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A sample processing method for immunoassay of whole blood tacrolimus.

Current sample processing (SP) methods for tacrolimus (FK506) immunoassays are mainly based on extraction of drug by organic solvent and divalent metal ions. Although these methods are effective for drug extraction and interference elimination, they suffer from drawbacks including inconvenience for operation, difficulties for automation and potential measurement bias. To overcome these limitations, this study describes a new SP reagent for blood cell lysis and protein denaturation. A TRFIA (time-resolved fluorescence immunoassay) was developed by using this SP reagent for whole blood FK506 quantification. Results show that blood samples could be turned into homogeneous solution after being treated by this SP reagent, and so could be directly applied to immunoassays without centrifugation. The analytical sensitivity of the FK506-TRFIA was 0.57 ng/mL, the within-run and between-run coefficient of variations (CVs) were both less than 10%. The FK506 values of 126 samples obtained by FK506-TRFIA correlated excellently with that obtained by ABBOTT FK506-CMIA (R = 0.982). Comparison studies also show that the FK506-TRFIA was highly resistant to endogenous interferences. These results suggest that the present SP method is a more promising chose for FK506 immunoassay, and in the meantime, its simplicity makes the whole-process immunoassay automation more feasible by obviating the necessary for centrifugation.

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The effect of the antisickling compound GBT1118 on the permeability of red blood cells from patients with sickle cell anemia.

Sickle cell anemia (SCA) is one of the commonest severe inherited disorders. Nevertheless, effective treatments remain inadequate and novel ones are avidly sought. A promising advance has been the design of novel compounds which react with hemoglobin S (HbS) to increase oxygen (O ) affinity and reduce sickling. One of these, voxelotor (GBT440), is currently in advanced clinical trials. A structural analogue, GBT1118, was investigated in the current work. As RBC dehydration is important in pathogenesis of SCA, the effect of GBT1118 on RBC cation permeability was also studied. Activities of P , the Gardos channel and the KCl cotransporter (KCC) were all reduced. Gardos channel and KCC activities were also inhibited in RBCs treated with Ca ionophore or the thiol reagent N-ethylmaleimide, indicative of direct effects on these two transport systems. Consistent with its action on RBC membrane transporters, GBT1118 significantly increased RBC hydration. RBC hemolysis was reduced in a nonelectrolyte lysis assay. Further to its direct effects on O affinity, GBT1118 was therefore found to reduce RBC shrinkage and fragility. Findings reveal important effects of GBT1118 on protecting sickle cells and suggest that this is approach may represent a useful therapy for amelioration of the clinical complications of SCA.

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Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology.

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 µg/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per µg of protein produced or $0.019 per µL of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

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A diffusion-based microfluidic device for single-cell RNA-seq.

Microfluidic devices provide a low-input and efficient platform for single-cell RNA-seq (scRNA-seq). Existing microfluidic devices have a complicated multi-chambered structure for handling the multi-step process involved in RNA-seq and dilution between steps is used to negate the inhibitory effects among reagents. This makes the device difficult to fabricate and operate. Here we present microfluidic diffusion-based RNA-seq (MID-RNA-seq) for conducting scRNA-seq with a diffusion-based reagent swapping scheme. This device incorporates cell trapping, lysis, reverse transcription and PCR amplification all in one simple microfluidic device. MID-RNA-seq provides high data quality that is comparable to existing scRNA-seq methods while implementing a simple device design that permits multiplexing. The robustness and scalability of the MID-RNA-seq device will be important for transcriptomic studies of scarce cell samples.

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Integration of marker-free selection of single cells at a wireless electrode array with parallel fluidic isolation and electrical lysis.

We present integration of selective single-cell capture at an array of wireless electrodes (bipolar electrodes, BPEs) with transfer into chambers, reagent exchange, fluidic isolation and rapid electrical lysis in a single platform, thus minimizing sample loss and manual intervention steps. The whole process is achieved simply by exchanging the solution in a single inlet reservoir and by adjusting the applied voltage at a pair of driving electrodes, thus making this approach particularly well-suited for a broad range of research and clinical applications. Further, the use of BPEs allows the array to be scalable to increase throughput. Specific innovations reported here include the incorporation of a leak channel to balance competing flow paths, the use of 'split BPEs' to create a distinct recapture and electrical lysis point within the reaction chamber, and the dual purposing of an ionic liquid as an immiscible phase to seal the chambers and as a conductive medium to permit electrical lysis at the split BPEs.

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Mouse Atherosclerosis Arr Esophageal inflammation, Lysis Buffer Antibody Arr Normal human tissue panel Atherosclerosis (Mouse) A Single Donor Human Atopic Anti-apoptotic marker in Mouse Atherosclerosis Arr Atherosclerosis (Human) A Breast cancer tissue arra Disease State Samples: Si Single Donor Human Atopic

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Effective gel-like floc matrix destruction and water seepage for enhancing waste activated sludge dewaterability under hybrid microwave-initiated Fe(II)-persulfate oxidation process.

Chemical conditioning before mechanical dewatering is an indispensable step to enhance the waste activated sludge (WAS) dewaterability and solid-liquid separation. Feasibility of utilizing Fe(II)/SO oxidation integrated with microwave irradiation (MW) in improving gel-like floc destruction, water seepage and WAS dewaterability was investigated. Comprehensive characterization of the treated WAS was conducted to explore the effects of MW on the catalyzing kinetics of Fe(II)/SO oxidation and reveal the underlying dewatering principle. The results demonstrated that MW-Fe(II)/SOprocess was more cost-efficient, reagent-saving than single Fe(II)/SO oxidation or MW irradiation in stimulating WAS dewaterability and the optimal conditions were 0.4/0.5 mmol-Fe(II)/SO g-TS (total solids) and 500 W with 94.6% capillary suction time (CST) reduction within 120 s of conditioning. Thermal effect of MW reduced the activation energy of SO decomposition and stimulated the generation of more SO· while athermal effect could create additional gel-network destruction and cell lysis, which reduced the water-binding energy and induced the seepage of more extracellular polymeric substances (EPS)-bound and cell water. Further analysis via fluorescence excitation-emission matrix combined with parallel factor analysis demonstrated that protein-like, humic- and fulvic-like substances in slime EPS (S-EPS) and loosely bound EPS (LB-EPS) together affected sludge dewaterability. Additionally, the hybrid process could further remove the released COD and ammonia, facilitating the subsequent advanced treatment.

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Arachidonic acid causes lysis of blood cells and ADP-dependent platelet activation responses in platelet function tests.

The use of arachidonic acid (AA) to stimulate platelets is considered as a specific approach to study aspirin treatment efficacy. However, very high concentrations of AA are used, and it has been previously reported that AA can induce cell lysis in other settings. Several clinical studies have reported decreased responses to AA in whole blood tests in the presence of clopidogrel. Our aim was to investigate whether unspecific effects contribute to AA-induced aggregation and platelet activation in light transmission aggregometry (LTA) in platelet-rich plasma (PRP), and in assays using whole blood, multiple electrode aggregometry (MEA, Multiplate®), and flow cytometry. We report that cell lysis, especially of red blood cells, does occur at concentrations of AA used in the clinical tests and that ADP is very important for the AA-induced platelet activation responses. In flow cytometry, very limited platelet activation was detected before reaching AA concentrations in the millimolar range, where cell lysis also occurred, making it problematic to develop a reliable flow cytometry assay using AA as reagent. We conclude that cell lysis and ADP release contribute to AA-induced platelet responses, most markedly in whole blood assays. This finding could potentially explain some differences between studies comparing methods using whole blood and PRP and also how clopidogrel treatment could influence AA-induced aggregation results in previously published studies. Our findings highlight some issues with AA as reagent for platelet activation, which also have an impact on how platelet activation assays using AA should be interpreted.

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A new DNA sensor system for specific and quantitative detection of mycobacteria.

In the current study, we describe a novel DNA sensor system for specific and quantitative detection of mycobacteria, which is the causative agent of tuberculosis. Detection is achieved by using the enzymatic activity of the mycobacterial encoded enzyme topoisomerase IA (TOP1A) as a biomarker. The presented work is the first to describe how the catalytic activities of a member of the type IA family of topoisomerases can be exploited for specific detection of bacteria. The principle for detection relies on a solid support anchored DNA substrate with dual functions namely: (1) the ability to isolate mycobacterial TOP1A from crude samples and (2) the ability to be converted into a closed DNA circle upon reaction with the isolated enzyme. The DNA circle can act as a template for rolling circle amplification generating a tandem repeat product that can be visualized at the single molecule level by fluorescent labelling. This reaction scheme ensures specific, sensitive, and quantitative detection of the mycobacteria TOP1A biomarker as demonstrated by the use of purified mycobacterial TOP1A and extracts from an array of non-mycobacteria and mycobacteria species. When combined with mycobacteriophage induced lysis as a novel way of effective yet gentle extraction of the cellular content from the model Mycobacterium smegmatis, the DNA sensor system allowed detection of mycobacteria in small volumes of cell suspensions. Moreover, it was possible to detect M. smegmatis added to human saliva. Depending on the composition of the sample, we were able to detect 0.6 or 0.9 million colony forming units (CFU) per mL of mycobacteria, which is within the range of clinically relevant infection numbers. We, therefore, believe that the presented assay, which relies on techniques that can be adapted to limited resource settings, may be the first step towards the development of a new point-of-care diagnostic test for tuberculosis.

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Interaction of the anti-tuberculous drug bedaquiline with artificial membranes and rat erythrocytes.

Bedaquiline (BDQ) is a new drug from the family of diarylquinolines, which has a potent bactericidal activity against Mycobacterium tuberculosis. This paper has examined the interaction of BDQ with model membranes (liposomes and BLM) and rat erythrocytes. It was shown that BDQ (1-10 mol%) changed the thermotropic phase behavior of DMPC liposomes, leading to the lateral phase separation in the lipid bilayer and the formation of membrane microdomains. BDQ (10-50 μM) was also demonstrated to cause permeabilization of lecithin liposomes loaded with the fluorescent dye sulforhodamine B. At the same time, it did not alter the ionic conductivity of BLM. A dynamic light scattering study showed that BDQ led to the emergence of two populations of light-scattering particles in the suspension of lecithin liposomes, suggesting that an aggregation of the vesicles took place. In rat erythrocytes, BDQ was found to induce changes in their size and shape, as well as aggregation and lysis of the cells.

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