Sequential viral introductions and spread of BA.1 across Pakistan provinces during the Omicron wave.

BACKGROUND: COVID-19 waves caused by specific SARS-CoV-2 variants have occurred globally at different times. We focused on Omicron variants to understand the genomic diversity and phylogenetic relatedness of SARS-CoV-2 strains in various regions of Pakistan. METHODS: We studied 276,525 COVID-19 cases and 1,031 genomes sequenced from December 2021 to August 2022. Sequences were analyzed and visualized using phylogenetic trees. RESULTS: The highest case numbers and deaths were recorded in Sindh and Punjab, the most populous provinces in Pakistan. Omicron variants comprised 93% of all genomes, with BA.2 (32.6%) and BA.5 (38.4%) predominating. The first Omicron wave was associated with the sequential identification of BA.1 in Sindh, then Islamabad Capital Territory, Punjab, Khyber Pakhtunkhwa (KP), Azad Jammu Kashmir (AJK), Gilgit-Baltistan (GB) and Balochistan. Phylogenetic analysis revealed Sindh to be the source of BA.1 and BA.2 introductions into Punjab and Balochistan during early 2022. BA.4 was first introduced in AJK and BA.5 in Punjab. Most recent common ancestor (MRCA) analysis revealed relatedness between the earliest BA.1 genome from Sindh with Balochistan, AJK, Punjab and ICT, and that of first BA.1 from Punjab with strains from KPK and GB. CONCLUSIONS: Phylogenetic analysis provides insights into the introduction and transmission dynamics of the Omicron variant in Pakistan, identifying Sindh as a hotspot for viral dissemination. Such data linked with public health efforts can help limit surges of new infections.

Nizatidine interacts with ct-DNA causing genotoxicity and cytotoxicity: an assessment by in vitro, in vivo, and in silico studies.

H2 receptor antagonists are the medication given for treating stomach ulcers, but lately, reports have shown their role in healing several malignant ulcers. The present work entails the interaction of H2 blocker nizatidine with calf thymus (ct)-DNA for determining the binding mode and energetics of the interaction. Multi-spectroscopic, calorimetric, viscometric and bioinformatic analysis revealed that nizatidine interacted with ct-DNA via groove-binding mode and is characterised by exothermic reaction. Moreover, assessment of genotoxic potential of nizatidine in vitro was carried out in peripheral human lymphocytes by alkaline comet assay. DNA damage occurred at high concentrations of nizatidine. Genotoxicity of nizatidine was also evaluated in vivo by assessing cytogenetic biomarkers viz. micronuclei formation and chromosomal aberration test. Nizatidine was able to induce micronuclei formation and chromosomal damage at high dose. Additionally, cytotoxic activity of nizatidine was determined in cancer cell lines, namely HeLa and HCT-116 and compared with the normal human cell line HEK-293 employing MTT assay. It was observed that nizatidine was more toxic towards HeLa and HCT-116 than HEK-293. Cell morphology analysis by compound inverted microscopy further strengthens the finding obtained through MTT assay.

End-to-End Approach to Surfactant Selection, Risk Mitigation, and Control Strategies for Protein-Based Therapeutics.

A survey performed by the AAPS Drug Product Handling community revealed a general, mostly consensus, approach to the strategy for the selection of surfactant type and level for biopharmaceutical products. Discussing and building on the survey results, this article describes the common approach for surfactant selection and control strategy for protein-based therapeutics and focuses on key studies, common issues, mitigations, and rationale. Where relevant, each section is prefaced by survey responses from the 22 anonymized respondents. The article format consists of an overview of surfactant stabilization, followed by a strategy for the selection of surfactant level, and then discussions regarding risk identification, mitigation, and control strategy. Since surfactants that are commonly used in biologic formulations are known to undergo various forms of degradation, an effective control strategy for the chosen surfactant focuses on understanding and controlling the design space of the surfactant material attributes to ensure that the desired material quality is used consistently in DS/DP manufacturing. The material attributes of a surfactant added in the final DP formulation can influence DP performance (e.g., protein stability). Mitigation strategies are described that encompass risks from host cell proteins (HCP), DS/DP manufacturing processes, long-term storage, as well as during in-use conditions.

Elucidating the molecular interaction of serum albumin with nizatidine and the role of ß-cyclodextrin: multi-spectroscopic and computational approach.

Nizatidine is a histamine H2 receptor antagonist which act by inhibiting the production of stomach acid, thereby, finds its application in treating various diseases related to the gastrointestinal tract. Studying albumin-drug interaction is important for understanding the pharmacokinetics and pharmacodynamics of therapeutic candidates. In the present work, the interaction of nizatidine with BSA was investigated by employing multi-spectroscopic and computational studies. The formation of BSA-nizatidine complex was characterised by UV-visible and fluorescence based-spectroscopic studies. Steady-state fluorescence demonstrated the static mode of quenching of BSA by nizatidine. The interaction was spontaneous and nizatidine binds to BSA with a stoichiometry of 1:1. Forster resonance energy transfer calculations revealed that there was a high possibility of energy transfer between nizatidine and BSA. The resultant secondary structural change in BSA on the addition of nizatidine was studied by circular dichroism spectroscopy. Moreover, synchronous and three-dimensional fluorescence spectroscopy was used to determine the conformational changes occurred in the structure of albumin on the binding of nizatidine. Competitive-site marker experiments suggested that nizatidine binds in the Sudlow site II of BSA. Additionally, the effect of ß-cyclodextrin as an inclusion compound on the interaction was studied. Furthermore, molecular modelling and simulation studies were performed to corroborate the results obtained above.Communicated by Ramaswamy H. Sarma.

Biophysical insights into the binding characteristics of bovine serum albumin with dipyridamole and the influence of molecular interaction with ß cyclodextrin.

The binding characteristic of anti-platelet drug dipyridamole has been investigated with a transport protein, serum albumin. A multi-spectroscopic approach has been employed, and the results were well supported by in silico molecular docking and simulation studies. The fluorescence quenching of serum albumin at three different temperatures revealed that the mechanism involved is static and the binding constant of the interaction was found to be of the order of 104 M-1. The reaction was found to be spontaneous and involved hydrophobic interactions. Synchronous, 3D fluorescence and CD spectroscopy indicated a change in conformation of bovine serum albumin (BSA) on interaction with DP. Using site-selective markers, the binding site of DP was found to be in subdomain IB. Molecular docking studies further corroborated these results. Molecular dynamic (MD) simulations showed lower RMSD values on interaction, suggesting the existence of a stable complex between DP and BSA. Furthermore, since ß-Cyclodextrin (ßCD) is used to improve the solubility of DP in ophthalmic solutions, therefore, the effect of (ßCD) on the interaction of BSA and DP was also studied, and it was found that in the presence of ßCD, the binding constant for BSA-DP interaction decreased. The present study is an attempt to characterize the transport of DP and to improve its bioavailability, consequently helping in dosage design to achieve optimum therapeutic levels.Communicated by Ramaswamy H. Sarma.

Evaluation of DNA interaction, genotoxicity and oxidative stress induced by iron oxide nanoparticles both in vitro and in vivo: attenuation by thymoquinone.

Iron oxide nanoparticles (IONPs) are known to induce cytotoxicity in various cancer cell lines through the generation of reactive oxygen species (ROS). However, the studies on its potential to induce toxicity in normal cell lines and in vivo system are limited and ambiguity still exists. Additionally, small molecules are known to interact with the DNA and cause damage to the DNA. The present study is designed to evaluate the potential interaction of IONPs with DNA along with their other toxicological effects and subsequent attenuation by thymoquinone both in vitro (primary lymphocytes) and in vivo (Wistar rats). IONPs were characterized by TEM, SEM-EDS, and XRD. The results from DNA interaction studies showed that IONPs formed a complex with DNA and also got intercalated between the base pairs of the DNA. The decrease in percent cell viability of rat's lymphocytes was observed along with an increase in ROS generation in a dose-dependent manner (50, 100, 200, 400 and 800 µg/ml of IONPs). The genetic damage in in vivo might be due to the generation of ROS as depletion in anti-enzymatic activity was observed along with an increase in lipid peroxidation in a dose-dependent manner (25, 50, 100 mg/kg of IONPs). Interestingly, supplementation of thymoquinone in combination with IONPs has significantly (P < 0.05) attenuated the genetic and oxidative damage in a dose-dependent manner both in vitro and in vivo. It can be concluded that thymoquinone has the potential to attenuate the oxidative stress and genetic toxicity in vitro and in vivo.

Elucidating the interaction of ticlopidine with serum albumin and its role in bilirubin displacement in vitro.

Ticlopidine is an anti-platelet drug that functions as a P2Y12 receptor antagonist. The present study provides a detailed characterization of interaction of ticlopidine with a model transport protein, bovine serum albumin (BSA) as well as an assessment of its bilirubin displacing ability using a multi-spectroscopic approach in combination with isothermal titration calorimetry. The value of binding constant determined using ITC studies was found to be 3.03 × 103 M-1 with a binding stoichiometry of approximately 1:1. Competitive site marker experiments indicate that ticlopidine binds to Sudlow site I, located in subdomain IIA of BSA. In addition, Circular dichroism and 3D fluorescence spectroscopy indicated structural and conformational changes in BSA on interaction with ticlopidine. Thermodynamic parameters suggested that the reaction was spontaneous, exothermic, entropically driven, and involved hydrophobic interactions. These results were well supported by those obtained through molecular docking studies. Additionally, the effect of ticlopidine on bilirubin and albumin interaction was evaluated using the peroxidase method as well as through fluorescence spectroscopy. Ticlopidine was found to displace bilirubin from serum albumin. Moreover, the binding constant of bilirubin-serum albumin interaction also decreased in presence of ticlopidine. The results indicated that ticlopidine is a competitive displacer of bilirubin in vitro and may contribute to the incidences hyperbilirubinemia associated with the usage of this drug.

Interaction of pirenzepine with bovine serum albumin and effect of ß-cyclodextrin on binding: A biophysical and molecular docking approach.

Studying the interaction of therapeutic molecules with serum albumin is important to understand their biopharmaceutics, pharmacokinetics and toxicity as well as their relation with the structure and function of protein. The biomolecular interaction of an anti-spasmodic drug, pirenzepine with bovine serum albumin (BSA) was investigated using multi-spectroscopic, calorimetric and docking studies. Fluorescence quenching of BSA on interaction with pirenzepine revealed the static mode of quenching. Pirenzepine exhibited a moderate binding to serum albumin with the binding constant value in the order of 104 M-1. Based on the Forster's non-radiative energy transfer theory, the average binding distance between BSA and pirenzepine was calculated. Competitive site marker experiments demonstrated that pirenzepine binds to the sudlow site III located in subdomain IB of BSA. Circular dichroic spectroscopy indicated secondary structural changes in BSA while three-dimensional fluorescence spectroscopy showed the microenvironmental perturbations in the structure of BSA on interaction with pirenzepine. Moreover, thermodynamic parameters obtained from isothermal titration calorimetry suggested that the interaction between pirenzepine and BSA was spontaneous and hydrophobic interactions played the major role in stabilizing the complex. Additionally, the effect of inclusion compound, ß-cyclodextrin on pirenzepine-BSA interaction was studied. As pirenzepine is involved in drug-drug interactions, ß-cyclodextrin forms an inclusion complex with pirenzepine and prevents drug-drug interactions, thereby, enhancing the therapeutic effect of pirenzepine. Some common metal ions have also been found to interfere with pirenzepine-BSA interaction. The above experimental results further corroborated the molecular modelling studies.

Molecular spectroscopic and thermodynamic studies on the interaction of anti-platelet drug ticlopidine with calf thymus DNA.

Ticlopidine is an anti-platelet drug which belongs to the thienopyridine structural family and exerts its effect by functioning as an ADP receptor inhibitor. Ticlopidine inhibits the expression of TarO gene in S. aureus and may provide protection against MRSA. Groove binding agents are known to disrupt the transcription factor DNA complex and consequently inhibit gene expression. Understanding the mechanism of interaction of ticlopidine with DNA can prove useful in the development of a rational drug designing system. At present, there is no such study on the interaction of anti-platelet drugs with nucleic acids. A series of biophysical experiments were performed to ascertain the binding mode between ticlopidine and calf thymus DNA. UV-visible and fluorescence spectroscopic experiments confirmed the formation of a complex between ticlopidine and calf thymus DNA. Moreover, the values of binding constant were found to be in the range of 103M-1, which is indicative of groove binding between ticlopidine and calf thymus DNA. These results were further confirmed by studying the effect of denaturation on double stranded DNA, iodide quenching, viscometric studies, thermal melting profile as well as CD spectral analysis. The thermodynamic profile of the interaction was also determined using isothermal titration calorimetric studies. The reaction was found to be endothermic and the parameters obtained were found to be consistent with those of known groove binders. In silico molecular docking studies further corroborated well with the experimental results.

Unravelling the interaction of pirenzepine, a gastrointestinal disorder drug, with calf thymus DNA: An in vitro and molecular modelling study.

Pirenzepine is an anti-ulcer agent which belongs to the anti-cholinergic group of gastrointestinal disorder drugs and functions as an M1 receptor selective antagonist. Drug-DNA interaction studies are of great significance as it helps in the development of new therapeutic drugs. It provides a deeper understanding into the mechanism through which therapeutic drugs control gene expression. Interaction of pirenzepine with calf-thymus DNA (Ct-DNA) was determined via a series of biophysical techniques. UV-visible absorption and fluorescence spectroscopy confirmed the formation of pirenzepine-Ct-DNA complex. The values of binding constant from various experiments were calculated to be in the order of 103 M-1 which is consistent with the groove binding mode. Various spectrofluorimetric experiments like competitive displacement of well known dyes with drug, iodide quenching experiments and the effect of Ct-DNA denaturation in presence of drug confirmed the binding of pirenzepine to the groove of Ct-DNA. The binding mode was further established by viscometric, circular dichroic and molecular modelling studies. Thermodynamic parameters obtained from isothermal titration calorimetric studies suggest that the interaction of pirenzepine with Ct-DNA is enthalpically driven. The value of TΔS and ΔH calculated from calorimetric studies were found to be 4.3 kcal mol-1 and -2.54 kcal mol-1 respectively, indicating that pirenzepine-Ct-DNA complex is mainly stabilized by hydrophobic interaction and hydrogen bonding. The binding energy calculated was -7.5 kcal mol-1 from modelling studies which was approximately similar to that obtained by isothermal titration calorimetric studies. Moreover, the role of electrostatic interaction in the binding of pirenzepine to Ct-DNA cannot be precluded.

Caffeic acid binds to the minor groove of calf thymus DNA: A multi-spectroscopic, thermodynamics and molecular modelling study.

Caffeic acid (CA) is a plant polyphenol which acts as an antioxidant and has various pharmacological effects. DNA is one of the major cellular targets of therapeutic molecules. Thus, studying the interaction of small molecules with DNA is of great importance. In the current article, we have studied the mode of binding of CA with calf thymus DNA (Ct-DNA) using a series of biophysical techniques. Formation of complex between CA and Ct-DNA is ascertained by analyzing the UV-vis absorbance and fluorescence emission spectra of CA upon successive addition of Ct-DNA. Binding constants of CA with Ct-DNA obtained using multiple experiments was in the order of 103 M-1 which is consistent with known groove binders. Analysis of thermodynamic parameters suggest that hydrogen bonding and van der Waal's forces played major role in the binding process. Competitive displacement studies confirmed that CA binds to the minor groove of Ct-DNA. These observations were further validated by KI quenching experiment, DNA melting studies, CD and viscosity measurements. In silico molecular docking further provided insight into the mode of binding of CA with Ct-DNA. Through in vitro experiments and in silico molecular docking studies, it was concluded that CA binds to the minor groove of Ct-DNA.