A new polymodal gating model of the proton-activated chloride channel.

The proton-activated chloride (PAC) channel plays critical roles in ischemic neuron death, but its activation mechanisms remain elusive. Here, we investigated the gating of PAC channels using its novel bifunctional modulator C77304. C77304 acted as a weak activator of the PAC channel, causing moderate activation by acting on its proton gating. However, at higher concentrations, C77304 acted as a weak inhibitor, suppressing channel activity. This dual function was achieved by interacting with 2 modulatory sites of the channel, each with different affinities and dependencies on the channel's state. Moreover, we discovered a protonation-independent voltage activation of the PAC channel that appears to operate through an ion-flux gating mechanism. Through scanning-mutagenesis and molecular dynamics simulation, we confirmed that E181, E257, and E261 in the human PAC channel serve as primary proton sensors, as their alanine mutations eliminated the channel's proton gating while sparing the voltage-dependent gating. This proton-sensing mechanism was conserved among orthologous PAC channels from different species. Collectively, our data unveils the polymodal gating and proton-sensing mechanisms in the PAC channel that may inspire potential drug development.

Identification and targeting of a unique NaV1.7 domain driving chronic pain.

Small molecules directly targeting the voltage-gated sodium channel (VGSC) NaV1.7 have not been clinically successful. We reported that preventing the addition of a small ubiquitin-like modifier onto the NaV1.7-interacting cytosolic collapsin response mediator protein 2 (CRMP2) blocked NaV1.7 function and was antinociceptive in rodent models of neuropathic pain. Here, we discovered a CRMP2 regulatory sequence (CRS) unique to NaV1.7 that is essential for this regulatory coupling. CRMP2 preferentially bound to the NaV1.7 CRS over other NaV isoforms. Substitution of the NaV1.7 CRS with the homologous domains from the other eight VGSC isoforms decreased NaV1.7 currents. A cell-penetrant decoy peptide corresponding to the NaV1.7-CRS reduced NaV1.7 currents and trafficking, decreased presynaptic NaV1.7 expression, reduced spinal CGRP release, and reversed nerve injury-induced mechanical allodynia. Importantly, the NaV1.7-CRS peptide did not produce motor impairment, nor did it alter physiological pain sensation, which is essential for survival. As a proof-of-concept for a NaV1.7 -targeted gene therapy, we packaged a plasmid encoding the NaV1.7-CRS in an AAV virus. Treatment with this virus reduced NaV1.7 function in both rodent and rhesus macaque sensory neurons. This gene therapy reversed and prevented mechanical allodynia in a model of nerve injury and reversed mechanical and cold allodynia in a model of chemotherapy-induced peripheral neuropathy. These findings support the conclusion that the CRS domain is a targetable region for the treatment of chronic neuropathic pain.

A peptidomimetic modulator of the CaV2.2 N-type calcium channel for chronic pain.

Transmembrane Cav2.2 (N-type) voltage-gated calcium channels are genetically and pharmacologically validated, clinically relevant pain targets. Clinical block of Cav2.2 (e.g., with Prialt/Ziconotide) or indirect modulation [e.g., with gabapentinoids such as Gabapentin (GBP)] mitigates chronic pain but is encumbered by side effects and abuse liability. The cytosolic auxiliary subunit collapsin response mediator protein 2 (CRMP2) targets Cav2.2 to the sensory neuron membrane and regulates their function via an intrinsically disordered motif. A CRMP2-derived peptide (CBD3) uncouples the Cav2.2-CRMP2 interaction to inhibit calcium influx, transmitter release, and pain. We developed and applied a molecular dynamics approach to identify the A1R2 dipeptide in CBD3 as the anchoring Cav2.2 motif and designed pharmacophore models to screen 27 million compounds on the open-access server ZincPharmer. Of 200 curated hits, 77 compounds were assessed using depolarization-evoked calcium influx in rat dorsal root ganglion neurons. Nine small molecules were tested electrophysiologically, while one (CBD3063) was also evaluated biochemically and behaviorally. CBD3063 uncoupled Cav2.2 from CRMP2, reduced membrane Cav2.2 expression and Ca2+ currents, decreased neurotransmission, reduced fiber photometry-based calcium responses in response to mechanical stimulation, and reversed neuropathic and inflammatory pain across sexes in two different species without changes in sensory, sedative, depressive, and cognitive behaviors. CBD3063 is a selective, first-in-class, CRMP2-based peptidomimetic small molecule, which allosterically regulates Cav2.2 to achieve analgesia and pain relief without negative side effect profiles. In summary, CBD3063 could potentially be a more effective alternative to GBP for pain relief.

Rescue Therapy for Failed Mechanical Thrombectomy in Acute Ischemic Stroke: A Pooled Analysis of the Society of Vascular and Interventional Neurology Registry.

OBJECTIVE: We aimed to evaluate the association between rescue therapy (RT) and functional outcomes compared to medical management (MM) in patients presenting after failed mechanical thrombectomy (MT). METHODS: This cross-sectional study utilized prospectively collected and maintained data from the Society of Vascular and Interventional Neurology Registry, spanning from 2011 to 2021. The cohort comprised patients with large vessel occlusions (LVOs) with failed MT. The primary outcome was the shift in the degree of disability, as gauged by the modified Rankin Scale (mRS) at 90 days. Additional outcomes included functional independence (90-day mRS score of 0-2), symptomatic intracranial hemorrhage (sICH), and 90-day mortality. RESULTS: Of a total of 7,018 patients, 958 presented failed MT and were included in the analysis. The RT group comprised 407 (42.4%) patients, and the MM group consisted of 551 (57.5%) patients. After adjusting for confounders, the RT group showed a favorable shift in the overall 90-day mRS distribution (adjusted common odds ratio = 1.79, 95% confidence interval [CI] = 1.32-2.45, p < 0.001) and higher rates of functional independence (RT: 28.8% vs MM: 15.7%, adjusted odds ratio [aOR] = 1.93, 95% CI = 1.21-3.07, p = 0.005) compared to the MM group. RT also showed lower rates of sICH (RT: 3.8% vs MM: 9.1%, aOR = 0.52, 95% CI = 0.28-0.97, p = 0.039) and 90-day mortality (RT: 33.4% vs MM: 45.5%, aOR = 0.61, 95% CI = 0.42-0.89, p = 0.009). INTERPRETATION: Our findings advocate for the utilization of RT as a potential treatment strategy for cases of LVO resistant to first-line MT techniques. Prospective studies are warranted to validate these observations and optimize the endovascular approach for failed MT patients. ANN NEUROL 2024;96:343-355.

Beliefs and Attitudes about Vermont's Buprenorphine Decriminalization Law among Clinicians Who Prescribe Buprenorphine.

BACKGROUND: On June 1, 2021, Vermont repealed all criminal penalties for possessing 224 milligrams or less of buprenorphine. We examined the potential impact of decriminalization with a survey of Vermont clinicians who prescribed buprenorphine within the past year. METHODS: All 638 Vermont clinicians with a waiver to prescribe buprenorphine were emailed the survey by Vermont Department of Health; 117 responded. We estimated the prevalence of the following four outcomes, for all responding clinicians and stratified by clinician demographics and practice characteristics: awareness of decriminalization, beliefs about the effects of decriminalization, support for decriminalization, and changes in practice resulting from decriminalization. RESULTS: 72 (62%) prescribers correctly stated that Vermont does not have criminal penalties for buprenorphine possession. 107 (91%) support decriminalization. 56 (48%) believe that, because buprenorphine is decriminalized, their patients are more likely to give, sell, or trade the buprenorphine that is prescribed to them to someone else. However, only 5 providers (4%) said they now prescribe to fewer patients. CONCLUSION: The great majority of Vermont clinicians who prescribe buprenorphine support its decriminalization and have not changed their prescribing practices because of decriminalization.

In 2021, Vermont repealed criminal penalties for buprenorphine possession.We surveyed Vermont (n = 117) buprenorphine prescribers about decriminalization.91% of providers support decriminalization.48% of providers believe decriminalization will increase diversion of medications.Only 4% of providers prescribe to fewer patients because of decriminalization.

Distinct Growth Responses of Tundra Soil Bacteria to Short-Term and Long-Term Warming.

Increases in Arctic temperatures have thawed permafrost and accelerated tundra soil microbial activity, releasing greenhouse gases that amplify climate warming. Warming over time has also accelerated shrub encroachment in the tundra, altering plant input abundance and quality, and causing further changes to soil microbial processes. To better understand the effects of increased temperature and the accumulated effects of climate change on soil bacterial activity, we quantified the growth responses of individual bacterial taxa to short-term warming (3 months) and long-term warming (29 years) in moist acidic tussock tundra. Intact soil was assayed in the field for 30 days using 18O-labeled water, from which taxon-specific rates of 18O incorporation into DNA were estimated as a proxy for growth. Experimental treatments warmed the soil by approximately 1.5°C. Short-term warming increased average relative growth rates across the assemblage by 36%, and this increase was attributable to emergent growing taxa not detected in other treatments that doubled the diversity of growing bacteria. However, long-term warming increased average relative growth rates by 151%, and this was largely attributable to taxa that co-occurred in the ambient temperature controls. There was also coherence in relative growth rates within broad taxonomic levels with orders tending to have similar growth rates in all treatments. Growth responses tended to be neutral in short-term warming and positive in long-term warming for most taxa and phylogenetic groups co-occurring across treatments regardless of phylogeny. Taken together, growing bacteria responded distinctly to short-term and long-term warming, and taxa growing in each treatment exhibited deep phylogenetic organization. IMPORTANCE Soil carbon stocks in the tundra and underlying permafrost have become increasingly vulnerable to microbial decomposition due to climate change. The microbial responses to Arctic warming must be understood in order to predict the effects of future microbial activity on carbon balance in a warming Arctic. In response to our warming treatments, tundra soil bacteria grew faster, consistent with increased rates of decomposition and carbon flux to the atmosphere. Our findings suggest that bacterial growth rates may continue to increase in the coming decades as faster growth is driven by the accumulated effects of long-term warming. Observed phylogenetic organization of bacterial growth rates may also permit taxonomy-based predictions of bacterial responses to climate change and inclusion into ecosystem models.

Where Are Sodium Ions in AOT Reverse Micelles? Fluoride Anion Probes Nanoconfined Ions by 19F Nuclear Magnetic Resonance Spectroscopy.

Confining water to nanosized spaces creates a unique environment that can change water's structural and dynamic properties. When ions are present in these nanoscopic spaces, the limited number of water molecules and short screening length can dramatically affect how ions are distributed compared to the homogeneous distribution assumed in bulk aqueous solution. Here, we demonstrate that the chemical shift observed in 19F NMR spectroscopy of fluoride anion, F-, probes the location of sodium ions, Na+, confined in reverse micelles prepared from AOT (sodium dioctyl sulfosuccinate) surfactants. Our measurements show that the nanoconfined environment of reverse micelles can lead to extremely high apparent ion concentrations and ionic strength, beyond the limit in bulk aqueous solutions. Most notably, the 19F NMR chemical shift trends we observe for F- in the reverse micelles indicate that the AOT sodium counterions remain at or near the interior interface between surfactant and water, thus providing the first experimental support for this hypothesis.

Characterizing fluid intake and physical activity in university students within the United States during the COVID-19 pandemic.

PURPOSE: This study determined fluid intake and physical activity behaviors among college students during the COVID-19 pandemic. METHODS: College students (n = 1014; females, 75.6%) completed an online survey during the Spring 2020 academic semester following the initial global response to the COVID-19 pandemic. Academic standing, habitation situation, and University/College responses to COVID-19 were collected. Participants completed the Godin Leisure-Time Exercise Questionnaire and a 15-item Beverage Questionnaire (BEVQ-15) to determine physical activity level and fluid intake behaviors, respectively. RESULTS: Females (1920 ± 960 mL) consumed significantly less fluid than males (2400 ± 1270 mL, p < 0.001). Living off-campus (p < 0.01) and living with a spouse/partner (p < 0.01) was associated with increased consumption of alcoholic beverages. 88.7% of participants reported being at least moderately active; however, Black/African American and Asian participants were more likely to be less active than their Caucasian/White counterparts (p < 0.05). Participants reporting no change in habitation in response to COVID-19 had a higher fluid intake (p = 0.002); however, the plain water consumption remained consistent (p = 0.116). While there was no effect of habitation or suspension of classes on physical activity levels (p > 0.05), greater self-reported physical activity was associated with greater fluid intake (std. ß = 0.091, p = 0.003). CONCLUSIONS: Fluid intake among college students during the initial response to the COVID-19 pandemic approximated current daily fluid intake recommendations. Associations between COVID-19-related disruptions (i.e., suspension of classes and changes in habitation) and increased alcohol intake are concerning and may suggest the need for the development of targeted strategies and programming to attenuate the execution of negative health-related behaviors in college students.

Application of the Theory of Planned Behaviour to inform development of a Dissemination and Implementation science training for nutrition practitioners.

OBJECTIVE: To determine nutrition practitioners' attitudes, behavioural control and normative beliefs to best inform the development and formulation of a nutrition-specific Dissemination and Implementation (D&I) science training. DESIGN: A cross-sectional survey aimed to assess Theory of Planned Behaviour (TPB) constructs and intention to use D&I science. A validated TPB questionnaire assessed constructs including perceived behavioural control, subjective, injunctive and descriptive normative beliefs, attitudes and intention to use D&I science. For analysis, Spearman's ρ, Kruskal-Wallis and Steel-Dwass tests were conducted for quantitative variables. SETTING: Online, 26-item Qualtrics survey. PARTICIPANTS: Cross-sectional sample of members (n 70) affiliated with the Society for Nutrition Education and Behaviour listserv. RESULTS: The major finding from this study was a significant positive correlation between perceived behavioural control score and intention (r = 0·315, P = 0·0119). CONCLUSIONS: D&I training interventions could formulate learning and teaching strategies to target perceived behavioural control (self-efficacy, knowledge and ability) to enhance intention. For example, application and experience-based learning techniques trainings could be strategies to increase knowledge and abilities.

Prospective Observational Cohort Study of Tenecteplase Versus Alteplase in Routine Clinical Practice.

BACKGROUND: A 10-hospital regional network transitioned to tenecteplase as the standard of care stroke thrombolytic in September 2019 because of potential workflow advantages and reported noninferior clinical outcomes relative to alteplase in meta-analyses of randomized trials. We assessed whether tenecteplase use in routine clinical practice reduced thrombolytic workflow times with noninferior clinical outcomes. METHODS: We designed a prospective registry-based observational, sequential cohort comparison of tenecteplase- (n=234) to alteplase-treated (n=354) stroke patients. We hypothesized: (1) an increase in the proportion of patients meeting target times for target door-to-needle time and transfer door-in-door-out time, and (2) noninferior favorable (discharge to home with independent ambulation) and unfavorable (symptomatic intracranial hemorrhage, in-hospital mortality or discharge to hospice) in the tenecteplase group. Total hospital cost associated with each treatment was also compared. RESULTS: Target door-to-needle time within 45 minutes for all patients was superior for tenecteplase, 41% versus 29%; adjusted odds ratio, 1.85 (95% CI, 1.27-2.71); P=0.001; 58% versus 41% by Get With The Guidelines criteria. Target door-in-door-out time within 90 minutes was superior for tenecteplase 37% (15/43) versus 14% (9/65); adjusted odds ratio, 3.62 (95% CI, 1.30-10.74); P=0.02. Favorable outcome for tenecteplase fell within the 6.5% noninferiority margin; adjusted odds ratio, 1.26 (95% CI, 0.89-1.80). Unfavorable outcome was less for tenecteplase, 7.3% versus 11.9%, adjusted odds ratio, 0.77 (95% CI, 0.42-1.37) but did not fall within the prespecified 1% noninferior boundary. Net benefit (%favorable-%unfavorable) was greater for the tenecteplase sample: 37% versus 27%. P=0.02. Median cost per hospital encounter was less for tenecteplase cases ($13 382 versus $15 841; P<0.001). CONCLUSIONS: Switching to tenecteplase in routine clinical practice in a 10-hospital network was associated with shorter door-to-needle time and door-in-door-out times, noninferior favorable clinical outcomes at discharge, and reduced hospital costs. Evaluation in larger, multicenter cohorts is recommended to determine if these observations generalize.

Urea Disrupts the AOT Reverse Micelle Structure at Low Temperatures.

Aside from its prominent role in the excretory system, urea is also a known protein denaturant. Here, we characterize urea as it behaves in confined spaces of AOT (sodium bis(2-ethylhexyl) sulfosuccinate) reverse micelles as a model of tight, confined spaces found at the subcellular level. Dynamic light scattering revealed that low temperatures (275 K) caused the smallest of the reverse micelle sizes, w0 = 10, to destabilize and dramatically increase in apparent hydrodynamic diameter. We attribute this to urea embedded into the surfactant interface as confirmed by 2D 1H-NOESY NMR spectroscopy. This increase in size in turn caused the hydrogen exchange between urea and water within the nanosized reverse micelles to increase as measured by 1D EXSY-NMR. A minimal enlarging effect and no increase in hydrogen exchange were observed when aqueous urea was introduced into w0 = 15 or 20 reverse micelles, suggesting that this effect is unique to particularly small-diameter spaces (∼7 nm).

Hepatitis B and Hepatitis D Viruses: A Comprehensive Update with an Immunological Focus.

Hepatitis B virus (HBV) and hepatitis delta virus (HDV) are highly prevalent viruses estimated to infect approximately 300 million people and 12-72 million people worldwide, respectively. HDV requires the HBV envelope to establish a successful infection. Concurrent infection with HBV and HDV can result in more severe disease outcomes than infection with HBV alone. These viruses can cause significant hepatic disease, including cirrhosis, fulminant hepatitis, and hepatocellular carcinoma, and represent a significant cause of global mortality. Therefore, a thorough understanding of these viruses and the immune response they generate is essential to enhance disease management. This review includes an overview of the HBV and HDV viruses, including life cycle, structure, natural course of infection, and histopathology. A discussion of the interplay between HDV RNA and HBV DNA during chronic infection is also included. It then discusses characteristics of the immune response with a focus on reactions to the antigenic hepatitis B surface antigen, including small, middle, and large surface antigens. This paper also reviews characteristics of the immune response to the hepatitis D antigen (including small and large antigens), the only protein expressed by hepatitis D. Lastly, we conclude with a discussion of recent therapeutic advances pertaining to these viruses.

Microvesicles and exosomes released by amnion epithelial cells under oxidative stress cause inflammatory changes in uterine cells†.

Extracellular vesicles play a crucial role in feto-maternal communication and provide an important paracrine signaling mechanism in pregnancy. We hypothesized that fetal cells-derived exosomes and microvesicles (MVs) under oxidative stress (OS) carry unique cargo and traffic through feto-maternal interface, which cause inflammation in uterine cells associated with parturition. Exosomes and MVs, from primary amnion epithelial cell (AEC) culture media under normal or OS-induced conditions, were isolated by optimized differential centrifugation method followed by characterization for size (nanoparticle tracking analyzer), shape (transmission electron microscopy), and protein markers (western blot and immunofluorescence). Cargo and canonical pathways were identified by mass spectroscopy and ingenuity pathway analysis. Myometrial, decidual, and cervical cells were treated with 1 × 107 control/OS-derived exosomes/MVs. Pro-inflammatory cytokines were measured using a Luminex assay. Statistical significance was determined by paired T-test (P < 0.05). AEC produced cup-shaped exosomes of 90-150 nm and circular MVs of 160-400 nm. CD9, heat shock protein 70, and Nanog were detected in exosomes, whereas OCT-4, human leukocyte antigen G, and calnexin were found in MVs. MVs, but not exosomes, were stained for phosphatidylserine. The protein profiles for control versus OS-derived exosomes and MVs were significantly different. Several inflammatory pathways related to OS were upregulated that were distinct between exosomes and MVs. Both OS-derived exosomes and MVs significantly increased pro-inflammatory cytokines (granulocyte-macrophage colony-stimulating factor, interleukin 6 (IL-6), and IL-8) in maternal cells compared with control (P < 0.05). Our findings suggest that fetal-derived exosomes and MVs under OS exhibited distinct characteristics and a synergistic inflammatory role in uterine cells associated with the initiation of parturition.

Extracellular vesicles from maternal uterine cells exposed to risk factors cause fetal inflammatory response.

BACKGROUND: Fetal cell-derived exosomes (extracellular vesicles, 40-160 nm) are communication channels that can signal parturition by inducing inflammatory changes in maternal decidua and myometrium. Little is known about maternal cell-derived exosomes and their functional roles on the fetal side. This study isolated and characterized exosomes from decidual and myometrial cells grown under normal and inflammatory/oxidative stress conditions and determined their impact on fetal membrane cells. METHODS: Decidual and myometrial cells were grown under standard culture conditions (control) or exposed for 48 h to cigarette smoke extract or tumor necrosis factor-α, as proxies for oxidative stress and inflammation, respectively. Exosomes were isolated from media (differential ultra-centrifugation followed by size exclusion chromatography), quantified (nano particle tracking analysis), and characterized in terms of their size and morphology (cryo-electron microscopy), markers (dot blot), and cargo contents (proteomics followed by bioinformatics analysis). Maternal exosomes (109/mL) were used to treat amnion epithelial cells and chorion trophoblast cells for 24 h. The exosome uptake by fetal cells (confocal microscopy) and the cytokine response (enzyme-linked immunosorbent assays for IL-6, IL-10, and TNF-α) was determined. RESULTS: Exosomes from both decidual and myometrial cells were round and expressed tetraspanins and endosomal sorting complexes required for transport (ESCRT) protein markers. The size and quantity was not different between control and treated cell exosomes. Proteomic analysis identified several common proteins in exosomes, as well as unique proteins based on cell type and treatment. Compared to control exosomes, pro-inflammatory cytokine release was higher in both amnion epithelial cell and chorion trophoblast cell media when the cells had been exposed to exosomes from decidual or myometrial cells treated with either cigarette smoke extract or tumor necrosis factor-α. In chorion trophoblast cells, anti-inflammatory IL-10 was increased by exosomes from both decidual and myometrial cells. CONCLUSION: Various pathophysiological conditions cause maternal exosomes to carry inflammatory mediators that can result in cell type dependent fetal inflammatory response. Video Abstract.

Postoperative delirium is associated with increased plasma neurofilament light.

While delirium is associated with cognitive decline and dementia, there is limited evidence to support causality for this relationship. Clarification of how delirium may cause cognitive decline, perhaps through evidence of contemporaneous neuronal injury, would enhance plausibility for a causal relationship. Dose-dependence of neuronal injury with delirium severity would further enhance the biological plausibility for this relationship. We tested whether delirium is associated with neuronal injury in 114 surgical patients recruited to a prospective biomarker cohort study. Patients underwent perioperative testing for changes in neurofilament light, a neuronal injury biomarker, as well as a panel of 10 cytokines, with contemporaneous assessment of delirium severity and incidence. A subset of patients underwent preoperative MRI. Initially we confirmed prior reports that neurofilament light levels correlated with markers of neurodegeneration [hippocampal volume (ΔR2 = 0.129, P = 0.015)] and white matter changes including fractional anisotropy of white matter (ΔR2 = 0.417, P < 0.001) with similar effects on mean, axial and radial diffusivity) in our cohort and that surgery was associated with increasing neurofilament light from preoperative levels [mean difference (95% confidence interval, CI) = 0.240 (0.178, 0.301) log10 (pg/ml), P < 0.001], suggesting putative neuronal injury. Next, we tested the relationship with delirium. Neurofilament light rose more sharply in participants with delirium compared to non-sufferers [mean difference (95% CI) = 0.251 (0.136, 0.367) log10 (pg/ml), P < 0.001]. This relationship showed dose-dependence, such that neurofilament light rose proportionately to delirium severity (ΔR2 = 0.199, P < 0.001). Given that inflammation is considered an important driver of postoperative delirium, next we tested whether neurofilament light, as a potential marker of neurotoxicity, may contribute to the pathogenesis of delirium independent of inflammation. From a panel of 10 cytokines, the pro-inflammatory cytokine IL-8 exhibited a strong correlation with delirium severity (ΔR2 = 0.208, P < 0.001). Therefore, we tested whether the change in neurofilament light contributed to delirium severity independent of IL-8. Neurofilament light was independently associated with delirium severity after adjusting for the change in inflammation (ΔR2 = 0.040, P = 0.038). These data suggest delirium is associated with exaggerated increases in neurofilament light and that this putative neurotoxicity may contribute to the pathogenesis of delirium itself, independent of changes in inflammation.

The relationship of C and N stable isotopes to high-latitude moss-associated N2 fixation.

Moss-associated N2 fixation by epiphytic microbes is a key biogeochemical process in nutrient-limited high-latitude ecosystems. Abiotic drivers, such as temperature and moisture, and the identity of host mosses are critical sources of variation in N2 fixation rates. An understanding of the potential interaction between these factors is essential for predicting N inputs as moss communities change with the climate. To further understand the drivers and results of N2 fixation rate variation, we obtained natural abundance values of C and N isotopes and an associated rate of N2 fixation with 15N2 gas incubations in 34 moss species collected in three regions across Alaska, USA. We hypothesized that δ15N values would increase toward 0‰ with higher N2 fixation to reflect the increasing contribution of fixed N2 in moss biomass. Second, we hypothesized that δ13C and N2 fixation would be positively related, as enriched δ13C signatures reflect abiotic conditions favorable to N2 fixation. We expected that the magnitude of these relationships would vary among types of host mosses, reflecting differences in anatomy and habitat. We found little support for our first hypothesis, with only a modest positive relationship between N2 fixation rates and δ15N in a structural equation model. We found a significant positive relationship between δ13C and N2 fixation only in Hypnales, where the probability of N2 fixation activity reached 95% when δ13C values exceeded - 30.4‰. We conclude that moisture and temperature interact strongly with host moss identity in determining the extent to which abiotic conditions impact associated N2 fixation rates.

Sodium Hydrogen Exchanger Regulatory Factor-1 (NHERF1) Regulates Fetal Membrane Inflammation.

The fetal inflammatory response, a key contributor of infection-associated preterm birth (PTB), is mediated by nuclear factor kappa B (NF-kB) activation. Na+/H+ exchanger regulatory factor-1 (NHERF1) is an adapter protein that can regulate intracellular signal transduction and thus influence NF-kB activation. Accordingly, NHERF1 has been reported to enhance proinflammatory cytokine release and amplify inflammation in a NF-kB-dependent fashion in different cell types. The objective of this study was to examine the role of NHERF1 in regulating fetal membrane inflammation during PTB. We evaluated the levels of NHERF1 in human fetal membranes from term labor (TL), term not in labor (TNIL), and PTB and in a CD1 mouse model of PTB induced by lipopolysaccharide (LPS). Additionally, primary cultures of fetal membrane cells were treated with LPS, and NHERF1 expression and cytokine production were evaluated. Gene silencing methods using small interfering RNA targeting NHERF1 were used to determine the functional relevance of NHERF1 in primary cultures. NHERF1 expression was significantly (p < 0.001) higher in TL and PTB membranes compared to TNIL membranes, and this coincided with enhanced (p < 0.01) interleukin (IL)-6 and IL-8 expression levels. LPS-treated animals delivering PTB had increased levels of NHERF1, IL-6, and IL-8 compared to phosphate-buffered saline (PBS; control) animals. Silencing of NHERF1 expression resulted in a significant reduction in NF-kB activation and IL-6 and IL-8 production as well as increased IL-10 production. In conclusion, downregulation of NHERF1 increased anti-inflammatory IL-10, and reducing NHERF1 expression could be a potential therapeutic strategy to reduce the risk of infection/inflammation associated with PTB.

Regional Requirements Influence Adoption of Exertional Heat Illness Preparedness Strategies in United States High Schools.

Background and objectives: Exertional heat stroke (EHS) continues to be a prevalent health issue affecting all athletes, including our pediatric populations. The purpose of this study was to evaluate the effect of a state policy requirement for EHS prevention and treatment on local high school policy adoption in the United States (US). Materials and Methods: Athletic trainers (ATs) from high schools across the US participated in an online survey (n = 365). This survey inquired about their compliance with nine components of an EHS policy which was then compared to their state requirements for the policies. Evaluation of the number of components adopted between states with a requirement versus states without a requirement was conducted with a Wilcoxon Sign Rank test. Finally, an ordinal logistic regression with proportional odds ratios (OR) with 95% confidence intervals (CI) were run to determine the effect of a state requirement and regional differences on the number of components adopted. Results: ATs working in states with a requirement reported adoption of more components in their heat modification policy compared to states that did not require schools to develop a heat modification policy (with requirement mean = 5.34 ± 3.68, median = 7.0; without requirement mean = 4.23 ± 3.59, median = 5.0; Z = -14.88, p < 0.001). ATs working in region 3 (e.g., hotter regions) reported adopting more components than those in region 1 (e.g., cooler regions) (OR = 2.25, 95% CI: 1.215-4.201, p = 0.010). Conclusions: Our results demonstrate a positive association between state policy requirements and subsequently increased local policy adoption for EHS policies. Additionally, the results demonstrate that regional differences exist, calling for the need for reducing disparities across the US. These findings may imply that policy adoption is a multifactorial process; furthermore, additional regional specific investigations must be conducted to determine the true determinants of high school policy adoption rates for EHS policies.

Emergency Medical Service Directors' Protocols for Exertional Heat Stroke.

Background and Objectives: Emergency Medical Service (EMS) protocols vary widely and may not implement best practices for exertional heat stroke (EHS). EHS is 100% survivable if best practices are implemented within 30 min. The purpose of this study is to compare EMS protocols to best practices for recognizing and treating EHS. Materials and Methods: Individuals (n = 1350) serving as EMS Medical or Physician Director were invited to complete a survey. The questions related to the EHS protocols for their EMS service. 145 individuals completed the survey (response rate = 10.74%). Chi-Squared Tests of Associations (χ2) with 95% confidence intervals (CI) were calculated. Prevalence ratios (PR) with 95% CI were calculated to determine the prevalence of implementing best practices based on location, working with an athletic trainer, number of EHS cases, and years of directing. All PRs whose 95% CIs excluded 1.00 were considered statistically significant; Chi-Squared values with p values < 0.05 were considered statistically significant. Results: A majority of the respondents reported not using rectal thermometry for the diagnosis of EHS (n = 102, 77.93%) and not using cold water immersion for the treatment of EHS (n = 102, 70.34%). If working with an athletic trainer, EMS is more likely to implement best-practice treatment (i.e., cold-water immersion and cool-first transport-second) (69.6% vs. 36.9%, χ2 = 8.480, p < 0.004, PR = 3.15, 95% CI = 1.38, 7.18). Conclusions: These findings demonstrate a lack of implementation of best-practice standards for EHS by EMS. Working with an athletic trainer appears to increase the likelihood of following best practices. Efforts should be made to improve EMS providers' implementation of best-practice standards for the diagnosis and management of EHS to optimize patient outcomes.

Exertional Heat Illness Preparedness Strategies: Environmental Monitoring Policies in United States High Schools.

Background and objectives: Environmental monitoring allows for an analysis of the ambient conditions affecting a physically active person's ability to thermoregulate and can be used to assess exertional heat illness risk. Using public health models such as the precaution adoption process model (PAPM) can help identify individual's readiness to act to adopt environmental monitoring policies for the safety of high school athletes. The purpose of this study was to investigate the adoption of policies and procedures used for monitoring and modifying activity in the heat in United States (US) high schools. Materials and Methods: Using a cross-sectional design, we distributed an online questionnaire to athletic trainers (ATs) working in high schools in the US. The questionnaire was developed based on best practice standards related to environmental monitoring and modification of activity in the heat as outlined in the 2015 National Athletic Trainers' Association Position Statement: Exertional Heat Illness. The PAPM was used to frame questions as it allows for the identification of ATs' readiness to act. PAPM includes eight stages: unaware of the need for the policy, unaware if the school has this policy, unengaged, undecided, decided not to act, decided to act, acting, and maintaining. Invitations were sent via email and social media and resulted in 529 complete responses. Data were aggregated and presented as proportions. Results: Overall, 161 (161/529, 30.4%) ATs report they do not have a written policy and procedure for the prevention and management of exertional heat stroke. The policy component with the highest adoption was modifying the use of protective equipment (acting = 8.2%, maintaining = 77.5%). In addition, 28% of ATs report adoption of all seven components for a comprehensive environmental monitoring policy. Conclusions: These findings indicate a lack of adoption of environmental monitoring policies in US high schools. Secondarily, the PAPM, facilitators and barriers data highlight areas to focus future efforts to enhance adoption.