Tuberculosis in kidney transplant candidates and recipients

Tuberculosis(TB)is the leading cause of infectious mortality and morbidity in the world,second only to coronavirus disease 2019.Patients with chronic kidney disease and kidney transplant recipients are at a higher risk of developing TB than the general population.Active TB is difficult to diagnose in this population due to close mimics.All transplant candidates should be screened for latent TB infection and given TB prophylaxis.Patients who develop active TB pre-or post-trans-plantation should receive multidrug combination therapy of antitubercular therapy for the recommended duration with optimal dose modification as per glomerular filtration rate.

The Essential Role of Zheng Qi in Promoting Health: From the Perspective of Chinese Medicine and Modern Medicine

The concept of Zheng Qi in traditional Chinese medicine (TCM) refers to the vital energy produced by the interaction of Yin and Yang forces in the body. Zheng Qi performs two main functions: Wei Qi (defensive Qi), which shields the body from external pathogens, and Ying Qi (nutritive Qi), which sustains the internal organs and enhances their functionality. In TCM, Chinese tonifying herbs can help restore the balance of Yin/Yang and Qi/Blood function in visceral organs (i.e., optimal physiological functions), thereby fostering the efficient production of Zheng Qi and enhancing health. To ensure the quality of Chinese herbal products, functional assays to measure Yin/Yang, Qi/Blood functions, and Zheng Qi production should be implemented. The efficacy of Yang and Qi herbs can be evaluated by their ability to increase mitochondrial ATP in cultured mouse cardiomyocytes, while Yin and Blood herbs are tested through their immunostimulatory effects on antigen-induced T/B cell proliferation in mouse splenocytes and the production of erythropoietin/nitric oxide in hepatocytes/vascular endothelial cells, respectively. Additionally, Zheng Qi’s effect can be gauged by examining natural killer cell activity and antigen-induced T/B cell proliferation in mice ex vivo. These assays act as biomarkers for assessing the quality and effectiveness of herbal health products within TCM theory.

Study on the processing technology of Polygonatum cyrtonema Hua decoction pieces based on softening method and drying method

Background:To optimize the steaming processing technology of Polygonatum cyrtonema Hua(PC)decoction pieces.Methods:The softening method and drying method of PC decoction pieces were studied with Polygonatum Polysaccharide(PCP),diosgenin combined extract as quantitative indexes.The process parameters such as softening time,drying temperature and drying time were determined,and the best processing technology of PC decoction pieces was optimized.Results:Among the three softening methods of PC,the infiltration method had the highest ranking,with an average comprehensive index of 0.9496,and the softening effect was the best.Among the three drying methods,the drying effect of the hot air drying method was the best,and the average comprehensive index was 0.8233.Conclusion:The infiltration method is the best softening method for PC decoction pieces,and the hot air drying method is the best drying method for PC decoction pieces.

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.

Current Status of Targets and Assays for Anti-HIV Drug Screening

HIV/AIDS is one of the most serious public health challenges globally. Despite the great efforts that are being devoted to prevent,treat and to better understand the disease,it is one of the main causes of morbidity and mortality worldwide. Currently,there are 30 drugs or combinations of drugs approved by FDA. Because of the side-effects,price and drug resistance,it is essential to discover new targets,to develop new technology and to find new anti-HIV drugs. This review summarizes the major targets and assays currently used in anti-HIV drug screening.

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.

STABLE EXPRESSION OF HUMAN CYTOCHROME CYP2B6 AND CYP1A1 IN CHINESE HAMSTER CHL CELLS:THEIR USE IN MICRONUCLEUS ASSAYS

With specific designed prmers. CYP2B6 and CYP1A1 cDNA were generatecl by reverse transcrlI7tion-Polymerase chain reaction(RT-PCR )technlque Performed on total RNAs isolated frorn hum1ln liver and 3-rnethylch(,lanthrene(3-Mtt)induc human amnion FL, cells. Cell llnes (CHL, 2B6 and CtHL-1A1 ) capableof expressing hunlan cytochome P 15O (CYP ) 2B6 and 1A1 were establishecl after transfection of corre-sponding eukaryotic reconlbinant expression plasmid with human CYP2ll6 and 1A1 cDNA lnserts respectlvely. These cell lines stably expressed the mRNAs and the enzymatic activltles cc)rresI’onding to ttYP2B6and CYP1A1, respectively’ Compared with Chinese hamster lung (CHL) cells, the n1icr()nucleus frecluencyin CHl,-2B6 cells is markedly lncreased when exPosed to nitrosamines,aflatoxln B, (AFB1) and cyclophos-Phamide (CPA). Thls is also in CHL-1A1 cells,when exposed to carcinogenic polycycllc aromatic hydrocar-bons.

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.

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.

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.

Primary Study of Egg Yolk Antibody for Detection of King Cobra Venom Antigens

Aim To study whether antivenom from laying hens can be used for the detection of venom antigens, Methods Chickens （white Leghorn） were immunized with detoxicated king cobra venom by formaldehyde and egg yolk immunoglobulin （IgY） isolated from yolk; IgY was labelled with the horseradish peroxidase （HRP）. Experimental condition and parameters were determined by chessboard test. The specificity, sensitivity, precision, and stability of this method were assayed in the experiment. Results This method could detect as low as 32 μg· L^-1 of the king cobra antigens. A good linear relation was found within 32 ～ 750 μg· L^-1 of king cobra venom concentrations （ r = 0. 963）. There was no cross reactivity for the reagents with Agkistrodon acutus Guenther venom or Vipera russelli siamensis Smith venom;slight cross reactivity .with Bungarus multicinctus Blyth venom or Bungarus fasciatus Chmeider venom; and notable cross reactivity with cobra venom. The average intra-assay relative standard deviation （RSD） was 1% - 3%, and the inter-assay RSD was less than 8%. The reagents （including IgY and HRP-IgY） were stable; no differences （P 〉 0.05） were observed for the detection of venom antigens when the reagents were stored at 37 ℃ up to 6 d. Conclusion IgY is a good reagent for diagnosis of snakebite after eliminating the genus cross reactivity.