Assay Reagents Research Articles

Investigation of in vitro and in vivo antioxidant and antidiabetic activities of Pinus halepensis extracts

Introduction: Pinus halepensis is a medicinal plant used in traditional medicine for treatment of various pathologies including diabetes. The objective of this study is to perform a phytochemical study and to evaluate the antioxidant and antidiabetic activities of extracts of the bark of P. halepensis. Methods: Total polyphenols, flavonoids and tannins were determined by the Folin Ciocalteu method, aluminum trichloride reagent (AlCl3) and vanillin assay. Evaluation of the antioxidant activity was carried out using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2’-azinobis- (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and ferric reducing agent (ferric reducing). The antidiabetic activity was first revealed by enzymatic inhibition tests through measuring the residual activities of α-amylase and α-glucosidase, and then, by oral tolerance tests of glucose and starch in male Wistar rats. To verify the safety of plant extracts, acute oral toxicity was determined. Results: The phytochemical analysis showed that the extracts of P. halepensis were rich in phenolic compounds. The anti-oxidation activity tests revealed a significant reducing power towards the radicals tested. In addition, P. halepensis inhibited the enzymes involved in diabetes (α-amylase and α-glucosidase) at very low concentrations. These effects were verified in the in vivo approach, in particular by using the starch tolerance test. Conclusion: P. halepensis extracts showed remarkable antioxidant and antidiabetic effects. However, further investigations are necessary to identify the main compounds of P. halepensis and to evaluate their antioxidant and antidiabetic effects.

Evaluation of Antiurolithiatic Potential of Piper cubeba Dried Fruits on Sodium Oxalate Induced Urolithiasis in Rats

Objectives: The main endeavor of the work is to assess the antiurolithiatic potentials of ethanolic extract of Piper cubeba dried fruits (EEPC). Methods: EEPC was prepared using soxhlation, subjected to phytochemical screening and determined total phenolic and flavonoid content using Folin- Ciocalteu reagent and aluminium chloride colorimetric assay methods. In vitro antioxidant activity was evaluated using methods like 2,2-diphenyl-1- picrylhydrazyl (DPPH) and lipid peroxidation assays using ascorbic acid as standard. Sodium oxalate (70 mg/kg, i.p) was given to rats for 5 days to promote urolithiasis. Cystone (750mg/kg, p. o), EEPC (100, 200 and 400 mg/kg, p.o) was administered to the respective group of rats from 6th to 15th day. On the 15th day, serum and urine were collected from individual animals and biochemical parameters like BUN, creatinine and uric acid in serum and sodium, chloride, potassium, calcium, phosphate, oxalate in urine and calcium, phosphate and oxalate in kidney homogenate have been measured. The kidney sections have been prepared and histopathologically tested to check the stones. Results: Preliminary phytochemical analysis discloses the existence of phenolics, tannins, steroids, terpenoids and flavonoids. The EEPC enriched with phenols and flavonoids which correlates with its antioxidant potentials. Following treatment with cystone and EEPC, concentrations of BUN, creatinine, uric acid in serum and sodium, chloride, potassium, calcium, phosphate, oxalate in urine and calcium, phosphate, oxalate in kidney homogenate (P<0.001 vs. control) were significantly reduced in urolithiasis caused by sodium oxalate in a dose-dependent way. Conclusion: The promising results suggest that EEPC would act as a potential agent in the management of urolithiasis along with its antioxidant properties.

An Automated High-Throughput Fluorescence In Situ Hybridization (FISH) Assay Platform for Use in the Identification and Optimization of siRNA-Based Therapeutics.

Since the revolutionary discovery of RNA interference (RNAi) more than 20 years ago, synthetic small interfering RNAs (siRNAs) have held great promise as therapeutic agents for treating human diseases by the specific knockdown of disease-causing gene products. To facilitate the development of siRNA therapeutics, a robust, high-throughput in vitro assay for measuring gene silencing is imperative during the initial siRNA lead sequence identification and, later, during the lead optimization with chemically modified siRNAs. There are several potential assays for measuring gene expression. Quantitative reverse transcription PCR (qRT-PCR) has been widely used to quantitate messenger RNA (mRNA). This method has a few disadvantages, however, such as the requirement for RNA isolation, complementary DNA (cDNA) generation, and PCR reaction, which are labor-intensive, limit the assay throughput, and introduce variability. We chose a high-content imaging assay, bDNA FISH, that combines the branched DNA (bDNA) technology with fluorescence in situ hybridization (FISH) to measure gene silencing by siRNAs because it is sensitive and robust with a short reagent procurement and assay development time. We also built a fully automated liquid-handling platform for executing bDNA FISH assays to increase throughput, and the system has a capacity of generating 192 concentration-response curves in a single run. We have successfully developed and executed the bDNA FISH assays for multiple targets using this automated platform to identify and optimize siRNA candidate molecules. Examples of the bDNA FISH assay for selected targets are presented.

Anti-Inflammatory and Cytotoxicity Effects of Cudrania tricuspidata Fruits Vinegar in a Co-Culture System with RAW264.7 Macrophages and 3T3-L1 Adipocytes.

Vinegar has been found to have in vitro improvement effect on inflammatory biomarkers, and clinically used to improve inflammation and obesity-related diseases. This study was designed to analyze in vitro anti-inflammatory effects of Cudrania tricuspidata fruits vinegar (CTFV) in a co-culture system with macrophages and adipocytes. We analyzed the physicochemical properties and polyphenolic ingredients of CTFV, and investigated in vitro anti-inflammatory effects of CTFV in a co-culture system with macrophages and adipocytes. The cells were cultured in the presence of CTFV for 24 h in contact with each other, then, harvested. The levels of monocyte chemoattractant protein (MCP)-1, tumour necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), nitric oxide (NO), and interleukin (IL)-6 were evaluated by using the Griess reagent, western blot, or enzyme-linked immunosorbent assay assays. We found that increasing levels for NO, iNOS, TNF-α, IL-6 and MCP-1 were caused by LPS treatment and co-culture using the contact method, whereas CTFV efficaciously attenuated inflammatory response by improving inflammatory parameters including NO, iNOS, TNF-α, IL-6 and MCP-1. The present study indicates that CTFV might provide a nutraceutical product or functional food resource for improving inflammation processed via the interaction of adipocytes and macrophages.

In Vitro Evaluation of Antioxidant Activity of Homeopathic Mother Tincture and Total Phenolic Content

Background: Antioxidants are those compounds which scavengers the free radicals, widely used in health protection and prescribed on large scale in biomedical health care units. In homeopathic system of medicine mother tinctures are prescribed. Mother tinctures of medicinal plants are having the potential to treat the various acute and chronic ailments.&#x0D; Objectives: The present study was accompanied to determine the total phenolic content and the antioxidant activity of homeopathic mother tincture of plant origin i.e. Baptisia tinctoria, Berberis aquifolium, Echinacea angustifolia, Hydrangea arborescens, Hydrastis canadensis, Kreosotum, and Thuja occidentalis in comparison with ascorbic acid.&#x0D; Methods: Antioxidant activity was evaluated by 2, 2-diphenyl-1-picrylhydrazyl (DPPH) inhibition assay method. A volume of 0.1 mM solution of DPPH was used while mother tinctures (1.25, 5, 2.5 μl volumes) were used for evaluation of antioxidant activity. Ascorbic acid was taken as a standard control and alcohol as negative control in antioxidant activity protocol. Total phenolic content was measured by Folin–Ciocalteu reagent assay. Total phenolic content of mother tinctures was measured in comparison with ascorbic acid.&#x0D; Results: All tested homeopathic mother tinctures of plant origin showed significant antioxidant potential except Hydrangea arborescens and Kreosotum. Alcohol which was used as negative control exhibited no antioxidant activity. Pulsatilla nigricans possess highest antioxidant potential 85±0.3 among other tested mother tinctures against DPPH free radical and 98.8±2 maximum no of phenolic contents.&#x0D; Conclusion: Homeopathic Mother Tinctures of plant origin showed antioxidant activity due to presence of phenolic content.

Irradiation effect of a submillimeter wave from 420 GHz gyrotron on amyloid peptides in vitro.

On using the far-infrared radiation system, whether the irradiation effect is thermal or non-thermal is controversial. We irradiated amyloid peptides that are causal factors for amyloidosis by using a submillimeter wave from 420 GHz gyrotron. Fluorescence reagent assay, optical and electron microscopies, and synchrotron-radiation infrared microscopy showed that the irradiation increased the fibrous conformation of peptides at room temperature for 30 min. The temperature increase on the sample was only below 5 K, and a simple heating up to 318 K hardly induced the fibril formation. Therefore, the amyloid aggregation was driven by the far-infrared radiation with little thermal effect.

Summary of the WHO hearing on the development of product-specific reference materials for coagulation factor VIII and factor IX products

In recent years, several modified recombinant factor (F) VIII and FIX therapeutics with extended half-life have been licensed internationally for the treatment of haemophilia. Safe and effective use of these products requires monitoring of factor activity in patient plasma. The potency of all FVIII and FIX products is currently assigned in International Units (IU) which anchors the relationship between potency labelling, dosing and clinical monitoring. However, varying degrees of discrepancies in factor activity assays are observed between and within the factor activity analytical methods (one-stage clotting and chromogenic), when measuring these modified products against plasma and plasma-derived (concentrate) International Standards (IS) or in-house reference standard traceable to the IS. Availability of product-specific reference reagents would mitigate assay discrepancies, facilitate independent testing of assay methods and reagents, and ensure long-term continuity of the IU related to each product. A hearing meeting was organised by the WHO to discuss the requirements for product-specific reference materials for these products and whether these reference materials should be produced by the WHO. Advantages and disadvantages of product-specific reference materials were identified and discussed.

Abstract 6495: Development of robust and reproducible controls to support clinical implementation of quantitative immunofluorescence assays for pharmacodynamic biomarkers

Abstract Robust pharmacodynamic (PD) assays are valuable to confirm putative drug mechanisms and downstream effects in support of early phase clinical trials. Evolving methods to achieve consistent performance across patients, clinical trials, and laboratories is critical for the success of clinical pharmacodynamic measurements. We have developed integral quality control tools to support clinical implementation of quantitative multiplex immunofluorescence assays (qmIFAs) that include xenograft tissue reference calibrators. These reference slides help to provide confirmation that the pharmacodynamic assay is reporting biomarker levels within designated target ranges with expected subcellular localization, helping to control for run-to-run issues with the assay procedures, reagents, or equipment; however, significant challenges are faced in producing these key reagents. Compared to conventional cell pellets, xenograft controls require more time, expense and specialized facilities; however, the tumor tissues are much more representative of the heterogeneity found in clinical biopsies. The challenges associated with xenograft controls include tissue and biomarker variability attributed to necrotic areas, fixation artifacts, and variable drug distribution. During our qualification process, xenografts undergo multiple rounds of selection and analysis to identify specimens that exhibit good tissue quality, adequate regions of interest (ROI), and appropriate target signal levels to support the intended biomarker measurements. Slide lots are produced, qualified, and stored in a manner that ensures adequate lot sizes, robust and reproducible slide to slide performance, and appropriate shelf life. Xenograft tissue reference standards representing a range of basal or drug-induced biomarker expression in appropriate models have been developed for several informative PD markers including Epithelial Mesenchymal Transition (EMT) markers such as E-Cadherin, Vimentin, and Beta-Catenin; DNA damage response markers γH2AX, pNBS1 and Rad51; apoptosis induction markers for colocalized γH2AX/cleaved Caspase 3; and the cell cycle markers pY15cdk and pHH3 among others and have been used as important tools to support ongoing clinical pharmacodynamic analyses of research biopsies at the NCI and other institutions (PMIDs 25964244, 29682208, 30792217, 31732654). In summary, generalized methods and workflows that will be made public have been developed by the NCI to optimize the production of qualified reference slide lots of sufficient reproducibility, stability, and quantity to support ongoing NCI-supported clinical trial pharmacodynamic assays for several key biomarkers. Funded by NCI Contract No HHSN261200800001E. Citation Format: Gabriel J. Benton, Katherine V. Ferry-Galow, Victor E. Lonsberry, Manisha Mohandoss, Howard Stotler, Debbie Trail, Kyle Georgius, Melinda Hollingshead, Deborah Wilsker, Robert Kinders, James H. Doroshow, Ralph E. Parchment. Development of robust and reproducible controls to support clinical implementation of quantitative immunofluorescence assays for pharmacodynamic biomarkers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6495.

Caracterización biofuncional de extractos de raíz de ginseng rojo, blanco y negro (Panax ginseng Meyer)

An organized biofunctional characterization of red, black, and white ginseng root extracts and fractions was performed. To assess antioxidant, antiglycation, enzyme inhibitory, antiamnestic, cytotoxic, thrombolytic, antibiofilm potentials in roots of three Panax ginseng preparations (red, black, white), powdered material was extracted and fractionated into different solvents. Antioxidant activity, phenolic and flavonoids contents were assessed by using free radical scavenging, Folin–Ciocalteu reagent and aluminum chloride colorimetric assays. Antiglycation and enzyme (alpha amylase, alpha glucosidase and acetylcholinesterse) inhibition activities were tested by prescribed methods along with cytotoxic (antihemolytic profile), thrombolytic activity, and biofilm growth inhibition assays. Red and black ginseng indicated maximum phenolic contents (37.26 ± 1.563 g GAE) and flavonoid contents (149.4 ± 2.032 g CE) respectively. The DPPH scavenging ability (62.84%) of red ginseng was higher than black and white ginseng. Significant antidiabetic activities were exhibited by black and white ginseng. The highest antihemolytic (71.2%) and thrombolytic (87%) actions were shown by black and red ginseng respectively. Red and white ginseng maximally inhibited the growth of Pasteurella multocida while in the case of Staphylococcus aureus, red ginseng showed optimum antibiofilm activity. The present study demonstrates that all three preparations of P. ginseng have effective yet variable functional characteristics that warrant further exploration.

Estimation of phenolic compounds content and antioxidant activity of leaves extracts of some selected non-traditional plants

The aim of the research is the determination of the total antioxidant activity and the content of phenolic compounds of the leaves of 12 species of non-traditional plants, namely, Amelanchier alnifolia (Nutt.) Nutt. ex M. Roem., Aronia mitschurinii A.K. Skvortsov &amp; Maitul., Castanea sativa Mill., Chaenomeles japonica (Thunb.) Lindl., Cornus mas L., Diospyros kaki L., Diospyros lotus L., Diospyros virginiana L., Lycium barbarum L., Lycium сhinense Mill., Pseudocydonia sinensis (Thouin) C.K. Schneid., Ziziphus jujuba Mill. Total phenolic content was evaluated using the Folin-Ciocalteu reagent assay. Antioxidant activity was measured using two different methods (DPPH – 2,2-diphenyl-1-picrylhydrazyl, MRAP – molybdenum reducing antioxidant power). Significant variability was observed in phenolic compounds content and total antioxidant activity. Total polyphenol content ranged from 38.02 (Z. jujuba) to 80.58 (C. sativa) mg GAE.g-1 DM, total flavonoid content from 22.47 (P. sinensis) to 54.61 (L. barbarum) mg QE.g-1 DM and phenolic acids content from 3.51 (A. mitschurinii) to 24.67 (Ch. japonica) mg CAE.g-1 DM. All tested samples exhibited DPPH• radical scavenging activities with values from 6.92 (A. mitschurinii) to 9.0 (C. mas) mg TEAC.g-1 DM. Antioxidant activity by molybdenum reducing antioxidant power method ranged from 109.43 (A. mitschurinii) to 322.95 (C. mas) mg TEAC.g-1 DM. Differences between the species of non-traditional plants were significant in all observed parameters. Obtained results of phytochemical composition demonstrated the possibility of leaves' use of non-traditional plants as sources of valuable bioactive compounds with health-promoting and disease-preventing properties.

Monoclonal secondary reagents do not outperform polyclonal secondary reagents for detection of anti-HLA IgG using single antigen flow beads assays.

Luminex single antigen flow beads (SAFB) assays are used to monitor anti-HLA antibodies, which are associated with allograft loss. The primary antibody binding level is evaluated by the mean fluorescence intensity (MFI) provided by the tracing secondary antibody. The aim of this study was to compare the MFI obtained with two secondary antibodies and to evaluate their sensitivity to detect newly developed de novo anti-HLA donor-specific antibodies (dnDSA). A total of 23 and 46 different sera from 20 HLA-sensitized kidney transplant recipients were tested with class I and II SAFB, respectively, as well as sera from 17 patients with dnDSA. The conventional secondary antibody (IgHPolyFab) was the phycoerythrin (PE)-conjugated goat anti-human IgG constant heavy chain (HC)-binding polyclonal-F(ab')2. The alternate secondary antibody was a PE-conjugated anti-human IgG Fc-specific monoclonal IgG (FcMonoIgG). MFI of negative control sera were drastically lower with the FcMonoIgG than with IgHPolyFab, requesting a unique positivity threshold to be defined for each secondary antibody. MFI obtained with positive control sera were also lower with FcMonoIgG. In kidney transplant recipient's sera, the number of antigenic specificities detected by each secondary antibody was comparable, for dnDSA as well as for nonDSA. In conclusion, decision thresholds must be refined when changing the secondary tracing reagent in SAFB assays. FcMonoIgG did not demonstrate any superiority over IgHPolyFab for monitoring HLA antibodies.

Loop-Mediated Isothermal Amplification (LAMP): A Rapid, Sensitive, Specific, and Cost-Effective Point-of-Care Test for Coronaviruses in the Context of COVID-19 Pandemic.

The rampant spread of COVID-19 and the worldwide prevalence of infected cases demand a rapid, simple, and cost-effective Point of Care Test (PoCT) for the accurate diagnosis of this pandemic. The most common molecular tests approved by regulatory bodies across the world for COVID-19 diagnosis are based on Polymerase Chain Reaction (PCR). While PCR-based tests are highly sensitive, specific, and remarkably reliable, they have many limitations ranging from the requirement of sophisticated laboratories, need of skilled personnel, use of complex protocol, long wait times for results, and an overall high cost per test. These limitations have inspired researchers to search for alternative diagnostic methods that are fast, economical, and executable in low-resource laboratory settings. The discovery of Loop-mediated isothermal Amplification (LAMP) has provided a reliable substitute platform for the accurate detection of low copy number nucleic acids in the diagnosis of several viral diseases, including epidemics like Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS). At present, a cocktail of LAMP assay reagents along with reverse transcriptase enzyme (Reverse Transcription LAMP, RT-LAMP) can be a robust solution for the rapid and cost-effective diagnosis for COVID-19, particularly in developing, and low-income countries. In summary, the development of RT-LAMP based diagnostic tools in a paper/strip format or the integration of this method into a microfluidic platform such as a Lab-on-a-chip may revolutionize the concept of PoCT for COVID-19 diagnosis. This review discusses the principle, technology and past research underpinning the success for using this method for diagnosing MERS and SARS, in addition to ongoing research, and the prominent prospect of RT-LAMP in the context of COVID-19 diagnosis.

Dehydrostephanine Isolated from Stephania venosa Possesses Anti-Inflammatory Activity in Lipopolysaccharide-Activated RAW264.7 Macrophages

Stephania venosa (Blume) Spreng. is a medicinal herb wildly used as a folklore medicine in Thailand. Many studies have reported that S. venosa tuber revealed a variety of pharmacological activities including anti-malarial, anti-microbial, anti-cancer, anti-oxidant, and anti-inflammatory activities. In this study, we investigated the effects of (–)-stephanine and dehydrostephanine isolated from S. venosa tuber on anti-inflammation in lipopolysaccharide (LPS)-activated RAW264.7 macrophages. RAW264.7 cells were treated with (–)-stephanine and dehydrostephanine in the presence of LPS and cell viability was determined by MTT assay. The levels of inflammatory mediators, nitric oxide (NO) and pro-inflammatory cytokines were determined by Griess reagent and enzyme-linked immunosorbent assay, respectively. Pre-treatment of dehydrostephanine significantly suppressed NO secretion in LPS-activated RAW264.7 cells with the half-maximal NO inhibitory concentration (IC50) value of 26.81±0.25 μM. However, (–)-stephanine had IC50 value on the inhibition of NO secretion of &gt;40 μM. In addition, dehydrostephanine at concentrations of 20 - 80 μM significantly reduced LPS-induced tumor necrosis factor-α, interleukin-1b, and interleukin-6 production in RAW264.7 cells. The present study showed that dehydrostephanine possesses the anti-inflammatory effect on LPS-activated RAW264.7 macrophages by suppression of inflammatory mediators. Dehydrostephanine may be a promising candidate compound for further investigation of a novel class of anti-inflammatory drug.

Glycyrrhizin, an HMGB1 inhibitor, Suppresses Interleukin-1β-Induced Inflammatory Responses in Chondrocytes from Patients with Osteoarthritis.

High mobility group box 1 (HMGB1) is increased in osteoarthritis (OA) tissue and chondrocytes stimulated with interleukin-1β (IL-1β). Suppression of HMGB1 expression is correlated with reduced inflammatory responses induced by IL-1β. This study aimed to investigate how inhibition of HMGB1 by glycyrrhizin might affect inflammatory responses and viability of OA patient-derived chondrocytes treated with IL-1β. The amounts of HMGB1 in the cartilage tissue and synovial fluid in patients with OA were assessed by Western blot and enzyme-linked immunosorbent assay (ELISA). Chondrocytes were extracted from OA patients and maintained in culture. The impact of glycyrrhizin on IL-1β-induced cell toxicity and inflammatory mediators and cytokines, including prostaglandin E2 (PGE2), nitric oxide (NO), proinflammatory cytokines, and metalloproteases (MMPs), were assessed by ELISA, Western blot, quantitative real-time polymerase chain reaction, and the Griess reagent assay. We confirmed that HMGB1 was significantly upregulated in specimens acquired from patients with OA. HMGB1 inhibition by glycyrrhizin improved cell viability of chondrocytes treated with IL-1β. Glycyrrhizin suppressed IL-1β-induced upregulation of HMGB1 and inflammatory mediators and cytokines, including PGE2, NO, proinflammatory cytokines, and MMPs. Our results indicate that glycyrrhizin may be a potential therapy for OA patients and these promising findings warrant further study for clinical application.

Chemical and Sensory Characterisation of Two Rubus rosifolius (Red Raspberry) Varieties.

Raspberries are economically important fruits, being highly valued for their taste and medicinal properties. Prior to our recent finding, the occurrence of different varieties of Rubus rosifolius growing in Jamaica had not been previously reported. Upon close observation of the plants, differences in various physical features pointed to the existence of two distinct plant morphotypes, which were described as Red “R” and Wine Red “WR.” With an aim to determine which variety may be more favourable for value-added food production, we undertook their physicochemical and sensory analysis. This characterisation led to the rationalisation of the differences in the perceived sensory properties of these biologically active fruits. Total phenolic content was determined using the Folin-Ciocalteu reagent assay, and the identification and quantification of anthocyanins were done via HPLC-MS and HPLC-UV, respectively. Proximate and physicochemical analyses were also carried out. The findings of the analyses were associated with those of a consumer sensory analysis. The WR fruits had a greater quantity of the deep red anthocyanin, cyanidin-3-glucoside (66.2 mg/100 g FW), and a significantly lower lightness value. They also received a significantly higher sweetness score, which is associated with their higher total sugar content (4.8 g/100 g) and maturity index (6.7). The R fruits had a higher quantity of the orange-coloured pelargonidin-3-rutinoside (17.2 mg/100 g FW) and significantly higher titratable acidity (1.3 g citric acid/100 mL), the latter being associated with its significantly more sour taste. The high total phenolic contents suggest a health-functional value of these R. rosifolius berry fruits. Our findings, which revealed that the WR variety was the preferred choice among consumers, may be used to guide future product-development endeavours of these commercially valuable fruits.

Surfactant-free, UV-curable core–shell microcapsules in a hydrophilic PDMS microfluidic device

We demonstrate a method to create surfactant-free core–shell microcapsules in a hydrophilic polydimethylsiloxane microfluidic device. An ultraviolet light curable polymer was used to encapsulate an oil core. These microcapsules ensure contamination-free compartmentation of the core material without any surfactant, while maintaining the monodispersed generation at a rate of 100 microcapsules per second. The device fabrication process is greatly simplified without the alignment of microchannels and hydrophobic/hydrophilic surface treatment. After drying, physically shaking the collection chamber can crack the capsule to release the liquid core material. Such solid microcapsules with a liquid core are ideal for the storage and delivery of oil-based materials in skincare products or reagents for biochemical assays.

Study on anti-inflammatory active components and mechanism of Corydalis Bungeanae Herba

Corydalis Bungeanae Herba is often used to treat a variety of inflammatory diseases in traditional Chinese medicine. In order to determine its chemical material basis, the components of Corydalis Bungeanae Herba were isolated by automated purification system. Flavonoids and alkaloids were prepared, and all such components were identified by mass spectrometry. The effects of the components on the production of inflammatory mediators and pharmacological mechanisms in the lipopolysaccharide(LPS)-induced RAW264.7 cell inflammation model were examined. Mouse macrophages(RAW264.7) were first treated with LPS. The relationship between cell viability and LPS concentration was observed. Then, the effects of flavonoids components and alkaloid components with different administration concentrations on cell viability were detected to determine the maximum administration concentration. Secondly, 2.5, 5, 10 and 20 μg·mL~(-1) flavonoids components and alkaloid components were added respectively to observe the effects and mechanism of different concentrations of flavonoids components and alkaloid components on LPS-induced inflammation of RAW264.7 macrophages. Griess reagent assay was used to detect NO content in cell supernatant. The inflammatory cytokines(TNF-α, IL-1β and IL-6) in cell supernatant were determined by ELISA method. Western blot method was used to detect the intracellular nuclear factor(NF-κB) IκBα phosphorylation(p-IκBα), p65 phosphorylation(p-p65) and protein expression of TLR4, TLR2. The results showed that the alkaloid components inhibited the production of NO, TNF-α, IL-1β and IL-6 in a dose-dependent mannerin the concentration range of 2.5-20 μg·mL~(-1). In inflammation upstream pathways, the inhibitory effect of the alkaloid components on the TLR2 expression level was weaker than that of TLR4. In inflammation downstream, alkaloid components significantly inhibited phosphorylation of IκBα and p65 in a dose-dependent manner. These data suggested that the alkaloid components were the material basis components of Corydalis Bungeanae Herba, and its anti-inflammatory mechanism might be related to inhibiting the transmission of inflammatory signals in TLRs/NF-κB signaling pathways dominated by TLR4, interfering with the activation of inflammatory genes and inhibiting their over expression, and down-regulating the secretion level of inflammatory factors.

ABTS radical-based single reagent assay for simultaneous determination of biologically important thiols and disulfides

Both the total amount of biothiols and thiol/disulfide ratio are wellness indicators of oxidative balance that play an important role in antioxidant defense system. Oxidized biothiols in disulfide form cannot be determined by conventional ABTS assay due to the biphasic kinetic pattern of the reaction between biothiols and ABTS radical cation (ABTS•+), necessitating the initial reduction of disulfides to thiols prior to measurement. In this study, direct simultaneous determination of biothiols (RSH) and their disulfides (RSSR) by using a single reagent of ABTS•+ was achieved without preliminary chemical reduction. Thus, conventional problems of preliminary operations arising from direct borohydride reduction of disulfides to thiols, followed by formaldehyde removal of borohydride excess and complications caused by formaldehyde-thiol reactions were effectively overcome with the use of a single reagent (ABTS•+). Box-Behnken statistical experimental design was employed to specify the optimal incubation temperature and time as 60 °C and 60 min, respectively. The detection limits (LOD) of the proposed assay for biothiols were compared to those of the widely used DTNB (Ellman) reference assay known to be nonresponsive to disulfides, and were found to be much lower (4–70 times). The proposed biothiol assay was successfully applied to some pharmaceutical samples and synthetic serum without preliminary treatment, and the results were highly compatible with the HPLC findings. The proposed assay was demonstrated to have superior features such as simplicity, rapidity and higher sensitivity over the widely applied Ellman thiols assay.

Impact and production of Non-HLA-specific antibodies in solid organ transplantation.

Organ transplantation is an effective way to treat end-stage organ disease. Extending the graft survival is one of the major goals in the modern era of organ transplantation. However, long-term graft survival has not significantly improved in recent years despite the improvement of patient management and advancement of immunosuppression regimen. Antibody-mediated rejection is a major obstacle for long-term graft survival. Donor human leucocyte antigen (HLA)-specific antibodies were initially identified as a major cause for antibody-mediated rejection. Recently, with the development of solid-phase-based assay reagents, the contribution of non-HLA antibodies in organ transplantation starts to be appreciated. Here, we review the role of most studied non-HLA antibodies, including angiotensin II type 1 receptor (AT1 R), K-α-tubulin and vimentin antibodies, in the solid organ transplant, and discuss the possible mechanism by which these antibodies are stimulated.

Evaluation of Oxidative Stress in Biological Samples Using the Thiobarbituric Acid Reactive Substances Assay.

Despite its limited analytical specificity and ruggedness, the thiobarbituric acid reactive substances (TBARS) assay has been widely used as a generic metric of lipid peroxidation in biological fluids. It is often considered a good indicator of the levels of oxidative stress within a biological sample, provided that the sample has been properly handled and stored. The assay involves the reaction of lipid peroxidation products, primarily malondialdehyde (MDA), with thiobarbituric acid (TBA), which leads to the formation of MDA-TBA2 adducts called TBARS. TBARS yields a red-pink color that can be measured spectrophotometrically at 532 nm. The TBARS assay is performed under acidic conditions (pH = 4) and at 95 °C. Pure MDA is unstable, but these conditions allow the release of MDA from MDA bis(dimethyl acetal), which is used as the analytical standard in this method. The TBARS assay is a straightforward method that can be completed in about 2 h. Preparation of assay reagents are described in detail here. Budget-conscious researchers can use these reagents for multiple experiments at a low cost rather than buying an expensive TBARS assay kit that only permits construction of a single standard curve (and thus can only be used for one experiment). The applicability of this TBARS assay is shown in human serum, low density lipoproteins, and cell lysates. The assay is consistent and reproducible, and limits of detection of 1.1 μM can be reached. Recommendations for the use and interpretation of the spectrophotometric TBARS assay are provided.