Quantitative Activity Measurements Research Articles

Quantitative characterization of immune cells by measuring cellular signal transduction pathway activity

For many diseases, including cancer, infections, and auto-immune diseases, the immune response is a major determinant of disease progression, response to therapy, and clinical outcome. Innate and adaptive immune responses are controlled by coordinated activity of different immune cell types. The functional activity state of immune cells is determined by Signal Transduction Pathways (STPs). A recently developed technology (Simultaneous Transcriptome-based Activity Profiling of Signal Transduction Pathways, STAP-STP) enables simultaneous and quantitative activity measurement of relevant STPs in immune cells based on mRNA-analysis. STAP-STP technology was used to analyze public transcriptome data of a variety of immune cell types in resting and activated functional state. In addition, a clinical study on rheumatoid arthritis (RA) was analyzed to illustrate utility of the technology. Per sample, activity of androgen and estrogen receptor, PI3K, MAPK, TGFβ, Notch, NFκB, JAK-STAT1/2, and JAK-STAT3 STPs was calculated, generating an STP activity profile (SAP) consisting of 9 activity scores. Each analyzed immune cell type, i.e. naive/resting and immune-activated CD4 + and CD8 + T cells, T helper cells, B cells, NK cells, monocytes, macrophages, and dendritic cells, had a reproducible and characteristic SAP, reflecting both cell type and its activity state. Analysis of clinical RA samples revealed increased TGFβ STP activity in whole blood samples. In conclusion, STAP-STP technology enables quantitative measurement of the functional activity state of immune cells of the innate and adaptive immune system. Aside from diagnostic applications, utility lies in unravelling abnormal immune function in disease and immunomodulatory drug development.

АКТИВНОСТЬ НАКОПЛЕНИЯ 18F-FDG И ЕЕ ПРОГНОСТИЧЕСКАЯ ЦЕННОСТЬ В ВИСЦЕРАЛЬНОЙ ЖИРОВОЙ ТКАНИ ПРИ ПЭТ/КТ ИССЛЕДОВАНИИ ПРИ ЭПИТЕЛИАЛЬНОМ РАКЕ ЯИЧНИКОВ И КОЛОРЕКТАЛЬНОМ РАКЕ

Quantitative activity measurements of 18F-FDG PET/CT accumulation in visceral adipose tissue in epithelial ovarian cancer and colorectal cancer in the abdominal cavity and pelvis with metastatic lesions of the omentum and lymph nodes in baseline and follow-up patients are presented

Characterization of Immunoactive and Immunotolerant CD4+ T Cells in Breast Cancer by Measuring Activity of Signaling Pathways That Determine Immune Cell Function.

Simple SummaryImmunotherapy enhances the immune response against cancer and is potentially curative. Unfortunately, few patients with breast cancer benefit from this therapy. It is not possible to predict which patients will benefit. A blood cell, called CD4+ T-cell, plays a role in the immune response and in resistance to immunotherapy. Its function is determined by activity of biochemical processes, called signal transduction pathways (STPs). We developed a new technology to measure activity of these STPs, which was used to investigate whether CD4+ T cells function abnormally in breast cancer patients. We show that in CD4+ T-cells from most of the investigated breast cancer patients a number of these STPs are overactive. The abnormal activity of a few notable STPs (Notch and TGFβ) suggests that CD4+ T-cells have changed into regulatory T-cells, which inhibit the immune response against cancer and have been associated with resistance to immunotherapy. We also provide evidence that this change in the CD4+ T- cells is caused by a factor produced by breast cancer cells. We conclude that this new technology can be used to measure STP activity in blood of patients with cancer and has the potential to better identify patients who will benefit from immunotherapy. Cancer immunotolerance may be reversed by checkpoint inhibitor immunotherapy; however, only a subset of patients responds to immunotherapy. The prediction of clinical response in the individual patient remains a challenge. CD4+ T cells play a role in activating adaptive immune responses against cancer, while the conversion to immunosuppression is mainly caused by CD4+ regulatory T cell (Treg) cells. Signal transduction pathways (STPs) control the main functions of immune cells. A novel previously described assay technology enables the quantitative measurement of activity of multiple STPs in individual cell and tissue samples. The activities of the TGFβ, NFκB, PI3K-FOXO, JAK-STAT1/2, JAK-STAT3, and Notch STPs were measured in CD4+ T cell subsets and used to investigate cellular mechanisms underlying breast cancer-induced immunotolerance. Methods: STP activity scores were measured on Affymetrix expression microarray data of the following: (1) resting and immune-activated CD4+ T cells; (2) CD4+ T-helper 1 (Th1) and T-helper 2 (Th2) cells; (3) CD4+ Treg cells; (4) immune-activated CD4+ T cells incubated with breast cancer tissue supernatants; and (5) CD4+ T cells from blood, lymph nodes, and cancer tissue of 10 primary breast cancer patients. Results: CD4+ T cell activation induced PI3K, NFκB, JAK-STAT1/2, and JAK-STAT3 STP activities. Th1, Th2, and Treg cells each showed a typical pathway activity profile. The incubation of activated CD4+ T cells with cancer supernatants reduced the PI3K, NFκB, and JAK-STAT3 pathway activities and increased the TGFβ pathway activity, characteristic of an immunotolerant state. Immunosuppressive Treg cells were characterized by high NFκB, JAK-STAT3, TGFβ, and Notch pathway activity scores. An immunotolerant pathway activity profile was identified in CD4+ T cells from tumor infiltrate and blood of a subset of primary breast cancer patients, which was most similar to the pathway activity profile in immunosuppressive Treg cells. Conclusion: Signaling pathway assays can be used to quantitatively measure the functional immune response state of lymphocyte subsets in vitro and in vivo. Clinical results suggest that, in primary breast cancer, the adaptive immune response of CD4+ T cells may be frequently replaced by immunosuppressive Treg cells, potentially causing resistance to checkpoint inhibition. In vitro study results suggest that this is mediated by soluble factors from cancer tissue. Signaling pathway activity analysis on TIL and/or blood samples may improve response prediction and monitoring response to checkpoint inhibitors and may provide new therapeutic targets (e.g., the Notch pathway) to reduce resistance to immunotherapy.

Clinical Conundrums When Integrating the QbTest into a Standard ADHD Assessment of Children and Young People.

The uptake of the QbTest in clinical practice is increasing and has recently been supported by research evidence proposing its effectiveness in relation to clinical decision-making. However, the exact underlying process leading to this clinical benefit is currently not well established and requires further clarification. For the clinician, certain challenges arise when adding the QbTest as a novel method to standard clinical practice, such as having the skills required to interpret neuropsychological test information and assess for diagnostically relevant neurocognitive domains that are related to attention-deficit hyperactivity disorder (ADHD), or how neurocognitive domains express themselves within the behavioral classifications of ADHD and how the quantitative measurement of activity in a laboratory setting compares with real-life (ecological validity) situations as well as the impact of comorbidity on test results. This article aims to address these clinical conundrums in aid of developing a consistent approach and future guidelines in clinical practice.

Analysis of signaling pathway activity in desmoid-type fibromatosis (DF) to identify druggable pathways.

e23558 Background: DF are locally invasive soft tissue tumors of fibroblast origin, with unpredictable course. CTTNB1 mutations are a hallmark of DF. Molecular-targeted therapies are lacking. Recently developed OncoSignal (www.philips.com/oncosignal) pathway activity tests enable quantitative measurement of activity of clinically relevant oncogenic signal transduction pathways (estrogen, progesterone & androgen receptors; PI3K, MAPK-AP1, TGFβ, Notch, Hedgehog, Wnt pathways), based on computational interpretation of expression levels of direct target genes of the pathway-associated transcription factor. Methods: OncoSignal was used to measure activity of signaling pathways on Affymetrix transcriptome data from sporadic DF (n = 128), generated before in our center (GEO database: GSE58697), and from presumably healthy fibroblasts (n = 63) derived from a number of different tissue types (public dataset GSE63626). Pathway activity was expressed on a normalized scale 0-100 (min and max pathway activity score depend on the cell/tissue type analyzed). Comparisons used Mann Whitney test. Results: Mean Notch (43.2+/-7.2 vs 11.2 +/- 5.2; p < .0001), PI3K (54.6 +/- 10.0 vs 48.1 +/- 6.6, p < .0001) and androgen receptor (AR) pathway activities (32.8 +/- 3.7 vs 21.7 +/- 2.4, p < .0001) were increased in DF compared to fibroblasts. MAPK-AP1 pathway activity was lower in DF (40.8+/-6.0 vs 70.2 +/- 9.5; p < .0001). Mean Wnt (p = 0.7), HH (p = 0.8) pathway activities were not different between DF and fibroblasts, however the range in Wnt pathway activity scores was larger in DF (median score 24.1, range 5.3-41.7) than in normal fibroblasts (median 26.0, range 14.6-33.0). Conclusions: The increased activity of Notch, PI3K pathways in DF is consistent with the mesenchymal nature. We confirm here the key role of AR pathway. The wide range of Wnt pathway expression was larger, suggesting the heterogeneity in TD biology, possibly related to the type of CTTNB1 mutations. Overall, the large inter-patient variation in pathway activities is in line with variety of TD course. We identified signaling pathways which may provide novel options for targeted drug treatment.

Abstract P5-02-14: The MAPK pathway is variably active across breast cancer subtypes, and increased activity may be associated with hormonal therapy resistance

Abstract Background: Based on preclinical research, the MAPK growth factor pathway is thought to be an oncogenic pathway driving breast cancer, potentially in association with the ER pathway. In patients it is unclear how functional activity of the MAPK pathway activity relates to activity of the ER pathway to drive cancer growth. We have reported before on the development of assays, e.g. for the ER pathway, that enable quantitative measurement of activity of signal transduction pathways based on Bayesian computational model inference of pathway activity from measurements of mRNA levels of target genes of the ER transcription factor (Cancer Res., 2014, vol. 74, no. 11, pp. 2936-2945). We now present (1) development of the MAPK-AP1 pathway model, (2) MAPK-AP1 pathway activity and its relation to ER pathway activity in breast cancer; (3) emergence of MAPK pathway activity associated with hormonal therapy resistance. Methods: The MAPK-AP1 pathway model was developed and biologically validated on Affymetrix mRNA data from samples (different cell types) with a known MAPK pathway activity. MAPK-AP1 and ER pathway activity scores were measured on Affymetrix expression data of primary untreated breast cancer samples (GSE45827, n=141) and of fulvestrant sensitive and resistant MCF7 cell line clones (GSE74391). Results: In clinical breast cancer samples, average MAPK pathway activity was increased in all subtypes compared to normal breast tissue, with a large dynamic range indicating that in each subtype there are samples with high and with low pathway activity; ER pathway activity in Luminal A and B subtypes ranged from high to low, and was low in all other subtypes. MAPK-AP1 pathway activity did not correlate with ER pathway activity. Fulvestrant-sensitive MCF7 cells had high ER and low MAPK pathway activity scores, while resistant MCF7 clones exhibited increased MAPK pathway activity and complete loss of ER pathway activity, explaining therapy resistance. Discussion: The MAPK-AP1 pathway is frequently activated in all breast cancer subtypes, independent of ER pathway activity. We hypothesize that in case of resistance to hormonal therapy measurement of both pathway activities may be informative as to the underlying resistance mechanism, i.e. loss of ER pathway activity and/or gain of MAPK pathway activity. Future clinical studies are needed to confirm the role of these signaling pathways in hormonal resistance. Citation Format: Anja van de Stolpe, Laurent Holtzer, Yvonne Wesseling-Rozendaal, Wim Verhaegh. The MAPK pathway is variably active across breast cancer subtypes, and increased activity may be associated with hormonal therapy resistance [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-02-14.

GLP-1 inhibits PKCβ2 phosphorylation to improve the osteogenic differentiation potential of hPDLSCs in the AGE microenvironment

Background and objectiveAdvanced glycation end products (AGEs) have been hypothesized as the etiologic factors of diabetic periodontitis. The discovery of incretins (including GLP-1 and GIP) provides a novel therapy for the treatment of diabetes. Recent reports have shown that glucagon-like peptide-1 (GLP-1) is an important modulator of bone growth and remodeling. The aim of this study was to clarify the mechanism of how GLP-1 weakens/inhibits the effect of AGEs in hPDLSCs (human periodontal ligament stem cells). Materials and methodsThe hPDLSCs were cultured under simulated conditions of osteogenic culture, AGEs, AGEs + GLP-1, AGEs + GLP-1 + PMA and AGEs + GLP-1 + LY333531. The phenomenon and related mechanism of cell osteogenesis under different microenvironments were evaluated by Alizarin red staining, ALP staining and quantitative activity measurement, RT-qPCR, western blotting and immunofluorescence staining. ResultsRT-qPCR showed that AGEs negatively regulated the expression of osteogenic differentiation markers (ALP, BSP, OPN, and Runx2); in contrast, GLP-1 increased the expression of these markers. Furthermore, the expression of RAGE and pPKCβ (PKC phosphorylation) in the AGE group was upregulated, while the expression of RAGE and pPKCβ was decreased in the GLP-1 group compared with the AGE group. ConclusionsAGEs impaired the osteogenic potential of hPDLSCs via PKCβ2. Our phenomenon showed that GLP-1 could reverse the function of AGEs on osteogenic potential. In addition, the mechanism of GLP-1 weakens/inhibits the effect of AGEs in hPDLSCs, possibly by inhibiting PKCβ2 phosphorylation.

Quantitative Signal Characteristics of Electrocorticography and Stereoelectroencephalography: The Effect of Contact Depth.

Patients undergoing epilepsy surgery often require invasive EEG, but few studies have examined the signal characteristics of contacts on the surface of the brain (electrocorticography, ECOG) versus depth contacts, used in stereoelectroencephalography (SEEG). As SEEG and ECOG have significant differences in complication rates, it is important to determine whether both modalities produce similar signals for analysis, to ultimately guide management of medically intractable epilepsy. Twenty-seven patients who underwent SEEG (19), ECOG (6), or both (2) were analyzed for quantitative measures of activity including spectral power and phase-amplitude coupling during approximately 1 hour of wakefulness. The position of the contacts was calculated by coregistering the postoperative computed tomography with a reconstructed preoperative MRI. Using two types of referencing schemes-local versus common average reference-the brain regions where any quantitative measure differed systematically with contact depth were established. Using even the most permissive statistical criterion, few quantitative measures were significantly correlated with contact depth in either ECOG or SEEG contacts. The factors that predicted changes in spectral power and phase-amplitude coupling with contact depth were failing to baseline correct spectral power measures, use of a local rather than common average reference, using baseline correction for phase-amplitude coupling measures, and proximity of other grey matter structures near the region where the contact was located. The signals recorded by ECOG and SEEG have very similar spectral power and phase-amplitude coupling, suggesting that both modalities are comparable from an electrodiagnostic standpoint in delineation of the epileptogenic network.

Motor behavior characteristics in various phases of bipolar disorder revealed through biomechanical analysis: Quantitative measures of activity and energy variables during gait and sit-to-walk

Abnormal motor behaviors influenced by high or low energy states are key signs and symptoms for mania/hypomania or depression, respectively. Clinical evaluation is currently based on qualitative, subjective self-reports. We aimed to objectively quantify activity and energy variables during gait and sit-to-walk in bipolar disorder. Gait and sit-to-walk were analyzed in 31 individuals with bipolar disorder (five hypomanic, 14 euthymic and 12 depressed) and 14 healthy controls using a motion capture system and two force platforms. The 9-item Patient Health Questionnaire and Altman Self Rating Mania Scale were administered to evaluate mood symptoms. During gait and sit-to-walk, the hypomanic group had 20–30% greater movement speed and produced 10–60% greater peak force, and 40–140% greater peak power in the knee or ankle compared to the euthymic, depressed and healthy groups. Biomechanical measures of activity and energy correlated with clinically defined hypomania. Our findings suggest that movement speed and force production could serve as objective activity and energy markers for hypomanic symptoms in individuals with bipolar disorder, but this study was based on a relatively small sample size, and the laboratory-based assessments are not directly transferable to a clinical setting.

Integrated PET/MR breast cancer imaging: Attenuation correction and implementation of a 16-channel RF coil.

This study aims to develop, implement, and evaluate a 16-channel radiofrequency (RF) coil for integrated positron emission tomography/magnetic resonance (PET/MR) imaging of breast cancer. The RF coil is designed for optimized MR imaging performance and PET transparency and attenuation correction (AC) is applied for accurate PET quantification. A 16-channel breast array RF coil was designed for integrated PET/MR hybrid imaging of breast cancer lesions. The RF coil features a lightweight rigid design and is positioned with a spacer at a defined position on the patient table of an integrated PET/MR system. Attenuation correction is performed by generating and applying a dedicated 3D CT-based template attenuation map. Reposition accuracy of the RF coil on the system patient table while using the positioning frame was tested in repeated measurements using MR-visible markers. The MR, PET, and PET/MR imaging performances were systematically evaluated using modular breast phantoms. Attenuation correction of the RF coil was evaluated with difference measurements of the active breast phantoms filled with radiotracer in the PET detector with and without the RF coil in place, serving as a standard of reference measurement. The overall PET/MR imaging performance and PET quantification accuracy of the new 16-channel RF coil and its AC were then evaluated in first clinical examinations on ten patients with local breast cancer. The RF breast array coil provides excellent signal-to-noise ratio and signal homogeneity across the volume of the breast phantoms in MR imaging and visualizes small structures in the phantoms down to 0.4 mm in plane. Difference measurements with PET revealed a global loss and thus attenuation of counts by 13% (mean value across the whole phantom volume) when the RF coil is placed in the PET detector. Local attenuation ranging from 0% in the middle of the phantoms up to 24% was detected in the peripheral regions of the phantoms at positions closer to attenuating hardware structures of the RF coil. The position accuracy of the RF coil on the patient table when using the positioning frame was determined well below 1 mm for all three spatial dimensions. This ensures perfect position match between the RF coil and its three-dimensional attenuation template during the PET data reconstruction process. When applying the CT-based AC of the RF coil, the global attenuation bias was mostly compensated to ±0.5% across the entire breast imaging volume. The patient study revealed high quality MR, PET, and combined PET/MR imaging of breast cancer. Quantitative activity measurements in all 11 breast cancer lesions of the ten patients resulted in increased mean difference values of SUVmax 11.8% (minimum 3.2%; maximum 23.2%) between nonAC images and images when AC of the RF breast coil was applied. This supports the quantitative results of the phantom study as well as successful attenuation correction of the RF coil. A 16-channel breast RF coil was designed for optimized MR imaging performance and PET transparency and was successfully integrated with its dedicated attenuation correction template into a whole-body PET/MR system. Systematic PET/MR imaging evaluation with phantoms and an initial study on patients with breast cancer provided excellent MR and PET image quality and accurate PET quantification.

Quantitative Activity Measurements of Brown Adipose Tissue at 7 T Magnetic Resonance Imaging After Application of Triglyceride-Rich Lipoprotein 59Fe-Superparamagnetic Iron Oxide Nanoparticle: Intravenous Versus Intraperitoneal Approach.

The aim of this study was to determine metabolic activity of brown adipose tissue (BAT) with in vivo magnetic resonance imaging (MRI) after intravenous (IV) and intraperitoneal (IP) injection of radioactively labeled superparamagnetic iron oxide nanoparticles (SPIOs) embedded into a lipoprotein layer. Fe-labeled SPIOs were either polymer-coated or embedded into the lipid core of triglyceride-rich lipoproteins (TRL-Fe-SPIOs). First biodistribution and blood half time analysis in thermoneutral mice after IP injection of either TRL-Fe-SPIOs or polymer-coated Fe-SPIOs (n = 3) were performed. In the next step, cold-exposed (24 hours), BAT-activated mice (n = 10), and control thermoneutral mice (n = 10) were starved for 4 hours before IP (n = 10) or IV (n = 10) injection of TRL-Fe-SPIOs. In vivo MRI was performed before and 24 hours after the application of the particles at a 7 T small animal MRI scanner using a T2*-weighted multiecho gradient echo sequence. R2* and ΔR2* were estimated in the liver, BAT, and muscle. The biodistribution of polymer-coated Fe-SPIOs and TRL-Fe-SPIOs was analyzed ex vivo using a sensitive, large-volume Hamburg whole-body radioactive counter. The amount of Fe-SPIOs in the liver, BAT, and muscle was correlated with the MRI measurements using the Pearson correlation coefficient. Tissue uptake of Fe-SPIOs was confirmed by histological and transmission electron microscopy analyses. Triglyceride-rich lipoprotein Fe-SPIOs exhibited a higher blood concentration after IP injection (10.1% ± 0.91% after 24 hours) and a greater [INCREMENT]R2* in the liver (103 ± 5.0 s), while polymer-coated SPIOs did not increase substantially in the blood stream (0.19% ± 0.01% after 24 hours; P < 0.001) and the liver (57 ± 4.08 s; P < 0.001). In BAT activity studies, significantly higher uptake of TRL-Fe-SPIOs was detected in the BAT of cold-exposed mice, with [INCREMENT]R2* of 107 ± 5.5 s after IV application (control mice: [INCREMENT]R2* of 22 ± 5.8 s; P < 0.001) and 45 ± 5.5 s after IP application (control mice: [INCREMENT]R2* of 11 ± 2.9 s; P < 0.01). Fe radioactivity measurements and [INCREMENT]R2* values correlated strongly in BAT (r > 0.85; P < 0.001) and liver tissue (r > 0.85; P < 0.001). Histological and transmission electron microscopy analyses confirmed the uptake of TRL-Fe-SPIOs within the liver and BAT for both application approaches. Triglyceride-rich lipoprotein-embedded SPIOs were able to escape the abdominal cavity barrier, whereas polymer-coated SPIOs did not increase substantially in the blood stream. Brown adipose tissue activity can be determined via MRI using TRL-Fe-SPIOs. The quantification of [INCREMENT]R2* using TRL-Fe-SPIOs is feasible and may serve as a noninvasive tool for the quantitative estimation of BAT activity.

Cherenkov luminescence measurements with digital silicon photomultipliers: a feasibility study.

BackgroundA feasibility study was done to assess the capability of digital silicon photomultipliers to measure the Cherenkov luminescence emitted by a β source. Cherenkov luminescence imaging (CLI) is possible with a charge coupled device (CCD) based technology, but a stand-alone technique for quantitative activity measurements based on Cherenkov luminescence has not yet been developed. Silicon photomultipliers (SiPMs) are photon counting devices with a fast impulse response and can potentially be used to quantify β-emitting radiotracer distributions by CLI.MethodsIn this study, a Philips digital photon counting (PDPC) silicon photomultiplier detector was evaluated for measuring Cherenkov luminescence. The PDPC detector is a matrix of avalanche photodiodes, which were read one at a time in a dark count map (DCM) measurement mode (much like a CCD). This reduces the device active area but allows the information from a single avalanche photodiode to be preserved, which is not possible with analog SiPMs. An algorithm to reject the noisiest photodiodes and to correct the measured count rate for the dark current was developed.ResultsThe results show that, in DCM mode and at (10–13) °C, the PDPC has a dynamic response to different levels of Cherenkov luminescence emitted by a β source and transmitted through an opaque medium. This suggests the potential for this approach to provide quantitative activity measurements. Interestingly, the potential use of the PDPC in DCM mode for direct imaging of Cherenkov luminescence, as a opposed to a scalar measurement device, was also apparent.ConclusionsWe showed that a PDPC tile in DCM mode is able to detect and image a β source through its Cherenkov radiation emission. The detector’s dynamic response to different levels of radiation suggests its potential quantitative capabilities, and the DCM mode allows imaging with a better spatial resolution than the conventional event-triggered mode. Finally, the same acquisition procedure and data processing could be employed also for other low light levels applications, such as bioluminescence.Electronic supplementary materialThe online version of this article (doi:10.1186/s40658-015-0134-z) contains supplementary material, which is available to authorized users.

Broad distribution and high proportion of protein synthesis active marine bacteria revealed by click chemistry at the single cell level

Marine bacterial and archaeal communities control global biogeochemical cycles through nutrient acquisition processes that are ultimately dictated by the metabolic requirements of individual cells. Currently lacking, however, is a sensitive, quick, and quantitative measurement of activity in these single cells. We tested the applicability of copper (I)-catalyzed cycloaddition, or “click”, chemistry to observe and estimate single-cell protein synthesis activity in natural assemblages and isolates of heterotrophic marine bacteria. Incorporation rates of the non-canonical methionine bioortholog L-homopropargylglycine (HPG) were quantified within individual cells by measuring fluorescence of alkyne-conjugated Alexa Fluor® 488 using epifluorescence microscopy. The method’s high sensitivity, along with a conversion factor derived from two Alteromonas spp. Isolates, revealed a broad range of cell-specific protein synthesis within natural microbial populations. Comparison with 35S-methionine microautoradiography showed that a large fraction of the natural marine bacterial assemblage (15-100%), previously considered inactive by autoradiography, were actively synthesizing protein. Data pooled from a large number of samples showed that cell-specific activity scaled logarithmically with cell volume. Assemblage activity distributions of each sample were fit to power-law functions, providing an illustrative and quantitative comparison of assemblages that demonstrate individual protein synthesis rates were commonly partitioned between cells in low- and high-metabolic states in our samples. The HPG method offers a simple potential approach to link individual cell physiology to the ecology and biogeochemistry of bacterial (micro)environments in the ocean.

Novel Point-Of-Use FXa Assay For Monitoring Anticoagulant Therapy By Oral Factor Xa Inhibitors and Heparin

Introduction A highly specific, rapid, robust and sensitive assay for Factor Xa (FXa) activity will greatly improve monitoring and optimization of anticoagulant therapy by direct FXa inhibitors and by low molecular weight (LMW) heparins. We have developed a new point-of-use diagnostic system that will provide results in less than 30 minutes, enabling rapid medical decisions based on quantitation of FXa activity and anticoagulant drugs in patients’ blood. Anticoagulant therapy with unfractionated or LMW heparins needs aggressive monitoring in pregnant women, patients with renal insufficiency, and neonates, in particular in cases of treatment-related side effects, including heparin-induced thrombocytopenia and bleeding tendency (Chest, 2008). New oral anticoagulants rivaroxaban (Xarelto®) and apixaban (Eliquis®) have been effective in reducing risk of stroke and systemic embolism in patients with nonvalvular atrial fibrillation, and in preventing and treating deep vein thrombosis. Both drugs directly inhibit FXa and have significant therapeutic potential as an alternative to warfarin. Unlike warfarin, these drugs do not require constant monitoring and dose adjustment to maintain anticoagulation within the therapeutic interval. However, excessive anticoagulation and consequent hemorrhage risks have been sufficiently common problems to warrant clinical development of specific antidotes for FXa inhibitors. Currently available anti-Xa heparin assays may not be appropriate for rivaroxaban and apixaban measurements. Additionally, recently published data indicate that new oral anticoagulants interfere with the measurement of common test parameters, expanding the need for more specific diagnostic tests (Clinical Chemistry, February 2013). This novel rapid and specific FXa assay will allow monitoring LMW heparins and the new FXa inhibitors, identify heparin-refractory patients as well as those requiring antidotes to oral Factor Xa inhibitors, in addition to directly measuring FXa activity in patients with inherited deficiency of Factor X, congenital antithrombin deficiency, and acquired FXa deficiencies. Method A diagnostic assay to measure FXa activity in human plasma using electrochemiluminescence (ECL) technology was developed at Wellstat Diagnostics, LLC. The assay utilizes a synthetic peptide containing an FXa substrate sequence that is recognized by specific antibodies (labeled with an ECL-active ruthenium chelate) only after specific cleavage by FXa protease. The resulting ECL signal is directly proportional to FXa activity within the physiological range. The assay has been adapted to also provide quantitative measurement of activity of anticoagulant drugs that inhibit FXa. In the modified format the resulting ECL signal is inversely proportional to the concentration of rivaroxaban, apixaban or heparin in plasma. Results The ECL-based assay measures activity of anticoagulant drugs on FXa in human plasma with high intra- and inter-assay reproducibility, accuracy and specificity. Using FX-deficient plasma spiked with Factor X in the range 1-100 ng/mL, we have demonstrated linear increases of ECL signal directly proportional to the concentration of spiked Factor X activated by a specific Russell Viper Venom activator. Serial dilutions of anticoagulant drugs rivaroxaban, apixaban and enoxaparin (LMW heparin) in normal donor plasma showed exponential decreases of ECL signal in the range of concentrations relevant to the therapeutic doses of all three drugs. The dynamic range of the drug detection was 15 - 500 ng/mL for rivaroxaban and apixaban, and 0.2 - 2 IU/mL of enoxaparin. The assay was specific to activity of FXa, while insensitive to variable concentrations of other clotting factors in human plasma, as well as to the presence of other drugs in plasma that don’t directly inhibit FXa. Conclusions The point-of-use FXa assay is shown to be highly specific, robust, rapid and reproducible for measurements of anticoagulant activity of apixaban, rivaroxaban and enoxaparin in human plasma. Use of this point-of-use system for monitoring anticoagulant therapy may be further extended to measure other blood clotting factors (for example, Factor IXa, VIIa, FVIII), for monitoring activity of drugs acting on these factors and for rapid diagnosis of coagulation disorders. Disclosures: No relevant conflicts of interest to declare.

A simple fluorometric assay for DNA exonuclease activity based on graphene oxide

A new assay platform for DNA exonuclease activity is developed based on the preferential binding of single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA) to graphene oxide. This approach allows a simple and quantitative activity measurement in a short time at low cost.

An audit of the maternity triage service at Birmingham Women's NHS Foundation Trust

Birmingham Women's NHS Foundation Trust established a triage service in 2004 in response to the large numbers of women who presented for an assessment. These assessments were not always labour...

Evaluation of Enzymatic Activity on Nanoscale Polystyrene-block-Polymethylmethacrylate Diblock Copolymer Domains

Understanding structural and functional changes of polymeric surface-bound proteins is extremely important as polymers play an increasingly significant role as arrays and substrates in proteomics applications. We carried out, for the first time, quantitative activity measurements of horseradish peroxidase (HRP) enzymes immobilized selectively on the polystyrene domains of microphase-separated polystyrene-block-polymethylmethacrylate ultrathin films. The specific enzymatic activity of HRP adsorbed on the diblock copolymer surface was evaluated and compared to that of HRP in free solution. We demonstrate that the polymeric surface-bound HRP molecules maintain approximately 85% of their activity in free solution. The unique advantages of diblock copolymer templates, involving nanoscale self-assembly and largely retained protein functionality, make the spontaneously constructed enzyme nanoarrays highly suitable as proteomics substrates. Our novel assembly method of providing functional enzymes on diblock copolymer thin films can be greatly beneficial for high-throughput and high-density protein assays.

Accuracy and uncertainty in radioactivity measurement for NAA

The basic assumptions of activation analysis are that the induced radioactivity is proportional to the amount of analyte, and that the quantity of radioactivity can be related simply to the number of counts observed. Quantitative measurement of activity (and of its uncertainty) is not always simple, especially when accuracy better than a few percent is sought. Recent work with 77Ge and 76As has demonstrated that the accuracy of half-lives in the literature is sometimes insufficient. Despite these and other problems, quantitative understanding and documentation of uncertainties can be accomplished, providing demonstrable quality assurance and supporting claims of traceability to the Systeme International.

Intérêt du E-test dans le suivi de l'antibiothérapie

Since the nineties, ETEST has become a widely accepted alternative quantitative susceptibility testing method for bacteria including difficult to assess organisms such as Mycobacterium. Reference dilution test using broth or agar are generally difficult to perform. Accurate quantitative susceptibility testing is very important to guide antibiotic therapy in many circumstances. ETEST method is rapid and easy, available for quantitative determination of antibiotic susceptibility testing, detection of resistance mechanisms, detection of synergy or antagonism in antibiotic combination tests and useful in many other situations. ETEST appears to be an excellent alternative to reference and standardized method for producing quantitative activity measurement.

Mental health nursing practice in acute psychiatric close-observation areas.

The provision of evidence-based therapeutic nursing care in close-observation units or psychiatric intensive care units, has been identified as a problem internationally. These areas of nursing practice have been the subject of considerable discussion particularly in relation to the management of aggression, violence, involuntary treatment, and seclusion. This study used a participatory action research framework to identify qualitative and quantitative measures of activity in the area. Quantitative data collected included rates of critical incidents, the use of prn medication, and the use of seclusion. These data were used as base-line data and were predicted as a measure of change. Qualitative data, collected by interview and focus groups, were used to reveal the experience of patients, relatives, and nurses in a close-observation area. Analysis of this data revealed three main themes: design and environment, lack of activity and structured time, and nursing care. The importance of this study is in demonstrating the multiple problems that exist in the provision of care in close-observation areas and the corresponding need for fundamental changes.