Screening and identification of potential MERS-CoV papain-like protease (PLpro) inhibitors; Steady-state kinetic and Molecular dynamic studies.

MERS-CoV belongs to the coronavirus group. Recent years have seen a rash of coronavirus epidemics. In June 2012, MERS-CoV was discovered in the Kingdom of Saudi Arabia, with 2,591 MERSA cases confirmed by lab tests by the end of August 2022 and 894 deaths at a case-fatality ratio (CFR) of 34.5% documented worldwide. Saudi Arabia reported the majority of these cases, with 2,184 cases and 813 deaths (CFR: 37.2%), necessitating a thorough understanding of the molecular machinery of MERS-CoV. To develop antiviral medicines, illustrative investigation of the protein in coronavirus subunits are required to increase our understanding of the subject. In this study, recombinant expression and purification of MERS-CoV (PLpro), a primary goal for the development of 22 new inhibitors, were completed using a high throughput screening methodology that employed fragment-based libraries in conjunction with structure-based virtual screening. Compounds 2, 7, and 20, showed significant biological activity. Moreover, a docking analysis revealed that the three compounds had favorable binding mood and binding free energy. Molecular dynamic simulation demonstrated the stability of compound 2 (2-((Benzimidazol-2-yl) thio)-1-arylethan-1-ones) the strongest inhibitory activity against the PLpro enzyme. In addition, disubstitutions at the meta and para locations are the only substitutions that may boost the inhibitory action against PLpro. Compound 2 was chosen as a MERS-CoV PLpro inhibitor after passing absorption, distribution, metabolism, and excretion studies; however, further investigations are required.

Role of site-directed mutagenesis and adjuvants in the stability and potency of anthrax protective antigen.

Anthrax is a zoonotic infection caused by the gram-positive, aerobic, spore-forming bacterium Bacillus anthracis. Depending on the origin of the infection, serious health problems or mortality is possible. The virulence of B. anthracis is reliant on three pathogenic factors, which are secreted upon infection: protective antigen (PA), lethal factor (LF), and edema factor (EF). Systemic illness results from LF and EF entering cells through the formation of a complex with the heptameric form of PA, bound to the membrane of infected cells through its receptor. The currently available anthrax vaccines have multiple drawbacks, and recombinant PA is considered a promising second-generation vaccine candidate. However, the inherent chemical instability of PA through Asn deamidation at multiple sites prevents its use after long-term storage owing to loss of potency. Moreover, there is a distinct possibility of B. anthracis being used as a bioweapon; thus, the developed vaccine should remain efficacious and stable over the long-term. Second-generation anthrax vaccines with appropriate adjuvant formulations for enhanced immunogenicity and safety are desired. In this article, using protein engineering approaches, we have reviewed the stabilization of anthrax vaccine candidates that are currently licensed or under preclinical and clinical trials. We have also proposed a formulation to enhance recombinant PA vaccine potency via adjuvant formulation.

Drug utilization and cost associated with inflammatory bowel disease management in Saudi Arabia.

BACKGROUND: There has been an increase in incidence and prevalence of inflammatory bowel disease (IBD) outside the western countries. Treatment costs are an essential component for healthcare planning and priority setting. The utilization patterns and annual administration and cost of IBD medications are largely unknown in countries with an increasing incidence of disease, Saudi Arabia being an example. AIM: To evaluate the use of non-biologic and biologic agents and their associated annual administration costs in a sample of patients with Crohn's disease (CD) and ulcerative colitis (UC) in Saudi Arabia. METHODS: Single-center retrospective chart review was performed to determine the use of biologic and non-biologic medications among IBD patients in a tertiary care hospital in Riyadh, Saudi Arabia. Daily and the annual acquisition cost of different IBD therapeutic agents was calculated. The utilization rates and cost of each type of medication by CD and UC patients were compared. RESULTS: Data of 258 CD patients and 249 UC patients were analyzed. Infliximab and adalimumab were the most commonly prescribed biologics among the study sample, however, their utilization rates were significantly higher among CD than UC patients (36.82% vs. 11.24%, and 20.54% vs. 9.64%, respectively, P < 0. 01). Azathioprine utilization rate was also higher among CD patients compared to their UC counterparts (71.71% vs. 40.16%, respectively, P < 0.01). However, the utilization rate of mesalazine in the UC patients was significantly higher than their CD counterparts (85.53% vs. 14.34% for CD, P < 0.01). The annual cost of biologics (including administration and lab test cost) ranged from 5572 USD for ustekinumab to 18,424 USD for vedolizumab. On the other hand, the annual cost of non-biologics ranged from 16 USD for prednisone to 527 USD for methotrexate. CONCLUSION: Biologics are extensively used in the management of IBD, particularly CD, and their utilization costs are significantly higher than non-biologics. Future studies should examine the cost effectiveness of IBD medications especially in countries with increasing incidence such as Saudi Arabia.

The status of licensed pharmacy workforce in Saudi Arabia: a 2030 economic vision perspective.

BACKGROUND: The economy of Saudi Arabia is currently undergoing a major transformation which will have an impact on employment in the pharmacy sector. However, quantitative data characterizing the pharmacy workforce in the Kingdom are currently not available. Therefore, the aim of this study was to determine the current status of the licensed pharmacy workforce in the pharmacy field in Saudi Arabia. METHODS: Descriptive statistics were performed on data from the Saudi Commission for Health Specialties (SCFHS) as of March 2017. RESULTS: The labor market for pharmacists in Saudi Arabia is dominated by expatriates. Saudi nationals constitute less than 20% of the pharmacists employed in the Kingdom. The underemployment of Saudis is most evident in the largest sectors of the pharmacy field, namely, private health care establishments, community pharmacies, and pharmaceutical companies. CONCLUSION: There is an unmet need to train Saudi citizens as pharmacists and retain them in the workforce. Addressing this issue should become an important objective in Saudi Arabia's Vision for 2030.

Aggregation and conformational stability evaluation of myoglobin in the presence of ionic surfactant.

Sodium lauroyl sarcosinate (SLS) is frequently used for the solubilization of inclusion bodies in vitro due to its structural similarity to lipid plasma membrane. There are many factors that could influence protein aggregation propensity, including overall protein surface charge and hydrophobicity. Here, the aggregation pathway of myoglobin protein was studied under different conditions (pH 3.5 and 7.4) in the presence of varying concentrations of SLS to evaluate the underlying forces dictating protein aggregation. Data obtained from Rayleigh light scattering, ThT binding assay, and far-UV CD indicated that SLS have different effects on the protein depending on its concentration and environmental conditions. In the presence of low concentrations of SLS (0.05-0.1 mM), no aggregation was detected at both pH conditions tested. Whereas, as we reach higher SLS concentrations (0.5-10.0 mM), myoglobin started forming larger-sized aggregates at pH 3.5 and not pH 7.4. These results suggest that electrostatics interactions as well as hydrophobic forces play an important role in SLS-induced myoglobin aggregation.

Shortage of psychotropic medications in community pharmacies in Saudi Arabia: Causes and solutions.

Background: Patients with mental disorders, such as depression and anxiety, who seek medical care in private psychiatric clinics in Riyadh, Saudi Arabia, have recently expressed concerns to doctors about difficulty in filling psychotropic medications, such as Amitriptyline and Aripiprazole, at retail community pharmacies. Objectives: The aim of this study was to investigate whether there is a shortage of some commonly prescribed psychotropic medications in retail community pharmacies in Saudi Arabia, and if so, to explore the possible reasons behind the shortage of these medications. Methods: The availability of 28 commonly prescribed psychotropic medications was checked in multiple retail community pharmacies in 4 different regions of Saudi Arabia. Further, potential reasons behind the shortage of some psychotropic medications in retail community pharmacies were also explored. Results: Amitriptyline, Amoxapine, Aripiprazole, Bupropion, Buspirone, Duloxetine, Haloperidol, Hydroxyzine, Lithium, Prochlorperazine, Procyclidine, Promethazine, Thioridazine, Trazodone, and Trifluoperazine were unavailable in over half of the 248 community pharmacies surveyed. Four possible reasons behind the shortage of these medications were reported by 31 pharmacists working in different retail community pharmacies' purchasing departments, with a majority (58.06%) reporting the primary reason for a shortage of these medications that they are slow-moving items with low profit margins. Conclusions: The findings of this study should expedite the reform process in both the Ministry of Health and the Saudi Food and Drug Authority (SFDA) to publish and enforce an essential list of medications for retail community pharmacies, which should include the most commonly prescribed psychotropic medications.

Denaturation induced aggregation in α-crystallin: differential action of chaotropes.

α-Crystallin is a member of small heat shock proteins and is believed to play an exceptional role in the stability of eye lens proteins. The disruption or denaturation of the protein arrangement or solubility of the crystallin proteins can lead to vision problems including cataract. In the present study, we have examined the effect of chemical denaturants urea and guanidine hydrochloride (GdnHCl) on α-crystallin aggregation, with special emphasis on protein conformational changes, unfolding, and amyloid fibril formation. GdnHCl (4 M) induced a 16 nm red shift in the intrinsic fluorescence of α-crystallin, compared with 4 nm shift by 8 M urea suggesting a major change in α-crystallin structure. Circular dichroism analysis showed marked increase in the ellipticity of α-crystallin at 216 nm, suggesting gain in ß-sheet structure in the presence of GdnHCl (0.5-1 M) followed by unfolding at higher concentration (2-6 M). However, only minor changes in the secondary structure of α-crystallin were observed in the presence of urea. Moreover, 8-anilinonaphthalene-1-sulfonic acid fluorescence measurement in the presence of GdnHCl and urea showed changes in the hydrophobicity of α-crystallin. Amyloid studies using thioflavin T fluorescence and congo red absorbance showed that GdnHCl induced amyloid formation in α-crystallin, whereas urea induced aggregation in this protein. Electron microscopy studies further confirmed amyloid formation of α-crystallin in the presence of GdnHCl, whereas only aggregate-like structures were observed in α-crystallin treated with urea. Our results suggest that α-crystallin is susceptible to unfolding in the presence of chaotropic agents like urea and GdnHCl. The destabilized protein has increased likelihood to fibrillate. Copyright © 2016 John Wiley & Sons, Ltd.

Development and Optimization of Sildenafil Orodispersible Mini-Tablets (ODMTs) for Treatment of Pediatric Pulmonary Hypertension Using Response Surface Methodology.

The availability of age-appropriate oral dosage forms for pediatric patients has remained a challenge. Orodispersible mini-tablets (ODMTs) are a promising delivery system for pediatric patients. The purpose of this work was the development and optimization of sildenafil ODMTs as a new dosage form for the treatment of pulmonary hypertension in children using a design-of-experiment (DoE) approach. A two-factor, three levels (32) full-factorial design was employed to obtain the optimized formulation. The levels of microcrystalline cellulose (MCC; 10-40% w/w) and partially pre-gelatinized starch (PPGS; 2-10% w/w) were set as independent formulation variables. In addition, mechanical strength, disintegration time (DT), and percent drug release were set as critical quality attributes (CQAs) of sildenafil ODMTs. Further, formulation variables were optimized using the desirability function. ANOVA analysis proved that MCC and PPGS had a significant (p < 0.05) impact on CQAs of sildenafil ODMTs with a pronounced influence of PPGS. The optimized formulation was achieved at low (10% w/w) and high (10% w/w) levels of MCC and PPGS, respectively. The optimized sildenafil ODMTs showed crushing strength of 4.72 ± 0.34 KP, friability of 0.71 ± 0.04%, DT of 39.11 ± 1.03 s, and sildenafil release of 86.21 ± 2.41% after 30 min that achieves the USP acceptance criteria for ODMTs. Validation experiments have shown that the acceptable prediction error (<5%) indicated the robustness of the generated design. In conclusion, sildenafil ODMTs have been developed as a suitable oral formulation for the treatment of pediatric pulmonary hypertension using the fluid bed granulation process and the DoE approach.

Design and Characterization of Baricitinib Incorporated PLA 3D Printed Pills by Fused Deposition Modeling: An Oral Pill for Treating Alopecia Areata.

This study aimed to develop three-dimensional (3D) baricitinib (BAB) pills using polylactic acid (PLA) by fused deposition modeling. Two strengths of BAB (2 and 4% w/v) were dissolved into the (1:1) PEG-400 individually, diluting it with a solvent blend of acetone and ethanol (27.8:18:2) followed by soaking the unprocessed 200 cm~6157.94 mg PLA filament in the solvent blend acetone-ethanol. FTIR spectrums of the 3DP1 and 3DP2 filaments calculated and recognized drug encapsulation in PLA. Herein, 3D-printed pills showed the amorphousness of infused BAB in the filament, as indicated by DSC thermograms. Fabricated pills shaped like doughnuts increased the surface area and drug diffusion. The releases from 3DP1 and 3DP2 were found to be 43.76 ± 3.34% and 59.14 ± 4.54% for 24 h. The improved dissolution in 3DP2 could be due to the higher loading of BAB due to higher concentration. Both pills followed Korsmeyer-Peppas' order of drug release. BAB is a novel JAK inhibitor that U.S. FDA has recently approved to treat alopecia areata (AA). Therefore, the proposed 3D printed tablets can be easily fabricated with FDM technology and effectively used in various acute and chronic conditions as personalized medicine at an economical cost.

Advances in Oral Drug Delivery.

The oral route is the most common route for drug administration. It is the most preferred route, due to its advantages, such as non-invasiveness, patient compliance and convenience of drug administration. Various factors govern oral drug absorption including drug solubility, mucosal permeability, and stability in the gastrointestinal tract environment. Attempts to overcome these factors have focused on understanding the physicochemical, biochemical, metabolic and biological barriers which limit the overall drug bioavailability. Different pharmaceutical technologies and drug delivery systems including nanocarriers, micelles, cyclodextrins and lipid-based carriers have been explored to enhance oral drug absorption. To this end, this review will discuss the physiological, and pharmaceutical barriers influencing drug bioavailability for the oral route of administration, as well as the conventional and novel drug delivery strategies. The challenges and development aspects of pediatric formulations will also be addressed.

Lignin nanoparticles as a promising vaccine adjuvant and delivery system for ovalbumin.

Vaccination is the most effective strategy of preventing and treating infectious diseases and the most significant issue in the development of potent vaccines is the sufficient immunogenicity and safety of vaccines. The main goal of the present study is to develop a potent and safe vaccine adjuvant that can also stabilize antigen formulations during preparation and storage. In this study, the model antigen ovalbumin (OVA) was encapsulated in polymeric nanoparticles based on lignin (OVA-LNPs). The nanoparticles had a particle size of 216 nm and a low polydispersity index. The nanoparticles were negatively charged (-26.7 mV) with high encapsulation efficiency 81.6% of OVA antigen. In vitro studies of the nanoparticles were tested against dendritic cells (DCs), specialized antigen-presenting cells (APCs). The results showed no cytotoxic effect from LNPs and a significantly higher percentage of dendritic cells have taken up the antigen when encapsulated inside LNPs in contrast to free OVA. The nanoparticle was administered intradermally to BALB/c mice and the resulting time-dependent systemic immune responses towards OVA were assessed by measuring the OVA-specific IgG titers using an enzyme-linked immunosorbent assay (ELISA). In vivo immunization with OVA-LNPs induced a stronger IgG antibody response than that induced by free OVA or alum adjuvanted OVA. Enhanced immunization by OVA-LNPs was attributed to the observed efficient uptake of the antigen by dendritic cells. These findings demonstrate that LNPs are promising to be used as vaccine adjuvant and delivery system for the induction of long-term immune responses.

Estimation Of Direct Medical Costs Of Middle East Respiratory Syndrome Coronavirus Infection: A Single-Center Retrospective Chart Review Study.

BACKGROUND: Among the countries affected by Middle East respiratory syndrome (MERS), Saudi Arabia was impacted the most, with 2,058 cases reported as of June 2019. However, the impact of the MERS epidemic on the Saudi economy is unknown. PURPOSE: The present study aimed to evaluate the direct medical costs associated with the management of MERS cases at a tertiary referral hospital in Riyadh, Saudi Arabia. METHODS: The study involved a retrospective chart review of confirmed cases of MERS coronavirus (MERS-CoV) infections in a tertiary care referral center in Riyadh, Saudi Arabia, from January 2015 to October 2018. The collected data included sociodemographic characteristics, medical information, and the cost of hospitalization of each patient as estimated by micro-costing. RESULTS: A complete set of relevant information was available only for 24 of 44 identified MERS-CoV cases. Patients were mostly females, and the mean age was 52 years. Diabetes, hypertension, and chronic kidney disease were the most frequent comorbidities. The length of hospital stay varied from 1 to 31 days, averaging 4.96 ± 7.29 days. Two of the 24 patients died. The total cost of managing a MERS case at the hospital ranged from $1278.41 to $75,987.95 with a mean cost of $12,947.03 ± $19,923.14. CONCLUSION: The findings of this study highlight the enormous expenses incurred by the Saudi health care system due to the MERS-CoV outbreak and the importance of developing an enforceable nationwide policy to control MERS-CoV transmission and infection.

A quercetin-based flavanoid (rutin) reverses amyloid fibrillation in ß-lactoglobulin at pH 2.0 and 358 K.

ß-lactoglobulin (BLG) is a well characterized milk protein and a model for folding and aggregation studies. Rutin is a quercetin based-flavanoid and a famous dietary supplement. It is a potential protector from coronary heart disease, cancers, and inflammatory bowel disease. In this study, amyloid fibrillation is reported in BLG at pH 2.0 and temperature 358 K. It is inhibited to some extent by rutin with a rate of 99.3 h-1 M-1. Amyloid fibrillation started taking place after 10 h of incubation and completed near 40 h at a rate of 16.6 × 10-3 h-1, with a plateau during 40-108 h. Disruption of tertiary structure of BLG and increased solvent accessibility of hydrophobic core seem to trigger intermolecular assembly. Increase in 7% ß-sheet structure at the cost of 10% α-helical structures and the electron micrograph of BLG fibrils at 108 h further support the formation of amyloid. Although it could not block amyloidosis completely, and even the time required to reach plateau remains the same, a decrease of growth rate from 16.6 × 10-3 to 13.5 × 10-3 h-1 was observed in the presence of 30.0 µM rutin. Rutin seems to block solvent accessibility of the hydrophobic core of BLG. A decrease in the fibril population was observed in electron micrographs, with the increase in rutin concentration. All evidences indicate reversal of fibrillation in BLG in the presence of rutin.

Sodium louroyl sarcosinate (sarkosyl) modulate amyloid fibril formation in hen egg white lysozyme (HEWL) at alkaline pH: a molecular insight study.

Amyloid fibril formation is responsible for several neurodegenerative diseases and are formed when native proteins misfold and stick together with different interactive forces. In the present study, we have determined the mode of interaction of the anionic surfactant sarkosyl with hen egg white lysozyme (HEWL) [EC No. 3.2.1.17] at two pHs (9.0 and 13.0) and investigated its impact on fibrillogenesis. Our data suggested that sarkosyl is promoting amyloid fibril formation in HEWL at the concentration range between 0.9 and 3.0 mM and no amyloid fibril formation was observed in the concentration range of 3.0-20.0 mM at pH 9.0. The results were confirmed by several biophysical and computational techniques, such as turbidity measurement, dynamic light scattering, Raleigh scattering, ThT fluorescence, intrinsic fluorescence, far-UV CD and atomic force microscopy. Sarkosyl was unable to induce aggregation in HEWL at pH 13.0 as confirmed by turbidity and RLS measurements. HEWL forms larger amyloid fibrils in the presence of 1.6 mM of sarkosyl. The spectroscopic, microscopic and molecular docking data suggest that the negatively charged carboxylate group and 12-carbon hydrophobic tail of sarkosyl stimulate amyloid fibril formation in HEWL via electrostatic and hydrophobic interaction. This study leads to new insight into the process of suppression of fibrillogenesis in HEWL which can be prevented by designing ligands that can retard the electrostatic and hydrophobic interaction between sarkosyl and HEWL.

Unraveling the molecular mechanism of the effects of sodium dodecyl sulfate, salts, and sugars on amyloid fibril formation in camel IgG.

Sodium dodecyl sulfate (SDS) is an anionic surfactant that can be used to stimulate protein fibrillation in vitro. Here, we investigated the effects of SDS on camel IgG aggregation at pH 3.5 and 7.4. SDS-induced amyloid fibril formation in camel IgG was examined by turbidity measurements, Rayleigh scattering, Thioflavin T (ThT) fluorescence, intrinsic fluorescence, circular dichroism (CD), and transmission electron microscopy (TEM). The results suggest that low SDS concentrations (0.2-2.0 mM) induce amyloid-like aggregates of camel IgG at pH 3.5, indicating an SDS/camel IgG ratio below 1000. However, in the presence of higher concentrations of SDS (2.5-10.0 mM), amyloid fibril formation was not observed. Furthermore, at the higher concentrations, the ß-sheet structure of camel IgG was transformed into a α-helical structure. The amyloid fibril formation was not observed in the presence of SDS at pH 7.4. Additionally, the role of salts and sugars was evaluated in the SDS-induced aggregation process. Interestingly, in the presence of 0.15 N of NaCl and (NH4)2SO4, SDS promoted camel IgG aggregation up to very high concentrations of SDS (0.2-10.0 mM; SDS/camel IgG ratio, 95-4750) and no suppression was observed. Moreover, osmoprotectants (trehalose and sucrose) were ineffective, neither promoting nor inhibiting the SDS-induced aggregation process. However, at pH 3.5, electrostatic and hydrophobic interactions, and hydrogen bonds were the major contributing factors in SDS-induced fibrillation. However, no aggregation was observed at pH 7.4 due to electrostatic repulsion between SDS and camel IgG because both of these molecules have overall similar charges.

Negatively charged food additive dye "Allura Red" rapidly induces SDS-soluble amyloid fibril in beta-lactoglobulin protein.

Recent studies have led to an increased interest to categorize small molecular inhibitors of protein fibrillation. In this study, we used spectroscopy, microscopy and gel electrophoresis techniques that provides an elaborated description of the Allura Red-induced amyloid fibrillation in the ß-LG protein at two pHs (7.4 and 3.5). The spectroscopy results show that ß-LG protein form aggregates in the presence of Allura Red (0.04-15.0mM) at pH 3.5 due to electrostatic and hydrophobic interactions. However, at pH 7.4, the ß-LG does not interact electrostatically with Allura Red and therefore no aggregation occurred. The Allura Red-induced aggregates have an amyloid-like structure that was confirmed by far-UV CD, Congo Red and transmission electron microscopy (TEM). The CD spectrum of ß-LG contains single minima at ∼218nm, which shifts towards higher wavelength minima at ∼225nm in the presence of Allura Red, characteristics of the cross ß-sheet structure. The TEM results suggest that ß-LG form long straight fibril when exposed to Allura Red at pH 3.5. The Allura Red-induced amyloid fibril is SDS-soluble confirmed by SDS-PAGE techniques. A far UV CD result shows the conversion of Allura Red induced cross ß-sheet structure into alpha-helical structure in the presence of increasing concentration of SDS. The results of this study suggest that the electrostatic, as well as hydrophobic interactions play an important role during Allura Red-induced ß-LG fibrillation.

Characterization of colchicine binding with normal and glycated albumin: In vitro and molecular docking analysis.

The transport of more than 90% of the drugs viz. anticoagulants, analgesics, and general anesthetics in the blood takes place by albumin. Hence, albumin is the prime protein needs to be investigated to find out the nature of drug binding. Serum albumin molecules are prone to glycation at elevated blood glucose levels as observed in diabetics. In this piece of work, glycation of bovine serum albumin (BSA) was carried out with glyceraldehyde and characterized by molecular docking and fluorometry techniques. Glycation of BSA showed 25% loss of free amino groups and decreased protein fluorescence (60%) with blue shift of 6 nm. The present study was also designed to evaluate the binding of colchicine (an anti-inflammatory drug) to native and glycated BSA and its ability to displace 8-analino-1-nephthalene sulfonic acid (ANS), from the BSA-ANS complex. Binding of ANS to BSA showed strong binding (Ka = 4.4 µM) with native conformation in comparison to glycated state (Ka = 8.4 µM). On the other hand, colchicine was able to quench the fluorescence of native BSA better than glycated BSA and also showed weaker affinity (Ka = 23 µM) for glycated albumin compared with native state (Ka = 16 µM). Molecular docking study showed that both glyceraldehyde and colchicine bind to common residues located near Sudlow's site I that explain the lower binding of colchicine in the glycated BSA. Based on our results, we believe that reduced drugs-binding affinity to glycated albumin may lead to drugs accumulation and precipitation in diabetic patients.

Unveiling the stimulatory effects of tartrazine on human and bovine serum albumin fibrillogenesis: Spectroscopic and microscopic study.

Amyloid fibrils are playing key role in the pathogenesis of various neurodegenerative diseases. Generally anionic molecules are known to induce amyloid fibril in several proteins. In this work, we have studied the effect of anionic food additive dye i.e., tartrazine (TZ) on the amyloid fibril formation of human serum albumins (HSA) and bovine serum albumin (BSA) at pHs7.4 and 3.5. We have employed various biophysical methods like, turbidity measurements, Rayleigh Light Scattering (RLS), Dynamic Light Scattering (DLS), intrinsic fluorescence, Congo red assay, far-UV CD, transmission electron microscopy (TEM) and atomic force microscopy (AFM) to decipher the mechanism of TZ-induce amyloid fibril formation in both the serum albumins at pHs7.4 and 3.5. The obtained results suggest that both the albumins forms amyloid-like aggregates in the presence of 1.0 to 15.0mM of TZ at pH3.5, but no amyloid fibril were seen at pH7.4. The possible cause of TZ-induced amyloid fibril formation is electrostatic and hydrophobic interaction because sulfate group of TZ may have interacted electrostatically with positively charged amino acids of the albumins at pH3.5 and increased protein-protein and protein-TZ interactions leading to amyloid fibril formation. The TEM, RLS and DLS results are suggesting that BSA forms bigger size amyloids compared to HSA, may be due to high surface hydrophobicity of BSA.

MERS-CoV papain-like protease (PLpro): expression, purification, and spectroscopic/thermodynamic characterization.

Within a decade, MERS-CoV emerged with nearly four times higher case fatality rate than an earlier outbreak of SARS-CoV and spread out in 27 countries in short span of time. As an emerging virus, combating it requires an in-depth understanding of its molecular machinery. Therefore, conformational characterization studies of coronavirus proteins are necessary to advance our knowledge of the matter for the development of antiviral therapies. In this study, MERS-CoV papain-like protease (PLpro) was recombinantly expressed and purified. Thermal folding pathway and thermodynamic properties were characterized using dynamic multimode spectroscopy (DMS) and thermal shift assay. DMS study showed that the PLpro undergoes a single thermal transition and follows a pathway of two-state folding with T m and van't Hoff enthalpy values of 54.4 ± 0.1 °C and 317.1 ± 3.9 kJ/mol, respectively. An orthogonal technique based on intrinsic tryptophan fluorescence also showed that MERS-CoV PLpro undergoes a single thermal transition and unfolds via a pathway of two-state folding with a T m value of 51.4 °C. Our findings provide significant understandings of the thermodynamic and structural properties of MERS-CoV PLpro.

Spectral and thermal properties of novel eye lens ζ-crystallin.

Eye lenses are exposed to thermal, solar radiations, dryness that enhances cataractogenesis. Some animal lenses contain novel proteins in bulk quantities. ζ-crystallin occurred in three ecologically divergent species, but it's physiological role not known. The truncated variant of ζ-crystallin causes hereditary cataract. Guinea pig ζ-crystallin is temperature-sensitive and rapidly aggregates at 41°C. Camels adopted to survive above 50°C, which raises an interesting question about how it retains lens proteins in the soluble state? Here, we have optimized expression and purification of recombinant camel ζ-crystallin. We have studied thermodynamic and spectroscopic properties using orthogonal techniques. Dynamic multimode spectroscopy results showed that camel ζ-crystallin unfolds via single transition with Tm value of 60.8±0.1°C and van't Hoff enthalpy of 714.7±7.1kJ/mol. Thermal-shift assay calculates Tm value of 62°C at pH 7. Additionally, the conformational stability of ζ-crystallin increases with ionic-strength. The influence of pH on ζ-crystallin was evaluated where the protein was found to be stable in the pH range of 6-9, but its stability drastically decreases below pH 6. Our results also showed that quaternary structure of ζ-crystallin drastically changed as a result of lowering pH. This study provides significant understandings onto the conformational, thermodynamic and unfolding pathway of camel ζ-crystallin.