Reduction of spectral interferences using ultraclean gold nanowire arrays in the LDI-MS analysis of a model peptide.

The surface chemistry of gold nanowires (AuNWs) has been systematically assessed in terms of contamination and cleaning processes. The nanomaterial's surface quality was correlated to its performance in the matrix-free laser desorption ionization mass spectrometry (LDI-MS) analysis of low molecular weight analytes. Arrays of AuNWs were deposited on glass slides by means of the lithographically patterned nanowire electrodeposition technique. AuNWs were then characterized in terms of surface chemical composition and morphology using X-ray photoelectron spectroscopy, scanning electron microscopy and atomic force microscopy. AuNWs were subjected to a series of well-known cleaning procedures with the aim of producing the best performing surfaces for the LDI-MS detection of leucine enkephalin, chosen as a model analyte with a molar mass below 1,000 g/mol. Prolonged cyclic voltammetry in 2 M sulfuric acid and, most of all, oxygen plasma cleaning for 5 min provided the best results in terms of simpler (interference-free) and more intense mass spectrometry spectra of the reference compound. The analyte always ionized as the sodiated adduct, and leucine enkephalin limits of detection of 0.5 and 2.5 pmol were estimated for the positive and negative analysis modes, respectively. This study points out the tight correlation existing between the chemical status of the nanostructure surface and the AuNW-assisted LDI-MS performance in terms of reproducibility of spectra, intensity of analyte ions and reduction of interferences.

Topological classification and enumeration of RNA structures by genus.

To an RNA pseudoknot structure is naturally associated a topological surface, which has its associated genus, and structures can thus be classified by the genus. Based on earlier work of Harer-Zagier, we compute the generating function Dg,σ (z) = ∑n dg,σ (n)zn for the number dg,σ (n) of those structures of fixed genus g and minimum stack size σ with n nucleotides so that no two consecutive nucleotides are basepaired and show that Dg,σ (z) is algebraic. In particular, we prove that dg,2(n) ∼ kg n3(g−1/2 )γ n2, where γ2 ≈ 1.9685. Thus, for stack size at least two, the genus only enters through the sub-exponential factor, and the slow growth rate compared to the number of RNA molecules implies the existence of neutral networks of distinct molecules with the same structure of any genus. Certain RNA structures called shapes are shown to be in natural one-to-one correspondence with the cells in the Penner-Strebel decomposition of Riemann's moduli space of a surface of genus g with one boundary component, thus providing a link between RNA enumerative problems and the geometry of Riemann's moduli space.

Monitoring of hemodynamic changes induced in the healthy breast through inspired gas stimuli with MR-guided diffuse optical imaging.

PURPOSE: The modulation of tissue hemodynamics has important clinical value in medicine for both tumor diagnosis and therapy. As an oncological tool, increasing tissue oxygenation via modulation of inspired gas has been proposed as a method to improve cancer therapy and determine radiation sensitivity. As a radiological tool, inducing changes in tissue total hemoglobin may provide a means to detect and characterize malignant tumors by providing information about tissue vascular function. The ability to change and measure tissue hemoglobin and oxygenation concentrations in the healthy breast during administration of three different types of modulated gas stimuli (oxygen/ carbogen, air/carbogen, and air/oxygen) was investigated. METHODS: Subjects breathed combinations of gases which were modulated in time. MR-guided diffuse optical tomography measured total hemoglobin and oxygen saturation in the breast every 30 s during the 16 min breathing stimulus. Metrics of maximum correlation and phase lag were calculated by cross correlating the measured hemodynamics with the stimulus. These results were compared to an air/air control to determine the hemodynamic changes compared to the baseline physiology. RESULTS: This study demonstrated that a gas stimulus consisting of alternating oxygen/carbogen induced the largest and most robust hemodynamic response in healthy breast parenchyma relative to the changes that occurred during the breathing of room air. This stimulus caused increases in total hemoglobin and oxygen saturation during the carbogen phase of gas inhalation, and decreases during the oxygen phase. These findings are consistent with the theory that oxygen acts as a vasoconstrictor, while carbogen acts as a vasodilator. However, difficulties in inducing a consistent change in tissue hemoglobin and oxygenation were observed because of variability in intersubject physiology, especially during the air/oxygen or air/carbogen modulated breathing protocols. CONCLUSIONS: MR-guided diffuse optical imaging is a unique tool that can measure tissue hemodynamics in the breast during modulated breathing. This technique may have utility in determining the therapeutic potential of pretreatment tissue oxygenation or in investigating vascular function. Future gas modulation studies in the breast should use a combination of oxygen and carbogen as the functional stimulus. Additionally, control measures of subject physiology during air breathing are critical for robust measurements.

Molybdenum nanowires by electrodeposition.

Metallic molybdenum (Mo(o)) wires with diameters ranging from 15 nanometers to 1.0 micrometers and lengths of up to 500 micrometers (0.5 millimeters) were prepared in a two-step procedure. Molybdenum oxide wires were electrodeposited selectively at step edges and then reduced in hydrogen gas at 500 degrees C to yield Mo(o). The hemicylindrical wires prepared by this technique were self-uniform, and the wires prepared in a particular electrodeposition (in batches of 10(5) to 10(7)) were narrowly distributed in diameter. Wires were obtained size selectively because the mean wire diameter was directly proportional to the square root of the electrolysis time. The metal nanowires could be embedded in a polystyrene film and lifted off the graphite electrode surface. The conductivity and mechanical resiliency of individual embedded wires were similar to those of bulk molybdenum.

Fabrication and use of nanometer-sized electrodes in electrochemistry.

Electrodes with electrochemical dimensions as small as 10 angstroms have been fabricated and used for electrochemical studies. These nanometer-scale electrodes have enabled the measurement of electron-transfer rate constants, k(het), that are two orders of magnitude faster than k(het) values accessible with any other electrochemical method.

Hydrogen sensors and switches from electrodeposited palladium mesowire arrays.

Hydrogen sensors and hydrogen-activated switches were fabricated from arrays of mesoscopic palladium wires. These palladium "mesowire" arrays were prepared by electrodeposition onto graphite surfaces and were transferred onto a cyanoacrylate film. Exposure to hydrogen gas caused a rapid (less than 75 milliseconds) reversible decrease in the resistance of the array that correlated with the hydrogen concentration over a range from 2 to 10%. The sensor response appears to involve the closing of nanoscopic gaps or "break junctions" in wires caused by the dilation of palladium grains undergoing hydrogen absorption. Wire arrays in which all wires possessed nanoscopic gaps reverted to open circuits in the absence of hydrogen gas.

Analysis of the published calorimetric evidence for electrochemical fusion of deuterium in palladium.

Estimates are given of the raw data that are the basis for the claims of excess power production by the electrochemical charging of palladium in deuterium oxide (D(2)O). Calorimetric results are also presented that show no anomalous power production in either 0.1M LiOD/D(2)O or 0.1M LiOH/H(2)O (LiOH is lithium hydroxide). Several possible sources of error in open-system calorimetry are discussed that can confound interpretation of temperature changes in terms of anomalous power production.

Acceleration of membrane recycling by axotomy of cultured aplysia neurons.

The rapid transition of a stationary axon into a motile growth cone requires the recruitment of membrane and its strategic insertion into the neurolemma. The source of membrane to support the initial rapid growth postaxotomy is not known. Using membrane capacitance measurements, we examined quantitative aspects of membrane dynamics following axotomy of cultured Aplysia neurons. Axotomy activates two processes in parallel: membrane retrieval and exocytosis. Unexpectedly, membrane retrieval is the dominant process in the majority of the experiments. Thus, while a growth cone is vigorously extending, the total neuronal surface area decreases. We suggest that the initial rapid extension phase of the newly formed growth cone postaxotomy is supported by a pool of intracellular membrane that is rapidly retrieved from the neurolemma.

The use of infliximab for treatment of hospitalized patients with acute severe ulcerative colitis.

BACKGROUND/AIM: The use of infliximab in severe ulcerative colitis (UC) is established; however, its role in severe acute UC requires clarification. The present multicentre case series evaluated infliximab in hospitalized patients with steroid-refractory severe UC. METHODS: Patients from six hospitals were retrospectively evaluated. Data collection included demographics, duration of disease and previous treatments. The primary end point was response to in-hospital infliximab; defined as discharge without colectomy. RESULTS: Twenty-one patients (median age 26 years) were admitted between May 2006 and May 2008 with severe UC requiring intravenous steroids and given infliximab (median time to infusion eight days). Sixteen (76%) patients were discharged home without colectomy; three of these underwent colectomy at a later date. Of the remaining 13 patients (62%), all but two did not require further courses of steroids; six patients had infliximab as a bridge to azathioprine and seven patients were maintained on regular infliximab. Five patients required in-hospital colectomy after the initial infliximab. CONCLUSIONS: In this real-life experience of infliximab in patients with steroid-refractory severe UC, infliximab appears to be a viable rescue therapy. The majority of patients were discharged without surgery and 62% maintained response either as a bridge to azathioprine or maintenance infliximab.

Review and clinical perspectives for the use of infliximab in ulcerative colitis.

Infliximab is a chimeric, monoclonal anti-tumour necrosis factor-alpha antibody. It has been previously demonstrated to be an effective treatment for patients with Crohn's disease who do not achieve the desired response with conventional treatments. Although the etiology of ulcerative colitis (UC) differs from that of Crohn's disease, randomized controlled trials have demonstrated that infliximab is also beneficial for the treatment of moderate to severe UC in patients who are either intolerant of or refractory to immunosuppressant agents or steroids, or those who are steroid-dependent. A review of the literature is followed by practical recommendations regarding infliximab that address the needs of clinicians and UC patients. Where there is a lack of evidence-based information, the expert panel provides its combined opinion derived from the members' clinical experiences.

The Mg2+ and Mg(2+)-nucleotide-regulated channel-kinase TRPM7.

TRPM7 is a member of the melastatin-related subfamily of TRP channels and represents a protein that contains both an ion channel and a kinase domain. The protein is ubiquitously expressed and represents the only ion channel known that is essential for cellular viability. TRPM7 is a divalent cation-selective ion channel that is permeable to Ca2+ and Mg2+, but also conducts essential metals such as Zn2+, Mn2+, and Co2+, as well as nonphysiologic or toxic metals such as Ni2+, Cd2+, Ba2+, and Sr2+. The channel is constitutively open but strongly downregulated by intracellular levels of Mg2+ and MgATP and other Mg-nucleotides. Reducing the cellular levels of these regulators leads to activation of TRPM7-mediated currents that exhibit a characteristic nonlinear current-voltage relationship with pronounced outward rectification due to divalent influx at physiologically negative voltages and monovalent outward fluxes at positive voltages. TRPM7 channel activity is also actively regulated following receptor-mediated changes in cyclic AMP (cAMP) and protein kinase A activity. This regulation as well as that by Mg-nucleotides requires a functional endogenous kinase domain. The function of the kinase domain is not completely understood, but may involve autophosphorylation of TRPM7 as well as phosphorylation of other target proteins such as annexin and myosin IIA heavy chain. Based on these properties, TRPM7 is currently believed to represent a ubiquitous homeostatic mechanism that regulates Ca2+ and Mg2+ fluxes based on the metabolic state of the cell. Physiologically, the channel may serve as a regulated transport mechanism for these ions that could affect cell adhesion, cell growth and proliferation, and even cell death under pathological stress such as anoxia.

The patch-clamp technique in the study of secretion.

One of the basic cellular functions of virtually every cell type is the exocytotic release of molecules synthesized, stored and packaged into intracellular vesicles or granules. Over decades much effort has been concentrated on elucidating the chain of events leading to exocytosis. Unfortunately, the nature of the process that ultimately induces membrane fusion is not known, nor has it been established definitively whether or not the final steps in the secretory cascade are identical in different cells. Although the fusion between vesicle and plasma membrane has been neatly documented by electron micrographs, it was only recently that the technique of time-resolved membrane capacitance measurement has provided a more detailed insight into mechanistic aspects of exocytosis, both in terms of the fusion event and the steps involved in stimulus-secretion coupling.

Calcium influx and its control by calcium release.

Changes in the concentration of intracellular Ca2+ are crucial for signal transduction in virtually every cell. In the past year, more of the diversity of receptor-mediated Ca2+ influx mechanisms has been shown, and it has been disclosed that one of the most effective Ca2+ influx pathways, known as 'capacitative Ca2+ entry', occurs via Ca(2+)-selective ion channels in the plasma membrane that are activated following depletion of intracellular Ca2+ stores. Although the exact activation mechanism of capacitative Ca2+ entry still remains a mystery, the identification of plasma membrane currents following store depletion and the characterization of their biophysical properties opens the possibility of unraveling the features and molecular components of the phenomenon of capacitative Ca2+ entry.

Shrinking nanowires by kinetically controlled electrooxidation.

Nanowires composed of antimony, gold, and bismuth telluride (Bi2Te3) were reduced in diameter by electrooxidation in aqueous solutions. When electrooxidation was carried out using low current densities (Jox < 150 microA cm(-2)), the mean wire diameter decreased in direct proportion to the oxidation time, as expected for a kinetically controlled process. Under these conditions, the diameter uniformity of nanowires remained constant as wires were shrunk from initial diameters of more than 120 nm to less than 40 nm, for Sb and Bi2Te3, and less than 60 nm for Au. Oxidized nanowires remained continuous for more than 100 microm. Electrooxidation at higher current densities rapidly introduced breaks into these nanowires. Electrochemical wire growth and shrinking by electrooxidation were integrated into a single electrochemical experiment that allowed the final mean diameter of nanowires to be specified with a precision of 5-10 nm.

In vivo prostate cancer detection and grading using restriction spectrum imaging-MRI.

BACKGROUND: Magnetic resonance imaging (MRI) is emerging as a robust, noninvasive method for detecting and characterizing prostate cancer (PCa), but limitations remain in its ability to distinguish cancerous from non-cancerous tissue. We evaluated the performance of a novel MRI technique, restriction spectrum imaging (RSI-MRI), to quantitatively detect and grade PCa compared with current standard-of-care MRI. METHODS: In a retrospective evaluation of 33 patients with biopsy-proven PCa who underwent RSI-MRI and standard MRI before radical prostatectomy, receiver-operating characteristic (ROC) curves were performed for RSI-MRI and each quantitative MRI term, with area under the ROC curve (AUC) used to compare each term's ability to differentiate between PCa and normal prostate. Spearman rank-order correlations were performed to assess each term's ability to predict PCa grade in the radical prostatectomy specimens. RESULTS: RSI-MRI demonstrated superior differentiation of PCa from normal tissue, with AUC of 0.94 and 0.85 for RSI-MRI and conventional diffusion MRI, respectively (P=0.04). RSI-MRI also demonstrated superior performance in predicting PCa aggressiveness, with Spearman rank-order correlation coefficients of 0.53 (P=0.002) and -0.42 (P=0.01) for RSI-MRI and conventional diffusion MRI, respectively, with tumor grade. CONCLUSIONS: RSI-MRI significantly improves upon current noninvasive PCa imaging and may potentially enhance its diagnosis and characterization.

Ca2+-induced Ca2+ release in Chinese hamster ovary (CHO) cells co-expressing dihydropyridine and ryanodine receptors.

Combined patch-clamp and Fura-2 measurements were performed on chinese hamster ovary (CHO) cells co-expressing two channel proteins involved in skeletal muscle excitation-contraction (E-C) coupling, the ryanodine receptor (RyR)-Ca2+ release channel (in the membrane of internal Ca2+ stores) and the dihydropyridine receptor (DHPR)-Ca2+ channel (in the plasma membrane). To ensure expression of functional L-type Ca+ channels, we expressed alpha2, beta, and gamma DHPR subunits and a chimeric DHPR alpha(i) subunit in which the putative cytoplasmic loop between repeats II and III is of skeletal origin and the remainder is cardiac. There was no clear indication of skeletal-type coupling between the DHPR and the RyR; depolarization failed to induce a Ca2+ transient (CaT) in the absence of extracellular Ca2+ ([Ca2+]o). However, in the presence of [Ca2+]o, depolarization evoked CaTs with a bell-shaped voltage dependence. About 30% of the cells tested exhibited two kinetic components: a fast transient increase in intracellular Ca2+ concentration ([Ca2+]i) (the first component; reaching 95% of its peak <0.6 s after depolarization) followed by a second increase in [Ca2+]i which lasted for 5-10 s (the second component). Our results suggest that the first component primarily reflected Ca2+ influx through Ca2+ channels, whereas the second component resulted from Ca2+ release through the RyR expressed in the membrane of internal Ca2+ stores. However, the onset and the rate of Ca2+ release appeared to be much slower than in native cardiac myocytes, despite a similar activation rate of Ca2+ current. These results suggest that the skeletal muscle RyR isoform supports Ca2+-induced Ca2+ release but that the distance between the DHPRs and the RyRs is, on average, much larger in the cotransfected CHO cells than in cardiac myocytes. We conclude that morphological properties of T-tubules and/or proteins other than the DHPR and the RyR are required for functional "close coupling" like that observed in skeletal or cardiac muscle. Nevertheless, some of our results imply that these two channels are potentially able to directly interact with each other.

Molybdenum disulfide nanowires and nanoribbons by electrochemical/chemical synthesis.

Molybdenum disulfide nanowires and nanoribbons have been synthesized by a two-step, electrochemical/chemical synthetic method. In the first step, MoO(x) wires (a mixture of MoO(2) and MoO(3)) were electrodeposited size-selectively by electrochemical step-edge decoration on a highly oriented pyrolytic graphite (HOPG) surface. Then, MoO(x) precursor wires were converted to MoS(2) by exposure to H(2)S either at 500-700 degrees C, producing "low-temperature" or LT MoS(2) nanowires that were predominantly 2H phase, or above 800 degrees C producing "high-temperature" or HT MoS(2) ribbons that were predominantly 3R phase. The majority of these MoS(2) wires and ribbons were more than 50 microm in length and were organized into parallel arrays containing hundreds of wires or ribbons. MoS(2) nanostructures were characterized by X-ray photoelectron spectroscopy, scanning and transmission electron microscopy, selected area electron diffraction, X-ray diffraction, UV-visible absorption spectrometry, and Raman spectroscopy. HT and LT MoS(2) nanowires were structurally distinct: LT MoS(2) wires were hemicylindrical in shape and nearly identical in diameter to the MoO(x) precursor wires from which they were derived. LT MoS(2) wires were polycrystalline, and the internal structure consisted of many interwoven, multilayer strands of MoS(2); HT MoS(2) ribbons were 50-800 nm in width and 3-100 nm thick, composed of planar crystallites of 3R-MoS(2). These layers grew in van der Waals contact with the HOPG surface so that the c-axis of the 3R-MoS(2) unit cell was oriented perpendicular to the plane of the graphite surface. Arrays of MoS(2) wires and ribbons could be cleanly separated from the HOPG surface and transferred to glass for electrical and optical characterization. Optical absorption measurements of HT MoS(2) nanoribbons reveal a direct gap near 1.95 eV and two exciton peaks, A1 and B1, characteristic of 3R-MoS(2). These exciton peaks shifted to higher energy by up to 80 meV as the wire thickness was decreased to 7 nm (eleven MoS(2) layers). The energy shifts were proportional to 1/ L( parallel)(2), and the effective masses were calculated. Current versus voltage curves for both LT and HT MoS(2) nanostructures were probed as a function of temperature from -33 degrees C to 47 degrees C. Conduction was ohmic and mainly governed by the grain boundaries residing along the wires. The thermal activation barrier was found to be related to the degree of order of the crystallites and can be tuned from 126 meV for LT nanowires to 26 meV for HT nanoribbons.

Novel technique for characterizing prostate cancer utilizing MRI restriction spectrum imaging: proof of principle and initial clinical experience with extraprostatic extension.

BACKGROUND: Standard magnetic resonance imaging (MRI) of the prostate lacks sensitivity in the diagnosis and staging of prostate cancer (PCa). To improve the operating characteristics of prostate MRI in the detection and characterization of PCa, we developed a novel, enhanced MRI diffusion technique using restriction spectrum imaging (RSI-MRI). METHODS: We compared the efficacy of our novel RSI-MRI technique with standard MRI for detecting extraprostatic extension (EPE) among 28 PCa patients who underwent MRI and RSI-MRI prior to radical prostatectomy, 10 with histologically proven pT3 disease. RSI cellularity maps isolating the restricted isotropic water fraction were reconstructed based on all b-values and then standardized across the sample with z-score maps. Distortion correction of the RSI maps was performed using the alternating phase-encode technique. RESULTS: 27 patients were evaluated, excluding one patient where distortion could not be performed. Preoperative standard MRI correctly identified extraprostatic the extension in two of the nine pT3 (22%) patients, whereas RSI-MRI identified EPE in eight of nine (89%) patients. RSI-MRI correctly identified pT2 disease in the remaining 18 patients. CONCLUSIONS: In this proof of principle study, we conclude that our novel RSI-MRI technology is feasible and shows promise for substantially improving PCa imaging. Further translational studies of prostate RSI-MRI in the diagnosis and staging of PCa are indicated.

Secretory responses of rat peritoneal mast cells to high intracellular calcium.

The patch-clamp technique was used to investigate the secretory responses of rat peritoneal mast cells at various intracellular calcium concentrations ([Ca2+]i). When Calcium was introduced into the cell with pipette-loaded dibromo-BAPTA, elevation of [Ca2+]i into the range 1-10 microM induced membrane capacitance increases indicative of exocytosis in a concentration-dependent manner. At higher concentrations a decrease of the response was observed. Cells that were exposed to micromolar [Ca2+]i underwent morphological alterations resulting in swelling, which is indicative of cytoskeletal alterations. The presence of dibromo-BAPTA (4 mM) strongly inhibited secretion induced by GTP-gamma-S, thus hampering the contribution of G-protein-mediated stimulation. Application of the Ca2+ ionophore ionomycin resulted in transient increases in [Ca2+]i which were parallelled by Ca2+-dependent secretion. Effective buffering of the cytosolic calcium level below 1 microM abolished the secretory response. Our results show that an increase in [Ca2+]i can trigger secretion, but only if it is high and sustained. During physiological stimulation, however, secretion proceeds at [Ca2+]i below 1 microM. It is, therefore, concluded that mast cell degranulation under physiological conditions is not simply a result of an increase in [Ca2+]i, but that other second messenger systems in conjunction with calcium act synergistically in order to ensure fast and efficient secretion.

Functional expression of the calcium release channel from skeletal muscle ryanodine receptor cDNA.

Combined patch-clamp and fura-2 measurements were performed to study the calcium release properties of Chinese hamster ovary (CHO) cells transfected with the rabbit skeletal muscle ryanodine receptor cDNA carried by an expression vector. Both caffeine (1-50 mM) and ryanodine (100 microM) induced release of calcium from intracellular stores of transformed CHO cells but not from control (non-transfected) CHO cells. The calcium responses to caffeine and ryanodine closely resembled those commonly observed in skeletal muscle. Repetitive applications of caffeine produced characteristic all-or-none rises in intracellular calcium. Inositol 1,4,5-trisphosphate (IP3) neither activated the ryanodine receptor channel nor interfered with the caffeine-elicited calcium release. These results indicate that functional calcium release channels are formed by expression of the ryanodine receptor cDNA.