Assay Reagents Research Articles

Air contamination of therapeutic drug monitoring assay reagents results in falsely high plasma ammonia levels.

Accumulating evidence shows that contamination of blood samples by atmospheric ammonia affects blood ammonia test levels; however, reports on the effect of ammonia contamination of assay reagents are limited. Here, we aimed to clarify the effect of ammonia contamination of assay reagents, particularly the therapeutic drug monitoring (TDM) reagents, on the detection levels of blood ammonia using enzymatic assays. Ammonia gas was measured in the refrigerator compartment of the automatic analyser and the reaction tank water, probe wash water and drain outlets connected to the automatic analyser. At different time points following the closure of the cold storage, ammonia levels in quality control plasma samples were measured using three commercial assay reagents to evaluate the effect of air contamination. The distribution of evaporated ammonia in the reagent was measured using the CicaLiquid NH3 assay kit containing the assay reagent most affected by air contamination. It was confirmed that ammonia gas was generated in the cold storage of the automatic analyser. More than half of the reagents detected >0.25 ppm ammonia, and the highest concentration was detected in the TDM reagent. The ammonia levels obtained using all three reagents increased significantly after 3h of air contamination. The effect was resolved by measuring a 'dummy' sample or mixing the reagents by inversion. We demonstrated that air contamination by TDM reagents placed in cold storage could result in significantly falsely high ammonia measurements. Preventing this effect would improve the accuracy of ammonia measurements.

Antioxidant and anti-inflammatory activities of Oroxylum indicum Kurz (L.) fruit extract in lipopolysaccharide-stimulated BV2 microglial cells


 
 
 
 Purpose: To investigate the anti-inflammatory effects and antioxidant activity of Oroxylum indicum (L.) Kurz fruit extract in lipopolysaccharide (LPS)-stimulated BV2 microglia.
 Methods: BV2 cells were treated with LPS for 24 h in the presence or absence of O. indicum fruit extract. Then, nitric oxide (NO), reactive oxygen species (ROS) and interleukin 6 (IL-16) levels were measured using Griess reagent assay, CM-H2DCFDA and enzyme-linked immunosorbent (ELISA) assays, respectively. The in vitro antioxidant property of the extract was also investigated by 1,1- diphenyl-2-picrylhydrazyl (DPPH) and 2,2’-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) assays.
 Results: Levels of IL-6, NO, and ROS in LPS-treated BV2 cells were significantly higher than those in control (p < 0.01). However, exposure of LPS-treated BV2 cells to O. indicum extract led to a marked decrease in the levels of these parameters, when compared to the untreated cells (p < 0.01). Results from DPPH and ABTS assays showed that the O. indicum extract exhibited good antioxidant properties, with total flavonoid and total phenolic contents of 115.58 ± 1.09 and 131.04 ± 2.37 mg/g of dried extract, respectively.
 Conclusion: The results demonstrate that O. indicum fruit exerts anti-oxidant and anti-inflammatory effects in LPS-stimulated BV2 cells. Thus O. indicum fruit might be beneficial in the development of novel anti-oxidative and anti-neuroinflammatory herbal medicines. However, the mechanisms by which O. indium fruits reduces NO and IL-6 needs to be further investigated.
 
 
 

Effect of 1-Deoxynojirimycin on insulin resistance in prediabetic mice based on next-generation sequencing and intestinal microbiota study

Ethnopharmacological relevance1-Deoxynojirimycin (DNJ), the major alkaloid in Morus alba L., is the main effective constituent in “Mulberry twig Alkaloids Tablets” launched in China in 2020. Prediabetes, characterized by insulin resistance, is regarded as the key period for reversing Type 2 diabetes mellitus (T2DM) through lifestyle intervention and glucose-lowering drugs. Besides the excellent activity as an α-glucosidase inhibitor, DNJ also improves insulin sensitivity in T2DM murine models, yet the mechanism is still unclear. Besides, the pharmaceutical effect of DNJ on prediabetes is also undocumented. Aim of the studyThe aim of this study was to investigate the pharmaceutical effect of DNJ on high-fat and streptozotocin (STZ)-induced prediabetes mice, and to elucidate the mechanism of insulin resistance ameliorated by DNJ. Materials and methodsOral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed to detect blood glucose level and insulin sensitivity in mice. The levels of circulating lipopolysaccharide (LPS), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) in the plasma of mice were measured by limulus reagent and enzyme-linked immunosorbent assay (ELISA), respectively. Next-generation sequencing (NGS) and intestinal microbiota sequencing were used to screen the alterations in the transcriptome of liver tissues and to assess the differences in intestinal flora composition, respectively. Expression of cytokine signaling pathway inhibitor 3 (SOCS3), insulin receptor substrate (IRS1), p-IRS1 (Tyr896), occludin, and toll like receptor 4 (TLR4)/NF-κB signaling pathway were confirmed by western blotting. ResultsOur study revealed that DNJ decreased the blood glucose level and improve insulin sensitivity in prediabetic mice. DNJ significantly reduced the relative risk of T2DM in prediabetic mice by approximately 83.7%. Mechanistically, DNJ treatment suppressed the circulating levels of LPS, IL-6, and TNF-α in plasma and decreased the inflammatory infiltration in liver and colon tissues. DNJ-treatment increased the abundance of Akkermansia, Bifidobacterium, and Lactobacillus, and decreased the abundance of Enterococcaceae and Lachnospiraceae. Moreover, DNJ suppressed the expression of SOCS3 and the activity of TLR4/NF-κB signaling pathway, meanwhile improving the expression of occludin and the ratio of p-IRS1 (Tyr896)/IRS1. ConclusionsDNJ effectively ameliorates glucose and lipid metabolism in prediabetic mice, and decreased the relative risk of progression into T2DM from prediabetes. The suppressed immune responses play essential roles in the improvement of insulin resistance by DNJ treatment. In conclusion, DNJ from Morus alba L. is a promising alternative agent in T2DM prevention.

Seroconversions After Withdrawal From Mycophenolate Mofetil in Solid Organ Transplant Recipients Without a Third Dose of BNT162b2 mRNA Coronavirus Disease 2019 Vaccine: A Case Series.

To control the burden of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, vaccines have emerged for preventing severe coronavirus disease 2019.1 Poor antibody response to 2 doses of vaccine against SARS-CoV-2 has been reported in solid organ transplants recipients.2-7 Mycophenolate mofetil is one of the key immunomodulators used by solid organ transplant recipients and it also provokes weak antibody responses to anti–SARS-CoV-2 vaccines.2 A third dose of BNT162b2 vaccine to solid organ transplant recipients has been considered as one alternative to achieve adequate antibody responses.3 In transplant patients, withdrawal of immunosuppressive drugs is impractical because of the risk of rejection of the transplanted organ, but other options include switching to other drugs, calcineurin inhibitor or steroid, reducing the dose of antimetabolites, or withdrawal from mycophenolate mofetil. We report the cases of 3 solid organ transplant recipients treated with mycophenolate mofetil at the Matsunami General Hospital (Gifu, Japan) who had each received a second dose of BNT162b2 mRNA (Pfizer/BioNTech) vaccine between March 2021 and October 2021 (Table 1). The SARS-CoV-2 S-immunoglobulin (Ig)G assay reagent kit (Fujirebio Co., Tokyo, Japan) was used to measure S receptor-binding domain IgG antibody titers. Titers >1.0 AU/mL were considered to represent seropositivity. None of the recipients received the third dose of vaccination or had developed prior clinically relevant and polymerase chain reaction–confirmed SARS-CoV-2 infection. All recipients completed their second dose of BNT162b2 mRNA vaccine at 21-d intervals. The institutional review board approval was obtained. TABLE 1. - Characteristics of solid organ transplant recipients who developed seroconversion after withdrawal from mycophenolic acid No. Age a/sex Organ/donor Years since transplant Days from second vaccination Immunosuppressive therapy Antibody titer b(AU/mL) Pre/post Pre/post 1 68/M Liver/deceased 16 103 Calcineurin inhibitor + mycophenolate mofetil/calcineurin inhibitor 0.2/2.4 2 67/F Kidney/deceased 15 110 Calcineurin inhibitor + mycophenolate mofetil/calcineurin inhibitor 0.8/36.7 3 63/M Liver/deceased 15 101 Calcineurin inhibitor + mycophenolate mofetil/calcineurin inhibitor <0.1/6.0 a Age at measurement.b RBD IgG antibody titer.F, female; M, male; RBD IgG, receptor-binding domain immunoglobulin G. In these 3 recipients, antibody titers 1 mo after the second vaccine administration showed seronegative antibody responses with a receptor-binding domain IgG antibody titer of 0.2, 0.8, and <0.1 AU/mL, respectively. The baseline characteristics and immunosuppression regimens are summarized in Table 1. All 3 recipients had received immunosuppression regimens including mycophenolate mofetil (1000 mg/d). After detailed discussions with attending physicians and solid organ transplant recipients regarding the potential risks and benefits, mycophenolate mofetil was tapered and terminated. The conversion to calcineurin inhibitor monotherapy was successful, with adequate control of rejection maintained. One month after withdrawal from mycophenolate mofetil, antibody titer had elevated to 2.4, 36.7, and 6.0 AU/mL, respectively, and seroconversion was confirmed in all 3 recipients. In solid organ transplant recipients requiring continuous immunosuppression, no specific alternatives to the standard immunization strategy have been proposed for these blunted antibody responses. Mycophenolate mofetil suppresses T-lymphocytic responses to allogeneic cells and also suppresses primary, but not secondary, antibody responses.8 In contrast, calcineurin inhibitors attenuated humoral immunity, cellular responses were preserved. We therefore assume that withdrawal from mycophenolic acid favors antibody production in solid organ transplant recipients. There may be cases where patients who have discontinued mycophenolate mofetil need to resume the drug afterward. In such cases, the possibility of a decline in antibody titers after restarting mycophenolate mofetil should be considered. The efficacy and safety of a third dose of mRNA coronavirus disease 2019 vaccine in solid organ transplant recipients have been studied.2,3 However, a large proportion of recipients failed to obtain sufficient antibody titers. Our therapeutic approach was well tolerated and led to serological recovery of antibody responses after withdrawal from mycophenolate mofetil without a third dose of vaccine. A point of notice for withdrawal of mycophenolate mofetil, we need to monitor the health of transplanted organs, particularly with respect to rejection by the host immune system. This case series could represent a useful alternative strategy for improving the immunogenicity of vaccines in solid organ transplant recipients. ACKNOWLEDGMENTS The authors are greatly indebted to the staff of Matsunami General Hospital and Matsunami Health Promotion Clinic for their generous cooperation in establishing the case report. They also gratefully acknowledge the special efforts of Matsunami Research Park.

Critical Reagents for Ligand-Binding Assays: Process Development Methodologies to Enable High-Quality Reagents

Development of biotherapeutics require pharmacokinetic/pharmacodynamic (PK/PD) and immunogenicity assays that are frequently in a ligand-binding assay (LBA) format. Conjugated critical reagents for LBAs are generated conjugation of the biotherapeutic drug or anti-drug molecule with a label. Since conjugated critical reagent quality impacts LBA performance, control of the generation process is essential. Our perspective is that process development methodologies should be integrated into critical reagent production to understand the impact of conjugation reactions, purification techniquesand formulation conditions on the quality of the reagent. In this article, case studies highlight our approach to developing process conditions for different molecular classes of critical reagents including antibodies and a peptide. This development approach can be applied to the generation of future conjugated critical reagents.

A rapid influenza diagnostic test based on detection of viral neuraminidase activity

Current methods used for diagnosis of acute infection of pathogens rely on detection of nucleic acids, antigens, or certain classes of antibodies such as IgM. Here we report a virus enzyme assay as an alternative to these methods for detection of acute viral infection. In this method, we used a luciferin derivative as the substrate for detection of the enzyme activity of influenza viral neuraminidase as a means for diagnosis of influenza. The resulting commercial test, the qFLU Dx Test, uses a different supply chain that does not compete with those for the current tests. The assay reagents were formulated as a master mix that accommodated both the neuraminidase and luciferase reactions, thereby enabling rapid and prolonged production of stable light signal in the presence of influenza virus in the sample. The assay was evaluated using depository throat swab specimens. As expected, the assay exhibited similar detection rates for all influenza types and subtypes except for A(H7N9), which exhibited lower detection rate due to lower viral titer in the specimens. When throat swab specimens were diluted with the sample buffer of the test kit and tested with the qFLU Dx Test. The sensitivity and specificity were 82.41% (95% confidence interval: 79.66–85.84%) and 95.39% (95% confidence interval: 94.32–96.46%), respectively, for these diluted specimens in comparison to a real-time polymerase chain reaction assay. The uniqueness of the qFLU Dx Test as an enzymatic assay makes it highly complementary with currently available methods.

Volumeless reagent delivery: a liquid handling method for adding reagents to microscale droplets without increasing volume.

The addition of reagents for assays in digital microfluidic (DMF) systems is traditionally done by merging of droplets containing different analytes or reagents in solution. However, this process significantly increases droplet volume after each step, resulting in dilution of the analyte and reagents. Here, we report a new technique for performing reagent additions to aqueous droplets without significantly increasing the droplet's volume: volume-less reagent delivery (VRD). VRD is enabled by a physical phenomenon we call "exclusive liquid repellency" (ELR), which describes an aqueous/oil/solid 3-phase system where the aqueous phase can be fully repelled from a solid phase (contact angle ∼180°). When performing VRD, a reagent of interest in solution is deposited onto the ELR solid surface and allowed to dry. The ELR surface containing the dried reagent is then immersed under oil, followed by introduction of an aqueous droplet. By dragging the aqueous droplet over the spot of dried reagent using paramagnetic particles or via a physical sliding wall, the droplet can then recover and reconstitute the reagent with negligible increase in its total volume, returning the ELR surface to its initial liquid repellent state in the process. We demonstrate that VRD can be performed across a wide range of reagent types including sugars, proteins (antibodies), nucleic acids (DNA), antibiotics, and even complex enzyme/substrate/buffer "kit" mixtures. We believe VRD is a flexible and powerful technique which can further the development of self-contained, multi-step assays in DMF and other microfluidic systems.

Can &amp;lt;I&amp;gt;Plasmodium falciparum&amp;lt;/I&amp;gt; Induce Homocysteinemia in Malaria Patients?

Background: Plasmodium falciparum has developed elaborate strategies to survive in the hostile intracellular environment of the infected host cell, including resistance to oxidative stress. Cysteine is a metabolic product of homocysteine and a precursor of the antioxidant glutathione used by Plasmodium falciparum to escape harmful oxidation. Objectives: In the present study we aimed to assess whether Plasmodium falciparum can induce homocysteinemia in malaria patients of Burkina Faso. Methods: Eighty-five (85) individuals including 25 affected by severe malaria, 44 by simple malaria, and 12 negative controls for P. falciparum infection were included in the present study. An enzymatic assay of plasma homocysteinemia was performed using the Homocysteine Enzymatic Assay reagent (ref 05385415 190) on the Roche/ Hitachi Cobas c. Results: The results of the present study show that the mean plasma homocysteine concentrations were 15.1 ± 8.4 μmol/L among patients with severe malaria, 14.0 ± 6.0 μmol/L in patients with uncomplicated malaria, and 12.6 ± 4.1 μmol/L in negative controls for malaria parasite. Conclusions: Our findings suggest high homocysteinemia in malaria patients, especially in those with severe malaria. Monitoring homocysteinemia in the latter group will be useful to avoid complications when an elevated plasma level of homocysteine is a known risk factor for cardiovascular diseases.

Validation of Microchip Based RT-PCR ABC Test (InfA/B &amp;amp; COVID-19) in Clinical Samples

To contain the rapid and global spread of SARS-CoV-2, it is essential to develop an accurate and sensitive test system to address pandemic bottlenecks, simplified sample collection, and no sample prep. While meeting the demand of testing large populations, the miniaturized volume of assay reagents and offering rapid results is the need in such scenarios. Moreover, in view of the reports of co-infections and overlapping symptoms of influenza caused by Influenza A or Influenza B, and COVID-19 caused by SARS-CoV-2, a test system with three targets can be supportive for accurate clinical diagnosis. In this presentation, we evaluated the performance of a test comprising Microchip RT-PCR Influenza and COVID-19 Detection System for identifying these three viral pathogens in nasal swabs and saliva specimens. A rapid and simplified total nucleic acid extraction method was developed and validated for the reliable, high-throughput simultaneous detection of respiratory viruses causing Influenza (type A and type B viruses) and COVID-19 (SARS-CoV-2 virus) using the microchip-based AriaDNATM platform deriving the name ABC Test. The test system was evaluated using 81 nasal swab samples, 77 clinical saliva samples, 5 blind CAP reference samples, and RNA standards. The limit of detection (LoD) was assessed using SARS-CoV-2, Influenza A, and Influenza B RNA standards. The multiplex ABC Test microchip displayed LoD of 14 copies/μL for SARS-CoV-2 and approximately 26 copies/μL for influenza A, and 140 copies/μL for influenza B, respectively. The ABC Test offers rapid multiplex one-step RT-PCR in 32 minutes for 45 cycles as the miniaturized reaction of 1.2 μL offering a highly sensitive, robust, and accurate assay for the detection of influenza A/B, and SARS-CoV-2.

Critical Examination of the Suitability of the Folin-Ciocalteu Reagent Assay for Quantitative Analysis of Polyphenols—The Case of Olive-Mill Wastewater

The suitability of the Folin-Ciocalteu reagent (FCR) and of HPLC for analyzing polyphenols is evaluated. FCR assay is commonly used although its flows, such as overestimating polyphenol content due to interference by oxidizable species, were previously reported. The wide range of oxidizable species present in biological systems seriously compromises this assay’s reliability. Adding small amounts of protein to olive-mill wastewater (OMWW) significantly increased the apparent polyphenol content indicated by the FCR assay. The commonly used “reference” polyphenol as a standard for “total polyphenols” quantification is problematic since each polyphenol responds differently to the FCR. Conversely, HPLC may underestimate polyphenol content. No single HPLC protocol is likely to detect the whole myriad of polyphenols which may be present in a polyphenol-containing system. In analyzing 5 OMWW samples both by FCR assay and HPLC, the polyphenol content indicated by the FCR assay was up to six-fold higher than that determined by HPLC.

CLINICAL AND LABORATORY PREDICTORS OF PAIN SYNDROME PERSISTENCE IN ELDERLY AND SENILE PATIENTS DURING LATE POSTOPERATIVE PERIOD

Changes in neurohumoral regulation and emotional perception of nociceptive impulses in elderly and senile patients result in some clinical peculiarities of pain syndrome manifestation. Recent studies have shown that more than 10% of geriatric patients experience pain not only as an irritant but also as a psychosomatic condition. Persistence of pain in the postoperative period in the elderly and senile patients can turn into its catastrophic and chronic character in the future thus impacting the quality of life of patients and increasing the health care economic expenses to provide the proper chronic pain management. Therefore, the aim of this study is to determine the main predictors of pain persistence in the elderly and senile patients in the late postoperative period. The study included 201 male and female patients who underwent surgery regardless of surgical profile. All patients were divided into 3 groups according to age: group 1 (n = 82) included the elderly individuals, group 2 (n = 21) included senile individuals, and the control group (n = 98) included the adult patients.&#x0D; To evaluate the presence and severity of pain syndrome, pain assessment was performed by applying 100-score visual-analog scale in 1 and 7 days. A detailed study of the perception of postoperative pain was performed by the McGill questionnaire, which was used only on day 7, because on day 1 patients could be weakened after surgery, and the results distorted by the action of drugs. All examined patients were measured for fasting cortisol in the morning on the 1st and 7th day. The blood collection procedure was performed according to the general requirements for medical and biological tests. Blood was drawn from a vein by a nurse in the direct presence of a doctor. The serum cortisol content was determined using a set of reagents for quantitative enzyme-linked immunosorbent assay. The study has demonstrated that age, score on the visual-analog scale and the evaluation scale of the McGill questionnaire for day 7, as well as blood cortisol concentration in day 1 after surgery are of definite prognostic value for the risk of persistence of postoperative pain syndrome. The chance of persistence of postoperative pain increases in 1.1 times for each year in older patients, 1.3 times for each score according to the visual-analog scale for 7 days, and in 11.4 times for each score on the evaluation scale of the McGill questionnaire, and in 2.4 times for each nmol/l of cortisol concentration in 1 day after surgery. Thus, the determined risk factors for persistence of postoperative pain include the patient's age, assessment of pain by applying a visual-analog scale, and the evaluation scale by the McGill questionnaire for 7 days and the concentration of blood cortisol in 1 day after surgery.

Estimation of Total Phenolic and Total Flavonoid Contents, In-Vitro Antioxidant Activity and Median Inhibitory Concentration of Methanol Root Extract of Ximenia americana, L. Olacaceae

Background: This research was carried out to estimate the total phenolic and flavonoid contents, assayed the in-vitro antioxidant potential, and estimate the median inhibitory concentration (IC50) of the methanol root extract of Ximenia americana, Linn.&#x0D; Study Design: It was an experimental study design.&#x0D; Methods: Total phenolic and flavonoid contents of the extract were determined by spectrophotometry using Folin-Ciocalteu reagent and Aluminum chloride colorimetric assay, readings were taken at 750 nm, and 415 nm wavelength respectively. The results were expressed in terms of Gallic acid and Rutin standards equivalent. The in-vitro antioxidant activity and the IC50 of the plant material were investigated by its ability to scavenge free radicals, using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging method. Similarly, the total percentage yield of the extract was estimated from the crude.&#x0D; Results: The crude root powder of Ximenia americana showed a total extract yield of 20.06%w/w. Total phenolic and flavonoid contents were determined to be 3.501 ± 0.774 mg GAE/g, and 10.644 ± 0.20 mg RE/g extract. The extract displayed concentration-dependent DPPH free radical scavenging activity, with the highest concentration (200 µg/mL) inhibiting 60.93% of DPPH free radical, and the IC50 of the extract was determined to be 69.01 µg/mL.&#x0D; Conclusions: Ximenia americana L methanol root extract has a yield of 20.06%w/w, and a total phenolic content of 3.501± 0.774 mg GAE/g, total flavonoids content of 10.644 ± 0.20 mg RE/g, and IC50 of 69.01 µg/mL, also, the extract possesses a potent antioxidant activity by its ability to inhibit DPPH activity.

Sulphite‐corrected, non‐phenolic and phenolic antioxidant capacities of fruit wines profiled by differential Folin‐Ciocalteu assay

SummaryThe Folin‐Ciocalteu reagent (FCR) assay is a reliable and robust, in vitro measure of antioxidant capacity (AOC) of foods but in winemaking its use has generally been confined to measuring total phenolics. However, the contributions of non‐phenolics and sulphites, and total AOC are also of interest for winemaking process control and human health functionality. Accordingly, we adapted the FCR assay and evaluated it with a range of wines made from grapes and other fruits, viz. peach, Chinese quince, blueberry, bayberry and red dragon fruit. The novel protocol avoided recognised interferences and revealed a wide variation among the wines in the contributions of phenolics, non‐phenolics and sulphites to their total AOC. Of the fruit‐derived antioxidants, phenolics accounted for 67.7–98.9% and non‐phenolics, 1.0–26.0% of the AOC, respectively. Direct reaction of sulphites with FCR accounted for 0.1–10.6% of the AOC.

Performance evaluation of the STA ECA-II reagent for specific assay of anti-IIa activity of argatroban

Argatroban is indicated in the management of patients with suspicion of Heparin-Induced Thrombocytopenia. Biological monitoring of argatroban can be carried out using the activated partial thromboplastin time (APTT) or the measurement of the anti-IIa activity. However, APTT is susceptible to many interferences making its use unsuitable. The objective of this work is to define and verify the performance of a dosing protocol for the anti-IIa activity of the argatroban from the STA ECA-II reagent (Diagnostica Stago) and compare them to the Hemoclot Thrombin Inhibitor proposed by Hyphen Biomed based on diluted thrombin time. Calibration curve shows a good correlation between anti-IIa activity and the coloration level of the reaction medium over the concentration range studied (0 to 2μg/mL). The analytical performances obtained at the native dilution of 1/10th are excellent, both in terms of repeatability and reproducibility with a coefficient of variation below 10%. Automatic relaunch at 1/20th for sample concentrations greater than 2μg/mL does not impact this performance level. We obtain a limit of quantification below 0.2μg/mL. The comparison of the methods gave consistent and well-correlated results on a wide range of concentrations (0.3 to 4μg/mL). The use of the STA ECA-II reagent is a valuable alternative to APTT for biological monitoring of anticoagulant action of argatroban.

Simple method to determine the concentration and incorporation ratio of ruthenium-labeled antibodies.

Aim: Ruthenium-labeled antibodies are commonly used detection reagents in bioanalysis assays and must be characterized to ensure quality. The aim of this work was to develop a method to determine the concentration and incorporation ratio (the degree of labeling [DOL]) of ruthenium-labeledantibodiesby UV/VIS spectroscopy. Materials & methods: Free SULFO-TAG compound was scanned using UV/VIS and showed an absorbance peak at 292nm. In contrast, antibodies demonstrate UV absorbance at 280nm. After experimentally determining the extinction coefficients at 280and 292nm of free ruthenium and antibody, we generated a formula based on the Beer-Lambert law that calculates both concentration and DOLof these ruthenium-labeled antibodies. Conclusion: The concentration and DOLvalues determined by our method were comparable to those determined from bicinchoninic acid and LC/MS for the same reagents. This method creates a faster and more accessible reagent characterization process that uses far less reagent than the more traditional alternatives.

Particle specific physical and chemical effects on antibacterial activities: A comparative study involving gold nanostars, nanorods and nanospheres

The present report compares the antibacterial properties of unique (sphere+rod)-shaped gold nanostar with its structural components, i.e., gold nanorods and nanosphere. Gold nanostars, nanorods, and nanospheres have been synthesized using literature reported protocols. Clinical isolates of gram-negative E. coli and gram-positive S. aureus bacteria have been used as model pathogens for the antibacterial studies. The antibacterial activities of the nanoparticles have been assessed using various techniques. To compare the antimicrobial activity of the nanoparticles, we have investigated the effects of varying gold concentrations of different nanoparticles on bacterial growth kinetics. The impacts of the leached/residual gold ions and the surface capping agent and charge of the particles on the antibacterial activity have also been assessed. Studies of the nanoparticle’s dose and exposure time show the highest antibacterial activity for nanostars and lowest for nanospheres for a given quantity of gold. Bradford’s Reagent Assay and confocal fluorescent microscopy studies confirm the trend of the particle-specific antibacterial activities. SEM/TEM and DNA extraction studies show that the bactericidal activities involve a combination of effects like the destruction of cell membrane, fragmentation of bacterial DNA, internalization of particles, etc. To assess the antibacterial impacts of the amount of leached/residual gold ions, their concentrations have been determined by inductively coupled plasma - optical emission (ICP-OES) and mass (ICP-MS) spectroscopic studies as well as the effects of the added gold ions have been studied. No discernible effects have been observed due to the variation in the leached/residual gold ions amount. The DCHF-DA fluorescence assay has been employed to determine the levels of reactive oxygen species (ROS) produced by different nanoparticle systems, which corresponds to the trend in antibacterial activities observed. The nature and magnitude of the surface charge have been determined by zeta potential measurements. Analyses of the impacts of various parameters/factors like gold ions, capping agents, surface charges, ROS levels, etc., show that the antibacterial activities of the gold nanoparticles of different morphology arise from both morphology-specific physical and chemical effects of the gold nanoparticles.

Abemaciclib in Combination with Copanlisib to Overcome Therapeutic Resistance in Mantle Cell Lymphoma

IntroductionAlthough novel therapeutic strategies including BTK and Bcl-2 inhibitors have dramatically improved the prognosis of MCL patients, resistance to these treatments is inevitable. We recently reported that the tumor suppressor gene CDKN2A were commonly deleted in ibrutinib-resistant tumors, leading to upregulation of CDK4/6 signaling. Among the other hallmarks are the mTOR/PI3K, Myc and OXPHOS pathways. Therefore, we attempt to exploit combinatory targeting of CDK4/6 and PI3K pathways to overcome therapy resistance using in vitro and PDX models.MethodsIbrutinib or venetoclax sensitive and resistant MCL cell lines were used in this preclinical study. 1x10 4 cells per well are seeded in 96-well plates and treated with abemaciclib monotherapy or in combination with copanlisib (PI3K inhibitor) in triplicate for 72h and then mixed with CellTiter-Glo Luminescent Cell viability Assay Reagent. For cell cycle assay, cells were seeded in 6 well plates and treated with vehicle or abemaciclib for 24h. Cells were fixed in 70% pre-cold ethanol and stained with propidium iodide. The cell cycle stages were quantified through the Novocyte Flow Cytometer. The molecular events at the protein level after treatment were determined by immunoblotting. For in vivo experiment, the combination of abemaciclib (25mg/kg, oral, daily) and copanlisib (5mg/kg, IP, three times a week) was assessed in Mino-venetoclax-resistant xenograft model. IC50 values were calculated using GraphPad Prism 8 for each cell line. Student's t-test was performed to compare the difference between vehicle and treated groups. Two-way analysis of variance (ANOVA) was conducted to analyze the tumor growth in vivo experiments. P values less than 0.05 were considered statistically significant.ResultsOur previous studies have identified a subset of MCL cells that were resistant to venetoclax (JeKo-1) or ibrutinib treatment (Maver-1 and Z-138). To overcome the resistance, we first treated MCL cell lines with abemaciclib and the result showed that abemaciclib as a single agent showed potent anti-MCL activity in a subset of MCL cell lines (IC 50 = 70-952 nM) including venetoclax sensitive- (Mino, Rec-1, Maver-1, and Z138) and primary resistant- MCL cells (JeKo-1). However, the cell lines Mino-ven-R and Rec-ven-R with acquired venetoclax resistance are highly resistant to abemaciclib treatment (IC 50 = 6.0 and 4.4 µM). PI3K/ATK pathway has been reported to be highly upregulated in Mino-ven-R and Rec-ven-R cells compared to their parental cells. To further increase the efficacy of the targeted therapy, we treated the resistant MCL cells with a combination of abemaciclib and copanlisib and the result showed synergistically enhanced cytotoxicity in ibrutinib or venetoclax-resistant MCL cell lines. Consistent with the role of CDK4/6 in cell cycle progression, inhibition of CDK4/6 with abemaciclib resulted in the cell cycle arrest at G1 phase in MCL cell lines.To validate whether abemaciclib in combination with copanlisib can overcome venetoclax resistance in vivo, we assessed the antitumor effect of abemaciclib in combination with copanlisib using a venetoclax-resistant xenograft models derived from Mino-ven-R cell line in immunodeficient NSG mice. As a result, abemaciclib (25 mg/kg, oral, daily), but not venetoclax (5 mg/kg, oral, daily) or copanlisib (5 mg/kg, IP, three times a week), significantly reduced tumor volume compared to the vehicle control (n = 5, p < 0.0001). Remarkably, the combination of abemaciclib and copanlisib also exhibited significantly in vivo synergistic efficacy compared with single-agent treatment (p<0.0001). Of note, the combination did not cause major decreases in body weight. Taken together, these results suggest that the combinatory therapy is effective in overcoming venetoclax resistance in MCL.ConclusionsCombinatory treatment with abemaciclib and copanlisib may achieve clinical actionable efficacy through overcoming the venetoclax-resistance in MCL that may become an effective treatment regimen for refractory/relapsed MCL patients in the future. DisclosuresWang: Dava Oncology: Honoraria; Imedex: Honoraria; CStone: Consultancy; Hebei Cancer Prevention Federation: Honoraria; OMI: Honoraria; Chinese Medical Association: Honoraria; Newbridge Pharmaceuticals: Honoraria; Moffit Cancer Center: Honoraria; Bayer Healthcare: Consultancy; Kite Pharma: Consultancy, Honoraria, Research Funding; Clinical Care Options: Honoraria; Physicians Education Resources (PER): Honoraria; AstraZeneca: Consultancy, Honoraria, Research Funding; Mumbai Hematology Group: Honoraria; InnoCare: Consultancy, Research Funding; Epizyme: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Genentech: Consultancy; DTRM Biopharma (Cayman) Limited: Consultancy; Acerta Pharma: Consultancy, Honoraria, Research Funding; Scripps: Honoraria; BGICS: Honoraria; CAHON: Honoraria; BeiGene: Consultancy, Honoraria, Research Funding; Anticancer Association: Honoraria; Miltenyi Biomedicine GmbH: Consultancy, Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; Juno: Consultancy, Research Funding; Loxo Oncology: Consultancy, Research Funding; Oncternal: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; VelosBio: Consultancy, Research Funding; BioInvent: Research Funding; Celgene: Research Funding; Lilly: Research Funding; Molecular Templates: Research Funding.

Effect of Tai Chi Chuan practice on immune system and pro-metastasis markers in early post-treatment breast cancer survivors

Purpose: To investigate the effect of Tai Chi Chuan (TCC) to improve immune system and decrease pro-metastasis markers in early post-treatment breast cancer survivors. Methods: 130 post-treatment breast cancer survivors were recruited and randomized 1:1 into TCC group and wait-list (control) group. The TCC group practiced for a 60-minutes session once per week, for a total of 52 weeks. 115 forms of Yang-style TCC were taught by a Tai Chi master. Blood samples were taken from each subject and complete blood count was performed. The expressions of NKG2D protein, P-selectin, and vascular endothelial growth factor (VEGF) in plasma were measured. Lymphocyte activity was measured by cell proliferation reagent and ATP assay. Images of lymphocyte colony formation were taken with an inverted microscope. Results: At 52 weeks, TCC group demonstrated a significantly higher WBC (p=0.001) , a significantly higher NKG2D value (p=0.001) and a significantly lower VEGF value (p=0.005) when compared to the wait-list group. However, there was a small, non-significant change for P-selectin values between the breast cancer survivor groups. After 72h incubation, TCC group had a significant increase in lymphocyte proliferation (p=0.001) and greater area of lymphocyte clusters or colonies (p=0.001). Conclusion: The practice of TCC could stimulate tumor immunosurveillance via NKG2D and activate the immune response. VEGF, a marker playing an important role in breast cancer and its metastases, was also reduced in those who practiced TCC. As an alternative for conventional exercise, post-treatment breast cancer survivors may select TCC in their rehabilitation program.

Supersensitive photon upconversion based immunoassay for detection of cardiac troponin I in human plasma

Background and aimsUpconverting nanoparticles (UCNPs) are attractive reporters for immunoassays due to their excellent detectability. Assays sensitive enough to measure baseline level of cardiac troponin I cTnI in healthy population could be used to identify patients at risk for cardiovascular disease. Aiming for a cTnI assay of such sensitivity, the surface chemistry of the nanoparticles as well as the assay reagents and the protocol were optimized for monodispersity of the UCNP antibody conjugates (Mab UCNPs) and to minimize their non-specific interactions with the solid support. Materials and methodsUCNPs were coated with poly(acrylic acid) via two-step ligand exchange and conjugated with monoclonal antibodies. The conjugates were applied in a microplate-based sandwich immunoassay using a combination of two capture antibodies to detect cTnI. Assay was evaluated according to guidelines of Clinical & Laboratory Standards Institute. ResultsThe limit of detection and limit of blank of the assay were 0.13 ng/L and 0.01 ng/L cTnI, respectively. The recoveries were >90% in spiked plasma in the linear range. The within- and between-run imprecisions were <10%. ConclusionThe results demonstrate that UCNPs enable quantification of cTnI concentrations expected in plasma of healthy individuals and could be used to identify patients at risk for cardiovascular disease.

A Hybrid Paper and 3D Printed Microfluidic Platform for Electrochemical Detection of Ag-Labeled Magnetic Particles

Here we present a hybrid microfluidic device (hyFlow) composed of a disposable paper electrode and a three-dimensional (3D) printed plastic chip for the electrochemical detection of a magnetic bead-silver nanoparticle (MB-AgNP) bioconjugate. Detection of micron-size magnetic conjugates in a paper-only fluidic device is a challenging task. Typically, these devices operate by capillary flow or use paper hollow channels and suffer from flow issues due to entrapment of MB conjugates in cellulose/ nitrocellulose fibers. The hyFlow system was designed to minimize such issues and transport the MB conjugate to the electrochemical detection area without using a syringe pump. Moreover, this device is capable of detecting the MB-AgNP conjugate in both preformed (off-chip) and in-situ formed (on-chip) formats. This portable device can store dried assay reagents in a buffered matrix containing 10% sucrose and 10% D(+)-trehalose and successfully resolvate and mix the reagents in less than 2 min to detect electrochemically. The hyFlow system could detect AgNP labels of preformed MB-AgNP conjugates down to concentrations as low as 12 pM, representing an estimated AgNP coverage of 22 AgNPs per MB.