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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 3 He 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 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.

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., (© 2016 The British Pharmacological Society.)

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.

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., (© 2011 American Physical Society)

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

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.

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.

A pathological feature of Type 2 diabetes is deposits in the pancreatic islets primarily composed of amylin (islet amyloid polypeptide). Although much attention has been paid to the expression and secretion of amylin, little is known about the enzymes involved in amylin turnover. Recent reports suggest that insulin-degrading enzyme (IDE) may have specificity for amyloidogenic proteins, and therefore we sought to determine whether amylin is an IDE substrate. Amylin-degrading activity co-purified with IDE from rat muscle through several chromatographic steps. Metalloproteinase inhibitors inactivated amylin-degrading activity with a pattern consistent with the enzymatic properties of IDE, whereas inhibitors of acid and serine proteases, calpains, and the proteasome were ineffective. Amylin degradation was inhibited by insulin in a dose-dependent manner, whereas insulin degradation was inhibited by amylin. Other substrates of IDE such as atrial natriuretic peptide and glucagon also competitively inhibited amylin degradation. Radiolabeled amylin and insulin were both covalently cross-linked to a protein of 110 kDa, and the binding was competitively inhibited by either unlabeled insulin or amylin. Finally, a monoclonal anti-IDE antibody immunoprecipitated both insulin- and amylin-degrading activities. The data strongly suggest that IDE is an amylin-degrading enzyme and plays an important role in the clearance of amylin and the prevention of islet amyloid formation.

Objectives: To describe a cluster of cases of proximal deep vein thrombosis among nursing home residents treated with megestrol acetate, to identify other risk factors for deep vein thrombosis development among these patients, and to alert physicians to this potential complication of megestrol acetate therapy., Design: Retrospective review., Setting: A 129-bed Maryland nursing home., Participants: Nineteen residents prescribed megestrol acetate between November 1997 July 1998, identified by computerized pharmacy records., Measurements: Demographic data (including identification of known risk factors for deep vein thrombosis development) were collected along with information on the indications for megestrol acetate therapy and its duration, diagnostic studies related to detection of deep vein thrombosis and their results, and patient outcomes following diagnosis of deep vein thrombosis., Results: Megestrol acetate was prescribed for 18 nutritionally at-risk patients and one with uterine cancer. Six (32%) patients developed deep vein thrombosis signs and symptoms, and all diagnoses were confirmed as proximal deep vein thromboses with Doppler studies. (No diagnosis of deep vein thrombosis were made among any nursing home patients not being treated with megestrol acetate during the observation period.) All patients diagnosed with deep vein thrombosis were hospitalized and anticoagulated, but none were diagnosed with pulmonary embolus or died. The length [median (range)] of megestrol acetate treatment was similar regardless of whether deep vein thrombosis developed [117 (57-244) versus 143 (2-294) days, respectively, P = 0.83]. Stratification by length of treatment in 50-day increments revealed that most patients who developed deep vein thrombosis did so after 50 days of treatment (P = 0.046)., Conclusion: A high incidence of deep vein thrombosis was identified among nursing home residents treated with megestrol acetate, even among ambulatory individuals with no other known risk factors. Because the efficacy of megestrol acetate treatment in nursing home residents with weight loss is unproven, the risk of deep vein thrombosis must be considered when prescribing megestrol acetate, and its use to treat nutritionally at-risk nursing home residents should be limited.

We investigated the effect of feeding by the western conifer-seed bug, Leptoglossus occidentalis Heidemann, on seed production in developing cones of coastal Douglas-fir, Pseudostuga menziesii (Mirb.) Franco, with respect to seed bug life stage and sex (nymphs, adult females, and adult males) and timing of feeding (early, mid-, and late season cone development). Feeding by females on caged cones for a 2-wk period during late season cone development reduced the proportion of full seeds in cones by approximately 70% compared with caged control cones. There was no significant difference among nymphs, adult females, and adult males with respect to the proportion of empty or partially fed-upon seeds produced during the same feeding period. Feeding by nymphs for 2 wk early in the season resulted in a threefold increase in the number of unextractable seeds fused to cones compared with the control. Weight measurements of harvested seeds indicated that radiography is an accurate tool to distinguish among Douglas-fir seeds that have sustained light, moderate, or severe damage. Determining the full impact of L. occidentalis on conifer seed production will require the development of a reliable method to distinguish between naturally aborted seeds and seeds emptied through feeding by seed bugs.

Background: Dermatofibrosarcoma protuberans (DFSP) is a spindle cell malignancy that has a high local recurrence rate after excision with minimal or no immediate tissue margin assessment. DFSP is exceedingly rare on the palms and soles., Objective: To report a case of a locally aggressive DFSP on the sole excised using Mohs micrographic surgery., Methods: Case report and review of the literature., Results: Mohs micrographic surgery unmasked tumor infiltration that extended around plantar aponeurosis and into underlying plantar muscle fascia., Conclusion: Mohs micrographic surgery should be considered the treatment of choice for DFSP, especially in acral locations. This technique allows the surgeon to trace out deep tumor extensions that may wrap around underlying tendon, a finding that may not be appreciated clinically.

Background: Two patients undergoing cutaneous surgery had thromboembolic strokes within 1 week after surgery. Both patients had been taking warfarin for prevention of thromboembolism and warfarin was stopped 3-7 days prior to surgery., Objective: To examine the rationale and problems associated with preoperative warfarin discontinuation., Methods: Review of the medical literature., Results: When warfarin is stopped prior to surgery and restarted soon after surgery, the patient is at increased risk for thromboembolism. Although it is commonly believed that continuing warfarin during surgery is associated with an increased bleeding risk, for cutaneous surgery, this risk is extremely low and can be easily managed., Conclusion: Warfarin should not be discontinued prior to cutaneous surgery because of the risk of thromboembolic stroke.

A major metabolic effect of insulin is inhibition of cellular proteolysis, but the proteolytic systems involved are unclear. Tissues have multiple proteolytic systems, including the ATP- and ubiquitin-dependent proteasome pathway. The effect of insulin on this pathway was examined in vitro and in cultured cells. Insulin inhibited ATP- and ubiquitin-dependent lysozyme degradation more than 90% by reticulocyte extract, in a dose-dependent manner (IC50 approximately 50 nM). Insulin did not reduce the conjugation of ubiquitin to lysozyme and was not itself ubiquitin-conjugated. In HepG2 cells, insulin increased ubiquitin-conjugate accumulation 80%. The association between the 26S proteasome and an intracellular protease, the insulin-degrading enzyme (IDE), was examined by a purification scheme designed to enrich for the 26S proteasome. Copurification of IDE activity and immunoreactivity with the proteasome were detected through several chromatographic steps. Glycerol gradient analysis revealed cosedimentation of IDE with the 20S proteasome and possibly with the 26S proteasome. The proteasome-associated IDE was displaced when the samples were treated with insulin. These results suggest that insulin regulates protein catabolism, at least in part, by decreasing ubiquitin-mediated proteasomal activity, and provides a new target for insulin action. The displacement of IDE from the proteasome provides a mechanism for this insulin action.

A 6-month clinical trial was conducted to evaluate two models of restorative nursing care designed to improve mobility in nursing home residents. The models were compared on number of residents enrolled, documentation of nursing assistant (NA) compliance, and nursing staff satisfaction. The designated model, which relied on one specially trained NA to perform restorative activities on the unit, resulted in higher rates of enrollment, compliance, and staff satisfaction compared with the integrated model, which relied on regular staff NAs who were trained to incorporate restorative activities into their daily routines.

In this study, we developed formulas to calculate nursing assistant turnover and stability rates, identified reasons for termination and facility-specific strategies to reduce turnover, and evaluated the effectiveness of implemented strategies. Although turnover remained relatively unchanged (23% in year 1, 28% in year 2), the stability rate remained high (76% in year 1, 75% in year 2). Tracking turnover rates without tracking stability yields an incomplete picture of a facility's efforts to attract and retain qualified employees. Achieving high stability rates in addition to low turnover rates are important goals, and we have included some recommendations.

Insulin degradation is a regulated process that plays a role in controlling insulin action by removing and inactivating the hormone. Abnormalities in insulin clearance and degradation are present in various pathological conditions including type 2 diabetes and obesity and may be important in producing clinical problems. The uptake, processing, and degradation of insulin by cells is a complex process with multiple intracellular pathways. Most evidence supports IDE as the primary degradative mechanism, but other systems (PDI, lysosomes, and other enzymes) undoubtedly contribute to insulin metabolism. Recent studies support a multifunctional role for IDE, as an intracellular binding, regulatory, and degradative protein. IDE increases proteasome and steroid hormone receptor activity, and this activation is reversed by insulin. This raises the possibility of a direct intracellular interaction of insulin with IDE that could modulate protein and fat metabolism. The recent findings would place intracellular insulin-IDE interaction into the insulin signal transduction pathway for mediating the intermediate effects of insulin on fat and protein turnover.

The insulin-degrading enzyme (IDE) plays an important role in the cellular metabolism of insulin. Recent studies have also suggested a regulatory role for this protein in controlling the activity of cytoplasmic protein complexes, including the proteasome [multicatalytic proteinase (MCP)] and the glucocorticoid and androgen receptors. Binding of IDE to these complexes increases their activity, whereas the addition of substrates for IDE inhibits activity. This provides a potential mechanism of action for internalized insulin and other IDE substrates in the control of protein turnover. To examine further the interactions, partially purified IDE-MCP complex was treated with EDTA or EGTA, and activity was measured in the absence and presence of various divalent cations (Ca2+, Mn2+, Co2+, and Zn2+) and insulin. EDTA treatment reduced MCP activity and eliminated the effect of insulin on the complex. Divalent cations partially or completely restored MCP activity, but did not restore the effect of insulin. EGTA treatment had a lesser effect on MCP activity, but abolished insulin inhibition of activity. Divalent cations restored the insulin effect. Inhibitors of IDE also blocked the insulin effect on MCP activity, as did treatment with SDS. These findings suggest that conformational changes in the complex may play a role in the insulin control of MCP activity.

Objective: To determine whether low airloss hydrotherapy reduces the incidence of new skin lesions associated with incontinence in hospitalized patients and results in more rapid healing of existing pressure sores compared with standard care. To assess subjectively patient and nursing satisfaction related to using low airloss hydrotherapy beds., Design: Randomized, prospective, unblinded study., Setting: Acute and chronic hospital wards., Participants: A total of 116 newly admitted, incontinent, hospitalized patients with and without existing pressure sores., Intervention: Low airloss hydrotherapy compared with treatment on hospital beds and mattresses ordered by the patient's attending physician., Measurements: Incidence rates of new skin lesion development, e.g., pressure sores, candidiasis, and chemical irritation; improvement in existing pressure sore size, volume, and status; subjective assessment of patient and nursing satisfaction., Results: Possible hypothermia was identified in two patients during the first week of the study, and patient and nursing dissatisfaction with low airloss hydrotherapy remained high throughout the first months of the study. Therefore, two major modifications in the initial protocol were made: (1) increased patient temperature monitoring for hypothermia was initiated in Week 2 of the study and (2) increased staff resources for in-service training on bed use began in Week 18 of the study. After the latter change, 58 subjects were randomized to low airloss hydrotherapy and 58 to standard care. Subjects were old (median age > or = 80 years), and almost all were bedbound or nonambulatory. The median (range) length of follow-up for subjects in the treatment group was significantly shorter than for those in the control group (4 (1-60) days versus 6 (1-62) days, respectively, P = .017) because there were more dropouts from the treatment group (24 (36%) of 58 versus 2 (3%) of 58, P = .0001). The major reasons dropout occurred were patient or family dissatisfaction (12 (21%)), new or worsened skin lesions thought to be related to bed use (4 (7%)), and hypothermia < 97 degrees F (4 (7%)). The total cumulative incidence of new truncal skin lesions within 9 days of enrollment was greater in the treatment than in the control group (48% versus 14%, respectively, P < 0.01). Too few patients with existing pressure sores were treated for too short a period of time to assess the effect of low airloss hydrotherapy on pressure sore healing. Because only 10 patients treated on low airloss hydrotherapy beds were able to complete satisfaction surveys meaningfully, interpretation of these data is difficult. Only nine (21%) of 44 nurses subjectively reported overall satisfaction using the low airloss hydrotherapy bed., Conclusions: This study shows the value of a rigorously designed clinically based evaluation of a new product developed for older patients. The results of the study led to re-engineering of the prototype low airloss hydrotherapy bed as well as a change in marketing strategy. Studies of products targeted to the prevention and treatment of pressure sores in older patients should be undertaken before generalized marketing begins.

Insulin decreases cellular protein degradation, but the mechanism of this action is poorly understood. We have shown that insulin can have an inhibitory effect on the action of the proteasome in vitro, which requires the presence of insulin degrading enzyme (IDE). In this study we have used an antibody which inhibits the activity of IDE to show that IDE is required for insulin inhibition of protein degradation in intact cells. The anti-IDE antibody blocked the insulin effect on cellular degradation of proteins prelabeled with radioactive amino acids. The anti-IDE antibody also decreased insulin inhibition of proteasome degradation of a specific substrate in intact cells. These data suggest that insulin works intracellularly via IDE to inhibit protein degradation by the proteasome. Thus, IDE may function as an intracellular mediator for insulin effects on protein degradation. This is a novel signal transduction mechanism for peptide hormones.

Objectives: To determine the accuracy of patient care staff estimates and documentation of food intake of residents in nursing homes., Design: Prospective, observed, unblinded cohort study., Setting: Three urban nursing home facilities., Subjects: Staff estimation and documentation of 27 nursing home residents' meal intake., Measurements: Actual amount consumed by 27 nursing home residents was ascertained by weighing food and caloric fluids on resident trays before and after one lunch time meal. Staff estimates and documentation of percent of meal consumed was compared with actual intake., Results: Patient care staff estimates differed from actual intake by approximately 20%, and in most instances intake was overestimated. Almost one-third of the residents at risk for nutritional problems were not identified correctly by staff. Chart documentation of meal intake frequently did not reflect either actual amount of meal consumed or the staff's estimation of what was eaten., Conclusion: Study findings indicate that the present system used to document nursing home residents' intake is inadequate and that a more accurate mechanism or an entirely different process for identifying residents at risk for nutritional problems should be developed and implemented.

The major purpose of this project was to test the feasibility of recruiting and training volunteers as lay health educators who could coordinate and reinforce the educational efforts of health care providers. A committee of health care professionals designed a 16-hour program. Twenty-five volunteers from T1 religious institutions and 4 retirement communities completed the 8-week program. The program was successful in identifying, recruiting, and training volunteers from racially and religiously diverse institutions. Favorable outcomes included participants' satisfaction and success in organizing numerous educational and screening programs in their communities.

Pressure-relieving strategies remain the foundation for the prevention and treatment of pressure sores. Although the published literature is inadequate, rational treatment decisions can be made if they are based on an understanding of the mechanisms by which pressure relief results with a particular device or strategy. This article reviews the theoretic and practical approaches to managing tissue loads so that rational choices of the most cost-effective strategies for preventing and treating pressure sores can be made when caring for patients.

Cellular homeostasis requires regulation of protein turnover. Protein degradation is an essential component of this process and is inhibited by insulin. The importance of cytosolic proteolysis in overall cellular protein degradation is increasingly apparent and an insulin effect on this system has been suggested but not proven. The present study shows that a membrane permeable substrate of the proteasome is degraded in HepG2 cells and that insulin inhibits its degradation both by isolated proteasomes and by intact cells. Inhibitors of the proteasome suppress degradation, and in the presence of these inhibitors insulin has no further effect. This is the first demonstration that insulin inhibition of cellular protein degradation is due to an effect on proteasomes.

Insulin-degrading enzyme (IDE) is a component of a cytosolic complex that includes multicatalytic proteinase (MCP), the major cytoplasmic proteolytic activity. Insulin, the primary substrate for IDE, inhibits the proteolytic activity of the IDE-MCP complex but not of purified MCP. This provides a regulatory role for IDE in cellular proteolysis and a potential mechanism for intracellular insulin action. To examine the specificity and to explore the mechanisms for the IDE-MCP interaction, we studied the functional interaction of a variety of peptides with the complex. Atrial natriuretic peptide (ANP), relaxin, glucagon, proinsulin, and insulin-like growth factor II (IGF-II) bind to and are degraded by IDE. These peptides have significant inhibitory effects on the chymotrypsin-like and trypsin-like MCP catalytic activities but not the peptidyl-glutamyl hydrolyzing activity. A panel of peptides that are not ligands of IDE had no effect. To explore the potential mechanism for the IDE control of MCP activity, dose response curves for insulin-like growth factor I (IGF-I) and IGF-II effects on MCP chymotrypsin-like activity were determined. IGF-II, which (similar to insulin) is a good substrate for IDE, had a substantial inhibitory effect, whereas IGF-I, which is bound but poorly degraded, had little inhibitory activity on MCP. Proinsulin, another ligand of IDE that is tightly bound but poorly degraded, had a partial effect on MCP activity, but inhibited the full insulin effect. These data suggest a requirement for both the binding and degradation of IDE ligands for the full inhibition of MCP. Insulin-sized degradation products, substrates of IDE, also inhibited MCP activity. Further examination of the insulin effect on MCP included kinetic studies. Insulin produced a noncompetitive inhibition of both the chymotrypsin-like and trypsin-like activities of MCP. These data suggest that the insulin-IDE effect on MCP is due to conformational changes in the IDE-MCP complex and provide an intracellular mechanism of action for insulin.

Objective: To compare in adults more than 50 years old the tolerability and immunogenicity of vaccination with recombinant hepatitis B surface antigen (HBs) compared with vaccination with recombinant hepatitis B protein PreS2 + S, and to investigate the safety and immunogenicity of a fourth vaccine dose in poor and non-responders., Design: Randomized, double-blind prospective study., Setting: General clinical research center for outpatient evaluation and vaccination., Subjects: Adults older than age 50 who were in general good health and with no known risk factors for acquiring or serologic evidence of hepatitis B virus infection., Intervention: Subjects were randomized to receive 10 mcg HBs (Recombivax, Merck, Sharp and Dohme), 12 mcg PreS2 + S, or 24 mcg PreS2 + S vaccine at 0, 1, and 6 months. Poor and non-responders (anti-Hbs < 10 mIU/mL at month 9 and/or 12) were encouraged to receive a fourth vaccine injection., Measurements: Diary records of temperature and local and systemic reactions following each vaccination were maintained by all subjects. Anti-HBs levels were measured by radioimmunoassay before the first injection, at 1, 2, 3, 6, 7, 9, and 12 months after for all subjects, and 1 month after the fourth injection for the group of poor and non-responders., Main Results: Twenty men and nine women (mean age +/- SD, 66 +/- 8.0 years) were enrolled. Ten subjects received HBs vaccine, nine received 12 mcg PreS2 + S vaccine, and 10 received 24 mcg PreS2 + S vaccine. One subject in the HBs group dropped out, and data were analyzed for the remaining 28 subjects. There were no differences in rates of side effects reported by each of the three groups. Overall, minor local adverse reactions occurred in 12 (40%) after at least one of the first three vaccinations. Systemic side effects occurred in five (17%) after the first vaccination, in one after the second, but in none after the third. The 24-mcg PreS2 + S vaccine was not more immunogenic than the HBs vaccine, and the 12-mcg PreS2 + S vaccine was judged inadequate. Nineteen of 22 (86%) poor and non-responders received a fourth vaccination. Minor local adverse reactions were reported by six (32%), and none reported a systemic side effect. For the 12 subjects receiving a fourth injection of HBs or 24 mcg PreS2 + S vaccine, the proportion of responders 1 month following the fourth injection was greater than for 1 month following the third injection (11 of 12 [92%] versus 12 of 19 [63%], respectively, P < .05)., Conclusion: For adults more than 50 years of age who have low anti-HBs levels after three vaccine injections, a fourth injection is well tolerated and results in improved immunogenic response.

Background: Acne keloidalis nuchae (AKN) is a chronic inflammatory disorder of the posterior aspect of the neck and occipital region of the scalp. Despite numerous medical and surgical treatment modalities, few offer cure or a superior cosmetic result for patients with extensive disease., Objective: Our purpose was to determine whether excision with second-intention healing is an effective therapeutic modality for AKN., Methods: Excision of the involved area to the level of the muscle fascia or deep subcutaneous tissue was performed in six patients. Postoperative sites healed by second intention., Results: Four of six patients had a horizontal elliptic excision of the involved area that included the posterior hairline, with good to excellent results. The other two, who received nonelliptic excision of affected scalp that spared the hairline, had slower wound healing and poor contraction., Conclusion: Best results were achieved in excision of AKN with second-intention healing when the excision was a horizontal ellipse of the posterior aspect of the scalp including the posterior hairline.

Background: Specific factors affect the outcome of full-thickness skin grafts after Mohs micrographic surgery. No recent clinical studies have systematically investigated the clinical factors that may affect the success of full-thickness skin grafts., Objective: Our objective was to analyze patient data to define factors that may influence the success of full-thickness skin grafts., Methods: We reviewed the clinical course of 117 patients who had full-thickness skin grafts after Mohs micrographic surgery. We analyzed these cases using patient variables, tumor variables, and skin graft variables and then correlated these variables to skin graft success., Results: Delay of skin graft placement (> 1 day) was significantly correlated with subsequent skin graft success (p = 0.015). Skin graft necrosis was more likely to develop in men than in women (p = 0.021)., Conclusion: A delay of 2 to 8 days between Mohs micrographic surgery and full-thickness skin graft placement resulted in a lower incidence of skin graft necrosis than no delay or a delay of 1 day.

To define ranges of plasma specific gravity useful for identifying volume depletion in older adults, plasma specific gravity was measured in 170 young adults (mean age 28 years) and 100 retirees (mean age 81 years), and ranges of values likely to be associated with volume depletion were defined. Subsequently, measurements of plasma specific gravity were made in 68 older emergency room (ER) patients (mean age 74 years), a few of whom had obvious reasons for being hypovolaemic, e.g. dehydrating diarrhoea, and these results were compared to those for the control groups. Ranges for plasma specific gravity useful for identifying volume depletion were designated as possible hypovolaemia (1.0265-1.0279), probable hypovolaemia (1.0280-1.0294), and hypovolaemia (> or = 1.0295). Using these definitions, there were more older ER patients compared to both young and old control group subjects, respectively, with probable hypovolaemia (21% vs. 5% and 8%; p < 0.03) and hypovolaemia (16% vs. 0% and 0%; p < 0.03). This study establishes ranges for plasma specific gravity for young and old adults likely to be associated with hypovolaemia, and shows that based upon measurement of plasma specific gravity, older ER patients may often be hypovolemic even in the absence of obvious fluid-wasting illnesses. Future studies are needed to identify the risk factors for hypovolaemia in ER patients, and more vigorously substantiate the findings of this study.

The insulin-degrading enzyme (IDE) and the multicatalytic proteinase (MCP) can be isolated as components of a cytosolic proteolytic complex. IDE is the primary enzyme involved in cellular degradation of insulin, and insulin has been shown to interact with cytosolic IDE. MCP is believed to be important in non-ubiquitin pathways of cellular protein degradation. Insulin has a dose- and time-dependent inhibitory effect on MCP degradation of N-succinyl-Leu-Leu-Val-Tyr 7-amino-4-methylcoumarin (LLVY), a substrate for MCP. Proinsulin also inhibits LLVY degradation in a dose-dependent manner. The effect of insulin is immediate as measured in a continuously monitored assay of LLVY degradation. Purification of the IDE-MCP complex using a variety of approaches, including affinity and conventional chromatography, retains the insulin effect on LLVY degradation as long as the complex remains intact. After ion-exchange chromatography, which separates IDE and MCP, insulin no longer has an inhibitory effect. Recombination of purified IDE and MCP does not restore the effect of insulin, but inclusion of additional components from the ion-exchange column does. These results support the existence of a functional cytosolic complex that contains IDE and MCP. Insulin interacts with IDE and alters the activity of MCP, suggesting a functional relationship between these two components and a mechanism for an intracellular action of insulin.

The insulin degrading enzyme (IDE) is the first recognized member of a new class of metalloproteinases. Studies on the purification and the properties of this enzyme have led to divergent results and conclusions from different laboratories. The present manuscript suggests that many of the divergent results may be due to the interaction of this enzyme with other proteins as part of a proteolytic complex. IDE co-isolates with the multicatalytic proteinase (MCP) during a wide variety of purification approaches including affinity chromatography and conventional purification approaches. Ion exchange chromatography will partially or completely separate IDE and MCP. The SDS-PAGE protein bands at various purification steps suggest the presence of a cytosolic proteolytic complex containing IDE, MCP and other unidentified components and raise the possibility of a functional interaction among these proteins.