Development of UV-visible spectrophotometric methods for the quantitative and in silico studies for cilazapril optimized by response surface methodology.

For cilazapril (CLZ), analytical methods based on donor-acceptor phenomenon that are simple, rapid with broad linear dynamic range for the quantification of drug are not available in the literature. Considering the requirement for the methods, in this study, two economic, potent analytical methods based on the complexation of CLZ with π-acceptors, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and 2,5-dichloro-3,6-dihydroxy-p-benzoquinone (CA) were developed, validated, and studied spectrophotometrically. Various analytical data were discussed. The effects of experimental variables were optimized from the results of in silico technique, i.e. Box-Behnken design under response surface methodology. Linear dynamic range was significantly good in the range of 6-60 µg mL-1 and 20-260 µg mL-1 for DDQ and CA methods. Moreover, molecular docking studies corroborated the experimental results. Further, the methods were supplemented by the pharmaceutical and biological application for the quantitative assay of CLZ. Collectively, the results of the reported method of the analysis suggest that the developed approach is simple, sensitive, accurate and precise.

Molecular Interactions Leading to Advancements in the Techniques for COVID-19 Detection: A Review.

Since 2019 the world has been in a combat with the highly contagious disease COVID-19 which is caused by the rapid transmission of the SARS-CoV-2 virus (severe acute respiratory syndrome coronavirus 2). Detection of this disease in an early stage helps to control its spread and management. To combat this epidemic with one-time effective medication, improved quick analytical procedures must be developed and validated. The requirement for accurate and precise analytical methods for the diagnosis of the virus and antibodies in infected patients has been a matter of concern. The global impact of this virus has motivated scientists and researchers to investigate and develop various analytical diagnostic techniques. This review includes the study of standard methods which are reliable and accredited for the analytical recognition of the said virus. For early detection of SARS-CoV-2 RNA, RT-PCR (Real-time reverse transcriptase-polymerase chain reaction) is an accurate method among other methods and, thus, considered as the "gold standard" technique. Here, we outline the most extensively used analytical methods for diagnosing COVID-19, along with a brief description of each technique and its analytical aspects/perspective.

Pharmacogenetic study of CES1 gene and enalapril efficacy.

Enalapril is an orally administered angiotensin-converting enzyme inhibitor which is widely prescribed to treat hypertension, chronic kidney disease, and heart failure. It is an ester prodrug that needs to be activated by carboxylesterase 1 (CES1). CES1 is a hepatic hydrolase that in vivo biotransforms enalapril to its active form enalaprilat in order to produce its desired pharmacological impact. Several single nucleotide polymorphisms in CES1 gene are reported to alter the catalytic activity of CES1 enzyme and influence enalapril metabolism. G143E, L40T, G142E, G147C, Y170D, and R171C can completely block the enalapril metabolism. Some polymorphisms like Q169P, E220G, and D269fs do not completely block the CES1 function; however, they reduce the catalytic activity of CES1 enzyme. The prevalence of these polymorphisms is not the same among all populations which necessitate to consider the genetic panel of respective population before prescribing enalapril. These genetic variations are also responsible for interindividual variability of CES1 enzyme activity which ultimately affects the pharmacokinetics and pharmacodynamics of enalapril. The current review summarizes the CES1 polymorphisms which influence the enalapril metabolism and efficacy. The structure of CES1 catalytic domain and important amino acids impacting the catalytic activity of CES1 enzyme are also discussed. This review also highlights the importance of pharmacogenomics in personalized medicine.

Optimizing Recombinant Cas9 Expression: Insights from E. coli BL21(DE3) Strains for Enhanced Protein Purification and Genome Editing.

The CRISPR-Cas9 system is a revolutionary tool in genetic engineering, offering unprecedented precision and efficiency in genome editing. Cas9, an enzyme derived from bacteria, is guided by RNA to edit DNA sequences within cells precisely. However, while CRISPR-Cas9 presents notable benefits and encouraging outcomes as a molecular tool and a potential therapeutic agent, the process of producing and purifying recombinant Cas9 protein remains a formidable hurdle. In this study, we systematically investigated the expression of recombinant SpCas9-His in four distinct Escherichia coli (E. coli) strains (Rosetta2, BL21(DE3), BL21(DE3)-pLysS, and BL21(DE3)-Star). Through optimization of culture conditions, including temperature and post-induction time, the BL21(DE3)-pLysS strain demonstrated efficient SpCas9 protein expression. This study also presents a detailed protocol for the purification of recombinant SpCas9, along with detailed troubleshooting tips. Results indicate successful SpCas9 protein expression using E. coli BL21(DE3)-pLysS at 0.5 mM IPTG concentration. Furthermore, the findings suggest potential avenues for further enhancements, paving the way for large-scale Cas9 production. This research contributes valuable insights into optimizing E. coli strains and culture conditions for enhanced Cas9 expression, offering a step forward in the development of efficient genome editing tools and therapeutic proteins.

Nuclear Magnetic Resonance Spectroscopy for Quantitative Analysis: A Review for Its Application in the Chemical, Pharmaceutical and Medicinal Domains.

Nuclear magnetic resonance (NMR) is a rapid and accurate analytical tool for qualification and quantification. The capacity of NMR of being quantitative can also justify the calibration of other analytical methods. In pharmaceutical domain, quantitative NMR (qNMR) can be applied in the identification and quantification of drug simultaneously. The early drug development stage requires a minimum sample for analysis. Thus, priority should be given to utilize this technique to attain results with least investment, rapid analysis time and minimum sample consumption. This technique is a significant phenomenon to identify impurities, drug substance, residual solvents of in-process control (IPC) samples and characterizing the formulations. From an analyst's perspective, qNMR proved to be a routine practice in pharmaceutical industry to qualify any drug product. The absolute and relative methods offer great help in quantifying the component of interest in the process control samples and finished products. This review highlights the evolution of NMR application in the pharmaceutical industry, where determining the purity of drug substance, drug product and establishing the identity of impurities and its level are the challenging aspects. NMR in medicinal field emerging as a numero uno for Covid-19 severity detection and its dire consequences, accelerated vaccine development and the mapping of SAR-COV-2 RNA and proteins via chemical shift assignments.

Circular dichroism assessment of an imidazole antifungal drug with plant based silver nanoparticles: Quantitative and DFT analysis.

Circular dichroism (CD) methods have been developed for the analysis of luliconazole (LUC) using plant based silver nanoparticles (P-AgNPs). Cleaner and natural approach have found significant attention in recent times owing to their exceptional physicochemical characteristics. Utilizing FTIR, SEM, and XRD, the produced nanoparticles were analyzed. The produced P-AgNPs were then used to assay LUC in formulation drugs. Four CD methods are developed as zero order and second order derivative methods. Methods I and II are based on a normal CD scan (zero order) that produced calibration range from 2 - 16 µgmL-1 at 232 nm (positive band) and 299 nm (negative band), respectively. Methods III and IV are the second order derivative methods that are developed at 232 nm (negative band) and at 251 nm (positive band). Density functional theory study was done to comprehend the feasibility of the developed methods and to optimize the structure and energy gap that validated the experimental procedure. The LUC assay methods using the proposed CD approach are simple, sensitive and precise with a limit of detection for methods I, II, III and IV of 0.527, 0.428, 0.250 and 0.30 µgmL-1 and limit of quantification of 1.75, 1.42, 0.833 and 1.0 µgmL-1, respectively. For intra- and inter-day precision, the recovery data ranged from 99.48 to 101% and 99.37 to 101%, respectively. The methods were used in dosage forms that produced a relative standard deviation of less than 2% and the true bias (θL and θU) within ±2%, demonstrating the potential use of the developed methods.

Development of low-cost in-house tetra-ARMS-PCR assay for the screening of five CBS mutations found in Pakistani homocystinuria patients.

BACKGROUND: Classical homocystinuria is an inborn amino acid metabolism disorder resulting from mutations in the Cystathionine-ß-Synthase (CBS) gene. These mutations lead to elevated homocysteine and methionine levels and reduced cysteine levels in the blood. Typically, diagnosis occurs after patients display symptoms, and various lab methods confirm it. DNA sequencing is the best option for early detection of genetic variants in asymptomatic suspected individuals. Unfortunately, its high cost can hinder its use, especially in low-income countries like Pakistan. OBJECTIVE: Aim of this study was to devise a robust low-cost diagnostic/screening assay based on Tetra-ARMS-PCR for five prevalent genetic variants found in Pakistani classical homocystinuria patients. MATERIALS AND METHODS: In the current study, T-ARMS-PCR assays were developed for five mutations (c.975G > C, c.770C > T, c.752T > C, c.1039 + 1G > T, c.451 + 1GG > TA), which were characterized previously in classical homocystinuria patients. These low-cost T-ARMS-PCR assays were then used to screen the affected individuals and their family members to identify their genotypes for pathogenic variations in the asymptomatic patients and carriers in their respective families. RESULTS: The outcomes were entirely consistent with those obtained from Sanger DNA sequencing, confirming the sensitivity, specificity, and reliability of the T-ARMS-PCR assay for detecting CBS mutations. CONCLUSION: T-ARMS-PCR has wide applications for low-income countries for the screening and early diagnosis of asymptomatic patients and carriers in the homocystinuria affected families as well as other inherited diseases.

Histological and Microscopic Analysis of Fats in Heart, Liver Tissue, and Blood Parameters in Experimental Mice.

The intake of various types and amounts of dietary fats influences metabolic and cardiovascular health. Hence, this study evaluated the impact of routinely consumed Pakistani dietary fats on their cardiometabolic impact. For this, we made four groups of mice, each comprising 5 animals: (1) C-ND: Control mice on a normal diet, (2) HFD-DG: High-fat diet mice on a normal diet plus 10% (w/w) desi ghee, (3) HFD-O: Mice on normal diet plus 10% (w/w) plant oil (4) HFD-BG: Mice on normal diet plus 10% (w/w) banaspati ghee. Mice were fed for 16 weeks, and blood, liver, and heart samples were collected for biochemical, histological, and electron microscopic analysis. The physical factors indicated that mice fed on HFD gained more body weight than the C-ND group. Blood parameters do not show significant differences, but overall, the glucose and cholesterol concentrations were raised in the mice fed with a fat-rich diet, with the highest concentrations in the HFD-BG group. The mice fed with HFD-BG and HFD-O had more lipid droplets in the liver, compared to HFD-DG and C-ND.