Assay Method Research Articles

Evaluation of Some Liver Enzymes in HIV/AIDS Patients on Antiretroviral Therapy in University of Abuja Teaching Hospital, Nigeria

Background: Hepatoxicity is a major complication and side effect of antiretroviral therapy (ART). Unfortunately, such studies in this area of evaluating the adverse drug reaction of ART on the liver hepatocytes are only few in number in Nigeria.Objective: To determine the levels of some liver enzymes in HIV/AIDS patients on antiretroviral therapy (ART) and as well evaluate their impact on liver hepatocytes.Methods: This is a hospital-based case-control study. A total of 153 subjects were recruited in the study, and they were divided into three groups: the control group (which consist of apparently healthy subjects), the study group 1 (consist of HIV positive subject not on ART), and study group 2 (consist of HIV/AIDS patients on ART). The liver enzymes Alanine amino Transferase (ALT), Asparate amino Transfarase (AST) and Alkaline Phosphatase (ALP) were determined using enzyme linked spectrophotometric assay methods, according to the manufacturer’s specifications.Results: The mean standard deviation (SD) of ALT and AST activity of the study group 1 showed significant differences when compared with that of the control group P<0.001, but the ALP has no significant differences P>0.05. In Study group 2, (HIV/AIDS patients on ART) showed significant differences of P<0.001 in ALT, AST and ALP.Conclusion: Some liver enzymes were elevated in subjects on antiretroviral therapy; this elevation could be due to hepatotoxicity of the (ART) used by the HIV/AIDS patients on the liver hepatocytes.International Journal of Human and Health Sciences Vol. 08 No. 02 Apr’24 Page: 126-131

Zingiber officinale-Mediated biosynthesis of bimetallic Gold/Silver (BAu/Ag) nanoalloys; an insight into antiviral and anticancer activities

Utilizing plant extracts in the green synthesis of bimetallic nanoalloys receives more attention due to its eco-friendly, cheap, single-step, easy-handling method, producing highly active and biocompatible nanoalloys. Therefore, the aqueous extract of ginger rhizome was used to form bimetallic gold/silver (Au/Ag) nanoalloys. There were 37.3 mg.g−1 of total soluble carbs, 0.75 mg.g−1 of proteins, and 0.03 mg.g−1 of phenolic compounds quantified in ginger rhizome. The production of BAu/Ag-NPs relies on these components. The as-formed BAu/Ag-NPs were characterized using UV–Vis spectroscopy. The results showed that the peak surface plasmon resonance (SPR) occurred between 440 and 545 nm. The existence of several functional groups involved in the reduction and stability of the generated nanoalloys was shown by Fourier-transform infrared (FT-IR). The nanoalloys, with an average size of 49.1 ± 16.2 nm, were observed to be spherical using transmission electron microscopy (TEM). Energy-dispersive X-ray (EDX) indicated that the synthesized nanoalloy mainly consisted of Au and Ag ions, which made up (31.2 % and 47.1 %) and (45.9 % and 28.6 %) of the sample’s atomic and weight percentages, respectively. The crystallography of the nanoalloys was confirmed using XRD analysis. When examined for their capacity to suppress viral replication, the synthesized nanoalloys showed remarkable results, with 78.2 % inhibition against HAV and 69.6 % against Cox-B4. In addition, the anticancer activity of nanoalloys against oral epithelial cells (OEC), MCF7 adenocarcinoma cells, and HepG2 hepatocarcinoma cells was investigated using the MTT assay method. While the nanoalloys exhibited moderate toxicity towards normal OEC (IC50: 502.5 ± 2.8 g.mL−1), they showed substantial cytotoxicity towards cancer cells, MCF7 and HepG2, with IC50 values of 212.6 ± 4.4 and 117.9 ± 6.6 µg.mL−1, respectively. According to these data, BAu/Ag nanoalloys show potential as antiviral and anticancer drugs that selectively kill cancer cells.

Correlation of serum fibroblast growth factor-23 levels and calcium phosphate products levels in chronic kidney disease; sub analysis of chronic kidney disease-mineral and bone disorder study

<em data-sider-select-id="0e59b1fc-fce5-41b4-b987-316daa778de4">Introduction: The body produces fibroblast growth factor-23 (FGF-23) to maintain normal phosphate levels when hyperphosphatemia occurs. Production of FGF-23 indirectly causes hypocalcemia. Phosphate and calcium disturbances also occur in chronic kidney disease (CKD), therefore this adaptation mechanism applies. This situation; however, only manifests in the early stages of CKD; if the estimated glomerular filtration rate (eGFR) is less than 30% of normal. This adaptation is no longer adequate and levels of calcium-phosphate (Ca×P) products and FGF-23 still rise. <em data-sider-select-id="f0dc3bf1-1598-419e-9de6-fe837eadc8e6">Objectives: In this study, the correlation between both the serum levels of FGF-23 and Ca×P products in CKD was analyzed. <em data-sider-select-id="a367eeb1-7c32-4bf0-98ef-e6ae52522dc7">Patients and Methods: A cross-sectional study including 78 subjects with CKD stages 3 to 5 dialysis was conducted. Serum FGF-23 levels were determined using the enzyme-linked immunosorbent assay (ELISA) method and Ca×P product levels were calculated using the formula calcium (mg/ dL) × phosphate (mg/dL). The Kolmogorov-Smirnov test and Spearman’s test were conducted in the statistical study. If the P value is less than 0.05, the statistical findings are significant. <em data-sider-select-id="e286c166-6c09-432e-b832-c902b1a13b81">Results: Serum FGF-23 levels and Ca×P product levels were shown to be significantly correlated. This analysis of the two correlations was independent of age and diabetes mellitus (DM). Based on stages of CKD, serum FGF-23 levels and Ca×P product levels were discovered to be significantly correlated only at stage 5 of non-dialysis. <em data-sider-select-id="9b3534d4-deaa-4ccf-8b82-745bb5380661">Conclusion: Increasing serum FGF-23 levels were correlated with increased Ca×P product levels, particularly in CKD stage 5 non-dialysis subjects. This correlation was independent of age and DM.

PHENOTYPIC DETECTION OF ESBL AND AMPC BETA LACTAMASES AMONG GRAM-NEGATIVE ISOLATES FROM CLINICAL SAMPLES IN A TERTIARY CARE HOSPITAL

Objectives: Drug-resistant Gram-negative bacilli expressing extended-spectrum beta-lactamases (ESBLs) and AmpC pose a serious therapeutic threat in nosocomial infections. Cost-effective screening methods are a boon to patients. This study aims to detect gram-negative bacilli and their antibiotic sensitivity patterns, as well as detect the ESBL and AmpC-producing isolates among Gram-negative bacilli. Methods: A prospective study was conducted with 150 samples. Gram-negative bacilli were isolated, and their antibiotic sensitivity tests were performed by the Kirby–Bauer disk diffusion method. Potential ESBL producers were screened using Ceftazidime disc, and AmpC producers were screened by Cefoxitin discs by the disc diffusion method. ESBL producers were confirmed by the combined disc diffusion assay method using ceftazidime and ceftazidime/Clavulanic acid disc. AmpC producers were confirmed by the Cefoxitin Cloxacillin Double Disc Synergy Test. Results: About 38% of 150 samples were gram-negative bacilli, of which 40.35% were Escherichia coli, followed by Pseudomonas aeruginosa (35.08%). Maximum sensitivity by E. coli was found toward imipenem, meropenem, and cotrimoxazole. P. aeruginosa showed maximum sensitivity toward piperacillin/tazobactam, imipenem, meropenem, and ceftazidime. 28.07% of Gram-negative isolates were ESBL producers, with E. coli (11 isolates) being the maximum, and 15.78% were AmpC producers, with E. coli (four isolates) being the maximum. Seven isolates were both ESBL and AmpC producers. Conclusion: Routine screening and timely reporting of ESBL and AmpC producers help in preventing the spread of multidrug-resistant strains. Antibiotic resistance surveillance helps in the implementation of strict infection control and prevention practices.

Fluorescence based methodology with greenness consideration for concurrent estimation of anti-allergic medication; application to dosage form

A green, sensitive and rapid spectrofluorimetric method for quantitative assay of an anti-allergic medication composed of montelukast and fexofenadine mixture in raw materials and dosage form was developed. The method was based on measuring the synchronous fluorimetric peak without interference, pre-separation or pre-extraction procedures. Montelukast was analyzed at 360 nm while fexofenadine was measured at 263 nm using Δλ = 20 nm for both drugs using ethanol as diluting solvent and acetate buffer of pH 4. The assay was rectilinear over the concentration range of 1.0–10.0 μg/mL for fexofenadine and 0.1–0.6 μg/mL for montelukast. The method was full validated according to ICH guidelines. The applicability of the method enables the assay of both drugs in raw materials, synthetic mixture as well as combined tablets. Moreover, the greenness of the method was assessed using different methods including; analytical eco-scale, GAPI and AGREE. All of these methods confirm that the proposed method is an eco-friendly method.

Neuron-Specific Enolase-What Are We Measuring?

Since the discovery of the neuron-specific protein by Moore and McGregor in 1965, tens of thousands of studies have investigated the basic and applied significance of neuron-specific enolase (NSE). This promising biomarker, according to many researchers, has not found widespread use in clinical practice, particularly in acute cerebrovascular accidents. Moreover, the several studies refuting the usefulness of serum NSE measurement in critically ill patients leads us to consider the reasons for such contradictory conclusions. In this article, we have analyzed the main directions in the study of NSE and expressed our perspective on the reasons for the contradictory results and the difficulties in implementing the results of these studies in clinical practice. In our opinion, the method of the enzyme-linked immunosorbent assay (ELISA) used in the majority of the studies is inappropriate for the evaluation of NSE as a marker of central nervous system damage, because it does not allow for the differentiation of heterodimers of enolases and the assessment of the enzymatic activity of this group of enzymatic proteins. Therefore, the methodological approach for the evaluation of NSE (γγ-enolase) as a biomarker needs to be elaborated and improved. Furthermore, the specificity of the applied research methods and the appropriateness of the continued use of the term "neuron-specific enolase" must be addressed.

A beginner’s guide to immunohistochemistry

Using the affinity between antibodies and antigens, immunohistochemistry (IHC) is a helpful method for analysing the cellular components within tissue sections placed on glass slides. The chemistry of antibodies was not discovered until 1941, and since then, their use has increased due to the discovery of enzymes and fluorochromes conjugated to antibodies, enabling observations through optical or fluorescence microscopy (FM). Hence, immunofluorescence (IF), or IHC with fluorescent antibodies, has been associated with the advancement of FM and fluorochromes. Specifically, the discovery of three fluorochromes that emit blue, red and green led to a rise in IF application in the context of visualizing numerous components in a single cell. The proximity ligation assay (PLA) method, so termed because the antibody is conjugated with short DNA strands called PLA probes, is the most recent development in the field of antibody conjugation. Protein–protein interactions and sub-localization of proteins can both be studied using this technique. The improvement of conjugation techniques has occurred in parallel with the development of sophisticated microscopies, including confocal and slide scanners. Confocal microscopy allows for the reconstruction of three-dimensional structures while a slide scanner can digitally convert up to 100 slides' worth of high-resolution pictures. The first step in the IHC process is the collection and fixation of tissues from animal models and humans. Paraformaldehyde is the most commonly used fixative. Fixation prevents antigen blocking and creates cross-links between proteins to preserve tissue structures. Fixation is followed by cutting tissues using microtome or cryostat, then slides are treated for antigen exposition (antigen retrieval) and then included in Triton X solution, which increases membrane permeability. Subsequently, incubations with enzyme- or fluorochrome-conjugated antibodies enable observations via optical microscopy or FM, respectively. In clinical practice and research, IHC and IF are still helpful methods for identifying the antigen-proteins linked to specific diseases.

In cases with raised sperm DNA fragmentation, can sperm selection by magnetic-activated cell sorting or testicular sperm aspiration help improve reproductive outcomes?

To evaluate the efficacy of magnetic-activated cell sorting (MACS) or testicular sperm aspiration (TESA) to improve reproductive outcomes in cases with elevated sperm DNA fragmentation undergoing assisted reproduction. This randomized controlled trial included couples with failed IVF cycles and sperm DNA fragmentation > 30%. Sperm DNA fragmentation was assessed using the sperm chromatin structure assay (SCSA) method. Participants were randomly assigned to either the MACS or TESA group. Testicular sperm retrieval was performed for the TESA group, while MACS involved sperm selection using magnetic beads. Extended blastocyst culture, freeze all policy of blastocysts by vitrification, and frozen embryo transfer were undertaken as per clinic's standard operating protocols. Blastocyst formation rate, implantation rate, miscarriage rate, multiple pregnancy rate, and live birth rate were analyzed and compared between MACS and TESA groups. There were no significant differences in female age, male age, or sperm DNA fragmentation index (DFI) between the MACS and TESA groups. The blastocyst conversion rate was slightly higher in the TESA group (39%) compared to the MACS group (32%). However, the MACS group had a higher implantation rate (50%) than the TESA group (35%). Miscarriage rates, multiple pregnancy rates, and live birth rates did not show statistically significant differences between the groups. A chi-squared test was conducted to compare categorical variables, and t-tests were done to compare continuous variables. In cases with raised sperm DNA fragmentation, sperm selection by MACS or TESA seems to offer comparable reproductive outcomes. There seems no superiority of one intervention over the other in cases with raised sperm DNA fragmentation undergoing assisted reproduction. Both interventions seem to be beneficial for couples seeking assisted reproduction with raised sperm DNA fragmentation.

Establishment and application of a rapid visual diagnostic method for Streptococcus agalactiae based on recombinase polymerase amplification and lateral flow strips

This study aims to establish a rapid diagnostic method for Streptococcus agalactiae (GBS) based on recombinase polymerase amplification (RPA) and lateral flow strips (LFS). The best primer pairs designed by SIP gene were screened according to the basic RPA reaction, then the probe was designed. The reaction condition was optimized based on the color development of the LFS detection line. To ascertain the reaction specificity, 10 common clinical pathogens and 10 clinical specimens of GBS were tested. Furthermore, the reaction sensitivity was assessed by utilizing a tenfold gradient dilution of GBS genomic DNA as templates. RPA–LFS method was compared to the qPCR assay and biochemical culture method for the Kappa consistency test. The RPA–LFS technique was able to complete the amplification process within 30 min and the results were observed on lateral flow strips. The method is highly sensitive, with a minimum detection limit of 1.31 ng for GBS. The RPA–LFS method showed consistent accuracy of results compared to qPCR and the culture–biochemical method. The establishment of this method is conducive to the development of on-site immediate detection, which can provide information for the timely development of a reasonable antimicrobial treatment plan, and has a greater potential for clinical application.

Survey of pharmaceutical industry's best practices around in vitro transporter assessment and Implications for Drug Development: Considerations from the IQ Transporter Working Group.

The IQ Transporter Working Group had a rare opportunity to analyse a cross-pharma collation of in vitro data and assay methods for the evaluation of drug transporter substrate and inhibitor potential. Experiments were generally performed in accordance with regulatory guidelines. Discrepancies, such as not considering the impact of pre-incubation for inhibition and free or measured in vitro drug concentrations, may be due to the retrospective nature of the dataset and analysis. Lipophilicity was a frequent indicator of cross-transport inhibition (P-gp, BCRP, OATP1B and OCT1) with high molecular weight ({greater than or equal to}500 Da) also common for OATP1B and BCRP inhibitors. A high level of overlap in in vitro inhibition across transporters was identified for BCRP, OATP1B1 and MATE1 suggesting that prediction of DDIs for these transporters will be common. In contrast inhibition of OAT1 did not coincide with inhibition of any other transporter. Neutrals, bases, and compounds with intermediate-high lipophilicity tended to be P-gp and/or BCRP substrates whilst compounds with MW <500 Da tended to be OAT3 substrates. Interestingly the majority of in vitro inhibitors were not reported to be followed up with a clinical study by the submitting company, whilst those compounds identified as substrates generally were. Approaches to metabolite testing were generally found to be similar to parent testing with metabolites generally being equally or less potent than parent compounds. However, examples where metabolites inhibited transporters in vitro were identified supporting the regulatory requirement for in vitro testing of metabolites to enable integrated clinical DDI risk assessment. Significance Statement A diverse dataset showed transporter inhibition often correlated with lipophilicity and molecular weight (>500 Da). Overlapping transporter inhibition was identified, particularly that inhibition of BCRP, OATP1B1 and MATE1 was frequent if the compound inhibited other transporters. In contrast inhibition of OAT1 did not correlate with the other drug transporters tested.

Current utility of first-line FT4 and TSH in screening for central hypothyroidism.

Thyroid testing strategies vary across laboratories. First-line combined thyroid stimulating hormone (TSH) and freeT4 (FT4) have historically been preferred by many laboratories as this detects individuals with undiagnosed central hypothyroidism who can be missed with a first-line TSH-only strategy. However, an up-to-date evaluation of the utility of this approach is lacking. We investigated the clinical utility of first-line TSH and FT4 in the detection of central hypothyroidism in current day practice. The All-Wales laboratory information system was queried to identify thyroid function tests in patients aged ≥16 years with decreased FT4 and inappropriate TSH (low-FT4). The 1-year incidence of low-FT4 was determined using mid-year population data. Clinical information of patients with low-FT4 was reviewed to determine causes of low-FT4 and the incidence of central hypothyroidism. The incidence of low-FT4 varied according to FT4 assay method (range: 98-301 cases/100,000 population/year). Fifteen new cases of central hypothyroidism were detected in two health boards, equivalent to 2 cases/100,000 population/year. Positive predictive value of low-FT4 for central hypothyroidism was 2%-4%. In a cross-section of primary care patients, low-FT4 was detected in 0.5% of all thyroid tests with assay-related differences in detection rates. Although low-FT4 is a common laboratory finding, the incidence of central hypothyroidism remains rare. With the currently increased rates of thyroid testing and increased use of medications that decrease FT4, low-FT4 has a much lower predictive value for central hypothyroidism than previously reported. Thyroid screening strategies will need to balance the yield from first line TSH and FT4 testing with the cost of investigating individuals with non-pathological laboratory abnormalities.

Human papillomavirus 16 specific immunoglobulin g antibodies and its correlates among women attendees of selected hospitals in Southern Kaduna, Kaduna State, Nigeria

Human papillomavirus type 16 (HPV-16) is one of the high-risk viruses that cause cervical cancers. Persistent infection with the virus leads to development of precancerous lesions of the cervix in infected women, which without medical intervention can progress to invasive cervical cancer. This study assessed HPV16-specific immunoglobulin G (IgG) antibodies in the serum of women attendees of General Outpatient Department (GOPD) at General Hospital Kafanchan and Kagarko in Kaduna State. This was a cross-sectional study. Structured, self-administered questionnaire was used to collect information and blood samples were also collected for testing. This was in addition to information collected from the laboratory analyzed. Five milliliters (5mls) of blood were aseptically collected from each of the 200 women, who had no history of HPV vaccination at the time of the study for the determination of HPV16-specific IgG antibodies using enzyme-linked immunosorbent assay method. The sero-positivity for HPV 16-specific IgG antibodies among the women was 24.5%. The sero-positivity among women who had their first sexual intercourse at age 13-19 years was 37.6%, and it was significantly different from those who had their sexual debut at ≥20 years (14.8 %) (p=0.001). Sero-positivity also increased from 9.2 % in women with one lifetime sexual partner to 60.0% in those with multiple sexual partners(p=0.001). The finding showed that the women in this study have been exposed to the HPV-16 virus. Further study with a larger population of women in this locality to determine the level of susceptibility or immunity to HPV-16 is strongly advocated, among others.

Hapten design, monoclonal antibody preparation and immunoassay development for direct detection of the toxic metabolite of nitrovin without derivatization from fish

Nitrovin, classified as a nitrofuran drug, has been banned in numerous countries due to its carcinogenic effect on human health. However, existing analytical techniques for its detection are laborious, requiring an overnight derivatization process lasting 16 h. In this study, novel haptens were designed to prepare antibodies against aminogunidine (AGD), the metabolite of nitrovin, and then developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) method to detect AGD from fish samples without derivatization for the first time. Computer-assisted molecular modeling predicted the efficiency of novel haptens by conformational studies and electronic analysis. The results were consistent with the predicted results in producing high-affinity monoclonal antibody (mAb) against AGD. The half-inhibition concentration (IC50) value of icELISA was 0.80 ng mL−1 for AGD, with a limit of detection of 0.27 μg kg−1 and recoveries of 89.8%–97.2%. The results showed that this new hapten design and synthesis provided a great prospect to obtain high-affinity mAb for AGD. This developed icELISA substantially avoided the conventional and lengthy derivatization process and was an effective method for the direct detection of AGD in fish tissue without derivatization.

Extracorporeal Shock Wave Therapy as Regenerative Medicine in Knee Osteoarthritis: Pre-Experiment for Matrix Metalloproteinase-3 Evaluation.

Knee osteoarthritis (KOA) is currently treated by regenerative therapies that aim to inhibit arthritic degeneration. Extracorporeal shock wave therapy (ESWT) is one of the physical regenerative approaches used for KOA management. However, little is known regarding the impact of shock wave treatment on matrix metalloproteinase-3 (MMP-3), which is one of the enzymes mediating cartilage degradation. To evaluate the effect of ESWT on MMP-3 levels and pain intensity in patients with KOA. Fourteen patients diagnosed with Kellgren Lawrence, grades 2 and 3 KOA were recruited for the study. ESWT piezo shockwave was applied once a week for six weeks. MMP-3 levels in the blood were measured pre-test, mid-test (three weeks after therapy) and post-test (one week after the last session) by enzyme-linked immunosorbent assay (ELISA). The perceived pain was recorded at each session by the Wong Becker Face Scale. The median pre-test, mid-test and post-test MMP-3 levels were 19.92 ng/mL, 15.89 ng/mL and 18.82 ng/mL, respectively, and there were significant differences between the pre-test and mid-test, and the pre-test and post-test values (p < 0.05). The pain scores also decreased significantly over the period of intervention. MMP-3 levels decreased significantly in KOA patients after ESWT, and the decline was most obvious after 3 weeks of therapy. Therefore, EWST should be considered as a suitable treatment option for KOA.

Paper-based radial chromatographic assay for rapid in situ measurement of gelatinized starch concentration

Starch gelatinization is pivotal in food processing, impacting viscosity, texture, and functionality. However, current methods for determining concentration and viscosity of gelatinized starch (GS), such as falling ball and rotational viscometers, are impractical for in situ measurements due to bulky equipment. Here, we introduce a paper-based radial chromatographic assay (PRCA) method for rapidly measuring the concentration of gelatinized starch (GS). We utilized Evans Blue (EB) as a chromatic agent to label GS and generate color-ring patterns on a nitrocellulose membrane based on the “Coffee-ring” effect. The resulting PRCA patterns varied depending on the starch concentrations, yielding a good linear correlation (R2 > 0.967) between grayscale value and viscosity. This method enables rapid measurement of starch concentration within 10s, even with varying ratios of amylose to amylopectin. PRCA demonstrates a detection limit for starch concentration between 0.027% and 0.051%, which is 20-fold lower than that of traditional rotational-viscometer. The intensity of PRCA patterns at pH 3 (45.8) was significantly lower than those at higher pH levels (5 to 9), consistent with the decreased viscosity measured by the viscosimeter. The intensity of PRCA patterns did not change with increasing temperature from 25°C to 90°C, despite the significant decrease in viscosity from 345 mPa•s to 195 mPa•s. Additionally, PRCA can monitor changes in starch viscosity during the hydrolysis process, which decreases from 744 mPa•s to 6 mPa•s. This capability provides an efficient means of measuring the concentration and viscosity of GS during food processing.

Novel method for screening probiotic candidates tolerant to human gastrointestinal stress

Tolerance to human gastrointestinal stressors is crucial for probiotics to exhibit their health benefits; however, there is no standardised method for screening their stress tolerance. In this study, we proposed a novel method for screening probiotic candidates tolerant to human gastrointestinal stress—gastrointestinal tolerance assay and culture (GTA-C) method—using black polyethylene terephthalate (PET) non-woven fabric as a scaffold to modify the specialized cellulose film (SCF) method. The modified SCF method showed excellent pH-based diffusion of medium components, had minimal effect on the growth of Escherichia coli, and improved the visibility of the colonies. Analysis of kimchi samples cultured using the SCF and modified SCF methods revealed that the modified method diversified the cultured bacteria. GTA in a simulated human fasting state using the modified SCF method showed that acid stress significantly affected the growth of four bacteria used as probiotics and that tolerance to acid stress may be species-dependent. Screening of probiotics in kimchi samples resulted in the identification of lactic acid bacteria tolerant to human gastrointestinal stress during fasting. Our results indicate that the modified SCF method (GTA-C method) is useful for screening probiotics resistant to the gastrointestinal environment during fasting.

Ultra-high performance liquid chromatography-tandem mass spectrometry assay for simultaneous determination of 22 marker compounds in traditional herbal medicine Ojeoksan

Traditional herbal medicines (THMs) have long been in use worldwide and are considered safe for use as tonics or complementary treatments for many diseases. Advanced quality control methods for THMs are required in the regulatory framework of modern medicines. In this study, an ultra-high performance liquid chromatography-tandem mass spectrometry assay was established for the simultaneous determination of 22 marker compounds in Ojeoksan (OJS), which is composed of 15 herbal substances. All marker compounds were analyzed within 20 min and successfully identified via scheduled multiple reaction monitoring. The method validation revealed excellent performance characteristics of the method such as specificity, linearity, sensitivity, precision, and accuracy, demonstrating its suitability for intended use. The developed method was applied to samples of commercial OJS tablet and soft-extract dosage forms. The 14 marker compounds corresponding to 12 component herbal substances were determined in the samples; ephedirine, albiflorin, paeoniflorin, ferulic acid, hesperidine, neohesperidin, cinnamic acid, platycodin D, 6-gingerol, atractylenolide III, glycyrrhizin, honokiol, decursin, and magnolol. A fast and easy assay method with sufficient discrimination power was established. As a novel assay, this method can contribute to the quality control of OJS products.

Correlation between two fructosamine assay methods according to the presence of hemoglobin variants

Correlation between two fructosamine assay methods according to the presence of hemoglobin variants

A reproducible and affordable method of conducting Luciferase assay

Commercially available glow luciferase assay kits are widely popular and convenient to use. However, concerning high-throughput screening, commercial kits are limited by huge running costs. As an alternative to commercial luciferase assay kits, this study presents a cost-effective and efficient methodology of performing a simple and rapid laboratory flash luciferase assay. The proposed luciferase assay method has a versatile use ranging from screening lysates in a microplate reader for quantitative assay as well as screening live cells qualitatively or quantitatively under an imaging system.

Assessing selection potential for Pacific oyster (Crassostrea gigas) to a North American OsHV-1 μvar: Comparing two experimental assay methods

Ostreid Herpesvirus - 1 is a global threat to Pacific oyster aquaculture as catastrophic farm losses have occurred where OsHV-1 microvariants (μvars) become endemic. Genetic selection has proven effective for increasing survival to OsHV-1 exposure in multiple Pacific oyster breeding programs, but a novel μvar detected in San Diego Bay California has the potential to severely impact commercial aquaculture along the North American Pacific coast as no OsHV-1 μvar selected population is available commercially. Using a Molluscan Broodstock Program population descending from imports originating in the Miyagi prefecture (Japan), we evaluated the potential for genetic selection to improve survival against the San Diego Bay OsHV-1 μvar using a laboratory trial. Two experimental methods were evaluated, a common garden method where all 15 full-sibling families were pooled, and a petri dish method, where each family was confounded within replicate. No striking differences were found between experimental methods, and both experiments displayed high heritability (0.69 and 0.64) of the tested trait, a broad range of estimated breeding values, and a moderate prediction accuracy for the survival trait. A significant genotype-by-environment interaction occurred, highlighting variability in laboratory-based disease challenges, but the two experiments yielded similar results with minor to moderate re-ranking of mean family survival. Overall, the Miyagi population of Pacific oysters curated by the Molluscan Broodstock Program is a promising population to use for artificial selection to increase survival against San Diego Bay OsHV-1 μvar in attempts to obviate farm losses if the virus expands its currently isolated range.