Generation of anti-idiotypic antibodies mimicking Cry2Aa toxin from an immunized mouse phage display library as potential insecticidal agents against Plutella xylostella.

This study tried to generate anti-idiotypic antibodies (Ab2s) which mimic Cry2Aa toxin using a phage-display antibody library (2.8 × 107 CFU/mL). The latter was constructed from a mouse immunized with F (ab')2 fragments digested from anti-Cry2Aa polyclonal antibodies. The F (ab')2 fragments and Plutella xylostella (P. xylostella) brush border membrane vesicles (BBMV) were utilized as targets for selection. Eight mouse phage-display single-chain variable fragments (scFvs) were isolated and identified by enzyme-linked immunoassay (ELISA), PCR and DNA sequencing after four rounds of biopanning. Among them, M3 exhibited the highest binding affinity with F (ab')2, while M4 bound the best with the toxin binding region of cadherin of P. xylostella (PxCad-TBR). Both of these two fragments were chosen for prokaryotic expression. The expressed M3 and M4 proteins with molecular weights of 30 kDa were purified. The M4 showed a binding affinity of 29.9 ± 2.4 nM with the PxCad-TBR and resulted in 27.8 ± 4.3 % larvae mortality against P. xylostella. Computer-assisted molecular modeling and docking analysis showed that mouse scFv M4 mimicked some Cry2Aa toxin binding sites when interacting with PxCad-TBR. Therefore, anti-idiotypic antibodies generated by BBMV-based screening could be useful for the development of new bio-insecticides as an alternative to Cry2Aa toxin for pest control.

Analysis of mutation profiles in extranodal NK/T-cell lymphoma: clinical and prognostic correlations.

The molecular pathogenesis of extranodal NK/T-cell lymphoma (NKTCL) remains obscured despite the next-generation sequencing (NGS) studies explored on ever larger cohorts in the last decade. We addressed the highly variable mutation frequencies reported among previous studies with comprehensive amplicon coverage and enhanced sequencing depth to achieve higher genomic resolution for novel genetic discovery and comparative mutational profiling of the oncogenesis of NKTCL. Targeted exome sequencing was conducted to interrogate 415 cancer-related genes in a cohort of 36 patients with NKTCL, and a total of 548 single nucleotide variants (SNVs) and 600 Copy number variances (CNVs) were identified. Recurrent amplification of the MCL1 (67%) and PIM1 (56%) genes was detected in a dominant majority of patients in our cohort. Functional mapping of genetic aberrations revealed that an enrichment of mutations in the JAK-STAT signaling pathway, including the cytokine receptor LIFR (copy number loss) upstream of JAK3, STAT3 (activating SNVs), and downstream effectors of MYC, PIM1 and MCL1 (copy number gains). RNA in situ hybridization showed the significant consistence of MCL1 RNA level and copy number of MCL1 gene. We further correlated molecular and clinical parameters with overall survival (OS) of these patients. When correlations were analyzed by univariate followed by multivariate modelling, only copy number loss of LIFR gene and stage (III-IV) were independent prognostic factors of reduced OS. Our findings identified that novel loss of LIFR gene significantly correlated with the adverse clinical outcome of NKTCL patients and provided therapeutic opportunities for this disease through manipulating LIFR.

Establishment of monoclonal antibody and scFv immuno-based assay for Cry2Aa toxin in spiked grain samples.

Bacillus thuringiensis (Bt) Cry toxins have been widely used in the development of genetically modified organisms (GMOs) for pest control. This work aimed to establish more cost effective methods for used Cry2Aa toxins. Three immunoassay methods (IC-ELISA, DAS-ELISA, and CLEIA) were successfully developed in this work. The mAb was used as the detecting antibody, for the IC-ELISA, the range of IC20 to IC80 was 1.11 µg/mL - 60.70 µg/mL, and an IC50 of 10.65 µg/mL. For the DAS-ELISA, the limit of detection (LOD) and limit of quantitation (LOQ) were 10.76 ng/mL and 20.70 ng/mL, respectively. For the CLEIA, the LOD and LOQ were 6.17 ng/mL and 7.40 ng/mL, respectively. The scFv-based detections were the most sensitive for detecting Cry2Aa. The LOD and LOQ for the DAS-ELISA were 118.75 ng/mL and 633.48 ng/mL, respectively. The LOD and LOQ for the CLEIA, read as 37.47 ng/mL and 70.23 ng/mL, respectively. The fact that Cry2Aa toxin was recovered in spiked grain samples further demonstrated that the approaches might be used to identify field samples. These methods provided good sensitivity, stability, and applicability for detecting Cry2Aa toxin, promising ultrasensitive monitoring and references for Cry toxins risk assessment.

Pathogenesis of COVID-19 described through the lens of an undersulfated and degraded epithelial and endothelial glycocalyx.

The glycocalyx surrounds every eukaryotic cell and is a complex mesh of proteins and carbohydrates. It consists of proteoglycans with glycosaminoglycan side chains, which are highly sulfated under normal physiological conditions. The degree of sulfation and the position of the sulfate groups mainly determine biological function. The intact highly sulfated glycocalyx of the epithelium may repel severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) through electrostatic forces. However, if the glycocalyx is undersulfated and 3-O-sulfotransferase 3B (3OST-3B) is overexpressed, as is the case during chronic inflammatory conditions, SARS-CoV-2 entry may be facilitated by the glycocalyx. The degree of sulfation and position of the sulfate groups will also affect functions such as immune modulation, the inflammatory response, vascular permeability and tone, coagulation, mediation of sheer stress, and protection against oxidative stress. The rate-limiting factor to sulfation is the availability of inorganic sulfate. Various genetic and epigenetic factors will affect sulfur metabolism and inorganic sulfate availability, such as various dietary factors, and exposure to drugs, environmental toxins, and biotoxins, which will deplete inorganic sulfate. The role that undersulfation plays in the various comorbid conditions that predispose to coronavirus disease 2019 (COVID-19), is also considered. The undersulfated glycocalyx may not only increase susceptibility to SARS-CoV-2 infection, but would also result in a hyperinflammatory response, vascular permeability, and shedding of the glycocalyx components, giving rise to a procoagulant and antifibrinolytic state and eventual multiple organ failure. These symptoms relate to a diagnosis of systemic septic shock seen in almost all COVID-19 deaths. The focus of prevention and treatment protocols proposed is the preservation of epithelial and endothelial glycocalyx integrity.

A phase II randomised trial of induction chemotherapy followed by concurrent chemoradiotherapy in locally advanced pancreatic cancer: the Taiwan Cooperative Oncology Group T2212 study.

BACKGROUND: The objective of this study was to evaluate the efficacy and safety of induction chemotherapy (ICT), GOFL (gemcitabine, oxaliplatin plus fluorouracil (5-FU)/leucovorin) versus modified FOLFIRINOX (irinotecan, oxaliplatin plus 5-FU/leucovorin), followed by concurrent chemoradiotherapy (CCRT) in locally advanced pancreatic adenocarcinoma (LAPC). METHODS: Chemo-naive patients with measurable LAPC were eligible and randomly assigned to receive biweekly ICT with either mFOLFIRINOX or GOFL for 3 months. Patients without systemic progression would have 5-FU- or gemcitabine-based CCRT (5040 cGy/28 fractions) and were then subjected to surgery or continuation of chemotherapy until treatment failure. The primary endpoint was 9-month progression-free survival (PFS) rate. RESULTS: Between July 2013 and January 2019, 55 patients were enrolled. After ICT, 21 (77.8%) of 27 patients who received mFOLFIRINOX and 17 (60.7%) of 28 patients who received GOFL completed CCRT. Of them, one and five had per-protocol R0/R1 resection. On intent-to-treat analysis, the 9-month PFS rate, median PFS and overall survival in mFOLFIRINOX and GOFL arms were 30.5% versus 35.9%, 6.6 (95% confidence interval: 5.9-12.5) versus 7.6 months (3.9-12.3) and 19.6 (13.4-22.9) versus 17.9 months (13.4-23.9), respectively. Grade 3-4 neutropenia and diarrhoea during induction mFOLFIRINOX and GOFL were 37.0% versus 21.4% and 14.8% versus 3.6%, respectively. CONCLUSION: Induction GOFL and mFOLFIRINOX followed by CCRT provided similar clinical outcomes in LAPC patients. GOV IDENTIFIER: NCT01867892.

Th1 and Th17 mucosal immune responses elicited by nasally inoculation in mice with virulence factors of Mycoplasma hyopneumoniae.

Nicotinamide Adenine Dinucleotide-Dependent (NADH) flavin oxidoreductase and NADH oxidase (NOX) are important virulence factors of Mycoplasma hyopneumoniae (Mhp), which are devoted to the function of adhesion, oxidative stress damage and apoptosis to host cells in our previous studies. Here, immune responses of NADH flavin oxidoreductase (NFOR) and NOX in mice and immune efficacy inoculated with intramuscular (IM), intranasal (IN), intramuscular unite intranasal (IM + IN) approaches were evaluated and compared. Cellular immunity levels, systemic immune and local mucosal immune responses were investigated by indirect enzyme-linked immunosorbent assay (iELISA) and quantitative reverse transcription PCR (qRT-PCR). Mice inoculated with NFOR and NOX by IM and IN or IM + IN could induce obvious secretion of specific immunoglobulin G (IgG) and secretory immunoglobulin A antibodies (sIgA) compared to those in negative control group. IM + IN inoculation resulted in systemic and local mucosal immune responses that were strongly produced. Moreover, Mhp NFOR and NOX could activate local mucosal immune responses mediated by Th1 and Th17 cells by IN. Our finding supported the notion that IM + IN was an effective immunization route for Mhp, which lays a foundation for more effective prevention of Mhp, and provides theoretical basis for the development of new subunit vaccines of Mhp.

NADH oxidase of Mycoplasma hyopneumoniae functions as a potential mediator of virulence.

BACKGROUND: Mycoplasma hyopneumoniae (M. hyopneumoniae) is the etiological agent of enzootic pneumonia, a highly infectious swine respiratory disease that distributed worldwide. The pathogenesis and virulence factors of M. hyopneumoniae are not fully clarified. As an important virulence factor of bacteria, nicotinamide adenine dinucleotide (NADH) oxidase (NOX) participates in host-pathogen interaction, however, the function of NOX involved in the pathogenesis of M. hyopneumoniae is not clear. RESULTS: In this study, significant differences in NOX transcription expression levels among different strains of M. hyopneumoniae differed in virulence were identified, suggesting that NOX may be correlated with M. hyopneumoniae virulence. The nox gene of M. hyopneumoniae was cloned and expressed in Escherichia coli, and polyclonal antibodies against recombinant NOX (rNOX) were prepared. We confirmed the enzymatic activity of rNOX based on its capacity to oxidize NADH to NAD+. Flow cytometry analysis demonstrated the surface localization of NOX, and subcellular localization analysis further demonstrated that NOX exists in both the cytoplasm and cell membrane. rNOX was depicted to mediate adhesion to immortalized porcine bronchial epithelial cells (hTERT-PBECs). Pre-neutralizing M. hyopneumoniae with anti-rNOX antibody resulted in a more than 55% reduction in the adhesion rate of high- and low-virulence M. hyopneumoniae strains to hTERT-PBECs. Moreover, a significant difference appeared in the decline in CCU50 titer between virulent (168) and virulence-attenuated (168L) strains. NOX not only recognized and interacted with host fibronectin but also induced cellular oxidative stress and apoptosis in hTERT-PBECs. The release of lactate dehydrogenase by NOX in hTERT-PBECs was positively correlated with the virulence of M. hyopneumoniae strains. CONCLUSIONS: NOX is considered to be a potential virulence factor of M. hyopneumoniae and may play a significant role in mediating its pathogenesis.

An assessment of the health risks associated with shared sanitation: a case study of the community ablution blocks in Durban, South Africa.

Shared sanitation facilities have been hailed as an innovative approach to solve the challenge with sanitation access. However, these facilities may act as hotspots for disease transmission due to unhygienic conditions. In this study we used quantitative (based on Escherichia coli contamination) techniques to assess the health risks associated with the use of community ablution blocks (CABs). The most contaminated surfaces were the cistern handle (5.7 Log10 cfu/cm2) and internal pull latch (5.8 Log10 cfu/cm2). Based on the E. coli contamination, at least two people out of 100 CAB users might be potentially infected when they touch "hot" surfaces. These risks were modelled assuming transfer of potentially pathogenic E. coli from these surfaces to the mouth. The incorporation of risk-reduction measures, such as wiping of these surfaces or washing of hands, could potentially result in significant reduction of infection risks. The most significant risk-reduction intervention was determined to be wiping of the contact surfaces, especially twice prior to contact. A combination of risk-reduction interventions could further reduce the risks. This study shows that contamination of contact surfaces within shared CABs could lead to increased risks of infections, requiring measures aimed at reducing the associated risks. The risk assessment framework used in this study could therefore be applied in similar settings to estimate associated health risks with the use of such facilities.

Analysis of the pan-Asian subgroup of patients in the NALA Trial: a randomized phase III NALA Trial comparing neratinib+capecitabine (N+C) vs lapatinib+capecitabine (L+C) in patients with HER2+metastatic breast cancer (mBC) previously treated with two or more HER2-directed regimens.

PURPOSE: Neratinib, an irreversible pan-HER tyrosine kinase inhibitor, has demonstrated systemic efficacy and intracranial activity in various stages of HER2+breast cancer. NALA was a phase III randomized trial that assessed the efficacy and safety of neratinib+capecitabine (N+C) against lapatinib+capecitabine (L+C) in HER2+ metastatic breast cancer (mBC) patients who had received ≥ 2 HER2-directed regimens. Descriptive analysis results of the Asian subgroup in the NALA study are reported herein. METHODS: 621 centrally assessed HER2+ mBC patients were enrolled, 202 of whom were Asian. Those with stable, asymptomatic brain metastases (BM) were eligible for study entry. Patients were randomized 1:1 to N (240 mg qd) + C (750 mg/m2 bid, day 1-14) with loperamide prophylaxis or to L (1250 mg qd) + C (1000 mg/m2 bid, day 1-14) in 21-day cycles. Co-primary endpoints were centrally assessed progression-free survival (PFS) and overall survival (OS). Secondary endpoints included time to intervention for central nervous system (CNS) disease, objective response rate, duration of response (DoR), clinical benefit rate, and safety. RESULTS: 104 and 98 Asian patients were randomly assigned to receive N+C or L+C, respectively. Median PFS of N+C and L+C was 7.0 and 5.4 months (P = 0.0011), respectively. Overall cumulative incidence of intervention for CNS disease was lower with N+C (27.9 versus 33.8%; P = 0.039). Both median OS (23.8 versus 18.7 months; P = 0.185) and DoR (11.1 versus 4.2 months; P < 0.0001) were extended with N+C, compared to L+C. The incidences of grade 3/4 treatment emergent adverse events (TEAEs) and TEAEs leading to treatment discontinuation were mostly comparable between the two arms. Diarrhea and palmar-plantar erythrodysesthesia were the most frequent TEAEs in both arms, similar to the overall population in incidence and severity. CONCLUSION: Consistent with the efficacy profile observed in the overall study population, Asian patients with HER2+ mBC, who had received ≥ 2 HER2-directed regimens, may also benefit from N+C. No new safety signals were noted. CLINICAL TRIAL REGISTRATION: NCT01808573.

Anti-idiotypic single-chain variable fragment antibody partially mimic the functionally spatial structure of Cry2Aa toxin.

The anti-idiotypic antibody is widely used in the field of immunology to simulate structural features or even induce the biological activity of antigens. In this study, we obtained seven anti-idiotypic single-chain variable fragments (scFv) antibodies of Cry2Aa toxin from a phage-displayed mutant library constructed using error-prone PCR technique. A mutant designated 2-12B showed the best binding ability amongst all anti-idiotypic scFv isolates to Plutella xylostella brush border membrane vesicles (BBMVs). 2-12B and Cry2Aa toxin shared a potential receptor of polycalin in P. xylostella BBMVs. Homology modeling and molecular docking demonstrated that 2-12B and Cry2Aa toxin have seven common binding amino acid residues in polycalin. Insect bioassay results suggested that 2-12 had insecticidal efficacy against P. xylostella larvae. These results indicated that the Cry2Aa anti-idiotypic scFv antibody 2-12B partially mimicked the structure and function of Cry2Aa toxin. The anti-idiotypic scFv antibody provides the basic material for the future study of surrogate molecules or new insecticidal materials.

The essential oils of Grewia Lasiocarpa E. Mey. Ex Harv.: chemical composition, in vitro biological activity and cytotoxic effect on Hela cells.

The chemical composition and biological activity of the essential oil extracted from the fresh leaves and stem bark of Grewia lasiocarpa was determined for the first time in this study. The essential oils were extracted by hydrodistillation and identified by GC-MS and FTIR. The antibacterial, antioxidant activity and total phenolic content of essential oils were determined. The major compounds identified were phytol (22.6%); α-farnesene (8.62%); n-hexadecanoic acid (7.24%); farnesol (4.61%) in the leaves, and 2-methylheptadecane (7.24%); heptacosane (7.60%); heptadecane, 2,6,10,14-tetramethyl (7.30%). The presence of aromatic, alkanes and phenolic compounds were revealed by FTIR analysis. The in silico oral prediction shows that some of the components are orally safe. The essential oil from the leaves showed cytotoxic activity at 1mg/mL(IC50 =555.70 µg/mL) against HeLa cells. The oils exhibited no significant antioxidant activity (IC50 >1 000 µg/mL) with <100 mg/g GAE of total phenol. The essential oils showed different degrees of activities against Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) and Klebsiella pneumoniae (ATCC 314588) at 10 µg/mL, 5 µg/mL and 2.5 µg/mL. These results might provide a future reference basis for further exploration of more of its medicinal application.

Kinetic and thermodynamic characterisation of HIV-protease inhibitors against E35D↑G↑S mutant in the South African HIV-1 subtype C protease.

Herein, we report the effect of nine FDA approved protease inhibitor drugs against a new HIV-1 subtype C mutant protease, E35D↑G↑S. The mutant has five mutations, E35D, two insertions, position 36 (G and S), and D60E. Kinetics, inhibition constants, vitality, Gibbs free binding energies are reported. The variant showed a decreased affinity for substrate and low catalytic efficiency compared to the wild type. There was a significant decrease in the binding of seven FDA approved protease inhibitors against the mutant (p < .0001). Amprenavir and ritonavir showed the least decrease, but still significant reduced activity in comparison to the wildtype (4 and 5 folds, respectively, p = .0021 and .003, respectively). Nelfinavir and atazanavir were the worst inhibitors against the variant as seen from the IC50, with values of 1401 ± 3.0 and 685 ± 3.0 nM, respectively. Thermodynamics data showed less favourable Gibbs free binding energies for the protease inhibitors to the mutant.

Biosurfactant: A new frontier for greener technology and environmental sustainability.

Petroleum hydrocarbons, oil, heavy metals pollution is becoming additional severe problem due to the growing call for crude oil and crude oil products related products in several fields of application. Such pollution have fascinated much considerations and attractions as it leads to ecological damages in both marines, aquatic and terrestrial ecosystems. Thus, different techniques including chemical surfactants and complex technologies have been proposed for their clean up from the environment, which in turn has detrimental effects on the environment. As of late, biosurfactant compounds have added much deliberation since they are considered as a reasonable option and eco-accommodating materials for remediation technology. The present society is confronting a few difficulties of usage, authorizing ecological protection and environmental change for the next generations. Biosurfactants hold the special property of minimizing and reducing the interfacial tension of liquids. Such features endure biosurfactants to afford a major part in emulsification, de-emulsification, biodegradability, foam formation, washing performance, surface activity, and detergent formulation, which have potential applications in the diverse industrial set-up. Conversations on cost-effective technologies, renewable materials, novel synthesis, downstream, upstream, emerging characterization techniques, molecular, and genetical engineering are substantial to produce biosurfactant of quality and quantity. Therefore, greater attention is being paid to biosurfactant production by identifying their environmental, and biotechnological applications. Be that as it may, the extravagant cost drew in with biosurfactants biotechnological synthesis and recovery can hamper their application in those areas. Notwithstanding these costs, biosurfactants can be used as these parts shows outstandingly high benefits that can at present beat the expenses incurred in the initial purification and downstream processes. Biosurfactant production by microorganisms is relatively considered one of the crucial know-how for improvement, growth, advancement, and environmental sustainability of the 21st century. There is a developing conversation around environmental safety and the significant role that biosurfactants will progressively play soon, for instance, the use of renewable by-products as substrates, potential reduction, re-use and recycling of waste and waste products. The review confers the usefulness of biosurfactants in the removal of environmental contaminants and, consequently, expanding environmental safety and drive towards greener technology.

Quantification of HBV core antibodies may help predict HBV reactivation in patients with lymphoma and resolved HBV infection.

BACKGROUND & AIMS: Absence or low anti-HBV surface antibody (anti-HBs) is associated with an increased risk of HBV reactivation in patients with lymphoma and resolved HBV infection receiving rituximab-containing chemotherapy. Quantification of anti-HBV core antibody (anti-HBc) is a new marker associated with the natural history and treatment response of chronic HBV infection. This study investigated whether baseline anti-HBc and anti-HBs levels may better predict HBV reactivation. METHODS: We prospectively measured the HBV DNA levels of patients with lymphoma and resolved HBV infection receiving rituximab-cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisolone-based chemotherapy and started an antiviral therapy upon HBV reactivation, defined as a greater than 10-fold increase in HBV DNA compared with previous nadir levels. Anti-HBs and anti-HBc were quantified by a double-sandwich assay. Receiver-operating-characteristic-curve analysis was used to determine the optimal baseline anti-HBc/anti-HBs levels for predicting HBV reactivation. RESULTS: HBV reactivation occurred in 24 of the 197 patients enrolled, with an incidence of 11.6/100 person-years. For the 192 patients with enough serum samples for analysis, low anti-HBs (<56.48 mIU/ml) and high anti-HBc (≥6.41 IU/ml) at baseline were significantly associated with high risk of HBV reactivation (hazard ratio [HR] 8.48 and 4.52, respectively; p <0.01). The multivariate analysis indicated that (1) patients with both high anti-HBc and low anti-HBs at baseline (36 of 192 patients) had an HR of 17.29 for HBV reactivation (95% CI 3.92-76.30; p <0.001), and (2) HBV reactivation may be associated with inferior overall survival (HR 2.41; 95% CI 1.15-5.05; p = 0.02). CONCLUSIONS: Baseline anti-HBc/anti-HBs levels may predict HBV reactivation in these patients with lymphoma and help optimize prophylactic antiviral therapy for high-risk patients. LAY SUMMARY: In this study, we identified a subgroup of patients with lymphoma and resolved hepatitis B virus infection that had a high risk of hepatitis B virus reactivation after receiving rituximab-containing chemotherapy. These findings will help optimize a preventive strategy, especially in hepatitis B virus endemic regions with limited healthcare resources. Clinical trial number: NCT 00931229.

An insight to the molecular interactions of the FDA approved HIV PR drugs against L38L↑N↑L PR mutant.

The aspartate protease of the human immune deficiency type-1 virus (HIV-1) has become a crucial antiviral target in which many useful antiretroviral inhibitors have been developed. However, it seems the emergence of new HIV-1 PR mutations enhances drug resistance, hence, the available FDA approved drugs show less activity towards the protease. A mutation and insertion designated L38L↑N↑L PR was recently reported from subtype of C-SA HIV-1. An integrated two-layered ONIOM (QM:MM) method was employed in this study to examine the binding affinities of the nine HIV PR inhibitors against this mutant. The computed binding free energies as well as experimental data revealed a reduced inhibitory activity towards the L38L↑N↑L PR in comparison with subtype C-SA HIV-1 PR. This observation suggests that the insertion and mutations significantly affect the binding affinities or characteristics of the HIV PIs and/or parent PR. The same trend for the computational binding free energies was observed for eight of the nine inhibitors with respect to the experimental binding free energies. The outcome of this study shows that ONIOM method can be used as a reliable computational approach to rationalize lead compounds against specific targets. The nature of the intermolecular interactions in terms of the host-guest hydrogen bond interactions is discussed using the atoms in molecules (AIM) analysis. Natural bond orbital analysis was also used to determine the extent of charge transfer between the QM region of the L38L↑N↑L PR enzyme and FDA approved drugs. AIM analysis showed that the interaction between the QM region of the L38L↑N↑L PR and FDA approved drugs are electrostatic dominant, the bond stability computed from the NBO analysis supports the results from the AIM application. Future studies will focus on the improvement of the computational model by considering explicit water molecules in the active pocket. We believe that this approach has the potential to provide information that will aid in the design of much improved HIV-1 PR antiviral drugs.

I36T↑T mutation in South African subtype C (C-SA) HIV-1 protease significantly alters protease-drug interactions.

The efficacy of HIV-1 protease (PR) inhibition therapies is often compromised by the emergence of mutations in the PR molecule that reduces the binding affinity of inhibitors while maintaining viable catalytic activity and affinity for natural substrates. In the present study, we used a recombinant HIV-1 C-SA PR and a recently reported variant for inhibition (Ki, IC50) and thermodynamic studies against nine clinically used inhibitors. This is the first time that binding free energies for C-SA PR and the mutant are reported. This variant PR harbours a mutation and insertion (I36T↑T) at position 36 of the C-SA HIV-1 PR, and did not show a significant difference in the catalytic effect of the HIV-1 PR. However, the nine clinically approved HIV PR drugs used in this study demonstrated weaker inhibition and lower binding affinities toward the variant when compared to the wild type HIV-1 PR. All the protease inhibitors (PIs), except Amprenavir and Ritonavir exhibited a significant decrease in binding affinity (p<0.0001). Darunavir and Nelfinavir exhibited the weakest binding affinity, 155- and 95-fold decreases respectively, toward the variant. Vitality values for the variant PR, against the seven selected PIs, confirm the impact of the mutation and insertion on the South African HIV-1 subtype C PR. This information has important clinical implications for thousands of patients in Sub-Saharan Africa.

Stress responses of Acinetobacter strain Y during phenol degradation.

Quantification of gene expression of Acinetobacter strain Y under 1000 mg/l of phenol was investigated using qPCR and proteomic analyses. The results show that Acinetobacter strain Y utilized 100 % of phenol within 18 h of exposure. The results of qPCR and proteomic analyses demonstrate a sequential expression of phenol-degrading genes of Acinetobacter strain Y via the ortho-pathway followed by the ß-ketoadipate pathway. Many stress-responsive proteins such as chaperones, chaperonins, porins and the enzymes involved in the signal transduction pathway were upregulated especially in the early stage. The stressed bacteria produced more ABC-type transporters, membrane receptors and efflux pumps to mitigate the impacts of phenol stress. The functions of TCA/glyoxylate cycle and oxidative phosphorylation processes were negatively affected. Many enzymes in the gluconeogenesis pathway were upregulated. This study demonstrates bacterial strategies of Acinetobacter strain Y via the energy saving mechanisms and the coordinated control between carbon (C)- and nitrogen (N)-limitations in coping with the stress by scavenging the reactive oxygen species.

Viruses in the environment - presence and diversity of bacteriophage and enteric virus populations in the Umhlangane River, Durban, South Africa.

Due to the continued persistence of waterborne viral-associated infections, the presence of enteric viruses is a concern. Notwithstanding the health implications, viral diversity and abundance is an indicator of water quality declination in the environment. The aim of this study was to evaluate the presence of viruses (bacteriophage and enteric viruses) in a highly polluted, anthropogenic-influenced river system over a 6-month period at five sampling points. Cytopathic-based tissue culture assays revealed that the isolated viruses were infectious when tested on Hep-G2, HEK293 and Vero cells. While transmission electron microscopy (TEM) revealed that the majority of the viruses were bacteriophages, a number of presumptive enteric virus families were visualized, some of which include Picornaviridae, Adenoviridae, Polyomaviridae and Reoviridae. Finally, primer specific nested polymerase chain reaction (nested-PCR)/reverse transcription-polymerase chain reaction (RT-PCR) coupled with BLAST analysis identified human adenovirus, polyomavirus and hepatitis A and C virus genomes in river water samples. Taken together, the complexity of both bacteriophage and enteric virus populations in the river has potential health implications. Finally, a systematic integrated risk assessment and management plan to identify and minimize sources of faecal contamination is the most effective way of ensuring water safety and should be established in all future guidelines.

Chlorine dioxide oxidation of Escherichia coli in water - A study of the disinfection kinetics and mechanism.

This study investigated the kinetics and mechanism of chlorine dioxide (ClO2) inactivation of a Gram-negative bacteria Escherichia coli (ATCC 35218) in oxidant demand free (ODF) water in detail as a function of disinfectant concentration (0.5-5.0 mg/L), water pH (6.5-8.5), temperature variations (4-37°C) and bacterial density (105-107 cfu/mL). The effects of ClO2 on bacterial cell morphology, outer membrane permeability, cytoplasmic membrane disruption and intracellular enzymatic activity were also studied to elucidate the mechanism of action on the cells. Increasing temperature and disinfectant concentration were proportional to the rate of cell killing, but efficacy was found to be significantly subdued at 0.5 mg/L and less dependent on the bacterial density. The bactericidal efficiency was higher at alkaline pH of 8 or above as compared to neutral and slightly acidic pH of 7 and 6.5 respectively. The disinfection kinetic curves followed a biphasic pattern of rapid inactivation within the initial 2 min which were followed by a tailing even in the presence of residual biocide. The curves were adequately described by the Cavg Hom model. Transmission Electron Microscopy images of the bacteria cells exposed to lethal concentrations of ClO2 indicated very little observable morphological damage to the outer membranes of the cells. ClO2 however was found to increase the permeability of the outer and cytoplasmic membranes leading to the leakage of membrane components such as 260 nm absorbing materials and inhibiting the activity of the intracellular enzyme ß-D-galactosidase. It is suggested that the disruption of the cytoplasmic membrane and subsequent efflux of intracellular components result in the inactivation of the Gram-negative bacteria.

Purification and characterization of naturally occurring HIV-1 (South African subtype C) protease mutants from inclusion bodies.

Human immunodeficiency virus (HIV) infections in sub-Saharan Africa represent about 56% of global infections. Many studies have targeted HIV-1 protease for the development of drugs against AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. Along with the wild type (C-SA) we also over-expressed and characterized two mutant forms from patients that had shown resistance to protease inhibitors. Using recombinant DNA technology, we constructed three recombinant plasmids in pGEX-6P-1 and expressed them containing a sequence encoding wild type HIV protease and two mutants (I36T↑T contains 100 amino acids and L38L↑N↑L contains 101 amino acids). These recombinant proteins were isolated from inclusion bodies by using QFF anion exchange and GST trap columns. In SDS-PAGE, we obtained these HIV proteases as single bands of approximately 11.5, 11.6 and 11.7 kDa for the wild type, I36T↑Tand L38L↑N↑L mutants, respectively. The enzyme was recovered efficiently (0.25 mg protein/L of Escherichia coli culture) and had high specific activity of 2.02, 2.20 and 1.33 µmol min(-1) mg(-1) at an optimal pH of 5 and temperature of 37 °C for the wild type, I36T↑T and L38L↑N↑L, respectively. The method employed here provides an easy and rapid purification of the HIV-1(C-SA) protease from the inclusion bodies, with high yield and high specific activities.