Flow characterization of a microfluidic device to selectively and reliably apply reagents to a cellular network

A three-dimensional microfluidic device has been successfully fabricated and the flow streams characterized for eventual use in studying communication in an in vitro network of nerve cells. The microfluidic system is composed of two layers of channels: a lower layer for the delivery of pharmacological solutions and an upper layer of channels used to direct the flow of the pharmacological solution streams and perfuse the cells with media and nutrients. Flow profiles have been characterized with computational fluid dynamics simulations, confocal fluorescence microscopy, and carbon-fiber amperometry, which have been used to map changes in flow profiles at different bulk flow rates. Ultimately, the microfluidic system and incorporated cell network will show how networked neurons adapt, compensate, and recover after being exposed to different chemical compounds.     

Crystallographic characterizations and new high-yield synthetic routes for the complete series of 6-X-B10H13 halodecaboranes (X = F, Cl, Br, I) via superacid-induced cage-opening reactions of closo-B10H10(2-)

The high-yield syntheses of 6-X-B 10H 13 [X = Cl (88%), Br (96%), I (84%)] resulted from the cage-opening reactions of the (NH 4 (+)) 2B 10H 10 (2-) salt with ionic-liquid-based superacidic hydrogen halides, while both the previously unknown 6-F-B 10H 13 (77%) derivative and 6-Cl-B 10H 13 (90%) were synthesized in high yields via the reactions of (NH 4 (+)) 2B 10H 10 (2-) with triflic acid in the presence of 1-fluoropentane and dichloromethane, respectively. Structural characterizations of 1- 4 confirm the predicted structures and indicate strong halogen back-bonding interactions with the B6 boron. The reaction of 6-Br-B 10H 13 with Bu 3SnH produced the parent B 10H 14 in 70% yield, and thus, this reaction, in conjunction with the haloacid-induced closo-B 10H 10 (2-) cage-opening reactions, has the potential to provide an alternative to the traditional diborane pyrolysis route to decaborane.     

Freeze-etching and vapor matrix deposition for ToF-SIMS imaging of single cells

Freeze-etching, the practice of removing excess surface water from a sample through sublimation into the vacuum of the analysis environment, has been extensively used in conjunction with electron microscopy. Here, we apply this technique to time-of-flight secondary-ion mass spectrometry (ToF-SIMS) imaging of cryogenically preserved single cells. By removing the excess water which condenses onto the sample in vacuo, a uniform surface is produced that is ideal for imaging by static SIMS. We demonstrate that the conditions employed to remove deposited water do not adversely affect cell morphology and do not redistribute molecules in the topmost surface layers. In addition, we found water can be controllably redeposited onto the sample at temperatures below -100 degrees C in vacuum. The redeposited water increases the ionization of characteristic fragments of biologically interesting molecules 2-fold without loss of spatial resolution. The utilization of freeze-etch methodology will increase the reliability of cryogenic sample preparations for SIMS analysis by providing greater control of the surface environment. Using these procedures, we have obtained high quality spectra with both atomic bombardment as well as C 60 (+) cluster ion bombardment.     

Short-chain alcohols promote accelerated membrane distention in a dynamic liposome model of exocytosis

We have used amperometric measurements in a model system consisting of two liposomes connected with a membrane nanotube to monitor catechol release during artificial exocytosis and thereby to elucidate the effect of small-chain alcohols on this dynamic membrane process. To determine the rate of membrane shape change, catechol release during membrane distention was monitored amperometrically, and the presence of alcohols in the buffer was shown to accelerate the membrane distention process in a concentration-dependent manner. Compression isotherms for the same lipid composition in the absence and presence of ethanol and 1-propanol were measured to determine how these short-chain alcohols affect the lipid packing in monolayers. The isotherms show a marked decrease in lipid packing density that is dependent on the particular alcohol and its concentration. Comparison of the electrochemical and isotherm results suggests a correlation between decreasing lipid packing density and increasing rates of membrane shape change.     

Degenerate two-phase incompressible flow III. Sharp error estimates

Summary. This is the third paper of a series in which we analyze mathematical properties and develop numerical methods for a degenerate elliptic-parabolic partial differential system which describes the flow of two incompressible, immiscible fluids in porous media. In this paper we consider a finite element approximation for this system. The elliptic equation for the pressure and velocity is approximated by a mixed finite element method, while the degenerate parabolic equation for the saturation is approximated by a Galerkin finite element method. A fully discrete approximation is analyzed. Sharp error estimates in energy norms are obtained for this approximation. The error analysis does not use any regularization of the saturation equation; the error estimates are derived directly from the degenerate equation. Also, the analysis does not impose any restriction on the nature of degeneracy. Finally, it respects the minimal regularity on the solution of the differential system. Received March 9, 1998 / Revised version received July 17, 2000 / Published online May 30, 2001     

Effects of cysteic acid groups on the gas-phase reactivity and dissociation of [M + 4H]4+ ions from insulin chain B

Gas-phase ion/molecule reactions and collision-induced dissociation (CID) were conducted on [M + 4H]4+ of insulin chain B. This Fourier transform mass spectrometry work involved ions from the oxidized peptide (with two cysteic acid residues) and its reduced form (with two cysteine residues). Kinetic behavior during deprotonation and hydrogen/deuterium exchange reactions indicates that insulin B (ox) ions have two distinct structural types. In contrast, insulin B (red) ions have only one major reacting population, which has a more compact structure than the oxidized ions. No significant differences in fragmentation patterns for the two insulin B (ox) populations were observed when CID was performed as a function of deprotonating reaction time. However, markedly different fragmentation was found between [M + 4H]4+ of insulin B (ox) and (red). Therefore, the presence of cysteic acid groups in insulin B (ox) significantly impacts dissociation and presumably structure. This suggests that some insulin B (ox) ions are zwitterionic, with the five basic sites protonated and one cysteic acid group deprotonated. Molecular dynamics calculations revealed several viable structures that are consistent with the experimental results. For example, the most stable form of the reduced ion, which is unprotonated at the His10, is very compact and has lost the alpha-helix of native insulin. Low energy structures for the oxidized ions include a zwitterion with an intraionic interaction between anionic Cyx7 and cationic His10, as well as a nonzwitterionic conformer that lacks a proton at Phe1; both structures retain the alpha-helix. These structures may account for the two experimentally observed isomers, although others are possible. In addition, experiments on oxidized insulin B were conducted from methanolic solution, which may denature the conformation, and pure aqueous solution, which may leave a native conformation. These differences in solvent composition had no effect on the gas-phase results.     

Amperometry and Electron Microscopy show Stress Granules Induce Homotypic Fusion of Catecholamine Vesicles

An overreactive stress granule (SG) pathway and long-lived, stable SGs formation are thought to participate in the progress of neurodegenerative diseases (NDs). To understand if and how SGs contribute to disorders of neurotransmitter release in NDs, we examined the interaction between extracellular isolated SGs and vesicles. Amperometry shows that the vesicular content increases and dynamics of vesicle opening slow down after vesicles are treated with SGs, suggesting larger vesicles are formed. Data from transmission electron microscopy (TEM) clearly shows that a portion of large dense-core vesicles (LDCVs) with double/multiple cores appear, thus confirming that SGs induce homotypic fusion between LDCVs. This might be a protective step to help cells to survive following high oxidative stress. A hypothetical mechanism is proposed whereby enriched mRNA or protein in the shell of SGs is likely to bind intrinsically disordered protein (IDP) regions of vesicle associated membrane protein (VAMP) driving a disrupted membrane between two closely buddled vesicles to fuse with each other to form double-core vesicles. Our results show that SGs induce homotypic fusion of LDCVs, providing better understanding of how SGs intervene in pathological processes and opening a new direction to investigations of SGs involved neurodegenerative disease.     

Concentration of stimulant regulates initial exocytotic molecular plasticity at single cells

Activity-induced synaptic plasticity has been intensively studied, but is not yet well understood. We examined the temporal and concentration effects of exocytotic molecular plasticity during and immediately after chemical stimulation (30 s K⁺ stimulation) via single cell amperometry. Here the first and the second 15 s event periods from individual event traces were compared. Remarkably, we found that the amount of catecholamine release and release dynamics depend on the stimulant concentration. No changes were observed at 10 mM K⁺ stimulation, but changes observed at 30 and 50 mM (i.e., potentiation, increased number of molecules) were opposite to those at 100 mM (i.e., depression, decreased number of events), revealing changes in exocytotic plasticity based on the concentration of the stimulant solution. These results show that molecular changes initiating exocytotic plasticity can be regulated by the concentration strength of the stimulant solution. These different effects on early plasticity offer a possible link between stimulation intensity and synaptic (or adrenal) plasticity.

Amperometric measurement of exocytosis (SCA) and vesicle content (IVIEC) over 15 s intervals reveals plasticity (none, potentiation, or depression), that is regulated by the concentration of stimulant solution (e.g., 30 s 10, 30, 50, and 100 mM K⁺).     

Dysfunction of vesicular storage in young-onset Parkinson's patient-derived dopaminergic neurons and organoids revealed by single cell electrochemical cytometry

Electrochemical cytometry based on nano-tip microelectrodes was used to quantify the vesicular storage at the single-cell level in human neurons and midbrain organoids which acted as disease models of young-onset Parkinson''s disease (YOPD). Human dopaminergic (DA) neurons and midbrain organoids were derived from an induced pluripotent stem cell line from one YOPD patient. We show a significant deficiency in vesicular catecholamine storage and a slower pore forming process on the surface of the microelectrode in the DA neurons derived from the YOPD patient. The upregulation of α-synuclein in both neurons and organoids derived from the YOPD patient is associated with vesicular storage dysfunction, revealing a correlation between the pathogenesis of YOPD and vesicular chemical storage deficiency, a novel chemical insight into the potential pathology of YOPD. Notably, efficacy evaluation and drug testing were performed with our platform to demonstrate that both amantadine, a clinical drug for Parkinson''s disease (PD), and phorbol 12-myristate 13-acetate, an attractive candidate, ameliorate the dysfunction of vesicular storage in DA neurons derived from the YOPD patient. Our platform offers promising avenues for new drug discovery for PD and other neurodegenerative disorders.

Deficient vesicular storage at the single-cell level in human neurons and midbrain organoids derived from an iPSC line from one YOPD patient was revealed via electrochemical cytometry at nanotip microelectrodes.