Specific protonation of acidic residues confers K+ selectivity to the gastric proton pump.

The gastric proton pump (H+,K+-ATPase) transports a proton into the stomach lumen for every K+ ion exchanged in the opposite direction. In the lumen-facing state of the pump (E2), the pump selectively binds K+ despite the presence of a 10-fold higher concentration of Na+. The molecular basis for the ion selectivity of the pump is unknown. Using molecular dynamics simulations, free energy calculations, and Na+ and K+-dependent ATPase activity assays, we demonstrate that the K+ selectivity of the pump depends upon the simultaneous protonation of the acidic residues E343 and E795 in the ion-binding site. We also show that when E936 is protonated, the pump becomes Na+ sensitive. The protonation-mimetic mutant E936Q exhibits weak Na+-activated ATPase activity. A 2.5-Å resolution cryo-EM structure of the E936Q mutant in the K+-occluded E2-Pi form shows, however, no significant structural difference compared with wildtype except less-than-ideal coordination of K+ in the mutant. The selectivity toward a specific ion correlates with a more rigid and less fluctuating ion-binding site. Despite being exposed to a pH of 1, the fundamental principle driving the K+ ion selectivity of H+,K+-ATPase is similar to that of Na+,K+-ATPase: the ionization states of the acidic residues in the ion-binding sites determine ion selectivity. Unlike the Na+,K+-ATPase, however, protonation of an ion-binding glutamate residue (E936) confers Na+ sensitivity.

Association between spatial access and hospitalization for ambulatory care sensitive conditions: A retrospective cohort study using claims data.

Background: Hospitalization for ambulatory care sensitive conditions (ACSCs) is potentially preventable with timely and effective primary care but may increase owing to poor access. Spatial access inequalities exist between Japan and other countries. This retrospective cohort study examined the association between admission for ACSC and spatial accessibility to primary care. Methods: We used claims data and spatial data of 50-74 years-old beneficiaries of the National Health Insurance program in a large city in Japan from April 2013-March 2014 and followed them until March 2015. We used a multilevel Poisson regression model to assess the association between the number of ACSC admissions, the distance to the nearest clinic, and the number of physicians in a given area, adjusting for age, gender, comorbidities, number of visits, and household income. Results: Among 126,666 eligible beneficiaries (mean age 65.8 years, 54% were women), 1,793 (1.4%) were hospitalized for ACSCs. The ACSC admission rate was significantly higher in those with a distance to the nearest clinic of >1 km than in those with <0.3 km (incident rate ratio [IRR] 1.32, 95% confidence interval [CI] 1.03-1.69). In the stratified analyses, a longer distance to the nearest clinic was associated with higher ACSC admission rates among women (≥0.3 km and <0.5 km: IRR 1.48, 95% CI 1.01-2.17; ≥0.5 km and <1 km: IRR 1.74, 95% CI 1.19-2.56; >1 km: IRR 1.98, 95% CI 1.29-3.03, respectively) and those aged ≥65 years (≥0.3 km and <0.5 km: IRR 1.38, 95% CI 1.07-1.79; ≥0.5 km and <1 km: IRR 1.38, 95% CI 1.06-1.80; >1 km: IRR 1.48, 95% CI 1.10-1.98). Conclusion: Unfavorable spatial access was associated with ACSC admissions, particularly among women and older adults.

A Na pump with reduced stoichiometry is up-regulated by brine shrimp in extreme salinities.

Brine shrimp (Artemia) are the only animals to thrive at sodium concentrations above 4 M. Salt excretion is powered by the Na+,K+-ATPase (NKA), a heterodimeric (αß) pump that usually exports 3Na+ in exchange for 2 K+ per hydrolyzed ATP. Artemia express several NKA catalytic α-subunit subtypes. High-salinity adaptation increases abundance of α2KK, an isoform that contains two lysines (Lys308 and Lys758 in transmembrane segments TM4 and TM5, respectively) at positions where canonical NKAs have asparagines (Xenopus α1's Asn333 and Asn785). Using de novo transcriptome assembly and qPCR, we found that Artemia express two salinity-independent canonical α subunits (α1NN and α3NN), as well as two ß variants, in addition to the salinity-controlled α2KK. These ß subunits permitted heterologous expression of the α2KK pump and determination of its CryoEM structure in a closed, ion-free conformation, showing Lys758 residing within the ion-binding cavity. We used electrophysiology to characterize the function of α2KK pumps and compared it to that of Xenopus α1 (and its α2KK-mimicking single- and double-lysine substitutions). The double substitution N333K/N785K confers α2KK-like characteristics to Xenopus α1, and mutant cycle analysis reveals energetic coupling between these two residues, illustrating how α2KK's Lys308 helps to maintain high affinity for external K+ when Lys758 occupies an ion-binding site. By measuring uptake under voltage clamp of the K+-congener 86Rb+, we prove that double-lysine-substituted pumps transport 2Na+ and 1 K+ per catalytic cycle. Our results show how the two lysines contribute to generate a pump with reduced stoichiometry allowing Artemia to maintain steeper Na+ gradients in hypersaline environments.

Deep learning driven de novo drug design based on gastric proton pump structures.

Existing drugs often suffer in their effectiveness due to detrimental side effects, low binding affinity or pharmacokinetic problems. This may be overcome by the development of distinct compounds. Here, we exploit the rich structural basis of drug-bound gastric proton pump to develop compounds with strong inhibitory potency, employing a combinatorial approach utilizing deep generative models for de novo drug design with organic synthesis and cryo-EM structural analysis. Candidate compounds that satisfy pharmacophores defined in the drug-bound proton pump structures, were designed in silico utilizing our deep generative models, a workflow termed Deep Quartet. Several candidates were synthesized and screened according to their inhibition potencies in vitro, and their binding poses were in turn identified by cryo-EM. Structures reaching up to 2.10 Å resolution allowed us to evaluate and re-design compound structures, heralding the most potent compound in this study, DQ-18 (N-methyl-4-((2-(benzyloxy)-5-chlorobenzyl)oxy)benzylamine), which shows a Ki value of 47.6 nM. Further high-resolution cryo-EM analysis at 2.08 Å resolution unambiguously determined the DQ-18 binding pose. Our integrated approach offers a framework for structure-based de novo drug development based on the desired pharmacophores within the protein structure.

An unusual conformation from Na+-sensitive non-gastric proton pump mutants reveals molecular mechanisms of cooperative Na+-binding.

The Na+,K+-ATPase (NKA) and non-gastric H+,K+- ATPase (ngHKA) share ~65 % sequence identity, and nearly identical catalytic cycles. These pumps alternate between inward-facing (E1) and outward-facing (E2) conformations and differ in their exported substrate (Na+ or H+) and stoichiometries (3 Na+:2 K+ or 1 H+:1 K+). We reported that structures of the NKA-mimetic ngHKA mutant K794S/A797P/W940/R949C (SPWC) with 2 K+ occluded in E2-Pi and 3 Na+-bound in E1·ATP states were nearly identical to NKA structures in equivalent states. Here we report the cryo-EM structures of K794A and K794S, two poorly-selective ngHKA mutants, under conditions to stabilize the E1·ATP state. Unexpectedly, the structures show a hybrid with both E1- and E2-like structural features. While transmembrane segments TM1-TM3 and TM4's extracellular half adopted an E2-like conformation, the rest of the protein assumed an E1 configuration. Two spherical densities, likely bound Na+, were observed at cation-binding sites I and III, without density at site II. This explains the E2-like conformation of TM4's exoplasmic half. In NKA, oxygen atoms derived from the unwound portion of TM4 coordinated Na+ at site II. Thus, the lack of Na+ at site II of K794A/S prevents the luminal portion of TM4 from taking an E1-like position. The K794A structure also suggests that incomplete coordination of Na+ at site III induces the halfway rotation of TM6, which impairs Na+-binding at the site II. Thus, our observations provide insight into the molecular mechanism of E2-E1 transition and cooperative Na+-binding in the NKA and other related cation pumps.

Three structures of PSI-LHCI from Chlamydomonas reinhardtii suggest a resting state re-activated by ferredoxin.

Photosystem I (PSI) from the green alga Chlamydomonas reinhardtii, with various numbers of membrane bound antenna complexes (LHCI), has been described in great detail. In contrast, structural characterization of soluble binding partners is less advanced. Here, we used X-ray crystallography and single particle cryo-EM to investigate three structures of the PSI-LHCI supercomplex from Chlamydomonas reinhardtii. An X-ray structure demonstrates the absence of six chlorophylls from the luminal side of the LHCI belts, suggesting these pigments were either physically absent or less stably associated with the complex, potentially influencing excitation transfer significantly. CryoEM revealed extra densities on luminal and stromal sides of the supercomplex, situated in the vicinity of the electron transfer sites. These densities disappeared after the binding of oxidized ferredoxin to PSI-LHCI. Based on these structures, we propose the existence of a PSI-LHCI resting state with a reduced active chlorophyll content, electron donors docked in waiting positions and regulatory binding partners positioned at the electron acceptor site. The resting state PSI-LHCI supercomplex would be recruited to its active form by the availability of oxidized ferredoxin.

In-Hospital Deaths From Ambulatory Care-Sensitive Conditions Before and During the COVID-19 Pandemic in Japan.

Importance: The COVID-19 pandemic may have played a role in the deterioration of access to medical care for ambulatory care-sensitive conditions (ACSCs). Objective: To ascertain whether the number of in-hospital deaths and in-hospital mortality rate associated with ACSC changed after the declaration of the COVID-19 national state of emergency in Japan. Design, Setting, and Participants: This cohort study used a difference-in-differences design to compare outcomes for ACSC in the period before (January 1, 2015, to December 31, 2019) vs the period after (January 1, 2020, to December 31, 2020) Japan declared a national state of emergency for the COVID-19 pandemic. Analyses used discharge summary data from 242 acute care hospitals across Japan. The sample comprised unscheduled hospitalizations of patients with ACSC during the study period (January 1, 2015, to December 31, 2020). Data analyses were performed between August 16 and December 7, 2022. Exposure: The declaration of the COVID-19 national state of emergency announced by the Japanese government in April 2020 was considered to be an exogenous shock. Main Outcomes and Measures: Primary outcomes were the ACSC-associated number of in-hospital deaths, number of hospitalizations, and in-hospital mortality rate. Results: A total of 28 321 ACSC-related hospitalizations were observed involving 15 318 males (54.1%), with a median (IQR) age of 76 (58-85) years. The number of in-hospital deaths was 2117 (7.5%). The number of hospitalizations decreased overall (incidence rate ratio [IRR], 0.84; 95% CI, 0.75-0.94), for chronic conditions (IRR, 0.84; 95% CI, 0.77-0.92), and for vaccine-preventable conditions (IRR, 0.58; 95% CI, 0.44-0.76). However, in-hospital deaths (IRR, 1.66; 95% CI, 1.15-2.39) and in-hospital deaths within 24 hours of hospital arrival (IRR, 7.27 × 106; 95% CI, 1.83 × 106 to 2.89 × 107) increased for acute conditions. The in-hospital mortality rate increased for acute conditions (IRR, 1.71; 95% CI, 1.16-2.54), and the 24-hour in-hospital mortality rates also increased overall (IRR, 1.87; 95% CI, 1.19-2.96), for acute conditions (IRR, 2.15 × 106; 95% CI, 5.25 × 105 to 8.79 × 106), and for vaccine-preventable conditions (IRR, 4.64; 95% CI, 1.28-16.77). Conclusions and Relevance: This cohort study found that in Japan, the number of in-hospital deaths increased after the declaration of the COVID-19 national state of emergency in 2020, particularly for acute ACSC and deaths within 24 hours of hospital admission. This finding suggests that access to good-quality primary care and inpatient care for patients with acute ACSC may have been compromised during the pandemic.

Clinical characteristics and treatment outcomes of severe asthma patients with a history of multiple biologic drugs use.

BACKGROUND: Asthma control has been shown to improve after clinical use of molecular-targeted biologic drugs. Although most patients have shown favorable responses to biologic drugs, some individuals need to switch to another biologic drug. To date, limited data are available regarding patients who received multiple biologic drugs. OBJECTIVE: We aimed to evaluate the characteristics and outcomes of patients treated with multiple biologic drugs. METHODS: We reviewed severe asthma patients who received biologic drugs between May 2009 and September 2019. Clinical characteristics of patients and changes in annualized asthma exacerbation rates, asthma control test (ACT), and oral corticosteroid (OCS) dose, before and after the use of the final biologic drug, were evaluated. RESULTS: Of the 105 patients who received biologic drugs, 20 patients received multiple biologic drugs. Twelve patients received two biologic drugs, six received three, and two received four. Patients who received multiple biologic drugs tended to have a significantly higher number of allergic or eosinophilic airway comorbidities (allergic rhinitis: p = 0.02, chronic rhinosinusitis with nasal polyps: p < 0.001). Approximately half of the patients changed to different treatments due to uncontrolled comorbidities. Annualized exacerbation rates, ACT, and OCS dose significantly improved after the latest biologic drug use (p = 0.035, p < 0.001, and p = 0.038, respectively). CONCLUSIONS: The results of this study indicated that allergic and eosinophilic airway comorbidities should be considered during the selection of biologic drugs. Furthermore, most patients who received multiple biologic drugs achieved disease control after switching to the optimal biologic drug.

Effects of cost sharing on long-term care service utilization among home-dwelling older adults in Japan.

OBJECTIVES: This study aimed to examine the effect of increased cost sharing on long-term care (LTC) service utilization among home-dwelling older adults, using nationwide long-term care insurance (LTCI) claims data in Japan. METHODS: In August 2015, the coinsurance rate for Japanese LTCI increased from 10% to 20% for higher-income beneficiaries. We analyzed 27,911,076 person-month observations between April 2015 and July 2016 from 1,983,163 home-dwelling older adults (aged ≥ 65 years). We employed a difference-in-differences approach to estimate the effect of the increased coinsurance rate on overall LTC service utilization and for each of the four main service subcategories. The control group comprised those whose coinsurance rates remained at 10%. RESULTS: The treatment group, whose coinsurance rate increased, accounted for 9.6% of all participants. The raised coinsurance rate caused statistically significant reductions of 0.46% (95% confidence interval [CI]: 0.36%, 0.56%) and $25.7 (95% CI: $23.7, $27.8) in the percentage of utilization of LTC services and total monthly LTC expenditures per person, respectively. Service utilization decreased in each of the four service subcategories. CONCLUSIONS: The increased coinsurance rate resulted in statistically significant but small reductions in LTC service utilization overall and in each service type among higher-income home-dwelling beneficiaries. Requiring more cost sharing from higher-income individuals may alleviate the fiscal burden on LTC systems without serious reductions in service utilization.

Structure and function of H+/K+ pump mutants reveal Na+/K+ pump mechanisms.

Ion-transport mechanisms evolve by changing ion-selectivity, such as switching from Na+ to H+ selectivity in secondary-active transporters or P-type-ATPases. Here we study primary-active transport via P-type ATPases using functional and structural analyses to demonstrate that four simultaneous residue substitutions transform the non-gastric H+/K+ pump, a strict H+-dependent electroneutral P-type ATPase, into a bona fide Na+-dependent electrogenic Na+/K+ pump. Conversion of a H+-dependent primary-active transporter into a Na+-dependent one provides a prototype for similar studies of ion-transport proteins. Moreover, we solve the structures of the wild-type non-gastric H+/K+ pump, a suitable drug target to treat cystic fibrosis, and of its Na+/K+ pump-mimicking mutant in two major conformations, providing insight on how Na+ binding drives a concerted mechanism leading to Na+/K+ pump phosphorylation.

Hospitalizations for Ambulatory Care Sensitive Conditions in a Large City of Japan: a Descriptive Analysis Using Claims Data.

BACKGROUND: Hospitalization for ambulatory care sensitive conditions (ACSCs) is an indicator of the quality of primary care in different health systems. In Japan, where patients can choose any healthcare facility with universal health coverage (UHC), data on these admissions are unknown. OBJECTIVE: To describe the current situation of ACSC admissions in a city of Japan. DESIGN: Retrospective observational study using claims data. PARTICIPANTS: Beneficiaries aged 0-74 years of the National Health Insurance (NHI) program in a large city in the Greater Tokyo Area. We extracted ACSC admissions from all inpatient claims between April 2013 and March 2017. MAIN MEASURES: We calculated age- and sex-specific annual ACSC admission rates for three categories: acute, chronic, and vaccine-preventable. We estimated the age-adjusted admission rates by ACSC category according to administrative districts and rate ratios using Poisson regression models. We also estimated medical expenditures and lengths of stay for ACSC admissions. KEY RESULTS: Of 91,350 hospitalization episodes, we identified 7666 (8.4%) that were ACSC admissions. Males had higher annual ACSC admission rates than females (p < 0.001), especially for chronic ACSCs. Admission rates were lowest in those aged 15-39 years and higher in the youngest (0-4 years) and oldest (70-74 years) age groups. Age-adjusted chronic ACSC admission rates were lower in a newly developed area (rate ratio [RR]: 0.79, 95% confidence interval [CI]: 0.71-0.87) and higher in a residential area (RR: 1.14, 95% CI: 1.04-1.24) than in the center of the city. Total medical expenditures for all ACSC admissions accounted for 5.8% of the total inpatient expenditures of NHI in the city. CONCLUSIONS: ACSC admission rates in Japan were higher for males than for females and showed a U-shaped trend in terms of age, as in other countries with UHC, and deferred by region. This study provided possible factors to reduce ACSC admissions.

Structural Basis for Binding of Potassium-Competitive Acid Blockers to the Gastric Proton Pump.

As specific inhibitors of the gastric proton pump, responsible for gastric acidification, K+-competitive acid blockers (P-CABs) have recently been utilized in the clinical treatment of gastric acid-related diseases in Asia. However, as these compounds have been developed based on phenotypic screening, their detailed binding poses are unknown. We show crystal and cryo-EM structures of the gastric proton pump in complex with four different P-CABs, tegoprazan, soraprazan, PF-03716556 and revaprazan, at resolutions reaching 2.8 Å. The structures describe molecular details of their interactions and are supported by functional analyses of mutations and molecular dynamics simulations. We reveal that revaprazan has a novel binding mode in which its tetrahydroisoquinoline moiety binds deep in the cation transport conduit. The mechanism of action of these P-CABs can now be evaluated at the molecular level, which will facilitate the rational development and improvement of currently available P-CABs to provide better treatment of acid-related gastrointestinal diseases.

Analysis of the diagnosis of Japanese patients with primary ciliary dyskinesia using a conditional reprogramming culture.

BACKGROUND: Primary ciliary dyskinesia (PCD) is diagnosed through multiple methods, including transmission electron microscopy (TEM), a high-speed video microscopy analysis (HSVA), immunofluorescence (IF), and genetic testing. A primary cell culture has been recommended to avoid the misdiagnosis of secondary ciliary dyskinesia derived from infection or inflammation and improve diagnostic accuracy. However, primary cells fail to differentiate into ciliated cells through repeated passages. The conditional reprogramming culture (CRC) method, a combination of a Rho-kinase inhibitor and fibroblast feeder cells, has been applied to cystic fibrosis. The goal of this study was to evaluate the value of CRC in diagnosing PCD in Japanese patients. METHODS: Eleven patients clinically suspected of having PCD were included. Airway epithelial cells were obtained from an endobronchial forceps biopsy and cultured at the air-liquid interface (ALI) combined with CRC. Ciliary movement, ultrastructure, and mutated ciliary protein evaluation were performed using HSVA, TEM, and IF, respectively. Genetic testing was performed on some patients. RESULTS: CRC yielded dense and well-differentiated ciliated cells with a high success rate (∼90%). In patients with PCD, the ciliary ultrastructure phenotype (outer dynein arm defects or normal ultrastructure) and IF findings (absence of the mutated ciliary protein) were confirmed after CRC. In DNAH11-mutant cases with normal ultrastructure by TEM, the HSVA revealed stiff and hyperfrequent ciliary beating with low bending capacity in CRC-expanded cells, thereby supporting the diagnosis. CONCLUSIONS: CRC could be a potential tool for improving diagnostic accuracy and contributing to future clinical and basic research in PCD.

Municipal Characteristics of In-Home Death Among Care-Dependent Older Japanese Adults.

Importance: The provision of in-home and community end-of-life care has emerged as an important policy issue for aging populations around the world. Despite most patients expressing the wish to die at home (as opposed to in the hospital), substantial geographic variation persists in the prevalence of in-home death at the end of life. Objective: To assess the association of municipal characteristics with variation in the place of death among care-dependent older people in Japan. Design, Setting, and Participants: In this cross-sectional study using multilevel logistic regression, analyses were performed using national claims data under Japan's long-term care insurance system. The study population included long-term care insurance beneficiaries 65 years and older who died in 2015, excluding those who died from external causes, such as unintentional injuries. Data analyses were conducted from January 1 to April 31, 2021. Exposures: Predisposing, enabling, and need factors at the individual and municipal levels according to a behavioral model. Main Outcomes and Measures: The outcome was whether individuals died at home or not. Results: This cross-sectional study analyzed 544 836 decedents (median [IQR] age, 87 [81-91] years; 300 142 [55.1%] female). The proportion of in-home deaths was 10.3%. In the multilevel null model, 7.2% of the variance in the place of death was associated with municipal-level characteristics. Municipal characteristics were associated with more of the variance than were prefectural characteristics (2.7%). The largest proportional change (7.3%) in variance at the municipality level was observed when enabling factors (ie, medical and long-term care resources) were added to the model. Municipalities with a high proportion of in-home death were more likely to have a higher supply of clinics, physicians, and care workers providing in-home services per population, as well as a lower number of hospital beds and long-term care facility workers per population. Conclusions and Relevance: In this cross-sectional study, municipal characteristics explained a substantial proportion of the geographic variance in in-home death in Japan. These results suggest that municipal policy makers need to ensure an adequate supply of clinics, physicians, and care workers providing in-home services to meet the preferences of care-dependent older people who wish to spend their final days at home.

National trends in the proportion of in-hospital deaths by cause of death among older adults with long-term care: a nationwide observational study in Japan from 2007 to 2017.

BACKGROUND: Japan has promoted end-of-life care at home and in long-term care facilities, and the total proportion of in-hospital deaths has decreased recently. However, the difference in trends of in-hospital deaths by the cause of death remains unclear. We investigated the variation in trends of in-hospital deaths among older adults with long-term care from 2007 to 2017, by cause of death and place of care. METHODS: Using the national long-term care insurance registry, long-term care claims data, and national death records, we identified people aged 65 years or older who died between 2007 and 2017 and used long-term care services in the month before death. Using a joinpoint regression model, we evaluated time trends of the proportion of in-hospital deaths by cause of death (cancer, heart diseases, cerebrovascular diseases, pneumonia, and senility) and place of care (home, long-term care health facility, or long-term care welfare facility). RESULTS: Of the 3,261,839 participants, the mean age was 87.0 ± 8.0 years, and 59.2% were female. Overall, the proportion of in-hospital deaths decreased from 66.2% in 2007 to 55.3% in 2017. By cause of death, the proportion of in-hospital deaths remained the highest for pneumonia (81.6% in 2007 and 77.2% in 2017) and lowest for senility (25.5% in 2007 and 20.0% in 2017) in all types of places of care. The joinpoint regression analysis showed the steepest decline among those who died of senility, especially among long-term care health facility residents. CONCLUSIONS: The findings of this nationwide study suggest that there was a decreasing trend of in-hospital deaths among older adults, although the speed of decline and absolute values varied widely depending on the cause of death and place of care.

Cryo-EM of the ATP11C flippase reconstituted in Nanodiscs shows a distended phospholipid bilayer inner membrane around transmembrane helix 2.

ATP11C is a member of the P4-ATPase flippase family that mediates translocation of phosphatidylserine (PtdSer) across the lipid bilayer. In order to characterize the structure and function of ATP11C in a model natural lipid environment, we revisited and optimized a quick procedure for reconstituting ATP11C into Nanodiscs using methyl-ß-cyclodextrin as a reagent for the detergent removal. ATP11C was efficiently reconstituted with the endogenous lipid, or the mixture of endogenous lipid and synthetic dioleoylphosphatidylcholine (DOPC)/dioleoylphosphatidylserine (DOPS), all of which retained the ATPase activity. We obtained 3.4 Å and 3.9 Å structures using single-particle cryo-electron microscopy (cryo-EM) of AlF- and BeF-stabilized ATP11C transport intermediates, respectively, in a bilayer containing DOPS. We show that the latter exhibited a distended inner membrane around ATP11C transmembrane helix 2, possibly reflecting the perturbation needed for phospholipid release to the lipid bilayer. Our structures of ATP11C in the lipid membrane indicate that the membrane boundary varies upon conformational changes of the enzyme and is no longer flat around the protein, a change that likely contributes to phospholipid translocation across the membrane leaflets.

Tryptase and IL-33 in Bronchoalveolar Lavage Fluid May Predict the Types of Eosinophilic Pneumonia and Disease Recurrence.

INTRODUCTION: Eosinophilic pneumonia (EP) is characterized by a marked accumulation of eosinophils in the lungs and blood. Eosinophils and mast cells play an important role in the pathogenesis of EP via release of biomarkers such as tryptase and interleukin (IL)-33. However, the potential role of these biomarkers is not fully understood. OBJECTIVES: We aimed to evaluate the differences among the levels of tryptase and IL-33 in bronchoalveolar lavage fluid (BALF) from several types of EP. We evaluated the differences between the levels of these biomarkers in the recurrent and nonrecurrent cases. METHOD: We prospectively collected the clinical data of patients with EP, diagnosed between 2006 and 2015 in our institution. Bronchoscopy was performed before steroid treatment; BALF was collected. The clinical characteristics of EP patients and the levels of tryptase and IL-33 in BALF were evaluated. RESULTS: We enrolled 15 patients with chronic EP (CEP), 5 with acute EP (AEP), 10 with drug-induced EP, and 6 with angiitis-related EP. Tryptase levels in the CEP group were significantly higher than that in the drug-induced EP group (p = 0.048), while the AEP group had the highest IL-33 levels. Recurrence of EP was noted in 67% of patients with CEP. The levels of tryptase and IL-33 were notably higher in the recurrent cases than that in the nonrecurrent CEP group (p = 0.004, p = 0.04, respectively). Furthermore, there was a positive correlation between the levels of tryptase and IL-33 in the BALF of patients with CEP (ρ = 0.69, p = 0.004). CONCLUSIONS: Tryptase and IL-33 in BALF are useful biomarkers for the assessment of EP types. These biomarkers could be used to predict disease recurrence.

Deaths in Nursing Homes during the COVID-19 Pandemic - Lessons from Japan.

To date, 35% of coronavirus disease 2019 (COVID-19) deaths in the United States have occurred among nursing home populations (https://jamanetwork.com/journals/jama-health-forum/fullarticle/2763666), compared with 14% in Japan (Werner et al. 2020). How did Japan manage such a low proportion of COVID-19 deaths in nursing homes? The similarity in case-fatality rates among patients with COVID-19 in nursing homes in the two countries (both approximately 16%) suggests that the infection rate in nursing homes in Japan was much lower than in the United States. Therefore, the pandemic unmasked long-standing problems with the quality and financing of US long-term care (LTC) services (Grabowski 2020; Werner et al. 2020). We compare differences between the LTC systems of Japan and the United States, focusing on the measures adopted to protect against COVID-19 in Japan.

Association of the Frequency of In-Home Care Services Utilization and the Probability of In-Home Death.

Importance: The provision of end-of-life care is an important policy issue associated with population aging around the world. Yet it is unclear whether the provision of in-home care services can allow patients the option of in-home death at end of life. Objective: To assess whether the frequent use of in-home care services can assist recipients to stay at home at the end of life. Design, Setting, and Participants: This cohort study of older adults in Japan's long-term care insurance system used national claims data. Participants were long-term care insurance beneficiaries aged 65 years or over who died in 2015, excluding those who died due to external causes such as accidents. Data analyses were conducted from October to December 2020. Exposures: Mean days of in-home care service used per week from the first day of the month before the month of death to the date of death. Main Outcomes and Measures: Primary outcome was whether the older person died at home (or not). To address lack of information on individual preference for place of death, we used an instrumental variable estimation with the full-time equivalent number of care workers providing in-home care services per older population at the municipality level in 2014. Results: Of the 572 059 decedents included in the study, 314 743 (55.0%) were women (median [IQR] age, 87 [81-91] years). The proportion of in-home deaths was 10.5% (60 175 decedents), and 81 675 decedents (14.3%) used in-home care services at least once prior to their death. Ordinary least squares and 2-stage least squares analyses both indicated that more frequent use of in-home care was associated with a higher probability of in-home deaths (ordinary least squares estimate, 5.0 percentage points; 95% CI, 4.9-5.1 points vs 2-stage least squares estimate, 3.6 percentage points; 95% CI, 2.3-4.9 points). Conclusions and Relevance: This retrospective cohort study using an instrumental variable approach demonstrated that more frequent use of in-home care services at the end of life was associated with a higher probability of in-home death. One policy implication of these results is that in order to meet the end-of-life preferences of patients, it is not only necessary to promote the provision of medical services at home but also to ensure an adequate supply of care workers.

Gastric proton pump with two occluded K+ engineered with sodium pump-mimetic mutations.

The gastric H+,K+-ATPase mediates electroneutral exchange of 1H+/1K+ per ATP hydrolysed across the membrane. Previous structural analysis of the K+-occluded E2-P transition state of H+,K+-ATPase showed a single bound K+ at cation-binding site II, in marked contrast to the two K+ ions occluded at sites I and II of the closely-related Na+,K+-ATPase which mediates electrogenic 3Na+/2K+ translocation across the membrane. The molecular basis of the different K+ stoichiometry between these K+-counter-transporting pumps is elusive. We show a series of crystal structures and a cryo-EM structure of H+,K+-ATPase mutants with changes in the vicinity of site I, based on the structure of the sodium pump. Our step-wise and tailored construction of the mutants finally gave a two-K+ bound H+,K+-ATPase, achieved by five mutations, including amino acids directly coordinating K+ (Lys791Ser, Glu820Asp), indirectly contributing to cation-binding site formation (Tyr340Asn, Glu936Val), and allosterically stabilizing K+-occluded conformation (Tyr799Trp). This quintuple mutant in the K+-occluded E2-P state unambiguously shows two separate densities at the cation-binding site in its 2.6 Å resolution cryo-EM structure. These results offer new insights into how two closely-related cation pumps specify the number of K+ accommodated at their cation-binding site.