Fabrication of microfluidic cavities using Si-to-glass anodic bonding.

We demonstrate the fabrication of ∼1.08 µm deep microfluidic cavities with characteristic size as large as 7 mm × 11 mm or 11 mm diameter, using a silicon-glass anodic bonding technique that does not require posts to act as separators to define cavity height. Since the phase diagram of 3He is significantly altered under confinement, posts might act as pinning centers for phase boundaries. The previous generation of cavities relied on full wafer-bonding which is more prone to failure and requires dicing post-bonding, whereas these cavities are made by bonding a pre-cut piece of Hoya SD-2 glass to a patterned piece of silicon in which the cavity is defined by etching. Anodic bonding was carried out at 425 °C with 200 V, and we observe that pressurizing the cavity to failure (>30 bars pressure) results in glass breaking, rather than the glass-silicon bond separation. In this article, we discuss the detailed fabrication of the cavity, its edges, and details of the junction between the coin silver fill line and the silicon base of the cavity that enables a low internal-friction joint. This feature is important for mass coupling torsional oscillator experimental assays of the superfluid inertial contribution where a high quality factor (Q) improves frequency resolution. The surface preparation that yields well-characterized smooth surfaces to eliminate pinning sites, the use of transparent glass as a cover permitting optical access, low temperature capability, and attachment of pressure-capable ports for fluid access may be features that are important in other applications.

The A-B transition in superfluid helium-3 under confinement in a thin slab geometry.

The influence of confinement on the phases of superfluid helium-3 is studied using the torsional pendulum method. We focus on the transition between the A and B phases, where the A phase is stabilized by confinement and a spatially modulated stripe phase is predicted at the A-B phase boundary. Here we discuss results from superfluid helium-3 contained in a single 1.08-µm-thick nanofluidic cavity incorporated into a high-precision torsion pendulum, and map the phase diagram between 0.1 and 5.6 bar. We observe only small supercooling of the A phase, in comparison to bulk or when confined in aerogel, with evidence for a non-monotonic pressure dependence. This suggests that an intrinsic B-phase nucleation mechanism operates under confinement. Both the phase diagram and the relative superfluid fraction of the A and B phases, show that strong coupling is present at all pressures, with implications for the stability of the stripe phase.

Anti-fibrotic actions of relaxin.

Fibrosis refers to the hardening or scarring of tissues that usually results from aberrant wound healing in response to organ injury, and its manifestations in various organs have collectively been estimated to contribute to around 45-50% of deaths in the Western world. Despite this, there is currently no effective cure for the tissue structural and functional damage induced by fibrosis-related disorders. Relaxin meets several criteria of an effective anti-fibrotic based on its specific ability to inhibit pro-fibrotic cytokine and/or growth factor-mediated, but not normal/unstimulated, fibroblast proliferation, differentiation and matrix production. Furthermore, relaxin augments matrix degradation through its ability to up-regulate the release and activation of various matrix-degrading matrix metalloproteinases and/or being able to down-regulate tissue inhibitor of metalloproteinase activity. Relaxin can also indirectly suppress fibrosis through its other well-known (anti-inflammatory, antioxidant, anti-hypertrophic, anti-apoptotic, angiogenic, wound healing and vasodilator) properties. This review will outline the organ-specific and general anti-fibrotic significance of exogenously administered relaxin and its mechanisms of action that have been documented in various non-reproductive organs such as the cardiovascular system, kidney, lung, liver, skin and tendons. In addition, it will outline the influence of sex on relaxin's anti-fibrotic actions, highlighting its potential as an emerging anti-fibrotic therapeutic. LINKED ARTICLES: This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc.

Phase diagram of the topological superfluid 3He confined in a nanoscale slab geometry.

The superfluid phases of helium-3 ((3)He) are predicted to be strongly influenced by mesoscopic confinement. However, mapping out the phase diagram in a confined geometry has been experimentally challenging. We confined a sample of (3)He within a nanofluidic cavity of precisely defined geometry, cooled it, and fingerprinted the order parameter using a sensitive nuclear magnetic resonance spectrometer. The measured suppression of the p-wave order parameter arising from surface scattering was consistent with the predictions of quasi-classical theory. Controlled confinement of nanofluidic samples provides a new laboratory for the study of topological superfluids and their surface- and edge-bound excitations.

Quantum transport in mesoscopic 3He films: experimental study of the interference of bulk and boundary scattering.

We discuss the mass transport of a degenerate Fermi liquid ^{3}He film over a rough surface, and the film momentum relaxation time, in the framework of theoretical predictions. In the mesoscopic regime, the anomalous temperature dependence of the relaxation time is explained in terms of the interference between elastic boundary scattering and inelastic quasiparticle-quasiparticle scattering within the film. We exploit a quasiclassical treatment of quantum size effects in the film in which the surface roughness, whose power spectrum is experimentally determined, is mapped into an effective disorder potential within a film of uniform thickness. Confirmation is provided by the introduction of elastic scattering centers within the film. The improved understanding of surface roughness scattering may impact on enhancing the conductivity in thin metallic films.

Modification of the 3He phase diagram by anisotropic disorder.

Motivated by the recent prediction that uniaxially compressed aerogel can stabilize the anisotropic A phase over the isotropic B phase, we measure the pressure dependent superfluid fraction of (3)He entrained in 10% axially compressed, 98% porous aerogel. We observe that a broad region of the temperature-pressure phase diagram is occupied by the metastable A phase. The reappearance of the A phase on warming from the B phase, before superfluidity is extinguished at T(c), is in contrast to its absence in uncompressed aerogel. The phase diagram is modified from that of pure (3)He, with the disappearance of the polycritical point (PCP) and the appearance of a region of A phase extending below the PCP of bulk (3)He, even in zero applied magnetic field. The expected alignment of the A phase texture by compression is not observed.

Anodically bonded submicron microfluidic chambers.

We demonstrate the use of anodic bonding to fabricate cells with characteristic size as large as 7 x 10 mm(2), with height of approximately 640 nm, and without any internal support structure. The cells were fabricated from Hoya SD-2 glass and silicon wafers, each with 3 mm thickness to maintain dimensional stability under internal pressure. Bonding was carried out at 350 degrees C and 450 V with an electrode structure that excluded the electric field from the open region. We detail fabrication and characterization steps and also discuss the design of the fill line for access to the cavity.

Putting the P in Ptilotus: a phosphorus-accumulating herb native to Australia.

BACKGROUND AND AIMS: Ptilotus polystachyus (green mulla mulla; ptilotus) is a short-lived perennial herb that occurs widely in Australia in arid and semi-arid regions with nutrient poor soils. As this species shows potential for domestication, its response to addition of phosphorus (P) and nitrogen (N) was compared to a variety of the domesticated exotic perennial pasture herb Cichorium intybus (chicory), 'Puna'. METHODS: Pots were filled with 3 kg of an extremely nutrient-deficient sterilized field soil that contained 3 mg kg(-1) mineral N and 2 mg kg(-1) bicarbonate-extractable P. The growth and P and N accumulation of ptilotus and chicory in response to seven rates of readily available phosphorus (0-300 mg P pot(-1)) and nitrogen (N) (0-270 mg N pot(-1)) was examined. KEY RESULTS: Ptilotus grew extremely well under low P conditions: shoot dry weights were 23, 6 and 1.7 times greater than for chicory at the three lowest levels of P addition, 0, 15 and 30 mg P pot(-1), respectively. Ptilotus could not downregulate P uptake. Concentrations of P in shoots approached 4% of dry weight and cryo-scanning electron microscopy and X-ray microanalysis showed 35-196 mM of P in cell vacuoles in a range of tissues from young leaves. Ptilotus had a remarkable tolerance of high P concentrations in shoots. While chicory exhibited symptoms of P toxicity at the highest rate of P addition (300 mg P pot(-1)), no symptoms were present for ptilotus. The two species responded in a similar manner to addition of N. CONCLUSIONS: In comparison to chicory, ptilotus demonstrated an impressive ability to grow well under conditions of low and high P availability. Further study of the mechanisms of P uptake and tolerance in ptilotus is warranted.

Effect of nelfinavir on insulin metabolism, proteasome activity and protein degradation in HepG2 cells.

HIV-1 protease inhibitors have revolutionized the treatment of HIV infection, but their use has been associated with lipodystrophy and insulin resistance. One suggestion for this has been the inhibition of insulin-degrading enzyme (IDE). We have previously demonstrated that insulin, through IDE, can inhibit the proteasome, thus decreasing cytosolic protein degradation. We examined whether the protease inhibitor nelfinavir inhibited IDE and its effect on protein degradation both in vitro and in whole cells. 125I-Insulin degradation was measured by trichloroacetic acid precipitation. Proteasome activities were measured using fluorogenic peptide substrates. Cellular protein degradation was measured by prelabelling cells with 3H-leucine and determining the release of TCA-soluble radioactivity. Nelfinavir inhibited IDE in a concentration-dependent manner with 50% inhibition at the maximal concentration tested, 100 microm. Similarly, the chymotrypsin-like and trypsin-like activities of the proteasome were decreased with an IC50 of approximately 3 microm. The ability of insulin to inhibit the proteasome was abrogated by nelfinavir. Treatment of HepG2 cells with 50 microm nelfinavir decreased 125I-insulin degradation and increased cell-associated radioactivity. Insulin alone maximally decreased protein degradation by 15%. Addition of 50 microm nelfinavir inhibited cellular protein degradation by 14% and blunted the effect of insulin. These data show that nelfinavir inhibits IDE, decreases insulin's ability to inhibit protein degradation via the proteasome and provides another possible mechanism for the insulin resistance seen in protease inhibitor-treated HIV patients.

The forehead flap for nasal reconstruction: how we do it.

The forehead flap is a useful technique to reconstruct deep and large nasal defects. It can safely be performed under local anesthesia in an outpatient setting. Advantages of this flap include the fact that it provides an excellent color and texture match to the missing nasal skin. Disadvantages include the fact that it is at least a two-stage procedure and that often patients require "touch up" surgeries to provide the best possible cosmetic outcome.

Insulin inhibition of the proteasome is dependent on degradation of insulin by insulin-degrading enzyme.

A consequence of insulin-dependent diabetes mellitus is the loss of lean muscle mass as a result of accelerated proteolysis by the proteasome. Insulin inhibition of proteasomal activity requires interaction with insulin-degrading enzyme (IDE), but it is unclear if proteasome inhibition is dependent merely on insulin-NIDE binding or if degradation of insulin by IDE is required. To test the hypothesis that degradation by IDE is required for proteasome inhibition, a panel of insulin analogues with variable susceptibility to degradation by IDE binding was used to assess effects on the proteasome. The analogues used were [Lys(B28), Pro(B29)]-insulin (lispro), [Asp(B10)]-insulin (Asp(B10)) and [Glu(B4), Gln(B16), Phe(B17)]-insulin (EQF). Lispro was as effective as insulin at inhibition of degradation of iodine-125 ((125)I)-labeled insulin, but Asp(B10) and EQF were somewhat more effective. All agents inhibited cross-linking of (125)I-insulin to IDE, suggesting that all were capable of IDE binding. In contrast, although insulin and lispro were readily degraded by IDE, Asp(B10) was degraded more slowly, and EQF degradation was undetectable. Both insulin and lispro inhibited the proteasome, but Asp(B10) was less effective, and EQF had little effect. In summary, despite effective IDE binding, EQF was poorly degraded by IDE, and was ineffective at proteasome inhibition. These data suggest that insulin inhibition of proteasome activity is dependent on degradation by IDE. The mechanism of proteasome inhibition may be the generation of inhibitory fragments of insulin, or by displacement of IDE from the proteasome.

Insulin inhibition of protein degradation in cells expressing wild-type and mutant insulin receptors.

The mechanism by which insulin decreases protein degradation is unknown. We examined insulin binding and degradation (125I[A14]insulin) and protein degradation (3H-leucine labeling) in Chinese hamster ovary (CHO) cells transfected with wild-type (WI) and mutant human insulin receptors. The deltaExon-16 mutant is missing the juxtamembrane domain that mediates endocytosis. The delta343 mutant receptor lacks the tyrosine kinase structural domain but retains the juxtamembrane internalization domain. The mutant deltaNPEY lacks the single NPEY sequence located 16 residues after the end of the transmembrane domain. Null transfected cells (NEO) not expressing human receptors were studied as controls. The WT and deltaNPEY cells equivalently internalized and degraded insulin; delta343 cells internalized and degraded insulin, but at a reduced rate; deltaExon-16 cells internalized and degraded significantly less insulin than the other mutants; NEO cells showed essentially no internalization and degradation. In contrast, all cell types showed the same efficacy at inhibition of protein degradation, albeit at different potencies. These results suggest insulin actions are mediated by multiple and redundant effector systems, but that receptor tyrosine kinase activity is not required for inhibition of protein degradation.

Relaxin stimulates bronchial epithelial cell PKA activation, migration, and ciliary beating.

Relaxin is an insulin-like serum protein secreted during pregnancy and found in many tissues, including the lung. Relaxin is reported to stimulate epithelial cell proliferation, but the effects of relaxin on airway epithelium are unknown. We tested the hypothesis that relaxin would stimulate the increased migration of bronchial epithelial cells (BEC) in response to wounding. Using monolayers of BEC in a wound-healing model, relaxin augmented wound closure with maximal closure occurring at 12 hr (1 micro M). Unlike cytokines, relaxin did not stimulate increased BEC interleukin-8 (IL-8) release. Relaxin caused a significant stimulation of ciliary beat frequency (CBF) in BEC. Because protein kinase (PKA) activation increases CBF and relaxin can elevate intracellular cAMP levels, we measured PKA activity in BEC treated with relaxin. Relaxin increased PKA activity 3-4 fold by approximately 4 hr, with a return to baseline levels by 8-10 hr. Relaxin-stimulated PKA activity differs temporally from the rapid (1 hr) beta-adrenergic activation of PKA in BEC. These data suggest that relaxin augments epithelial repair by increasing airway cell migration and CBF via PKA-dependent mechanisms.

Insulin inhibits peroxisomal fatty acid oxidation in isolated rat hepatocytes.

Inhibition by insulin of long chain fatty acid oxidation in mitochondria is mediated in part by elevating malonyl-CoA levels, which inhibit carnitine palmitoyl-transferase I. Whether insulin alters peroxisomal oxidation has not been studied. We present data which show that insulin inhibits the oxidation of palmitic acid by peroxisomes (IC(50) = 8.5 x 10(-11) M) at hormone concentrations 100-fold less than those needed for mitochondrial inhibition (IC(50) = 1.3 x 10(-8) M). We used a purified peroxisome preparation to study the mechanism of insulin action. Insulin had a direct effect in the peroxisome preparations to decrease oxygen consumption, fatty acyl-CoA oxidizing system activity and acyl-CoA oxidase by approximately 40%, 30% and 15%, respectively. Since insulin degrading enzyme (IDE) is an insulin-binding protein known to be in peroxisomes, we studied the effect of an inhibitory anti-IDE antibody on the ability of insulin to inhibit the fatty acyl-CoA oxidizing system. The antibody eliminated the inhibitory effect of insulin. We conclude that insulin inhibits peroxisomal fatty acid oxidation by a mechanism requiring IDE.

The effect of stitch type on flap tip blood flow.

BACKGROUND: The 3-point corner stitch is generally used for apposition of skin flap tips to recipient wound corners. This stitch theoretically provides better blood flow, leading to improved flap tip survival, than alternative suture techniques. However, this assumption is not based on human experimental data. OBJECTIVE: We tested in patients whether certain stitch types influence flap tip blood flow and necrosis. METHODS: Flap tips were closed with either a 3-point corner stitch, a vertical loop stitch at the tip, or two vertical loop stitches adjacent to the tip. Blood flow was indirectly measured by means of the laser Doppler imager, and flap tips were observed for subsequent necrosis. RESULTS: The 3-point corner stitch resulted in a higher overall mean percent flux ratio implying greater blood flow than the other stitch types used. However, none of the stitch types resulted in a large number of necrotic flap tips. CONCLUSION: The 3-point corner stitch provides increased blood flow to flap tips that may be critical when flap tip survival is problematic.

Insulin and analogue effects on protein degradation in different cell types. Dissociation between binding and activity.

In adult animals, the major effect of insulin on protein turnover is inhibition of protein degradation. Cellular protein degradation is under the control of multiple systems, including lysosomes, proteasomes, calpains, and giant protease. Insulin has been shown to alter proteasome activity in vitro and in vivo. We examined the inhibition of protein degradation by insulin and insulin analogues (Lys(B28),Pro(B29)-insulin (LysPro), Asp(B10)-insulin (B10), and Glu(B4),Gln(B16),Phe(B17)-insulin (EQF)) in H4, HepG2, and L6 cells. These effects were compared with receptor binding. Protein degradation was examined by release of trichloroacetic acid-soluble radioactivity from cells previously labeled with [(3)H]leucine. Short- and intermediate-lived proteins were examined. H4 cells bound insulin with an EC(50) of 4.6 x 10(-9) m. LysPro was similar. The affinity of B10 was increased 2-fold; that of EQF decreased 15-fold. Protein degradation inhibition in H4 cells was highly sensitive to insulin (EC(50) = 4.2 x 10(-11) and 1.6 x 10(-10) m, short- and intermediate-lived protein degradation, respectively) and analogues. Despite similar binding, LysPro was 11- to 18-fold more potent than insulin at inhibiting protein degradation. Conversely, although EQF showed lower binding to H4 cells than insulin, its action was similar. The relative binding potencies of analogues in HepG2 cells were similar to those in H4 cells. Examination of protein degradation showed insulin, LysPro, and B10 were equivalent while EQF was less potent. L6 cells showed no difference in the binding of the analogues compared with insulin, but their effect on protein degradation was similar to that seen in HepG2 cells except B10 inhibited intermediate-lived protein degradation better than insulin. These studies illustrate the complexities of cellular protein degradation and the effects of insulin. The effect of insulin and analogues on protein degradation vary significantly in different cell types and with different experimental conditions. The differences seen in the action of the analogues cannot be attributed to binding differences. Post-receptor mechanisms, including intracellular processing and degradation, must be considered.

Improving rates of advance directive discussions among managed care nursing home enrollees.

BACKGROUND: Discussions about advance directives should be offered to all nursing home residents. Managed Medicare programs for nursing home residents allow for the development of performance improvement initiatives to ensure that these discussions occur and are documented. PURPOSE: To assess the effectiveness of an intervention to increase discussion and documentation of advance directives for enrollees in a managed Medicare program for nursing home residents, and to evaluate whether this intervention affected preferences for cardiopulmonary resuscitation (CPR) and hospitalization among enrollees. SUBJECTS: Participants were 4,248 enrollees in a managed Medicare program in 1996, and 6,598 enrollees in 1997, in Georgia, Maryland, Massachusetts, Minnesota, Arizona, and Florida. DESIGN: Descriptive study of a quality improvement initiative. METHODS: A chart review was conducted in the fall of 1996 to determine the prevalence of documented advance directive discussions among all enrollees, and the preferences regarding CPR and hospitalization. Because the discussion rates varied across sites, and were lower than expected, each site developed strategies to improve advance directive discussion and documentation. One year later, a similar survey was conducted to determine the efficacy of the interventions, as well as to assess the impact, if any, on rates of desire for CPR and hospitalization. RESULTS: Documented discussions of advance directives increased across the six sites from 73% to 85% (P < 0.001). The overall percentage of patients desiring CPR did not change following the intervention (18%). However, there were geographical differences in the desire for CPR among enrollees, with those in Minnesota (8%), Arizona (11%), and Florida (12%) desiring it the least, and those in Massachusetts (20%), Georgia (29%), and Maryland (29%) desiring it the most. The overall percentage of desire for hospitalization decreased from 65% to 62% (P < 0.001). Enrollees in Georgia were most likely to want hospitalization (87%), and enrollees in Minnesota were the least likely to want hospitalization (57%). CONCLUSIONS: In a managed care program, documentation of advance directive discussions can be increased with focused efforts. Overall, most enrollees did not desire CPR, but a majority desired hospitalization. Despite the similarity of interventions and program philosophy across sites, significant geographic variations in desire for CPR and hospitalization remained.