Influence of humectants on the thermotropic behaviour and nanostructure of fully hydrated lecithin bilayers.

Humectants are used widely in topical formulations as they provide cosmetic and health benefits to skin. Of particular interest to our laboratories is the interaction of humectants in phospholipid based topical skin care formulations. This study probed the effects of three exemplary humectants on a fully hydrated lecithin system (DPPC) by use of X-ray scattering and differential scanning calorimetry. While the three humectants affected the nanostructure of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC, bilayers in a similar manner, leading to an increased membrane order, differences in the effect on the thermal behaviour of DPPC suggest that betaine and sarcosine interacted via a different mechanism compared to acetic monoethanolamide, AMEA. At concentrations above 0.4 M, betaine and sarcosine stabilised the gel phase by depletion of the interfacial water via the preferential exclusion mechanism. At the same time, a slight increase in the rigidity of the membrane was observed with an increase in the membrane thickness. Overall, the addition of betaine or sarcosine resulted in an increase in the pre- and main transition temperatures of DPPC. AMEA, on the other hand, decreases both transition temperatures, and although the interlamellar water layer was also decreased, there was evidence from the altered lipid chain packing, that AMEA molecules are present also at the bilayer interface, at least at high concentrations. Above the melting point in the fluid lamellar phase, none of the humectants induced significant structural changes, neither concerning the bilayer stacking order nor its overall membrane fluidity. An humectant-induced increase in the Hamaker constant is the most plausible explanation for the observed reduction of the inter-bilayer distances, both in the gel and fluid phase.

Population Vitamin D Stores Are Increasing in Tasmania, and This Is Associated With Less BMD Loss Over 10 Years.

CONTEXT: Vitamin D deficiency is a common, modifiable determinant of musculoskeletal health. OBJECTIVE: There are limited data that examine the longitudinal change in population 25-hydroxyvitamin D (25[OH]D) and none that evaluate the long-term skeletal outcomes of longitudinal vitamin D status. METHODS: A prospective cohort analysis was conducted of community-dwelling adults aged 50 to 80 years who had 25(OH)D assessed by radioimmunoassay and bone mineral density (BMD) by dual-energy x-ray absorptiometry at baseline (n = 1096), 2.5 (n = 870), and 10 (n = 565) years. Sun exposure was quantified by questionnaire and supplement use at clinic review. 25(OH)D less than 50 nmol/L was considered deficient. Participants were provided with their 25(OH)D results. RESULTS: Over 10 years 25(OH)D increased (52.2 ±â€…17.0 to 63.5 ±â€…23.6 nmol/L, P < .001). Participants with baseline deficiency had larger 25(OH)D increases than baseline sufficient participants (19.2 ±â€…25.3 vs 1.6 ±â€…23.3 nmol/L, P < .001). Longitudinal change in 25(OH)D was associated with baseline summer (ß = 1.46, P < .001) and winter (ß = 1.29, P = .003) sun exposure, change in summer (ß = 1.27, P = .002) and winter (ß = 1.47, P < .001) sun exposure, and vitamin D supplement use (ß = 25.0-33.0, P < .001). Persistent vitamin D sufficiency was associated with less BMD loss at the femoral neck (ß = 0.020, P = .027), lumbar spine (ß = 0.033, P = .003), and total hip (ß = 0.023, P = .021) compared to persistent vitamin D deficiency. Achieving vitamin D sufficiency was associated with less BMD loss at the lumbar spine (ß = 0.045, P < .001) compared to persistent vitamin D deficiency. CONCLUSIONS: Population 25(OH)D concentration increased because of a combination of increased sun exposure and supplement use. Maintaining or achieving vitamin D sufficiency was associated with less BMD loss over 10 years.

Influence of the geometry of fluorescently labelled DNA constructs on fluorescence anisotropy assay.

DNA-encoded chemical libraries (DECLs) are powerful tools for modern drug discovery. A DECL is a pooled mixture of small molecule compounds, each of which is tagged with a unique DNA sequence which functions as a barcode. After incubation with a drug target and washing to remove non-binders, the bound molecules are eluted and submitted for DNA sequencing to determine which molecules are binding the target. While the DECL technology itself is ultra-high throughput, the following re-synthesis of identified compounds for orthogonal validation experiments remains the bottleneck. Using existing DNA-small molecule conjugates directly for affinity measurements, as opposed to complete compound resynthesis, could accelerate the discovery process. To this end, we have tested various geometries of fluorescently-labelled DNA constructs for fluorescence anisotropy (FA) experiments. Minimizing the distance between the fluorescent moiety and ligand can maximize the correlation between ligand-protein interaction and corresponding change in fluorophore rotational freedom, thus leading to larger, easier to interpret changes in FA values. However, close proximity can also cause artifacts due to potentially promiscuous interactions between fluorophore and protein. By balancing these two opposite effects, we have identified applicable fluorescently labelled DNA constructs displaying either a single ligand or pairs of fragments for affinity measurement using a FA assay.

People-centred care versus clinic-based DOT for continuation phase TB treatment in Armenia: a cluster randomized trial.

BACKGROUND: WHO's directly observed therapy (DOT) strategy for tuberculosis (TB) treatment depends upon a well-organized healthcare system. This study sought to evaluate the effectiveness of self-administered drug intake supported by a family member versus in-clinic DOT. METHODS: This open-label, nationally-representative stratified cluster randomized controlled non-inferiority trial with two parallel equal arms involved drug-susceptible pulmonary TB patients in the continuation treatment phase. We randomly assigned outpatient-TB-centres (52 clusters) to intervention and control arms. The intervention included an educational/counseling session to enhance treatment adherence; weekly visits to outpatient-TB-centres to receive medication, and daily SMS medication reminders and phone calls to track adherence and record side effects. Controls followed clinical DOT at Outpatient-TB-centres. Both groups participated in baseline and 4-5 months follow-up surveys. The trial's non-inferiority comparisons include: treatment success as the clinical (primary) outcome and medication adherence (self-reported), knowledge, depressive symptoms, stigma, quality of life, and social support as non-clinical (secondary) outcomes. RESULTS: Per-protocol analysis showed that the intervention (n = 187) and control (n = 198) arms achieved successful treatment outcome of 92.0 and 92.9%, respectively, indicating that the treatment success in the intervention group was non-inferior to DOT. Knowledge, depression, stigma, quality of life, and social support also showed non-inferiority, demonstrating substantial improvement over time for knowledge (change in the intervention = 1.05: 95%CL (0.49, 1.60); change in the control = 1.09: 95%CL (0.56, 1.64)), depression score (change in the intervention = - 3.56: 95%CL (- 4.99, - 2.13); change in the control = - 1.88: 95% CL (- 3.26, - 0.49)) and quality of life (change in the intervention = 5.01: 95%CL (- 0.64, 10.66); change in the control = 7.29: 95%CL (1.77, 12.81)). The intervention resulted in improved treatment adherence. CONCLUSIONS: This socially empowering alternative strategy might be a preferable alternative to DOT available to patients in Armenia and in other countries. Further research evaluating cost effectiveness of the intervention and generalizability of the results is warranted. TRIAL REGISTRATION: Clinicaltrials.gov: NCT02082340, March 10, 2014.

Coordinating an Oncology Precision Medicine Clinic Within an Integrated Health System: Lessons Learned in Year One.

Precision medicine is a term describing strategies to promote health and prevent and treat disease based on an individual's genetic, molecular, and lifestyle characteristics. Oncology precision medicine (OPM) is a cancer treatment approach targeting cancer-specific genetic and molecular alterations. Implementation of an OPM clinical program optimally involves the support and collaboration of multiple departments, including administration, medical oncology, pathology, interventional radiology, genetics, research, and informatics. In this review, we briefly introduce the published evidence regarding OPM's potential effect on patient outcomes and discuss what we have learned over the first year of operating an OPM program within an integrated health care system (Aurora Health Care, Milwaukee, WI) comprised of multiple hospitals and clinics. We also report our experience implementing a specific OPM software platform used to embed molecular panel data into patients' electronic medical records.

Varying redox potential affects P release from stream bank sediments.

Sediments in streams that drain agricultural watersheds may be sinks that can adsorb P from the stream or sources that can release P to the stream. Sediment characteristics and environmental factors, including the oxidation-reduction (redox) potential of the water associated with the sediment, determine whether P will be adsorbed or released by the sediment. We investigated P adsorption and release by four sediments [three Holocene-age sediments (Camp Creek, Roberts Creek and Gunder) as well as Pre-Illinoian-age Till] that occur in Walnut Creek, a second-order stream in Jasper County, Iowa, that is representative of many small streams in the glaciated upper Midwest of the US. The effects of two redox potentials on phosphorus buffering capacity (PBC) and equilibrium phosphorus concentration (EPC) were evaluated in batch adsorption experiments. We also simulated aerobic and anerobic conditions over a 24-day period and measured solution-phase P concentrations in stirred systems where the sediments were isolated from the water by dialysis tubing. The batch experiment indicated that the EPCs of the three Holocene-age sediments were similar to one another and increased with decreasing redox potential. In the stirred flow reactors, more dissolved P was released from the Camp Creek and Roberts Creek sediments under anaerobic conditions than from the other sediments. This observation suggests that these two sediments, which are younger and higher in the stratigraphic sequence, are more likely to be P sources in suboxic settings. The P buffering capacity was greatest in the till. Where it is in contact with the stream water, the till is likely to serve as an adsorbing sink for P in the water column.

Synthesis and Formation Mechanism of All-Organic Block Copolymer-Directed Templating of Laser-Induced Crystalline Silicon Nanostructures.

This report describes the generation of three-dimensional (3D) crystalline silicon continuous network nanostructures by coupling all-organic block copolymer self-assembly-directed resin templates with low-temperature silicon chemical vapor deposition and pulsed excimer laser annealing. Organic 3D mesoporous continuous-network resin templates were synthesized from the all-organic self-assembly of an ABC triblock terpolymer and resorcinol-formaldehyde resols. Nanosecond pulsed excimer laser irradiation induced the transient melt transformation of amorphous silicon precursors backfilled in the organic template into complementary 3D mesoporous crystalline silicon nanostructures with high pattern fidelity. Mechanistic studies on laser-induced crystalline silicon nanostructure formation revealed that the resin template was carbonized during transient laser-induced heating on the milli- to nanosecond timescales, thereby imparting enhanced thermal and structural stability to support the silicon melt-crystallization process at temperatures above 1250 °C. Photoablation of the resin material under pulsed excimer laser irradiation was mitigated by depositing an amorphous silicon overlayer on the resin template. This approach represents a potential pathway from organic block copolymer self-assembly to alternative functional hard materials with well-ordered 3D morphologies for potential hybrid photovoltaics, photonic, and energy storage applications.

Is whole-colon investigation by colonoscopy, computerised tomography colonography or barium enema necessary for all patients with colorectal cancer symptoms, and for which patients would flexible sigmoidoscopy suffice? A retrospective cohort study.

BACKGROUND: For patients referred to hospital with suspected colorectal cancer (CRC), it is current standard clinical practice to conduct an examination of the whole colon and rectum. However, studies have shown that an examination of the distal colorectum using flexible sigmoidoscopy (FS) can be a safe and clinically effective investigation for some patients. These findings require validation in a multicentre study. OBJECTIVES: To investigate the links between patient symptoms at presentation and CRC risk by subsite, and to provide evidence of whether or not FS is an effective alternative to whole-colon investigation (WCI) in patients whose symptoms do not suggest proximal or obstructive disease. DESIGN: A multicentre retrospective study using data collected prospectively from two randomised controlled trials. Additional data were collected from trial diagnostic procedure reports and hospital records. CRC diagnoses within 3 years of referral were sourced from hospital records and national cancer registries via the Health and Social Care Information Centre. SETTING: Participants were recruited to the two randomised controlled trials from 21 NHS hospitals in England between 2004 and 2007. PARTICIPANTS: Men and women aged ≥ 55 years referred to secondary care for the investigation of symptoms suggestive of CRC. MAIN OUTCOME MEASURE: Diagnostic yield of CRC at distal (to the splenic flexure) and proximal subsites by symptoms/clinical signs at presentation. RESULTS: The data set for analysis comprised 7380 patients, of whom 59% were women (median age 69 years, interquartile range 62-76 years). Change in bowel habit (CIBH) was the most frequently presenting symptom (73%), followed by rectal bleeding (38%) and abdominal pain (29%); 26% of patients had anaemia. CRC was diagnosed in 551 patients (7.5%): 424 (77%) patients with distal CRC, 122 (22%) patients with cancer proximal to the descending colon and five patients with both proximal and distal CRC. Proximal cancer was diagnosed in 96 out of 2021 (4.8%) patients with anaemia and/or an abdominal mass. The yield of proximal cancer in patients without anaemia or an abdominal mass who presented with rectal bleeding with or without a CIBH or with a CIBH to looser and/or more frequent stools as a single symptom was low (0.5%). These low-risk groups for proximal cancer accounted for 41% (3032/7380) of the cohort; only three proximal cancers were diagnosed in 814 low-risk patients examined by FS (diagnostic yield 0.4%). LIMITATIONS: A limitation to this study is that changes to practice since the trial ended, such as new referral guidelines and improvements in endoscopy quality, potentially weaken the generalisability of our findings. CONCLUSIONS: Symptom profiles can be used to determine whether or not WCI is necessary. Most proximal cancers were diagnosed in patients who presented with anaemia and/or an abdominal mass. In patients without anaemia or an abdominal mass, proximal cancer diagnoses were rare in those with rectal bleeding with or without a CIBH or with a CIBH to looser and/or more frequent stools as a single symptom. FS alone should be a safe and clinically effective investigation in these patients. A cost-effectiveness analysis of symptom-based tailoring of diagnostic investigations for CRC is recommended. TRIAL REGISTRATION: Current Controlled Trials ISRCTN95152621. FUNDING: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 21, No. 66. See the NIHR Journals Library website for further project information.

Moving Mountains for Mental Health and Well-Being.

Today, for mental health, it is the best of times, and it is the worst of times. The barriers to achieving the mental health system we need are not just a chasm of a poorly organized system of care but a mountain range of issues that stop us from bringing mental health and well:being into the 21st century on a par with the rest of health care. This article reviews the progress that has been made, describes the continuing concerns and outlines a path forward toward holistic mental health and well-being in the workplace. We have been starting to move these mountains, but it is a heavy lift and will require unprecedented dialogue, engagement of diverse stakeholders and actions on many fronts to get us to the other side.

Phosphorus source-sink relationships of stream sediments in the Rathbun Lake watershed in southern Iowa, USA.

The surface waters of Rathbun Lake watershed in southern Iowa are impacted by agricultural sources of sediments and nutrients, including phosphorus (P). Because stream sediments often play an important role in regulating P concentrations in stream water, we investigated sediment-water column P relationships in four creeks within the watershed and then evaluated the relationship between sediment properties and indicators of the risk of P loss. Based on Mehlich-3-extractable P (17 to 68 mg kg(-1)) and degree of P saturation (2 to 12 %), stream bank and bed sediments at the four sites were unlikely to serve as major sources of P. However, equilibrium P concentrations, which ranged from 0.02 to 0.12 mg L(-1), indicated that bed sediments could release P to the water column depending on dissolved P (DP) concentrations in the stream water and the time of year. The likelihood of P desorption from the sediments increased with increasing pH (r = 0.92, p < 0.01) and sand content (r = 0.78, p < 0.05), but decreased with clay content (r = -0.72, p < 0.05) and iron (Fe) (r = -0.93, p < 0.001) associated with organic matter. From these results, we speculate that changes in land use within the riparian areas may, at least initially, have little effect on P concentrations in the streams. Low concentrations of DP relative to total P (TP) in these streams, however, suggest that P loads to Rathbun Lake can be reduced if P inputs from eroded bank sediments are controlled.

Vitamin D Reduces Inflammation-induced Contractility and Remodeling of Asthmatic Human Airway Smooth Muscle.

BACKGROUND: Although multiple clinical studies have found an association between vitamin D (Vit D) deficiency and asthma, a recent clinical study suggested lack of therapeutic effect of Vit D supplementation. Nonetheless, the mechanisms by which Vit D influences airway structure and function in the context of inflammation and asthma remains undefined. In this regard, Vit D effects on airway smooth muscle (ASM) are important, given the role of this cell type in the hypercontractility and remodeling. We assessed the mechanisms by which Vit D modulates the enhancing effects of proinflammatory cytokines tumor necrosis factor-α (TNF-α) and IL-13 on intracellular Ca(2+) ([Ca(2+)]i) levels and remodeling in nonasthmatic versus asthmatic human ASM. METHODS: Human ASM was enzymatically isolated from surgical lung specimens of patients with clinically defined mild to moderate asthma versus no asthma. Cells were treated with 10 ng/ml TNF-α and 50 ng/ml IL-13 in the presence or absence of 100 nM calcitriol. MEASUREMENTS AND MAIN RESULTS: Interestingly, Vit D receptor (VDR) and retinoic X receptor-α levels were maintained, even increased, in subjects with asthma when treated with TNF-α and IL-13. Compared with untreated cells, calcitriol blunted the heightened effect of TNF-α on [Ca(2+)]i response to histamine in ASM. Calcitriol particularly blunted TNF-α and IL-13 effects on collagen and fibronectin deposition, especially in asthmatic ASM. Calcitriol stimulated VDR/retinoic X receptor dimerization and VDR activity even in subjects with asthma and with IL-13, highlighting retained functionality. Expression of Class I histone deacetylases 1-3 (HDAC) and overall HDAC activity were lower in IL-13-exposed ASM, but calcitriol enhanced HDAC expression/activity. CONCLUSIONS: In asthmatic ASM, Vit D functionality is maintained, allowing calcitriol to reduce the procontractile and proremodeling effects of inflammatory cytokines, particularly IL-13, which is relevant to asthma. These findings highlight a potential role for Vit D in asthma pathogenesis, particularly in the context of airway structure and functional changes early in disease.

Soluble guanylate cyclase modulators blunt hyperoxia effects on calcium responses of developing human airway smooth muscle.

Exposure to moderate hyperoxia in prematurity contributes to subsequent airway dysfunction and increases the risk of developing recurrent wheeze and asthma. The nitric oxide (NO)-soluble guanylate cyclase (sGC)-cyclic GMP (cGMP) axis modulates airway tone by regulating airway smooth muscle (ASM) intracellular Ca(2+) ([Ca(2+)]i) and contractility. However, the effects of hyperoxia on this axis in the context of Ca(2+)/contractility are not known. In developing human ASM, we explored the effects of novel drugs that activate sGC independent of NO on alleviating hyperoxia (50% oxygen)-induced enhancement of Ca(2+) responses to bronchoconstrictor agonists. Treatment with BAY 41-2272 (sGC stimulator) and BAY 60-2770 (sGC activator) increased cGMP levels during exposure to 50% O2. Although 50% O2 did not alter sGCα1 or sGCß1 expression, BAY 60-2770 did increase sGCß1 expression. BAY 41-2272 and BAY 60-2770 blunted Ca(2+) responses to histamine in cells exposed to 50% O2. The effects of BAY 41-2272 and BAY 60-2770 were reversed by protein kinase G inhibition. These novel data demonstrate that BAY 41-2272 and BAY 60-2770 stimulate production of cGMP and blunt hyperoxia-induced increases in Ca(2+) responses in developing ASM. Accordingly, sGC stimulators/activators may be a useful therapeutic strategy in improving bronchodilation in preterm infants.

Hyperoxia-induced changes in estradiol metabolism in postnatal airway smooth muscle.

Supplemental oxygen, used to treat hypoxia in preterm and term neonates, increases the risk of neonatal lung diseases, such as bronchopulmonary dysplasia (BPD) and asthma. There is a known sex predilection for BPD, but the underlying mechanisms are not clear. We tested the hypothesis that altered, local estradiol following hyperoxia contributes to pathophysiological changes observed in immature lung. In human fetal airway smooth muscle (fASM) cells exposed to normoxia or hyperoxia, we measured the expression of proteins involved in estrogen metabolism and cell proliferation responses to estradiol. In fASM cells, CYP1a1 expression was increased by hyperoxia, whereas hyperoxia-induced enhancement of cell proliferation was blunted by estradiol. Pharmacological studies indicated that these effects were attributable to upregulation of CYP1a1 and subsequent increased metabolism of estradiol to a downstream intermediate 2-methoxyestradiol. Microarray analysis of mouse lung exposed to 14 days of hyperoxia showed the most significant alteration in CYP1a1 expression, with minimal changes in expression of five other genes related to estrogen receptors, synthesis, and metabolism. Our novel results on estradiol metabolism in fetal and early postnatal lung in the context of hyperoxia indicate CYP1a1 as a potential mechanism for the protective effect of estradiol in hyperoxia-exposed immature lung, which may help explain the sex difference in neonatal lung diseases.

Prevention of thrombogenesis from whole human blood on plastic polymer by ultrathin monoethylene glycol silane adlayer.

In contemporary society, a large percentage of medical equipment coming in contact with blood is manufactured from plastic polymers. Unfortunately, exposure may result in undesirable protein-material interactions that can potentially trigger deleterious biological processes such as thrombosis. To address this problem, we have developed an ultrathin antithrombogenic coating based on monoethylene glycol silane surface chemistry. The strategy is exemplified with polycarbonate--a plastic polymer increasingly employed in the biomedical industry. The various straightforward steps of surface modification were characterized with X-ray photoelectron spectroscopy supplemented by contact angle goniometry. Antithrombogenicity was assessed after 5 min exposure to whole human blood dispensed at a shear rate of 1000 s(-1). Remarkably, platelet adhesion, aggregation, and thrombus formation on the coated surface was greatly inhibited (>97% decrease in surface coverage) compared to the bare substrate and, most importantly, nearly nonexistent.

Zoledronic acid for prevention of bone loss in patients receiving primary therapy for lymphomas: a prospective, randomized controlled phase III trial.

UNLABELLED: In patients with newly diagnosed lymphoma, low bone mineral density (BMD) is common at diagnosis and worsens with therapy. Our randomized phase III trial demonstrates that 2 doses of zoledronic acid (ZA) and supplementation with calcium and vitamin D effectively prevent further bone loss. BACKGROUND: Patients with lymphoma are at risk of development of bone mineral density (BMD) loss from therapy with high-dose corticosteroids and alkylating agents. Zoledronic acid (ZA), a bisphosphonate, may prevent this complication of therapy. We evaluated the effect of ZA on the change in BMD and surrogate biomarkers in patients with lymphoma receiving initial chemotherapy. PATIENTS AND METHODS: Our phase III trial randomized 74 patients with newly diagnosed lymphoma and a baseline BMD of ≥ -2.0 to receive oral calcium and vitamin D daily with or without ZA at enrollment and at 6 months after enrollment. BMD was evaluated at baseline and 1 year after enrollment. Secondary biomarker endpoints were collected at baseline and at 3, 6, 9, and 12 months after enrollment. RESULTS: Forty-three percent of patients had baseline osteopenia. Fifty-three patients were evaluable for response: 24 received ZA and had stable BMD during the observation period, whereas 29 patients in the control group had decreased BMD (P < .05 at lumbar spine and bilateral femoral neck). Twenty-one randomized patients were not evaluable for response because of lymphoma progression or death, withdrawn consent/incomplete testing, or ineligibility. Bone biomarkers were higher in the control group at all intervals after treatment (P < .001). No fractures or intervention-related toxicities were observed during this trial. CONCLUSIONS: Newly diagnosed patients with lymphoma are at risk of low BMD, which may worsen with therapy. Treatment with ZA effectively stabilizes BMD and prevents bone loss. Our data suggest that BMD testing and prophylaxis should be considered as an early intervention for a preventable problem.

Oxygen dose responsiveness of human fetal airway smooth muscle cells.

Maintenance of blood oxygen saturation dictates supplemental oxygen administration to premature infants, but hyperoxia predisposes survivors to respiratory diseases such as asthma. Although much research has focused on oxygen effects on alveoli in the setting of bronchopulmonary dysplasia, the mechanisms by which oxygen affects airway structure or function relevant to asthma are still under investigation. We used isolated human fetal airway smooth muscle (fASM) cells from 18-20 postconceptual age lungs (canalicular stage) to examine oxygen effects on intracellular Ca(2+) ([Ca(2+)](i)) and cellular proliferation. fASM cells expressed substantial smooth muscle actin and myosin and several Ca(2+) regulatory proteins but not fibroblast or epithelial markers, profiles qualitatively comparable to adult human ASM. Fluorescence Ca(2+) imaging showed robust [Ca(2+)](i) responses to 1 µM acetylcholine (ACh) and 10 µM histamine (albeit smaller and slower than adult ASM), partly sensitive to zero extracellular Ca(2+). Compared with adult, fASM showed greater baseline proliferation. Based on this validation, we assessed fASM responses to 10% hypoxia through 90% hyperoxia and found enhanced proliferation at <60% oxygen but increased apoptosis at >60%, effects accompanied by appropriate changes in proliferative vs. apoptotic markers and enhanced mitochondrial fission at >60% oxygen. [Ca(2+)](i) responses to ACh were enhanced for <60% but blunted at >60% oxygen. These results suggest that hyperoxia has dose-dependent effects on structure and function of developing ASM, which could have consequences for airway diseases of childhood. Thus detrimental effects on ASM should be an additional consideration in assessing risks of supplemental oxygen in prematurity.

Interfacial behavior of immortalized hypothalamic mouse neurons detected by acoustic wave propagation.

The attachment of immortalized hypothalamic murine neurons onto the surface of an acoustic wave device yields both positive series resonant frequency (f(s)) and motional resistance (R(m)) shifts as opposed to commonly reported negative f(s) and positive R(m) shifts observed for other cell types. These unique shifts have been confirmed by a variety of experiments in order to verify the source and the validity of the signals. These studies involved monitoring responses to solution flow, the absence of serum proteins, the effect of reducing specific cell -surface interactions and the disruption of the neuronal cytoskeleton components. For the adhesion and deposition of neurons, f(s) and R(m) shifts are positively correlated to the amount of adhered neurons on the sensor surface, whereas non-adhered neurons do not produce any significant change in the monitored parameters. In the absence of serum proteins, initial cell adhesion is followed by subsequent cell death and removal from the sensor surface. The presence of the peptide, GRGDS is observed to significantly reduce cell-surface specific interactions compared to the control of SDGRG and this produces f(s) and R(m) responses that are opposite in direction to that observable for cell adhesion. Cytoskeletal studies, using the drugs nocodazole (10 µM), colchicine (1 µM), cytochalasin B (10 µM) and cytochalasin D (2 µM) all elicit neuronal responses that are validated by phalloidin actin-filament staining. These results indicate that the responses are associated with a wide range of cellular changes that can be monitored and studied using the acoustic wave method in real time, under optimal physiological conditions.

Synchronization of the circadian rhythm generator and the effects of glucagon on hypothalamic mouse neurons detected by acoustic wave propagation.

A thickness shear mode acoustic wave sensor has been used to study the reaction of clonal, immortalized hypothalamic murine neurons in response to glucagon and serum shock in a label free, continuous and real time manner under physiological conditions. Two cell lines were examined; these were the mHypoE-38s and the mHypoE-46s. The technique possesses sufficient sensitivity to detect minor neuronal changes and is capable of discerning subtle differences in cellular behaviors under both stimuli. The kinetics and magnitude of the changes observed here are significantly different compared to those instigated upon causing depolarization, cytoskeletal modifications and surface-adhesion specific interaction alterations with the same cells. Interestingly, this technique has the sensitivity and capability of observing all such changes at the neuronal level without the necessity for invasive interrogation. Under the influence of glucagon, the neurons display both short- and long-term changes, in particular the resonant frequency shifts by -23 ± 8 Hz (n = 13, std. dev.) and the motional resistance decays at a rate of approximately 10 Ω h(-1) over a 2 hour interval. The effect of synchronizing the neurons prior to glucagon stimulation did not influence the cellular changes observed. The process of partial and full synchronization of the cells resulted in different responses. For full synchronization, the addition of the serum bolus triggered resonant frequency and motional resistance shifts of +75 Hz and +18.5 Ω respectively, which decayed back to baseline levels after 30 minutes. The duration of this decay closely matched the time required for full synchronization in a separate study. The changes observed for partial synchronization were significantly different from full synchronization as the baseline levels in both resonant frequency and motional resistance were not re-achieved indicative of the cell-sensor system detecting the difference between full and partial synchronization. Preliminary qualitative immunocytochemistry and RT-PCR studies on these cells support the results obtained with the TSM sensor for the glucagon receptor study.

Calcium-activated and voltage-gated potassium channels of the pancreatic islet impart distinct and complementary roles during secretagogue induced electrical responses.

Glucose-induced ß-cell action potential (AP) repolarization is regulated by potassium efflux through voltage gated (Kv) and calcium activated (K(Ca)) potassium channels. Thus, ablation of the primary Kv channel of the ß-cell, Kv2.1, causes increased AP duration. However, Kv2.1(-/-) islet electrical activity still remains sensitive to the potassium channel inhibitor tetraethylammonium. Therefore, we utilized Kv2.1(-/-) islets to characterize Kv and K(Ca) channels and their respective roles in modulating the ß-cell AP. The remaining Kv current present in Kv2.1(-/-) ß-cells is inhibited with 5 µM CP 339818. Inhibition of the remaining Kv current in Kv2.1(-/-) mouse ß-cells increased AP firing frequency by 39.6% but did not significantly enhance glucose stimulated insulin secretion (GSIS). The modest regulation of islet AP frequency by CP 339818 implicates other K(+) channels, possibly K(Ca) channels, in regulating AP repolarization. Blockade of the K(Ca) channel BK with slotoxin increased ß-cell AP amplitude by 28.2%, whereas activation of BK channels with isopimaric acid decreased ß-cell AP amplitude by 30.6%. Interestingly, the K(Ca) channel SK significantly contributes to Kv2.1(-/-) mouse islet AP repolarization. Inhibition of SK channels decreased AP firing frequency by 66% and increased AP duration by 67% only when Kv2.1 is ablated or inhibited and enhanced GSIS by 2.7-fold. Human islets also express SK3 channels and their ß-cell AP frequency is significantly accelerated by 4.8-fold with apamin. These results uncover important repolarizing roles for both Kv and K(Ca) channels and identify distinct roles for SK channel activity in regulating calcium- versus sodium-dependent AP firing.

Depolarization of surface-attached hypothalamic mouse neurons studied by acoustic wave (thickness shear mode) detector.

Isolation of neurons from animal tissue is an important aspect of understanding basic biochemical processes such as the action of hormones and neurotransmitters. In the present work, the focus is on an effort to evaluate the utility of acoustic wave physics for the study of such cells. Immortalised hypothalamic neuronal cells from mouse embryos were cultured on the surface of the gold electrode of a 9.0 MHz thickness-shear mode acoustic wave sensor. These cells, which are clonal, are imposed on the surface of the device at a confluence in the range of 80-100%. The coated sensor is incorporated into a flow-injection configuration such that electrolytes can be introduced in order to examine their effects through measurement by network analysis. Both series resonance frequency, fs, and motional resistance, R(m), were measured in a number of experiments involving the injection of KCl and NaCl into the sensor-neuron system. The various responses to these electrolytes were interpreted in terms of changes in cellular structure associated with the depolarization process. The sensor-neuron system was found to elicit different responses to the addition of KCl and NaCl. Preliminary findings indicate that the TSM sensor does not purely measure changes in the membrane potential upon KCl addition. Typical changes in fs for 15 mM, 30 mM and 60 mM KCl additions were 54 +/- 15, 80 +/- 26 and 142 +/- 58 Hz (mean +/- standard deviation) respectively. Typical changes in R(m) for these KCl additions were 7 +/- 3, 13 +/- 4 and 23 +/- 6 Omega, respectively. These results were concluded after 17 runs at each concentration. Despite the large relative standard deviations, the dependence of f(s) and R(m) with respect to concentration was apparent. Controls performed by coating the TSM sensor with laminin or a cell attachment matrix showed no significant changes in either f(s) or R(m) for the same solutions tested on the sensor-neuron system.