Protocol for fluorescent live-cell staining of tardigrades.

Tardigrades are microscopic organisms with exceptional resilience to environmental extremes. Most protocols to visualize the internal anatomy of tardigrades rely on fixation, hampering our understanding of dynamic changes to organelles and other subcellular components. Here, we provide protocols for staining live tardigrade adults and other postembryonic stages, facilitating real-time visualization of structures including lipid droplets, mitochondria, lysosomes, and DNA.

Gold Tripyrrindione: Redox Chemistry and Reactivity with Dichloromethane.

The identification of ligands that stabilize Au(III) centers has led to the isolation of complexes for applications in catalysis, gold-based therapeutics, and functional materials. Herein, we report the coordination of gold by tripyrrin-1,14-dione, a linear tripyrrole with the scaffold of naturally occurring metabolites of porphyrin-based protein cofactors (e.g., heme). Tripyrrindione H3TD2 binds Au(III) as a trianionic tridentate ligand to form square planar complex [Au(TD2)(H2O)], which features an adventitious aqua ligand. Two reversible ligand-based oxidations of this complex allow access to the other known redox states of the tripyrrindione framework. Conversely, (spectro)electrochemical measurements and DFT analysis indicate that the reduction of the complex is likely metal-based. The chemical reduction of [Au(TD2)(H2O)] leads to a reactive species that utilizes dichloromethane in the formation of a cyclometalated organo-Au(III) complex. Both the aqua and the organometallic Au(III) complexes were characterized in the solid state by microcrystal electron diffraction (MicroED) methods, which were critical for the analysis of the microcrystalline sample of the organo-gold species. Overall, this study illustrates the synthesis of Au(III) tripyrrindione as well as its redox profile and reactivity leading to gold alkylation chemistry.

Robust identification of environmental exposures and community characteristics predictive of rapid lung disease progression.

Environmental exposures and community characteristics have been linked to accelerated lung function decline in people with cystic fibrosis (CF), but geomarkers, the measurements of these exposures, have not been comprehensively evaluated in a single study. To determine which geomarkers have the greatest predictive potential for lung function decline and pulmonary exacerbation (PEx), a retrospective longitudinal cohort study was performed using novel Bayesian joint covariate selection methods, which were compared with respect to PEx predictive accuracy. Non-stationary Gaussian linear mixed effects models were fitted to data from 151 CF patients aged 6-20 receiving care at a CF Center in the midwestern US (2007-2017). The outcome was forced expiratory volume in 1 s of percent predicted (FEV1pp). Target functions were used to predict PEx from established criteria. Covariates included 11 routinely collected clinical/demographic characteristics and 45 geomarkers comprising 8 categories. Unique covariate selections via four Bayesian penalized regression models (elastic-net, adaptive lasso, ridge, and lasso) were evaluated at both 95 % and 90 % credible intervals (CIs). Resultant models included one to 6 geomarkers (air temperature, percentage of tertiary roads outside urban areas, percentage of impervious nonroad outside urban areas, fine atmospheric particulate matter, fraction achieving high school graduation, and motor vehicle theft) representing weather, impervious descriptor, air pollution, socioeconomic status, and crime categories. Adaptive lasso had the lowest information criteria. For PEx predictive accuracy, covariate selection from the 95 % CI elastic-net had the highest area under the receiver-operating characteristic curve (mean ± standard deviation; 0.780 ± 0.026) along with the 95 % CI ridge and lasso methods (0.780 ± 0.027). The 95 % CI elastic-net had the highest sensitivity (0.773 ± 0.083) while the 95 % CI adaptive lasso had the highest specificity (0.691 ± 0.087), suggesting the need for different geomarker sets depending on monitoring goals. Surveillance of certain geomarkers embedded in prediction algorithms can be used in real-time warning systems for PEx onset.

Localized Evaporative Cooling Explains Observed Ocular Surface-Temperature Patterns.

Purpose: We determined interblink corneal surface-temperature decline and tear-film evaporation rates of localized tear breakup cold regions (LCRs) and localized tear unbroken warm regions (LWRs) of the corneal surface, as well as that of the overall average corneal surface. Methods: Each subject underwent 4 inter-day visits where the interblink corneal surface-temperature history of the right eye was measured using a FLIR A655sc infrared thermographer. Corneal surface temperature history was analyzed to determine the overall, LCR, and LWR temperature-decline rates. Evaporation rates of LCR and LWR regions were determined from the measured LCR and LWR temperature data using the physical model of Dursch et al. Results: Twenty subjects completed the study. Mean (SD) difference of LCR temperature-decline rate was -0.08 (0.07)°C/s faster than LWR (P < 0.0001). Similarly, evaporation rates of LCR and LWR were statistically different (P < 0.0001). At ambient temperature, mean LCR and LWR evaporation rates were 76% and 27% of pure water evaporation flux, respectively. There was no statistically significant difference between the inter-day measured temperature-decline rates and the interblink starting temperature. Conclusions: Significant differences in corneal temperature-decline rate and evaporation rate between LCR and LWR were quantified using infrared thermography. In agreement with literature, LCRs and LWRs correlate directly with fluorescein break-up areas and unbroken tear areas, respectively. Because lipid-evaporation protection is diminished in breakup areas, higher local evaporation rates and faster local cooling rates occur in LCRs relative to LWRs. Our results confirm this phenomenon clinically for the first time.

Using RE-AIM Framework to Evaluate Recovery Opioid Overdose Team Plus: A Peer-Led Post-overdose Quick Response Team.

Peer recovery coaches utilize their lived experiences to support overdose survivors, a role gaining prominence across communities. A convergent mixed methods design, informed by the RE-AIM (Reach, Effectiveness, Adoption, Implementation, Maintenance) framework, was used to evaluate the Recovery Opioid Overdose Team Plus (ROOT +), through an iterative evaluation using web-based surveys and qualitative interviews. Reach: Over 27 months, ROOT + responded to 83% of suspected overdose referrals (n = 607) and engaged with 41% of survivors (n = 217) and 7% of survivors' family/friends (n = 38). Effectiveness: Among those initially engaged with ROOT +, 36% of survivors remained engaged, entered treatment, or were in recovery at 90 days post-overdose (n = 77). Adoption: First responders completed 77% of ROOT + referrals (n = 468). Implementation: Barriers included lack of awareness of ROOT + , working phones, and access to treatment from community partner interviews (n = 15). Maintenance: Adaptations to ROOT + were made to facilitate implementation. Peer-led teams are promising models to engage with overdose survivors.

Ectodermal dysplasia and cholesteatoma: A cross-sectional analysis of otologic issues.

OBJECTIVES: Previous studies have also associated cleft palate with increased cholesteatoma risk. Despite this close relation, the incidence of cholesteatoma and associated otologic issues in patients with ectodermal dysplasia types highly associated with cleft palate (EDT-ACPs) has not been formally analyzed. This study provides insight to guide clinicians caring for patients with ED types associated with cleft palate. METHODS: Individuals with TP63 disorders and Goltz syndrome/Focal Dermal Hypoplasia in the National Foundation for Ectodermal Dysplasia database were contacted for participation in an online REDCap survey from Sept-Dec '22. Descriptive statistics were generated using SAS JMP Pro 17 statistical software. RESULTS: 65 individuals participated in the survey (response rate approx. 18 %). The median [IQR] age was 22 [14, 43], 41 (63 %) were female, and Ectrodactyly-Ectodermal Dysplasia-Cleft Lip/Palate Syndrome (EEC) was most common (n = 26, 40 %). We found that, among our respondents with a history of cleft palate, the incidence of cholesteatoma was 39 %. Among respondents without a history of cleft palate, the incidence of cholesteatoma was 13 % CONCLUSIONS: Otologic issues, such as cholesteatoma, can have permanent implications including hearing loss that can be minimized by early identification and treatment. The estimated incidence of cholesteatoma among our participants is far above the estimated incidence of cholesteatoma in the general population with and without a history of cleft palate, suggesting an independent contribution of EDT-ACPs to the risk of cholesteatoma.

Polyploidy and environmental stress response: a comparative study of fern gametophytes.

Climate change is rapidly altering natural habitats and generating complex patterns of environmental stress. Ferns are major components of many forest understories and, given their independent gametophyte generation, may experience unique pressures in emerging temperature and drought regimes. Polyploidy is widespread in ferns and may provide a selective advantage in these rapidly changing environments. This work aimed to understand whether the gametophytes of allopolyploid ferns respond differently to climate-related physiological stress than their diploid parents. The experimental approach involved a multifactorial design with 27 treatment combinations including exposure to multiple levels of drought and temperature over three treatment durations, with recovery measured at multiple timepoints. We measured Chl fluorescence from over 2000 gametophytes to evaluate stress avoidance and tolerance in diploid and polyploid species. Polyploids generally showed a greater ability to avoid and/or tolerate a range of stress conditions compared with their diploid counterparts, suggesting that polyploidy may confer enhanced flexibility and resilience under climate stress. Overall, these results suggest that polyploidy may provide some resilience to climate change in mixed ploidy populations. However, all species remain susceptible to the impacts of extreme drought and heat stress.

ArtSpective™ for Perinatal Substance Use: Feasibility of an Arts-Based Intervention Addressing Stigma.

OBJECTIVE: The purpose of this proof-of-concept pilot study was to test the initial feasibility and limited efficacy of ArtSpective™ for perinatal substance use (PSU), a novel, arts-based intervention designed to decrease stigmatizing attitudes toward PSU. METHODS: Using a pre-post mixed methods quasi-experimental design, ArtSpective™ for PSU was pilot tested for proof of concept among a convenience sample of 11 undergraduate and graduate students with experience in maternal-infant nursing from a Midwestern U.S. nursing school. As a proof-of-concept pilot study, we evaluated feasibility (acceptability, demand, and implementation) and limited efficacy. Participants completed presurveys and postsurveys that included satisfaction items, demographic items, and an adapted version of the Attitudes About Drug Use in Pregnancy Scale and participated in a focus group. Focus group data were analyzed using constant comparative methods, and survey data were analyzed using descriptive statistics and Wilcoxon signed rank tests. RESULTS: Participants reported high demand and satisfaction with the intervention and provided recommendations to improve scalability. ArtSpective™ demonstrated significant improvement in stigma toward PSU (pre vs. post: p = .003; d = .633). CONCLUSION: ArtSpective™ for PSU demonstrated initial feasibility and limited efficacy for improving nurse attitudes toward PSU. Efforts are needed to further test this novel intervention, adapt it to augment existing educational interventions, and improve its scalability.

Interferon signaling in the nasal epithelium distinguishes among lethal and common cold coronaviruses and mediates viral clearance.

All respiratory viruses establish primary infections in the nasal epithelium, where efficient innate immune induction may prevent dissemination to the lower airway and thus minimize pathogenesis. Human coronaviruses (HCoVs) cause a range of pathologies, but the host and viral determinants of disease during common cold versus lethal HCoV infections are poorly understood. We model the initial site of infection using primary nasal epithelial cells cultured at an air-liquid interface (ALI). HCoV-229E, HCoV-NL63, and human rhinovirus-16 are common cold-associated viruses that exhibit unique features in this model: early induction of antiviral interferon (IFN) signaling, IFN-mediated viral clearance, and preferential replication at nasal airway temperature (33 °C) which confers muted host IFN responses. In contrast, lethal SARS-CoV-2 and MERS-CoV encode antagonist proteins that prevent IFN-mediated clearance in nasal cultures. Our study identifies features shared among common cold-associated viruses, highlighting nasal innate immune responses as predictive of infection outcomes and nasally directed IFNs as potential therapeutics.

Subjective versus Psychophysical Measures of Chemosensory Alterations following COVID-19.

INTRODUCTION: Olfactory dysfunction is a common symptom of COVID-19. However, subjective perception of olfactory function does not always correlate well with more objective measures. This study seeks to clarify associations between subjective and psychophysical measures of olfaction and gustation in patients with subjective chemosensory dysfunction following COVID-19. METHODS: Adults with persistent COVID-19-associated chemosensory disturbance were recruited for a prospective, longitudinal cohort study at a tertiary care institution. Participants provided subjective measures of olfactory and gustatory function and underwent psychophysical assessment using Sniffin' Sticks olfactory and Monell gustatory tests. RESULTS: Data analysis (n = 65) showed a statistically significant association between subjective and psychophysical measures of olfaction (p < 0.001). For each one-point increase in subjectively-reported olfactory ability, there is, on average, a 0.11 (95% CI: 0.06, 0.16; p < 0.001) point increase in TDI score while adjusting for age at baseline assessment, sex, and follow-up time. For each one-point increase in subjectively-reported olfactory ability, there is, on average, a 0.04 (95% CI: 0.02, 0.06; p < 0.001) point and 0.05 (95% CI: 0.03, 0.07; p < 0.001) point increase in discrimination and identification scores, respectively, when adjusting for age at baseline assessment, sex, and follow-up time. CONCLUSION: Subjective olfaction shows a mild to moderate association with psychophysical measures, but it fails to comprehensively assess persistent COVID-19-associated chemosensory deficits. The lack of significant association between subjective olfaction and threshold limits the utility of subjective olfaction in tracking recovery. These findings support the push for more widespread psychophysical chemosensory testing.

SARS-CoV-2 nsp15 endoribonuclease antagonizes dsRNA-induced antiviral signaling.

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 has caused millions of deaths since its emergence in 2019. Innate immune antagonism by lethal CoVs such as SARS-CoV-2 is crucial for optimal replication and pathogenesis. The conserved nonstructural protein 15 (nsp15) endoribonuclease (EndoU) limits activation of double-stranded (ds)RNA-induced pathways, including interferon (IFN) signaling, protein kinase R (PKR), and oligoadenylate synthetase/ribonuclease L (OAS/RNase L) during diverse CoV infections including murine coronavirus and Middle East respiratory syndrome (MERS)-CoV. To determine how nsp15 functions during SARS-CoV-2 infection, we constructed a recombinant SARS-CoV-2 (nsp15mut) expressing catalytically inactivated nsp15, which we show promoted increased dsRNA accumulation. Infection with SARS-CoV-2 nsp15mut led to increased activation of the IFN signaling and PKR pathways in lung-derived epithelial cell lines and primary nasal epithelial air-liquid interface (ALI) cultures as well as significant attenuation of replication in ALI cultures compared to wild-type virus. This replication defect was rescued when IFN signaling was inhibited with the Janus activated kinase (JAK) inhibitor ruxolitinib. Finally, to assess nsp15 function in the context of minimal (MERS-CoV) or moderate (SARS-CoV-2) innate immune induction, we compared infections with SARS-CoV-2 nsp15mut and previously described MERS-CoV nsp15 mutants. Inactivation of nsp15 had a more dramatic impact on MERS-CoV replication than SARS-CoV-2 in both Calu3 cells and nasal ALI cultures suggesting that SARS-CoV-2 can better tolerate innate immune responses. Taken together, SARS-CoV-2 nsp15 is a potent inhibitor of dsRNA-induced innate immune response and its antagonism of IFN signaling is necessary for optimal viral replication in primary nasal ALI cultures.

Defining Populations and Predicting Future Suitable Niche Space in the Geographically Disjunct, Narrowly Endemic Leafy Prairie-Clover (Dalea foliosa; Fabaceae).

Conservation actions for rare species are often based on estimates of population size and number, which are challenging to capture in natural systems. Instead, many definitions of populations rely on arbitrarily defined distances between occurrences, which is not necessarily biologically meaningful despite having utility from a conservation management perspective. Here, we introduce a case study using the narrowly endemic and highly geographically disjunct leafy prairie-clover (Dalea foliosa), for which we use nuclear microsatellite loci to assess the current delimitations of populations and management units across its entire known range. We model future potential suitable niche space for the species to assess how currently defined populations could fare under predicted changes in climate over the next 50 years. Our results indicate that genetic variation within the species is extremely limited, particularly so in the distal portions of its range (Illinois and Alabama). Within the core of its range (Tennessee), genetic structure is not consistent with populations as currently defined. Our models indicate that predicted suitable niche space may only marginally overlap with the geology associated with this species (limestone glades and dolomite prairies) by 2070. Additional studies are needed to evaluate the extent to which populations are ecologically adapted to local environments and what role this could play in future translocation efforts.

Colloidal Stability of PFSA-Ionomer Dispersions. Part I. Single-Ion Electrostatic Interaction Potential Energies.

Charged colloidal particles neutralized by a single counterion are increasingly important for many emerging technologies. Attention here is paid specifically to hydrogen fuel cells and water electrolyzers whose catalyst layers are manufactured from a perfluorinated sulfonic acid polymer (PFSA) suspended in aqueous/alcohol solutions. Partially dissolved PFSA aggregates, known collectively as ionomers, are stabilized by the electrostatic repulsion of overlapping diffuse double layers consisting of only protons dissociated from the suspended polymer. We denote such double layers containing no added electrolyte as "single ion". Size-distribution predictions build upon interparticle interaction potential energies from the Derjaguin-Landau-Verwey-Overbeek (DLVO) formalism. However, when only a single counterion is present in solution, classical DLVO electrostatic potential energies no longer apply. Accordingly, here a new formulation is proposed to describe how single-counterion diffuse double layers interact in colloidal suspensions. Part II (Srivastav, H.; Weber, A. Z.; Radke, C. J. Langmuir 2024 DOI: 10.1021/acs.langmuir.3c03904) of this contribution uses the new single-ion interaction energies to predict aggregated size distributions and the resulting solution pH of PFSA in mixtures of n-propanol and water. A single-counterion diffuse layer cannot reach an electrically neutral concentration far from a charged particle. Consequently, nowhere in the dispersion is the solvent neutral, and the diffuse layer emanating from one particle always experiences the presence of other particles (or walls). Thus, in addition to an intervening interparticle repulsive force, a backside osmotic force is always present. With this new construction, we establish that single-ion repulsive pair interaction energies are much larger than those of classical DLVO electrostatic potentials. The proposed single-ion electrostatic pair potential governs dramatic new dispersion behavior, including dispersions that are stable at a low volume fraction but unstable at a high volume fraction and finite volume-fraction dispersions that are unstable with fine particles but stable with coarse particles. The proposed single-counterion electrostatic pair potential provides a general expression for predicting colloidal behavior for any charged particle dispersion in ionizing solvents with no added electrolyte.

Colloidal Stability of PFSA-Ionomer Dispersions Part II: Determination of Suspension pH Using Single-Ion Potential Energies.

Perfluorosulfonic acid (PFSA) ionomers serve a vital role in the performance and stability of fuel-cell catalyst layers. These properties, in turn, depend on the colloidal processing of precursor inks. To understand the colloidal structure of fuel-cell catalyst layers, we explore the aggregation of PFSA ionomers dissolved in water/alcohol solutions and relate the predicted aggregation to experimental measurements of solution pH. Not all side chains contribute to measured pH because of burying inside particle aggregates. To account for the measured degree of dissociation, a new description is developed for how PFSA aggregates interact with each other. The developed single-counterion electrostatic repulsive pair potential from Part I is incorporated into the Smoluchowski collision-based kinetics of interacting aggregates with buried side chains. We demonstrate that the surrounding solvent mixture affects the degree of aggregation as well as the pH of the system primarily through the solution dielectric permittivity, which drives the strength of the interparticle repulsive energies. Successful pH prediction of Nafion ionomer dispersions in water/n-propanol solutions validates the numerical calculations. Nafion-dispersion pH measurements serve as a surrogate for Nafion particle-size distributions. The model and framework can be leveraged to explore different ink formulations.

Maternal blood pressure and birth weight associations in U.S.-born and foreign-born Latinas.

OBJECTIVES: Research suggests that acculturating to the United States is detrimental for immigrants' health. Consistent with this pattern, higher levels of U.S. acculturation among Latina-American women have been associated with giving birth to lower birth weight babies. The mechanisms that shape this shift in pregnancy health are not clear, but researchers have begun to consider the role of physiological systems that are sensitive to social experience. The present study examined the association of cultural orientation with blood pressure (BP) trajectories over the course of pregnancy. METHOD: In a study of 1,011 U.S.- and foreign-born Latina-American women, cultural orientation was assessed and multiple BP measures were collected throughout pregnancy. Postpregnancy data, including gestational age-adjusted birth weight, were extracted from medical records. Bayesian structural equation models examined average BP and slopes of BP change during pregnancy while accounting for psychosocial stress, support, and pregnancy health-related factors (e.g., maternal age, smoking). RESULTS: We found evidence that greater U.S. orientation was associated with higher diastolic blood pressure (DBP) and steeper increases in DBP, which was associated with less fetal growth. CONCLUSIONS: This is the first evidence that BP may mediate the association between cultural orientation and pregnancy outcomes in Latina-American women. These findings advance our understanding of the biopsychosocial pathways through which acculturation to the U.S. links with health. As scholars seek to better understand the influence of U.S. acculturation on health, focusing on the cardiovascular system and other physiological systems that are sensitive to social experience is warranted and likely to prove valuable. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Pediatric Multidrug-Resistant Disseminated Tuberculosis Presenting as Small Finger Tuberculous Osteomyelitis: A Case Report.

CASE: We report a case in the United States of a 12-year-old girl with multidrug-resistant tuberculous (MDR-TB) osteomyelitis of the hand managed with surgical debridement and second-line anti-TB therapy. The disease course was complicated by dissemination and multifocal progression. CONCLUSION: Despite early intervention, multidrug resistance makes TB treatment challenging and facilitated progression to disseminated disease in this case. We review the difficulties in diagnosis and treatment of pediatric MDR-TB.

Current Medicare reimbursement for complex endovascular aortic repair is inadequate based on results from a multi-institutional cost analysis.

OBJECTIVE: Complex endovascular juxta-, para- and suprarenal abdominal aortic aneurysm repair (comEVAR) is frequently accomplished with commercially available fenestrated (FEVAR) devices or off-label use of aortoiliac devices with parallel branch stents (chEVAR). We sought to evaluate the implantable vascular device costs incurred with these procedures as compared with standard Medicare reimbursement to determine the financial viability of comEVAR in the modern era. METHODS: Five geographically distinct institutions with high-volume, complex aortic centers were included. Implantable aortoiliac and branch stent device cost data from 25 consecutive, recent, comEVAR in the treatment of juxta-, para-, and suprarenal aortic aneurysms at each center were analyzed. Cases of rupture, thoracic aneurysms, reinterventions, and physician-modified EVAR were excluded, as were ancillary costs from nonimplantable equipment. Data from all institutions were combined and stratified into an overall cost group and two, individual cost groups: FEVAR or chEVAR. These groups were compared, and each respective group was then compared with weighted Medicare reimbursement for Diagnosis-Related Group codes 268/269. Median device costs were obtained from an independent purchasing consortium of >3000 medical centers, yielding true median cost-to-institution data rather than speculative, administrative projections or estimates. RESULTS: A total of 125 cases were analyzed: 70 FEVAR and 53 chEVAR. Two cases of combined FEVAR/chEVAR were included in total cost analysis, but excluded from direct FEVAR vs chEVAR comparison. Median Medicare reimbursement was calculated as $35,755 per case. Combined average implantable device cost for all analyzed cases was $28,470 per case, or 80% of the median reimbursement ($28,470/$35,755). Average FEVAR device cost per case ($26,499) was significantly lower than average chEVAR cost per case ($32,122; P < .002). Device cost was 74% ($26,499/$35,755) of total reimbursement for FEVAR and 90% ($32,122/$35,755) for chEVAR. CONCLUSIONS: Results from this multi-institutional analysis show that implantable device cost alone represents the vast majority of weighted total Medicare reimbursement per case with comEVAR, and that chEVAR is significantly more costly than FEVAR. Inadequate Medicare reimbursement for these cases puts high-volume, high-complexity aortic centers at a distinct financial disadvantage. In the interest of optimizing patient care, these data suggest a reconsideration of previously established, outdated, Diagnosis-Related Group coding and Medicare reimbursement for comEVAR.