Quantification Of Receptors Research Articles

Expression mapping, quantification, and complex formation of GluD1 and GluD2 glutamate receptors in adult mouse brain.

In the cerebellum, GluD2 is exclusively expressed in Purkinje cells, where it regulates synapse formation and regeneration, synaptic plasticity, and motor learning. Delayed cognitive development in humans with GluD2 gene mutations suggests extracerebellar functions of GluD2. However, extracerebellar expression of GluD2 and its relationship with that of GluD1 are poorly understood. GluD2 mRNA and protein were widely detected, with relatively high levels observed in the olfactory glomerular layer, medial prefrontal cortex, cingulate cortex, retrosplenial granular cortex, olfactory tubercle, subiculum, striatum, lateral septum, anterodorsal thalamic nucleus, and arcuate hypothalamic nucleus. These regions were also enriched for GluD1, and many individual neurons coexpressed the two GluDs. In the retrosplenial granular cortex, GluD1 and GluD2 were selectively expressed at PSD-95-expressing glutamatergic synapses, and their coexpression on the same synapses was shown by SDS-digested freeze-fracture replica labeling. Biochemically, GluD1 and GluD2 formed coimmunoprecipitable complex formation in HEK293T cells and in the cerebral cortex and hippocampus. We further estimated the relative protein amount by quantitative immunoblotting using GluA2/GluD2 and GluA2/GluD1 chimeric proteins as standards for titration of GluD1 and GluD2 antibodies. Intriguingly, the relative amount of GluD2 was almost comparable to that of GluD1 in the postsynaptic density fraction prepared from the cerebral cortex and hippocampus. In contrast, GluD2 was overwhelmingly predominant in the cerebellum. Thus, we have determined the relative extracerebellar expression of GluD1 and GluD2 at regional, neuronal, and synaptic levels. These data provide a molecular-anatomical basis for possible competitive and cooperative interactions of GluD family members at synapses in various brain regions.

Investigation of Allocar TTM Targeting CD70 As a Potential Therapy for an Array of Hematological Malignancies

Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) is an exciting new therapy showing great promise in hematologic malignancies. Recent approval of two CD19-targeting CAR Ts, Kymriah® and Yescarta®, has been followed with promising results from BCMA clinical trials, showing that activity can extend to other targets. While these treatments show excellent potential for patients with an otherwise poor prognosis, a number of patients are still subject to relapse. These data suggest the potential utility of expanding the number of targets available for hematological malignancies. Autologous chimeric antigen receptor (CAR) T therapies use a patient's own T cells to produce the therapy. This approach has inherent challenges, including requiring significant time for production, complex supply chain logistics, separate GMP manufacturing for each patient, and variability in performance of patient-derived cells. Given rapid disease progression, combined with limitations associated with the autologous approach, many patients may not be able to receive CAR T therapy. Allogeneic CAR T (AlloCAR T) cell therapy, or "off-the-shelf" therapy which utilizes cells from healthy donors, may provide greater convenience with readily available CAR T therapy on-demand, reliable product consistency, and accessibility at greater scale for more patients. To create an allogeneic product, mRNAs encoding Transcription Activator-Like Effector Nucleases (TALEN®) designed to specifically disrupt the TRAC and CD52 genes are introduced to CAR Ts. These modifications are intended to minimize the risk of graft-versus-host disease and to confer resistance to ALLO-647, an anti-CD52 antibody that can be used as part of the conditioning regimen to deplete host alloreactive immune cells potentially leading to increased persistence and efficacy of the infused allogeneic cells. CD70 is a TNF superfamily member expressed on subsets of activated lymphocytes and is being explored as a CAR T target for renal cell carcinoma but may also be suitable for targeting hematological malignancies. To evaluate the potential of CD70 as a heme target, expression of CD70 RNA was compared to that of three known targets, CD19, BCMA, and FLT3, across a range of hematological malignancies by interrogating The Cancer Genome Atlas (TCGA) database. CD19 was expressed in lymphomas and leukemias, BCMA was expressed primarily in MM, and FLT3 expression was largely limited to AML. CD70 expression was observed across all 4 cancer types, indicating the potential broad utility CD70 CAR T cells. To determine cell-surface expression of these four targets, flow cytometry and receptor quantification was performed on a panel of heme cell lines. Good correlation was seen between RNA and the cell-surface protein expression of CD70. CAR T cells against each of the 4 targets were generated and evaluated in vitro. All CAR T cells exhibited robust specific activity against cells expressing their corresponding antigens. To ensure clinical relevance of results with cell lines, studies are currently ongoing to evaluate CD70 expression on primary patient samples and activity of CD70 CAR T cells against these same samples. Based on our studies, CD70 has a broad expression profile across a range of hematological malignancies and potent and selective CD70 CAR T activity has been demonstrated. Given these results, an allogeneic CD70 CAR T is expected to be clinically useful against a range of hematological tumor types either as a single agent or in combination with other CAR Ts. Disclosures Panowski: Allogene Therapeutics: Employment, Equity Ownership. Srinivasan:Allogene Therapeutics: Employment, Equity Ownership. Van Blarcom:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Sommer:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Cheng:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Pertel:Allogene Therapeutics: Employment, Equity Ownership. Santaguida:Notable Labs: Employment. Galetto:Cellectis Inc: Employment. Sasu:Allogene Therapeutics, Inc.: Employment, Equity Ownership.

Expression mapping, quantification, and complex formation of GluD1 and GluD2 glutamate receptors in adult mouse brain

Expression mapping, quantification, and complex formation of GluD1 and GluD2 glutamate receptors in adult mouse brain

Targeted Proteomics Allows Quantification of Ethylene Receptors and Reveals SlETR3 Accumulation in Never-Ripe Tomatoes.

Ethylene regulates fruit ripening and several plant functions (germination, plant growth, plant-microbe interactions). Protein quantification of ethylene receptors (ETRs) is essential to study their functions, but is impaired by low resolution tools such as antibodies that are mostly nonspecific, or the lack of sensitivity of shotgun proteomic approaches. We developed a targeted proteomic method, to quantify low-abundance proteins such as ETRs, and coupled this to mRNAs analyses, in two tomato lines: Wild Type (WT) and Never-Ripe (NR) which is insensitive to ethylene because of a gain-of-function mutation in ETR3. We obtained mRNA and protein abundance profiles for each ETR over the fruit development period. Despite a limiting number of replicates, we propose Pearson correlations between mRNA and protein profiles as interesting indicators to discriminate the two genotypes: such correlations are mostly positive in the WT and are affected by the NR mutation. The influence of putative post-transcriptional and post-translational changes are discussed. In NR fruits, the observed accumulation of the mutated ETR3 protein between ripening stages (Mature Green and Breaker + 8 days) may be a cause of NR tomatoes to stay orange. The label-free quantitative proteomics analysis of membrane proteins, concomitant to Parallel Reaction Monitoring analysis, may be a resource to study changes over tomato fruit development. These results could lead to studies about ETR subfunctions and interconnections over fruit development. Variations of RNA-protein correlations may open new fields of research in ETR regulation. Finally, similar approaches may be developed to study ETRs in whole plant development and plant-microorganism interactions.

Targeted Treatment Options of Recurrent Radioactive Iodine Refractory Hürthle Cell Cancer.

Objective: To evaluate the efficacy and treatment rationale of Hürthle cell carcinoma (HCC) following a patient with progressive and metastatic HCC. HCC was recently shown to harbor a distinct genetic make-up and the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kiase (PI3K)/AKT signaling pathways are potential targets for anti-cancer agents in the management of recurrent HCC. The presence or absence of gene variants can give a rationale for targeted therapies that could be made available in the context of drug repurposing trials. Methods: Treatment included everolimus, sorafenib, nintedanib, lenvatinib, and panitumumab. Whole genome sequencing (WGS) of metastatic tumor material obtained before administration of the last drug, was performed. We subsequently evaluated the rationale and efficacy of panitumumab in thyroid cancer and control cell lines after epidermal growth factor (EGF) stimulation and treatment with panitumumab using immunofluorescent Western blot analysis. EGF receptor (EGFR) quantification was performed using flow cytometry. Results: WGS revealed a near-homozygous genome (NHG) and a somatic homozygous TSC1 variant, that was absent in the primary tumor. In the absence of RAS variants, panitumumab showed no real-life efficacy. This might be explained by high constitutive AKT signaling in the two thyroid cancer cell lines with NHG, with panitumumab only being a potent inhibitor of pEGFR in all cancer cell lines tested. Conclusions: In progressive HCC, several treatment options outside or inside clinical trials are available. WGS of metastatic tumors might direct the timing of therapy. Unlike other cancers, the absence of RAS variants seems to provide insufficient justification of single-agent panitumumab administration in HCC cases harboring a near-homozygous genome.

Intervention changes acoustic peak frequency and mesolimbic neurochemistry in the Pink1-/- rat model of Parkinson disease.

The neural mechanisms underlying behavioral therapy for vocal acoustic deficits in patients with Parkinson disease is unknown. A primary hypothesis is that voice therapy may modulate mesolimbic brainstem regions, including the ventral tegmental area (VTA). The VTA is implicated in ultrasonic call peak frequency, involved in rewarding behaviors, and impacted by Parkinsonism. We tested the hypothesis that chronic (daily) behavioral vocal exercise of male Pink1-/- rats would alter ultrasonic vocalization acoustics and mesolimbic neurochemistry (catecholamines, GABA, mu-opioid receptor) compared to three different controls: sham-exercised Pink1-/-, unexercised Pink1-/-, and unexercised wildtype (WT) rats. A sub-hypothesis is that sham-exercise rats may exhibit changes to VTA neurochemistry as a result of a type or rewarding intervention. Results demonstrate that average bandwidth (frequency range) of ultrasonic vocalizations did not differ between WT, Pink1-/- no exercise vs. sham and vocal-exercise rats. However, average peak frequency is significantly reduced in vocal-exercised Pink1-/- rats compared to Pink1-/- no exercise, and WT groups. Unexpectedly, there were no significant acoustic differences between the vocal- and sham-exercised groups. There were no differences in catecholamine protein concentrations or tyrosine hydroxylase mRNA expression in the VTA between any of the groups. However, there was significant upregulation of all GABA-related genes in both vocal- and sham-exercised Pink1-/- rats (Gad1, Gad2, Gls, Glul); this finding was confirmed with follow up quantitative Western blotting for GAD. Additionally, there were differential results for mu-opioid receptor quantification in the VTA: vocal-exercised Pink1-/- rats showed increased mRNA expression for mu-opioid receptors whereas Western blotting indicated decreased protein levels in all Pink1-/- rats compared to WT controls suggesting the possible onset of pathology in this model. These data demonstrate modulatory effects of a rewarding behavioral paradigm on ultrasonic vocalization peak frequency. The results suggest that neuromodulators such as GABA and opioid activity, as well as the rewarding aspects of therapy may play a key role in shaping vocal treatments.

Isophthaloyl-Based Selective Fluorescence Receptor for Zn (II) Ion in Semi-Aqueous Medium.

A novel Isophthaloyl-based symmetrical (12E,21E)-N1',N3'-bis(2-hydroxybenzylidene) isophthalohydrazide, receptor (1) was synthesized and characterized using various spectroscopic technique. The reorganization ability of receptor (1) was evaluated in semi-aqueous medium and shows significant enhancement in fluorescence intensity for Zn (II) ion over various metal ions in CH3CN:H2O (1:1, v/v). The 1:2 binding stoichiometry between receptor (1) and Zn (II) ion was established using Job's plot and the proposed complex structure was calculated by applying Density Functional Theory (DFT) method. The binding constant (Ka) of receptor (1) with Zn (II) ion was established with the Benesi-Hildebrand plot, Scatchard and Connor's plot and the values are 1.00 × 104M-1, 1.05× 104M-1 and 1.05× 104M-1 respectively. The limit of detection (LOD) and limit of quantification (LOQ) of receptor (1) and Zn (II) ion was 0.292μM and 0.974μM respectively. The binding mode was due to photo-induced electron transfer (PET) and the coordination of Zn (II) ion with C = N hydroxyl group of receptor (1). Electrochemical analysis of metal free receptor (1) and with Zn (II) ion also confirmed the formation of complex.

11C]JNJ54173717, a novel P2X7 receptor radioligand as marker for neuroinflammation: human biodistribution, dosimetry, brain kinetic modelling and quantification of brain P2X7 receptors in patients with Parkinson's disease and healthy volunteers.

The P2X7 receptor (P2X7R) is an ATP-gated ion channel predominantly expressed on activated microglia and is important in neurodegenerative diseases including Parkinson's disease (PD). In this first-in-human study, we investigated [11C]JNJ54173717 ([11C]JNJ717), a selective P2X7R tracer, in healthy volunteers (HV) and PD patients. Biodistribution, dosimetry, kinetic modelling and short-term test-retest variation (TRV), as well as possible genotype effects, were investigated. Biodistribution and radiation dosimetry studies were performed in threeHV (mean age 30 ± 2years, two women) using whole-body PET/CT. The most appropriate kinetic model was determined in 11HV (mean age 62 ± 10years, six women) and 10 PD patients (mean age 64 ± 8years, three women; mean UPDRS motor score 21 ± 8) using 90-min dynamic simultaneous PET/MR scans. The total volume of distribution (VT) was calculated using a one-tissue and a two-tissue compartment model (1TCM, 2TCM) and Logan graphical analysis, and its time stability was assessed. Seven subjects underwent retest scans (mean age 60 ± 13years, fourHV, one woman). A group analysis was performed to compare PD patients and HV. Finally, 13 exons of P2X7R were genotyped in all subjects included in the second part of the study. The mean effective dose was 4.47 ± 0.32μSv/MBq, with the highest absorbed doses to the gallbladder, liver and small intestine. A reversible 2TCM was the most appropriate kinetic model with relatively homogeneous VT values in the grey and white matter. Average VT values were 3.4 ± 0.8 in HV and 3.3 ± 0.7 in PD patients, with no significant difference between the groups, but a possible genotype effect (rs3751143) was identified which can affect VT. Average TRV was 10-15%. The stability of VT over time allowed a reduction in scan time to 70min. [11C]JNJ717 is safe and suitable for quantifying P2X7R expression in human brain. In this pilot study, no significant differences in P2X7R binding were found between HV and PD patients. The results also suggest that genotype effects need to be incorporated in future P2X7R PET analyses.

Single-Cell Receptor Quantification of an In Vitro Coculture Angiogenesis Model Reveals VEGFR, NRP1, Tie2, and PDGFR Regulation and Endothelial Heterogeneity

Angiogenesis, the formation of new blood vessels from pre-existing ones, is essential for both normal development and numerous pathologies. Systems biology has offered a unique approach to study angiogenesis by profiling tyrosine kinase receptors (RTKs) that regulate angiogenic processes and computationally modeling RTK signaling pathways. Historically, this systems biology approach has been applied on ex vivo angiogenesis assays, however, these assays are difficult to quantify and limited in their potential of temporal analysis. In this study, we adopted a simple two-dimensional angiogenesis assay comprised of human umbilical vein endothelial cells (HUVECs) and human dermal fibroblasts (HDFs) and examined temporal dynamics of a panel of six RTKs and cell heterogeneity up to 17 days. We observed ~2700 VEGFR1 (vascular endothelial growth factor receptor 1) per cell on 24-h-old cocultured HDF plasma membranes, which do not express VEGFR when cultured alone. We observed 4000–8100 VEGFR2 per cell on cocultured HUVEC plasma membranes throughout endothelial tube formation. We showed steady increase of platelet-derived growth factor receptors (PDGFRs) on cocultured HDF plasma membranes, and more interestingly, 1900–2900 PDGFRβ per plasma membrane were found on HUVECs within the first six hours of coculturing. These quantitative findings will offer us insights into molecular regulation during angiogenesis and help assess in vitro tube formation models and their physiological relevance.

Quantitative Rodent Brain Receptor Imaging

Positron emission tomography (PET) is a non-invasive imaging technology employed to describe metabolic, physiological, and biochemical processes in vivo. These include receptor availability, metabolic changes, neurotransmitter release, and alterations of gene expression in the brain. Since the introduction of dedicated small-animal PET systems along with the development of many novel PET imaging probes, the number of PET studies using rats and mice in basic biomedical research tremendously increased over the last decade. This article reviews challenges and advances of quantitative rodent brain imaging to make the readers aware of its physical limitations, as well as to inspire them for its potential applications in preclinical research. In the first section, we briefly discuss the limitations of small-animal PET systems in terms of spatial resolution and sensitivity and point to possible improvements in detector development. In addition, different acquisition and post-processing methods used in rodent PET studies are summarized. We further discuss factors influencing the test-retest variability in small-animal PET studies, e.g., different receptor quantification methodologies which have been mainly translated from human to rodent receptor studies to determine the binding potential and changes of receptor availability and radioligand affinity. We further review different kinetic modeling approaches to obtain quantitative binding data in rodents and PET studies focusing on the quantification of endogenous neurotransmitter release using pharmacological interventions. While several studies have focused on the dopamine system due to the availability of several PET tracers which are sensitive to dopamine release, other neurotransmitter systems have become more and more into focus and are described in this review, as well. We further provide an overview of latest genome engineering technologies, including the CRISPR/Cas9 and DREADD systems that may advance our understanding of brain disorders and function and how imaging has been successfully applied to animal models of human brain disorders. Finally, we review the strengths and opportunities of simultaneous PET/magnetic resonance imaging systems to study drug-receptor interactions and challenges for the translation of PET results from bench to bedside.

Multiplexing Angiogenic Receptor Quantification via Quantum Dots.

Clinical and biomedical research seeks single-cell quantification to better understand their roles in a complex, multicell environment. Recently, quantification of vascular endothelial growth factor receptors (VEGFRs) provided important insights into endothelial cell characteristics and response in tumor microenvironments. However, existing technologies for quantifying plasma membrane receptor tyrosine kinases (RTKs) lack multiplexing capabilities, limiting detailed characterization. Here, we use the unique spectral properties of quantum dots (Qdots) to optimize and dually quantify VEGFR1 and VEGFR2 on human umbilical vein endothelial cells (HUVECs). To enable this quantification, we reduce nonspecific binding between Qdot-conjugated antibodies and cells via buffer optimization. Second, we identify optimal labeling conditions by examining Qdot-conjugated antibody binding to five receptors: VEGFRs (VEGFR1 and VEGFR2), their coreceptor neuropilin1 (NRP1), and platelet-derived growth factor receptor (PDGFRα and PDGFRβ). We establish that 800-20 000 is the dynamic range where accurate Qdot-enabled quantification can be achieved. Through these optimizations, we demonstrate measurement of 1 100 VEGFR1 and 6 900 VEGFR2 per HUVEC. We induce ∼90% upregulation of VEGFR1 and ∼30% downregulation of VEGFR2 concentration via 24 h VEGF-A165 treatment. We observe no change in VEGFR1 or VEGFR2 concentration with 24 h VEGF-B167 treatment. We further apply Qdots to analyze HUVEC heterogeneity and observe that 24 h VEGF-A165 treatment induces a ∼15% decrease in VEGFR2 heterogeneity, but little to no change in VEGFR1 heterogeneity. We observe that VEGF-B167 induces little to no change in either VEGFR1 or VEGFR2 heterogeneity. Overall, we demonstrate experimental and analytical strategies for quantifying two or more RTKs at single-level using Qdots, which will help provide new insights into biological systems.

Quantification of IGF-1 Receptor Is Useful in the Differential Diagnosis of Essential Thrombocytosis from Reactive Thrombocytosis

To make a definite diagnosis of essential thrombocytosis (ET) from reactive thrombocytosis (RT), the most reliable criteria are the presence of driver mutations, namely JAK2, CALR, or MPL gene mutations. In the absence of these driver mutations, so-called triple-negative ET, the differential diagnosis could be difficult; bone marrow biopsy could be helpful, but in some cases it may be difficult, to do gene sequence analysis to find other clonal marker gene mutations than the driver mutations, only very few were found. We studied IGF-1R quantification by flow cytometry in mononuclear cells (MNC) from peripheral blood, a substitute for performing endogenous erythroid colony formation (EEC), in 33 patients with ET (untreated or off treatment with hydroxyurea), 28 patients with RT, and 16 normal volunteer controls. We found IGF-1R levels significantly elevated in ET patients compared to RT patients or controls. A cutoff value of 253 was chosen from the logistic regression to predict each patient's group, a value ≥253 meant that a patient belonged to the ET group (sensitivity of 68.6% and specificity 96.4.%). Therefore, we suggest adding quantification of IGF-1R in blood MNC by flow cytometry is useful in differentiating ET from RT , we suggest this maybe added to the minor criteria for diagnosing ET. Funding Statement: JCW received grants from Celgen Corp. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Declaration of Interests: The author declares no conflicting interests with the commercial funder relating to using products, consultancy, patents, product development, or market products. The other authors declare no competing financial interests. Ethics Approval Statement: Peripheral blood was obtained from patients with written informed consent, the protocol for which was approved by the IRB of Brookdale University Hospital.

Tissue-specific quantification and localization of androgen and estrogen receptors in prostate cancer

Androgens and estrogens, working together, promote prostate cancer (PRCA) initiation and progression, with androgens acting via androgen receptor (AR) and estrogens acting primarily through estrogen receptor α (ERα). While the interplay between these steroid hormones has been established, the interaction between steroid hormone receptors in prostatic disease remains unstudied. The goal of this study was to objectively determine the incidence, stage specificity, and tissue/cell type specificity of AR and ERα expression, both independently and simultaneously, during the progression of PRCA. Using multiplexed immunohistochemistry and multispectral imaging analysis, AR, ERα, and smooth muscle α-actin expression was detected and quantitated in benign prostate tissue (BPT), high-grade prostatic intraepithelial neoplasia (HGPIN), PRCA, and metastasis (MET) from patient specimens (n=340). Epithelial AR expression was significantly increased in HGPIN, PRCA, and MET compared with BPT, whereas ERα expression in epithelial and stromal cells was highest in HGPIN. With analysis of AR and ERα coexpression, we identified a unique population of double-positive (AR+/ERα+) cells that increased in HGPIN specimens in both the stroma and the epithelium. Double-negative (AR-/ERα-) cells significantly decreased across PRCA progression, from 65% in BPT to 30% in MET. Preliminary analysis of this AR+/ERα+ population indicates potential cell type specificity in smooth muscle α-actin-negative stromal cells. This study demonstrates stage-, tissue-, and cell type-specific AR and ERα expression changes during PRCA progression, both independently and coexpressed. A more complete understanding of steroid hormones and their receptors in the initiation and progression of prostatic disease may elucidate improved strategies for PRCA prevention or therapy.

High-resolution imaging and single-cell analysis via laser ablation-inductively coupled plasma-mass spectrometry for the determination of membranous receptor expression levels in breast cancer cell lines using receptor-specific hybrid tracers

This work evaluates the possibility of placement of high-resolution imaging and single-cell analysis via laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) within precision medicine by assessing the suitability of LA-ICP-MS as a micro-analytical technique for the localization and quantification of membranous receptors in heterogeneous cell samples that express both the membrane-bound receptors C-X-C chemokine receptor type 4 (CXCR4) and epidermal growth factor receptor (EGFR). Staining of the breast cancer cell lines MDA-MB-231 X4 and MDA-MB-468 was achieved using receptor-specific hybrid tracers, containing both a fluorophore and a DTPA single-lanthanide chelate. Prior to LA-ICP-MS imaging, fluorescence confocal microscopy (FCM) imaging was performed to localize the receptors, hereby enabling direct comparison. Based on the different expression levels of CXCR4 and EGFR, a distinction could be made between the cell lines using both imaging modalities. Furthermore, FCM and LA-ICP-MS demonstrated complementary characteristics, as a more distinct discrimination could be made between both cell lines based on the EGFR-targeting hybrid tracer via LA-ICP-MS, due to the intrinsic CXCR4-related green fluorescent protein (GFP) signal present in the MDA-MB-231 X4 cells. Employing state-of-the-art LA-ICP-MS instrumentation in bidirectional area scanning mode for sub-cellular imaging of MDA-MB-231 X4 cells enabled the specific binding of the CXCR4-targeting hybrid tracer to the cell membrane to be clearly demonstrated. The stretching of cells over the glass substrate led to a considerably higher signal response for pixels at the cell edges, relative to the more central pixels. The determination of the expression levels of CXCR4 and EGFR for the MDA-MB-468 cell line was performed using LA-ICP-MS single-cell analysis (sc-LA-ICP-MS) and external calibration, based on the quantitative ablation of Ho-spiked dried gelatin droplet standards. Additionally, a second calibration approach was applied based on spot ablation of highly homogeneous dried gelatin gels in combination with the determination of the ablated volume using atomic force microscopy (AFM) and yielded results which were in good agreement with the expression levels determined via flow cytometry (FC) and mass cytometry (MC). Hybrid tracers enable a direct comparison between (i) FCM and LA-ICP-MS imaging for the evaluation of the microscopic binding pattern and between (ii) FC, MC and sc-LA-ICP-MS for the quantification of receptor expression levels in single cells.

Lorcaserin treatment decreases body weight and reduces cardiometabolic risk factors in obese adults: A six-month, randomized, placebo-controlled, double-blind clinical trial.

Lorcaserin is a serotonin 2c receptor agonist that promotes weight loss while contributing to the prevention and improvement of type 2 diabetes and improvement of atherogenic lipid profiles, without higher rates of major cardiovascular events. The full spectrum of possible lorcaserin-induced improvements in cardiometabolic health remains to be clarified. Thus, we investigated the way in which lorcaserin treatment may alter cardiovascular disease risk, either independently or through changes in body weight. We measured, for the first time, lipid particle quantification, lipid peroxidation, appetite-regulating hormones and mRNA expression of the 5-hydroxytryptamine 2c receptor (5-HT2c receptor). A total of 48 obese participants were enrolled in this six-month, randomized (1:1), placebo-controlled, double-blinded clinical trial. Lorcaserin treatment reduced fat mass (P < 0.001), the fatty liver index (P < 0.0001) and energy intake (P < 0.03) without affecting energy expenditure or lean mass. Total low-density lipoprotein (LDL) (P < 0.04) and small LDL particles (P < 0.03) decreased, while total high-density lipoprotein (HDL) P < 0.02) increased and heart rate significantly decreased with lorcaserin treatment. No mRNA expression of the 5-HT2c receptor was observed in peripheral organs. These data suggest that lorcaserin treatment for six months improves cardiometabolic health in obese individuals, acting mainly through the brain.

Rapid diagnostics for point-of-care quantification of soluble transferrin receptor.

BackgroundIron deficiency (ID) and anaemia are major health concerns, particularly in young children. Screening for ID based on haemoglobin (Hb) concentration alone has been shown to lack sensitivity and specificity. The American Academy of Pediatrics (AAP) recommends soluble transferrin receptor (sTfR) as a promising approach to screen for iron deficiency. However, in most settings, assessment of iron status requires access to centralized laboratories. There is an urgent need for rapid, sensitive, and affordable diagnostics for sTfR at the point-of-care.MethodsAn immunochromatographic assay-based point-of-care screening device was developed for rapid quantification of sTfR from a drop of serum within a few minutes. Performance optimization of the assay was done in sTfR-spiked buffer and commercially available sTfR calibrator, followed by a small-scale proof-of-concept validation with archived serum samples.FindingsOn preliminary testing with archived serum samples and comparison with Ramco ELISA, a correlation of 0.93 (P < 0.0001) was observed, demonstrating its potential for point-of-care assessment of iron status.InterpretationThe analytical performance of the point-of-care sTfR screening device indicates the potential for application in home-use test kits and field settings, especially in low- and middle-income settings. An added advantage of sTfR quantification in combination with our previously reported serum ferritin diagnostics is in integration of Cook's equation as a quantitative and minimally-invasive indicator of total body iron stores.FundThrasher Research Fund (Early Career Award #13379), NIH R03 EB 023190, NSF grant #1343058, and Nutrition International (project #10-8007-CORNE-01).

Accurate Estrogen Receptor Quantification in Patients with Negative and Low-Positive Estrogen-Receptor-Expressing Breast Tumors: Sub-Analyses of Data from Two Clinical Studies.

Accurate assessment of estrogen receptor (ER) expression is crucial to ensure that patients with early breast cancer are accurately identified for appropriate treatment with endocrine therapy. Reverse transcriptase polymerase chain reaction (RT-PCR), compared with immunohistochemistry (IHC), may provide a more precise indication of ER status. Data were pooled and analyzed from two independent, but similarly designed, studies that examined ER status by IHC and the 21-gene Recurrence Score that employs RT-PCR-based methodology. Tumor tissue from patients with early stage breast cancer where ER status could be determined by both IHC and RT-PCR was included. ER status by IHC staining was defined as ER-negative (< 1%), ER-low+ (1-10%), or ER+ (> 10%). ER status by RT-PCR was defined as ER-negative (≤ 6.5) or ER+ (> 6.5). Recurrence Score results from the 21-gene assay were reported on a continuous scale from 0 to 100. A sub-analysis examined the association between ER expression (Allred score 2-7) and response to a 14-day pre-surgery pulse with an aromatase inhibitor. A separate sub-analysis examined the association between ER expression and human epidermal growth factor receptor 2 (HER2) expression. Tumor specimens from 192 patients (aged 25-92years) were included in the pooled analysis. Correlation between IHC- and RT-PCR-measured ER was strong for IHC-defined ER-negative and ER+ samples (r = 0.646 [95% CI 0.553-0.720]). There was 100% concordance for ER+ tumors; however, 56% of the ER-low+ tumors were negative by RT-PCR. Allred score correlated better with ER status measured by RT-PCR at pre-treatment (r = 0.83) than at post-treatment (r = 0.76). The majority (77%) of ER-negative and ER-low+ tumors were HER2-negative. RT-PCR provided a more accurate assessment of ER expression in patients with ER-low+ tumors, and data support dual testing for patients with ER-low+ status to ensure appropriate treatment planning as it pertains to endocrine therapy. Genomic Health, Inc.

PET imaging for better understanding of normal and pathological neurotransmission

Positron emission tomography imaging is still an expanding field of preclinical and clinical investigations exploring the brain and its normal and pathological functions. In addition to technological improvements in PET scanners, the availability of suitable radiotracers for unexplored pharmacological targets is a key factor in this expansion. Many radiotracers (or radiopharmaceuticals, when administered to humans) have been developed by multidisciplinary teams to visualize and quantify a growing numbers of brain receptors, transporters, enzymes and other targets. The development of new PET radiotracers still represents an exciting challenge, given the large number of neurochemical functions that remain to be explored. In this article, we review the development context of the first preclinical radiotracers and their passage to humans. The main current contributions of PET radiotracers are described in terms of imaging neuronal metabolism, quantification of receptors and transporters, neurodegenerative and neuroinflammatory imaging. The different approaches to functional imaging of neurotransmission are also discussed. Finally, the contributions of PET imaging to the research and development of new brain drugs are described.

Cholinergic activity regulates the secretome of epicardial adipose tissue: Association with atrial fibrillation.

Botulinum toxin injection on epicardial fat, which inhibits acetylcholine (ACh) release, reduced the presence of atrial fibrillation (AF) in patients after heart surgery. Thus, we wanted to study the profile of the released proteins of epicardial adipose tissue (EAT) under cholinergic activity (ACh treatment) and their value as AF predictors. Biopsies, explants, or primary cultures were obtained from the EAT of 85 patients that underwent open heart surgery. The quantification of muscarinic receptors (mAChR) by real-time polymerase chain reaction or western blot showed their expression in EAT. Moreover, mAChR Type 3 was upregulated after adipogenesis induction (p < 0.05). Cholinergic fibers in EAT were detected by vesicular ACh transporter levels and/or acetylcholinesterase activity. ACh treatment modified the released proteins by EAT, which were identified by nano-high-performance liquid chromatography and TripleTOF analysis. These differentially released proteins were involved in cell structure, inflammation, or detoxification. After testing the plasma levels of alpha-defensin 3 (inflammation-involved protein) of patients who underwent open heart surgery ( n = 24), we observed differential levels between the patients who developed or did not develop postsurgery AF (1.58 ± 1.61 ng/ml vs. 6.2 ± 5.6 ng/ml; p < 0.005). The cholinergic activity on EAT might suggest a new mechanism for studying the interplay among EAT, autonomic nervous system dysfunction, and AF.

Combining fluorescence-based image segmentation and automated microfluidics for ultrafast cell-by-cell assessment of biomarkers for HER2-type breast carcinoma.

Immunohistochemistry (IHC) is one of the main clinical techniques for biomarker assessment on tissue biopsies. It consists in chromogenic labeling with specific antibodies, followed by optical imaging, and it is used for diagnosis and therapeutic targeting. A well-known drawback of IHC is its limited robustness, which often precludes quantitative biomarker assessment. We combine microfluidic immunostaining, fluorescence imaging, and image-based cell segmentation to create an ultrafast procedure for accurate biomarker assessment via IHC. The experimental protocol is very simple and based on fast delivery of reagents in a microfluidic chamber created by clamping a half-chamber patterned in a silicon chip on top of a tumor tissue section. Also, the imaging procedure simply requires a standard fluorescence microscope, already widely used in clinical practice. The image processing is based on local-contrast enhancement and thresholding of the obtained fluorescence image, with subsequent Voronoi segmentation. To assess the experimental and analytical procedure on robust biological controls, we apply our method to well-characterized cell lines, which guarantee higher reproducibility than whole-tissue samples and therefore enable to disentangle the technical variability from the biological variability. To increase the potential translationality, we address the detection and quantification of the human epidermal growth factor receptor 2 (HER2) protein, which is a biomarker for HER2-type breast carcinoma diagnosis and therapy. We report both ultrafast immunofluorescence staining (5min per sample) of two breast cancer biomarkers and ultrafast cell segmentation (1min per sample = processing of thousands of cells). This provides a quantitative, cell-based immunofluorescent signal, with which we propose a potential diagnostic criterion to separate HER2-positive and HER2-negative breast cancer cells at high sensitivity and specificity.