Enzyme inhibition assay for pyruvate dehydrogenase complex: Clinical utility for the diagnosis of primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is usually diagnosed by the presence of characteristic histopathological features of the liver and/or antimitochondrial antibodies (AMA) in the serum traditionally detected by immunofluorescence. Recently, new and more accurate serological assays for the detection of AMA, such as enzyme-linked immunosorbent assay (ELISA), immunoblotting, and enzyme inhibition assay, have been developed. Of these, the enzyme inhibition assay for the detection of anti-pyruvate dehydrogenase complex (PDC) antibodies offers certain advantages such as objectivity, rapidity, simplicity, and low cost. Since this assay has almost 100% specificity, it may have particular applicability in screening the at-risk segment of the population in developing countries. Moreover, this assay could be also used for monitoring the disease course in PBC. Almost all sera of PBC-suspected patients can be confirmed for PBC or non-PBC by the combination results of immunoblotting and enzyme inhibition assay without histopathological examination. For the development of a "complete" or "gold standard" diagnostic assay for PBC, similar assays of the enzyme inhibition for anti-2-oxoglutarate dehydrogenase complex (OGDC) and anti-branched chain oxo-acid dehydrogenase complex (BCOADC) antibodies will be needed in future.

Preliminary Validation of Tumor Cell Attachment Inhibition Assay for Developmental Toxicants With Mouse S180 Cells

This study was designed to explore the possibility of using ascitic mouse sarcoma cell line (S180) to validate the mouse tumor cell attachment assay for developmental toxicants, and to test the inhibitory effects of various developmental toxicants. The results showed that 2 of 3 developmental toxicants under consideration, sodium pentobarbital and ethanol, significantly inhibited S180cells attachment to Concanavalin A-coaed surfaces. Inhibition was dependent on concentration, and the IC50 (the concentration tha reduced attachment by 50% ), of these 2 chemicals was 1.2×10-3mol/L and 1 .0 mol/L, respectively. Anoher developmental toxiant, hydmiortisone, did not show inhibitory activity. Two non-developmental toxicants, sodium chloride and glycine were also tested and these did not decrease attachment rates. The main results reported here were generally sindlar to those obtained with ascitic mouse ovdrian tumor cells as a model. Therefore, this study added further evidence to the conclusion that cell specificity does not lindt attachment inhibition to Con A-coated surfaces, so S180 cell may serve as an altemative cell model, especially when other cell lines are unavailable. Furthermore, after optimal validation, it can be suggested that an S180 cell attachment assay may be a candidate for a series of assays to detect developmental toxicants.

Assessment of in vitro sensitivity of Plasmodium vivax fresh isolates

Objective:To compare the applicability of the SYBK Grcen-Ⅰ assay with the standard schizont maturalion assay,for determination of sensitivity of Plasmodium vivax(P.vivax) to chloroquine and a new antifolale WR 99210.Methods:The study was conducted at Mae Tao Clinic for migrant workers,Tak Province during April 2009 to July 2010.A total of 64 blood samples(1 mL blood collected into sodium heparinized plastic tube) were collected from patients with monoinfection with P.vivax malaria prior to treatment with standard regimen of a 3-day chloroquine. In vitro sensitivity of P.vivax isolates was evaluated by schizont maturation inhibition and SYBR Green-Ⅰ assays.Results:A total of 30 out of 64 blood samples collected from patients with P.vivax malaria were successfully analyzed using both the microscopic schizont maturation inhibition and SYBR Green-I assays.The failure rates of the schizont maturation inhibition assay(50%) and the SYBR Green-I assay(54%) were similar(P=0.51).The median IC_(10)s,IC_(50)s and IC_(90)s of both chloroquine and WR99210 were not significantly different from the clinical isolates of P.vivax tested.Based on the cut-off of 100 nM,the prevalences of chloroquine resistance determined by schizont maturation inhibition and SYBR Green-I assays were 19 and 11 isolates,respectively.The strength of agreement between the two methods was very poor for both chloroquine and WR992I0.Conclusions:On the basis of this condition and its superior sensitivity,the microscopic method appears better than the SYUK Green-I Green assay for assessing in vitro sensitivity of fresh P.vivax isolates to antimalarial drugs.

In vitro assessment of the synergism between extracts of Cocos nucifera husk and some standard antibiotics

Objective: To evaluate the interactions between the crude extracts of Cocos nucifera(C.nucifera) and six front line antibiotics(ampicillin sodium salt, penicillin G sodium,amoxicillin, chloramphenicol, ciprofloxacin and tetracycline hydrochloride), against some bacterial pathogens linked with human infection.Methods: The pulverized husk of C.nucifera was dissolved in 95% n-hexane and extracted using Soxhlet extraction method and sterile distilled water(aqueous).The antibacterial susceptibility of the crude extracts of C.nucifera was tested against environmental and clinical strains(6) obtained from the South African Bureau of Standards(SABS), Vibrio(6) and Listeria pathogens(6).The agar-well diffusion method was used for screening the extracts for their antibacterial activity.The minimum inhibitory concentration and minimum bactericidal concentration of the extracts were determined.Time-kill assay was used to evaluate bactericidal and/or bacteriostatic activity.The synergistic effect of the crude extracts and antibiotics was assessed and evaluated by adopting the checkerboard methods.Results: With the time-kill assay, the highest bactericidal activity was observed on Vibrio fluvialis EL041 with a-5.6 ± 0.2 log_(10)CFU/mL decrease in cell density as a result of the combination of the extracts and chloramphenicol at two-fold minimum inhibitory concentrations.Synergisms using the time-kill assay constituted about 72%, while indifference constituted about 28%.The checkerboard method revealed synergistic interaction in 67% of the combinations, and indifference in 33%.There was no specificity in the observed synergy to a particular class of antibiotics.Conclusions: This investigation suggests the crude extracts of C.nucifera to be a potential broad spectrum antimicrobial compound.Therefore, further study is needed to isolate the pure compounds from these crude extracts.

Synthesized flavanoid-derived ligand reduced dengue virus type-2 replication in vitro

Objective: To investigate the antiviral property of a lead ligand, YK51 that was synthesized based on the flavanoid of a natural product toward dengue virus type-2(DENV2)replication.Methods: c RNA was isolated from HepG2 cells inoculated with 1 000 median tissue culture infective dose of DENV2 and treated with different doses of the ligand followed by RT-PCR to quantify the virus gene copies. Confocal microscopy of actin and tubulin redistribution was also performed.Results: The quantitative RT-PCR result showed reduction of the DENV2 gene copies as the ligand concentration was increased. The confocal microscopy result showed increase in the tubulin intensity(79.6%) of infected BHK21 cells treated with the ligand,compared with the non-treated cells(54.8%). The 1.5-fold increase in the intensity of tubulin suggested that the ligand inhibitory effect stabilized the cellular microtubule structure.Conclusions: The synthesized ligand YK51 reduced DENV2 viral load by inhibiting virus replication thus is highly potential to be developed as antiviral agent.

Degradation and detoxification of microcystin-LR in drinking water by sequential use of UV and ozone

Microcystins （MCs） produced by cyanobacteria are strong hepatotoxins and classified as possible carcinogens. MCs pose a considerable threat to human health through tainted drinking and surface waters. Herein filtrated water from a waterworks in Harbin, China, was spiked with microcysfin-LR （MC-LR） extracted from a toxic scum of microcystis aeruginosa, and the spiked sample waters were treated using UV irradiation with consequent ozonation process （UV/O3）, compared with ozonation at a dose range commonly applied in water treatment plants, UV irradiation at 254 nm and UV irradiation combined with ozonation （UV＋O3）, respectively. The remaining of toxins were analyzed using high-performance liquid chromatography and also determined using a protein phosphatase type 2A inhibition assay, which was utilized to evaluate the reduction in toxicity. Results indicated that in comparison to other three processes （O3, UV, and UV＋O3）, UV/O3 process could effectively decrease both the concentration and toxicity of MC-LR at 100 μg/L level after 5 min UV irradiation with consequent 5 min ozonation at 0.2 mg/L （below 1 μg/L ）, while 0.5 mg/L ozone dose was required for the level below 0.1 μg/L. The addition of an UV treatment step to the existing treatment train may induce significant transformation of micropollutants and breaks down the natural organic matters into moieties unfavorable for ozone decomposition, stabilizing the ozone residual. These findings suggested that sequential use of UV and ozone may be a suitable method for the removal of these potentially hazardous microcystins from drinking water.

Organophosphate esters cause thyroid dysfunction via multiple signaling pathways in zebrafish brain

Organophosphate esters(OPEs)are widespread in various environmental media,and can disrupt thyroid endocrine signaling pathways.Mechanisms by which OPEs disrupt thyroid hormone(TH)signal transduction are not fully understood.Here,we present in vivo-in vitro-in silico evidence establishing OPEs as environmental THs competitively entering the brain to inhibit growth of zebrafish via multiple signaling pathways.OPEs can bind to transthyretin(TTR)and thyroxine-binding globulin,thereby affecting the transport of TH in the blood,and to the brain by TTR through the blood-brain barrier.When GH3 cells were exposed to OPEs,cell proliferation was significantly inhibited given that OPEs are competitive inhibitors of TH.Cresyl diphenyl phosphate was shown to be an effective antagonist of TH.Chronic exposure to OPEs significantly inhibited the growth of zebrafish by interfering with thyroperoxidase and thyroglobulin to inhibit TH synthesis.Based on comparisons of modulations of gene expression with the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases,signaling pathways related to thyroid endocrine functions,such as receptor-ligand binding and regulation of hormone levels,were identified as being affected by exposure to OPEs.Effects were also associated with the biosynthesis and metabolism of lipids,and neuroactive ligand-receptor interactions.These findings provide a comprehensive understanding of the mechanisms by which OPEs disrupt thyroid pathways in zebrafish.

Optical substrates for drug-metabolizing enzymes: Recent advances and future perspectives

Drug-metabolizing enzymes(DMEs),a diverse group of enzymes responsible for the metabolic elimination of drugs and other xenobiotics,have been recognized as the critical determinants to drug safety and efficacy.Deciphering and understanding the key roles of individual DMEs in drug metabolism and toxicity,as well as characterizing the interactions of central DMEs with xenobiotics require reliable,practical and highly specific tools for sensing the activities of these enzymes in biological systems.In the last few decades,the scientists have developed a variety of optical substrates for sensing human DMEs,parts of them have been successfully used for studying target enzyme(s)in tissue preparations and living systems.Herein,molecular design principals and recent advances in the development and applications of optical substrates for human DMEs have been reviewed systematically.Furthermore,the challenges and future perspectives in this field are also highlighted.The presented information offers a group of practical approaches and imaging tools for sensing DMEs activities in complex biological systems,which strongly facilitates high-throughput screening the modulators of target DMEs and studies on drug/herb-drug interactions,as well as promotes the fundamental researches for exploring the relevance of DMEs to human diseases and drug treatment outcomes.

Accelerated Evolution of H7N9 Subtype Influenza Virus under Vaccination Pressure

No avian H7 N9 outbreaks have occurred since the introduction of H7 N9 inactivated vaccine in the fall of 2017.However,H7 N9 is still prevalent in poultry.To surveil the prevalence,genetic characteristics,and antigenic changes of H7 N9,over7000 oropharyngeal and cloaca swab specimens were collected from live poultry markets and farms in 15 provinces of China from 2017 to 2019.A total of 85 influenza virus subtype H7 N9 strains were isolated and 20 representative strains were selected for genetic analysis and antigenicity evaluation.Results indicated the decreased prevalence of low-pathogenic H7 N9 strains while highly-pathogenic H7 N9 strains became dominated since the introduction of vaccine.Phylogenetic analysis showed that strains from 2019 formed an independent small branch and were genetically distant to strains isolated in 2013–2018.Analysis of key amino acid sites showed that the virus strains may adapt to the host environment evolutionally through mutation.Our analysis predicted additional potential glycosylation sites for HA and NA genes in the 2019 strains.Sequence analysis of HA gene in strains isolated from 2018 to 2019 showed that there were an increased nucleotide substitution rate and an increased mutation rate in the first and second nucleotides of coding codons within the open reading frame.The hemagglutination inhibition(HI)assay showed that H7-Re1 and H7-Re2 exhibited a lower HI titer for isolates from 2019,while H7-Re3 and r LN79 showed a high HI titer.The protective effect of the vaccine decreased after15 months of use.Overall,under vaccination pressure,the evolution of influenza virus subtype H7 N9 has accelerated.

Evaluation of antiplasmodial properties in 15 selected traditional medicinal plants from India

Objective:The objective of this study was to evaluate the in vitro antiplasmodial properties against malaria parasite in 15 plants mentioned in Indian traditional medicine texts.Methods:In vitro antiplasmodial activity of methanolic extracts obtained from Indian traditional medicinal plants was evaluated on Plasmodium falciparum of FCK2 and INDO strains using schizont maturation inhibition assay and parasite lactate dehydrogenase inhibition assay.Results:Methanolic extracts of Adhatoda zeylanica,Embelia ribes,Piper nigrum and Plumbago zeylanica exhibited more than 50%inhibition in both the stains in schizont maturation inhibition assay.Methanolic extracts of seven medicinal plants exhibited antiplasmodial activity at half maximal inhibitory concentration(IC50)<100 mg/m L,and methanolic extracts of five medicinal plants exhibited antiplasmodial activity at IC50<50 mg/m L in P.falciparum lactate dehydrogenase(PfLDH)inhibition assay.A.zeylanica,E.ribes and P.nigrum exhibited promising antiplasmodial activity in PfLDH inhibition assay.A.zeylanica and E.ribes exhibited improved activity against resistant in comparison to sensitive strain.Conclusion:A.zeylanica and E.ribes were the most promising extracts from this study and deserve further investigation of their antiplasmodial properties.

Comparative antitumor and anti-proliferative activities of Hippophae rhamnoides L. leaves extracts

Objective:To evaluate the antitumor and anti-proliferative activities of methanol,aqueous,acetone,ethyl acetate,ethanol,chloroform and n-hexane extracts of Hippophae rhamnoides leaves.Methods:Antitumor activities were evaluated by using the antitumor potato disc assay by using inoculums(Agrobacterium tumefaciens)with three different concentrations of test samples(10,100 and 1000 mg/L).Anti-proliferative activity was evaluated by the given method of methyl thiazolyl tetrazolium assay.The concentrations of the extract ranging from 0.039 to 10 mg/mL were tested against HeLa cells.Results:Highest tumors inhibition activity(60.9%and 55.8%)was shown by methanol and ethanol extracts,with EC_(50) values of 424.41 and 434.61 mg/L respectively.At 10 mg/mL,The highest cell inhibition 75.61%was observed in methanol extract and the lowest 36.59%were calculated in n-hexane extract.The difference in tumor and cell inhibition(%)may be due to the different concentration of active compounds responsible for antitumor and anti-proliferative activities.All extracts have considerable level of tumor and cell inhibitiory effect in a dose dependent manner.Conclusions:Our finding showed that Hippophae rhamnoides leaves are a potent natural source of antitumor and antiproliferative agent.