Novel Tocolytic Strategy: Modulating Cx43 Activity by S-Nitrosation.

Currently available tocolytics are ineffective at significantly delaying preterm birth. This is due in part to our failure to better understand the mechanisms that drive spontaneous preterm labor (sPTL). Cyclic nucleotides are not the primary contributors to myometrial quiescence, but instead nitric oxide (NO)-mediated protein S-nitrosation (SNO) is integral to the relaxation of the tissue. Connexin-43 (Cx43), a myometrial "contractile-associated protein" that functions as either a gap junction channel or an hemichannel (HC), was the focus of this study. Protein analysis determined that Cx43 is downregulated in sPTL myometrium. Furthermore, Cx43 is S-nitrosated by NO, which correlates with an increase of phosphorylated Cx43 at serine 368 (Cx43-pS368 -gap junction inhibition) as well as an increase in the HC open-state probability (quiescence). Pharmacologic inhibition of Cx43 with 18ß-glycyrrhetinic acid (18ß-GA) exhibits a negative inotropic effect on the myometrium in a dose-dependent manner, as does administration of nebivolol, an NO synthase activator that increases total protein SNOs. When 18ß-GA and nebivolol were coadministered at their IC50 values, the effect on contractile dynamics was additive and all but eliminated contractions. The development of new tocolytics demands a better understanding of the underlying mechanisms of sPTL. Here it has been shown that 18ß-GA and nebivolol leverage dysregulated pathways in the myometrium, resulting in a novel approach for the treatment of sPTL. SIGNIFICANCE STATEMENT: Although there are many known causes of preterm labor (PTL), the mechanisms of "spontaneous" PTL (sPTL) remain obfuscated, which is why treating this condition is so challenging. Here we have identified that connexin-43 (Cx43), an important contractile-associated protein, is dysregulated in sPTL myometrium and that the pharmacologic inhibition of Cx43 and its S-nitrosation with 18ß-glycyrrhetinic acid and nebivolol, respectively, significantly blunts contraction in human myometrial tissue, presenting a novel approach to tocolysis that leverages maladjusted pathways in women who experience sPTL.

The cellular bases of choroid fissure formation and closure.

Defects in choroid fissure (CF) formation and closure lead to coloboma, a major cause of childhood blindness. Despite genetic advances, the cellular defects underlying coloboma remain poorly elucidated due to our limited understanding of normal CF morphogenesis. We address this deficit by conducting high-resolution spatio-temporal analyses of CF formation and closure in the chick, mouse and fish. We show that a small ventral midline invagination initiates CF formation in the medial-proximal optic cup, subsequently extending it dorsally toward the lens, and proximally into the optic stalk. Unlike previously supposed, the optic disc does not form solely as a result of this invagination. Morphogenetic events that alter the shape of the proximal optic cup also direct clusters of outer layer and optic stalk cells to form dorsal optic disc. A cross-species comparison suggests that CF closure can be accomplished by breaking down basement membranes (BM) along the CF margins, and by establishing BM continuity along the dorsal and ventral surfaces of the CF. CF closure is subsequently accomplished via two distinct mechanisms: tissue fusion or the intercalation of various tissues into the inter-CF space. We identify several novel cell behaviors that underlie CF fusion, many of which involve remodeling of the retinal epithelium. In addition to BM disruption, these include NCAD downregulation along the SOX2+ retinal CF margin, and the protrusion or movement of partially polarized retinal cells into the inter-CF space to mediate fusion. Proximally, the inter-CF space does not fuse or narrow and is instead loosely packed with migrating SOX2+/PAX2+/Vimentin+ astrocytes until it is closed by the outgoing optic nerve. Taken together, our results highlight distinct proximal-distal differences in CF morphogenesis and closure and establish detailed cellular models that can be utilized for understanding the genetic bases of coloboma.

Correction to: Pioglitazone reduces cold-induced brown fat glucose uptake despite induction of browning in cultured human adipocytes: a randomised, controlled trial in humans.

The baseline insulin data given in Table 1 for the placebo group were incorrectly reported as 51 ± 10 pmol/l instead of 48 ± 10 pmol/l. This mistake also impacts on data reported in Table 4.

Pioglitazone reduces cold-induced brown fat glucose uptake despite induction of browning in cultured human adipocytes: a randomised, controlled trial in humans.

AIMS/HYPOTHESIS: Increasing brown adipose tissue (BAT) activity is a possible therapeutic strategy to increase energy expenditure and glucose and lipid clearance to ameliorate obesity and associated comorbidities. The thiazolidinedione (TZD) class of glucose-lowering drugs increase BAT browning in preclinical experimental models but whether these actions extend to humans in vivo is unknown. The aim of this study was to determine the effect of pioglitazone treatment on adipocyte browning and adaptive thermogenesis in humans. METHODS: We first examined whether pioglitazone treatment of cultured human primary subacromioclavicular-derived adipocytes induced browning. Then, in a blinded, placebo-controlled, parallel trial, conducted within the Baker Institute clinical research laboratories, 14 lean male participants who were free of cardiometabolic disease were randomised to receive either placebo (lactose; n = 7, age 22 ± 1 years) or pioglitazone (45 mg/day, n = 7, age 21 ± 1 years) for 28 days. Participants were allocated to treatments by Alfred Hospital staff independent from the study via electronic generation of a random number sequence. Researchers conducting trials and analysing data were blind to treatment allocation. The change in cold-stimulated BAT activity, assessed before and after the intervention by [18F]fluorodeoxyglucose uptake via positron emission tomography/computed tomography in upper thoracic and cervical adipose tissue, was the primary outcome measure. Energy expenditure, cardiovascular responses, core temperature, blood metabolites and hormones were measured in response to acute cold exposure along with body composition before and after the intervention. RESULTS: Pioglitazone significantly increased in vitro browning and adipogenesis of adipocytes. In the clinical trial, cold-induced BAT maximum standardised uptake value was significantly reduced after pioglitazone compared with placebo (-57 ± 6% vs -12 ± 18%, respectively; p < 0.05). BAT total glucose uptake followed a similar but non-significant trend (-50 ± 10% vs -6 ± 24%, respectively; p = 0.097). Pioglitazone increased total and lean body mass compared with placebo (p < 0.05). No other changes between groups were detected. CONCLUSIONS/INTERPRETATION: The disparity in the actions of pioglitazone on BAT between preclinical experimental models and our in vivo human trial highlight the imperative to conduct human proof-of-concept studies as early as possible in BAT research programmes aimed at therapeutic development. Our clinical trial findings suggest that reduced BAT activity may contribute to weight gain associated with pioglitazone and other TZDs. TRIAL REGISTRATION: ClinicalTrials.gov NCT02236962 FUNDING: This work was supported by the Diabetes Australia Research Program and OIS scheme from the Victorian State Government.

Two-Color Pump-Probe Measurement of Photonic Quantum Correlations Mediated by a Single Phonon.

We propose and demonstrate a versatile technique to measure the lifetime of the one-phonon Fock state using two-color pump-probe Raman scattering and spectrally resolved, time-correlated photon counting. Following pulsed laser excitation, the n=1 phonon Fock state is probabilistically prepared by projective measurement of a single Stokes photon. The detection of an anti-Stokes photon generated by a second, time-delayed laser pulse probes the phonon population with subpicosecond time resolution. We observe strongly nonclassical Stokes-anti-Stokes correlations, whose decay maps the single phonon dynamics. Our scheme can be applied to any Raman-active vibrational mode. It can be modified to measure the lifetime of n≥1 Fock states or the phonon quantum coherences through the preparation and detection of two-mode entangled vibrational states.

Preservation of skeletal muscle mitochondrial content in older adults: relationship between mitochondria, fibre type and high-intensity exercise training.

KEY POINTS: Ageing is associated with an upregulation of mitochondrial dynamics proteins mitofusin 2 (Mfn2) and mitochondrial dynamics protein 49 (MiD49) in human skeletal muscle with the increased abundance of Mfn2 being exclusive to type II muscle fibres. These changes occur despite a similar content of mitochondria, as measured by COXIV, NDUFA9 and complexes in their native states (Blue Native PAGE). Following 12 weeks of high-intensity training (HIT), older adults exhibit a robust increase in mitochondria content, while there is a decline in Mfn2 in type II fibres. We propose that the upregulation of Mfn2 and MiD49 with age may be a protective mechanism to protect against mitochondrial dysfunction, in particularly in type II skeletal muscle fibres, and that exercise may have a unique protective effect negating the need for an increased turnover of mitochondria. ABSTRACT: Mitochondrial dynamics proteins are critical for mitochondrial turnover and maintenance of mitochondrial health. High-intensity interval training (HIT) is a potent training modality shown to upregulate mitochondrial content in young adults but little is known about the effects of HIT on mitochondrial dynamics proteins in older adults. This study investigated the abundance of protein markers for mitochondrial dynamics and mitochondrial content in older adults compared to young adults. It also investigated the adaptability of mitochondria to 12 weeks of HIT in older adults. Both older and younger adults showed a higher abundance of mitochondrial respiratory chain subunits COXIV and NDUFA9 in type I compared with type II fibres, with no difference between the older adults and young groups. In whole muscle homogenates, older adults had higher mitofusin-2 (Mfn2) and mitochondrial dynamics protein 49 (MiD49) contents compared to the young group. Also, older adults had higher levels of Mfn2 in type II fibres compared with young adults. Following HIT in older adults, MiD49 and Mfn2 levels were not different in whole muscle and Mfn2 content decreased in type II fibres. Increases in citrate synthase activity (55%) and mitochondrial respiratory chain subunits COXIV (37%) and NDUFA9 (48%) and mitochondrial respiratory chain complexes (∼70-100%) were observed in homogenates and/or single fibres. These findings reveal (i) a similar amount of mitochondria in muscle from young and healthy older adults and (ii) a robust increase of mitochondrial content following 12 weeks of HIT exercise in older adults.

Chronic ephedrine administration decreases brown adipose tissue activity in a randomised controlled human trial: implications for obesity.

AIMS/HYPOTHESIS: Brown adipose tissue (BAT) activation increases energy expenditure and may have therapeutic potential to combat obesity. The primary activating and adaptive signal for BAT is via ß-adrenergic signalling. We previously demonstrated that human BAT is acutely responsive to oral administration of the sympathomimetic, ephedrine. Here we aimed to determine whether adaptive thermogenesis can be induced via chronic treatment with ephedrine. METHODS: Twenty-three healthy young men, recruited from the general public in Melbourne, Australia, who were non-smokers, physically inactive and non-medicated with no prior history of cardiovascular disease or diabetes were recruited for this study. They were assigned to receive either 1.5 mg kg(-1) day(-1) ephedrine ('active' group; n = 12, age 23 ± 1 years, BMI 24 ± 1 kg/m(2)) or placebo (n = 11; 22 ± 2 years, 23 ± 2 kg/m(2)) for 28 days in a randomised (computer-generated random order sequence), placebo-controlled, parallel-group trial. Participants and all investigators were blinded to treatments. Body composition was measured before and after the intervention by dual energy X-ray absorptiometry. BAT activity, measured via (18)F-fluorodeoxyglucose positron emission tomography-computed tomography, in response to a single dose of 2.5 mg/kg ephedrine, was the primary outcome measure to be determined before and after the 28 day treatment period. RESULTS: Twenty-eight individuals were randomised and consented to the study. Twenty-three completed the trial and only these participants were included in the final analyses. After 28 days of treatment, the active group lost a significant amount of total body fat (placebo 1.1 ± 0.3 kg, ephedrine -0.9 ± 0.5 kg; p < 0.01) and visceral fat (placebo 6.4 ± 19.1 g, ephedrine -134 ± 43 g; p < 0.01), with no change in lean mass or bone mineral content compared with the placebo group. In response to acute ephedrine, BAT activity (change in mean standardised uptake value: placebo -3 ± 7%, ephedrine -22 ± 6%) and the increase in systolic blood pressure were significantly reduced (p < 0.05) in the active group compared with placebo. CONCLUSIONS/INTERPRETATION: Chronic ephedrine treatment reduced body fat content, but this was not associated with an increase in BAT activity. Rather, chronic ephedrine suppressed BAT glucose disposal, suggesting that chronic ephedrine treatment decreased, rather than increased, BAT activity. TRIAL REGISTRATION: ClinicalTrials.gov NCT02236962 FUNDING: This study was funded by the National Health and Medical Research Council of Australia Program Grant (1036352) and the OIS scheme from the Victorian State Government.

Optimized Fixation and Phalloidin Staining of Basally Localized F-Actin Networks in Collectively Migrating Follicle Cells.

The follicular epithelial cells of the Drosophila egg chamber have become a premier model to study how cells globally orient their actin-based machinery for collective migration. The basal surface of each follicle cell has lamellipodial and filopodial protrusions that extend from its leading edge and an array of stress fibers that mediate its adhesion to the extracellular matrix; these migratory structures are all globally aligned in the direction of tissue movement. To understand how this global alignment is achieved, one must be able to reliably visualize the underlying F-actin; however, dynamic F-actin networks can be difficult to preserve in fixed tissues. Here, we describe an optimized protocol for the fixation and phalloidin staining of the follicular epithelium. We also provide a brief primer on relevant aspects of the image acquisition process to ensure high quality data are collected.

Demographics and risk of isolation due to sea level rise in the United States.

Within coastal communities, sea level rise (SLR) will result in widespread intermittent flooding and long-term inundation. Inundation effects will be evident, but isolation that arises from the loss of accessibility to critical services due to inundation of transportation networks may be less obvious. We examine who is most at risk of isolation due to SLR, which can inform community adaptation plans and help ensure that existing social vulnerabilities are not exacerbated. Combining socio-demographic data with an isolation metric, we identify social and economic disparities in risk of isolation under different SLR scenarios (1-10 ft) for the coastal U.S. We show that Black and Hispanic populations face a disproportionate risk of isolation at intermediate levels of SLR (4 ft and greater). Further, census tracts with higher rates of renters and older adults consistently face higher risk of isolation. These insights point to significant inequity in the burdens associated with SLR.

Heat Acclimation with or without Normobaric Hypoxia Exposure Leads to Similar Improvements in Endurance Performance in the Heat.

BACKGROUND: Combining the key adaptation of plasma volume (PV) expansion with synergistic physiological effects of other acclimation interventions to maximise endurance performance in the heat has potential. The current study investigated the effects of heat acclimation alone (H), combined with normobaric hypoxia exposure (H+NH), on endurance athletic performance. METHODS: Well-trained participants completed a heat-stress trial (30 °C, 80% relative humidity (RH), 20.8% fraction of inspired oxygen (FiO2)) of a 75 min steady-state cycling (fixed workload) and a subsequent 15 min cycling time trial for distance before and after intervention. Participants completed 12 consecutive indoor training days with either heat acclimation (H; 60 min·day-1, 30 °C, 80% RH; 20.8% FiO2) or heat acclimation and overnight hypoxic environment (H+NH; ~12 h, 60% RH; 16% FiO2 simulating altitude of ~2500 m). Control (CON) group trained outdoors with average maximum daily temperature of 16.5 °C and 60% RH. RESULTS: Both H and H+NH significantly improved time trial cycling distance by ~5.5% compared to CON, with no difference between environmental exposures. PV increased (+3.8%) and decreased (-4.1%) following H and H+NH, respectively, whereas haemoglobin concentration decreased (-2%) and increased (+3%) in H and H+NH, respectively. CONCLUSION: Our results show that despite contrasting physiological adaptations to different environmental acclimation protocols, heat acclimation with or without hypoxic exposure demonstrated similar improvements in short-duration exercise performance in a hot environment.

In situ 24 kHz coherent imaging of morphology change in laser percussion drilling.

We observe sample morphology changes in real time (24 kHz) during and between percussion drilling pulses by integrating a low-coherence microscope into a laser micromachining platform. Nonuniform cut speed and sidewall evolution in stainless steel are observed to strongly depend on assist gas. Interpulse morphology relaxation such as hole refill is directly imaged, showing dramatic differences in the material removal process dependent on pulse duration/peak power (micros/0.1 kW, ps/20 MW) and material (steel, lead zirconate titanate PZT). Blind hole depth precision is improved by over 1 order of magnitude using in situ feedback from the imaging system.

Short-term endurance training does not alter the oxidative capacity of human subcutaneous adipose tissue.

Endurance training results in adaptations that enhance regulation of energy storage and expenditure at rest and during exercise. While processes involved in skeletal muscle oxidative remodelling are well described, it is unknown whether oxidative capacity of human subcutaneous white adipose tissue (WAT) is modified by endurance training. Since human WAT retains rudimentary characteristics required for upregulation of oxidative function, we hypothesised that 10 days of intense endurance training would promote changes in WAT that favour an increase in oxidative capacity. Eleven untrained males (age 22 +/- 1 years, body mass 81 +/- 5 kg, peak oxygen uptake (VO(2peak)) 3.7 +/- 0.2 l/min) undertook a 10-day endurance training protocol. Subcutaneous adipose tissue biopsies were taken from the abdomen prior to and 1 day after completion of training and analysed for fatty acid oxidative capacity, citrate synthase activity, and mitochondrial content via electron microscopy and gene expression analyses. There was a reduction in whole-body rates of carbohydrate oxidation, and concomitant increases in fat oxidation rate measured during 20-min of submaximal cycling (70% of pre-training VO(2peak)) and an increase in basal GLUT4 protein in skeletal muscle. Despite these training-induced adaptations, there were no changes in WAT of ex-vivo fat oxidation rate, maximal citrate synthase activity, mitochondrial volume or in selected genes involved in adipose tissue oxidative capacity. We conclude that 10 days training in previously untrained subjects results in adaptations in skeletal muscle but does not increase the oxidative capacity of WAT.

Biocompatible liquid-crystal elastomers mimic the intervertebral disc.

The hierarchical and anisotropic mechanical behavior requirement of load-bearing soft tissues limits the utility of conventional elastomeric materials as a replacement for soft-tissue materials. Liquid-crystal elastomers (LCEs) have the potential to excel in this regard owing to its unique combination of mesogenic order in an elastomeric network. In this study, the mechanical behavior of the LCEs relevant to load-bearing biomedical applications was explored. LCEs with different network orientations (i.e., mesogen alignments) were investigated by fabricating the LCEs with polydomain and monodomain configurations. The polydomain and monodomain LCEs with the same degree of network crosslinking demonstrated diverse mechanical behavior, ranging from highly stiff and elastic nature to high damping capacity, depending on the loading direction with respect to the network alignment. The LCEs were also capable of matching the anisotropic mechanical behavior of an intervertebral disc. Additional studies were conducted on the in vivo biological response of LCEs upon subcutaneous implantation, as well as on the effect of the exposure to an in vitro simulated physiological environment on the mechanical behavior. The LCEs' mechanical response was negligibly affected when exposed to biomedically relevant conditions. Furthermore, the solid and porous LCEs did not show any adverse effect on the surrounding tissues when implanted subcutaneously in rats. The biological response allows for tissue ingrowth and helps illustrate their utility in implantable biological devices. Finally, the utility of LCEs to mimic the mechanical function of biological tissue such as intervertebral disc was demonstrated by fabricating a proof of concept total disc replacement device.

Anderson localisation in steady states of microcavity polaritons.

We present an experimental signature of the Anderson localisation of microcavity polaritons, and provide a systematic study of the dependence on disorder strength. We reveal a controllable degree of localisation, as characterised by the inverse-participation ratio, by tuning the positional disorder of arrays of interacting mesas. This constitutes the realisation of disorder-induced localisation in a driven-dissipative system. In addition to being an ideal candidate for investigating localisation in this regime, microcavity polaritons hold promise for low-power, ultra-small devices and their localisation could be used as a resource in quantum memory and quantum information processing.

Trends in inferior turbinate surgery: analysis of patients using the Medicare database.

BACKGROUND: The aim of this study was to characterize trends in turbinate reduction procedures from 2000 to 2015. METHODS: Annual procedure data were obtained for the period 2000-2015 and detailed Medicare provider and payment data were obtained for 2012-2015. Turbinate procedures analyzed included turbinate soft tissue mucosal ablation (TMA), turbinate soft tissue submucosal ablation (TSMA), turbinate excision (TE), and turbinate submucous resection (TSR). TMA and TSMA were grouped as turbinate soft tissue ablation (TA) for analysis. From 2012 to 2015, the type and location-facility (F) or nonfacility (NF)-of the providers performing the procedures were assessed. RESULTS: From 2000 to 2015, the total number of turbinate reduction procedures increased by an average of 3.8% annually. TSR had the highest annual increase at 5.4%. TE is the only procedure to show a decrease, by an average of -2.3% annually. From 2012 to 2015, the number of turbinate reduction procedures changed by -1.6% and 107.7% at F and NF locations, respectively. NF TSMA and TSR had the largest increases at 121.6% and 260.1%, respectively. Of the NF TA procedures, there was an average annual increase of 50% by non-otolaryngologists. For TA, the average F charge was 78.0% more than the NF charge, and the average NF otolaryngologist charge 11.5% more than the non-otolaryngologist charge. CONCLUSION: The number of turbinate reduction procedures increased steadily between 2000 and 2015, with the majority being TSRs. This is consistent with previous studies demonstrating that TSR leads to better outcomes. There has been a significant increase in turbinate reduction procedures performed in outpatient/ambulatory settings by otolaryngologists, non-otolaryngologists, and midlevel providers.

Shortcomings in the diagnosis of chronic rhinosinusitis: evaluating diagnosis by otolaryngologists and primary care physicians.

BACKGROUND: Chronic rhinosinusitis (CRS) is a prevalent illness in the United States that accounts for 18-22 million physician visits annually. The American Academy of Otolaryngology-Head and Neck Surgery (AAO-HNS) has defined diagnostic criteria, but a recent study demonstrated that nearly all patients diagnosed by nonspecialists did not meet these criteria. In this study we aimed to evaluate the diagnostic rate of CRS by primary care physicians and otolaryngologists. METHODS: We retrospectively reviewed a random sample of adult patients diagnosed with CRS in 2016, based on ICD-10 codes from primary care and otolaryngology departments. Patients with previous CRS diagnosis, previous sinus surgery, and related comorbidities were excluded. RESULTS: A total of 502 patients with a new CRS diagnosis were analyzed (308 from primary care, 194 from otolaryngology). The percentage of diagnoses meeting the criteria was significantly higher from otolaryngology (28.9% vs 0.97%, p < 0.0001), but was low in both cohorts. Symptom duration <12 weeks was higher in primary care (81.6% vs 53.6%, p < 0.0001), as was lack of evidence of inflammation (97.4% vs 50.0%, p < 0.0001). Having <2 of the required symptoms was significantly higher in otolaryngology (63.8% vs 50.8%, p = 0.013). The most commonly unevaluated symptom was decreased sense of smell (97.7% in primary care, 69.1% in otolaryngology encounters). CONCLUSION: CRS diagnoses commonly do not meet the diagnostic criteria outlined by the AAO-HNS in both primary care and otolaryngology. As a specialty, we should aim to improve our adherence to the guidelines and educate our primary care colleagues to better identify patients with CRS and initiate appropriate treatment.

Adjuvant radiation therapy improves patient survival in early-stage merkel cell carcinoma: A 15-year single-institution study.

OBJECTIVES/HYPOTHESIS: Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine neoplasm of the skin. Growing evidence supports the benefit of postoperative adjuvant radiation therapy (RT) for locoregional control, but whether it improves overall survival (OS) has been debated. Our objective was to compare the OS of MCC patients who received postoperative RT with those who received surgery alone. STUDY DESIGN: Retrospective case series. METHODS: Cases of MCC between 2001 and 2016 at the University of California, Los Angeles Health System were reviewed. We identified 87 unique cases of MCC. Among the patients, 74% were identified as male and 26% as female. The average age at diagnosis was 71.2 years. The median survival was 48.0 months. The OS of all the patients at 2 years, 5 years, and 10 years was 54%, 46%, and 26%, respectively. Univariate analysis showed that stage, T stage, N stage, and M stage were significant determinants of OS. The inclusion of RT was not found to be a determinant; however, when restricting the analysis to early-stage MCC (stages I and II), postoperative adjuvant RT was associated with significantly improved OS. A Cox regression model confirmed that inclusion of RT was an independent prognosticator of OS even when controlled for overall stage and negative margin status. The small sample size and retrospective nature of this study limit its statistical power. CONCLUSIONS: MCC is an aggressive tumor with a poor prognosis for survival especially in elderly patients. In this study, we found that RT during early-stage MCC improves OS. Prospective randomized control trials are necessary to validate the observed benefit for MCC patients. LEVEL OF EVIDENCE: 4 Laryngoscope, 1862-1866, 2018.