Omicron extensively but incompletely escapes Pfizer BNT162b2 neutralization.

The emergence of the SARS-CoV-2 variant of concern Omicron (Pango lineage B.1.1.529), first identified in Botswana and South Africa, may compromise vaccine effectiveness and lead to re-infections1. Here we investigated Omicron escape from neutralization by antibodies from South African individuals vaccinated with Pfizer BNT162b2. We used blood samples taken soon after vaccination from individuals who were vaccinated and previously infected with SARS-CoV-2 or vaccinated with no evidence of previous infection. We isolated and sequence-confirmed live Omicron virus from an infected person and observed that Omicron requires the angiotensin-converting enzyme 2 (ACE2) receptor to infect cells. We compared plasma neutralization of Omicron relative to an ancestral SARS-CoV-2 strain and found that neutralization of ancestral virus was much higher in infected and vaccinated individuals compared with the vaccinated-only participants. However, both groups showed a 22-fold reduction in vaccine-elicited neutralization by the Omicron variant. Participants who were vaccinated and had previously been infected exhibited residual neutralization of Omicron similar to the level of neutralization of the ancestral virus observed in the vaccination-only group. These data support the notion that reasonable protection against Omicron may be maintained using vaccination approaches.

Omicron infection enhances Delta antibody immunity in vaccinated persons.

The extent to which Omicron infection1-9, with or without previous vaccination, elicits protection against the previously dominant Delta (B.1.617.2) variant is unclear. Here we measured the neutralization capacity against variants of severe acute respiratory syndrome coronavirus 2 in 39 individuals in South Africa infected with the Omicron sublineage BA.1 starting at a median of 6 (interquartile range 3-9) days post symptom onset and continuing until last follow-up sample available, a median of 23 (interquartile range 19-27) days post symptoms to allow BA.1-elicited neutralizing immunity time to develop. Fifteen participants were vaccinated with Pfizer's BNT162b2 or Johnson & Johnson's Ad26.CoV2.S and had BA.1 breakthrough infections, and 24 were unvaccinated. BA.1 neutralization increased from a geometric mean 50% focus reduction neutralization test titre of 42 at enrolment to 575 at the last follow-up time point (13.6-fold) in vaccinated participants and from 46 to 272 (6.0-fold) in unvaccinated participants. Delta virus neutralization also increased, from 192 to 1,091 (5.7-fold) in vaccinated participants and from 28 to 91 (3.0-fold) in unvaccinated participants. At the last time point, unvaccinated individuals infected with BA.1 had low absolute levels of neutralization for the non-BA.1 viruses and 2.2-fold lower BA.1 neutralization, 12.0-fold lower Delta neutralization, 9.6-fold lower Beta variant neutralization, 17.9-fold lower ancestral virus neutralization and 4.8-fold lower Omicron sublineage BA.2 neutralization relative to vaccinated individuals infected with BA.1. These results indicate that hybrid immunity formed by vaccination and Omicron BA.1 infection should be protective against Delta and other variants. By contrast, infection with Omicron BA.1 alone offers limited cross-protection despite moderate enhancement.

Rapid epidemic expansion of the SARS-CoV-2 Omicron variant in southern Africa.

The SARS-CoV-2 epidemic in southern Africa has been characterized by three distinct waves. The first was associated with a mix of SARS-CoV-2 lineages, while the second and third waves were driven by the Beta (B.1.351) and Delta (B.1.617.2) variants, respectively1-3. In November 2021, genomic surveillance teams in South Africa and Botswana detected a new SARS-CoV-2 variant associated with a rapid resurgence of infections in Gauteng province, South Africa. Within three days of the first genome being uploaded, it was designated a variant of concern (Omicron, B.1.1.529) by the World Health Organization and, within three weeks, had been identified in 87 countries. The Omicron variant is exceptional for carrying over 30 mutations in the spike glycoprotein, which are predicted to influence antibody neutralization and spike function4. Here we describe the genomic profile and early transmission dynamics of Omicron, highlighting the rapid spread in regions with high levels of population immunity.

SARS-CoV-2 BA.4/5 infection triggers more cross-reactive FcγRIIIa signaling and neutralization than BA.1, in the context of hybrid immunity.

SARS-CoV-2 variants of concern (VOCs) differentially trigger neutralizing and antibody-dependent cellular cytotoxic (ADCC) antibodies with variable cross-reactivity. Omicron BA.4/5 was approved for inclusion in bivalent vaccination boosters, and therefore the antigenic profile of antibodies elicited by this variant is critical to understand. Here, we investigate the ability of BA.4/5-elicited antibodies following the first documented (primary) infection (n = 13) or breakthrough infection after vaccination (n = 9) to mediate neutralization and FcγRIIIa signaling across multiple SARS-CoV-2 variants including XBB.1.5 and BQ.1. Using a pseudovirus neutralization assay and a FcγRIIIa crosslinking assay to measure ADCC potential, we show that unlike SARS-CoV-2 Omicron BA.1, BA.4/5 infection triggers highly cross-reactive functional antibodies. Cross-reactivity was observed both in the absence of prior vaccination and in breakthrough infections following vaccination. However, BQ.1 and XBB.1.5 neutralization and FcγRIIIa signaling were significantly compromised compared to other VOCs, regardless of prior vaccination status. BA.4/5 triggered FcγRIIIa signaling was significantly more resilient against VOCs (<10-fold decrease in magnitude) compared to neutralization (10- to 100-fold decrease). Overall, this study shows that BA.4/5 triggered antibodies are highly cross-reactive compared to those triggered by other variants. Although this is consistent with enhanced neutralization and FcγRIIIa signaling breadth of BA.4/5 vaccine boosters, the reduced activity against XBB.1.5 supports the need to update vaccines with XBB sublineage immunogens to provide adequate coverage of these highly antibody evasive variants. IMPORTANCE: The continued evolution of SARS-CoV-2 has resulted in a number of variants of concern. Of these, the Omicron sublineage is the most immune evasive. Within Omicron, the BA.4/5 sublineage drove the fifth wave of infection in South Africa prior to becoming the dominant variant globally. As a result this spike sequence was approved as part of a bivalent vaccine booster, and rolled out worldwide. We aimed to understand the cross-reactivity of neutralizing and Fc mediated cytotoxic functions elicited by BA.4/5 infection following infection or breakthrough infection. We find that, in contrast to BA.1 which triggered fairly strain-specific antibodies, BA.4/5 triggered antibodies that are highly cross-reactive for neutralization and antibody-dependent cellular cytotoxicity potential. Despite this cross-reactivity, these antibodies are compromised against highly resistant variants such as XBB.1.5 and BQ.1. This suggests that next-generation vaccines will require XBB sublineage immunogens in order to protect against these evasive variants.

Genomic characterization of multi drug resistant ESBL-producing Escherichia coli isolates from patients and patient environments in a teaching hospital in Ghana.

BACKGROUND: ESBL-producing Escherichia coli pose a growing health risk in community and healthcare settings. We investigated the resistome, virulome, mobilome, and genetic relatedness of multidrug-resistant (MDR) E. coli isolates from patients and their environment in a Ghanaian teaching hospital. MATERIALS AND METHODS: Twenty-three MDR ESBL-producing or carbapenem-resistant E. coli isolates from a collection of MDR Gram-negative bacteria (GNB) from patients and environments were selected for genomic analyses. Whole genome sequencing and bioinformatics tools were used to analyze genomic characteristics and phylogeny. RESULTS: The prevalence and incidence of rectal carriage of ESBL E. coli among patients were 13.65% and 11.32% respectively. The ß-lactamase genes, blaTEM-1B (10 isolates) and blaCTX-M-15 (12 isolates) were commonly associated with IncFIB plasmid replicons and co-occurred with aminoglycoside, macrolide, and sulfamethoxazole/trimethoprim resistance. Insertion sequences, transposons, and class I integrons were found with blaCTX-M-15. Carriage and environmental isolates carried multiple virulence genes, with terC being the most prevalent in 21 isolates. Seventeen sequence types (STs) were identified, including a novel ST (ST13846). Phylogenetic analysis grouped the isolates into four main clusters, with one outlier. High genetic relatedness was observed between two carriage isolates of ST940 and between a carriage isolate and an environmental isolate of ST648. Isolates with different STs, collected at different times and locations, also showed genetic similarities. CONCLUSION: We identified ESBL-producing E. coli with diverse genomic characteristics circulating in different hospital directorates. Clonal relatedness was observed among isolates from patients and the environment, as well as between different patients, suggesting transmission within and between sources.

Household Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 From Adult Index Cases With and Without Human Immunodeficiency Virus in South Africa, 2020-2021: A Case-Ascertained, Prospective, Observational Household Transmission Study.

BACKGROUND: In South Africa, 19% of adults are living with human immunodeficiency virus (HIV; LWH). Few data on the influence of HIV on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) household transmission are available. METHODS: We performed a case-ascertained, prospective household transmission study of symptomatic adult index SARS-CoV-2 cases LWH and not living with HIV (NLWH) and their contacts from October 2020 to September 2021. Households were followed up 3 times a week for 6 weeks to collect nasal swabs for SARS-CoV-2 testing. We estimated household cumulative infection risk (HCIR) and duration of SARS-CoV-2 positivity (at a cycle threshold value <30 as proxy for high viral load). RESULTS: HCIR was 59% (220 of 373), not differing by index HIV status (60% LWH vs 58% NLWH). HCIR increased with index case age (35-59 years: adjusted OR [aOR], 3.4; 95% CI, 1.5-7.8 and ≥60 years: aOR, 3.1; 95% CI, 1.0-10.1) compared with 18-34 years and with contacts' age, 13-17 years (aOR, 7.1; 95% CI, 1.5-33.9) and 18-34 years (aOR, 4.4; 95% CI, 1.0-18.4) compared with <5 years. Mean positivity was longer in cases LWH (adjusted hazard ratio, 0.4; 95% CI, .1-.9). CONCLUSIONS: Index HIV status was not associated with higher HCIR, but cases LWH had longer positivity duration. Adults aged >35 years were more likely to transmit and individuals aged 13-34 to be infected SARS-CoV-2 in the household. As HIV infection may increase transmission, health services must maintain HIV testing and antiretroviral therapy initiation.

Selection Analysis Identifies Clusters of Unusual Mutational Changes in Omicron Lineage BA.1 That Likely Impact Spike Function.

Among the 30 nonsynonymous nucleotide substitutions in the Omicron S-gene are 13 that have only rarely been seen in other SARS-CoV-2 sequences. These mutations cluster within three functionally important regions of the S-gene at sites that will likely impact (1) interactions between subunits of the Spike trimer and the predisposition of subunits to shift from down to up configurations, (2) interactions of Spike with ACE2 receptors, and (3) the priming of Spike for membrane fusion. We show here that, based on both the rarity of these 13 mutations in intrapatient sequencing reads and patterns of selection at the codon sites where the mutations occur in SARS-CoV-2 and related sarbecoviruses, prior to the emergence of Omicron the mutations would have been predicted to decrease the fitness of any virus within which they occurred. We further propose that the mutations in each of the three clusters therefore cooperatively interact to both mitigate their individual fitness costs, and, in combination with other mutations, adaptively alter the function of Spike. Given the evident epidemic growth advantages of Omicron overall previously known SARS-CoV-2 lineages, it is crucial to determine both how such complex and highly adaptive mutation constellations were assembled within the Omicron S-gene, and why, despite unprecedented global genomic surveillance efforts, the early stages of this assembly process went completely undetected.

Early assessment of the clinical severity of the SARS-CoV-2 omicron variant in South Africa: a data linkage study.

BACKGROUND: The SARS-CoV-2 omicron variant of concern was identified in South Africa in November, 2021, and was associated with an increase in COVID-19 cases. We aimed to assess the clinical severity of infections with the omicron variant using S gene target failure (SGTF) on the Thermo Fisher Scientific TaqPath COVID-19 PCR test as a proxy. METHODS: We did data linkages for national, South African COVID-19 case data, SARS-CoV-2 laboratory test data, SARS-CoV-2 genome data, and COVID-19 hospital admissions data. For individuals diagnosed with COVID-19 via TaqPath PCR tests, infections were designated as either SGTF or non-SGTF. The delta variant was identified by genome sequencing. Using multivariable logistic regression models, we assessed disease severity and hospitalisations by comparing individuals with SGTF versus non-SGTF infections diagnosed between Oct 1 and Nov 30, 2021, and we further assessed disease severity by comparing SGTF-infected individuals diagnosed between Oct 1 and Nov 30, 2021, with delta variant-infected individuals diagnosed between April 1 and Nov 9, 2021. FINDINGS: From Oct 1 (week 39), 2021, to Dec 6 (week 49), 2021, 161 328 cases of COVID-19 were reported in South Africa. 38 282 people were diagnosed via TaqPath PCR tests and 29 721 SGTF infections and 1412 non-SGTF infections were identified. The proportion of SGTF infections increased from two (3·2%) of 63 in week 39 to 21 978 (97·9%) of 22 455 in week 48. After controlling for factors associated with hospitalisation, individuals with SGTF infections had significantly lower odds of admission than did those with non-SGTF infections (256 [2·4%] of 10 547 vs 121 [12·8%] of 948; adjusted odds ratio [aOR] 0·2, 95% CI 0·1-0·3). After controlling for factors associated with disease severity, the odds of severe disease were similar between hospitalised individuals with SGTF versus non-SGTF infections (42 [21%] of 204 vs 45 [40%] of 113; aOR 0·7, 95% CI 0·3-1·4). Compared with individuals with earlier delta variant infections, SGTF-infected individuals had a significantly lower odds of severe disease (496 [62·5%] of 793 vs 57 [23·4%] of 244; aOR 0·3, 95% CI 0·2-0·5), after controlling for factors associated with disease severity. INTERPRETATION: Our early analyses suggest a significantly reduced odds of hospitalisation among individuals with SGTF versus non-SGTF infections diagnosed during the same time period. SGTF-infected individuals had a significantly reduced odds of severe disease compared with individuals infected earlier with the delta variant. Some of this reduced severity is probably a result of previous immunity. FUNDING: The South African Medical Research Council, the South African National Department of Health, US Centers for Disease Control and Prevention, the African Society of Laboratory Medicine, Africa Centers for Disease Control and Prevention, the Bill & Melinda Gates Foundation, the Wellcome Trust, and the Fleming Fund.

Characterization, Pathogenicity, Phylogeny, and Comparative Genomic Analysis of Pseudomonas tolaasii Strains Isolated from Various Mushrooms in China.

Since 2016, devastating bacterial blotch affecting the fruiting bodies of Agaricus bisporus, Cordyceps militaris, Flammulina filiformis, and Pleurotus ostreatus in China has caused severe economic losses. We isolated 102 bacterial strains and characterized them polyphasically. We identified the causal agent as Pseudomonas tolaasii and confirmed the pathogenicity of the strains. A host range test further confirmed the pathogen's ability to infect multiple hosts. This is the first report in China of bacterial blotch in C. militaris caused by P. tolaasii. Whole-genome sequences were generated for three strains: Pt11 (6.48 Mb), Pt51 (6.63 Mb), and Pt53 (6.80 Mb), and pangenome analysis was performed with 13 other publicly accessible P. tolaasii genomes to determine their genetic diversity, virulence, antibiotic resistance, and mobile genetic elements. The pangenome of P. tolaasii is open, and many more gene families are likely to emerge with further genome sequencing. Multilocus sequence analysis using the sequences of four common housekeeping genes (glns, gyrB, rpoB, and rpoD) showed high genetic variability among the P. tolaasii strains, with 115 strains clustered into a monophyletic group. The P. tolaasii strains possess various genes for secretion systems, virulence factors, carbohydrate-active enzymes, toxins, secondary metabolites, and antimicrobial resistance genes that are associated with pathogenesis and adapted to different environments. The myriad of insertion sequences, integrons, prophages, and genome islands encoded in the strains may contribute to genome plasticity, virulence, and antibiotic resistance. These findings advance understanding of the determinants of virulence, which can be targeted for the effective control of bacterial blotch disease.

Genomic analysis of antibiotic-resistant Enterococcus spp. reveals novel enterococci strains and the spread of plasmid-borne Tet(M), Tet(L) and Erm(B) genes from chicken litter to agricultural soil in South Africa.

Manure from food animals exposed to antibiotics is often used as soil fertiliser, potentially releasing antibiotic-resistant bacteria (ARB) with diverse antibiotic-resistance genes (ARGs) into the soil. To determine the impact of chicken litter application on the soil resistome, Enterococcus spp. isolated from chicken litter and soil samples collected before and after the soil amendment were characterised, using whole-genome sequencing and bioinformatics tools. Nineteen Enterococcus spp. isolates from the three sources were sequenced on Illumina Miseq platform to ascertain the isolates' resistome, mobilome, virulome, clonality, and phylogenomic relationships. Multilocus sequence typing (MLST) analysis revealed eight novel sequence types (STs) (ST1700, ST1752, ST1753, ST1754, ST1755, ST1756, ST1004, and ST1006). The isolates harboured multiple resistance genes including those conferring resistance to inter alia macrolides-lincosamide-streptogramin (erm(B), lnu(B), lnu(G), lsaA, lsaE, eat(A), msr(C)), tetracycline (tet(M), tet(L), tet(S)), aminoglycosides (aac(6')-Ii, aac(6')-Iih, ant(6)-Ia, aph(3')-III, ant(9)-Ia), fluoroquinolones (efmA, and emeA), vancomycin (VanC {VanC-2, VanXY, VanXYC-3, VanXYC-4, VanRC}), and chloramphenicol (cat). The litter-amended soil harboured new ARB (particularly E. faecium) and ARGs (ant(6)-Ia, aac(6')-Ii, aph(3')-III), lnu(G), msr(C), and eat(A), efmA) that were not previously detected in the soil. The identified ARGs were associated with diverse mobile genetic elements (MGEs) such as insertion sequences (IS6, ISL3, IS256, IS30), transposons (Tn3 and Tn916) and plasmids (repUS43, repUS1, rep9b, and rep 22). Twenty-eight virulence genes encoding adherence/biofilm formation (ebpA, ebpB, ebpC), antiphagocytosis (elrA) and bacterial sex pheromones (Ccf10, cOB1, cad, and camE), were detected in the genomes of the isolates. Phylogenomic analysis revealed a close relationship between a few isolates from litter-amended soil and the chicken litter isolates. The differences in the ARG and ARB profiles in the soil before and after the litter amendment and their association with diverse MGEs indicate the mobilisation and transmission of ARGs and ARB from the litter to the soil.

Di-2-picolylamine triggers caspase-independent apoptosis by inducing oxidative stress in human liver hepatocellular carcinoma cells.

Di-2-picolylamine (DPA) is an organic compound that has been shown to possess antioxidant properties when conjugated to form a metal complex. The basis of this study was to determine the effects of DPA on the proliferation and apoptosis of human hepatocellular carcinoma cells and elucidate the possible mechanisms. The methylthiazol tetrazolium assay served to measure cell viability and generated an IC50 of 1591 µM. Luminometry was used to investigate caspase activity and ATP concentration. It was observed that the decreased cell viability was associated with reduced ATP levels. Despite increased Bax and caspase 9 activity, cell death was caspase independent as indicated by the reduction in caspase 3/7 activity. This was associated with the downregulation poly(ADP-ribose) polymerase cleavage (Western blotting). However, the Hoescht assay depicted nuclear condensation and apoptotic body formation with elevated DPA levels suggesting DNA damage in HepG2 cells. DNA damage assessed by the comet assay confirmed an increased comet tail formation. The presence of oxidative stress was investigated by quantifying reactive species (malondialdehyde and nitrates concentration) and Western blotting to confirm the expression of antioxidant proteins. The DPA increased lipid peroxidation (RNS), a marker of oxidative stress, consequently causing cell death. The accompanying upregulation of stress-associated proteins superoxide dismutase (SOD2), nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and Hsp70 verifies oxidative stress.

Toxicogenicity and mechanistic pathways of aflatoxin B1 induced renal injury.

The study investigated the toxicogenic effects, molecular mechanisms and proteomic assessment of aflatoxin B1 (AFB1 ) on human renal cells. Hek293 cells were exposed to AFB1 (0-100 µM) for 24 h. The effect on cell viability was assessed using the methylthiazol tetrazolium (MTT) assay, which also produced the half maximal inhibitory concentration (IC50 ) used in subsequent assays. Free radical production was evaluated by quantifying malondialdehyde (MDA) and nitrate concentration, while DNA fragmentation was determined using the single cell gel electrophoresis (SCGE) assay and DNA gel electrophoresis. Damage to cell membranes was ascertained using the lactate dehydrogenase (LDH) assay. The concentration of ATP, reduced glutathione (GSH), necrosis, annexin V and caspase activity was measured by luminometry. Western blotting and quantitative PCR was used to assess the expression of proteins and genes associated with apoptosis and oxidative stress. The MTT assay revealed a reduction in cell viability of Hek293 cells as the AFB1 concentration was increased, with a half maximum inhibitory concentration (IC50 ) of 32.60 µM. The decreased viability corresponded to decreased ATP concentration. The upregulation of Hsp70 indicated that oxidative stress was induced in the AFB1 -treated cells. While this implies an increased production of free radicals, the accompanying upregulation of the antioxidant system indicates the activation of defense mechanisms to prevent cellular damage. Thus, membrane damage associated with increased radical formation was prevented as indicated by the reduced LDH release and necrosis. In addition, cytotoxic effects were evident as AFB1 activated the intrinsic pathway of apoptosis with corresponding increased DNA fragmentation, p53 and Bax upregulation and increased caspase activity, but externalization of phosphatidylserine (PS), a major hallmark of apoptosis, did not occur in AFB1 treated renal cells. The results suggest that AFB1 induced oxidative stress leading to cell death by the intrinsic pathway of apoptosis in renal cells.

Influenza Viruses: Harnessing the Crucial Role of the M2 Ion-Channel and Neuraminidase toward Inhibitor Design.

As a member of the Orthomyxoviridae family of viruses, influenza viruses (IVs) are known causative agents of respiratory infection in vertebrates. They remain a major global threat responsible for the most virulent diseases and global pandemics in humans. The virulence of IVs and the consequential high morbidity and mortality of IV infections are primarily attributed to the high mutation rates in the IVs' genome coupled with the numerous genomic segments, which give rise to antiviral resistant and vaccine evading strains. Current therapeutic options include vaccines and small molecule inhibitors, which therapeutically target various catalytic processes in IVs. However, the periodic emergence of new IV strains necessitates the continuous development of novel anti-influenza therapeutic options. The crux of this review highlights the recent studies on the biology of influenza viruses, focusing on the structure, function, and mechanism of action of the M2 channel and neuraminidase as therapeutic targets. We further provide an update on the development of new M2 channel and neuraminidase inhibitors as an alternative to existing anti-influenza therapy. We conclude by highlighting therapeutic strategies that could be explored further towards the design of novel anti-influenza inhibitors with the ability to inhibit resistant strains.

Quantitative microbial risk assessment for waterborne pathogens in a wastewater treatment plant and its receiving surface water body.

BACKGROUND: Access to safe water for drinking and domestic activities remains a challenge in emerging economies like South Africa, forcing resource-limited communities to use microbiologically polluted river water for personal and household purposes, posing a public health risk. This study quantified bacterial contamination and the potential health hazards that wastewater treatment plant (WWTP) workers and communities may face after exposure to waterborne pathogenic bacteria in a WWTP and its associated surface water, respectively. RESULTS: Escherichia coli (Colilert®-18/ Quanti-Tray® 2000) and enterococci (Enterolert®/ Quanti-Tray® 2000) were quantified and definitively identified by real-time polymerase chain reaction targeting the uidA and tuf genes, respectively. An approximate beta-Poisson dose-response model was used to estimate the probability of infection (Pi) with pathogenic E. coli. Mean E. coli concentration ranged from 2.60E+ 02/100 mL to 4.84E+ 06/100 mL; enterococci ranged from 2.60E+ 02/100 mL to 3.19E+ 06/100 mL across all sampled sites. Of the 580 E. coli isolates obtained from this study, 89.1% were intestinal, and 7.6% were extraintestinal pathogenic E. coli. The 579 enterococci obtained were 50.4% E. faecalis (50.4%), 31.4% E. faecium, 3.5%, E. casseliflavus and 0.7% E. gallinarum. The community health risk stemming from the use of the water for recreational and domestic purposes revealed a greater health risk (Pi) from the ingestion of 1 mL of river water from upstream (range, 55.1-92.9%) than downstream (range, 26.8-65.3%) sites. The occupational risk of infection with pathogenic E. coli for workers resulting from a once-off unintentional consumption of 1 mL of water was 0% (effluent) and 23.8% (raw influent). Multiple weekly exposures of 1 mL over a year could result in a Pi of 1.2 and 100% for the effluent and influent, respectively. CONCLUSION: Our findings reveal that there is a potentially high risk of infection for WWTP workers and communities that use river water upstream and downstream of the investigated WWTP.

The molecular effect of 1,4,7-triazacyclononane on oxidative stress parameters in human hepatocellular carcinoma (HepG2) cells.

Antibiotic resistance poses a great threat to human, animal and environmental health. ß-Lactam antibiotics have been successful in combating bacterial infections. However, the overuse, inappropriate prescribing, unavailability of new antibiotics and regulation barriers have exacerbated bacterial resistance to these antibiotics. 1,4,7-Triazacyclononane (TACN) is a cyclic organic tridentate inhibitor with strong metal-chelating abilities that has been shown to inhibit ß-lactamase enzymes and may represent an important breakthrough in the treatment of drug-resistant bacterial strains. However, its cytotoxicity in the liver is unknown. This study aimed to determine the effect of TACN on oxidative stress in HepG2 cells. The HepG2 cells were treated with 0 to 500 µM TACN for 24 hours to obtain an IC50 for use in subsequent assays. Free radicals were measured using the thiobarbituric acid reactive substance and nitric oxide synthase assays, respectively, while antioxidant levels were assessed using luminometry (glutathione [GSH] and adenosine triphosphate [ATP]) and Western blot analysis (SOD, catalase, GPx-1, HSP70 and Nrf2). Percentage survival fluctuated as TACN concentration increased with a calculated IC50 of 545 µM. A slight increase in HSP70 and Nrf2 expression indicated the presence of stress and a response against it, respectively. However, free radical production was not increased as indicated by decreased malondialdehyde levels and reactive nitrogen species. Glutathione levels increased slightly, while ATP levels were marginally altered. The results suggest that TACN does not induce oxidative stress in HepG2 cells and can be exploited as a potential inhibitor.

Molecular Insights Into Di(2-Picolyl) Amine-Induced Cytotoxicity and Apoptosis in Human Kidney (HEK293) Cells.

Di(2-picolyl) amine (DPA) is a pyridine derivative known to chelate metal ions and thus has potential anticancer properties; however, its effect on normal cells remains unchartered necessitating further research. This study, therefore, investigated the mechanistic effects of DPA-induced cytotoxicity and apoptosis in the HEK293 cell line. Methods required that an half the maximum inhibition concentration (IC50) was derived using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Analyses aimed to assess oxidative stress, membrane damage, and DNA fragmentation by means of biochemical assays were performed. Luminometry analysis was carried out to understand the mechanism of apoptosis induction by determining the levels of adenosine triphosphate (ATP) and the activities of caspase-8, -9, and -3/7. Western blotting was used to ascertain the expression of apoptotic and stress-related proteins. An IC50 of 1,079 µM DPA was obtained. Antioxidant effect correlated with a minimum increase in reactive oxygen species induced lipid peroxidation. The increase in initiator caspase-8 and -9 and executioner caspase-3/7 activities by DPA-induced apoptosis albeit prompting a decline in the levels of ATP. Furthermore, DPA brought about the following consequences on HEK293 cells: markedly elevated tail lengths of the comets, poly (ADP-ribose) polymerase 1 cleavage, and apoptotic body formation observed in the late stages. The cytotoxic effects of DPA in HEK293 cells may be mediated by induction of apoptosis via the caspase-dependent mechanism.

Understanding the Hsp90 N-terminal Dynamics: Structural and Molecular Insights into the Therapeutic Activities of Anticancer Inhibitors Radicicol (RD) and Radicicol Derivative (NVP-YUA922).

Heat shock protein 90 (Hsp90) is a crucial component in carcinogenesis and serves as a molecular chaperone that facilitates protein maturation whilst protecting cells against temperature-induced stress. The function of Hsp90 is highly dependent on adenosine triphosphate (ATP) binding to the N-terminal domain of the protein. Thus, inhibition through displacement of ATP by means of competitive binding with a suitable organic molecule is considered an attractive topic in cancer research. Radicicol (RD) and its derivative, resorcinylic isoxazole amine NVP-AUY922 (NVP), have shown promising pharmacodynamics against Hsp90 activity. To date, the underlying binding mechanism of RD and NVP has not yet been investigated. In this study, we provide a comprehensive understanding of the binding mechanism of RD and NVP, from an atomistic perspective. Density functional theory (DFT) calculations enabled the analyses of the compounds' electronic properties and results obtained proved to be significant in which NVP was predicted to be more favorable with solvation free energy value of -23.3 kcal/mol and highest stability energy of 75.5 kcal/mol for a major atomic delocalization. Molecular dynamic (MD) analysis revealed NVP bound to Hsp90 (NT-NVP) is more stable in comparison to RD (NT-RD). The Hsp90 protein exhibited a greater binding affinity for NT-NVP (-49.4 ± 3.9 kcal/mol) relative to NT-RD (-28.9 ± 4.5 kcal/mol). The key residues influential in this interaction are Gly 97, Asp 93 and Thr 184. These findings provide valuable insights into the Hsp90 dynamics and will serve as a guide for the design of potent novel inhibitors for cancer treatment.

1,4,7-Triazacyclononane Restores the Activity of ß-Lactam Antibiotics against Metallo-ß-Lactamase-Producing Enterobacteriaceae: Exploration of Potential Metallo-ß-Lactamase Inhibitors.

Metallo-ß-lactamase (MBL)-producing Enterobacteriaceae are of grave clinical concern, particularly as there are no metallo-ß-lactamase inhibitors approved for clinical use. The discovery and development of MBL inhibitors to restore the efficacy of available ß-lactams are thus imperative. We investigated a zinc-chelating moiety, 1,4,7-triazacyclononane (TACN), for its inhibitory activity against clinical carbapenem-resistant Enterobacteriaceae MICs, minimum bactericidal concentrations (MBCs), the serum effect, fractional inhibitory concentration indexes, and time-kill kinetics were determined using broth microdilution techniques according to Clinical and Laboratory Standards Institute (CSLI) guidelines. Enzyme kinetic parameters and the cytotoxic effects of TACN were determined using spectrophotometric assays. The interactions of the enzyme-TACN complex were investigated by computational studies. Meropenem regained its activity against carbapenemase-producing Enterobacteriaceae, with the MIC decreasing from between 8 and 64 mg/liter to 0.03 mg/liter in the presence of TACN. The TACN-meropenem combination showed bactericidal effects with an MBC/MIC ratio of ≤4, and synergistic activity was observed. Human serum effects on the MICs were insignificant, and TACN was found to be noncytotoxic at concentrations above the MIC values. Computational studies predicted that TACN inhibits MBLs by targeting their catalytic active-site pockets. This was supported by its inhibition constant (Ki ), which was 0.044 µM, and its inactivation constant (Kinact), which was 0.0406 min-1, demonstrating that TACN inhibits MBLs efficiently and holds promise as a potential inhibitor.IMPORTANCE Carbapenem-resistant Enterobacteriaceae (CRE)-mediated infections remain a significant public health concern and have been reported to be critical in the World Health Organization's priority pathogens list for the research and development of new antibiotics. CRE produce enzymes, such as metallo-ß-lactamases (MBLs), which inactivate ß-lactam antibiotics. Combination therapies involving a ß-lactam antibiotic and a ß-lactamase inhibitor remain a major treatment option for infections caused by ß-lactamase-producing organisms. Currently, no MBL inhibitor-ß-lactam combination therapy is clinically available for MBL-positive bacterial infections. Hence, developing efficient molecules capable of inhibiting these enzymes could be a promising way to overcome this phenomenon. TACN played a significant role in the inhibitory activity of the tested molecules against CREs by potentiating the activity of carbapenem. This study demonstrates that TACN inhibits MBLs efficiently and holds promises as a potential MBL inhibitor to help curb the global health threat posed by MBL-producing CREs.

Mechanistic Insights into Oxidative Stress and Apoptosis Mediated by Tannic Acid in Human Liver Hepatocellular Carcinoma Cells.

The study investigated the cytotoxic effect of a natural polyphenolic compound Tannic acid (TA) on human liver hepatocellular carcinoma (HepG2) cells and elucidated the possible mechanisms that lead to apoptosis and oxidative stress HepG2 cell. The HepG2 cells were treated with TA for 24 h and various assays were conducted to determine whether TA could induce cell death and oxidative stress. The cell viability assay was used to determine the half maximal inhibitory concentration (IC50), caspase activity and cellular ATP were determined by luminometry. Microscopy was employed to determine deoxyribonucleic acid (DNA) integrity, while thiobarbituric acid (TBARS) and nitric oxide synthase (NOS) assays were used to elucidate cellular reactive oxygen species (ROS) and reactive nitrogen species (RNS), respectively. Western blotting was used to confirm protein expression. The results revealed that tannic acid induced caspase activation and increased the presence of cellular ROS and RNS, while downregulating antioxidant expression. Tannic acid also showed increased cell death and increased DNA fragmentation. In conclusion, TA was able to induce apoptosis by DNA fragmentation via caspase-dependent and caspase-independent mechanism. It was also able to induce oxidative stress, consequently contributing to cell death.

Diversity and Proliferation of Metallo-ß-Lactamases: a Clarion Call for Clinically Effective Metallo-ß-Lactamase Inhibitors.

The worldwide proliferation of life-threatening metallo-ß-lactamase (MBL)-producing Gram-negative bacteria is a serious concern to public health. MBLs are compromising the therapeutic efficacies of ß-lactams, particularly carbapenems, which are last-resort antibiotics indicated for various multidrug-resistant bacterial infections. Inhibition of enzymes mediating antibiotic resistance in bacteria is one of the major promising means for overcoming bacterial resistance. Compounds having potential MBL-inhibitory activity have been reported, but none are currently under clinical trials. The need for developing safe and efficient MBL inhibitors (MBLIs) is obvious, particularly with the continuous spread of MBLs worldwide. In this review, the emergence and escalation of MBLs in Gram-negative bacteria are discussed. The relationships between different class B ß-lactamases identified up to 2017 are represented by a phylogenetic tree and summarized. In addition, approved and/or clinical-phase serine ß-lactamase inhibitors are recapitulated to reflect the successful advances made in developing class A ß-lactamase inhibitors. Reported MBLIs, their inhibitory properties, and their purported modes of inhibition are delineated. Insights into structural variations of MBLs and the challenges involved in developing potent MBLIs are also elucidated and discussed. Currently, natural products and MBL-resistant ß-lactam analogues are the most promising agents that can become clinically efficient MBLIs. A deeper comprehension of the mechanisms of action and activity spectra of the various MBLs and their inhibitors will serve as a bedrock for further investigations that can result in clinically useful MBLIs to curb this global menace.