Search

Background: Biomarkers are needed to monitor disease progression, target engagement and efficacy in Huntington's disease (HD). Cerebrospinal fluid (CSF) is an ideal medium to research such biomarkers due to its proximity to the brain., Objective: To investigate the safety and feasibility of research lumbar punctures (LP) in HD., Methods: HDClarity is an ongoing international biofluid collection initiative built on the Enroll-HD platform, where clinical assessments are recorded. It aims to recruit 1,200 participants. Biosamples are collected following an overnight fast: blood via venipuncture and CSF via LP. Participants are healthy controls and HD gene expansion carriers across the disease spectrum. We report on monitored data from February 2016 to September 2019., Results: Of 448 participants screened, 398 underwent at least 1 sampling visit, of which 98.24% were successful (i.e., CSF was collected), amounting to 10,610 mL of CSF and 8,200 mL of plasma. In the total 572 sampling visits, adverse events were reported in 24.13%, and headaches of any kind and post-LP headaches in 14.86% and 12.24%, respectively. Frequencies were less in manifest HD; gender, age, body mass index and disease burden score were not associated with the occurrence of the events in gene expansion carriers. Headaches and back pain were the most frequent adverse events., Conclusion: HDClarity is the largest CSF collection initiative to support scientific research into HD and is now stablished as a leading resource for HD research. Our data confirm that research LP in HD are feasible and acceptable to the community, and have a manageable safety profile.

Advancements in flow cytometers with capability to measure 15 or more parameters have enabled us to characterize cell populations at unprecedented levels of detail. Beyond discovery research, there is now a growing demand to dive deeper into evaluating the immune response in clinical trials for immune modulating compounds. However, for high-volume, complex flow cytometry data generated in clinical trials, conventional manual gating remains the standard of practice. Traditional manual gating is resource intense and becomes a bottleneck and an impractical method to complete high volumes of flow cytometry data analysis. Current efforts to automate "manual gating" have shown that computational algorithms can facilitate the analysis of daunting multi-parameter data; however, a greater degree of precision in comparison with traditional manual gating is needed for wide-scale adoption of automated gating methods. In an effort to more closely follow the manual gating process, our automated gating pipeline was created to include negative controls (Fluorescence Minus One [FMO]) to enhance the reliability of gate placement. We demonstrate that use of an automated pipeline, heavily relying on FMO controls for population discrimination, can analyze multi-parameter, large-scale clinical datasets with comparable precision and accuracy to traditional manual gating., Competing Interests: Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Aim: Ki67 is a prognostic and/or predictive biomarker in patients with malignancies. Flow cytometry is a powerful technology for single-cell multiparameter analysis., Results: We developed and validated a multicolor quantitative flow cytometry assay for detection of intracellular Ki67 expression in various immune cell subsets from human blood. The assay was optimized and showed excellent precisions. Assessment of the sample stability indicated that percentage changes from the fresh sample for the reportable results of interest were within 20%, up to 72 h after blood collection in the Cyto-Chex ® BCT tube., Conclusion: The validated assay is sufficiently robust to analyze clinical samples. Easy access to peripheral blood enables continuous monitoring of Ki67 expression in blood as a biomarker, for example, for immunotherapy studies.

Cholesteryl ester transfer protein (CETP) is a target of therapeutic intervention for coronary heart disease. Anacetrapib, a potent inhibitor of CETP, has been shown to reduce LDL-cholesterol by 40% and increase HDL-cholesterol by 140% in patients, and is currently being evaluated in a phase III cardiovascular outcomes trial. HDL is known to possess anti-inflammatory properties, however with such large increases in HDL-cholesterol, it is unclear whether CETP inhibition perturbs HDL functionality such as anti-inflammatory effects on endothelial cells. The purpose of the present study was to determine whether CETP inhibition by anacetrapib affects the anti-inflammatory properties of HDL. HDL was isolated from either hamsters treated with vehicle or anacetrapib for 2weeks, or from normal human subjects treated either placebo, 20mg, or 150mg anacetrapib daily for 2weeks. Anacetrapib treatment increased plasma HDL cholesterol levels by 65% and between 48 and 82% in hamsters and humans, respectively. Pre-incubation of human aortic endothelial cells with HDL isolated from both control and anacetrapib treated hamsters suppressed TNFα induced expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin. Similar results were obtained with human HDL samples pre and post treatment with placebo or anacetrapib. Further, HDL inhibited TNFα-induced MCP-1 secretion, monocyte adhesion and NF-κB activation in endothelial cells, and the inhibition was similar between control and anacetrapib treated groups. These studies demonstrate that anacetrapib treatment does not impair the ability of HDL to suppress an inflammatory response in endothelial cells., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The function of a particular protein is dependent upon its localization and milieu. The ability to track the "fate" of a protein is a valuable tool to elucidate its function. We present the use of HaloTag technology to study the localization and fate of human Proprotein Convertase Subtilisin-like Kexin type 9 (PCSK9).The role of PCSK9 in the regulation of circulating low density lipoprotein-cholesterol (LDL-c) levels is ascribed to binding of circulating PCSK9 to the LDL receptor (LDLR) and subsequent lysosomal degradation of LDLR. However, hints in the literature indicate that intracellular PCSK9 may act on the LDLR, possibly during processing of newly synthesized protein. To address this question, the source and fate of intracellular PCSK9 requires further investigation.We applied HaloTag technology to distinguish the source of intracellular PCSK9 and showed that newly synthesized intracellular PCSK9 has unique localization from the PCSK9 after re-uptake. This suggests different functions of PCSK9 while interacting with the LDLR.

The activity of the ERK has complex spatial and temporal dynamics that are important for the specificity of downstream effects. However, current biochemical techniques do not allow for the measurement of ERK signaling with fine spatiotemporal resolution. We developed a genetically encoded, FRET-based sensor of ERK activity (the extracellular signal-regulated kinase activity reporter, EKAR), optimized for signal-to-noise ratio and fluorescence lifetime imaging. EKAR selectively and reversibly reported ERK activation in HEK293 cells after epidermal growth factor stimulation. EKAR signals were correlated with ERK phosphorylation, required ERK activity, and did not report the activities of JNK or p38. EKAR reported ERK activation in the dendrites and nucleus of hippocampal pyramidal neurons in brain slices after theta-burst stimuli or trains of back-propagating action potentials. EKAR therefore permits the measurement of spatiotemporal ERK signaling dynamics in living cells, including in neuronal compartments in intact tissues.

JIP scaffold proteins are implicated in the regulation of protein kinase signal transduction pathways. To test the physiological role of these scaffold proteins, we examined the phenotype of compound mutant mice that lack expression of JIP proteins. These mice were found to exhibit severe defects in N-methyl-D-aspartic acid (NMDA) receptor function, including decreased NMDA-evoked current amplitude, cytoplasmic Ca(++), and gene expression. The decreased NMDA receptor activity in JIP-deficient neurons is associated with reduced tyrosine phosphorylation of NR2 subunits of the NMDA receptor. JIP complexes interact with the SH2 domain of cFyn and may therefore promote tyrosine phosphorylation and activity of the NMDA receptor. We conclude that JIP scaffold proteins are critically required for normal NMDA receptor function.

Cell-substrate contacts, called focal adhesions (FAs), are dynamic in rapidly moving cells. We show that supervillin (SV)--a peripheral membrane protein that binds myosin II and F-actin in such cells--negatively regulates stress fibers, FAs, and cell-substrate adhesion. The major FA regulatory sequence within SV (SV342-571) binds to the LIM domains of two proteins in the zyxin family, thyroid receptor-interacting protein 6 (TRIP6) and lipoma-preferred partner (LPP), but not to zyxin itself. SV and TRIP6 colocalize within large FAs, where TRIP6 may help recruit SV. RNAi-mediated decreases in either protein increase cell adhesion to fibronectin. TRIP6 partially rescues SV effects on stress fibers and FAs, apparently by mislocating SV away from FAs. Thus, SV interactions with TRIP6 at FAs promote loss of FA structure and function. SV and TRIP6 binding partners suggest several specific mechanisms through which the SV-TRIP6 interaction may regulate FA maturation and/or disassembly.

During interphase, the centrosome concentrates cell stress response molecules, including chaperones and proteasomes, into a proteolytic center. However, whether the centrosome functions as proteolytic center during mitosis is not known. In this study, cultured mammalian cells were treated with the proteasome inhibitor MG 132 and spindle morphology in mitotic cells was characterized in order to address this issue. Proteasome inhibition during mitosis leads to the formation of additional asters that cause the assembly of multipolar spindles. The cause of this phenomenon was investigated by inhibiting microtubule-based transport and protein synthesis. These experimental conditions prevented the formation of supernumerary asters during mitosis. In addition, the expression of dsRed without proteasome inhibition led to the fragmentation of spindle poles. These experiments showed that the formation of extra asters depends on intact microtubule-based transport and protein synthesis. These results suggest that formation of supernumerary asters is due to excessive accumulation of proteins at the spindle poles and consequently fragmentation of the centrosome. Together, this leads to the conclusion that the centrosome functions as proteolytic center during mitosis and proteolytic activity at the spindle poles is necessary for maintaining spindle pole integrity., (Copyright 2005 Wiley-Liss, Inc.)

1. DDT1 MF-2 smooth muscle cells responded to the bath application of histamine or ATP with an increase in the cytosolic Ca2+ concentration ([Ca2+]c) and the whole-cell K+ current, IK(BA). 2. In cell-attached patches, histamine (100 microM) activated currents through a 200 pS K+ channel ('BKA' channel). In the absence of agonists, the BKA channel was activated by excision of the patch. Both histamine and patch excision increased the channel activity (NPo; where N is the number of channels per patch and Po is the open probability) by reducing the long closures between the bursts of openings. 3. In inside-out patches, the BKA channel had a conductance of 201 +/- 4 pS (symmetrical solutions of 150 mM KCl, 2 mM MgCl2 and 2 mM EGTA). Replacement of K+ in the patch electrode by Na+, Li+ or Cs+ prevented the flow of inward currents and reduced the outward K+ conductance to 113 pS. 4. NPo was insensitive to changes in [Ca2+]c from 10 nM to 1 microM. NPo was also not modified either by cytosolic Na+, ATP, GTP, GTP gamma S, dithiothreitol or TEA (10 mM) or by extracellular 4-aminopyridine (5 mM), glibenclamide (20 microM) or TEA (10 mM). The BKA channel was blocked by 5 mM intracellular BaCl2 or by 10 nM extracellular iberiotoxin. 5. In cell-attached patches, BKA channel activity could be induced by 1 microM cytochalasin B, applied either through the patch pipette or in the bath solution. The effects of cytochalasin B, of patch excision, or of histamine on NPo were not additive but saturative. 6. Whole DDT1 MF-2 cells had resting potentials of -10 mV, dominated by the chloride conductance; the resting potential changed to -82 mV when the K+ conductance was increased by cytochalasin B or by histamine. The effects of cytochalasin B and histamine on IK(BA) were not additive but saturative. 7. We discuss the hypothesis that the interaction between the cytoskeleton and the BKA channel promotes the long channel closures; depolymerization of F-actin may constitute a mechanism by which the agonists histamine or ATP disinhibit BKA channel activity.