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.

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.

Crystal structures of the gastric proton pump.

The gastric proton pump-the H+, K+-ATPase-is a P-type ATPase responsible for acidifying the gastric juice down to pH 1. This corresponds to a million-fold proton gradient across the membrane of the parietal cell, the steepest known cation gradient of any mammalian tissue. The H+, K+-ATPase is an important target for drugs that treat gastric acid-related diseases. Here we present crystal structures of the H+, K+-ATPase in complex with two blockers, vonoprazan and SCH28080, in the luminal-open state, at 2.8 Å resolution. The drugs have partially overlapping but clearly distinct binding modes in the middle of a conduit running from the gastric lumen to the cation-binding site. The crystal structures suggest that the tight configuration at the cation-binding site lowers the pK a value of Glu820 sufficiently to enable the release of a proton even into the pH 1 environment of the stomach.

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.

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.

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.

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.

Isoform-selective regulation of mammalian cryptochromes.

CRY1 and CRY2 are essential components of the circadian clock controlling daily physiological rhythms. Accumulating evidences indicate distinct roles of these highly homologous proteins, in addition to redundant functions. Therefore, the development of isoform-selective compounds represents an effective approach towards understanding the similarities and differences of CRY1 and CRY2 by controlling each isoform individually. We conducted phenotypic screenings of circadian clock modulators, and identified KL101 and TH301 that selectively stabilize CRY1 and CRY2, respectively. Crystal structures of CRY-compound complexes revealed conservation of compound-binding sites between CRY1 and CRY2. We further discovered a unique mechanism underlying compound selectivity in which the disordered C-terminal region outside the pocket was required for the differential effects of KL101 and TH301 against CRY isoforms. By using these compounds, we found a new role of CRY1 and CRY2 as enhancers of brown adipocyte differentiation, providing the basis of CRY-mediated regulation of energy expenditure.

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.

Crystal structure of a human plasma membrane phospholipid flippase.

ATP11C, a member of the P4-ATPase flippase, translocates phosphatidylserine from the outer to the inner plasma membrane leaflet, and maintains the asymmetric distribution of phosphatidylserine in the living cell. We present the crystal structures of a human plasma membrane flippase, ATP11C-CDC50A complex, in a stabilized E2P conformation. The structure revealed a deep longitudinal crevice along transmembrane helices continuing from the cell surface to the phospholipid occlusion site in the middle of the membrane. We observed that the extension of the crevice on the exoplasmic side is open, and the complex is therefore in an outward-open E2P state, similar to a recently reported cryo-EM structure of yeast flippase Drs2p-Cdc50p complex. We noted extra densities, most likely bound phosphatidylserines, in the crevice and in its extension to the extracellular side. One was close to the phosphatidylserine occlusion site as previously reported for the human ATP8A1-CDC50A complex, and the other in a cavity at the surface of the exoplasmic leaflet of the bilayer. Substitutions in either of the binding sites or along the path between them impaired specific ATPase and transport activities. These results provide evidence that the observed crevice is the conduit along that phosphatidylserine traverses from the outer leaflet to its occlusion site in the membrane and suggest that the exoplasmic cavity is important for phospholipid recognition. They also yield insights into how phosphatidylserine is incorporated from the outer leaflet of the plasma membrane into the transmembrane.

Epidemiology of Food Choking Deaths in Japan: Time Trends and Regional Variations.

BACKGROUND: With increasing age globally, more people may become vulnerable to food choking. We investigated the nationwide epidemiology of food choking deaths in Japan. METHODS: Using Japanese Vital Statistics death data between 2006 and 2016, we identified food choking deaths based on the 10th revision of the International Statistical Classification of Diseases code W79 (Inhalation and ingestion of food causing obstruction of respiratory tract) as a primary diagnosis. We assessed the demographics of people with food choking deaths; temporal trends of food choking deaths by the year (overall and by age group), the day of year; and prefecture variations. RESULTS: Overall, 52,366 people experienced food choking deaths (median age, 82 years, 53% were male, and 57% occurred at home). The highest numbers occurred January 1-3, and were lowest in June. Despite a stable total number of cases at around 4,000 yearly, from 2006 to 2016 the incidence proportion declined from 16.2 to 12.1 per 100,000 population among people aged 75-84 years. Among people ≥85 years, the incidence proportion peaked at 53.5 in 2008 and decreased to 43.6 in 2016. The number of food choking deaths varied by prefecture. CONCLUSIONS: There are temporal and regional variations of food choking deaths in Japan, possibly due to the consumption of Japanese rice cake (mochi), particularly over the New Year's holiday.

Place of death associated with types of long-term care services near the end-of-life for home-dwelling older people in Japan: a pooled cross-sectional study.

BACKGROUND: Many older people wish to die at home. However, there is still a huge gap between the place where older adults wish to die and the place where they, in fact, do die. We aimed to assess the association between each type of long-term care (LTC) services that home-dwelling older individuals utilized at their end of life and place of death. METHODS: A pooled cross-sectional study at the point of death was used for the analysis. Participants included beneficiaries of long-term care insurance in Japan, aged 65 years and above, who passed away between January 2008 and December 2013, excluding those who died due to external factors and those who were using residential services at their time of death. We conducted a multivariate Poisson regression analysis with robust standard errors adjusting for potential confounders and examined the association between the use of each type of LTC service for home-dwelling recipients, including in-home services, day services, and short-stay services, with the interaction terms being time of death (exposure) and home death (outcome). We calculated the adjusted probability of home deaths for each combination pattern of LTC services for home-dwelling recipients using standard marginalization. RESULTS: We analyzed 2,035,657 beneficiaries. The use of in-home services, day services, and short-stay services were associated with an increased probability of home deaths; the incident rate ratio (IRR) was 13.40 (with a 95% confidence interval (CI): 13.23-13.57) for in-home services, the IRR was 6.32 (6.19-6.45) for day services, and the IRR was 1.25 (1.16-1.34) for short-stay services. Those who used day or short-stay services with in-home services exhibited a higher probability of home deaths than those who used only day or short-stay services. CONCLUSIONS: We demonstrated that home-dwelling older persons who used LTC services near end-of-life had a higher probability of home deaths as compared to those who did not. Our findings can clarify the importance of providing and integrating such services to support care recipients who wish to die at home as well as for the benefit of their informal caregivers.

[The relationship between death from renal insufficiency and diuretic therapy among older adults at two nursing homes catering atomic bomb survivors].

AIM: In Geriatrics Gerontology International we previously reported the efficacy of reducing diuretics to prevent falls and fractures in older adults. We have since noticed another important problem, regarding the diuretic therapy for older adults with decreased muscle and water volumes. We performed a study on renal insufficiency and diuretic therapy in an attempt to confirm the need for case control study between standard diuretic therapy administered, according to guidelines and "NY-mode" diuretic therapy, which involves the administration of the mineral-corticoid receptor inhibitor spironolactone at 12.5 mg orally every other day. METHODS: We reviewed the causes of death among 1,855 residents living at 2 atomic bomb survivors nursing homes, with a focus on the death certification and diuretic therapy status of 48 older adults who died due to renal insufficiency. We also evaluated the relationship between the estimated glomerular filtration rate of 407 residents using serum creatinine data and the level of independence in daily life of disabled older adults. RESULTS: We found that deaths due to chronic renal insufficiency were concentrated within certain period in two nursing homes examined and in patients receiving standard diuretic therapy (continuous daily loop diuretics or combination of loop diuretics with mineral-corticoid receptor inhibitor). Older adults with a relatively low level of independence in daily life showed a relatively higher estimated glomerular filtration rate, depending on their muscle volume. CONCLUSION: These results suggest the need for a case control study of standard diuretic therapy and "NY-mode" therapy among older adults with decreased muscle and water volumes to not only prevent falls and fall-related fractures but also protect the kidney from damage.

Systematic comparison of molecular conformations of H+,K+-ATPase reveals an important contribution of the A-M2 linker for the luminal gating.

Gastric H(+),K(+)-ATPase, an ATP-driven proton pump responsible for gastric acidification, is a molecular target for anti-ulcer drugs. Here we show its cryo-electron microscopy (EM) structure in an E2P analog state, bound to magnesium fluoride (MgF), and its K(+)-competitive antagonist SCH28080, determined at 7 Å resolution by electron crystallography of two-dimensional crystals. Systematic comparison with other E2P-related cryo-EM structures revealed that the molecular conformation in the (SCH)E2·MgF state is remarkably distinguishable. Although the azimuthal position of the A domain of the (SCH)E2·MgF state is similar to that in the E2·AlF (aluminum fluoride) state, in which the transmembrane luminal gate is closed, the arrangement of transmembrane helices in the (SCH)E2·MgF state shows a luminal-open conformation imposed on by bound SCH28080 at its luminal cavity, based on observations of the structure in the SCH28080-bound E2·BeF (beryllium fluoride) state. The molecular conformation of the (SCH)E2·MgF state thus represents a mixed overall structure in which its cytoplasmic and luminal half appear to be independently modulated by a phosphate analog and an antagonist bound to the respective parts of the enzyme. Comparison of the molecular conformations revealed that the linker region connecting the A domain and the transmembrane helix 2 (A-M2 linker) mediates the regulation of luminal gating. The mechanistic rationale underlying luminal gating observed in H(+),K(+)-ATPase is consistent with that observed in sarcoplasmic reticulum Ca(2+)-ATPase and other P-type ATPases and is most likely conserved for the P-type ATPase family in general.

Cryo-EM structure of gastric H+,K+-ATPase with a single occupied cation-binding site.

Gastric H(+),K(+)-ATPase is responsible for gastric acid secretion. ATP-driven H(+) uptake into the stomach is efficiently accomplished by the exchange of an equal amount of K(+), resulting in a luminal pH close to 1. Because of the limited free energy available for ATP hydrolysis, the stoichiometry of transported cations is thought to vary from 2H(+)/2K(+) to 1H(+)/1K(+) per hydrolysis of one ATP molecule as the luminal pH decreases, although direct evidence for this hypothesis has remained elusive. Here, we show, using the phosphate analog aluminum fluoride (AlF) and a K(+) congener (Rb(+)), the 8-Å resolution structure of H(+),K(+)-ATPase in the transition state of dephosphorylation, (Rb(+))E2~AlF, which is distinct from the preceding Rb(+)-free E2P state. A strong density located in the transmembrane cation-binding site of (Rb(+))E2~AlF highly likely represents a single bound Rb(+) ion, which is clearly different from the Rb(+)-free E2AlF or K(+)-bound (K(+))E2~AlF structures. Measurement of radioactive (86)Rb(+) binding suggests that the binding stoichiometry varies depending on the pH, and approximately half of the amount of Rb(+) is bound under acidic crystallization conditions compared with at a neutral pH. These data represent structural and biochemical evidence for the 1H(+)/1K(+)/1ATP transport mode of H(+),K(+)-ATPase, which is a prerequisite for generation of the 10(6)-fold proton gradient in terms of thermodynamics. Together with the released E2P-stabilizing interaction between the ß subunit's N terminus and the P domain observed in the (Rb(+))E2~AlF structure, we propose a refined vectorial transport model of H(+),K(+)-ATPase, which must prevail against the highly acidic state of the gastric lumen.

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.

Characteristics of first-time users of the nursing small-scale multifunctional home care service: a pooled cross-sectional study using Japanese long-term care insurance claims data from 2012 to 2019.

OBJECTIVES: In April 2012, the Japanese government launched a new nursing service called the nursing small-scale multifunctional home care (NSMHC) to meet the nursing care demands of individuals with moderate-to-severe activities of daily living (ADLs) dysfunction and who require medical care, thereby allowing them to continue living in the community. We aimed to preliminarily analyse the characteristics of first-time users of NSMHC service. DESIGN: This pooled cross-sectional study used the Japanese long-term care insurance (LTCI) claims data from the users' first use of NSMHC (from April 2012 to December 2019). SETTING: NSMHC includes nursing home visits, home care, daycare, overnight stays and medical treatment. PARTICIPANTS: The study population included LTCI beneficiaries who received their first long-term care requirement certification in Japan from April 2012 onwards, died between April 2012 and December 2019, and used any LTCI service at least once. RESULTS: Among the 836 563 individuals who used any LTCI service at least once, 3957 (0.47%) used NSMHC. We analysed 3634 individuals without any missing data regarding long-term care requirement certification. Most individuals were aged 80 years or older, with 64.3% requiring care level 3 or above, indicating complete assistance with ADLs. Regarding ADLs in individuals with dementia, 70.6% were at level 2 or below, indicating they can live almost independently even with dementia. A large proportion of NSMHC users availed the service approximately 6 months before death, with no prior use of any LTCI services; they continued using the service for around 4 months, although some people continued to use NSMHC until their month of death. CONCLUSIONS: Using individual data on nationwide LTCI, we described the characteristics of first-time users of NSMHC among those who died within 7.5 years from the first certification of care needs. Further studies are needed to investigate the effect of NSMHC use on user outcomes.

Inter-subunit interaction of gastric H+,K+-ATPase prevents reverse reaction of the transport cycle.

The gastric H(+),K(+)-ATPase is an ATP-driven proton pump responsible for generating a million-fold proton gradient across the gastric membrane. We present the structure of gastric H(+),K(+)-ATPase at 6.5 A resolution as determined by electron crystallography of two-dimensional crystals. The structure shows the catalytic alpha-subunit and the non-catalytic beta-subunit in a pseudo-E(2)P conformation. Different from Na(+),K(+)-ATPase, the N-terminal tail of the beta-subunit is in direct contact with the phosphorylation domain of the alpha-subunit. This interaction may hold the phosphorylation domain in place, thus stabilizing the enzyme conformation and preventing the reverse reaction of the transport cycle. Indeed, truncation of the beta-subunit N-terminus allowed the reverse reaction to occur. These results suggest that the beta-subunit N-terminus prevents the reverse reaction from E(2)P to E(1)P, which is likely to be relevant for the generation of a large H(+) gradient in vivo situation.

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.

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.