND METHODS: This descriptive cohort study was conducted among 280 students of MBBS at Rawalpindi Medical University for a period of 1 month (30 days). Physically healthy students who owned Android smartphones were included in the study. Students with diagnosed sleep disorders and students taking sleep medication were excluded from the study. ST was recorded using a smartphone application. Sleep quality was assessed at the end of 30 days using the PSQI questionnaire. Data entry and analysis was done using SPSS v23.0. RESULTS: Total and mean ST were calculated for every participant. The mean screen time of 242 individuals was 147.50±51.09 hours. The mean PSQI score was 6.68±2.3. 65.70% of the participants had a poor sleep quality (PSQI>5). Pearson's correlation revealed that long total ST was associated with decreased sleep quality (R=0.356, p<0.001). CONCLUSION: Our findings are in accordance with previous scientific literature largely based on self-reported ST measurements and affirm that excessive ST deteriorates sleep quality and hence has numerous adverse physical and psychological manifestations.

Nanoclay based study on protein stability and aggregation and its implication in human health.

Protein aggregation is the major cause of several acute amyloid diseases such as Parkinson's, Huntington's, Alzheimer's, Lysozyme Systemic amyloidosis, Diabetes-II etc. While these diseases have attracted much attention but the cure is still unavailable. In the present study, Human Serum Albumin (HSA) and Human Lysozyme (HL) were used as the model proteins to investigate their aggregations. Nanoclays are hydrous silicates found in clay fraction of soil and known as natural nanomaterials. They have long been used in several applications in health-related products. In the present paper, the different types of nanoclays (MMT K-10, MMT K-30, Halloysite, Bentonite) were used to inhibit the process of HSA and HL aggregation. Aggregation experiments were evaluated using several biophysical tools such as Turbidity measurements, Intrinsic fluorescence, 1-anilino-8-naphthalene sulfonate (ANS), Thioflavin T (Th T), congo red (CR) binding assays and Circular dichroism. Results demonstrated that all the nanoclays inhibit the DTT-induced aggregation. However, bentonite and MMT K-10 were progressively intense and potent as they slowed down nucleation stage which can be perceived using several biophysical techniques. Hence, nanoclays can be used as an artificial chaperone and might provide effective treatment against several protein aggregation related disorders.

Electromyographic Activity of Hip Musculature During Functional Exercises in Participants With and Without Chronic Ankle Instability.

OBJECTIVE: The main objective of the present study was to investigate the electromyographic (EMG) activity of gluteus medius (Gmed) and gluteus maximus (Gmax) muscles during functional exercises in subjects with chronic ankle instability (CAI) vs healthy controls. METHODS: Seventeen subjects (age, 24.4 ± 2.03 years) with CAI and 17 healthy controls (age, 24.6 ± 2.57 years) were recruited for the present study. For all participants, after testing maximum voluntary isometric contraction of the Gmed and Gmax muscle, EMG activity of these muscles was recorded during functional exercises, such as the Y Balance Test and the single-leg squat with and without Swiss ball. RESULTS: EMG activity of Gmed and Gmax was found to be significantly (P < .05) reduced during all functional exercises in subjects with CAI when compared with healthy controls. No significant differences (P > .05) were observed in the EMG activity of both muscles across different functional exercises. CONCLUSION: Our findings indicate that EMG activity of hip muscles is significantly reduced in CAI subjects, which might give an indication regarding the inclusion of hip muscle strengthening (Gmax and Gmed) in the rehabilitation of CAI. Moreover, Gmed and Gmax muscle activity did not vary during the different functional exercises within each group, which might indicate that activation pattern of these muscles are not sensitive to the type of functional task.

Effect of cetyltrimethylammonium bromide (CTAB) on the conformation of a hen egg white lysozyme: A spectroscopic and molecular docking study.

The interactions between cetyltrimethylammonium bromide (CTAB) and hen egg white lysozymes (HEWL) was carried out to investigate protein-surfactant interaction mechanisms while both exist in the overall same charged state. The interactions between CTAB and the HEWL were examined with circular dichroism (CD), dynamic light scattering (DLS), fluorescence spectroscopy, and computational docking at a pH9.0 at room temperature. The far-UV CD and fluorescence results revealed that CTAB at concentrations from 0.15 to 10.0mM influenced the secondary as well as the tertiary structure of HEWL. The secondary structure of the HEWL was retained, while the tertiary structure of the HEWL was disrupted in the CTAB-treated samples at pH9.0. The hydrodynamic radii of the HEWL were also expanded in the presence of CTAB. Molecular docking studies showed that CTAB formed one electrostatic and four hydrophobic interactions, as well as one carbon hydrogen bond with HEWL. The data obtained from spectroscopic and computational studies demonstrated that the positively charged head and 18­carbon alkyl chain of the CTAB interacted through weak electrostatic and strong hydrophobic interactions.

Phytochemical analysis and In-vitro Biochemical Characterization of aqueous and methanolic extract of Triphala, a conventional herbal remedy.

Triphala, an Indian ayurvedic triherbal formulation, is an equiproportional mixture of fruits of three herbs, amalaki (Emblica officinalis), haritaki (Terminalia chebula) and bibhitaki (Terminalia bellerica). The present study focused on phytocompounds detection and comparative analysis of various biochemical activities in the aqueous and methanolic extracts of triphala and its constituting herbs. Antioxidant activity was determined by 1, 1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), super oxide dismutase (SOD), catalase assay. Antibacterial potential was determined by broth dilution and agar well diffusion assays. Results revealed the presence of valuable bioactive compounds such as flavonoids, alkaloids, phenols, etc which might be responsible for biochemical activities. Extracts exhibited satisfactory radical-scavenging activity comparable with ascorbic acid. Methanolic extracts demonstrated higher antioxidant activity compared to aqueous extract. Extracts showed promising antibacterial potential against tested strain comparable to ampicillin. Hence, it can be concluded that triphala may be a promising candidate in pharmaceuticals and future medicine.

Trypsin Inhibitors from Cajanus cajan and Phaseolus limensis Possess Antioxidant, Anti-Inflammatory, and Antibacterial Activity.

Protease inhibitors are one of the most promising and investigated subjects for their role in pharmacognostic and pharmacological studies. This study aimed to investigate antioxidant, anti-inflammatory, and antimicrobial activities of trypsin inhibitors (TIs) from two plant sources (Cajanus cajan and Phaseolus limensis). TI was purified from C. cajan (PUSA-992) by ammonium sulfate precipitation followed by ion exchange chromatography. TI from Phaseolus limensis (lima bean trypsin inhibitor; LBTI) was procured from Sigma-Aldrich, St. Louis, Missouri, United States. The antioxidant activity was analyzed by ferric ion reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH). The anti-inflammatory property of TIs was determined by inhibition of albumin denaturation assay. Ascorbic acid and aspirin were used as standards for antioxidant and anti-inflammatory assays, respectively. These TIs were tested against various bacterial and fungal strains. The TIs showed DPPH radical-scavenging activity in a concentration-dependent manner with IC50 values comparable to ascorbic acid. The FRAP values were also observed comparable to ascorbic acid and followed the trend of dose-dependent manner. The half maximal inhibitory concentration (IC50) values of CCTI and LBTI in anti-inflammatory test showed that LBTI is more potent than CCTI. The TIs showed potent antibacterial activity, but apparently no action against fungi. This study has reported the biological properties of CCTI and LBTI for the first time. The results show that TIs possess the ability to inhibit diseases caused by oxidative stress, inflammation, and bacterial infestation.

Panchakola Reduces Oxidative Stress in MCF-7 Breast Cancer and HEK293 Cells.

A large number of studies have proven the efficacy of ayurveda in the field of health and wellness. Panchakola, an ayurvedic formulation, is a general health tonic primarily used to cure fever, inflammation, pain, indigestion, and so on. We investigated effects of panchakola on oxidative stress in MCF-7 breast cancer and human embryonic kidney 293 (HEK293) cells. This work was performed to assess the antineoplastic and free radical-scavenging potential of aqueous extract of panchakola, a polyherbal formulation, in normal and breast cancer cell lines (i.e., HEK and MCF-7, respectively) using MTT assay. Activities of antioxidant enzyme, nitric oxide scavenger, superoxide dismutase, glutathione S-transferase, and glutathione peroxidase were assessed in cell lines incubated with and without panchakola. The outcome was analyzed by spectrophotometer. The results demonstrated increased cytotoxicity in MCF-7 (IC50 16.446 µg/ml) comparable to the results obtained with standard anticancer control (curcumin) with IC50 10.265 µg/ml in MCF-7 cell line. Further, the results obtained from antioxidant assays suggested increased antioxidant activity in MCF-7 cells as compared to normal HEK cells. The results derived from this study suggested panchakola is a strong contender in the field of phytomedicines to fight cancer and free radical-related diseases.

Structural and Biophysical Characterization of Cajanus cajan Protease Inhibitor.

CONTEXT: A large number of studies have proven that Protease inhibitors (PIs), specifically serine protease inhibitors, show immense divergence in regulation of proteolysis by targeting their specific proteases and hence, they play a key role in healthcare. OBJECTIVE: We aimed to access in-vitro anticancer potential of PI from Cajanus cajan (CCPI). Also, crystallization of CCPI was targetted alongwith structure determination and its structure-function relationship. MATERIALS AND METHODS: CCPI was purified from Cajanus cajan seeds by chromatographic techniques. The purity and molecular mass was determined by SDS-PAGE. Anticancer potential of CCPI was determined by MTT assay in normal HEK and cancerous A549 cells. The crystallization screening of CCPI was performed by commercially available screens. CCPI sequence was subject to BLASTp with homologous PIs. Progressive multiple alignment was performed using clustalw2 and was modelled using ab initio protocol of I-TASSER. RESULTS: The results showed ~14kDa CCPI was purified in homogeneity. Also, CCPI showed low cytotoxic effects of in HEK i.e., 27% as compared with 51% cytotoxicity in A549 cells. CCPI crystallized at 16°C using 15% PEG 6000 in 0.1M potassium phosphate buffer (pH 6.0) in 2-3weeks as rod or needles visualized as clusters under the microscope. The molecular modelling revealed that it contains 3 beta sheets, 3 beta hairpins, 2 ß-bulges, 6 strands, 3 helices, 1helix-helix interaction, 41 ß-turns and 27 γ-turns. DISCUSSION AND CONCLUSION: The results indicate that CCPI may help to treat cancer in vivo aswell. Also, this is the first report on preliminary crystallization and structural studies of CCPI.

A review on protein misfolding, aggregation and strategies to prevent related ailments.

This review aims to highlight the fundamental mechanism of protein misfolding leading to protein aggregation and associated diseases. It also aims to anticipate novel therapeutic strategies with which to prevent or treat these highly debilitating conditions linked to these pathologies. The failure of a protein to correctly fold de novo or to remain correctly folded can have profound consequences on a living system especially when the cellular quality control processes fail to eliminate the rogue proteins. The core cause of over 20 different human diseases which have now been designated as 'conformational diseases' including neurodegenerative diseases such as Alzheimer's disease (AD), Huntington's disease (HD) and Parkinson's disease (PD) etc. A comprehensive study on protein misfolding, aggregation, and the outcomes of the effects of cytotoxic aggregates will lead to understand the aggregation-mediated cell toxicity and serves as a foundation for future research in development of promising therapies and drugs. This review has also shed light on the mechanism of protein misfolding which leads to its aggregation and hence the neurodegeneration. From these considerations, one could also envisage the possibility that protein aggregation may be exploited by nature to perform specific physiological functions in differing biological contexts.

Trypsin inhibitors demonstrate antioxidant activities, inhibit A549 cell proliferation, and increase activities of reactive oxygen species scavenging enzymes.

OBJECTIVES: Protease inhibitors are one of the most promising and investigated subjects for their role in pharmacognostical and pharmacological studies. This study aimed to investigate antineoplastic and antioxidant activity of trypsin inhibitors (TIs) isolated from three plant sources and their inhibitory role in the cell line. MATERIALS AND METHODOLOGY: TIs were obtained from different plant sources. Antineoplastic potential on adenocarcinoma human alveolar basal epithelial cell line (A549) and normal Human Embryonic Kidney (HEK) was determined using MTT assay. Activities of antioxidant enzyme, nitric oxide scavenger, superoxide dismutase, glutathione S-transferase, and glutathione peroxidase were assessed in cell lines incubated with and without TIs. The outcome was analyzed by spectrophotometer. RESULTS: TIs showed the higher cytotoxicity on A549 cells as compared to normal HEK cell line. TIs exhibited fair increase in antioxidant enzyme activity in A549 cells as compared to control. This might be one of the strategies of antineoplastic effect in cancer cells. CONCLUSIONS: This study has reported the antioxidant and antineoplastic properties of these TIs for the first time in A549 cells (to the best of our knowledge). The results show that TIs possess ability to prevent cancer and diseases caused due to oxidative stress. Therefore, we conclude that TIs can be used as supplements along with the conventional drugs for increased efficacy in the treatment of diseases such as cardiovascular disease, atherosclerosis, and cancer.

pH induced single step shift of hydrophobic patches followed by formation of an MG state and an amyloidogenic intermediate in Lima Bean Trypsin Inhibitor (LBTI).

Lima Bean Trypsin Inhibitor (LBTI) is 83 residues monomeric protein of 9.0 KDa, consisting of six antiparallel ß-strands and can undergo concentration dependant dimerization. We have tried to characterize folding intermediates of LBTI under equilibrium denaturation conditions. We have used various spectroscopic and microscopic techniques to understand the folding and misfolding pathways. LBTI forms molten globule structure at pH 2 and amyloidiogenic intermediate state (Ia) at pH 4. pH induced Shifting of surface exposed hydrophobic patches and that followed by withdrawal of the lone tyrosine residue (Y69) towards nonpolar environment have been reported. Denaturation profile of native and molten globule (MG) states of LBTI in presence of guanidine hydrochloride show sigmoidal curves with non-coincidental and irreversible behaviour in both states. Concentration dependent amyloid fibril formation was confirmed by Thioflavin T and Congo Red binding and its morphology was studied by transmission electron microscopy (TEM). This is the first report on biophysical characterization of folding intermediates of LBTI and its aggregation behaviour to the best of our knowledge.

Biophysical insight into structure-function relation of Allium sativum Protease Inhibitor by thermal, chemical and pH-induced modulation using comprehensive spectroscopic analysis.

In this study, we have analyzed the structural and functional changes in the nature of Allium sativum Protease Inhibitor (ASPI) on undergoing various denaturation with variable range of pH, temperature and urea (at pH 8.2). ASPI being anti-tryptic in nature has native molecular mass of ∼15kDa. The conformational stability, functional parameters and their correlation were estimated under different conditions using circular dichroism, fluorescence and activity measurements. ASPI was found to fall in belongs to α+ß protein. It demonstrated structural and functional stability in the pH range 5.0-12.0 and up to70°C temperature. Further decrease in pH and increase in temperature induces unfolding followed by aggregation. Chemical induced denaturation was found to be cooperative and transitions were reversible and sigmoid. Tm (midpoint of denaturation), ΔCp (constant pressure heat capacity change) and ΔHm (van't Hoff enthalpy change at Tm were calculated to be 41.25±0.2°C, 1.3±0.07kcalmol-1K-1 and 61±2kcalmol-1 respectively for thermally denatured ASPI earlier. The reversibility of the protein was confirmed for both thermally and chemically denatured ASPI. The results obtained from trypsin inhibitory activity assay and structural studies are found to be in a significant correlation and hence established structure-function relationship of ASPI.

A novel multicopper oxidase (laccase) from cyanobacteria: Purification, characterization with potential in the decolorization of anthraquinonic dye.

A novel extracellular laccase enzyme produced from Spirulina platensis CFTRI was purified by ultrafiltration, cold acetone precipitation, anion exchange and size exclusion chromatography with 51.5% recovery and 5.8 purification fold. The purified laccase was a monomeric protein with molecular mass of ~66 kDa that was confirmed by zymogram analysis and peptide mass fingerprinting. The optimum pH and temperature of the enzyme activity was found at 3.0 and 30°C using ABTS as substrate but the enzyme was quite stable at high temperature and alkaline pH. The laccase activity was enhanced by Cu+2, Zn+2 and Mn+2. In addition, the dye decolorization potential of purified laccase was much higher in terms of extent as well as time. The purified laccase decolorized (96%) of anthraquinonic dye Reactive blue- 4 within 4 h and its biodegradation studies was monitored by UV visible spectra, FTIR and HPLC which concluded that cyanobacterial laccase can be efficiently used to decolorize synthetic dye and help in waste water treatment.