Early Results of an Initiative to Assess Exposure to Firearm Violence in Ambulatory Care: Descriptive Analysis of Electronic Health Record Data.

BACKGROUND:  Current research on firearm violence is largely limited to patients who received care in emergency departments or inpatient acute care settings or who died. This is because standardized disease classification codes for firearm injury only represent bodily trauma. As a result, research on pathways and health impacts of firearm violence is largely limited to people who experienced acute bodily trauma and does not include the estimated millions of individuals who were exposed to firearm violence but did not sustain acute injury. Assessing and collecting data on exposure to firearm violence in ambulatory care settings can expand research and more fully frame the public health issue. OBJECTIVE: The aim of the study is to evaluate the demographic and clinical characteristics of patients who self-reported exposure to firearm violence during a behavioral health visit. METHODS: This study assessed early data from an initiative implemented in 2022 across a national network of ambulatory behavioral health centers to support trauma-informed care by integrating structured data fields on trauma exposure into an electronic health record behavioral health patient assessment form (SmartForm), as such variables are generally not included in standard outpatient medical records. We calculated descriptive statistics on clinic characteristics, patient demographics, and select clinical conditions among clinics that chose to implement the SmartForm and among patients who reported an exposure to firearm violence. Data on patient counts are limited to positive reports of exposure to firearm violence, and the representativeness of firearm exposure among all patients could not be calculated due to unknown variability in the implementation of the SmartForm. RESULTS: There were 323 of 629 (51%) clinics that implemented the SmartForm and reported at least 1 patient exposed to firearm violence. In the first 11 months of implementation, 3165 patients reported a recent or past exposure to firearm violence across the 323 clinics. Among patients reporting exposure, 52.7% (n=1669) were male, 38.8% (n=1229) were Black, 45.7% (n=1445) had posttraumatic stress disorder, 37.5% (n=1186) had a substance abuse disorder (other than nicotine), and 11.7% (n=371) had hypertension. CONCLUSIONS: Current research on firearm violence using standardized data is limited to acute care settings and death data. Early results from an initiative across a large network of behavioral health clinics demonstrate that a high number of clinics chose to implement the SmartForm, resulting in thousands of patients reporting exposure to firearm violence. This study demonstrates that collecting standardized data on firearm violence exposure in ambulatory care settings is feasible. This study further demonstrates that resultant data from ambulatory settings can be used for meaningful analysis in describing populations affected by firearm violence. The results of this study hold promise for further collection of structured data on exposure to firearm violence in ambulatory settings.

The (Re-)Emergence and Spread of Viral Zoonotic Disease: A Perfect Storm of Human Ingenuity and Stupidity.

Diseases that are transmitted from vertebrate animals to humans are referred to as zoonotic diseases. Although microbial agents such as bacteria and parasites are linked to zoonotic events, viruses account for a high percentage of zoonotic diseases that have emerged. Worryingly, the 21st century has seen a drastic increase in the emergence and re-emergence of viral zoonotic disease. Even though humans and animals have coexisted for millennia, anthropogenic factors have severely increased interactions between the two populations, thereby increasing the risk of disease spill-over. While drivers such as climate shifts, land exploitation and wildlife trade can directly affect the (re-)emergence of viral zoonotic disease, globalisation, geopolitics and social perceptions can directly facilitate the spread of these (re-)emerging diseases. This opinion paper discusses the "intelligent" nature of viruses and their exploitation of the anthropogenic factors driving the (re-)emergence and spread of viral zoonotic disease in a modernised and connected world.

Skeletal Muscle Mitochondrial Respiration and Exercise Intolerance in Patients With Heart Failure With Preserved Ejection Fraction.

Importance: The pathophysiology of exercise intolerance in patients with heart failure with preserved ejection fraction (HFpEF) remains incompletely understood. Multiple lines of evidence suggest that abnormal skeletal muscle metabolism is a key contributor, but the mechanisms underlying metabolic dysfunction remain unresolved. Objective: To evaluate the associations of skeletal muscle mitochondrial function using respirometric analysis of biopsied muscle fiber bundles from patients with HFpEF with exercise performance. Design, Setting, and Participants: In this cross-sectional study, muscle fiber bundles prepared from fresh vastus lateralis biopsies were analyzed by high-resolution respirometry to provide detailed analyses of mitochondrial oxidative phosphorylation, including maximal capacity and the individual contributions of complex I-linked and complex II-linked respiration. These bioenergetic data were compared between patients with stable chronic HFpEF older than 60 years and age-matched healthy control (HC) participants and analyzed for intergroup differences and associations with exercise performance. All participants were treated at a university referral center, were clinically stable, and were not undergoing regular exercise or diet programs. Data were collected from March 2016 to December 2017, and data were analyzed from November 2020 to May 2021. Main Outcomes and Measures: Skeletal muscle mitochondrial function, including maximal capacity and respiration linked to complex I and complex II. Exercise performance was assessed by peak exercise oxygen consumption, 6-minute walk distance, and the Short Physical Performance Battery. Results: Of 72 included patients, 50 (69%) were women, and the mean (SD) age was 69.6 (6.1) years. Skeletal muscle mitochondrial function measures were all markedly lower in skeletal muscle fibers obtained from patients with HFpEF compared with HCs, even when adjusting for age, sex, and body mass index. Maximal capacity was strongly and significantly correlated with peak exercise oxygen consumption (R = 0.69; P < .001), 6-minute walk distance (R = 0.70; P < .001), and Short Physical Performance Battery score (R = 0.46; P < .001). Conclusions and Relevance: In this study, patients with HFpEF had marked abnormalities in skeletal muscle mitochondrial function. Severely reduced maximal capacity and complex I-linked and complex II-linked respiration were associated with exercise intolerance and represent promising therapeutic targets.

Characterization of a Near Full-Length Hepatitis E Virus Genome of Subtype 3c Generated from Naturally Infected South African Backyard Pigs.

Eight genotypes of the hepatitis E virus (Orthohepevirus A; HEV) designated HEV-1 to HEV-8 have been reported from various mammalian hosts. Notably, domestic pigs and wild boars are the natural reservoirs of HEV-3 and HEV-4 genotypes with zoonotic propensity. Since HEV infection in domestic pigs is usually subclinical, it may remain undetected, facilitating zoonotic spillover of HEV to the exposed human populations. A previous study from our group in 2021, using deep sequencing of a pooled saliva sample, generated various swine enteric virus genomes, including a near full-length swine HEV genome (7040 nt; 97.7% genome coverage) from five-month-old grower pigs at a backyard pig farm in the uMgungundlovu District, KwaZulu-Natal, South Africa. In the present study, we describe the further characterization, including genotyping and subtyping of the swine HEV isolate using phylogenetics and 'HEVnet Typing Tool'. Our analyses confirmed that the South African swine HEV genome characterized in this study belonged to HEV genotype 3 subtype 3c (HEV-3c). While HEV-3c infections in domestic pigs have been previously reported from Brazil, Germany, Italy, and the Netherlands, they only generated partial genome sequences of open reading frame 1 (ORF1) and/or ORF2. To our knowledge, this is the first near full-length swine HEV-3c genome generated from naturally infected domestic pigs (Sus scrofa domesticus) in South Africa. However, due to the gap in the information on the HEV-3c genome sequences in various geographical locations worldwide, including South Africa, the epidemiology of the South African swine HEV genome characterized in this study remains inconclusive. Molecular and genomic surveillance of HEV in domestic pig populations in South Africa would be useful to determine their prevalence, circulating subtypes, and zoonosis risk.

Metagenomic Analysis of RNA Fraction Reveals the Diversity of Swine Oral Virome on South African Backyard Swine Farms in the uMgungundlovu District of KwaZulu-Natal Province.

Numerous RNA viruses have been reported in backyard swine populations in various countries. In the absence of active disease surveillance, a persistent knowledge gap exists on the diversity of RNA viruses in South African backyard swine populations. This is the first study investigating the diversity of oral RNA virome of the backyard swine in South Africa. We used three samples of backyard swine oral secretion (saliva) collected from three distantly located backyard swine farms (BSFs) in the uMgungundlovu District, KwaZulu-Natal, South Africa. Total viral RNA was extracted and used for the library preparation for deep sequencing using the Illumina HiSeq X instrument. The FASTQ files containing paired-end reads were analyzed using Genome Detective v 1.135. The assembled nucleotide sequences were analyzed using the PhyML phylogenetic tree. The genome sequence analysis identified a high diversity of swine enteric viruses in the saliva samples obtained from BSF2 and BSF3, while only a few viruses were identified in the saliva obtained from BSF1. The swine enteric viruses belonged to various animal virus families; however, two fungal viruses, four plant viruses, and five unclassified RNA viruses were also identified. Specifically, viruses of the family Astroviridae, according to the number of reads, were the most prevalent. Of note, the genome sequences of Rotavirus A (RVA) and Rotavirus C (RVC) at BSF2 and RVC and Hepatitis E virus (HEV) at BSF3 were also obtained. The occurrence of various swine enteric viruses in swine saliva suggests a high risk of diarrhoeic diseases in the backyard swine. Of note, zoonotic viruses in swine saliva, such as RVA, RVC, and HEV, indicate a risk of zoonotic spillover to the exposed human populations. We recommend the implementation of biosecurity to ensure sustainable backyard swine farming while safeguarding public health.

The impact of the COVID-19 pandemic on children with medical complexity.

BACKGROUND: Descriptions of the COVID-19 pandemic's indirect consequences on children are emerging. We aimed to describe the impacts of the pandemic on children with medical complexity (CMC) and their families. METHODS: A one-time survey of Canadian paediatricians using the Canadian Paediatric Surveillance Program (CPSP) was conducted in Spring 2021. RESULTS: A total of 784 paediatricians responded to the survey, with 70% (n = 540) providing care to CMC. Sixty-seven (12.4%) reported an adverse health outcome due to a COVID-19 pandemic-related disruption in healthcare delivery. Disruption of the supply of medication and equipment was reported by 11.9% of respondents (n = 64). Respondents reported an interruption in family caregiving (47.5%, n = 252) and homecare delivery (40.8%, n = 218). Almost 47% of respondents (n = 253) observed a benefit to CMC due to COVID-19 related changes in healthcare delivery, including increased availability of virtual care and reduction in respiratory illness. Some (14.4%) reported that CMC were excluded from in-person learning when their peers without medical complexity were not. CONCLUSION: Canadian paediatricians reported that CMC experienced adverse health outcomes during the COVID-19 pandemic, including disruptions to family caregiving and community supports. They also describe benefits related to the pandemic including the expansion of virtual care. These results highlight the need for healthcare, community and education policymakers to collaborate with families to optimize their health.

The HIV-1 Gag Protein Displays Extensive Functional and Structural Roles in Virus Replication and Infectivity.

Once merely thought of as the protein responsible for the overall physical nature of the human immunodeficiency virus type 1 (HIV-1), the Gag polyprotein has since been elucidated to have several roles in viral replication and functionality. Over the years, extensive research into the polyproteins' structure has revealed that Gag can mediate its own trafficking to the plasma membrane, it can interact with several host factors and can even aid in viral genome packaging. Not surprisingly, Gag has also been associated with HIV-1 drug resistance and even treatment failure. Therefore, this review provides an extensive overview of the structural and functional roles of the HIV-1 Gag domains in virion integrity, functionality and infectivity.

An overview of influenza A virus genes, protein functions, and replication cycle highlighting important updates.

The recent research findings on influenza A virus (IAV) genome biology prompted us to present a comprehensive overview of IAV genes, protein functions, and replication cycle. The eight gene segments of the IAV genome encode 17 proteins, each having unique functions contributing to virus fitness in the host. The polymerase genes are essential determinants of IAV pathogenicity and virulence; however, other viral components also play crucial roles in the IAV replication, transmission, and adaptation. Specific adaptive mutations within polymerase (PB2, PB1, and PA) and glycoprotein-hemagglutinin (HA) and neuraminidase (NA) genes, may facilitate interspecies transmission and adaptation of IAV. The HA-NA interplay is essential for establishing the IAV infection; the low pH triggers the inactivation of HA-receptor binding, leading to significantly lower NA activities, indicating that the enzymatic function of NA is dependent on HA binding. While the HA and NA glycoproteins are required to initiate infection, M1, M2, NS1, and NEP proteins are essential for cytoplasmic trafficking of viral ribonucleoproteins (vRNPs) and the assembly of the IAV virions. The mechanisms that enable IAV to exploit the host cell resources to advance the infection are discussed. A comprehensive understanding of IAV genome biology is essential for developing antivirals to combat the IAV disease burden.

A systematic review of influenza A virus prevalence and transmission dynamics in backyard swine populations globally.

BACKGROUND: Backyard swine farming is critical to generating subsistence and food security in rural and peri-urban households in several developing countries. The objective of this systematic review was to analyze the molecular and serological prevalence of influenza A virus (IAV) in backyard swine populations globally. RESULTS: We identified 34 full-text research articles in NCBI-PubMed and Google Scholar databases that have reported IAV sero- and/or virological prevalence in backyard swine up to 11 July 2021. The highest number of studies were reported from Asia (n = 11) followed by North America (n = 10), South America (n = 6), Africa (n = 6), and Europe (n = 1). While the maximum number of studies (44.12%) reported human-to-swine transmission of IAV, swine-to-human (5.88%), poultry-to-swine (5.88%), and wild birds-to-swine (2.94%) transmissions were also reported. An overall higher IAV seroprevalence (18.28%) in backyard swine was detected compared to the virological prevalence (1.32%). The human-origin pandemic A(H1N1)pdm09 virus clade 1A.3.3.2 was the more frequently detected IAV subtype in virological studies (27.27%) than serological studies (18.92%). In addition, the avian-origin highly pathogenic H5N1 and H5N8 viruses were also detected, which further substantiated the evidence of avian-swine interactions in the backyards. CONCLUSION: Human-swine and avian-swine interactions in backyards may transmit IAV between species. Monitoring the circulation and evolution of IAV in backyard swine would help stakeholders make informed decisions to ensure sustainable backyard swine farming and public safety.

Review of genome sequencing technologies in molecular characterization of influenza A viruses in swine.

The rapidly evolving antigenic diversity of influenza A virus (IAV) genomes in swine makes it imperative to detect emerging novel strains and track their circulation. We analyzed in our review the sequencing technologies used for subtyping and characterizing swine IAV genomes. Google Scholar, PubMed, and International Nucleotide Sequence Database Collaboration (INSDC) database searches identified 216 studies that have utilized Sanger, second-, and third-generation sequencing techniques to subtype and characterize swine IAV genomes up to 31 March 2021. Sanger dideoxy sequencing was by far the most widely used sequencing technique for generating either full-length (43.0%) or partial (31.0%) IAV genomes in swine globally; however, in the last decade, other sequencing platforms such as Illumina have emerged as serious competitors for the generation of whole-genome sequences of swine IAVs. Although partial HA and NA gene sequences were sufficient to determine swine IAV subtypes, whole-genome sequences were critical for determining reassortments and identifying unusual or less frequently occurring IAV subtypes. The combination of Sanger and second-generation sequencing technologies also greatly improved swine IAV characterization. In addition, the rapidly evolving third-generation sequencing platform, MinION, appears promising for on-site, real-time sequencing of complete swine IAV genomes. With a higher raw read accuracy, the use of the MinION could enhance the scalability of swine IAV testing in the field and strengthen the swine IAV disease outbreak response.

Structural effects of HIV-1 subtype C integrase mutations on the activity of integrase strand transfer inhibitors in South African patients.

HIV-1 integrase enzyme is responsible for the integration of viral DNA into the host genomic DNA. Integrase strand transfer inhibitors (INSTIs) are highly potent antiretroviral agents that inhibit this process, and are internationally approved for the treatment of both naïve and treated HIV-1 patients. However, their long-term efficacy is threatened by development of drug resistance strains resulting in resistance mutations. This work aimed to examine the effect of INSTI resistance-associated mutations (RAMs) and polymorphisms on the structure of HIV-1 subtype C (HIV-1C) integrase. Genetic analysis was performed on seven HIV-1C infected individuals with virologic failure after at least 6 months of INSTI-based antiretroviral therapy, presenting at the King Edward VIII hospital in Durban, South Africa. These were compared with sequences from 41 INSTI-naïve isolates. Integrase structures of selected isolates were modeled on the SWISS model online server. Molecular docking and dynamics simulations were also conducted using AutoDock-Vina and AMBER 18 force fields, respectively. Only one INSTI-treated isolate (14.28%) harboured major mutations (G140A + Q148R) as well as the E157Q minor mutation. Interestingly, S119T and V151I were only found in patients failing raltegravir (an INSTI drug). Molecular modeling and docking showed that RAMs and polymorphisms associated with INSTI-based therapy affect protein stability and this is supported by their weakened hydrogen-bond interactions compared to the wild-type. To the best of our knowledge, this is the first study to identify a double mutant in the 140's loop region from South African HIV-1C isolates and study its effects on Raltegravir, Elvitegravir, and Dolutegravir binding.Communicated by Ramaswamy H. Sarma.

Understanding the co-evolutionary molecular mechanisms of resistance in the HIV-1 Gag and protease.

Due to high human immunodeficiency virus type 1 (HIV-1) subtype C infections coupled with increasing antiretroviral treatment failure, the elucidation of complex drug resistance mutational patterns arising through protein co-evolution is required. Despite the inclusion of potent protease inhibitors Lopinavir (LPV) and Darunavir (DRV) in second- and third-line therapies, many patients still fail treatment due to the accumulation of mutations in protease (PR) and recently, Gag. To understand the co-evolutionary molecular mechanisms of resistance in the HIV-1 PR and Gag, we performed 100 ns molecular dynamic simulations on multidrug resistant PR's when bound to LPV, DRV or a mutated A431V NC|p1 Gag cleavage site (CS). Here we showed that distinct changes in PR's active site, flap and elbow regions due to several PR resistance mutations (L10F, M46I, I54V, L76V, V82A) were found to alter LPV and DRV drug binding. However, binding was significantly exacerbated when the mutant PRs were bound to the NC|p1 Gag CS. Although A431V was shown to coordinate several residues in PR, the L76V PR mutation was found to have a significant role in substrate recognition. Consequently, a greater binding affinity was observed when the mutated substrate was bound to an L76V-inclusive PR mutant (Gbind: -62.46 ± 5.75 kcal/mol) than without (Gbind: -50.34 ± 6.28 kcal/mol). These data showed that the co-selection of resistance mutations in the enzyme and substrate can simultaneously constrict regions in PR's active site whilst flexing the flaps to allow flexible movement of the substrate and multiple, complex mechanisms of resistance to occur. Communicated by Ramaswamy H. Sarma.

Molecular dynamic mechanism(s) of inhibition of bioactive antiviral phytochemical compounds targeting cytochrome P450 3A4 and P-glycoprotein.

P-glycoprotein (ABCB1) and cytochrome P450 3A4 (CYP3A4) metabolize almost all known human immunodeficiency virus' protease inhibitor drugs (PIs). Over induction of these proteins' activities has been linked to rapid metabolism of PIs which are then pumped out of the circulatory system, eventually leading to drug-resistance in HIV-positive patients. This study aims to determine, with the use of computational tools, the inhibitory potential of four phytochemical compounds (PCs) (epigallocatechin gallate (EGCG), kaempferol-7-glucoside (K7G), luteolin (LUT) and ellagic acid (EGA)) in inhibiting the activities of these drug-metabolizing proteins. The comparative analysis of the MM/GBSA results revealed that the binding affinity (ΔGbind) of EGCG and K7G for CYP3A4 and ABCB1 are higher than LUT and EGA and fall between the ΔGbind of the inhibitors of CYP3A4 and ABCB1 (Ritonavir (strong inhibitor) and Lopinavir (moderate inhibitor)). The structural analysis (RMSD, RMSF, RoG and protein-ligand interaction plots) also confirmed that EGCG and K7G showed similar inhibitory activities with the inhibitors. The study has shown that EGCG and K7G have inhibitory activities against the two proteins and assumes they could decrease intracellular efflux of PIs, consequently increasing the optimal concentration of PIs in the systemic circulation.Communicated by Ramaswamy H. Sarma.

Deciphering transmission dynamics and spillover of avian influenza viruses from avian species to swine populations globally.

Genome sequences of eleven avian influenza virus (AIV) subtypes have been reported in swine populations from seven countries until August 2020. To unravel the transmission dynamics and spillover events of AIVs from avian reservoirs to swine, full-length hemagglutinin (HA) sequences of AIV subtypes (n = 11) reported from various avian species and swine were retrieved from the 'Influenza Research Database'. Phylogenetic analysis identified closely related avian and swine AIV sequences suggesting potential spillover events from multiple domestic and wild avian species, including chicken, duck, pigeon, goose, quail, and aquatic birds to swine. Furthermore, N-linked glycosylation analysis of these closely related AIV sequences supported the possibility of multiple spillover events of highly pathogenic H5N1 and low pathogenic H9N2 viruses from various avian species to swine. The principal coordinate analysis further validated these findings for H5N1 and H9N2 viruses; however, spillover events of the other nine AIV subtypes were limited. Interestingly, the presence of potential mammalian adaptation markers, particularly in some of the swine H5N1, H7N9, and H9N2 viruses, suggested that these viruses may have already adapted in swine. The occurrence and circulation of these AIVs in swine, especially the H5N1 and H9N2 viruses with numerous spillover events from the avian reservoirs to swine, pose a significant threat in terms of their reassortment with endemic swine viruses or circulating human influenza viruses within the swine which may facilitate the emergence of a novel influenza virus strain with pandemic potential.

HIV-1 integrase strand transfer inhibitors: a review of current drugs, recent advances and drug resistance.

Antiretroviral therapy has been imperative in controlling the human immunodeficiency virus (HIV) epidemic. Most low- and middle-income countries have used nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors extensively in the treatment of HIV. However, integrase strand transfer inhibitors (INSTIs) are becoming more common. Since their identification as a promising therapeutic drug, significant progress has been made that has led to the approval of five INSTIs by the US Food and Drug Administration (FDA), i.e. dolutegravir (DTG), raltegravir (RAL), elvitegravir (EVG), bictegravir (BIC) and cabotegravir (CAB). INSTIs have been shown to effectively halt HIV-1 replication and are commended for having a higher genetic barrier to resistance compared with NRTIs and NNRTIs. More interestingly, DTG has shown a higher genetic barrier to resistance compared with RAL and EVG, and CAB is being used as the first long-acting agent in HIV-1 treatment. Considering the increasing interest in INSTIs for HIV-1 treatment, we focus our review on the retroviral integrase, development of INSTIs and their mode of action. We also discuss each of the INSTI drugs, including potential drug resistance and known side effects.

Acquired HIV-1 Protease Conformational Flexibility Associated with Lopinavir Failure May Shape the Outcome of Darunavir Therapy after Antiretroviral Therapy Switch.

Understanding the underlying molecular interaction during a therapy switch from lopinavir (LPV) to darunavir (DRV) is essential to achieve long-term virological suppression. We investigated the kinetic and structural characteristics of multidrug-resistant South African HIV-1 subtype C protease (HIV-1 PR) during therapy switch from LPV to DRV using enzyme activity and inhibition assay, fluorescence spectroscopy, and molecular dynamic simulation. The HIV-1 protease variants were from clinical isolates with a combination of drug resistance mutations; MUT-1 (M46I, I54V, V82A, and L10F), MUT-2 (M46I, I54V, L76V, V82A, L10F, and L33F), and MUT-3 (M46I, I54V, L76V, V82A, L90M, and F53L). Enzyme kinetics analysis shows an association between increased relative resistance to LPV and DRV with the progressive decrease in the mutant HIV-1 PR variants' catalytic efficiency. A direct relationship between high-level resistance to LPV and intermediate resistance to DRV with intrinsic changes in the three-dimensional structure of the mutant HIV-1 PR as a function of the multidrug-resistance mutation was observed. In silico analysis attributed these structural adjustments to the multidrug-resistance mutations affecting the LPV and DRV binding landscape. Though DRV showed superiority to LPV, as a lower concentration was needed to inhibit the HIV-1 PR variants, the inherent structural changes resulting from mutations selected during LPV therapy may dynamically shape the DRV treatment outcome after the therapy switch.

Design and synthesis of quinoline-pyrimidine inspired hybrids as potential plasmodial inhibitors.

Presently, artemisinin-based combination therapy (ACT) is the first-line therapy of Plasmodium falciparum malaria. With the emergence of malaria parasites that are resistant to ACT, alternative antimalarial therapies are urgently needed. In line with this, we designed and synthesised a series of novel N-(7-chloroquinolin-4-yl)-N'-(4,6-diphenylpyrimidin-2-yl)alkanediamine hybrids (6a-7c) and evaluated their inhibitory activity against the NF54 chloroquine-susceptible strain as a promising class of antimalarial compounds. The antiplasmodial screening revealed that seven analogues showed promising to good activity with half-maximal inhibitory concentration (IC50) = 0.32 µM-4.30 µM. Compound 7a with 1,4-diamine butyl linker and 4-hydroxyl phenyl on fourth and sixth position of pyrimidine core showed the most prominent activity with an IC50 value of 0.32 ± 0.06 µM, with a favourable safety profile of 9.79 to human kidney epithelial (HEK293) cells. The remaining six analogues showed moderate activity with IC50 values ranging from 7.50 µM to 83.01 µM. We further investigated the binding affinities of the molecules to two essential cytosolic P. falciparum heat shock protein 70 homologues; PfHsp70-1 and PfHsp70-z. Compound 7a exhibited the highest binding affinity for both PfHsp70s with KD in a lower nanomolar range (4.4-11.4 nM). Furthermore, molecular docking revealed that compounds 6, 6k, 7b and 7a exhibited better fitness in PfHsp70-1 with compound 7a showing the highest and lowest binding scores of -9.8 kcal/mol. Therefore, we speculate that PfHsp70-1 is one of the targets of these inhibitors. The bioisoteric replacement of the groups at phenyl ring at the fourth and sixth position of the pyrimidine core had a constructive association with antiplasmodial activity. The promising antiplasmodial activity of the synthesised analogues illustrates how crucial molecular hybridisation is as a strategy in the development of quinoline-pyrimidine hybrids as prospective antiprotozoal agents.

Recombinant expression of HIV-1 protease using soluble fusion tags in Escherichia coli: A vital tool for functional characterization of HIV-1 protease.

HIV-1 protease expression in the laboratory is demanding because of its high cytotoxicity, making it difficult to express in bacterial expression systems such as Escherichia coli. To overcome these challenges, HIV-1 protease fusion with solubility enhancing tags helps to mitigate its cytotoxic effect and drive its expression as a soluble protein. Therefore, this review focuses on the expression of bioactive HIV-1 protease using solubility-enhancing fusion tags in Escherichia coli and summarises the characteristic features of the different common fusion tags that have been used in the expression of HIV-1 protease. This review will assist researchers with their choice of protein fusion tag for HIV-1 protease expression.

Modulatory influences of antiviral bioactive compounds on cell viability, mRNA and protein expression of cytochrome P450 3A4 and P-glycoprotein in HepG2 and HEK293 cells.

The induction of cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (ABCB1) influence drug plasma, and eventually decreases the drugs' therapeutic effects. The effects of Plant-derived compounds (PCs) on drug-metabolising proteins are largely unknown. This study investigated the cytotoxicity, cell viability profiles and regulatory influences of four PCs (epigallocatechin gallate (EGCG), kaempferol-7-glucoside (K7G), luteolin (LUT) and ellagic acid (EGA)) on the mRNA and protein expressions of CYP3A4 and ABCB1 in HepG2 and HEK293 cells. After treatment with the PCs (0-400 µM) for 24 h, 80% (IC20) and 50% (IC50) cell viability were determined. The PCs were not toxic to HepG2 (ATP levels increased at IC20, insignificant change in LDH (lactate dehydrogenase) with the exception of LUT, and ABCB1 protein expressions decreased. The PCs decreased CYP3A4 at IC20 (except LUT), EGCG and K7G at IC20 decreased mRNA expression. For HEK293 cells, no significant change in ATP, except for EGCG IC20 and K7G IC50 which decreased and increased, respectively. LDH decreased at IC20, but LUT IC50 significant increase LDH. ABCB1 protein expression increased at both IC20 and IC50, but LUT and EGA at IC50 decreased mRNA expression. The PCs at IC20, and IC50 of LUT, K7G and of EGCG may enhance drug bioavailability.