Subjects <70 years and >70 years had significantly higher mortality with a serum 25(OH)D less than 30 nmol·L−1 (11.8% and 55%), than with a serum 25(OH)D greater than 30 nmol·L−1 (2.2% and 25%). Unvaccinated Caucasian adults with a low vitamin D state have higher mortality due to SARS CoV-2 pneumonia, which is not explained by confounders and is not closely linked with elevated serum CRP.COVID-19 and a low vitamin D state share common risk factors, which might explain why vitamin D deficiency has been linked with higher COVID-19 mortality. Moreover, measures of serum vitamin D may become lower during systemic inflammatory responses, further confounding the association via reverse causality. In this prospective study (recruited over 12 months), we examined whether the association between a low vitamin D state and in-hospital mortality due to SARS-CoV-2 pneumonia in unvaccinated subjects is explained by (i) the presence of shared risk factors  or (ii) a reduction in serum 25(OH)D due to COVID-19 . In this cohort of 232 (mean age = 56 years) patients , we failed to find an association between serum vitamin D and levels of CRP, or other inflammatory markers. However, the hazard ratio for mortality for subjects over 70 years of age (13.2) and for subjects with a serum 25(OH)D level less than 30 nmol·L Retrospective studies ,2 and st2). The primary outcome was mortality. We also measured length of admission, duration of oxygen therapy and admission to the intensive care unit. After signing an IRB approved consent form, background data including age, gender, body mass index (BMI) and comorbidities were recorded. The investigating team believed that patients who take chronic corticosteroid therapy might have lower measures of CRP, lower measures of 25(OH)D and increased mortality due to COVID pneumonia, and these patients were not recruited into the study.The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Research Ethics Committee of Connolly Hospital Dublin. All subjects (except those on chronic systemic corticosteroid therapy) were eligible to enroll in a prospective observational study examining serum 25(OH)D and CRP levels and COVID disease severity. The cohort was defined as the patient population admitted to our hospital with COVID-19 pneumonia between March 2020 and April 2021. From a total of 972 hospitalized patients, 232 agreed to enroll . To asse® containing EDTA, sodium citrate and no additive. Whole blood with EDTA was analysed for full blood count (FBC) using a Beckman Coulter UniCel® DxH 800 automated analyser ; the D-dimer levels were assessed on a Sysmex CS-2500 analyser with a Siemens kit  on the day of collection. Total serum 25(OH)D levels were analysed on a Roche Cobas 6000′s module e602  using an electrochemiluminescence immunoassay method with between-run and within-run CVs <8.9%. C-reactive protein (CRP), ferritin, troponin and total calcium and magnesium were measured, using commercially available diagnostic kits on the Roche Cobas 6000 automated analyser. The between-run and within-run CVs for these assays ranged between 1% and 8%. Venous blood was collected into BD Vacutainer tubesBiochemical (including serum 25(OH)D levels) and haematological laboratory parameters during the hospital stay were measured and the peak result was recorded. Serum 25(OH)D levels were measured and recorded within 24 h of admission. SARS-CoV-2 infection was confirmed in all patients using reverse transcription-polymerase chain reaction (RT-PCR) on samples which were obtained from nasopharyngeal swabs. −1 is associated with an increased risk of death within 30 days of admission. Our sample size was based on our estimated 20% mortality rate in hospitalized patients with SARS-CoV-2 pneumonia [−1 is approximately 7% [−1) [We hypothesized that a serum 25(OH)D level less than 30 nmol·Lneumonia  and we aately 7% , we assu 7% [−1) .n) and relative frequencies (%). Continuous variable data were tested for normality using Shapiro—Wilk’s test, a visual inspection of their histograms, and via assessment of their skewness and kurtosis. For all analyses, a p value of <0.05 was considered statistically significant.All statistical analyses were performed using SPSS statistical software . Categorical variables were expressed as absolute numbers D concentration of 30–49.9 nmol·L−1, while D50 was defined as a serum 25(OH)D concentration ≥50 nmol·L−1. Parametric and non-parametric analyses were used to compare various laboratory biomarkers between COVID-19 patients in the three vitamin D categories. For normally distributed continuous variables, mean levels and standard deviation (SD) were compared between the three groups using analysis of variance (ANOVA) tests; for non-normally distributed continuous variables, median levels and interquartile ranges (IQR) were compared between the three groups using Kruskal—Wallis tests.Descriptive statistics were used to define patients’ demographic and comorbidity characteristics. Vitamin D status was divided into three categories: D30 as a serum 25(OH)D concentration <30 nmol·LPearson’s Chi-square test was used to assess the association between the three vitamin D status categories and outcome measures such as extended duration of O2 requirement , ICU admission and mortality. Binary logistic regression analysis was used to determine if age, gender, presence of diabetes mellitus, obesity (BMI ≥ 30 kg.m−2) or renal disease predicted the likelihood of having a 25(OH)D level < 30 nmol·L−1. −1) predicted clinical outcomes such as O2 requirement >24 h, ICU admission and mortality after controlling for confounders. Study participants were dichotomised into D30 (<30 nmol·L−1) and other (≥30 nmol·L−1) groups for these analyses. Multivariate logistic regression analyses were finally used to assess whether vitamin D deficiency (25(OH)D < 30 nmol·L2 requirement >24 h, ICU admission, mortality) were associated with measures of 25(OH)D. Multivariate logistic regression analyses were used to determine whether any observed associations between background status  and 25(OH)D and between 25(OH)D and clinical outcomes persisted after adjusting for confounders. We performed additional analyses, including Yates’ continuity correction, to detect differences in mortality rates with a 2 × 2 crosstabulation of mortality in the over 70 years group and the under 70 years age groups vs. (<30 nmol/L) and (>30 nmol/L) serum 25(OH)D measurements. A Mann—Whitney U test was employed to detect significant differences in serum 25(OH)D levels between survivors and non-survivors in older and younger age groups. Univariate analyses were used to determine whether serum measures of CRP or clinical outcome  of participants were D30 (25(OH)D <30 nmol·L−1), 26% (n = 60) were D40 (25(OH)D of 30–49 nmol·L−1), while 36 % (n = 84) were D50 (25 (OH) ≥ 50 nmol·L−1).The socio-demographic, anthropometric and clinical characteristics of the patient cohort are summarised in −1) and D40 (158 mg·L−1) groups than in the D50 group (91 mg·L −1), although this trend did not reach statistical significance (p = 0.39) (p = 0.45). There was a statistically significant difference in lymphocyte count across the three vitamin D groups (p = 0.036). Lymphocyte levels were higher in the D50 group (1 × 109) and the D30 group (1 × 109) than in the D40 group (0.68 × 109)  (The biometric associations between serum 25 (OH) and other biochemical and hematological indices are described in  = 0.39) . Median ctively) .p = 0.249) (p = 0.013). Additionally, significantly lower median 25(OH)D concentrations were seen in patients who required ICU admission (33 nmol·L−1) compared to those who did not require ICU admission (42 nmol·L−1) (p = 0.031). Subjects in the D30 group experienced higher mortality (22%) than patients in either the D40 group (6.5%) or the D50 group (13%) on univariate analysis (p = 0.037)  . Both th= 0.037) .−1 (−2) and renal disease). As shown in −1 (OR: 0.520 (95% CI: 0.285–0.951) (p = 0.034)), but none of the other parameters was independently associated with a 25(OH)D ≤ 30 nmol·L−1.Binary logistic regression analysis was performed to assess the association between a set of putative predictor variables and the likelihood of a 25(OH)D ≤ 30 nmol·L−1 . The mod−1 was strongly predictive of mortality (OR: 4.63 (95% CI: 1.53–13.97) (p = 0.006)). Age ≥70 was also a strong predictor of mortality (OR: 13.32 (95% CI: 4.24–41.81) (p < 0.001)). Diabetes mellitus, male gender and obesity were not independently associated with mortality.Multivariate logistic regression was performed to assess the association between vitamin D status and clinical outcomes after adjustment for the potential confounders included in p = 0.01)) and diabetes mellitus (OR: 2.82 (95% CI: 1.21–6.53) (p = 0.016)) were independent predictors of ICU admission however. A negative association between age ≥70 years (OR: 0.29 (95% CI: 0.11–0.73) (p = 0.009)) and ICU admission was also observed. A measure of 25(OH)D <30 nmol·L−1 was not a significant predictor of ICU admission (OR: 1.18 (95% CI: 0.58–2.39) (p = 0.632)). After adjusting for male gender, 25(OH)D <30 nmol·L−1 was not a significant predictor of supplemental O2 >24 h (OR: 2.11 (95% CI: 0.85–5.39) (p = 0.116)). Male gender itself however was a significant predictor of requirement for supplemental O2 >24 h (OR: 3.22 (95% CI: 1.26–8.22) (p = 0.014)).Male gender (OR: 2.90 (95% CI: 1.29–6.51)  and the effect persisted after allowing for gender, BMI, and the presence of diabetes mellitus. While uncertainty exists about what measures accurately reflect vitamin D status, here we employed 25(OH)D because it is in widespread clinical use and is used in many epidemiologic studies, although measures of 25(OH)D may not accurately reflect vitamin D status of patients who receive large bolus injections of vitamin D2 [In this prospective, observational study of unvaccinated Caucasian patients hospitalized for SARS-CoV-2 pneumonia, a serum 25(OH)D lower than 30 nmol·Ltamin D2 . Neverthtamin D2 ,16. −1 [Here, we confirm that advancing age (greater than 70 years) was a significant independent risk for mortality with a hazard ratio of greater than 13. Indeed, the mean age of our population was 56 years, which may explain why the mortality rate in our study (15%) is somewhat lower than that reported in other prospective studies. For example, in one ICU study with a mean age of 65 years, the 28 day mortality was 33% for subjects with 25(OH)D measures less than 38 nmol·L−1 . The mor−1 .Our decision to not recruit patients prescribed systemic corticosteroid therapy is supported by a recent large study of 7845 hospitalized patients, in whom corticosteroid therapy alone (within 2 weeks of a positive SARS-CoV-2 PCR test) was associated with significantly higher mortality (17%), compared to the mortality of subjects who took neither vitamin D supplements nor corticosteroids (12.5%) . Interes−1 and none of those died, we cannot make a meaningful comment about the level of 25(OH)D, if any, where immunocompetent adults are protected from severe COVID. Indeed, many of our subjects with a level greater than 50 nmol·L−1 suffered pneumonia, respiratory failure and death due to COVID, indicating that some subjects with a serum 25(OH)D significantly greater than 30 nmol·L−1 do suffer severe COVID.The strength of this study is that we have prospectively recruited unvaccinated patients with their first episode of COVID pneumonia at a single centre and we did not recruit subjects who took systemic corticosteroid therapy, because of the apparent reduction in measures of serum 25(OH)D and CRP seen in such subjects ,20. We a−1 in these older adults [While oral vitamin D supplementation data for the current cohort are unavailable, data from the Irish National Adult Nutrition Survey (NANS) in 2009 show a steady age-related increase in the use of vitamin D supplements from the 18–24 year age group (10% of men and 12% of women) to the 50–64 year age group (18% of men and 27% of women) . In older adults ,25. Recent data suggest that supplementation rates (and by extrapolation vitamin D levels) pre-admission to hospital are significantly higher in subjects with less severe disease . Since wp = 0.006), even after adjustment for age, gender, presence of diabetes mellitus and BMI. In a German study of hospitalised patients, even after adjustment for age, gender and underlying disease, the risk of mechanical ventilation was 6.1 times higher and risk of death was 14.7 times higher in those with 25(OH)D < 30 nmol·L−1 than in those with 25(OH)D > 30 nmol·L−1 (p < 0.001 for both) [−1 in asymptomatic SARS-CoV-2 positive patients versus 36 nmol·L−1 in severely-ill COVID-19 patients requiring ICU admission (p = 0.0001) [−1) was present in 97% of patients requiring ICU admission versus 33% of asymptomatic patients, with a case fatality rate of 21% in those with 25(OH)D < 50 nmol·L−1 vs. 3.1% in those with 25(OH)D > 50 nmol·L−1 [Our data are consistent with the results of a number of prospective observational studies of hospitalized COVID-19 patients that also demonstrate lower vitamin D levels are significantly associated with 28 day mortality ,28, in p 0.0001) . Vitaminnmol·L−1 . In Bostnmol·L−1 . It is u−1  than in those with serum 25(OH)D ≥ 30 nmol·L−1  (p = 0.032)  compared to the remainder of the study population  (p < 0.001) (−1 was only 2.2% (2/92 patients)—significantly lower than the 22.9% mortality rate (32/140 patients) observed amongst the remainder of the study population (p < 0.001). Thus, in this study population, patients aged >70 years with 25(OH)D levels <30 nmol·L−1 represented just 8.6% of the overall cohort, but accounted for one third of the deaths. This finding is mirrored in the analysis of younger subjects: in our study population, younger than 70 years with a serum 25(OH)D <30 nmol·L−1 mortality was significantly higher (8/68 = 11.8%) compared to the population of patients aged less than 70 years with 25(OH)D >30 nmol·L−1 (2/92 = 2.2%) (p = 0.032) (−1) compared to those under 70 years (42.8 SD ± 28.1 nmol·L−1) (p = 0.002). We did not specifically study supplementation rates, but it is likely that higher oral supplementation rates and greater time spent outdoors in summertime might account for some of the difference. Interestingly, the average measures of serum 25(OH)D in survivors over the age of 70 years (56.9 ± 29.3 nmol·L−1) did not differ significantly from those over 70 years who died (54.0 ± 39.1 nmol·L−1) (p = 0.729). In contrast, the mean serum 25(OH)D was significantly lower in subjects younger than 70 years who died (mean 24.5 ± 12.4 nmol·L−1) compared to subjects younger than 70 years who survived (mean 44.1 ± 28.4 nmol·L−1) (p = 0.032).The mortality rate  for subjects younger than 70 years was lower than for subjects over 70 years of age (24/72 = 33.3%). Although this study was not designed to compare younger and older subjects, we made some interesting observations: First, in the subjects aged 70 years and over, overall mortality was 33% (24/72), but was significantly higher for subjects with a serum 25(OH)D < 30 nmol·L= 0.032) . Overall< 0.001) . Convers= 0.032) . Second,−1) in healthy subjects who were injected with LPS (a surrogate for gram-negative septic shock syndrome) [A low serum vitamin D state has been proposed in some studies to arise as a consequence of systemic inflammation . For example, Smolders et al. demonstrated small but significant drops in serum 25(OH)D (~6 nmol·Lyndrome) . Althougyndrome) ,33,34. Cyndrome) . A recenyndrome) ; however−1 [n = 4) subjects with 25(OH)D levels greater than 100 nmol·L−1, and while none died, we cannot make any comment about what level, if any, protects against COVID mortality. Nevertheless, our data add to a growing body of literature which now meets many of the Bradford Hill criteria supporting a causal link between low vitamin D status and COVID-19 disease severity and death [This prospective study demonstrates that a low vitamin D state is associated with increased mortality rates in patients with COVID pneumonia—and that this effect is not confined to older age groups. There appears to be an incremental rise in mortality rates in both younger and older age groups according to vitamin D status and age. In this regard, a recent meta-analysis incorporating data from eight studies in which 25(OH)D was measured prior to infection or on the first day of hospitalisation has shown a significant inverse correlation between these pre- and peri-infection 25(OH)D levels and mortality, proposing a threshold for minimal mortality risk at ~125 nmol·L−1 . This stnd death .2 D3) impact cellular function via both nuclear receptor-dependent and nuclear receptor-independent mechanisms [2 D3-bound vitamin D receptors (VDR) forming a heterodimer complex with retinoid X receptors  and subsequently binding to vitamin D response elements regulating gene transcription [2 D3 are mediated by a membrane-associated rapid response steroid-binding protein, identified as PDIA3, with impacts on cell survival and immune responses [The current data suggest that unvaccinated subjects, who become hospitalized with COVID pneumonia, have increased mortality with increasing age and with lower serum 25(OH)D levels. Vitamin D has a multitude of effects on the human immune system. Metabolites of vitamin D chanisms . The nuccription . In contesponses ,41. If vesponses .


It is formed as a high-temperature phase in the Ir-Gasystem. Single-crystal X-ray diffraction analysis confirms the tetragonalsymmetry  with a = 6.4623(1) Å and c = 6.5688(2) Å and reveals strong disorder in thecrystal structure, reflected in the huge values and anisotropy ofthe atomic displacement parameters. A model for the real crystal structureof ht-IrGa3 is derived by the split-positionapproach from the single-crystal X-ray diffraction data and confirmedby an atomic-resolution transmission electron microscopy study. Temperature-dependentelectrical resistivity measurements evidence semiconductor behaviorwith a band gap of 30 meV. A thermoelectric characterization was performedfor ht-IrGa3 and for the solid solutionIrGax3–Znx.The compound IrGa The electronicstructures show a strong d(T)–p(Tr) hybridization with a sharpgradient of the density of states near the Fermi level, fulfillingthe appropriate conditions for a high Seebeck coefficient. On thebasis of this particular feature, numerous studies have been carriedout regarding the thermoelectric properties of T(8)Tr3 intermetallic compounds and their substitutional derivatives:e.g., RuIn3,8 RuGa39 and FeGa3.11 On the other hand, the isostructural intermetallic compounds T(9)Tr3 with group 9 transition metals  should show metallic behavior.1 In recent theoretical studies on FeGa3-type representatives, CoGa3 and RhGa3 showno band gap, indicating metallic character. However, for the isoelectroniccompound IrGa3 a narrow band gap of ∼0.15–0.07eV was predicted, suggesting a semiconductor behavior and, thus, anauspicious candidate for a thermoelectric material.13 The IrGa3 compound has been known since 1958.14 A later publication assumed it to be a dimorph.15 Experimental evidence of the phase relationshipand the thermoelectric properties for IrGa3 has not yetbeen reported.Intermetallic compoundshave been the focus of thermoelectric materialsresearch in the last decades, with surprising singular features beingfrequently unveiled during their investigation, thereby contributingto the understanding of physical and chemical properties. Rather unexpectedly,materials with the composition Tht-IrGa3 is the subject of the presentstudy.The influence of nonisoelectronic substitution on the thermoelectricbehavior is investigated on the solid solution IrGax3–Znx.The physicochemical and thermoelectric characterizationof 3 andIrGax3–Znx  samples wereprepared from the elementsiridium , gallium , and zinc . For theliquid–solid reaction the starting materials were placed inprefired Al2O3 crucibles . Thecrucible was sealed in a Ta ampule and jacketed in quartz glass. Theampule was placed hanging in a vertical furnace for heat treatmentusing the following temperature–time profile: heating in 12h to 900 °C, followed by a dwell of 168 h at this temperature,and final quick quenching by breaking of the quartz jacket in ice/water(samples 1 and 2). Additionally, the IrGa3 sample 2 wasground in a mortar, cold-pressed to a pellet (diameter of 8 mm), sealedin a quartz glass ampule, and annealed for a second time with thesame temperature–time profile as before.IrGaAfter heat treatment,powdered samples with a particle size of <50 μm were densifiedto pellets via spark plasma sintering (SPS) at a maximum temperatureof 250 °C  using a tungstencarbide mold (inner diameter 8 mm) and applying a force of 340 kNduring 30 min. Appropriate bar-shaped specimens for chemical characterizationand physical property measurements were cut from the pellets.2 and elemental zinc as standards.For metallographic analyses, piecesof as-cast and/or sintered samples were embedded in a conductive resin, ground and polished using microsizeddiamond powders. The sample homogeneity and local chemical compositionwere analyzed on a scanning electron microscope (CAMECA SX100 ElectronMicroprobe) by standard-based wavelength dispersive X-ray spectroscopy(WDXS). The spectral lines Ga Kβ, Ir Lβ, and Zn Kαwere employed using IrGaht-IrGa3 sample 1 usingelectron backscatter diffraction (EBSD) on a JSM-7800F field emissionscanning electron microscope , equipped withan EBSD system .The crystallite orientation (texture) analysis was performed onSPS-compacted 1 radiation, λ = 1.54056Å, 5° ≤ 2θ ≤ 100°; Δθ= 0.005°). Qualitative phase analysis was done with the programpackage WinXPOW;16 all crystallographiccalculations were carried out with the program package WinCSD.17 The lattice parameters were determined by least-squaresrefinement on 65 reflection positions in the range of 15° <2θ < 100° using LaB6 (a =4.1569 Å) as an internal standard.Powder X-raydiffraction (PXRD) analyses were used at differentsteps of the experiments. For phase identification and lattice parameterdetermination, PXRD data were collected on an Image Plate GuinierCamera Huber G670 .The thermal behaviorof the samples was evaluated on a DSC 404Cdifferential scanning calorimeter (DSC)  in the temperature range from RT to 1020 °C , equipped with an Oxford Cryosystems N-HeliX low-temperaturedevice . Intensity data reduction comprisedcorrection of Lorentz–polarization and absorption effects.Reconstructions of precession photos from collected images were performedwith the software CrysAlisPRO.18Single-crystal X-ray diffraction was performedon selected crystalswith nonregular shape fixed with Apiezon H grease  at the top of a glass capillary or at a microloop  for low-temperature diffraction experiments. Theintensity data collection at room temperature was performed on a RigakuAFC7 diffractometer system . The temperature-dependent single-crystalX-ray diffraction data in the range 30 ≤ 3 was ground in liquid nitrogen to enhance the brittlenessand avoid structural changes caused by grinding. The obtained powderwas suspended in butanol and transferred onto a TEM grid coated witha holey-carbon film. Initial TEM investigations were performed ona FEI Tecnai F30-G2 instrument with supertwin lens  with a point resolution of around 2.0 Å and informationlimit of around 1.2 Å and equipped with a slow scan CCD camerafor imaging. The sample was ground and suspended in ethanol and transferredonto a TEM grid. The investigated particles were oriented for studyin the [001] and [110] zones.For transmission electron microscopy (TEM) investigations,polycrystallineIrGaAtomic-resolution scanning TEM(STEM) and high-resolution TEM (HR-TEM)analyses were carried out by a JEM-ARM300F double spherical aberrationcorrected microscope . The maximumHR-TEM resolution is 0.5–0.7 Å, and the maximum STEM resolutionis 0.5 Å. A CCD camera with a 4k × 4k pixel array (GatanUS4000) was used as the imaging system for TEM.S, and thermalconductivity κ at low temperature (5 K ≤ T ≤ 350 K) were simultaneously measured on a Physical PropertiesMeasurement System , using thethermal transport option (TTO). The Hall effect was also measuredon a PPMS with a standard five-point ac technique and by sweepingthe magnetic fields up to 9 T. Five 25 μm Pt wires were contactedto the sample by spot-welding. The Hall carrier concentration wascalculated as 1/(RHe),with e being the electron charge and RH the Hall coefficient.Appropriatelyshaped specimens were cut for the thermoelectricproperty measurements from SPS-compacted materials. The electricalresistivity ρ, Seebeck coefficient T ≤ 550K), ρ and S were measuredon ZEM-3 equipment  on 7 × 1.5 × 1.2mm3 bar-shaped specimens. The thermal conductivity wascalculated by using the equation κ = α × d × Cp. The thermal diffusivity(α) was measured on a SPS-sintered pellet  using the laser flash technique .The density (d) was determined by the Archimedeanmethod with a balance equipped with a density determination attachment and using ethanol asimmersion medium. The densities of all the analyzed samples were around97% of the theoretical values, indicating a high compaction degree.The specific heat (Cp) was measured ona DSC equipment .For characterization atelevated temperature Ga3 compounds are theGa-richest phases in the corresponding binary systems. In the caseof FeGa3, the two-phase region solid + melt of the phasediagram offers an advantageous thermodynamic condition for crystalgrowth of this compound.20 However, in the Ir-Ga system, the Ga-richest phase is Ir2Ga9.21 IrGa3 wasreported to be a dimorph,15 with no furtherinformation about the stability range. To determine the latter, theregion of the phase diagram around 75 atom % Ga was investigated indetail .For single-crystalX-ray diffraction analysis, appropriate ≈ 6.56Å 3.No supkl with k + l = 2n, hhl with l = 2n, 0k0 with k = 2n, 00l with l = 2n) indicate the possible space groups P42nm, P4̅n2, and P42/mnm. Assuming the isotypism of ht-IrGa3 andFeGa3, the centrosymmetric space group P42/mnm (No. 136) was chosen for structuresolution and refinement. Atomic coordinates from FeGa3 wereused as the starting model for ht-IrGa3. The structure refinement in the isotropic model for the atomicdisplacement parameter (ADP) results in the residual value R(F) = 0.122, the large difference betweenthe ADPs for iridium and gallium, Biso(Ga) ≈ 4 × Biso(Ir), andthe high residual electron density of ca. 11 e Å–3 in the vicinity of Ga1 and Ga2.The reflection conditions in bothcollected data sets (0ht-IrGa3 with the anisotropic model for the ADPsresults in R(F) ≈ 0.068. It reveals huge valuesof ADP and their anisotropywith B22 ≈ B33 ≫ B11 for Ga1 and B33 ≈ 2 × (B11 = B22) for Ga2  for Ga1 and Ga2 Tr3 compounds also show systematicallylarger ADPs for Tr than those for T.27 To describe the strong anisotropyof ADPs for the Ga sites in RuGa3, a split model for theGa1 in the 4c site was even used.9It is worth noting that reportedcrystallographic data of otherTB(Ir) = 0.9 Å2; B(Ga) = 1.5 Å2). The distributionof DED reveals several maxima in the vicinity of the atomic positionsof the ideal structure . Inthe DED distribution, each of the initial sites has two closely locatedsplit positions .In order to understand the reasons for the unusual atomic displacement,the difference electron density (DED) was calculated from the experimentaldata of crystal 1 for the ideal model with the more realistic ADPsin an isotropic approximation . The “main”additional sites are the same, and the differences in the list ofthe less occupied split positions can be understood by consideringthe different thermal histories of the samples.An analogous treatment revealed a very similar situation for thecrystal 2  and filled by an Ir2 dumbbell. The atomic coordination of Ir (d(Ir–Ir) = 2.89 Å and eight Gaatoms with d1(Ir–Ga2) = 2.42 Å(2×), d2(Ir–Ga1) = 2.43 Å(2×), d3(Ir–Ga2) = 2.56 Å(4×) and d4(Ga2–Ga2) = 2.77Å. According to the tabulated covalent radii rcov(Ir) = 1.26 Å and rcov(Ga) = 1.25 Å,28 the observed Ir–Gadistances are comparable with rcov(Ir)+ rcov(Ga) = 2.51 Å. Thus, they arein the range of covalent bond interactions. The Ir–Ir distanceof 2.89 Å is longer than that for a single-bond distance of 2.52Å. An analysis of the interatomic distances in IrGa3 shows comparable chemical bonding scenario to the prototype FeGa3.29The ideal crystalstructure of IrGaon of Ir 6, bottomht-IrGa3 reveals a huge variety of Ir–Gaand Ga–Ga distances, illustrating the material to be in an in-transformation state. Only the Ir–Ir distanceremains more or less stable, being the consequence of two-center T–Tbonding.29In comparison with the ideal structure, the real structure of in-transformation state) due to the stability region of ht-IrGa3 at higher temperature (cf. phase diagramabove).The unusual features ofthe crystal structure model developed fromthe single-crystal X-ray diffraction data are confirmed by the transmissionelectron study of sample 1. Modulated regions are recognizable alreadyin the nonfiltered HRTEM image 7, top. Aht-IrGa3 (sample 1). While thegeneral contrast distribution was found to be in agreement with theideal structure  resembles the basic structural motive of the FeGangements 9, top. Tht-IrGa3 should have an influence on the electronictransport,which was predicted to be a semiconductor, and on the thermal conductivity,especially its lattice part, which—due to the strong deviationsfrom the periodic arrangement—should be strongly suppressed.The complexity of the real crystal structure of ht-IrGa3. From the Arrhenius plot,a band gap of Eg = 0.03 eV is obtained.The calculated band-gap value is 0.15–0.07 eV.13 The reduction in Eg is most probablythe effect of the structural disorder.For the measurements of thermoelectric properties, a compactedand reannealed (900 °C + quenching) sample 1 was used. The latticeparameters of the material after SPS are within 3–5 estimatedstandard deviations equal to those before sintering 1. The ArS identifies ht-IrGa3 as an n-type semiconductor,with |S| < 35 μV K–1 upto 350 K Tr3 compounds at T = 300 K: κL(ht-IrGa3) ≈ 0.7 < κL(FeGa3) ≈ 3.231 < κL(OsGa3) ≈ 3.52 < κL(RuIn3) ≈5.57 < κL(RuGa3) ≈ 7 W m–1 K–1.9 At the same time, ReGa2Gewith κ = 1.1 W m–1 K–1,32 Fe0.99Co0.01Ga2.991Ge0.09 with κ = 1.37 W m–1 K–1,11 and Ru0.99Ir0.01In3 with κ = 1.55 W m–1 K–1 30 at T = 300 K are compounds of the FeGa3 type withthe lowest thermal conductivity reported, ascribable to the chemicalsubstitutions. In comparison, a low total thermal conductivity resultsalready in binary ht-IrGa3 with κ300 K = 1.10 W m–1 K–1. This can be understood by considering structural disorder in thematerial and appearance of new kinds of atomic interactions (inhomogeneityof bonding33), which reduces the latticethermal conductivity.34 The reduction inthermal conductivity is not as strong as could be expected from thestructural disorder and bonding reasons, e.g., in the transition-metal-containingintermetallic clathrates.35 However, incontrast to the temperature-dependent behavior of κ(T(8)Tr3) with lower values by a temperature increase, κ(IrGa3) increases continuously with temperature increase in thewhole range from 5 to 350 K . The appropriatevalue for a good thermoelectric material should be around 1019 cm–3.36 A suitabletuning of the charge carrier concentration can be achieved throughthe introduction of holes by substituting Ga with Zn. The thermoelectricproperties of the solid solution IrGax3–Znx were studied for x = 0.08, 0.16, 0.24, 0.32.The charge carrier concentration in pristine x3–Znx and the correspondingcompositions according to WDXS are shown in x ≤ 0.32, the lattice parameter c evolves with a slightly negative slope, whereas the lattice parameter a clearly grows with increasing Zn content  increasescontinuously with an increase in temperature up to T = 350 K. This behavior is also observed for the solid solution IrGax3–Znx.When the thermoelectric properties of FeGa3-type compoundsare reviewed, a similar trend is observed in isostructural ReGa2Ge32 and Fe0.5Co0.5Ga2.65Ge0.35,37 whose thermal conductivities steadily increase from room temperatureupward. The latter is shown for comparison with the analogous behaviorof κ in IrGax3–Znx, exemplified with IrGa2.76Zn0.24  present exceptionally lowthermal conductivity, which may originate from the atomic disorderand the covalent interactions in the ttdp unit, inanalogy to FeGa3.29As was mentioned previously, κ under ambient conditions. ht-IrGa3 presents semiconductor-like behavior with a bandgap of Eg = 0.03 eV and shows very lowlattice thermal conductivity, as assessed by the complex local atomicarrangements in ht-IrGa3. Hole dopingvia substitution of Ga by Zn increases the figure of merit zT, but it is not sufficient to consider these materialsas being thermoelectric.Thesingle-crystal X-ray diffraction and atomic resolution TEMand STEM study of the intermetallic compound


While the presence of simple phenylsubstituents around the Bi center results in a polymeric structurewith three Bi centers in the repeating monomer, substituents at the ortho- and meta-positions lead to cationicmono- and dinuclear fluorobismuthonium complexes, respectively. Preparationof all compounds is accomplished by fluoride abstraction from theparent triaryl Bi(V) difluorides using NaBArF 2]4–). Structuralparameters were obtained via single crystal X-ray diffraction (XRD),and their behavior in solution was studied by NMR spectroscopy. Trinuclearand binuclear complexes are held together through one bridging fluoride(μ-F) between two Bi(V) centers. In contrast, the presence ofMe groups in both ortho-positions of the aryl ringprovides the adequate steric encumbrance to isolate a unique mononuclearnonstabilized fluorotriarylbismuthonium cation. This compound featuresa distorted tetrahedral geometry and is remarkably stable at roomtemperature both in solution  and in thesolid state.A series of cationicfluorotriarylbismuthonium salts bearing differentlysubstituted aryl groups (Ar = 9,9-Me Although these complexes were characterizedby 1H NMR and IR spectroscopy,11 structural characterization by X-ray diffraction (XRD) eluded experimentalists.In this work, the cationic iodotriphenylbismuthonium structure wasreported to be thermally unstable, rapidly leading to decomposition.More than 30 years later, Hoge reported the reaction of Ph3BiF2 with (C2F5)3PF2 resulting in a fluorotriphenylbismuthonium cation, whichforms a polymeric structure in the solid state, as characterized byXRD analysis (12 Such long chains feature bridgingμ-F between Bi(V) centers, uniting the triarylbismuthonium cationsin a polymeric linear disposition (F–Bi–F angles closeto linearity [175.5(2)°]). It was also demonstrated that replacingthe Ph group in Ph3BiF2 with 2-[(dimethylamino)methyl]phenylleads to isolation of a monomeric fluorobismuthonium cation 3BiF2 by Seppelt resulted in a solid state structure of(C6F5)3BiF2·2Bi(C6F5)3, formed by association of a Bi(V)unit with two trigonal-pyramidal Bi(III) molecules (16 This situation highlights the high Lewis acidity of Bi(III)bearing electron-withdrawing groups, to the point that allows coordinationof the F at the σ* C–Bi bonds. Another example of suchneutral halogen···Bi interactions can be found in thestudy reported by Auer and Mehring in Bi(III) complexes.17 In there, intermolecular donor–acceptorBi–Cl···Bi interactions of Bi–Cl wereobserved for Ar2BiCl .Thedevelopment of cationic halogenated organopnictogen(V) compoundshas witnessed a revival in recent years, due to their applicationas Lewis acids in catalysis.18 and structure.19 Due to thelimited number of triarylhalobismuthonium cations and the even morelimited structural information, we decided to fill this gap in thearea by preparing a family of novel cationic fluorotriarylbismuthoniumsalts, whose structures were analyzed both in solution (NMR) and insolid state (XRD). Variation of the substituents on the aryl ringresulted in the isolation of tri-, di-, or mononuclear fluorobismuthoniumspecies. This systematic study culminated with the preparation andisolation of a genuine fluorotriarylbismuthonium cation, without hypervalentcoordination or additional stabilization  and its pentavalent tetrachlorinated bismuth(V).19 In order to investigate the behavior of thecorresponding fluorinated analog, oxidation of XantBis (1) was attempted using XeF2. Gratifyingly, compound 2 was formed exclusively and could be isolated in 95% yield2]4–), one of thefluorine atoms was abstracted, leading to the cationic dinuclear bismuthoniumsalt 3 in 96% isolated yield (path a). Interestingly,despite the use of 2 equiv of NaBArF, only one fluorineis removed to form 2, suggesting a high stability ofthe corresponding salt. Colorless single crystals of compound 3 suitable for XRD analysis were obtained by layering a concentrateddichloromethane solution of 3 with hexane at +5 °C-9,9-dimethylxanthene) , X-ray Single Crystals Analysis section). However, when lookingat the single units of this polymer  Å; Bi2–F3, 2.1212(15)Å] compared to the distance between the bridging fluorine andBi atoms . Based on the close bond lengths of Bi1–F1 and Bi2–F1,the positive charge is likely to be shared by both bismuth centers.A deviation from the linearity can be observed for the Bi1–F1–Bi2angle of 159.68(8)°. The bridging and terminal fluorides canbe clearly observed in the 19F NMR spectrum as a tripletat −105.36 ppm and a doublet at −156.04 ppm with 2JF–F =98.2 Hz and 2JF–F = 98.0 Hz coupling constants, respectively.The XRD analysis reveals thatcomplex  polymer 2, bottom polymer 2, top, ey Gabbaï1C.14 The6) (path b), leading to a 93% yield of 4. Colorlesssingle crystals suitable for X-ray diffraction were obtained upondiffusion of hexane into a dichloromethane solution of 4. This compound exhibits a similar polymeric structure as the cationiccomplex 3, albeit with some interesting differences inthe single units , whilethe three rings in structure 4 are disposed in a nearlyplanar geometry .In orderto study the influence of the anion, we carried out thefluoride abstraction with silver hexafluoroantimonate (AgSbF6) 1, path ble units 3. While 3 and 4, the F abstractionusingNaBArF and AgSbF6 has led to F-bridged intramolecularconnections. However, with the precedents from Seppelt and Hoge,16 we hypothesized that such behavior could also be observed in anintermolecular fashion. In order to have a broader spectrum beyondthe simple unsubstituted difluorotriphenylbismuth (8),aryls substituted with tBu- and Me-substituents havealso been synthesized. Based on literature described protocols forthe syntheses of complexes 6 and 7,21 the respective bismuth(III) species were obtained as colorless solidsin 60% and 45% yield, respectively  Å] (ortho)H···F hydrogeninteractions, the tBu groups in the meta-position are in the plane delineated by the quasi-linear F1–Bi–F1axis . The Bi–C bondsof Bi complex 9 are similar to 8 and onlyslightly shorter to compared to 6 .17 Complex 9 showsa singlet at −160.92 ppm in the 19F NMR spectrum,similar to that of 8. This suggests that in 9, the tBu groups exert no significant electronicinfluence on the F atoms.The Bi center in complexes 29(3) Å] 5. As a rtBu groups at the ortho-positionfailed,a decrease in steric demand was envisaged. Hence, trimesitylbismuthine(7),21 bearing methyl groupsin the ortho- and para-positions,was synthesized instead bismuthdifluoride (9). TheF1–Bi–F1* angle of 179.00(5)° deviates slightlyfrom linearity. Comparing trimesitylbismuthdifluoride with its lighterisostructural analogs,22 it can be observed that all complexescrystallize in the monoclinic space group C2/c. However, a slight trend can be observed. The solid statestructure of compound 10 reveals a Bi–F bond distanceof 2.1222(10) Å, which is longer than the reported Sb and P analogues Å;9 P–F, 1.673(2) Å).22 The 19F NMR spectrum of 10 shows a singlet at a higher chemical shift (−100.41 ppm)compared to 8 and 9, presumably due to theinfluence of the methyl group in the ortho-position.Considering the (ortho-Me)H···F distance of complex 10, it maybe assumed that the interaction between H and F is contributing tothe increase in the chemical shift compared to those of complexes 8 17 and 9 , in which the H···Fdistances are longer. Interestingly, whereas the same chemical shiftcan also be observed for the fluorine atoms in the lighter Mes3SbF2,9 the signal forMes3PF2 appears at −25.7 ppm.22Whereas attempts to synthesize triarylbismuth(III)bearing  instead 4. Oxidat3BiF2 with 1.0 or 0.5 equiv ofNaBArF in CH2Cl2 at 25 °C resultedin clear 19F NMR spectra of the crude: a triplet at −123.01ppm (2JF–F = 91.0 Hz)and a doublet at −162.01 ppm (2JF–F = 88.9 Hz) in addition to the [BArF]− signal at −62.87 ppm . Crystallization of the compound from the reaction mixture resultedin the isolation of a 1D coordination polymer, which is assembledvia intermolecular Bi···F interactions of single cationictrinuclear bismuth units such as complex 11  Å, and Bi2–F3,2.173(2) Å, which are shorter compared to the polymeric structurereported by Hoge et al.  and to Ph3BiF2 .20 With respect to the central Ph3BiF2 subunit,the terminal Bi–F bonds are significantly shorter with Bi2–F2of 2.078(2) Å and Bi1–F1 of 2.068(2) Å, respectively.Such distances manifest that the dicationic character of 11 is located at the terminal Ph3BiF moieties, while thecentral Ph3BiF2 unit remains rather neutral.The cationic FBiPh3 moieties are also evident by the longerbond lengths between the terminal Bi atoms and the bridging fluorineatoms .The F2–Bi2–F3 angle of 179.44(9)° of the centralPh3BiF2 moiety deviates slightly from linearity,whereas the F1–Bi1–F2 angle of 178.50(8)° and F3–Bi3–F4of 177.35(8)° of the Ph3BiF moieities are slightlymore distorted. The angles of the bismuth atoms bridged by fluorinecan be described by Bi1–F2–Bi2 of 175.11(12)° andBi2–F3–Bi3 of 171.72(11)° as slightly bent. Inthe analysis of the entire polymer  Å; Bi3–F1, 2.855(2) Å] than in complexes 3 and 4, indicating that the intermolecular interactionsare weaker.This monomeric unit represents a dicationic bismuthonium salt withthree BiPh polymer 6, bottom8 using either 0.5 or 1equiv of NaBArF led to the isolation of the same compound 11 .24 The symmetric F–Bi–F–Bi–Farrangement is evident by its characteristic signals in 19F NMR. The two terminal fluorine ligands appear as a sharp doubletsignal at −162.06 ppm with a 2JF–F = 102.9 Hz, similar to thatobtained for 3, 4, and 11.The bridging fluorine atom can be observed as a triplet peak at −152.78ppm in the 19F NMR.When the F abstraction is carriedout with complex edyield 3. Here,12 reveals that each Bi centeradopts a trigonal-bipyramidal geometry with two fluorides trans to each other in apical positions and aromatic ringsin equatorial positions  to 1.0 equiv of NaBArF. This time, monomericcomplex 13 was isolated in 92% as an off white solid Å; Bi1–F2, 2.056(2) Å], which highlightsthe cationic nature of this structure. While the Bi–F bondlengths are only slightly shorter compared to the neutral parent compound 10 , an increase in the (ortho-Me)H···Fbond distances can be noticed ,considering the ortho-Me substituents, which areoriented parallel to the fluorine. As a result of the elongation andthe reduced H···F electron interaction, a singlet signalcan be observed at −177.11 ppm in CDCl3, CD2Cl2, and toluene-d8. Interestingly, by using CD3CN as solvent,the singlet appears at −148 ppm, probably due to possible coordinationof the solvent. However, the singlet signal in both 19FNMR and XRD analyses indicates that no polymeric chain or furtherinteractions with another Bi complex are formed . The use of Me groups in the ortho- and para-positions gives the structure a high steric demandcompared to complexes 11 and 12. In contrastto compound 12, in which the aryl groups bearing tBu substituents in the meta-position arearranged perpendicular to the F–Bi–F axis, the mesitylligands exhibit an oblique orientation to the Bi–F bond. Thisdescribed propeller-like orientation leads to the prevention of furtherpolymerization. As already indicated, the peculiarity of this structureresults from its steric congestion, unlike base stabilization. However,comparison of the monomeric cationic Bi species 13 withits lighter analogs, such as [Mes3PF][FB(C6F5)3]25 and [Mes3SbF][OTf],9 reveals an increase in thePn–F bond length as the pnictogen becomes heavier . An interesting observationto note is that the Bi–F bond is only slightly longer thanthe Sb–F bond . However, this result must be treated with cautionsince OTf as anion is weakly coordinated to the antimony center andconsequently noninnocent. Unfortunately, a comparison cannot be madewith the arsenic analog, as this structure is not yet known in theliterature. Furthermore, the monomeric Bi(V) cation 13 cannot be compared with the monomeric fluorobismuthonium salt reportedby Hoge et al.,12 since no single crystalstructure has been reported. However, when comparing complex 13 with the single unit of the polymeric bismuthonium cationreported by Hoge, differences in the Bi–C and Bi–F bondlengths can be observed. Whereas the Bi–C bond distances ofthe polymeric structure by Hoge are shorter , the Bi–F bond lengths are elongated and even longer than the Bi–Fdistances in the triarylbismuthdifluoride complexes 9 and 10 .The XRD analysis reveals that the Bi atom adoptsa pseudotetrahedralgeometry. The Bi–F bond distance of 2.009(5) Å is consistentwith the distances between the terminal fluorine atoms and the bismuthcenter of compounds 11–13 were synthesized andfully characterized. In the trinuclear and dinuclear complexes, μ-Fbridges are observed connecting two different Bi(V) atoms and holdingthe complex together in the solid state. In the case of the polymericstructure 13, consisting of trinuclear bismuth units,we observed a dinuclear species in solution. By introducing steric tBu groups in the meta-position, trimerizationcould be prevented and a snowflake-like dinuclear structure was obtained,which exhibits the same pattern both in solution and in the solidstate. By further fine-tuning the ligand scaffold, the presence ofMe groups in both ortho-positions of the aryl ledto the isolation of a mononuclear fluorotriarylbismuthonium cationthat is not stabilized by the introduction of additional electroniceffects. With this unique mononuclear fluorotrimesitylbismuthoniumcation, we provide a fundamental contribution to the spectrum of halopnictoniumcations. Overall, these results represent a step forward in understandingthe subtle differences between solid state and in solution analysisof high-valent arylbismuth–fluoride complexes. The study presentedhere sheds light onto the influence of substituents on the aryl groupsin speciation of the complexes. This structural study serves as aroadmap for catalyst design and is essential for the efficient developmentof catalytic C–F bond formation strategies.18bIn summary, a series of mono-, di-, and trinuclear fluorobismuthoniumcations


Tmc1 mutations reduced the Ca2+ influx into the hair bundle. Two mutations (Tmc1 p.D528N or Tmc1 p.E520Q) also decreased channel conductance and two (Tmc1 p. D569N or Tmc1 p.W554L) lowered expression. These mutations endorse TMC1 as the pore of the MET channel. The MET channel also contains accessory subunits, LHFPL5 and TMIE. MET currents were small in Lhfpl5 or Tmie knockout mice. Nevertheless, MET channels could still be activated by hair bundle displacement; single-channel conductance was unaffected in Lhfpl5−/− but reduced in −/−,Tmie suggesting TMIE likely contributes to the pore.We studied the role of TMC1 as the central component of the hair cell mechanotransducer (MET) channel by characterizing transduction in mice harboring mutations in the pore region. All  2+ permeability of the MET channel, triggering hair cell apoptosis and deafness. In addition, Tmc1 p.E520Q and Tmc1 p.D528N reduced channel conductance, whereas Tmc1 p.W554L and Tmc1 p.D569N lowered channel expression without affecting the conductance. Tmc1 p.M412K and Tmc1 p.T416K reduced only the Ca2+ permeability. The consequences of these mutations endorse TMC1 as the pore of the MET channel. The accessory subunits, LHFPL5 and TMIE, are thought to be involved in targeting TMC1 to the tips of the stereocilia. We found sufficient expression of TMC1 in outer hair cells of Lhfpl5 and Tmie knockout mice to determine the properties of the channels, which could still be gated by hair bundle displacement. Single-channel conductance was unaffected in Lhfpl5−/− but was reduced in −/−Tmie, implying TMIE very likely contributes to the pore. Both the working range and half-saturation point of the residual MET current in Lhfpl5−/− were substantially increased, suggesting that LHFPL5 is part of the mechanical coupling between the tip-link and the MET channel. Based on counts of numbers of stereocilia per bundle, we estimate that each PCDH15 and LHFPL5 monomer may contact two channels irrespective of location.Transmembrane channel-like protein 1 (TMC1) is thought to form the ion-conducting pore of the mechanoelectrical transducer (MET) channel in auditory hair cells. Using single-channel analysis and ionic permeability measurements, we characterized six missense mutations in the purported pore region of mouse TMC1. All mutations reduced the Ca Caenorhabditis elegans indicated an assembly of two copies each of the pore-forming TMC1 subunit, the transmembrane (TM) inner ear protein TMIE, and the calcium-binding protein CALM-1 homologous with CIB2  channel is activated by tension in a tip-link extending from the side wall of one stereocilium to the tip of the adjacent shorter stereocilium , 2, wher41114ith CIB2 . The locith CIB2 .2+-activated Cl− channel TMEM16, has a large extent of the pore exposed to the membrane lipid. This feature is related to the purported phospholipid scramblase activity of TMEM16 as a template for the TMC1 model  ethane-N,N,N′,N′-tetra-acetic acid)]. This procedure severs most of the tip-links that transmit force to the MET channel, leaving one or two channels activatable by bundle deflection. We first used this method while studying transduction in turtle auditory hair cells  mice were obtained from Karen Steel, Kings College, London, United Kingdom, and Walter Marcotti, Sheffield University, United Kingdom. The −/−Tmie (transmembrane inner ear) mouse strain was B6.B(CBA)sr-Tmie/J . The −/−Lhfpl5 (lipoma MMGIC fusion partner-like 5) mouse strain was the B6.129-tm1KjnLhfpl5/Kjn . All mutants used for characterizing hair cell transduction were bred on a −/−Tmc2 background to circumvent complications due to different channel properties of TMC2 (−/−Tmc2 mice (B6.129S5-tm1LexTmc2/Mmucd) were obtained from the Mutant Mouse Regional Resource Center . Neonatal mice up to postnatal day (P)8 were killed by decapitation according to the animal protocol approved by the Institutional Animal Care and Use Committee at the University of Wisconsin–Madison. of TMC2 . Tmc2−/−d, the distance along the basilar membrane from the apex divided by the total length of the basilar membrane, which in P6 mice has a mean of 5.2 mm. Recordings were made at an apical low-frequency position, d = 0.2 to 0.3, and a basal high-frequency position, d = 0.8. Since the magnitude of the MET current varies with OHC position along the cochlea, it is essential that mutant and control properties be compared at the same cochlear location. The recording chamber was mounted on the stage of a Leica DMLFS top-focusing microscope and viewed with 40× (NA = 0.8) objective and a 2× optivar. The chamber was perfused with saline: 152 mM NaCl, 6 mM KCl, 1.5 mM CaCl2, 2 mM Na-pyruvate, 8 mM d-glucose, and 10 mM Na-Hepes, pH 7.4. Patch electrodes were filled with : 138 mM CsCl, 3.5 mM MgCl2, 5 mM Na2ATP, 0.5 mM Na2GTP, 10 mM Tris phosphocreatine, 1 mM BAPTA, 10 mM Cs-Hepes, pH 7.2, and were connected to an Axopatch 200B amplifier. Electrode series resistances with 60% compensation were at best 3 MΩ, which, with a ∼5 pF cell capacitance, gave a recording time constant of 15 µs. All MET currents were smoothed with a Frequency Device LPO3 8-pole filter at 3 kHz. Stereociliary bundles were stimulated with a fluid jet or with a glass probe driven by a piezoactuator. For both types of stimulator, the amplitude of motion was calibrated by projecting the bundle image onto a pair of photodiodes and measuring the change in photocurrent /XS), where XO is the half-saturating displacement and XS is a slope factor; the 10 to 90% working range (WR) is 4.4 × XS. According to the gating spring model, XS. = kBT/Z, where kB is the Boltzmann constant and Z is the gating sensitivity  and outer hair cells (OHCs) in isolated organs of Corti of mice between P2 to P8, as previously documented , 28. Theocurrent , 30. Thesitivity . I–X reltrations . Experim2+  of cochleae of 30-d-old CD1 mice. Maintenance and treatment of mice during preparation was in accord with the UK Animals (Scientific Procedures) Act of 1986. P30 mice were anesthetized with sodium pentobarbitone, decapitated, the cochleae removed and fixed through holes in the round window, and the cochlear apex with 2.5% glutaraldehyde (GTA) in 0.1 sodium cacodylate buffer plus 2 mM CaClechnique . Cochlea2+ permeability  to 53 ± 3 pS   = 4.3 ± 0.4 (n = 8) in +/+;Tmc2−/−Tmc1 and 0.6 ± 0.04 (n = 7) in Tmc1 p.D528N/D528N−/−;Tmc2   = 2.0 ± 0.4 (n = 10) in Tmc1 p.E520Q/E520Q−/−;Tmc2. The similar effects of D528N and E520Q on channel conductance are consistent with these residues being in the external neck of the pore to restrict the influx of ions. The Tmc1 p.D528N/D528N mutation also drastically increased (by 67-fold) the half-blocking concentration of the aminoglycoside antibiotic dihydrostreptomycin , suggesting a tight electrostatic control of access to the pore.Our studies of hair-cell MET channels in mice harboring mutations of Tmc2−/−) A–D. The 4 cells) E and F. Tmc1 p.D569N and Tmc1 p.W554L, which are located at the intracellular interface of TM7, reduced TMC1 expression with no alteration in single-channel conductance , 86.2 ± 8.2 pS , and 87.2 ± 4.5 ; the value for neither mutant was significantly different from control (Tmc1 p.G411R (using amino acid numbering in mouse)  . These eeethoven ] and Tmc;Tmc2+/+ ; the latTmc1 p.M412K mutation on transduction, and the mutation has been claimed to reduce single-channel conductance , but histograms of many traces from this experiment exhibit predominantly an ∼14-pA level (Lower).MET currents from OHCs at the base of the cochlea, where high-frequency sounds are detected, are up to twice as large in amplitude as those at the low-frequency apex A–C. In cfter P12 . The molfter P12 . A possifter P12 , 25, 26 fter P12 D. But arfter P12 D, the mofter P12 E, Upper,pA level E, Lower.The notion of generating large-amplitude channels using multiple channels of smaller conductance is consistent with analysis of MET current noise, which yields almost identical single-channel current amplitudes at both apex and base . This ob−/−Tmie , encouraging us to search for single channels  in +/+;Tmie+/+;Tmc2−/−Tmc1 measured at the same location. To confirm that we were indeed studying TMC1-containing MET channels, we crossed −/−Tmie with Tmc1 p.D528N/D528N mice, which we had already found to reduce the TMC1 channel conductance. MET currents in apical OHCs recorded from Tmc1 p.D528N/D528N−/−; Tmc2−/−; Tmie had a reduced size of −3.3 ± 0.4 pA (n = 4 OHCs). The ratio of the MET channel current in Tmc1 p.D528N/D528N−/−;Tmc2−/−; Tmie relative to +/+;Tmie−/−;Tmc2−/−Tmc1 was 0.63, identical to the ratio of channel current amplitudes of Tmc1 p.D528N/D528N+/+;Tmc2−/−;Tmie to +/+;Tmie+/+;Tmc2−/−Tmc1, which was also 0.63. From these results, we conclude that MET channels can still be gated in the absence of TMIE, but they have a 25% smaller amplitude. A 20% reduction in channel amplitude from 58 pS to 46 pS was reported with the deafness-linked mutation Tmie p.R82C/R82C , but these were 30-fold smaller than the maximum currents of 1,000 pA in +/+;Lhfpl5+/+;Tmc2−/−Tmc1 of similar age (n = 51 channel events in 2 OHCs) at −84-mV holding potential, equivalent to a conductance 75 ± 11 pS. Control recordings in +/+;Lhfpl5+/+;Tmc2−/−Tmc1 mice made at the same cochlear location (d = 0.2) gave a unitary current of 6.8 ± 0.5 pA (120 traces in 5 OHCs). There was no difference between the Lhfpl5 knockout and control . Thus, as with Tmie knockouts, MET channels without LHFPL5 still possess an ion conduction pathway across the membrane that can be opened by hair bundle deflection; such channels also displayed marked adaptation  than the value of 70 ± 8 nm in four +/+;Lhfpl5+/+;Tmc2−/−Tmc1mice. The half-saturation XO was also significantly increased, 153 ± 13 nm in four +/+;Lhfpl5−/−;Tmc2−/−Tmc1 mice compared to 58 ± 8 nm in four +/+;Lhfpl5+/+;Tmc2−/−Tmc1 mice. One explanation for the difference is that the mechanical connection from the tip-link to the MET channel was weakened in the absence of LHFPL5, thereby reducing the gating sensitivity or single-channel gating force  over 60 missense or deletion mutations in the  hearing ; 2) Tmc1 channel , 7 ; 3)  channel  and cryo channel ; 4) the  channel , 54; and channel .n = 19), which are denoted as being components of the transducing staircase , suggesting some channels may be stimulated without direct connection to PCDH15. However, if each 6.7-pA channel corresponds to one half of the dimeric complex , then fo82+ plus BAPTA: if its sole effect is to sever the tip-links, then a minimum of three to four channels should remain, implying BAPTA has some other action, for example on the connection between PCDH15 and LHFPL5. The precise effect of low Ca2+ plus BAPTA remains to be clarified, as do the exact channel conductance values to reconcile the differences in the literature and whether the gradient in channel conductance exists. An alternate method of MET channel isolation, such as recording from cell-attached patches on single stereocilia, might resolve the discrepancies.These calculations of channel numbers also raise concerns about whether the apparent gradient in channel conductance is minor or artifactual. It is conceivable that for high-frequency OHCs, in which the stereocilia are narrower, multiple channels contribute to the records, resulting in conductances of double or triple the unitary value. A further concern is the exact action of low CaTmc1 mutations revealed two mutations, Tmc1 p.D528N and Tmc1 p.E520Q, which each produced about a 35% decrease in single MET channel conductance, arguing that they are located around the entrance of the pore (Tmc1 mutations studied reduced the Ca2+-permeability of the MET channel and, when compounded with a reduced MET current, decreased Ca2+ influx into the hair bundle (2+ permeability. Associated with a severely reduced Ca2+ influx was an early apoptosis of the cochlear hair cells, especially at the base. For example, by P30, Tmc1 p.D528N showed substantial loss of OHCs and IHCs (Tmc1 p.T416K had less hair cell loss at P30, and Tmc1 p.W554L had no hair cell loss until P60. Even though hair cells harboring the Tmc1 mutations exhibited MET currents in the first few postnatal days, all mutations eventually led to loss of transduction and deafness by P30. The decreased Ca2+ influx into the hair bundle is likely to be a major causal factor: it can lead to decreased actin polymerization and shortening or regression of the transducing stereocilia (Tmc1 mutants, OHC bundles are transformed from a typical “V” pattern to a rounded shape by the onset of hearing at P12 (−/−Cib2-lacking MET currents (2+ in determining hair bundle shape is not yet understood.Our studies of hair cell MET currents in mouse harboring the pore B and C. r bundle C. The gland IHCs . In contreocilia , 59, as g at P12 , 28. Discurrents , 16. The


Lactobacillus, a natural and beneficial commensal component of the gut microbiome. First, we used intestinal epithelial cells generated from human induced pluripotent stem cells to confirm that VHH 1E4 showed neutralizing activity against GII.17 norovirus. We then expressed VHH 1E4 as a cell-wall–anchored form in Lactobacillus paracasei BL23. Flow cytometry confirmed the expression of VHH 1E4 on the surface of lactobacilli, and L. paracasei that expressed VHH 1E4 inhibited the replication of GII.17 norovirus in vitro. We then orally administered VHH 1E4-expressing L. paracasei BL23 to germ-free BALB/c mice and confirmed the presence of lactobacilli with neutralizing activity in the intestine for at least 10 days after administration. Thus, cell-wall-anchored VHH-displaying lactobacilli are attractive oral nanobody deliver vectors for passive immunization against norovirus infection.Passive administration of neutralizing antibodies (Abs) is an attractive strategy for the control of gastrointestinal infections. However, an unanswered practical concern is the need to assure the stability of sufficient amounts of orally administered neutralizing Abs against intestinal pathogens  in the harsh environment of the gastrointestinal tract. To this end, we expressed a single-domain Ab  against norovirus on the cell surface of  Noroviruses are non-enveloped RNA viruses that are divided into seven genogroups according to their capsid sequences. The 28 genotypes of viruses in the GI and GII genogroups can infect humans causing brutal illness . Human nlactobacilli  Tween 20 and successively incubated with mouse anti-E-tag antibody   and horseradish peroxidase-labeled goat anti-mouse antibody  . Proteins were detected by using the ECL Plus Western Blotting Detection System  [Two ml of an MRS growth culture from escribed . The memlthcare) . L. paracasei BL23 transformants were grown in MRS broth containing 5 µg/mL erythromycin until the cultures reached an OD600 of 1.0. Samples of the bacterial cultures (400 µL each) were centrifuged. The resulting cell pellets were washed twice with PBS, resuspended in 400 µL PBS containing 1% bovine serum albumin. The cells in 100-µL quantities of this suspension were pelleted again, and the resulting pellet was resuspended in 100 µL PBS containing 1% bovine serum albumin and 1 µg/mL mouse anti-E-tag antibody and incubated on ice for 30 min. After one wash with PBS, the cells were resuspended in 100 µL of buffer containing alkaline phosphatase-conjugated rabbit anti-mouse IgG (1 µg/mL) and then incubated on ice for 30 min. After two washes with PBS, the cells were suspended in 200 µL diethanolamine buffer; 100 µL of bacterial suspension from each clone was added to a well of a 96-well microtiter plate, followed by 100 µL of diethanolamine buffer containing 1 mg/mL p-nitrophenyl phosphate . Plates were incubated for 10–30 min at room temperature, after which the absorbance at 405 nm was read in a Varioskan Flash multimode reader (Thermo Scientific). Lactobacillus paracasei cells containing the empty plasmid (pIAV7) were used as a negative control.For a semi-quantitative assay to verify surface expression of the VHH, Lactobacillus paracasei BL23 transformants were grown in MRS broth containing 5 µg/mL erythromycin until the cultures reached an OD600 of 1.0. The cultures were serially diluted in PBS and plated on MRS plates supplemented with or without 5 µg/mL erythromycin. The plates were incubated at 37 °C for 2 days. The percentage of plasmid persistence was calculated as A1/A0 × 100%, where A1 and A0 are the viable counts of the antibiotic plate and antibiotic-free plate, respectively.L. paracasei strains BL23-pIAV7 and BL23-1E4 were grown in MRS broth containing 5 µg/mL erythromycin in an anaerobic system  until the cultures reached an OD600 of 1.0 and then diluted in buffer to 3 × 106 cells per 50 μL. Neutralization assay of human noroviruses was performed as described previously [6 genome equivalents per 100 µL with and without BL23-pIAV7 or BL23-1E4 (3 × 106 cells) in base medium [2 and 37 °C. The inoculum was then removed, and the IECs were washed three times with 150 µL of base medium. We then added 100 μL of differentiation medium [2 incubator at 37 °C. The supernatants were collected, with one wash, in the same way as the reference samples . The inhibitory activity of L. paracasei BL23-pIAV7 or BL23-1E4 against norovirus replication was shown as the percentage inhibition relative to untreated L. paracasei BL23-pIAV7 or BL23-1E4. Recombinant VHH 1E4 was expressed in E. coli system and purified by the method described previously, and 5 μg of 1E4 was used as a positive control [The eviously . Human ieviously . Human ne medium  containin medium  containi control . L. paracasei BL23-pIAV7 or BL23-1E4 at the Institute of Medical Science of The University of Tokyo. The animal study protocol was approved by the Animal Care and Use Committee of The University of Tokyo .Germ-free female BALB/c mice (4–7 weeks old) were purchased from Japan SLC and used for oral administration of Lactobacillus paracasei BL23-pIAV7 or BL23-1E4 was grown at 37 °C for 15 h in MRS broth containing 5 µg/mL erythromycin in an anaerobic system  without shaking until the culture reached an OD600 of 1.0. The bacteria were counted, and doses of L. paracasei BL23-pIAV7 or BL23-1E4 (1 × 109 cells per 200 mL) were administered orally to germ-free mice (n = 3). L. paracasei BL23-pIAV7 or BL23-1E4; fecal pellets were weighed and added to PBS to achieve 100 mg feces per milliliter of PBS. These mixtures were then diluted 500-fold and 2000-fold by using PBS, and aliquots were spread on MRS agar plates ; plates were incubated at 37 °C for 48 h in an anaerobic system  without shaking. Single colonies of L. paracasei were picked and then grown at 37 °C for 15 h in MRS broth containing 5 µg/mL erythromycin in an anaerobic system (BBL GasPak Anaerobic Systems) without shaking until the cultures reached an OD600 of 1.0. Bacteria were counted, and L. paracasei BL23-pIAV7 or BL23-1E4 (3 × 106 cells per 50 μL) underwent flow cytometry and neutralization assay of human norovirus.For flow cytometry and neutralization assay of human norovirus, feces from germ-free mice were collected at 4, 8, 24, 48, 72, 120, 240, and 336 h (2 weeks) after oral administration of 7 cells were transferred into a 2-mL tube. A total of 2 μL rabbit anti-E tag antibody  was added, and cells were incubated for 30 min at 37 °C. After two wash with PBS, the cells were resuspended in 100 µL PBS containing 2 μL of Brilliant Violet 421-labelled goat anti-rabbit IgG  and then incubated for 30 min at 37 °C. The cells were washed twice with PBS and then fixed in 1 mL of 1% formaldehyde (Sigma–Aldrich) at room temperature. A total of 12.5 μL thiazole orange  was added before bacterial cells were analyzed in a Attune NxT flow cytometer (Thermo Fisher Scientific). The number of live L. paracasei BL23 in the gut of germ-free BALB/c mice after oral administration was calculated as the percentage of E-tag-positive cells per thiazole orange-positive cells.For flow cytometry, bacteria were washed with PBS, and 5 × 10t-tests .Results were compared using unpaired two-tailed Student’s We directly fused VHH 1E4 to the signal peptide, leaving one alanine residue at the N-terminal for correct cleavage of the signal peptide a. We conL. paracasei BL23 cells, expression of VHH 1E4 was verified by Western blot analysis of the culture supernatant and cell fractions  needed for assessment of neutralization activity by the VHH 1E4-expressing recombinant L. paracasei BL23 strain, we changed the antibiotic treatment in the in vitro propagation assay using human iPSC-derived IECs to erythromycin instead of the penicillin–streptomycin originally described. GII.17 norovirus has the ability to survive in low pH  and replicates in iPSC-derived IECs [L. paracasei BL23 pIAV7 (the negative control strain) might result in acidic pH in the cell culture, there was no pH-effect on GII.17 norovirus propagation in human iPSC-derived IEC in vitro when compared with that of PBS ; this dosage was equivalent to 5 μg of recombinant 1E4  environment after oral delivery. To isolate L. paracasei BL23 from feces, we collected feces from inoculated mice from 4 to 340 h for a total of 2 weeks after oral administration and then cultured the samples anaerobically on MRS plates containing erythromycin. This assay confirmed the presence of L. paracasei BL23 in the feces of germ-free mice for as long as 240 h (10 days) after oral administration  norovirus. Because norovirus infection is associated with severe complications in infants, young children, and the elderly, a cold-chain-free lyophilized powder containing live bacteria, when mixed with a suitable excipient, may be useful for oral immunotherapy and prophylaxis against this pathogen. VHH-displaying L. paracasei BL23 represents an attractive approach for the prevention and treatment of norovirus infection in both developed and developing countries. We developed a nanobody-displaying


CBS encodes a pyridoxal 5′-phosphate-dependent enzyme that catalyses the condensation of homocysteine and serine to form cystathionine. Due to its implication in some cancers and in the cognitive pathophysiology of Down syndrome, the identification of pharmacological inhibitors of this enzyme is urgently required. However, thus far, attempts to identify such molecules have only led to the identification of compounds with low potency and limited selectivity. We consequently developed an original, yeast-based screening method that identified three FDA-approved drugs of the 8-hydroxyquinoline family: clioquinol, chloroxine and nitroxoline. These molecules reduce CBS enzymatic activity in different cellular models, proving that the molecular mechanisms involved in yeast phenotypic rescue are conserved in mammalian cells. A combination of genetic and chemical biology approaches also revealed the importance of copper and zinc intracellular levels in the regulation of CBS enzymatic activity—copper promoting CBS activity and zinc inhibiting its activity. Taken together, these results indicate that our effective screening approach identified three new potent CBS inhibitors and provides new findings for the regulation of CBS activity, which is crucial to develop new therapies for CBS-related human disorders. The standard solutions and cell lysate samples (20 µL) were then extracted with 100 µL of methanol and 50 µL of the IS solution. The samples were mixed and centrifuged at 10,000× g at 10 °C for 15 min to remove the precipitated proteins. The supernatants were collected and dried under a gentle stream of nitrogen (45 °C). The derivatization step was performed by dissolving the dried extract in 100 µL of a freshly prepared butanol solution containing 5% acetyl chloride and kept at 60 °C for 30 min. The solvent was then removed under a gentle stream of nitrogen (60 °C). The dried samples were dissolved in 100 µL of water containing 0.1% formic acid and 10 mmol/L TCEP (to reduced the disulphide bond of cysteine) and injected into the LC-MS/MS system. Samples (10 µL) were injected onto an Acquity BEH-C18 column  held at 60 °C, and compounds were separated with a linear gradient of mobile phase B (0.1% formic acid in methanol) in mobile phase A (0.1% formic acid in water) at a flow rate of 400 µL/min. Mobile phase B was kept constant at 1% for 0.5 min, linearly increased from 1% to 95% for 4.5 min, kept constant for 1 min, returned to the initial condition over 0.5 min, and kept constant for 1.5 min before the next injection. Targeted compounds were then detected by the mass spectrometer with the electrospray interface operating in positive ion mode  flow, 650 L/h; desolvatation gas temperature, 350 °C; source temperature, 120 °C). Multiple reaction monitoring mode was applied for MS/MS detection, as detailed in Cystathionine, methionine, 2, 10 mM Hepes, 2.7 mM KCl, 1 mM K2HPO4, 1.8 mM CaCl2 pH7.4 supplemented with 10 mM glucose) containing 100 µM of 7-azido-4-methylcoumarin (AzMC) fluorescent probe (Sigma), which selectively reacts with H2S to form a fluorescent compound. Fluorescent AzMC signal acquisition (λEx = 365 nm and λEm = 450 nm) was performed on a Flexstation 3 microplate reader using SoftMax Pro 5.4.5 software . Values were expressed as a percent of the corresponding controls.Following a 24 h treatment, cells were washed once with 1X PBS and incubated for 2 h in a saline buffer  was performed on a Flexstation 3 microplate reader with SoftMax Pro 5.4.5 software . Values were expressed as a percent of the corresponding controls.The cytotoxicity of all tested compounds was examined using a cell counting kit . Briefly, following measurement of HEx = 365 nm and λEm = 450 nm) was performed on a Flexstation 3 microplate reader with SoftMax Pro 5.4.5 software .HepG2 transfection and cell lysis were performed as described above. The equivalent of 50 µg of total proteins was then incubated with CBS substrates (10 mM cysteine and 10 mM homocysteine) and with 25 µM of the fluorescent probe AzMC diluted in 100 mM Tris–HCl pH 8 along with 50 µM of the tested compound for 2 h. AzMC fluorescent signal acquisition  was added at the same time as the drugs. Phase contrast and fluorescent images were collected with the 20× lens every 2 h for 48 h using phase contrast of the white light channel and the green fluorescence channel (300 ms exposure). Cell proliferation was assessed by determination of cell confluency analysed on the phase-contrast images captured every 2 h. Toxicity was assessed as the intensity of fluorescence of cells loaded with cytotox dye normalized to confluency at each measurement time point. Images were analysed and data were generated using Incucyte 2019B Rev2 analysis software, Sartorius, Goettingen, Germany).2S production measurement. Reactive oxygen species (ROS) levels in HepG2 cells were monitored using the cell-permeable reagent 2′-7′-dichlorofluorescein , which is oxidized by ROS to form a fluorescent compound. Briefly, following drug treatment, cells were incubated with 10 µM DCFDA for 30 min. Tert-butyl hydroperoxide  or H2O2 were used as positive controls. Fluorescent signal acquisition (λEx = 485 nm and λEm = 535 nm) was performed on a Flexstation 3 microplate reader with SoftMax Pro 5.4.5 software .Cells were plated in Greiner Bio-One  black 96-well plates with transparent flat bottoms and treated as previously described for HStatistical analysis was performed using GraphPad Prism software 8.0.2 . Results present data obtained in a representative experiment from at least 3 independent experiments, except for amino acid dosage results, which are presented as the mean of at least 3 independent experiments.