Syntaxin clusters and cholesterol affect the mobility of Syntaxin1a.

Syntaxin1a (Syx1a) is essential for stimulated exocytosis in neuroendocrine cells. The vesicle docking process involves the formation of nanoscale Syx1a domains on the plasma membrane and the Syx1a clusters disintegrate during the fusion process. Syx1a nanodomains are static yet Syx1a molecules dynamically enter and leave the domains; the process by which these clusters maintain this balance is unclear. In this work, the dynamics of the Syx1a molecules is elucidated relative to the cluster position through a labeling strategy that allows both the bulk position of the Syx clusters to be visualized concurrent with the trajectories of single Syx1a molecules on the surface of PC12 cells. Single Syx1a molecules were tracked in time relative to cluster positions to decipher how Syx1a moves within a cluster and when clusters are not present. Syx1a is mobile on the plasma membrane, more mobile at the center of clusters, and less mobile near the edges of clusters; this depends on the presence of the N-terminal Habc domain and cholesterol, which are essential for proper exocytosis. Simulations of the dynamics observed at clusters support a model where clusters are maintained by a large cage (r = 100 nm) within which Syx1a remains highly mobile within the cluster (r = 50 nm). The depletion of cholesterol dramatically reduces the mobility of Syx1a within clusters and less so over the rest of the plasma membrane. This suggests that fluidity of Syx1a supramolecular clusters is needed for function.

Phospholipase D1 produces phosphatidic acid at sites of secretory vesicle docking and fusion.

Phospholipase D1 (PLD1) activity is essential for the stimulated exocytosis of secretory vesicles where it acts as a lipid-modifying enzyme to produces phosphatidic acid (PA). PLD1 localizes to the plasma membrane and secretory vesicles, and PLD1 inhibition or knockdowns reduce the rate of fusion. However, temporal data resolving when and where PLD1 and PA are required during exocytosis is lacking. In this work, PLD1 and production of PA are measured during the trafficking, docking, and fusion of secretory vesicles in PC12 cells. Using fluorescently tagged PLD1 and a PA-binding protein, cells were imaged using TIRF microscopy to monitor the presence of PLD1 and the formation of PA throughout the stages of exocytosis. Single docking and fusion events were imaged to measure the recruitment of PLD1 and the formation of PA. PLD1 is present on mobile, docking, and fusing vesicles and also colocalizes with Syx1a clusters. Treatment of cells with PLD inhibitors significantly reduces fusion, but not PLD1 localization to secretory vesicles. Inhibitors also alter the formation of PA; when PLD1 is active, PA slowly accumulates on docked vesicles. During fusion, PA is reduced in cells treated with PLD1 inhibitors, indicating that PLD1 produces PA during exocytosis.

Exosome secretion kinetics are controlled by temperature.

When multivesicular endosomes (MVEs) fuse with the plasma membrane, exosomes are released into the extracellular space where they can affect other cells. The ability of exosomes to regulate cells nearby or further away depends on whether they remain attached to the secreting cell membrane. The regulation and kinetics of exosome secretion are not well characterized, but probes for directly imaging single MVE fusion events have allowed for visualization of the fusion and release process. In particular, the design of an exosome marker with a pH-sensitive dye in the middle of the tetraspanin protein CD63 has facilitated studies of individual MVE fusion events. Using TIRF microscopy, single fusion events were measured in A549 cells held at 23-37°C and events were identified using an automated detection algorithm. Stable docking precedes fusion almost always and a decrease in temperature was accompanied by decrease in the rate of content loss and in the frequency of fusion events. The loss of CD63-pHluorin fluorescence was measured at fusion sites and fit with a single or double exponential decay, with most events requiring two components and a plateau because the loss of fluorescence was typically incomplete. To interpret the kinetics, fusion events were simulated as a localized release of tethered/untethered exosomes coupled with the membrane diffusion of CD63. The experimentally observed decay required three components in the simulation: 1) free exosomes, 2) CD63 membrane diffusion from the endosomal membrane into the plasma membrane, and 3) tethered exosomes. Modeling with slow diffusion of the tethered exosomes (0.0015-0.004 µm2/s) accurately fits the experimental data for all temperatures. However, simulating with immobile tethers or the absence of tethers fails to replicate the data. Our model suggests that exosome release from the fusion site is incomplete due to postfusion, membrane attachment.

Phosphatidic Acid Accumulates at Areas of Curvature in Tubulated Lipid Bilayers and Liposomes.

Phosphatidic acid (PA) is a signaling lipid that is produced enzymatically from phosphatidylcholine (PC), lysophosphatidic acid, or diacylglycerol. Compared to PC, PA lacks a choline moiety on the headgroup, making the headgroup smaller than that of PC and PA, and PA has a net negative charge. Unlike the cylindrical geometry of PC, PA, with its small headgroup relative to the two fatty acid tails, is proposed to support negatively curved membranes. Thus, PA is thought to play a role in a variety of biological processes that involve bending membranes, such as the formation of intraluminal vesicles in multivesicular bodies and membrane fusion. Using supported tubulated lipid bilayers (STuBs), the extent to which PA localizes to curved membranes was determined. STuBs were created via liposome deposition with varying concentrations of NaCl (500 mM to 1 M) on glass to form supported bilayers with connected tubules. The location of fluorescently labeled lipids relative to tubules was determined by imaging with total internal reflection or confocal fluorescence microscopy. The accumulation of various forms of PA (with acyl chains of 16:0-6:0, 16:0-12:0, 18:1-12:0) were compared to PC and the headgroup labeled phosphatidylethanolamine (PE), a lipid that has been shown to accumulate at regions of curvature. PA and PE accumulated more at tubules and led to the formation of more tubules than PC. Using large unilamellar liposomes in a dye-quenching assay, the location of the headgroup labeled PE was determined to be mostly on the outer, positively curved leaflet, whereas the tail labeled PA was located more on the inner, negatively curved leaflet. This study demonstrates that PA localizes to regions of negative curvature in liposomes and supports the formation of curved, tubulated membranes. This is one way that PA could be involved with curvature formation during a variety of cell processes.

Biochemically prepared C-reactive protein conformational states differentially affect C1q binding.

C-reactive protein (CRP) is commonly measured as an inflammatory marker in patient studies for coronary heart disease, autoimmune disease and recent acute infections. Due to a correlation of CRP to a vast number of disease states, CRP is a well-studied protein in medical literature with over 16000 references in PubMed [1]. However, the biochemical and structural variations of CRP are not well understood in regards to their binding of complement immune response proteins. Conformations of CRP are thought to affect disease states differently, with a modified form showing neoepitopes and activating the complement immune response through C1q binding. In this work, we compare the unfolding of CRP using chemical denaturants and identify which states of CRP bind a downstream complement immune response binding partner (C1q). We used guanidine HCl (GndHCl), urea/EDTA, and 0.01% SDS with heat to perturb the pentameric state. All treatments give rise to a monomeric state in non-denaturing polyacrylamide gel electrophoresis experiments, but only treatment with certain concentrations of denaturant or dilute SDS with heat maintains CRP function with a key downstream binding partner, C1q, as measured by enzyme-linked immunosorbent assays. The results suggest that the final form of modified CRP and its ability to mimic biological binding is dependent on the preparation method.

Membrane dynamics are slowed for Alexa594-labeled membrane proteins due to substrate interactions.

The addition of fluorescent dyes to proteins, lipids and other biological molecules can affect a range of processes such as mobility, molecular interactions, localization, and, ultimately, function. The dynamics of a protein can be dramatically affected if the label interacts non-specifically with the substrate or with other molecules in the system. To test how dye-substrate interactions affect protein diffusion, fluorescence recovery after photobleaching (FRAP) measurements were designed to explicitly determine the role of the dye on the diffusion of a transmembrane protein, Syntaxin1a, expressed on the cell surface. Syntaxin1a, was tagged with EGFP on the extracellular side and an EGFP nanobody with or without a dye label was attached. FRAP was performed on Syx1a-EGFP and the choice of cell growth substrate affected mobility in the presence of a dye labeled nanobody. This work provides evidence for choosing fibronectin (Fn) over poly-L-lysine (PLL) in FRAP and single molecule tracking measurements when using Alexa594, a common probe for red fluorescent measurements. Alexa594-labeled nanobody but not unlabeled nanobody, dramatically reduced the mobility of Syx1a-EGFP when cells were cultured on PLL. However, when Fn was used, the mobility returned. Mobility measured by single molecule tracking measurements align with the FRAP measurements with Fn coated surfaces being more mobile than PLL.

A Phase II and Biomarker Study of Sorafenib Combined with Modified FOLFOX in Patients with Advanced Hepatocellular Carcinoma.

PURPOSE: Sorafenib is a standard first-line treatment for advanced hepatocellular carcinoma (HCC). The phase III SHARP trial showed a median time-to-progression (mTTP) of 5.5 months, overall response rate (ORR) of 2%, and median overall survival (mOS) of 10.7 months with sorafenib. FOLFOX4 has shown modest activity in advanced HCC. We evaluated the combination of sorafenib and modified (m)FOLFOX in a single-arm, multicenter phase II study. PATIENTS AND METHODS: The study included Child-Pugh A patients with advanced HCC and no prior systemic therapies. Patients received sorafenib 400 mg twice a day for 2 weeks, followed by concurrent mFOLFOX [5-fluorouracil (5-FU) 1,200 mg/m2/day for 46 hours, leucovorin 200 mg/m2, and oxaliplatin 85 mg/m2 biweekly]. The primary endpoint was mTTP with an alternative hypothesis of 7 months, and secondary endpoints included ORR, mOS, and circulating biomarkers. RESULTS: The study enrolled 40 patients: HCV/EtOH/HBV, 43%/28%/13%; Child-Pugh A5, 70%. Notable grade 3/4 adverse events (AE) included AST/ALT elevation (28%/15%), diarrhea (13%), hyperbilirubinemia (10%), hand-foot syndrome (8%), and bleeding (8%). mTTP was 7.7 months [95% confidence interval (CI): 4.4-8.9], ORR 18%, and mOS 15.1 months (7.9-16.9). Sorafenib + mFOLFOX increased plasma PlGF, VEGF-D, sVEGFR1, IL12p70, and CAIX and CD4+ and CD8+ effector T lymphocytes and decreased plasma sVEGFR2 and s-c-KIT and regulatory T cells (Tregs). Shorter TTP was associated with high baseline sVEGFR1. Shorter TTP and OS were associated with increases in Tregs and CD56Dim natural killer (NK) cells after sorafenib alone and plasma sMET after combination treatment (all P < 0.05). CONCLUSIONS: Sorafenib + mFOLFOX met the prespecified endpoint with encouraging efficacy but moderate hepatotoxicity. Thus, this regimen may be effective in select patients with adequate liver reserve. Biomarker evaluations suggested a correlation between time-to-progression (TTP) and angiogenic biomarkers and circulating Tregs.

Development of a sandwich ELISA with potential for selective quantification of human lactoferrin protein nitrated through disease or environmental exposure.

Lactoferrin (LF) is an important multifunctional protein that comprises a large fraction of the protein mass in certain human fluids and tissues, and its concentration is often used to assess health and disease. LF can be nitrated by multiple routes, leading to changes in protein structure, and nitrated proteins can negatively impact physiological health via nitrosative stress. Despite an awareness of the detrimental effects of nitrated proteins and the importance of LF within the body, cost-effective methods for detecting and quantifying nitrated lactoferrin (NLF) are lacking. We developed a procedure to selectively quantify NLF using sandwich enzyme-linked immunosorbent assay (ELISA), utilizing a polyclonal anti-LF capture antibody paired with a monoclonal anti-nitrotyrosine detector antibody. The assay was applied to quantify NLF in samples of pure LF nitrated via two separate reactions at molar ratios of excess nitrating agent to the total number of tyrosine residues between 10/1 and 100/1. Tetranitromethane (TNM) was used as a laboratory surrogate for an environmental pathway selective for production of 3-nitrotyrosine, and sodium peroxynitrite (ONOO-) was used as a surrogate for an endogenous nitration pathway. UV-vis spectroscopy (increased absorbance at 350 nm) and fluorescence spectroscopy (emission decreased by > 96%) for each reaction indicate the production of NLF. A lower limit of NLF detection using the ELISA method introduced here was calculated to be 0.065 µg mL-1, which will enable the detection of human-physiologically relevant concentrations of NLF. Our approach provides a relatively inexpensive and practical way to assess NLF in a variety of systems. Graphical abstract We developed a procedure to selectively quantify nitrated lactoferrin (NLF) protein using a sandwich enzyme-linked immunosorbent assay (ELISA) and verified results against several spectroscopic techniques. Our approach provides an inexpensive and practical way to assess NLF in a variety of systems.

Single Lipid Molecule Dynamics on Supported Lipid Bilayers with Membrane Curvature.

The plasma membrane is a highly compartmentalized, dynamic material and this organization is essential for a wide variety of cellular processes. Nanoscale domains allow proteins to organize for cell signaling, endo- and exocytosis, and other essential processes. Even in the absence of proteins, lipids have the ability to organize into domains as a result of a variety of chemical and physical interactions. One feature of membranes that affects lipid domain formation is membrane curvature. To directly test the role of curvature in lipid sorting, we measured the accumulation of two similar lipids, 1,2-Dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DHPE) and hexadecanoic acid (HDA), using a supported lipid bilayer that was assembled over a nanopatterned surface to obtain regions of membrane curvature. Both lipids studied contain 16 carbon, saturated tails and a head group tag for fluorescence microscopy measurements. The accumulation of lipids at curvatures ranging from 28 nm to 55 nm radii was measured and fluorescein labeled DHPE accumulated more than fluorescein labeled HDA at regions of membrane curvature. We then tested whether single biotinylated DHPE molecules sense curvature using single particle tracking methods. Similar to groups of fluorescein labeled DHPE accumulating at curvature, the dynamics of single molecules of biotinylated DHPE was also affected by membrane curvature and highly confined motion was observed.

Conformational Changes in C-Reactive Protein Affect Binding to Curved Membranes in a Lipid Bilayer Model of the Apoptotic Cell Surface.

C-reactive protein (CRP) is a serum protein that binds to damaged membranes through a phosphatidylcholine binding site. The membrane binding process can initiate the complement immune response and facilitates the clearance of apoptotic cells, likely aiding in the protection of autoimmunity. The initiation of an immune response relies on a conformation change from a native, pentameric form to a modified form, where the modified form binds complement proteins (i.e., C1q) and regulatory proteins substantially better than the native form. In vitro, this reactivity is observed when CRP is monomeric, and a modified form has also been observed at sites of inflammation. Despite evidence that the monomeric form has much higher affinities for almost all proteinaceous binding partners, the role of CRP conformation on lipid binding is yet unknown. In this work, we mimic the outer leaflet of apoptotic cell membranes using a nanopatterned substrate to create curved, supported lipid bilayers and then characterize how CRP conformation affects the interactions between CRP and target membranes. In this assay, the chemical composition and shape are separately tunable parameters. The lipids consisted primarily of palmitoyloleoylphosphatidylcholine, with and without lysophosphatidylcholine, and the curvature had a radius of 27-55 nm. Using this model system combined with quantitative fluorescence microscopy methods, CRP binding to lipid membranes was measured as a function of different conformations of CRP. The modified form of CRP bound curved membranes, but the pentameric form did not for the range of curvatures measured. Unlike most other curvature-sensing proteins, modified CRP accumulated more at a moderate curvature, rather than highly curved or flat regions, suggesting that the membrane bound form does not solely depend on a defect binding mechanism. The presence of lysophosphatidylcholine, a component of apoptotic membranes, increased CRP binding to all types of membranes. Overall, our results show that CRP interactions vary with protein form, lipid composition, and membrane shape. The mechanism by which CRP recognizes damaged membranes depends on the combination of all three.

A phase 2 and biomarker study of cabozantinib in patients with advanced cholangiocarcinoma.

BACKGROUND: Advanced cholangiocarcinoma carries a poor prognosis, and no standard treatment exists beyond first-line gemcitabine/platinum-based chemotherapy. A single-arm, phase 2 and biomarker study of cabozantinib, a multikinase inhibitor with potent activity against vascular endothelial growth factor receptor 2 (VEGFR2) and MET, was performed for patients with advanced refractory cholangiocarcinoma. METHODS: Previously treated patients with unresectable or metastatic cholangiocarcinoma received cabozantinib (60 mg orally and daily on a continuous schedule). The primary endpoint was progression-free survival (PFS). Tumor MET expression and plasma biomarkers were evaluated. RESULTS: The study enrolled 19 patients with cholangiocarcinoma (female, 68%; median age, 67 years; intrahepatic vs extrahepatic, 84% vs 16%). The median PFS was 1.8 months (95% confidence interval, 1.6-5.4 months), and the median overall survival (OS) was 5.2 months (95% confidence interval, 2.7-10.5 months). Grade 3/4 adverse events occurred in 89% of the patients and included neutropenia (5%), hyperbilirubinemia (5%), epistaxis (5%), bowel perforation (5%), enterocutaneous fistulas (5%), and hypertension (11%). One patient with 3 + MET expression in the tumor stayed on treatment for 278 days, but the MET expression did not correlate with the outcomes in the overall study population. Plasma vascular endothelial growth factor, placental growth factor, and stromal cell-derived factor 1α increased and soluble VEGFR2 and angiopoietin 2 decreased after treatment (all P values < .01). Plasma tissue inhibitor of matrix metalloproteinase 1 was inversely correlated with PFS, and soluble MET (sMET) and interleukin 6 were inversely correlated with OS. CONCLUSIONS: In unselected patients with cholangiocarcinoma, cabozantinib demonstrated limited activity and significant toxicity. In the first clinical trial to assess the role of MET inhibition in cholangiocarcinoma, 1 patient with a MET-high tumor had a prolonged benefit from treatment. Baseline plasma soluble MET was associated with OS. Any further development of this drug in cholangiocarcinoma should include a dose reduction and a biomarker-driven approach. Cancer 2017;123:1979-1988. © 2017 American Cancer Society.

Polymer mediated layer-by-layer assembly of different shaped gold nanoparticles.

Gold nanoparticles (GNPs) have a wide range of properties with potential applications in electronics, optics, catalysis, and sensing. In order to demonstrate that dense, stable, and portable samples could be created for these applications, multiple layers of GNPs were assembled via drop casting on glass substrates by layer-by-layer (LBL) techniques. Two cationic polyelectrolytes, poly(diallyldimethylammonium chloride) and polyethyleneimine, one anionic polyelectrolyte, poly(sodium 4-styrene sulfonate), and one neutral polymer, polyvinylpyrrolidone, were combined with four different shapes of GNPs (spherical, rod, triangular prismatic, and octahedral) to prepare thin films. A subset of these polymer nanoparticle combinations were assembled into thin films. Synthesized GNPs were characterized via dynamic light scattering, UV-vis spectroscopy, and transmission electron microscopy and the LBL thin films were characterized using UV-vis spectroscopy and atomic force microscopy. Sensing applications of the nanoparticles in solution and thin films were tested by monitoring the localized surface plasmon resonance of the GNPs. LBL thin films were prepared ranging from 25 to 100 layers with optical densities at plasmon from 0.5 to 3.0. Sensitivity in solutions ranged from 14 to 1002nm/refractive index units (RIU) and films ranged from 18.8 to 135.1nm/RIU suggesting reduced access to the GNPs within the films.

Fluorescence quantum yield measurements of fluorescent proteins: a laboratory experiment for a biochemistry or molecular biophysics laboratory course.

Fluorescent proteins are commonly used in cell biology to assess where proteins are within a cell as a function of time and provide insight into intracellular protein function. However, the usefulness of a fluorescent protein depends directly on the quantum yield. The quantum yield relates the efficiency at which a fluorescent molecule converts absorbed photons into emitted photons and it is necessary to know for assessing what fluorescent protein is the most appropriate for a particular application. In this work, we have designed an upper-level, biochemistry laboratory experiment where students measure the fluorescence quantum yields of fluorescent proteins relative to a standard organic dye. Four fluorescent protein variants, enhanced cyan fluorescent protein (ECFP), enhanced green fluorescent protein (EGFP), mCitrine, and mCherry, were used, however the methods described are useful for the characterization of any fluorescent protein or could be expanded to fluorescent quantum yield measurements of organic dye molecules. The laboratory is designed as a guided inquiry project and takes two, 4 hr laboratory periods. During the first day students design the experiment by selecting the excitation wavelength, choosing the standard, and determining the concentration needed for the quantum yield experiment that takes place in the second laboratory period. Overall, this laboratory provides students with a guided inquiry learning experience and introduces concepts of fluorescence biophysics into a biochemistry laboratory curriculum.

Membrane curvature based lipid sorting using a nanoparticle patterned substrate.

Cellular membranes contain a variety of shapes that likely act as motifs for sorting lipids and proteins. To understand the sorting that takes place within cells, a continuous, fluid bilayer with regions of membrane curvature was designed and characterized using confocal fluorescence and total internal reflection fluorescence microscopy techniques. A supported lipid bilayer was formed over fluorescently labelled nanoparticles deposited on a glass surface. The lipid composition and membrane shape are separately controlled and the nanoparticle dimensions (d = 40-200 nm) determine the extent of curvature. The bulk membrane is fluid as demonstrated by fluorescence recovery after photobleaching (FRAP) using dye labelled lipids. In bilayers that contain fluorescently labelled, single-tailed lipids, accumulation is observed at regions of curvature, yet the molecules retain fluidity. Using single particle imaging methods, lipids are observed to visit regions of curvature and exchange with the surrounding flat membrane. The nanoparticle patterned substrate described here allows for quantitative measurement of the transient interactions between fluorescently labelled biomolecules and regions of membrane curvature.

Efficacy, safety, pharmacokinetics, and biomarkers of cediranib monotherapy in advanced hepatocellular carcinoma: a phase II study.

PURPOSE: We conducted a single-arm phase II study of cediranib, a pan-VEGFR tyrosine kinase inhibitor, in patients with advanced hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN: Patients with histologically confirmed measurable advanced HCC and adequate hematologic, hepatic, and renal functions received cediranib 30-mg orally once daily (4 weeks/cycle). The primary endpoint was progression-free survival (PFS) rate at 3 months. Other endpoints included response rates, overall survival (OS), pharmacokinetics (PK), and biomarkers for cediranib. RESULTS: Cediranib treatment resulted in an estimated 3-month PFS rate of 77% (60%, 99%). Median PFS was 5.3 (3.5,9.7) months, stable disease was seen in 5/17 patients (29%), and median OS was 11.7 (7.5-13.6) months. Grade 3 toxicities included hypertension (29%), hyponatremia (29%), and hyperbilirubinemia (18%). Cediranib PK were comparable to those seen in cancer patients with normal hepatic function. Plasma levels of VEGF and PlGF increased and sVEGFR1, sVEGFR2, and Ang-2 decreased after cediranib treatment. PFS was inversely correlated with baseline levels of VEGF, sVEGFR2, and bFGF and with on-treatment levels of bFGF and IGF-1, and directly associated with on-treatment levels of IFN-γ. OS was inversely correlated with baseline levels of sVEGFR1, Ang-2, TNF-α, CAIX, and CD34(+)CD133(+)CD45(dim) circulating progenitor cells and on-treatment levels of sVEGFR2. CONCLUSIONS: Despite the limitations of primary endpoint selection, cediranib at 30-mg daily showed a high incidence of toxicity and preliminary evidence of antitumor activity in advanced HCC. Hepatic dysfunction did not seem to affect the steady-state PK of cediranib. Exploratory studies suggested proangiogenic and inflammatory factors as potential biomarkers of anti-VEGF therapy in HCC.

Single molecule tracking of P-glycoprotein in live cells reveals dynamic heterogeneity.

P-glycoprotein transports chemotherapy drugs from the plasma membrane and allows cancer cells to survive treatment. We transiently transfected PGP labeled with enhanced green fluorescent protein (PGP-EGFP) into MES-SA cells and used single molecule tracking techniques to characterize the dynamics on the surface of live cells. PGP exhibits freely diffusive behavior at short times and is confined at long times with a transition to anomalous diffusion at 0.7 s.

C-reactive protein (CRP) aptamer binds to monomeric but not pentameric form of CRP.

Native C-reactive protein (CRP) is composed of five identical subunits arranged in a pentameric structure (pCRP). Binding of pCRP to damaged cell membranes produces a second isoform, modified CRP, which has similar antigenicity to isolated monomeric subunits of CRP (mCRP). Emerging evidence indicates that modified CRP plays a role in inflammation and atherosclerosis, however, there are very few techniques that can distinguish the different isoforms of CRP. Here we show that an RNA aptamer binds specifically to mCRP and not to pCRP. Using this aptamer, we describe a simple, fast, and sensitive assay to detect nanomolar concentrations of mCRP using fluorescence anisotropy. In addition, we show that this aptamer can be used to detect mCRP in polyacrylamide gels and bound to a surface using total internal reflection fluorescence microscopy. The biological activity of the mCRP we prepared by heating pCRP with 0.1% sodium dodecyl sulfate was confirmed by observing binding to the complement protein, C1q. This probe provides an important tool for CRP research and has the potential to improve clinical diagnostics that predict risk for cardiovascular disease.

Sensing muscle ischemia: coincident detection of acid and ATP via interplay of two ion channels.

Ischemic pain--examples include the chest pain of a heart attack and the leg pain of a 30 s sprint--occurs when muscle gets too little oxygen for its metabolic need. Lactic acid cannot act alone to trigger ischemic pain because the pH change is so small. Here, we show that another compound released from ischemic muscle, adenosine tri-phosphate (ATP), works together with acid by increasing the pH sensitivity of acid-sensing ion channel number 3 (ASIC3), the molecule used by sensory neurons to detect lactic acidosis. Our data argue that ATP acts by binding to P2X receptors that form a molecular complex with ASICs; the receptor on sensory neurons appears to be P2X5, an electrically quiet ion channel. Coincident detection of acid and ATP should confer sensory selectivity for ischemia over other conditions of acidosis.

Direct measurement of relative and collective diffusion in a dilute binary colloidal suspension.

Experimental characterization of the dynamics of multicomponent fluids is a problem of general importance to the field of complex fluids. We demonstrate a new experimental approach, termed two-color Fourier imaging correlation spectroscopy, which allows direct measurement of the partial dynamic structure factors, S(11)(k,tau), S(22)(k,tau), and S(12)(k,tau), where 1, 2 label the component species of a binary colloidal suspension. Linear combinations of the partial dynamic structure factors yield the characteristic time-correlation functions of the binary fluid. These are the correlation functions of concentration fluctuations S(CC)(k,tau), number density fluctuations S(NN)(k,tau), and cross-correlations between number density and concentration fluctuations S(NC)(k,tau). Test measurements are performed on a dilute symmetric mixture of fluorescently labeled 0.5 and 1.0 microm polystyrene spheres. From these data, we determine generalized collective and relative diffusion coefficients, and compare them to the predictions for an ideal mixture of noninteracting particles.

Cancer patients' descriptions of their nursing care.

AIM: This paper reports a study of cancer patients' descriptions of nurses and nursing care. BACKGROUND: Nurses lament their poor representation in the media, and campaigns to improve their portrayal have been initiated. Media portrayal of nurses might be more realistic if patients' descriptions of nursing care were incorporated. METHOD: Qualitative data from an instrument development study were analysed. A total of 461 patients answered the question, 'In general, how do you feel about nurses?' The data were analysed by the constant comparative method and grounded theory coding techniques. FINDINGS: A typology of four concepts reflecting cancer patients' descriptions of their nursing care emerged from the data. The concepts were: laudable, caring, professional and outcomes. The concept laudable refers to commendable qualities of the nurse and nursing care. Caring refers to the nurse showing compassion, concern and kindness. Professional refers to the nurse as meeting expected standards of knowledge, skill and demeanour. Outcomes refer to the affective, cognitive, or physical effects attributed to nursing care. Both positive and negative instances of the concepts were included in the analysis. Examples of each concept using patients' own words are given. CONCLUSIONS: These cancer patients held nurses in relatively high esteem. These findings could be disseminated to the public press as an example of what patients' value about nurses and nursing care. They also could be used in media efforts to recruit and retain nurses.