Classification and antioxidant assays of polyphenols:a review

Polyphenols are widely recognized as the effective antioxidants,which are divided into flavonoids,phenolic acids,stilbenes,lignans,tannins and so on.They could regulate internal functions and protect the body from diseases related to oxidative damage.Due to the fact that their antioxidant capacity is influenced by the structure,stability and bioavailability,the detection of their bioactivity should be considered comprehensively.Currently,the methods for measuring the antioxidant capacity of phenolic compounds are divided into chemical,cell-based and in vivo assays.The chemical assays include 2,2-diphenyl-l-picrylhydrazyl(DPPHI),2,2"-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS),ferric reducing/antioxidant power(FRAP),oxygen radical absorbance capacity(ORAC),peroxyI radical scavenging capacity(PSC),which are rapid identification method,but their reaction mechanism has a great gap with the internal body response.The cell-based assays are more consistent with biological reaction,but still do not take the bioavailability into consideration.The in vivo assays,which commonly utilized Caenorhabditis elegans or rats as models,are more representative,but these methods are more complex and spend longer.This review summarizes the antioxidant evaluation methods of phenolic compounds and discusses their advantages and limitations comparatively,which could help discriminate and select the appropriate assay in the actual operation,and facilitate the development of comprehensive approaches as well.

The Inhibition Effect of Tert-Butyl Alcohol on the TiO_2 Nano Assays Photoelectrocatalytic Degradation of Different Organics and Its Mechanism

The inhibition effect of tert-butyl alcohol(TBA), identified as the·OH radical inhibitor, on the TiO_2 nano assays(TNA) photoelectrocatalytic oxidation of different organics such as glucose and phthalate was reported. The adsorption performance of these organics on the TNA photoelectrode was investigated by using the instantaneous photocurrent value, and the degradation property was examined by using the exhausted reaction. The results showed that glucose exhibited the poor adsorption and easy degradation performance, phthalate showed the strong adsorption and harddegradation, but TBA showed the weak adsorption and was the most difficult to be degraded. The degradation of both glucose and phthalate could be inhibited evidently by TBA. But the effect on glucose was more obvious. The different inhibition effects of TBA on different organics could be attributed to the differences in the adsorption and the degradation property. For instance, phthalate of the strong adsorption property could avoid from the capture of·OH radicals by TBA in TNA photoelectrocatalytic process.

Performance and correlation of interferon gamma release assays and tuberculin skin test in HIV-infected children and adolescents with immune reconstitution

Objective:To evaluate the performance of interferon gamma release assays and tuberculin skin test in HIV-infected children and adolescents with immune reconstitution.Methods:A cross-sectional study was conducted in HIV-infected patients aged 5-18 years receiving antiretroviral treatment with CD4 T-lymphocytes>25%or>500 cells/mm3 for at least 6 months.QuantiF ERON-TB Gold,T-SPOT.TB,and tuberculin skin test were performed in each patient.Results:A total of 50 patients were enrolled with median age of 13.7 years,CD4 counts of 753(IQR:587-989)cells/mm3.Among 27 patients with tuberculosis(16)or tuberculosis exposure(11),8(29.6%)were positive to at least one test,2(7.4%)were positive QuantiFERON-TB Gold,3(11.1%)positive T-SPOT.TB,and 7(25.9%)had tuberculin skin test≥5 mm.Among 23 patients without history of tuberculosis or exposure,all had negative interferon gamma release assays,while 2(8.7%)had positive tuberculin skin test.Conclusions:All tests had low sensitivity despite immune reconstitution.

Endophytic actinobacteria of medicinal plant Aloe vera: Isolation, antimicrobial, antioxidant, cytotoxicity assays and taxonomic study

Objective: To explore the new sources of novel bioactive compounds having pharmaceutical and agricultural interest and to search the endophytic actinobacteria from medicinal plants. Methods: NAF-1 an endophyte actinobacteria was isolated from leaves of medicinal plant Aloe vera collected in Marrakesh, Morocco using Bennett agar as selective medium. NAF-1 was tested for its antimicrobial activity against five pathogenic bacteria such as Staphylococcus aureus PIC 53156, Micrococcus luteus ATCC381, Bacillus subtilis ATCC 14579, Pseudomonas aeruginosa DSM 50090 and Escherichia coli ATCC 8739 and four human clinic fungi belonging to the Candida, Aspergillus and Microsporum genera. Several antioxidant activities were studied such as DPPH free radical scavenging, β-carotene and linoleic acid and reducing power assays. The total of phenol and flavonoid was also calculated. Using Artemia salina shrimp assay, the cytotoxicity of NAF-1 crude extract was determined. Results: The results revealed that the actinobacteria showed a high activity(≥20 mm) against only Gram positive bacteria but it had a moderate activity(between 13 and 15 mm) against Human clinic fungi. The isolate also exhibited a LD50 of 14.20 μg/mL in the cytotoxicity assay. The result showed that the crude extract presented an interesting free radical-scavenging activity with IC50 value of(5.58 ± 0.26) μg/mL and a high value of phenolic and flavonoid compounds with(15.41 ± 0.18) μg GAE/mg extract and(11.41± 0.06) μg QE/mg extract respectively. Moreover, the taxonomic position of our endophyte actinobacteria using the morphological and physiological criteria and using 16 S r RNA gene sequence(polyphasic approach) showed that the NAF-1 isolate was similar to Streptomyces hydrogenans which was never described as an endophyte actinobacteria. Conclusions: This isolated strain appears promising resources of bioactive agents and can be exploited to produce therapeutic agents active against pathogenic disease.

Interferon-gamma release assays in tuberculous uveitis:a comprehensive review

·Tuberculous uveitis(TBU)comprises a broad clinical spectrum of ocular manifestations,making its diagnosis challenging.Ophthalmologists usually require evidence from investigations to confirm or support a clinical diagnosis of TBU.Since direct isolation of the causative organism from ocular specimens has limitations owing to the small volume of the ocular specimens,resultant test positivities are low in yield.Immunodiagnostic tests,including the tuberculin skin test and interferon-gamma release assays(IGRAs),can help support a clinical diagnosis of TBU.Unlike the tuberculin skin test,IGRAs are in vitro tests that require a single visit and are not affected by prior Bacillus Calmette-Guerin vaccination.Currently,available IGRAs consist of different techniques and interpretation methods.Moreover,newer generations have been developed to improve the sensitivity and ability to detect active tuberculosis.This narrative review collates salient practice points as a reference for general ophthalmologists,such as evidence for the utilization of IGRAs in patients with suspected TBU,and summarizes basic knowledge and details of clinical applications of these tests in a clinical setting.

Exploration of natural enzyme inhibitors with hypoglycemic potentials amongst Eucalyptus Spp. by in vitro assays

AIM: To investigate the presence and potency of natural enzyme inhibitors with hypoglycemic potentials amongst Eucalyptus Spp. by in vitro assays.METHODS: The leaf extracts of the three different Eucalyptus species [E. globulus(EG), E. citriodora(EC), E. camaldulensis(ECA)] were subjected to in vitro assay procedures to explore the prevalence of natural enzyme inhibitors(NEIs) after preliminary qualitative and quantitative phytochemical evaluations, to study their inhibitory actions against the enzymes like α-amylase, α-glucosidase, aldose reductase, angiotensin converting enzyme and dipeptidyl peptidase 4 playing pathogenic roles in type 2 diabetes. The antioxidant potential and total antioxidant capacity of the species were also evaluated.RESULTS: Major bioactive compounds like polyphenols(341.75 ± 3.63 to 496.85 ± 3.98) and flavonoids(4.89 ± 0.01 to 7.15 ± 0.02) were found in appreciable quantity in three species. Based on the IC50 values of the extracts under investigation, in all assays the effectivity was in the order of EG > ECA > EC. The results of the ferric reducing antioxidant power assay showed that the reducing ability of the species was also in the order of EG > ECA > EC. A strong correlation(R2 = 0.81-0.99) was found between the phenolic contents and the inhibitory potentials of the extracts against the targeted enzymes.CONCLUSION: These results show immense hypoglycemic potentiality of the Eucalyptus Spp. and a remarkable source of NEIs for a future phytotherapeutic approach in Type 2 diabetes.

Development of PDMS-based Microfluidic Device for Cell-based Assays

In a single step photolithography, muhi-level microfluidic device is fabricated by printing novel architectures on a film photomasks. The whole fabrication process is executed by classical PCB technology without the need to access clean room facilities. Different levels of protruding features on PCB master are produced by exposing a photomask with specifically arranged "windows and rims" architectures, followed by chemical wet etching. Poly(dimethylsiloxane)(PDMS) is then molded against the positive relief master to generate microfluidic device featured with multi-level sandbag structure and peripheral microchannels. This sandbag structure is an analog to traditional dam or weir for particle entrapment. The microstructure does not collapse when subjected to applied pressure, which is suitable for operation on elastic PDMS substrate.Typical immunocytochemcial staining assays were performed in the microdevice to demonstrate the applicability of the sandbag structure for cellular analysis. This simplified microfabrication process employs low-cost materials and minimal specialized equipment and can reproducibly produce mask lines with about 20 μm in width, which is sufficient for most microfluidic applications.

Stability of fluorochrome based assays to measure subcellular sperm functions

Aim： To evaluate the long-term stability of the fluorescence signals of new fluorescence-based semen analysis assays for clinical application. Methods： Semen samples from 87 unselected infertile patients were used to perform the following assays： （i） detection of active caspase-3 （n = 17）; （ii） integrity of the mitochondrial membrane potential （MMP） （n = 17）; （iii） externalization of phosphatidylserine （EPS） （n = 16）; and （iv） detection of intact acrosomes via CD46 （n = 37）. After the assays, 4% paraformaldehyde was added to all aliquots. The fluorescence intensity of each sample was evaluated by flow cytometry on days 0, 3, 7, 10 and 14. Results： Differences of up to ± 5% positive spermatozoa from the value measured at day 0 were estimated as acceptable deviation. The Caspase-3 FLICA^TM showed mean differences 〈 5% at day 3, 7 and 10. At day 14 the mean difference was 7.6%. In contrast, the disrupted MMP and the EPS detection showed differences 〉 5% at day 3. The CD46-FITC labeling displayed absolute differences 〈 5% CD46-positive spermatozoa at days 3, 7, 10 and 14. Conclusion： Although immediate analysis of the fluorescence signals is recommended, it is possible to evaluate caspase-3 activation up to 10 days and CD46 up to 14 days after staining of sperm. The FACS evaluation of MMP and EPS detection should be conducted on the same day.

Study on Immunochemical Assays for the Organophosphorus Insecticide Chlorpyrifos

The anti-chlorpyrifos polyclonal antibodies were obtained by using the artificial immuneantigen to immune in New Zealands white rabbits. The enzyme-tagged antibodies wereprepared by coupling horseradish peroxidase (HRP) to the purified antibody with themodified sodium periodate method. The indirect competitive enzyme linked immuno-sorbentassays (ELISA) and the HRP-tagged antibody direct ELISA (E-Ab) were established, respectively.The limit of detection (LOD) for the indirect ELISA and E-Ab were 0.0033 and 0.0042 gmL-1, respectively. The linear detection ranged well from 0.005 to 2.0 g mL-1.

Development and Validation of Multiplex One-Step Real-Time TaqManqRT-PCR Assays for Detection and Quantification of Arboviral Encephalitis Viruses

Arboviral encephalitis is a group of animal and human illness that is mostly caused by several distinct families of viruses including orthobunya virus, phlebovirus, flaviviruses, and the alphaviruses. Although specific signs and symptoms vary by the type of central nervous system (CNS), initial signs and symptoms are very similar. Therefore rapid immunologic and molecular tools for differential diagnosis of arboviral encephalitis viruses are important for effective case management and control of the spread of encephalitis. The qRT-PCR assay, especially multiplex PCR, has the potential to produce considerable savings in time and resources in the laboratory detection. Meanwhile, the use of IC can prevent false negatives effectively by monitoring the processes of nucleic acid extraction and amplification. This report describes the development of a panel of internally controlled multiplex one-step real-time RT-PCR assays in which two virus specific-probe sets were used in the same reaction for the detection of 15 species arboviral encephalitis viruses: the comparative sensitivity of multiplex one-step qRT-PCR assays to single plex one-step qRT-PCR assays as well as one-step RT-PCR assays for detection of each viral species. And total of 150 human serum samples were detected to evaluate the multiplex one-step qRT-PCR assays. These multiplex one-step real-time RT-PCR assays with IC were evaluated in terms of sensitivity, linearity, precision, specificity, and also field samples including serum and vector. These assays can detect and differentiate arboviral encephalitis viruses by high throughput, sensitive, and specific way. It is useful for clinical management and outbreak control of arboviral encephalitis viruses and vector surveillance.

Molecular Evaluation of the Enterotoxigenicity of <i>Clostridium difficile</i>and <i>Clostridium perfringens</i>Swine Isolates by PCR Assays

Clostridium difficile and C. perfringens are enteric pathogens affecting a variety of mammals. This study evaluated the molecular enterotoxigenicity of Clostridium swine isolates by PCRs. One hundred and ten swine faeces were analyzed by culture assay. The faecal samples were from sixty-seven healthy animals and 43 with gastrointestinal tract disease. C. difficile strains were PCR-screened for the presence of tcdA/tcdB and cdtA/cdtB genes. All C. perfringens isolates were tested for the characterization of the toxinotype. Overall, sixty-five swine resulted positive: 38 for C. difficile and 17 for C. perfringens. One sample tested C. perfringens and C. difficile-positive, at the same time: on the whole, 39 C. difficile strains were isolated. Thirty-eight C. difficile isolates (all from healthy animals) resulted tcdA/tcdB and cdtA/cdtB-negative by PCRs and toxins A/B-negative by immunological tests. All C. perfringens strains were type A;eight were also cpb2-positive. In the sample (diarrhoeic), with double infection, C. difficile tested tcdA/tcdB and cdtA/cdtB-positive by PCRs and toxins A/B-positive by immunoassays;C. perfringens resulted cpb2-positive. The molecular genotypeing/toxinotyping should be applied to establish a final diagnosis and to assess properly the full implications and the epidemiological impact of these findings in particular in samples of healthy animals and aid in the development of effective intervention methods for controlling clostridial disease outbreaks.

A multi-center performance evaluation of different hepatitis C virus core antigen assays for clinical infection screening

Objective To evaluate the performance for three commercial hepatitis C virus(HCV) core antigen assays in HCV infection screening,and to provide clues for further improving the sensitivity and specificity of the assays.Methods Key performance indicators including the lower limit of detection(LOD),diagnostic sensitivity.

Thermal Shift Assays在重组森林脑炎病毒丝氨酸蛋白酶纯化中的应用

应用Thermal Shift Assays技术,高通量优化了重组森林脑炎病毒丝氨酸蛋白酶纯化过程中所使用的缓冲液中盐的种类和pH,显著改善了该重组蛋白在纯化过程中出现可溶性聚集体的现象。最终获得的重组蛋白质单体量由原先的75%提高到99%以上,极大提高了该纯化蛋白质的均一性和质量,为后续的蛋白质结晶研究奠定了基础。

Diagnostic Value of T-cell Interferon-y Release Assays on Synovial Fluid for Articular Tuberculosis： A Pilot Study

Background： Tuberculosis （TB） remains a major global public health challenge. Articular T/3 is an important form of extrapulmonary tuberculosis, and its diagnosis is difficult because of the low sensitivity of traditional methods. The aim of this study was to analyze the diagnostic value of T-SPOT.TB on synovial fluid for the diagnosis of articular TB. Methods： Patients with suspected articular TB were enrolled consecutively between August 2011 and December 2015. T-SPOT.TB was performed on both synovial fluid mononuclear cells （SFMCs） and peripheral blood mononuclear cells （PBMCs）. The final diagnosis of articular TB was independent of the T-SPOT.TB result. The diagnostic sensitivity, specificity, predictive value, and likelihood ratio of T-SPOT.TB on SFMCs and PBMCs were analyzed. Results： Twenty patients with suspected articular TB were enrolled. Six were diagnosed with articular TB, and 14 patients were diagnosed with other diseases. Sensitivity and specificity were 83% and 86% for T-SPOT.TB on SFMCs, and 67% and 69% for T-SPOT.TB on PBMCs, respectively. The positive predictive value （PPV） and negative predictive value （NPV） of T-SPOT.TB on SFMCs were 71% and 92%, respectively. The PPV and NPV were 50% and 82% for T-SPOT.TB on PBMCs. Conclusion： Sensitivity, specificity, and NPV of T-SPOT.TB on SFMCs appeared higher than that on PBMCs, indicating that T-SPOT. TB on SFMCs might be a rapid and accurate diagnostic test for articular TB.

Clinical practice guidelines for multigene assays in patients with early-stage breast cancer: Chinese Society of Breast Surgery (CSBrS) practice guidelines 2021

The role of multigene assays in chemotherapy decision-making in patients with early invasive breast cancer has been widely recognized.In 2017,the American Society of Clinical Oncology(ASCO)clinical guidelines for multigene profiling assays focused on increasing the intensity of recommendations for the clinical use of MammaPrint®.[1]The 8th edition of the American Joint Committee on Cancer(AJCC)staging system,officially launched in 2018,established the concept of prognostic staging for the first time,adding the use of non-anatomical information to evaluate the prognosis.Initially,Oncotype Dx®was recommended for suitable patients based on Level I evidence.Subsequently,five testing techniques,Oncotype Dx®,MammaPrint®,EndoPredict®,PAM50®,and BCI,were formally incorporated into the system.[2]To assist breast disease specialists in China in their selection of appropriate multigene profiling assays and detection methods for patients,and also to instill caution on decision-making with reference to multigene assays,the Chinese Society of Breast Surgery(CSBrS)has,through literature investigation and expert discussion,provided information on the key clinical problems and guidelines for the use of multigene assays,evaluating the evidence with reference to the Grades of Recommendations Assessment Development and Evaluation(GRADE)system.Combined with the availability of these assays in China,the clinical practice guidelines for multigene assays were formulated and published.The purpose of this guideline is to provide a reference for clinicians specializing in breast diseases in China.

Fluorescent glycan nanoparticle-based FACS assays for the identification of genuine drug-resistant cancer cells with differentiation potential

Herein we develop a unique differentiated-uptake strategy capable of efficient and high-purity isolation of genuine drug-resistant(DR)cells from three types of drug-surviving cancer cells,which include paclitaxel-surviving human ovarian OVCAR-3 cancer cells and human lung carcinoma A549/Taxol cells,and doxorubicin-surviving human immortalized myelogenous leukemia K562/ADR cells.By using this strategy which relies on fluorescent glycan nanoparticle(FGNP)-based fluorescence-activated cell sorting(FACS)assays,two subpopulations with distinct fluorescences existing in drug-surviving OVCAR-3 cells were separated,and we found that the lower fluorescence(LF)subpopulation consisted of DR cells,while the higher fluorescence(HF)subpopulation was comprised of non-DR cells.Besides,the DR cells and their progenies were found distinct in their increased expression of drug-resistant genes.More intriguingly,by using the FGNP-based FACS assay to detect DR/non-DR phenotypes,we found that the DR phenotype had a potential to differentiate into the non-DR progeny,which demonstrates the differentiation feature of stem-like cancer cells.Further research disclosed that the assay can quantitatively detect the degree of drug resistance in DR cells,as well as the reversal of drug resistance that are tackled by various therapeutic methods.The strategy thus paves the way to develop theranostic approaches associated with chemotherapy-resistance and cancer stemness.

Design of Smart Polymer-Protein Conjugates and Smart Magnetic Nanoparticles for Use in Microfluidic Diagnostic Assays

1 Results In this talk,I will describe the design,synthesis and application of smart polymers for use in microfluidic diagnostic devices.We are synthesizing a variety of temperature- and pH-responsive polymers using RAFT living free radical polymerization techniques.This allows us to control molecular weight and to achieve a narrow MW distribution of the polymers. Furthermore,RAFT polymers have reactive end groups that are used to conjugate the polymers to proteins.We are also using those groups to bind...

Biopotency Assays:an Integrated Application to Quality Control of Chinese Materia Medica

The current quality control（QC） pattern for Chinese materia medica（CMM） lacks suitable methods and indicators to evaluate their safety and efficacy effectively, which impedes the smooth development of CMM. In this review, main problems of the current QC pattern for CMM, principally focused on the content determination of constituents,were summarized and the inspiration from the QC of biological products was introduced. With the aim at introducing a suitable tool to the QC of CMM, biopotency assay and its feasibility in the QC pattern for CMM were analyzed and confirmed by relevant researches with years of practice. From the applications of biopotency assays in the QC of CMM in the last 10 years, we propose that biopotency assays should be an integral part of the QC pattern for CMM, for these assays can make the QC indicators related to the clinical safety and efficacy, supplementing the existed QC system of CMM.

The role of SLC12A family of cation-chloride cotransporters and drug discovery methodologies

The solute carrier family 12(SLC12)of cation-chloride cotransporters(CCCs)comprises potassium chloride cotransporters(KCCs,e.g.KCC1,KCC2,KCC3,and KCC4)-mediated Cl^(-)extrusion,and sodium potassium chloride cotransporters(N[K]CCs,NKCC1,NKCC2,and NCC)-mediated Cl^(-)loading.The CCCs play vital roles in cell volume regulation and ion homeostasis.Gain-of-function or loss-of-function of these ion transporters can cause diseases in many tissues.In recent years,there have been considerable advances in our understanding of CCCs'control mechanisms in cell volume regulations,with many techniques developed in studying the functions and activities of CCCs.Classic approaches to directly measure CCC activity involve assays that measure the transport of potassium substitutes through the CCCs.These techniques include the ammonium pulse technique,radioactive or nonradioactive rubidium ion uptakeassay,and thallium ion-uptake assay.CCCs'activity can also be indirectly observed by measuring gaminobutyric acid(GABA)activity with patch-clamp electrophysiology and intracellular chloride concentration with sensitive microelectrodes,radiotracer^(36)Cl^(-),and fluorescent dyes.Other techniques include directly looking at kinase regulatory sites phosphorylation,flame photometry,22Nat uptake assay,structural biology,molecular modeling,and high-throughput drug screening.This review summarizes the role of CCCs in genetic disorders and cell volume regulation,current methods applied in studying CCCs biology,and compounds developed that directly or indirectly target the CCCs for disease treatments.