3D Amplified Single-Cell RNA and Protein Imaging Identifies Oncogenic Transcript Subtypes in B-Cell Acute Lymphoblastic Leukemia.

Simultaneous in situ detection of transcript and protein markers at the single-cell level is essential for gaining a better understanding of tumor heterogeneity and for predicting and monitoring treatment responses. However, the limited accessibility to advanced 3D imaging techniques has hindered their rapid implementation. Here, we present a 3D single-cell imaging technique, termed 3D digital rolling circle amplification (4DRCA), capable of the multiplexed and amplified simultaneous digital quantification of single-cell RNAs and proteins using standard fluorescence microscopy and off-the-shelf reagents. We generated spectrally distinguishable DNA amplicons from molecular markers through an integrative protocol combining single-cell RNA and protein assays and directly enumerated the amplicons by leveraging an open-source algorithm for 3D deconvolution with a custom-built automatic gating algorithm. With 4DRCA, we were able to simultaneously quantify surface protein markers and cytokine transcripts in T-lymphocytes. We also show that 4DRCA can distinguish BCR-ABL1 fusion transcript positive B-cell acute lymphoblastic leukemia cells with or without CD19 protein expression. The accessibility and extensibility of 4DRCA render it broadly applicable to other cell-based diagnostic workflows, enabling sensitive and accurate single-cell RNA and protein profiling.

Electrocatalytic Access to Azetidines via Intramolecular Allylic Hydroamination: Scrutinizing Key Oxidation Steps through Electrochemical Kinetic Analysis.

Azetidines are prominent structural scaffolds in bioactive molecules, medicinal chemistry, and ligand design for transition metals. However, state-of-the-art methods cannot be applied to intramolecular hydroamination of allylic amine derivatives despite their underlying potential as one of the most prevalent synthetic precursors to azetidines. Herein, we report an electrocatalytic method for intramolecular hydroamination of allylic sulfonamides to access azetidines for the first time. The merger of cobalt catalysis and electricity enables the regioselective generation of key carbocationic intermediates, which could directly undergo intramolecular C-N bond formation. The mechanistic investigations including electrochemical kinetic analysis suggest that either the catalyst regeneration by nucleophilic cyclization or the second electrochemical oxidation to access the carbocationic intermediate is involved in the rate-determining step (RDS) of our electrochemical protocol and highlight the ability of electrochemistry in providing ideal means to mediate catalyst oxidation.

Sessile droplet array for sensitive profiling of multiple extracellular vesicle immuno-subtypes.

The sensitive detection of the multiple immuno-subtypes of cancer-specific extracellular vesicles (EVs) has emerged as a promising method for multiclass cancer diagnosis; however, its limitations in sensitivity, accessibility, and multiple detection of EV subtypes have hindered its further implementation. Here, we present a platform for sensitive EV detection enabled by sessile droplet array (eSD) that exploits enhanced immuno-capture of EVs via evaporation-driven radial flows in a sessile droplet. Compared to a micro-well without internal flows, this platform demonstrates significantly enhanced EV capture and detection by detecting low levels of EVs with a detection limit of 384.7 EVs per microliter, which is undetectable in the micro-well. In addition, using a small sample consumption of ∼0.2 µL plasma per droplet, the platform detects EV immuno-subtypes against seven different antibodies in patient plasma samples of different cancer types (liver, colon, lung, breast and prostate cancers). Further, using the profiling data, the platform exhibits a sensitivity of 100% (95% confidence interval (CI): 83-100%) and a specificity of 100% (95% CI: 40-100%) for the diagnosis of cancer, and classified cancer types with an overall accuracy of 96% (95% CI: 86-100%) using a two-staged algorithm based on quadratic discriminant analysis technique for machine learning.

Is measurement of central venous pressure required to estimate systemic vascular resistance? A retrospective cohort study.

BACKGROUND: The clinical range of central venous pressure (CVP) (typically 5 to 15 mmHg) is much less than the range of mean arterial blood pressure (60 to 120 mmHg), suggesting that CVP may have little impact on estimation of systemic vascular resistance (SVR). The accuracy and feasibility of using an arbitrary CVP rather than actual CVP for the estimation of SVR during intraoperative period is not known. METHODS: Using vital records obtained from patients who underwent neurological and cardiac surgery, the present study retrospectively calculated SVR using fixed values of CVP (0, 5, 10, 15, and 20 mmHg) and randomly changing values of CVP (5 to 15 mmHg) and compared these calculated SVRs with actual SVR, calculated using actual CVP. Differences between actual SVR and SVRs based on fixed and random CVPs were quantified as root mean square error (RMSE) and mean absolute percentage error (MAPE). Bland-Altman analysis and four-quadrant plot analysis were performed. RESULTS: A total of 34 patients are included, including 18 who underwent neurosurgery and 16 who underwent cardiac surgery; 501,380 s (139.3 h) of data was analyzed. The SVR derived from a fixed CVP of 10 mmHg (SVRf10) showed the highest accuracy (RMSE: 115 and 104 [dynes/sec/cm- 5] and MAPE: 6.3 and 5.7% in neurological and cardiac surgery, respectively). The 95% limits of agreement between SVRf10 and actual SVR were - 208.5 (95% confidence interval [CI], - 306.3 to - 148.1) and 242.2 (95% CI, 181.8 to 340.0) dynes/sec/cm- 5 in neurosurgery and - 268.1 (95% CI, - 367.5 to - 207.7) and 163.2 (95% CI, 102.9 to 262.6) dynes/sec/cm- 5 in cardiac surgery. All the SVRs derived from the fixed CVPs (regardless of its absolute value) showed excellent trending ability (concordance rate > 0.99). CONCLUSIONS: SVR can be estimated from a fixed value of CVP without causing significant deviation or a loss of trending ability. However, caution is needed when using point estimates of SVR when the actual CVP is expected to be out of the typical clinical range. TRIAL REGISTRATION: This study was registered Clinical Research Information Service, a clinical trial registry in South Korea ( KCT0006187 ).

A retrospective comparison for prediction of optimal length of right subclavian vein catheterization in infants: landmark-based estimation vs. linear regression model.

BACKGROUND: The optimal insertion length for right subclavian vein catheterization in infants has not been determined. This study retrospectively compared landmark-based and linear regression model-based estimation of optimal insertion length for right subclavian vein catheterization in pediatric patients of corrected age < 1 year. METHODS: Fifty catheterizations of the right subclavian vein were analyzed. The landmark related distances were: from the needle insertion point (I) to the tip of the sternal head of the right clavicle (A) and from A to the midpoint (B) of the perpendicular line drawn from the sternal head of the right clavicle to the line connecting the nipples. The optimal length of insertion was retrospectively determined by reviewing post-procedural chest radiographs. Estimates using a landmark-based equation (IA + AB - intercept) and a linear regression model were compared with the optimal length of insertion. RESULTS: A landmark-based equation was determined as IA + AB - 5. The mean difference between the landmark-based estimate and the optimal insertion length was 1.0 mm (95% limits of agreement -18.2 to 20.3 mm). The mean difference between the linear regression model (26.681 - 4.014 × weight + 0.576 × IA + 0.537 × AB - 0.482 × postmenstrual age) and the optimal insertion length was 0 mm (95% limits of agreement -16.7 to 16.7 mm). The difference between the estimates using these two methods was not significant. CONCLUSION: A simple landmark-based equation may be useful for estimating optimal insertion length in pediatric patients of corrected age < 1 year undergoing right subclavian vein catheterization.

One-Step Microfluidic Purification of White Blood Cells from Whole Blood for Immunophenotyping.

One-step purification of white blood cells (WBCs) is essential to automate blood sample preparation steps for WBC analysis, but conventional methods such as red blood cell (RBC) lysis and density-gradient centrifugation typically require harsh chemical or physical treatment, followed by repeated manual washing steps. Alternative microfluidic separation methods show limited separation performances due to the trade-off between purity and throughput. Herein, an integrated microfluidic device is developed to decouple the trade-off by synergistically combining a slant array ridge-based WBC enrichment unit as a throughput enhancer and a slant, asymmetric lattice-based WBC washing unit as a purity enhancer. The enrichment unit can maintain a high sample-infusion throughput while lowering the flow rate into the washing unit, thus enabling WBC-selective washing without significant influence by the overwhelming number of RBCs and inertial forces. The device delivers efficient separation performances by rejecting 99.9% of RBCs as well as 99.9% of blood plasma from canine and human whole blood in a single round of purification at a high throughput of 60 µL/min. The purified WBC population well preserves the composition of lymphocyte subpopulations, the major components of the adaptive immune system, thus providing the potential for the integrated device to be used as an essential sample-preparation tool for immunologic investigations.

Integrated microfluidic pneumatic circuit for point-of-care molecular diagnostics.

Developing simple, portable, rapid, and easy-to-use diagnostic technologies is essential for point-of-care (POC) blood molecular testing. Integrated microfluidic devices that include the functionalities of blood separation, microfluidic pumping, and molecular detection are desirable for POC testing; however, current technologies still rely on off-chip sample processing or require bulky equipment. We report a fully-integrated microfluidic diagnostic device, i.e., an integrated pneumatic microfluidic circuit (iPC), that can autonomously pump whole blood, continuously sort blood plasma, and readily enable blood plasma proteomic analysis. The iPC contains vacuum pillars as a vacuum source and waste reservoir, as well as microchannels connecting the pillars as a plasma separator or a flow stabilizer. We combined the iPC and a miniaturized fluorescence microscope to create a portable diagnostic platform that enables fluorescence-based biomarker detection. First, we performed systematic parametric studies to establish design rules for determining the transport and distribution of fluid streams in the iPC. We then demonstrated the capability of the iPC-based diagnostic platform by successfully separating blood plasma from microliter quantities of whole blood while simultaneously quantifying thrombin in blood samples using an aptamer beacon within 5 min of sample injection. Our platform holds potential as a rapid, field-deployable, essentially universal diagnostic tool in POC settings.

Pumpless Microflow Cytometry Enabled by Viscosity Modulation and Immunobead Labeling.

Major challenges of miniaturizing flow cytometry include obviating the need for bulky, expensive, and complex pump-based fluidic and laser-based optical systems while retaining the ability to detect target cells based on their unique surface receptors. We addressed these critical challenges by (i) using a viscous liquid additive to control flow rate passively, without external pumping equipment, and (ii) adopting an immunobead assay that can be quantified with a portable fluorescence cell counter based on a blue light-emitting diode. Such novel features enable pumpless microflow cytometry (pFC) analysis by simply dropping a sample solution onto the inlet reservoir of a disposable cell-counting chamber. With our pFC platform, we achieved reliable cell counting over a dynamic range of 9-298 cells/µL. We demonstrated the practical utility of the platform by identifying a type of cancer cell based on CD326, the epithelial cell adhesion molecule. This portable microflow cytometry platform can be applied generally to a range of cell types using immunobeads labeled with specific antibodies, thus making it valuable for cell-based and point-of-care diagnostics.

Soil moisture could enhance electrokinetic remediation of arsenic-contaminated soil.

Electrokinetic remediation (EKR) is the most efficient technique for remediation of fine-grained soil. The primary removal mechanisms of heavy metal in EKR are the electromigration and electroosmosis flow under appropriate electric gradients. Most EKR studies have researched the variation according to the electrolyte and electric voltage. Also, EKR could be influenced by the migration velocity of ions, while few studies have investigated the effect of moisture content. In this study, soil moisture was controlled by using tap water and NaOH as electrolytes to enhance electromigration and electroosmosis flow. In both electrolytes, the higher moisture content led to the more As removal efficiency, but there were no differences between tap water and NaOH. Therefore, tap water was the most cost-effective electrolyte to remove As from fine-grained soil.

Pharmacokinetics of a new once-daily controlled-release formulation of aceclofenac in Korean healthy subjects compared with immediate-release aceclofenac and the effect of food: a randomized, open-label, three-period, crossover, single-centre study.

BACKGROUND: A new controlled-release formulation of aceclofenac 200 mg (Clanza CR®) developed by Korea United Pharm., Inc., South Korea, for once-daily (od) dosing provides biphasic aceclofenac release consisting of immediate release of 85 mg followed by sustained release of 115 mg. Food has been known to affect the rate and extent of absorption of several drugs, in both immediate-release and controlled-release formulations. OBJECTIVE: The aim of this study was to evaluate the relative bioavailability of a new controlled-release formulation of aceclofenac (200 mg od; Clanza CR®) in comparison with immediate-release aceclofenac (100 mg twice daily [bid], Airtal®) and to assess the effect of food on the pharmacokinetics of the new controlled-release aceclofenac formulation. METHODS: This study was designed as a randomized, open-label, three treatment-period, crossover, single-centre study with a 1-week washout in 41 healthy adults. The three treatments consisted of immediate-release aceclofenac 100 mg bid administered under fasting conditions; controlled-release aceclofenac 200 mg od administered under fasting conditions; and controlled-release aceclofenac 200 mg od administered immediately after a standardized high-fat breakfast. Plasma concentrations of aceclofenac were determined using a high-performance liquid chromatography method. RESULTS: In the fasted state, the 90% confidence intervals (CIs) of the least squares geometric mean ratios (GMRs) for the area under the plasma concentration-time curve from time zero to 24 hours (AUC(24)) and the peak plasma concentration (C(max)) of aceclofenac for the controlled-release and immediate-release formulations of aceclofenac were all within the bioequivalence criteria range of 0.8-1.25. The 90% CIs of the GMRs for the AUC(24) and C(max) of aceclofenac for the controlled-release formulation of aceclofenac in the fed and fasted states were also within the bioequivalence range. Both aceclofenac formulations were well tolerated in all subjects, and no serious adverse effects were observed. CONCLUSION: The results demonstrate that controlled-release aceclofenac 200 mg is equivalent to immediate-release aceclofenac 100 mg when administered at the same total daily dose. Additionally, the bioavailability of controlled-release aceclofenac was not affected by high-fat foods.