Assay Reagents Research Articles

Digital PCR-based cell-free DNA K-Ras mutations analysis and clinical implication in pancreatic cancer.

e16215 Background: Pancreatic cancer is one of the most fatal cancers worldwide. In addition to patients presenting later in the disease, limitations in current testing modalities pose a challenge to early diagnosis and treatment. Somatic mutations of the K-Ras gene have been suggested as a key driver of pancreatic carcinogenesis and thus proposed as a biomarker for diagnosis and therapy. Majority of these studies utilize tissue-based methods for analyzing K-Ras mutations. In recent years, liquid biopsy assay, in particular, analysis of cell-free DNA (cfDNA), has emerged as a promising noninvasive diagnostic approach in oncology, with the respect to the identification of minimal residual diseases, monitoring treatment response, detection recurrence and metastasis, and identification of chemo-resistance mechanisms. The application of digital PCR based plasma cfDNA K-Ras mutation assay in pancreatic cancer has not been reported. Methods: We included patients who underwent evaluation of a pancreatic lesion detected by conventional radiography (CT/MRI) and confirmed by endoscopic ultrasound (EUS) and biopsy. The biopsies from pancreatic tissues were formalin-fixed and paraffin-embedded (FFPE) for pathological diagnosis, and the total DNA was extracted from the FFPE slides. In addition, matched blood from the same patient was collected at the time of biopsy, and plasma cfDNA was extracted. Both DNA quality and concentration were evaluated. 4.0 ng of DNA was used for droplet digital PCR (ddPCR) analysis. K-Ras G12D, G12V, G12R, G12C, G12A, G12S, and G13D mutations were analyzed by multiplexed assay reagents. The sensitivity cut-off of the multiplexed K-Ras G12/G13 assay was 1.0%. Results: cfDNA could be detected in the initiation of pathological diagnosis. Among the twelve patients with pancreatic lesions, eight (67%) were pancreatic ductal adenocarcinoma (PDAC) (head (4), body (2), tail (2) of the pancreas), one was acute pancreatitis, one was a tail cyst and two were unspecified non-malignant lesions. All the eight PDAC (100%) FFPE tissues exhibited K-Ras G12/13 mutations (Scores: 4.2-41.5%), while none of the non-malignant lesions (0%) demonstrated K-Ras mutations. Interestingly, K-Ras mutations were detected from seven of the eight PDAC patient’s plasma cfDNA (87.5%) (Scores: 1.2-20.7%) while the plasma cfDNA from patients with non-malignant lesions did not show any positive K-Ras mutations. Conclusions: Digital PCR-based plasma cfDNA assay for K-Ras mutation is a promising tool for diagnosis of PDAC and is comparable to tissue-based assays. Larger prospective studies can substantiate this and explore their roles in the prediction and early detection of recurrence of PDAC.

A one-step in vitro continuous flow assessment of protein release from core-shell polymer microcapsules designed for therapeutic protein delivery

Developing accurate methods for the assessment of therapeutic protein release from polymer drug delivery systems (microcapsules, microspheres, nanoparticles, 3D-printed systems) is of paramount importance for new formulation development. The most straightforward method for protein release assessment is spectrophotometric analysis of the release medium surrounding the formulation. However, direct spectrophotometric analysis is inapplicable to formulations releasing interfering compounds (co-encapsulated drugs, additives) absorbing light in the same spectrum as proteins. Conventional protein release assays also require frequent release medium sampling and replacement, which reduces their accuracy. We propose a one-step method to assess protein release from core/shell microcapsules eliminating the need for sampling and allowing selective real-time protein quantitation in the release medium. To prevent spectral interferences, released protein is differentiated from interfering compounds by employing a colorimetric protein assay reagent, forming a colour complex selectively with the protein, as the release medium. To eliminate sampling, we employed a continuous flow closed loop set-up, where the release medium is constantly circulating between microcapsule-containing tank and spectrophotometer. A series of colorimetric protein assay reagents (bromocresol green, tetrabromophenol blue, eosin B, eosin Y, biuret) were evaluated in terms of their applicability as the release medium in described system. Only biuret reagent was found compatible with proposed method due to formation of color complex stable over extended period of time and low adsorption to microcapsules. Presented method allowed effective evaluation of albumin release from alginate-polyethersulfone microcapsules with accuracy equal to conventional ‘sample and separate’ technique. Albumin release followed first-order kinetics with plateau reached after 19 h.

A Tail Fiber Engineering Platform for Improved Bacterial Transduction-Based Diagnostic Reagents.

Bacterial transduction particles were critical to early advances in molecular biology and are currently experiencing a resurgence in interest within the diagnostic and therapeutic fields. The difficulty of developing a robust and specific transduction reagent capable of delivering a genetic payload to the diversity of strains constituting a given bacterial species or genus is a major impediment to their expanded utility as commercial products. While recent advances in engineering the reactivity of these reagents have made them more attractive for product development, considerable improvements are still needed. Here, we demonstrate a synthetic biology platform derived from bacteriophage P1 as a chassis to target transduction reagents against four clinically prevalent species within the Enterobacterales order. Bacteriophage P1 requires only a single receptor binding protein to enable attachment and injection into a target bacterium. By engineering and screening particles displaying a diverse array of chimeric receptor binding proteins, we generated a potential transduction reagent for a future rapid phenotypic carbapenem-resistant Enterobacterales diagnostic assay.

Zerumbone Suppresses the LPS-Induced Inflammatory Response and Represses Activation of the NLRP3 Inflammasome in Macrophages

Zerumbone is a natural product isolated from the pinecone or shampoo ginger, Zingiber zerumbet (L.) Smith, which has a wide range of pharmacological activities, including anti-inflammatory effects. However, the effects of zerumbone on activation of the NLRP3 inflammasome in macrophages have not been examined. This study aimed to examine the effects of zerumbone on LPS-induced inflammatory responses and NLRP3 inflammasome activation using murine J774A.1 cells, murine peritoneal macrophages, and murine bone marrow-derived macrophages. Cells were treated with zerumbone following LPS or LPS/ATP treatment. Production of nitric oxide (NO) was measured by Griess reagent assay. The levels of IL-6, TNF-α, and IL-1β secretion were analyzed by ELISA. Western blotting analysis was performed to determine the expression of inducible NO synthase (iNOS), COX-2, MAPKs, and NLRP3 inflammasome-associated proteins. The activity of NF-κB was determined by a promoter reporter assay. The assembly of NLRP3 was examined by immunofluorescence staining and observed by confocal laser microscopy. Our experimental results indicated that zerumbone inhibited the production of NO, PGE2 and IL-6, suppressed the expression of iNOS and COX-2, repressed the phosphorylation of ERK, and decreased the activity of NF-κB in LPS-activated J774A.1 cells. In addition, zerumbone suppressed the production of IL-1β and inhibited the activity of NLRP3 inflammasome in LPS/ATP- and LPS/nigericin-activated J774A.1 cells. On the other hand, we also found that zerumbone repressed the production of NO and proinflammatory cytokines in LPS-activated murine peritoneal macrophages and bone marrow-derived macrophages. In conclusion, our experimental results demonstrate that zerumbone effectively attenuates the LPS-induced inflammatory response in macrophages both in vitro and ex vivo by suppressing the activation of the ERK-MAPK and NF-κB signaling pathways as well as blocking the activation of the NLRP3 inflammasome. These results imply that zerumbone may be beneficial for treating sepsis and inflammasome-related diseases.

Factor IX assay discrepancies in the setting of liver gene therapy using a hyperfunctional variant factor IX‐Padua

BackgroundLimited information exists regarding the factor IX (FIX) coagulant activity (FIX:C) measured by different assays following FIX‐Padua gene therapy. ObjectiveAssess for the first time FIX:C in five commonly used coagulation assays in plasma samples from hemophilia B subjects receiving FIX‐Padua gene transfer. MethodsFIX:C was compared between central (n = 1) and local laboratories (n = 5) in the study, and across four commonly used FIX:C one‐stage assays and one FIX:C chromogenic assay. For comparison, samples of pooled congenital FIX‐deficient plasma spiked with purified recombinant human FIX (rHFIX)‐Padua protein or rHFIX (nonacog alfa) to obtain FIX:C concentrations from ~20% to ~40% were tested. ResultsFIX:C results at local laboratories strongly correlated with central laboratory results. However, absolute values at the central laboratory were consistently lower than those at local laboratories. Across five different FIX:C assays, a consistent pattern of FIX:C was observed for subjects receiving fidanacogene elaparvovec‐expressed gene transfer. Use of Actin FSL activated partial thromboplastin time (APTT) reagent in the central laboratory resulted in lower FIX:C values compared with other APTT reagents tested. The chromogenic assay determined lower FIX:C than any of the one‐stage assays. The rHFIX‐Padua protein–spiked samples showed similar results. In contrast, FIX:C results for rHFIX‐nonacog alfa measured within 25% of expected for all one‐stage assays and below 25% in the chromogenic assay. ConclusionsAssay‐based differences in FIX:C were observed for fidanacogene elaparvovec transgene product and rHFIX‐Padua protein, suggesting the variable FIX:C determined with different assay reagents is inherent to the FIX‐Padua protein and is not specific to gene therapy–derived FIX‐Padua.

Lumit&[trade] Immunoassays for Simple and Rapid Determination of Cytokine Release from Cell Culture Models

Abstract Rapid (< 70 min), add-and-read assays for cytokine determination were developed with Lumit™ immunoassay technology. Separate antibodies labeled with complementary SmBiT or LgBiT subunits bind to their shared cytokine target, resulting in proximity-driven reconstitution of NanoBiT luciferase and luminescence signal. Lumit immunoassays for human IL-1β, IL-2, IL-4, IL-6, IL-10, IFN-γ, and TNF-α exhibit LLODs < 10 pg/ml and linearity > 3 logs of analyte in 96-well format. Maximal S/B ratios are greater than 1000, and an assay signal T1/2 of 2 h accommodates batch plate processing. 384-well, assay performance (Z′ > 0.7) on treated human peripheral blood mononuclear cells (hPBMC) indicates the assay format is amenable to screening applications. Assay reagents were added directly to cell wells for multiple culture models. For hPBMC, LPS, R848, or a combination of PMA and ionomycin (CSC) produced dose-and time-dependent release of cytokines with a wide range of responses, all measurable without sample dilution. In a mix of purified CD8+ T cells and target Raji B cells treated with the bispecific T-cell engager Blincyto®, IL-2, IFN-γ, and TNF-α release was observed with an EC50 of ~0.2 ng/ml and max S/B ratios of 82-, 168-, and 31-fold, respectively. For Th2 cells, significant release of IL-4 was observed in response to CSC treatment. An alternative to the no-transfer assay method, split-sample analysis, can be performed to easily determine the levels of multiple cytokines released in the same cell well. The implementation of this novel detection chemistry will enable simple and rapid assay of cytokine release from cells for both low-and high-throughput applications, including quantitative assessments of T cell activation and differentiation.

Increasing robustness, reliability and storage stability of critical reagents by freeze-drying.

Aim: Stabilization of critical reagents by freeze-drying would facilitate storage and transportation at ambient temperatures, and simultaneously enable constant reagent performance for long-term bioanalytical support throughout drug development. Freeze-drying as a generic process for stable performance and storage of critical reagents was investigated by establishing an universal formulation buffer and lyophilization process. Results: Using a storage-labile model protein, formulation buffers were evaluated to preserve reagent integrity during the freeze-drying process, and to retain functional performance after temperature stress. Application to critical reagents used in pharmacokinetics and anti-drug antibodies assays demonstrated stable functional performance of the reagents after 11month at +40°C. Conclusion: Stabilization and storage of critical assay reagents by freeze-drying is an attractive alternative to traditional deep freezing.

Adventures in critical reagent lot changes in ligand-binding assays: redevelopment, bridging and additional processing requirements.

Aim: Critical reagents have significant impact on ligand-binding assay performance. The critical reagents selected during method development should be well-evaluated, as the quality of these reagents will dictate performance of the assay over time. Critical reagents in ligand-binding assays are almost always produced using a biological system, so batch yield, purity and performance tendto vary greatly. Due to the essential nature of critical reagents in the assay, changes in critical reagents can have dramatic impact on the assay and results, so close monitoring of assay performance is required. Methodology & results: We present here three examples of critical reagent lot changes that required creative solutions to maintain assay performance. The first case study is an example of the impact of different lots of analyte within a quantitative assay that resulted in the need to redevelop the assay in a different format. Case study two outlines an assay where a surrogate matrix is the critical reagent in anassay and the difficulties encountered over the course of several years and lot changes. The third case study covers an immunogenicity assay with a commercial detection that did not have sufficient quantity to cover the entire study lifecycle. As a result of the reagent change, a new assay was developed. Discussion & conclusion: A robust plan for critical reagent generation and lifecycle management should be adapted in order to avoid costly delays and rework. The performance of an assay depends on the continuity of the critical reagent supply. Reagents should be carefully selected to include the binding and performance properties required for an assay.

Enhanced nail delivery of voriconazole-loaded nanomicelles by thioglycolic acid pretreatment: A study of protein dynamics and disulfide bond rupture

This work aimed to select an effective penetration enhancer (PE) for nail pretreatment, develop voriconazole (VOR)-loaded nanomicelles, and evaluate their ability to deliver VOR to the nail. A complete analysis of nail protein dynamics, bond rupture, and microstructure was performed. Alternative methods as electron paramagnetic resonance (EPR) and the Ellman's reagent (DTNB) assay were also evaluated. Nanomicelles were produced and characterized. The PE hydrated the hooves, following the order: urea ≈ cysteine ≈ glycolic acid < thioglycolic acid (TGA) < NaOH. SEM images and methylene blue assay showed enlarged pores and roughness of porcine hooves after incubation with NaOH and TGA. EPR was demonstrated to be the most sensitive technique. DTNB assay quantified higher thiol groups for samples treated with TGA (p < 0.05). A stratigraphic analysis with Raman spectroscopy demonstrated that hooves treated with TGA presented a higher SH/SS ratio at the edges, affecting protein secondary structure. In vitro permeation studies demonstrated significant VOR permeation (29.44 ± 6.13 µg/cm2), 10-fold higher than previous studies with lipid nanoparticles. After TGA pretreatment, VOR permeation was further enhanced (3-fold). TGA pretreatment followed by VOR-loaded nanomicelles demonstrates a promising approach for onychomycosis treatment. The novel methods for protein analysis were straightforward and helpful.

Development, deployment and in-field demonstration of mobile coronavirus SARS-CoV-2 Nucleic acid amplification test.

IntroductionThe evolving SARS-CoV-2 coronavirus pandemic presents a series of challenges to clinical diagnostic services. Many proprietary PCR platforms deployed outside centralised laboratories have limited capacity to upscale when public health demands increase. We set out to develop and validate an open-platform mobile laboratory for remote area COVID-19 diagnosis, with a subsequent field trial.Gap StatementIn regional Western Australia, molecular diagnostic support is limited to near point-of-care devices. We therefore aimed to demonstrate open-platform capability in a rapidly deployable format within the context of the COVID-19 pandemic.MethodologyWe compared, selected and validated components of a SARS-CoV-2 RT-PCR assay in order to establish a portable molecular diagnostics laboratory. The optimal combination of PCR assay equipment, reagents and consumables required for operation to national standards in regional laboratories was identified. This comprised RNA extraction and purification (QuickGene-480, Kurabo, Japan), a duplex RT-PCR assay (Logix Smart COVID-19, Co-Diagnostics, USA), a Myra liquid handling robot (Biomolecular Systems, Australia) and a magnetic induction thermal cycler (MIC, Biomolecular Systems).ResultsThe 95 and 99% limits of detection were 1.01 copies μl−1 (5.05 copies per reaction) and 2.80 copies μl−1 (14.00 copies per reaction) respectively. The Co-Diagnostics assay amplified both SARS-CoV-1 and −2 RNA but showed no other cross reactivity. Qualitative results aligned with the reference laboratory SARS-CoV-2 assay (sensitivity 100% [95 % CI 96.48–100%], specificity 100% [95% CI 96.52–100%]). In field trials, the laboratory was operational within an hour of arrival on-site, can process up to 36 samples simultaneously, produces results in two and a half hours from specimen reception, and performed well during six consecutive runs during a 1 week deployment.ConclusionOur mobile laboratory enables an adaptive response to increased test demand, and unlike many proprietary point-of-care PCR systems, rapid substitution with an alternative assay if gene targets change or reagent supply chains fail. We envisage operation of this RT-PCR assay as a standby capability to meet varying regional test demands under public health emergency operations guidance.

Disease Modeling with 3D Cell-Based Assays Using a Novel Flowchip System and High-Content Imaging.

There is an increasing interest in using three-dimensional (3D) cell structures for modeling tumors, organs, and tissue to accelerate translational research. We describe here a novel automated organoid assay system (the Pu·MA System) combined with microfluidic-based flowchips that can facilitate 3D cell-based assays. The flowchip is composed of sample wells, which contain organoids, connected to additional multiple wells that can hold various assay reagents. Organoids are positioned in a protected chamber in sample wells, and fluids are exchanged from side reservoirs using pressure-driven flow. Media exchange, sample staining, wash steps, and other processes can be performed without disruption to or loss of 3D sample. The bottom of the sample chamber is thin, optically clear plastic compatible with high-content imaging (HCI). The whole system can be kept in an incubator, allowing long-term cellular assays to be performed. We present two examples of use of the system for biological research. In the first example, cytotoxicity effects of anticancer drugs were evaluated on HeLa and HepG2 spheroids using HCI and vascular endothelial growth factor expression. In the second application, the flowchip system was used for the functional evaluation of Ca2+ oscillations in neurospheroids. Neurospheres were incubated with neuroactive compounds, and neuronal activity was assessed using Ca2+-sensitive dyes and fast kinetic fluorescence imaging. This novel assay system using microfluidics enables automation of 3D cell-based cultures that mimic in vivo conditions, performs multidosing protocols and multiple media exchanges, provides gentle handling of spheroids and organoids, and allows a wide range of assay detection modalities.

Sensitive quantification of carbon monoxide in vivo\xa0reveals a protective role of circulating hemoglobin in CO\xa0intoxication

Carbon monoxide (CO) is a gaseous molecule known as the silent killer. It is widely believed that an increase in blood carboxyhemoglobin (CO-Hb) is the best biomarker to define CO intoxication, while the fact that CO accumulation in tissues is the most likely direct cause of mortality is less investigated. There is no reliable method other than gas chromatography to accurately determine CO content in tissues. Here we report the properties and usage of hemoCD1, a synthetic supramolecular compound composed of an iron(II)porphyrin and a cyclodextrin dimer, as an accessible reagent for a simple colorimetric assay to quantify CO in biological samples. The assay was validated in various organ tissues collected from rats under normal conditions and after exposure to CO. The kinetic profile of CO in blood and tissues after CO treatment suggested that CO accumulation in tissues is prevented by circulating Hb, revealing a protective role of Hb in CO intoxication. Furthermore, hemoCD1 was used in vivo as a CO removal agent, showing that it acts as an effective adjuvant to O2 ventilation to eliminate residual CO accumulated in organs, including the brain. These findings open new therapeutic perspectives to counteract the toxicity associated with CO poisoning.

Time-related performance characteristics of high-sensitivity troponin I assay using manufacturer’s controls and reagents

BackgroundTroponin is a widely used cardiac protein biomarker for acute coronary syndrome. Its increasing importance drives an increasing need to assess, in real-world conditions, the performance of the tests to measure it. We evaluated the performance characteristics of high-sensitivity troponin I assay reagents and ancillary agents on the Abbott ARCHITECT ci4100, ARCHITECT i2000SR and Alinity ci using historical quality control data spanning 5 years. Design and methodRetrospective diagnostic hs-TnI quality control data were collected between 2015 and 2019 from the Abbott ARCHITECT ci4100, ARCHITECT i2000SR and Alinity ci located in the University College Dublin Clinical Research Centre Core Lab facility. Descriptive statistics for bias and variability were generated. Linear regression models were used to calculate the mean hs-TnI concentrations over Abbott quality control or reagent lot age and over time from the last calibration of the analysers. ResultsMeasurement bias on all three systems ranged between -2.49% and 3.98%. The total CV was ≤8.80%, with a within-lot variability for the reagents and controls of ≤5.45% and ≤7.13%, respectively. The between-lot CVs for reagents and controls were ≤7.16% and 6.19%, respectively. The effect of control or reagent age did not greatly affect stability over time. Results were also stable over different times from the last calibration of the analysers. ConclusionThe results of this study show that the Abbott hs-TnI assay on the ARCHITECT i2000sr and ARCHITECT ci4000 systems is quite stable in a core laboratory environment. The Alinity ci system exhibits similar performance characteristics.

Compounds DRG and DAG, Two Phenol Glycosides, Inhibit TNF-α-stimulated Inflammatory Response through Blocking NF-kB/AKT/JNK Signaling Pathways in MH7A Cells.

Fourteen constituents were recently isolated from the roots of Dendropanax dentiger with cyclooxygenase-2 (COX-2) inhibitory effects. However, the effect of 14 constituents on rheumatoid arthritis (RA) and their action mechanism remain unclear. The study aimed to explore the anti-RA effect and potential mechanism of these constituents in tumor necrosis factor α (TNF-α)-stimulated human RA fibroblast-like synoviocytes (MH7A cells). The cell viability, nitric oxide (NO) production, inflammatory cytokine levels, and protein expressions were measured by cell counting kit-8 (CCK-8), Griess reagent, ELISA, and Western blot assays, respectively. Results showed that 14 constituents (40 μM) have no cytotoxicity for MH7A cells. Among them, two phenols including 3,4-dimethoxyphenyl-1-O-α-L-rhamnopyranosyl-(1→6)-O-β-D-glucopyranoside (DRG) and 3,4-dimethoxyphenol-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside (DAG) were shown to significantly inhibit the NO production with IC50 values of 5.25±0.34 and 5.35±0.31 μM, respectively. They also remarkably decreased the release of interleukin (IL)-2, 6, 8, and interferon (IFN)-γ, as well as prominently reduced the phosphorylation protein levels of p65, IkBα, AKT, and JNK at a concentration of 10 μM. Taken together, DRG and DAG could inhibit TNF-α-induced inflammatory response through blocking NF-kB/AKT/JNK signaling pathways in MH7A cells, thus could be promising against RA and other inflammation-related agents.

Prognostic role of KL-6 in SSc-ILD patients with pleuroparenchymal fibroelastosis.

Krebs von den Lungen-6 (KL-6) is a high-molecular-weight (200kDa) glycoprotein proposed as a diagnostic biomarker for differentiating interstitial lung disease (ILD). Systemic sclerosis (SSc) is a rare immune-mediated disorder, and ILD is the leading cause of morbidity and mortality. Pleuroparenchymal fibroelastosis (PPFE) has been described to have a poor prognosis in SSc-ILD patients. This study undertook to compare serial changes in KL-6 in SSc-ILD patients with and without PPFE, to verify its prognostic value as a disease biomarker. Twenty-five SSc-ILD patients (median IQR, 62 (56-58); 20% males) were retrospectively enrolled. 12 SSc-ILD patients (48%) had also a radiological diagnosis of PPFE. Serum KL-6 concentrations were measured by KL-6 reagent assay (Fujirebio Europe, Ghent, Belgium). Serum KL-6 measurements were increased in SSc-ILD patients with and without PPFE compared with healthy controls (P<.0001). Comparative analysis of the rate of variation of KL-6 over the 6years of follow-up was performed by serial two-yearly KL-6 measurements: Δ1(t1-t0), Δ2(t2-t1) and Δ3(t3-t2). In SSc-ILD patients with PPFE pattern, Δ3 was significantly different than those without PPFE pattern (P=.0020). Serum KL-6 levels were significantly different (P=.0455) either at Δ2 and Δ3 in the PPFE group. In SSc-ILD patients with PPFE, at t3 serum KL-6 concentrations were inversely correlated with FEV1 (r=-.76; P=.037) and FVC percentages (r=-.79; P=.028). These results suggest that serial measurements of KL-6 in the follow-up of these patients may help to monitor disease progression. In real life, in SSc-ILD patients PPFE should be always evaluated at CT and when present should suggest a tight follow-up to monitor its evolution.

Temperature-regulated gold nanoparticle sensors for immune chromatographic rapid test kits with reproducible sensitivity: a study.

Immune‐chromatographic kits are being used since several years in the rapid detection of infectious diseases. It is also called the lateral flow technique, and is used for antigen or antibody detection. There are a series of steps involved in the development of these immune‐chromatographic test kits. Still, the preparation of gold nanoparticles (AuNPs) is an important quality variable for the immune‐chromatographic test kit sensitivity. The immune chromatographic test must be specific in detection for specific antigen and antibody; this implies that the test kit should not show a false result. Secondly, the test kit should be sensitive enough to give a readable result, and the intensity of the test line should increase or decrease with the concentration of an analytic sample. Various factors can influence the performance of a test. Temperature differences in AuNPs preparation can alter the assay kinetics and contribute to assay variability. Other factors such as assay components, manufacturing processes and reagent variation also contribute to assay precision and accuracy. It is important to note that assay reproducibility is the combined effect of individual sources of variability. The authors have synthesized AuNPs by immediately controlling the reaction temperature. Different batches of Malaria rapid test kit were developed and the test kit sensitivity was analysed. It was found that test kits designed with temperature‐controlled AuNPs sensor had reproducible uniformity in terms of batch to batch sensitivity than AuNPs synthesized by conventional Turkevich and Fern process.

Arctigenin Triggers Apoptosis and Autophagy via PI3K/Akt/mTOR Inhibition in PC-3M Cells.

Arctigenin (ARG), a natural lignans compound isolated from Arctium lappa L. In this study, the anti-tumor effect of ARG on prostate cancer cell PC-3M and the mechanism of apoptosis and autophagy induced by phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway were discussed, and further confirmed by the joint treatment of ARG and PI3K inhibitor LY294002. Here, the effect of ARG on cell viability was evaluated in PC-3M cells by Cell Counting Kit-8 reagent (CCK-8) assay. After the treatment of ARG, colony formation assay was used to detect the anti-proliferation effect. Annexin V-fluoresceine isothiocyanate/propidium iodide (FITC/PI) kit and 4',6-diamidino-2-phenylindole (DAPI) staining were used to detect the apoptosis level, and cell cycle changes were analyzed by flow cytometry. The expression of autophagy was detected by acridine orange staining. In addition, the expression levels of apoptosis and autophagy-related proteins were analyzed by Western blot. The result showed that different concentrations of ARG inhibited the proliferation of PC-3M cells. DAPI staining and flow cytometry showed that ARG induced PC-3M cell apoptosis and arrested cell in G0/G1 phase. Acridine orange staining showed that ARG induced autophagy in PC-3M cells. Western blot experiments showed that ARG inhibited the expression of Bcl-2, promoted the expression of Bax and cleaved caspase-3. At the same time, the expression of autophagy-related proteins LC3B-II and Beclin-1 increased after ARG treatment, but P62 decreased. In addition, further studies have shown that treatment with LY294002 enhanced the effects of ARG on the expression of proteins associated with apoptosis and autophagy, indicating that ARG may induce apoptosis and autophagy through PI3K/Akt/mTOR pathway.

Sorghum halepense (L.) Pers rhizomes inhibitory potential against diabetes and free radicals

BackgroundOwing to the side effects of synthetic medicine and less effectiveness against different syndromes, the researchers have focused on phytotherapy to overcome these problems. The purpose of this project was to study the in vitro phytochemical, cytotoxic, total phenolic, antioxidant and antidiabetic activities of the methanol extract of the rhizome of Sorghum halepense (L.) Pers and its n-hexane, chloroform and aqueous fractions. Thereafter, to conduct in vivo evaluation of the effective extract for its antidiabetic and antioxidant characteristics.MethodsCytotoxic, total phenolic content and antidiabetic properties were ascertained by brine shrimps lethality, Folin- Ciocalteu reagent and alpha-amylase inhibition assays respectively while antioxidant activities were investigated through DPPH, ABTS and H2O2 assays. The methanolic extract was assessed in vivo for its antidiabetic and antioxidant activities by using Wistar albino rats.ResultsThe phytochemical investigation of the methanolic extract and its unlike fractions revealed the availability of alkaloids, cardiac glycosides, flavonoids, terpenes, steroids, carbohydrate and proteins while lack of saponins and gums in methanolic extract. Steroids and carbohydrates were only present in aqueous and chloroform fraction respectively while both fractions contained proteins and alkaloids. Cardiac glycosides and flavonoids were absent in aqueous and chloroform fractions respectively. The highest brine shrimps lethality (70.5 ± 1.2), total phenolic content (28.30 ± 1.3 mg GAE/g), free radicals scavenging potential i.e. DPPH (40.02%), ABTS (40.48%) and H2O2 (50.85%) and alpha amylase inhibition (61.87%) was shown by the methanolic extract. The in vivo results did not disclose any sign of acute toxicity. The diabetic control showed a noteworthy (P < 0.05) decline in weight, HDL and glutathione and a raised level of bilirubin, blood glucose, urea, creatinine, triglyceride, LDL, VLDL, ALT, ALP, AST, SOD, catalase. The mentioned alterations were restored considerably (P < 0.05) by treatment of diabetic rats with methanolic extract of Sorghum halepense (L.) Pers (150 and 300 mg/kg b.w.).ConclusionIt is concluded that the extract of rhizomes of Sorghum halepense (L.) Pers is an effective fount of antioxidant and anti-diabetic compounds. Further analysis is needed to sharpen its pharmacological activities.

Serum Concentrations of KL-6 in Patients with IPF and Lung Cancer and Serial Measurements of KL-6 in IPF Patients Treated with Antifibrotic Therapy.

Simple SummaryEvaluation of the prognostic significance of serial measurements of serum concentrations of Krebs von den Lungen-6 (KL-6) showed that an annual increment in KL-6 exceeding a threshold amount was an independent risk factor for progressive disease and poor prognosis. No literature data are available on long-term KL-6 measurements in the follow-up of IPF patients treated with Nintedanib. The aim of this study is to serially analyze KL-6 in idiopathic pulmonary fibrosis (IPF) patients after 24 months of Nintedanib and to investigate its biomarker potential in patients with IPF and lung cancer with respect to fibrotic hypersensitivity pneumonitis patients, pulmonary fibrosis associated with autoimmune diseases group and healthy controls.Background: Krebs von den Lungen-6 (KL-6) was suggested as ILD biomarker including idiopathic pulmonary fibrosis (IPF). Lung cancer is one of the most severe comorbidity of IPF patients. This study aims to serially analyze KL-6 in IPF patients after 24 months of Nintedanib and to first investigate the biomarker behavior in IPF associated with adenocarcinoma. Materials and methods: One hundred and forty-two ILD patients (median (IQR), 69 (63–75) years; 86 males) were retrospectively enrolled. Serial serum samples were collected from IPF patients before starting antifibrotic therapy and after 12 months. Serum KL-6 levels were measured by KL-6 reagent assay (Fujirebio Europe, UK). Results: Increased KL-6 concentrations were identified in IPF-LC patients than IPF, fibrotic hypersensitivity pneumonitis, and pulmonary fibrosis associated with autoimmune disease groups. A cut-off value was calculated to distinguish IPF and IPF-LC patients. IPF patients monitored for 24 months with Nintedanib showed persisted increased levels of KL-6 with a progressive decline of FVC percentages. Conclusion: This preliminary study offers a first demonstration that very high serum concentrations of KL-6 in IPF-LC patients are associated with poor prognosis. Moreover, serial evaluation of serum KL-6 in IPF patients over 24 months of Nintedanib treatment revealed that most patients experienced a stabilization of lung function parameters and of serum concentrations of KL-6.

An AIE‐based enzyme‐activatable fluorescence indicator for Western blot assay: Quantitative expression of proteins with reproducible stable signal and wide linear range

AbstractWestern blot is a commonly used experimental method to analyze the protein expression. However, the most commonly used chromogenic indicator based on chemiluminescence is limited by narrow linear range and unstable quantitative reproducibility, whereas the recently developed fluorescent indicator suffers from poor detection limit. Herein, we report an enzyme‐activatable fluorescence indicator to quantify proteins with reproducible stable signal and wide linear range, through introducing the hydrophilic alkaline phosphatase (ALP)‐triggered phosphoric acid moiety into our established aggregation‐induced emission (AIE) building block of quinoline‐malononitrile (QM). In this strategy, the indicator DQM‐ALP disperses well in both aqueous and lipid environments to exhibit initial “off” fluorescence, but when exposing to the ALP‐coupled secondary antibody on the PVDF membrane, the specific enzymatic turnover would liberate hydrophobic AIE luminogen (AIEgen) QM‐OH to emit strong luminescence, thereby achieving an ideal “off‐on” state for sensitively imaging proteins with high signal‐to‐noise (S/N) ratio. Moreover, benefiting from the excellent signal stability of AIE fluorophore, DQM‐ALP indicator exhibits superior quantitative analysis of protein expression with high reproducibility. Upon taking advantage of the AIEgens to reduce high concentration‐induced luminance quenching, the linear quantification range is extremely expanded. In contrast with the traditional chemiluminescent indicator, the AIE‐based enzyme‐activatable indicator DQM‐ALP not only greatly improves the signal stability for quantitative reproducibility, but also expands the linear quantification range, and further provides a practical alternative reagent for fluorescence Western blot assay.