Fluorescence-Based Proteoliposome Methods to Monitor Redox-Active Transition Metal Transmembrane Translocation by Metal Transporters.

Transmembrane transition metal transporter proteins are central gatekeepers in selectively controlling vectorial metal cargo uptake and extrusion across cellular membranes in all living organisms, thus playing key roles in essential and toxic metal homeostasis. Biochemical characterization of transporter-mediated translocation events and transport kinetics of redox-active metals, such as iron and copper, is challenged by the complexity in generating reconstituted systems in which vectorial metal transport can be studied in real time. We present fluorescence-based proteoliposome methods to monitor redox-active metal transmembrane translocation upon reconstitution of purified metal transporters in artificial lipid bilayers. By encapsulating turn-on/-off iron or copper-dependent sensors in the proteoliposome lumen and conducting real-time transport assays using small unilamellar vesicles (SUVs), in which selected purified Fe(II) and Cu(I) transmembrane importer and exporter proteins have been reconstituted, we provide a platform to monitor metal translocation events across lipid bilayers in real time. The strategy is modular and expandable toward the study of different transporter families featuring diverse metal substrate selectivity and promiscuity.

County-level political group density, partisan polarization, and individual-level mortality among adults in the United States: A lagged multilevel study.

Objective: To investigate the associations between county-level political group density, partisan polarization, and individual-level mortality from all causes and from coronary heart disease (CHD) in the United States. Methods: Using data from five survey waves (1998-2006) of the General Social Survey-National Death Index dataset and the County Presidential Election Return 2000 dataset, we fit weighted Cox proportional hazards models to estimate the associations between (1) political group density and (2) partisan polarization measured at the county level in 2000 (n = 313 counties) categorized into quartiles with individual-level mortality (n = 14,983 participants) from all causes and CHD, controlling for individual- and county-level factors. Maximum follow-up was from one year after the survey up until 2014. We conducted these analyses using two separate measures based on county-level vote share differences and party affiliation ideological extremes. Results: In the overall sample, we found no evidence of associations between county-level political group density and individual-level mortality from all causes. There was evidence of a 13% higher risk of dying from heart disease in the highest quartile of county-level polarization (hazards ratio, HR = 1.13; 95% CI = 0.74-1.71). We observed heterogeneity of effects based on individual-level political affiliation. Among those identifying as Democrats, residing in counties with high (vs. low) levels of polarization appeared to be protective against mortality, with an associated 18% lower risk of dying from all causes (HR = 0.82, 95% CI = 0.71-0.94). This association was strongest in areas with the highest concentrations of Democrats. Conclusions: Among all study participants, political group density and polarization at the county level in 2000 were not linked to individual-level mortality. At the same time, we found that Democratic party affiliation may be protective against the adverse effects of high polarization, particularly in counties with high concentrations of Democrats. Future research should further explore these associations to potentially identify new structural interventions to address political determinants of population health.

Implementing Integrated Early Childhood Mental Health Services in Primary Care: Relationships, Vision, and Sustainability.

The Massachusetts Multi-City Young Children's System of Care Project was a federally funded program to provide integrated early childhood mental health (ECMH) services in primary care for families of very young children (birth-six years old) with Serious Emotional Disturbances across three cities in Massachusetts, U.S.A. This study describes lessons learned from the implementation of this program and makes recommendations for best practices to improve the delivery and efficacy of ECMH services in primary care settings. Staff and leadership (n = 35) from 11 agencies (primary care practices, community service agencies, and local health departments) that co-implemented this program participated in focus groups and semi-structured key informant interviews. Thematic analysis was used to characterize specific facilitators and barriers to successfully implementing system-wide programming for ECMH. Four main themes were identified: (1) Strong multilevel working relationships are critical for integration, (2) Capacity-building activities can be leveraged to improve implementation, (3) Financial challenges are a primary barrier to building efficacious systems of care, and (4) Flexibility and resourcefulness can help overcome logistical challenges in integration. Implementation lessons learned may serve as guidance for other states and institutions in the U.S. seeking to improve the integration of ECMH services into primary care. They may also provide strategies to adapt and scale these interventions to improve the mental health and well-being of young children and their families.

Plastic recognition and electrogenic uniport translocation of 1st-, 2nd-, and 3rd-row transition and post-transition metals by primary-active transmembrane P1B-2-type ATPase pumps.

Transmembrane P1B-type ATPase pumps catalyze the extrusion of transition metal ions across cellular lipid membranes to maintain essential cellular metal homeostasis and detoxify toxic metals. Zn(ii)-pumps of the P1B-2-type subclass, in addition to Zn2+, select diverse metals (Pb2+, Cd2+ and Hg2+) at their transmembrane binding site and feature promiscuous metal-dependent ATP hydrolysis in the presence of these metals. Yet, a comprehensive understanding of the transport of these metals, their relative translocation rates, and transport mechanism remain elusive. We developed a platform for the characterization of primary-active Zn(ii)-pumps in proteoliposomes to study metal selectivity, translocation events and transport mechanism in real-time, employing a "multi-probe" approach with fluorescent sensors responsive to diverse stimuli (metals, pH and membrane potential). Together with atomic-resolution investigation of cargo selection by X-ray absorption spectroscopy (XAS), we demonstrate that Zn(ii)-pumps are electrogenic uniporters that preserve the transport mechanism with 1st-, 2nd- and 3rd-row transition metal substrates. Promiscuous coordination plasticity, guarantees diverse, yet defined, cargo selectivity coupled to their translocation.

Alignment of state-level policies and public attitudes towards abortion legality and government restrictions on abortion in the United States.

BACKGROUND: Contextual factors can shape public opinion towards abortion. We investigated the association between the state-level abortion legislative climate and individual attitudes towards abortion legality and government restrictions of abortion access in the United States. METHODS: Data come from the 2020 Cooperative Congressional Election Study (n = 61,000). Using multivariable logistic regression with generalized estimating equations, we explored whether state-level abortion policy climates (based on the Guttmacher Institute's 2020 rating of state abortion policies) were associated with individual attitudes (1) towards abortion legality, and (2) towards government restriction of abortion access, controlling for individual socio-demographic factors. RESULTS: Eighty-eight percent of participants supported the legality of abortion in some or all circumstances. Conversely, 30% of the sample opposed all federal government restrictions on abortion. More than 60% of the sample lived in highly abortion-restrictive states. Participants living in states with a more supportive abortion legislative climate were more likely to support the legality of abortion in some or all circumstances (AOR = 1.07, (95% CI 1.05, 1.09). Participants in states with more supportive abortion policies were more likely to oppose federal governmental restrictions (AOR = 1.03, 95% CI 1.02, 1.04). Low religiosity, higher educational attainment, and politically liberal views were associated with increased support for abortion legality and increased opposition to government restrictions on abortion. CONCLUSIONS: State-level abortion policy contexts were positively associated with public attitudes towards abortion. While attitudes towards abortion legality are favorable across the country; there is also strong support at least one type of government restriction on abortion access. Results highlight a disconnect between multifaceted public attitudes towards abortion and polarized state contexts, suggesting that policymaking on abortion represents a higher level of polarization than exists at the individual level. Policymakers and legislators should more carefully consider the desires of the public when designing abortion legislation.

PcoB is a defense outer membrane protein that facilitates cellular uptake of copper.

Copper (Cu) is one of the most abundant trace metals in all organisms, involved in a plethora of cellular processes. Yet elevated concentrations of the element are harmful, and interestingly prokaryotes are more sensitive for environmental Cu stress than humans. Various transport systems are present to maintain intracellular Cu homeostasis, including the prokaryotic plasmid-encoded multiprotein pco operon, which is generally assigned as a defense mechanism against elevated Cu concentrations. Here we structurally and functionally characterize the outer membrane component of the Pco system, PcoB, recovering a 2.0 Å structure, revealing a classical ß-barrel architecture. Unexpectedly, we identify a large opening on the extracellular side, linked to a considerably electronegative funnel that becomes narrower towards the periplasm, defining an ion-conducting pathway as also supported by metal binding quantification via inductively coupled plasma mass spectrometry and molecular dynamics (MD) simulations. However, the structure is partially obstructed towards the periplasmic side, and yet flux is permitted in the presence of a Cu gradient as shown by functional characterization in vitro. Complementary in vivo experiments demonstrate that isolated PcoB confers increased sensitivity towards Cu. Aggregated, our findings indicate that PcoB serves to permit Cu import. Thus, it is possible the Pco system physiologically accumulates Cu in the periplasm as a part of an unorthodox defense mechanism against metal stress. These results point to a previously unrecognized principle of maintaining Cu homeostasis and may as such also assist in the understanding and in efforts towards combatting bacterial infections of Pco-harboring pathogens.

Assessing plasma circulating tumor human papillomavirus (HPV) DNA in determining treatment response in HPV-associated oropharyngeal cancer.

BACKGROUND: Human papillomavirus (HPV)-mediated oropharyngeal squamous cell carcinoma is a subset of head and neck cancer with a unique mechanism of carcinogenesis. Local disease is treated definitively with a multimodal approach. Navigating recurrences can be challenging, as they are sometimes indiscernible from de novo primary malignancies. Identification of dynamic biomarkers that are specific to HPV-mediated disease may assist in disease monitoring. We present a 78-year-old man who developed a squamous cell carcinoma in the lung 7 years after completing definitive chemoradiation for his p16+ head and neck squamous cell carcinoma. METHODS: A novel assay for plasma circulating tumor HPV DNA was employed and provided a tool for longitudinal disease monitoring during therapy. CONCLUSION: We bring attention to a novel assay and highlight its potential for use in the treatment paradigm of HPV-mediated oropharyngeal carcinoma.

Predictors of Service Utilization of Young Children and Families Enrolled in a Pediatric Primary Care Mental Health Promotion and Prevention Program.

Understanding early childhood mental health service utilization in community-based clinical settings is important. Project Linking Actions for Unmet Needs in Children's Health (Project LAUNCH) provided mental health-related services for young children and families within pediatric medical homes. Using data from the Project LAUNCH evaluation (n = 106), we implemented negative binomial regression models to determine if baseline variables were associated with service utilization, defined as the number of encounters between the family and the team. Past-year homelessness emerged as a significant predictor of service utilization. Encounters for families with children who experienced homelessness within the last 12 months occurred at a rate 34.5% lower than those who had not experienced homelessness. Results highlight the importance of addressing homelessness as a barrier to mental health service utilization for families. Screening for recent housing insecurity and developing interventions that integrate housing support services into mental health programs may inform strategies to increase attendance for families with young children.

Engaging and Supporting Young Children and their Families in Early Childhood Mental Health Services: The Role of the Family Partner.

This study explores the role of family partners, peer professionals with lived experiences of raising a child with behavioral health needs, and their value in primary and community-care based mental health services for young children aged 0-8 years. Interviews and focus groups were conducted with staff, leadership, and caregiver participants (n = 38) from two early childhood mental health programs and analyzed using thematic analysis. Five interdependent themes emerged: (1) the centrality of lived experience to the family partner role; (2) the importance of the family partner in family engagement and relationship building; (3) the value added by the family partner in navigating systems; (4) the ability of the family partner to build skills and empower caregivers; (5) the role of the family partner in alleviating caregiver stress and other mental health concerns. Adapting and expanding the role of family partners will improve effective mental health care for children and their caregivers.

Splenic Artery Embolization in Subcapsular Splenic Hematoma Secondary to Dengue Hemorrhagic Fever.

Dengue hemorrhagic fever (DHF) is a common syndrome of dengue viral infection but complications such as sub-capsular splenic hematoma leading to capsular rupture in dengue are rare. We report a case of a young male who presented with fever, breathlessness, and acute abdomen. His CT of the abdomen revealed subcapsular splenic hematoma measuring 16.7 cm × 13.0 cm × 11 cm. His laboratory parameters were suggestive of anemia, thrombocytopenia, acute kidney injury, coagulopathy, and hepatopathy because of which instead of splenectomy, splenic artery embolization with ultrasound-guided splenic hemorrhage drainage was performed for his management as his clinical condition deteriorated. This case report sensitizes newer modalities of treatment of subcapsular splenic hematoma with splenic arterial embolization.

Is divisive politics making Americans sick? Associations of perceived partisan polarization with physical and mental health outcomes among adults in the United States.

OBJECTIVES: To investigate whether changes in perceived partisan polarization since the 2016 US presidential election and current perceptions of polarization are associated with the onset of physical and mental health conditions in adults. METHODS: We surveyed a nationally-representative sample (n = 2752) of US adults between December 2019 and January 2020. We used multivariable logistic regression to estimate associations between perceived polarization and the incidence of hypertension, high cholesterol, obesity, diabetes, and anxiety, depressive, and sleep disorders in or after 2016 and current self-rated health. Our secondary exposure variables measured perceptions of mass and elite polarization at the state and national level. Perceived mass polarization measured perceptions of the partisan gap between Democrat and Republican voters; perceived elite polarization measured perceptions of the partisan gap between Democrat and Republican elected officials. RESULTS: Participants reporting an increase in polarization had 52-57% higher odds of developing depressive disorders (OR = 1.52, 95% CI: 1.01, 2.29, P = 0.047) and anxiety disorders (OR = 1.57, 95% CI: 1.07, 2.29, P = 0.02) compared to participants who perceived no change in polarization. Those reporting high (vs. low) levels of perceived state-level mass polarization had a 49% higher odds of incident depressive disorders (P = 0.03). Participants who perceived high levels of state-level elite polarization reported a 71% higher odds of incident depressive disorders (P = 0.004) and a 49% higher odds of incident sleep disorders (P = 0.03). CONCLUSIONS: Perceptions of partisan polarization may represent important factors that are linked to the onset of mental health and sleep disorders.

A single point mutation converts a proton-pumping rhodopsin into a red-shifted, turn-on fluorescent sensor for chloride.

The visualization of chloride in living cells with fluorescent sensors is linked to our ability to design hosts that can overcome the energetic penalty of desolvation to bind chloride in water. Fluorescent proteins can be used as biological supramolecular hosts to address this fundamental challenge. Here, we showcase the power of protein engineering to convert the fluorescent proton-pumping rhodopsin GR from Gloeobacter violaceus into GR1, a red-shifted, turn-on fluorescent sensor for chloride in detergent micelles and in live Escherichia coli. This non-natural function was unlocked by mutating D121, which serves as the counterion to the protonated retinylidene Schiff base chromophore. Substitution from aspartate to valine at this position (D121V) creates a binding site for chloride. The binding of chloride tunes the pK a of the chromophore towards the protonated, fluorescent state to generate a pH-dependent response. Moreover, ion pumping assays combined with bulk fluorescence and single-cell fluorescence microscopy experiments with E. coli, expressing a GR1 fusion with a cyan fluorescent protein, show that GR1 does not pump ions nor sense membrane potential but instead provides a reversible, ratiometric readout of changes in extracellular chloride at the membrane. This discovery sets the stage to use natural and laboratory-guided evolution to build a family of rhodopsin-based fluorescent chloride sensors with improved properties for cellular applications and learn how proteins can evolve and adapt to bind anions in water.

Stabilization of supramolecular membrane protein-lipid bilayer assemblies through immobilization in a crystalline exoskeleton.

Artificial native-like lipid bilayer systems constructed from phospholipids assembling into unilamellar liposomes allow the reconstitution of detergent-solubilized transmembrane proteins into supramolecular lipid-protein assemblies called proteoliposomes, which mimic cellular membranes. Stabilization of these complexes remains challenging because of their chemical composition, the hydrophobicity and structural instability of membrane proteins, and the lability of interactions between protein, detergent, and lipids within micelles and lipid bilayers. In this work we demonstrate that metastable lipid, protein-detergent, and protein-lipid supramolecular complexes can be successfully generated and immobilized within zeolitic-imidazole framework (ZIF) to enhance their stability against chemical and physical stressors. Upon immobilization in ZIF bio-composites, blank liposomes, and model transmembrane metal transporters in detergent micelles or embedded in proteoliposomes resist elevated temperatures, exposure to chemical denaturants, aging, and mechanical stresses. Extensive morphological and functional characterization of the assemblies upon exfoliation reveal that all these complexes encapsulated within the framework maintain their native morphology, structure, and activity, which is otherwise lost rapidly without immobilization.

Autonomous navigation of stratospheric balloons using reinforcement learning.

Efficiently navigating a superpressure balloon in the stratosphere1 requires the integration of a multitude of cues, such as wind speed and solar elevation, and the process is complicated by forecast errors and sparse wind measurements. Coupled with the need to make decisions in real time, these factors rule out the use of conventional control techniques2,3. Here we describe the use of reinforcement learning4,5 to create a high-performing flight controller. Our algorithm uses data augmentation6,7 and a self-correcting design to overcome the key technical challenge of reinforcement learning from imperfect data, which has proved to be a major obstacle to its application to physical systems8. We deployed our controller to station Loon superpressure balloons at multiple locations across the globe, including a 39-day controlled experiment over the Pacific Ocean. Analyses show that the controller outperforms Loon's previous algorithm and is robust to the natural diversity in stratospheric winds. These results demonstrate that reinforcement learning is an effective solution to real-world autonomous control problems in which neither conventional methods nor human intervention suffice, offering clues about what may be needed to create artificially intelligent agents that continuously interact with real, dynamic environments.

Environmental impact of utilization of "produced water" from oil and gas operations in turfgrass systems.

This study attempted to use produced water (PW) to irrigate turfgrass species, Cynodon dactylon and Paspalum sp. Assessment on established grasses, heavy metal accumulation and germination tests for weeds and turf grass seeds were conducted to evaluate the impact of PW irrigation. C. dactylon depicted lower tolerance while Paspalum sp. showed better tolerance capacity towards PW. C. dactylon grown from seeds under greenhouse conditions were not able to tolerate more than 30% concentration of PW (4.5% salinity). In comparison to tap water irrigated turf grass, Paspalum sp. was found to accumulate higher concentrations of V and Pb in shoots and Cr, Ni and As in roots. The results of seed germination tests recommended that irrigation with PW is to be performed after turfgrass establishment. Germination tests also revealed that PW could encourage growth of the weed-Chloris virgata while it could discourage growth of Amaranthus viridis and Launaea mucronata. This study suggests that PW could be used for turfgrass as an alternative water resource but only after further research on the long-term scale.

The Combined Use of SBRT and Immunotherapy-a Literature Review.

PURPOSE OF REVIEW: The purpose of this paper is to review all recent and relevant data regarding the combined use of immunotherapy (particularly immune checkpoint inhibitors) and radiotherapy. RECENT FINDINGS: The use of radiotherapy, specifically stereotactic body radiation therapy (SBRT), and immunotherapy may be synergistic in the treatment of non-small cell lung cancer (NSCLC). There have been many preclinical and clinical studies that have shown that the combination of SBRT and immunotherapy is both safe and effective. In many cases, the benefits are greater than either SBRT or immunotherapy alone. Several ongoing trials are testing the combination of SBRT and immunotherapy for the treatment of all stages of NSCLC. CONCLUSION: The combined use of SBRT and immunotherapy is a promising new development. The techniques may synergistically work better than either alone.

Investigation of Lewis Acid-Carbonyl Solution Interactions via Infrared-Monitored Titration.

Lewis acid-activation of carbonyl-containing substrates is broadly utilized in organic synthesis. In order to facilitate the development of novel reaction pathways and understand existing methods, it is necessary to determine the solution interactions between Lewis acids and Lewis bases. Herein, we report the application of in situ infrared spectroscopy and solution conductivity toward the identification of the solution structures formed when a range of carbonyl compounds are combined with catalytically active metal halide Lewis acids under synthetically relevant conditions. These data are consistent with formation of Lewis acid-dependent complexes, where metals of low relative Lewis acidity display no ground state interaction with carbonyls. Conversely, we observed the formation of polyligated complexes when stronger Lewis acids (SnCl4, TiCl4, ZrCl4, FeCl3, and AlCl3) were treated with ketones, aldehydes, and esters. This collection of observations is intended to assist the synthetic chemist in the design of new catalysts and the development of novel methods.

Management of local recurrences and regional failure in early stage non-small cell lung cancer after stereotactic body radiation therapy.

Stereotactic body radiation therapy (SBRT) is a very effective way to treat early stage non-small cell lung cancer (NSCLC) and small oligometastatic lung lesions with consistently high rates of local control, but both local and regional/distant recurrences still occur. The management of recurrences remains unsettled and may entail repeat SBRT, conventionally fractionated external beam RT (EF-EBRT), chemotherapy or surgery. Most patients with local recurrences [within the initial planning target volume (PTV)] can be salvaged successfully with good cancer specific survival. Nonetheless, proximity of the initial SBRT delivery to organs at risk (ribs, blood vessels, airways) may make retreatment more difficult. With attention to detail and careful patient selection, both surgery and reirradiation can be performed safely and effectively. Strategies for management of regional (nodal) recurrences may require conventional therapies tailored to the patterns of failure. The role of immunotherapy in salvage has not been elucidated as yet. We review here data on the available literature concerning salvage of SBRT lung patients.