A geological record of VPD is needed for paleoclimate studies of past greenhouse spikes which attempt to constrain future climate, but at present there are few quantitative atmospheric moisture proxies that can be applied to fossil material. Here we show that VPD leaves a permanent record in the slope (S) of least-squares regressions between stable isotope ratios of carbon and oxygen (13C and 18O) found in cellulose and pedogenic carbonate. Using previously published data collected across four continents we show that S can be used to reconstruct VPD within and across biomes. As one application, we used S to estimate VPD of 0.46 kPa ± 0.26 kPa for cellulose preserved tens of millions of years ago—in the Eocene (45 Ma) Metasequoia from Axel Heiberg Island, Canada—and 0.82 kPa ± 0.52 kPa—in the Oligocene (26 Ma) for pedogenic carbonate from Oregon, USA—both of which are consistent with existing records at those locations. Finally, we discuss mechanisms that contribute to the positive correlation observed between VPD and S, which could help reconstruct past climatic conditions and constrain future alterations of global carbon and water cycles resulting from modern climate change.The drying power of air, or vapour pressure deficit (VPD), is an important measurement of potential plant stress and productivity. Estimates of VPD values of the past are integral for understanding the link between rising modern atmospheric carbon dioxide (pCO Changes in VPD reflect the potential for the atmosphere to extract water from terrestrial ecosystems. VPD is often monitored as a proxy for plant water stress because it is a principal control on stomatal water loss and photosynthetic carbon fixation2. VPD is not a meteorological parameter for climate studies because it is a relative metric of stress that varies among plant species, as inferred from their leaf functional traits, and from interactions between roots, soils, and microorganisms in the rhizosophere, which together govern responses to climate at local to global scales5. However, VPD does reflect the effect of temperature and precipitation on relative humidity6 and transpiration demand, which stimulates stomatal closure to minimize water loss, and thus the flow of water and nutrients from the soil through plants and ultimately to the atmosphere8. Stomatal closure in turn affects carbon (C) isotopes of plant cellulose11, which decays into soil organic matter and respired CO2, and as a result the ratio of stable carbon isotopes (δ13C) can be passed on to pedogenic carbonates9. Oxygen (O) isotopes are also impacted by VPD, responding as a function of stomatal closure as well as independently of transpiration demands, and stable oxygen isotope ratios (δ18O) can be used to isolate VPD-imposed stress from other environmental factors that control δ13C of cellulose13. Numerous processes are known to affect the fractionation of C and O isotopes in plants and soils. Here, we explore the mechanisms that relate VPD with changes in δ13C and δ18O measured in cellulose and pedogenic carbonates and make a case for using those ratios as a proxy for climatic conditions.Vapour pressure deficit, or VPD, is the difference between the amount of moisture in the air and how much moisture the air can hold when it is saturated, with the latter depending on ambient temperature13C values in C3 plants reflect the ratio of intercellular (ci) to atmospheric (ca) partial pressure of CO2 and CO2 fixation by RuBisCo, which yield δ13C fractionations of − 4.4‰ and − 27‰, respectively14  while the ratio of ci and ca is approximately related to δ13C values recorded in cellulose and other compounds. Decreased stomatal conductance combined with evaporative enrichment of leaf water 18O causes both 13C and 18O to increase simultaneously in cellulose  at sites with markedly different soil moisture indices. The difference in slope values between species indicated a stronger dependence on ci/ca which suggests a species-dependent relationship between slope and VPD, e.g., Fagus reacted more strongly in terms of stomatal downregulation of gas exchange to moisture conditions than did Picea17. Consistent with Saurer et al., a mechanistic model proposed by Barbour et al. can be used to relate slope of the δ18O:δ13C relationship to annual VPD in cellulose of Pinus trees under varying stomatal conductance (gs) and photosynthetic capacity (Vcmax)8. Here, we summarize the main outputs of that model  is identical to the slope found in P. sylvestris by Saurer et al.17 which support the hypothesis that δ13C:δ18O slope can be used to infer VPD-induced stress for different species of conifers.Previous studies have identified possible mechanisms by which δdel Fig.  to illus12 predict with a conceptual model the occurrence of negative slope between δ13C and δ18O when ci, gs and δ18O increase (while δ13C decreases and Vcmax is held constant), or when Vcmax decreases and gs is held constant. On the other hand, drought-induced changes in stomatal conductance (high VPD) increase both δ13C  and δ18O (via changes to ca/ci), so positive correlations between δ13C and δ18O are expected for time-series data from modern tree-ring cellulose23. Moreover, a positive linear relationship implies that ci/ca depends linearly on ei/ea and is influenced by changes in VPD17. Indeed, experimental studies have shown that ci/ca decreases linearly with increased VPD in C3 plants24 which supports the hypothesis that VPD controls the slope value of δ13C and δ18O during periods of water stress when other factors are held constant. Building on those findings, we examined the relationship between δ18O and δ13C slope and annual average VPD, and then compared our data compilation to the modelled response to VPD flux  when both gs and Vcmax varies while gs varies over a limited range  as well as Larix sibirica and Tsuga canadensis  forms in soil where potential evaporation exceeds evapotranspiration, most often in arid to subhumid regions which receive less than ~ 100 cm of precipitation annually26. The sources of C in pedogenic carbonate are from autotrophic root-respired CO2, heterotrophic decomposition of organic matter by soil microbes and from the diffusion of atmospheric CO2 into the soil matrix28  the proportion of C3–C4 plants growing at the site; (2) root and microbial respiration rates, which are sensitive to changes in VPD; and (3) the CO2 concentration of the atmosphere29. The carbon isotopic signature of water stress in C3 plants is passed on to soil-respired CO2 because the original δ13C isotope composition of the plant community is preserved (± 2‰) in soil-respired CO2 generated during aerobic decay of soil organic matter30. Soil-respired CO2 then equilibrates with soil water to form pedogenic carbonate during seasonal drying of the soil31.Changes in VPD are expected to cause changes in δ32. Pedogenic carbonate is assumed to be in O isotopic equilibrium with soil water and thus carbonate δ18O values are used to constrain paleotemperature and/or paleoelevation34. Oxygen isotope ratios of pedogenic carbonate do not carry a plant signal because plant compounds show little O isotopic exchange with soil water during decomposition35. Therefore, the decay of cellulose into soil organic matter and respired CO2 is expected to pass the carbon isotopic signature of the plant community to pedogenic carbonates  are strong predictors of VPD, such that S may be used to infer climatic conditions at spatiotemporal scales that go beyond those of tree-ring studies. Given that profound changes in atmospheric moisture are predicted with climate change7, VPD records would be useful for inferring past climate conditions and reducing uncertainties in future climate projections. We posit that atmospheric VPD is preserved in isotope ratios of soil carbonate, just as in cellulose, such that suitably preserved fossil wood and paleosol carbonate can be used as a proxy for VPD of past environments. As proof of concept, we use previously published data to develop S-to-VPD transfer functions using fossil cellulose from Arctic Metasequoia during the Eocene39 and pedogenic carbonate formed during the Oligocene in calcareous paleosols from Oregon40, both of which show a positive correlation between δ13C and δ18O similar to those found for modern cellulose and carbonate samples.In the following sections, we show that cellulose and carbonate δP < 0.05) correlation between δ13C and δ18O measured in α-cellulose isolated from individual trees (needle-bearing taxa only) from 1950-present which had n > 8 data points and met model assumptions for simple linear regression. For carbonate we considered datasets reporting positive and significant correlation between δ13C and δ18O in nodular calcite gathered from individual soil profiles which also had n > 8 data points and met assumptions for simple linear regression (see Methods for details). A positive correlation between δ13C and δ18O was found when annual average VPD  exceeded ~ 0.3 kPa. Non-significant (P > 0.05) and/or negative correlations of δ13C and δ18O were noted in cases where VPDannual was less than ~ 0.3 kPa and/or when original authors noted that drought stress was not a significant factor influencing isotope ratios . The slope of the δ13C:δ18O relationship (S) in both modern plant cellulose (Sc) and pedogenic carbonate (Sk) is correlated with VPDannual of the contemporary atmospheric systems . The coefficient of determination of the correlation between Sk and VPDannual according to Eq. . Additionally, a negative correlation between Sk and annual relative humidity was observed  tree-ring cellulose of Metasequoia from Axel Heiberg Island, Nunavut, Canada10 was 0.55 . This gives paleo-VPD values of 0.46 kPa ± 0.26 kPa  Piceoxylon from Lac De Gras, Canada , which indicates a paleo-VPD estimate of 0.30 kPa ± 0.26 kPa. It should be noted that the original authors concluded that if the first 8 “juvenile” tree rings are excluded from the analysis the remaining samples are not significantly correlated, so caution is necessary in interpreting this dataset. Nevertheless, as a second test of the VPD proxy the Piceoxylon dataset, which included juvenile rings predicted VPD of 0.30 kPa ± 0.26 kPa, which is consistent with the mean annual temperature estimate of 11.4 °C ± 1.8 °C derived from transfer functions and modeled RH values ranging from 64 to 83%23 for early Eocene polar forests. The propagated error for the transfer function is nearly as large as the estimate for VPD and the correlation coefficient is low so, here too, cautious interpretation is necessary, but assuming atmospheric CO2 of 915 ppmv44 and low VPD across Arctic Canada during that period, we conclude that our record captured the early Eocene “hothouse” climate described in previous studies.The transfer functions generated from modern tree-ring cellulose isotope training datasets Eqs.  and 2 ca Eocene 4 Ma tree-da Table  show Sc 40 had Sk of 0.82 . This gives a VPD of 0.82 kPa ± 0.52 kPa for late Oligocene (26 Ma) calcareous paleosols of the Turtle Cove Member of the John Day Formation in central Oregon  VPD was derived from pedogenic carbonate from the Hannold Hill Member of the Tornillo Formation, Big Bend National Park, Texas, USA47. The Sk was 0.444  which allowed for a paleo-VPD estimate of 0.56 kPa ± 0.52 kPa. Propagated error (± 0.52 kPa) was nearly as large as the VPD estimate of this early Eocene sample so caution with this estimate is also necessary, but a lower Sk value in this sample compared to the Oligocene example discussed above supports the hypothesis that early Eocene VPD was less than Early Oligocene VPD. The Tornillo Formation is assumed to be to be North America’s most southerly exposure of early Paleogene continental deposits and as such cannot be directly compared to Oligocene VPD because of differences in latitude and age. However, the predicted VPD of the Tornillo Formation sample is an estimate consistent with the conclusion of the previous study47 about a decrease in humidity, precipitation and temperature after the Paleocene-Eocene Thermal Maximum (PETM), which is thought to have decreased the production of kaolinite and increased the accumulation of calcite in these paleosols compared to the older underlying kaolinite-rich PETM paleosols.Late Oligocene (26 Ma) pedogenic carbonate from the Turtle Cove Member of the John Day Formation in central Oregon13C and δ18O regression slopes (S) occur in response to increasing aridity, assessed as increasing VPD, in both fossil plant samples and in paleosols that were buried tens of millions of years ago. Positive correlations between δ13C and δ18O ratios found in cellulose samples, which reflect species or genus-level responses to VPD, are similar to those found for pedogenic carbonate samples, which reflect ecosystem-scale responses to VPD. Taken together, our compiled data indicate that changes in S identified for both modern and paleo samples are directly related to VPD, and thus S may be used to constrain climatic conditions at spatiotemporal scales that go beyond those of tree-ring studies.Using a compilation of isotopic data gathered from modern cellulose and carbonate samples we found a persistent record of vapour pressure deficit (VPD) preserved in plants and soils across four continents. A remarkably consistent shift in δ13C and δ18O relationship to vary with atmospheric moisture deficit in pedogenic carbonate? Laboratory studies of pedogenic calcite precipitation under variable temperature and relative humidity conditions show that δ13C and δ18O slope is steeper under both higher temperature and low relative humidity during elevated CO2 concentrations38. Soil temperature, relative humidity, soil CO2 concentration and the saturation state of evaporating fluids (with respect to CaCO3) are factors that determine trends in the positive linear correlation of δ13C and δ18O in pedogenic carbonate38. In this way, slope steepness is increased with high evaporation rates and reduced with lower evaporation rates  caused by the combined effect of physiological fractionation and root contribution to the soil carbon pool. However, it should be noted that slope is strongly dependent on the timing of calcite precipitation during fluid evaporation , and the steepest slopes in laboratory-precipitated calcite are from samples with the greatest soil CO2 concentrations and evaporation rates38 both of which are highly variable in the vadose zone during pedogenic carbonate formation. Despite these uncertainties, a recent dual-isotope mechanistic model of natural pedogenic carbonates show that specific covariance of δ13C and δ18O can result from a shared climatic driver like VPD, which is responsible for the change in both isotope systems48. Here, we find further evidence of that relationship with nearly identical slopes found for modern cellulose and carbonate data  should be considered. Careful sampling of paleosol carbonate with micrite concentrations of 70% or greater can ensure measurement of primary and not diagenetic δ18O values56.Alteration after burial may compromise application of these transfer functions to fossils. For example, δ13C:δ18O ratios is complex and many processes are at play, most notably photosynthetic capacity, stomatal conductance, RuBisCO fractionation, and the amount, type and timing of water inputs, all of which have been shown to alter δ13C values or δ18O values or both57. We therefore do not expect that a single mechanism would adequately explain the consistent increase in the isotope ratios in plant molecules and pedogenic carbonates. As predicted by theory, plants and soils respond differently to VPD , which is to be expected given the large variation in source water across time and space. Biochemical oxygen isotope fractionation during cellulose synthesis can vary between 26‰ and 31‰ depending on temperature and VPD61, but another potential source of variation could possibly result from RuBisCO fractionation4 (− 27‰), which may select light isotopologues of CO2 for chemical reduction regardless of whether CO2 is enriched or depleted with respect to heavy C or O isotopes63. This “ternary effect” 63 is expected to be maximized when the leaf-to-air vapour mole fraction difference is greatest and the effect is thought to be most pronounced on factors derived by the difference, most notably mesophyll resistance to CO2 assimilation63. In this scenario, light isotopes of both C and O may be selected simultaneously which could theoretically contribute to correlations between δ13C and δ18O in cellulose. However, the potential net fractionation effect of this process should be much smaller than the large effect of VPD on evaporative enrichment. Indeed, a fractionation of − 4.4‰ is produced by stomatal resistance to diffusion of CO2 from the air into leaves4 , rather than completely submerged during growth67. Additionally, trees like Metasequoia cannot form woody coals unless their roots are aerated as well as their leaves68. Positive covariance of δ13C and δ18O in the Metasequoia sample implies that both roots and leaves were coupled to the atmosphere and thereby suitable for paleo-VPD estimation.Finally, it is important to note that the use of ant Fig. . These s13C and δ18O in response to VPD which is recorded in modern and fossil cellulose and carbonate samples. The most likely mechanisms that contribute to the correlation of δ13C and δ18O under varying VPD in plants are changes to stomatal conductance and evaporative enrichment of leaf and soil water. A third possible contribution is from leaf-level RuBisCO selection of light isotopologues of CO2 when the isotopic composition of the ambient air is significantly different from inside the leaf, although that effect appears to be small and unlikely to vary with VPD. Together, our results suggest that the slope of δ13C and δ18O regressions in modern cellulose and pedogenic carbonate is directly related to VPD, and thus δ13C:δ18O slope may be used to infer paleo-VPD conditions at spatiotemporal scales that go beyond those of tree-ring studies. This hypothesis is supported by a comparison of our S-to-VPD transfer functions applied to two fossil sites for which climate reconstructions have been previously reported. Our findings highlight the interconnectivity of the soil–plant–atmosphere system in response to atmospheric water deficit and could pave the way for the use of well-preserved fossil wood and pedogenic carbonate to estimate VPD during past climates and to improve Earth system models and their predictions of future climate.A compilation of previously published data reveals positive correlations between δ3 plant cellulose21 and modern soil carbonate22  and climate data from previously published isotopic studies of plant cellulose (n = 23) and pedogenic carbonate (n = 31) from around the world (Supplementary data). Cellulose was chosen in this study because cellulose is commonly preserved in the fossil record43, and because cellulose reflects overall trends in bulk soil organic matter variation across ecosystems64. Pedogenic carbonate was chosen because it is also widely observed and analyzed in the fossil record of soils34.We use modern climate records Table  along wi18 and ecosystem48 levels, for which fractionation steps have been mechanistically described  positive correlation between δ13C and δ18O and met all assumptions for simple linear regression . Datasets that passed all criteria were included in the transfer function dataset  and carbonate n = 13) stable C and O isotope datasets Tables  and S4 t3 stable ed Figs. . For celset Fig. . For cardy Table .annual,], MAThigh and MATlow) as provided by the original authors or gathered from the closest weather station to each location , MAT, and saturation vapour pressure  and displayed in kPa using the following formula2We use modern meteorological data  was computed for both cellulose and pedogenic carbonate datasets that met inclusion criteria and model assumptions for ordinary least-squares regression.A partial-least squares regression was performed on each modern dataset, and the slope of the δ18O:δ13C slope and annual average VPD on the modelled slopes versus VPD calibrated for P. radiata reported by Barbour et al.8 , whereas δ13C:δ18O slopes (S) are used as previously suggested for paleo-VPD estimates. We then fit least-squares regressions to the scenarios proposed by Barbour et al.8 to get slopes of 0.11 when gs alone varies, 0.17 when both gs and Vcmax vary, and 1.34 when gs and Vcmax vary while gs varies over a limited range  the standard error of modern VPD measurements when calculated from average climate statistics (± 0.13 kPa)42; and C) the standard error of the transfer functions. We compared the δ13C:δ18O relationship in modern and fossil samples  there was a positive correlation between δ13C and δ18O; B) it met all the inclusion criteria  le Table  for compSupplementary Information.Supplementary Information.


Escherichia coli). The study aimed to verify the protective capacity of a CE culture in broilers using the seeder bird model against EEC exposure of the chicks.Administration of a competitive exclusion culture (CE culture) has the potential to induce protective effects in very young chicks against caecal colonisation by EEC (= extended-spectrum β-lactamases [ESBL] and AmpC-type [AmpC] beta-lactamases producing 10 units (P < 0.05) on day 9 of life to 2.5–3.0 log10 units (P < 0.05) on day 37. A slightly higher protective level of the CE culture in layer birds than in broilers raises the question on reasons for possible differences in the efficacy of CE culture in broiler and layer breeds. Whether the diet’s protein content has an impact on both normal intestinal flora composition and the efficacy of CE cultures against EEC or other pathogens remains open and needs further elucidation.Introduction of infected seeder birds resulted in rapid and strong caecal colonisation of four different EEC challenge strains tested in untreated contact broilers. Compared to controls the broilers pre-treated with the CE culture showed a considerable decrease in caecal load of different EEC challenge strains from about 3.0–3.5 logOur findings suggest that CE cultures of undefined composition can be valuable to reduce the intestinal colonisation by EEC in newly hatched broilers. Escherichia coli) in the broiler production [The high prevalence worldwide of EEC  resulted in a reduced (P < 0.05) caecal colonisation of the EEC challenge strains at all days of examination until the end of the experiment .The chickens used in this study were shown to be free of EEC strains after hatching. In experiment 1, introduction of 4 White Leghorn seeder birds infected with EEC 1478 N (group 2) and 1479 N (group 4) into a group of 20 untreated control birds resulted in rapid and very high-level caecal colonisation of both EEC challenge organisms in the untreated contact birds  on day 37 of life the broilers. The total caecal count of EEC challenge strains 1478 N and 1479 N in pre-treated broilers . To better verify the results obtained in broilers, experiment 3 was carried out under identical conditions using further EEC strains for challenge. Four Ross 308 broiler seeder birds infected with EEC 1475 N or 1476 N were placed each in a group of pre-treated or untreated contact broiler chickens on day 2 of life of the birds (Table 10 units) than in experiment 2. Pretreatment of the contact chicks with the CE culture (groups 1 and 3) resulted in a reduced (P < 0.05) caecal colonisation of the EEC challenge strains at all days of examination until the end of the experiment (Table P < 0.05) difference between treated and untreated chickens amounted also in this broiler experiment to not more than 2.0–2.5 log10 units between days 16 and 37 of life. Also in this experiment, the EEC challenge strains were not detected in liver of the broilers neither in untreated nor in pre-treated chicks throughout the whole trial (data not shown). The observed considerable increase in the difference in caecal counts of EEC challenge organisms between pre-treated and untreated White Leghorn birds from day 9 to day 37 of life (experiment 1) was not seen in Ross 308 broiler chickens (experiments 2 and 3).Using the same experimental design, 4 Ross 308 broiler seeder birds (experiment 2) infected either with EEC 1478 N or 1479 N were placed each in a group of pre-treated or untreated contact broiler chickens on day 2 of life of the birds  and Ross 308 broiler chicks  were hatched at the facilities of the Friedrich-Loeffler-Institute. After hatching, samples from the incubators  were collected and examined for the occurrence of EEC exactly as described , 30. Exp5 cfu/ bird via crop instillation in all experiments. Doses for infection were adjusted as described, all strains had been stored in a Cryobank system (Mast Diagnostica) at − 20 °C [A commercially available CE culture  was used to pretreat the birds on day 1 of age after dissolving and applied via crop instillation . Four EE − 20 °C .5 cfu per bird on day 1 of age. In experiment 2, two groups of 20 Ross 308 broiler contact chickens each were pre-treated on day 1 of life with the CE culture  from a sample proven to be positive only after enrichment was rated as log10 = 1.0. A sample which demonstrated no EEC growth after enrichment was rated as log10 = 0. Data were calculated using multifactorial variance analysis followed by “Multiple Range Test”. The factors taken into account were group and time. P values < 0.05 were regarded as statistically significant .Viable bacterial counts were transformed into logarithmic form. For statistical purposes a viable count of log


Type 2 diabetes mellitus (T2DM) is believed to be associated with low-grade chronic inflammation. The current study aims to investigate the involvement of leptin and TNF-α) is a cytokine that is released by adipocytes and inflammatory cells in response to chronic inflammation. Type 2 diabetes mellitus (T2DM) is believed to be associated with low-grade chronic inflammation. The current study aims to investigate the involvement of leptin and TNF-Methodology. This is a cross-sectional study involving 63 healthy volunteers and 65 patients with T2DM. Body composition was measured, and fasting venous blood samples were analyzed for blood glucose, glycosylated hemoglobin (HbA1c), basal insulin, leptin, and TNF-α) is a cytokine that is released by adipocytes and inflammatory cells in response to chronic inflammation. Type 2 diabetes mellitus (T2DM) is believed to be associated with low-grade chronic inflammation. The current study aims to investigate the involvement of leptin and TNF-α) is a cytokine that is released by adipocytes and inflammatory cells in response to chronic inflammation. Type 2 diabetes mellitus (T2DM) is believed to be associated with low-grade chronic inflammation. The current study aims to investigate the involvement of leptin and TNF- Diabetes and obesity are very common associated metabolic disorders that are linked to chronic inflammation. Leptin is one of the important adipokines released from adipocytes, and its level increases with increasing body mass index (BMI). Tumor necrosis factor alpha (TNF-α) is a cytokine that is released by adipocytes and inflammatory cells in response to chronic inflammation. Type 2 diabetes mellitus (T2DM) is believed to be associated with low-grade chronic inflammation. The current study aims to investigate the involvement of leptin and TNF-p=0.008). In obese diabetic patients, the serum level of TNF-α) is a cytokine that is released by adipocytes and inflammatory cells in response to chronic inflammation. Type 2 diabetes mellitus (T2DM) is believed to be associated with low-grade chronic inflammation. The current study aims to investigate the involvement of leptin and TNF-p=0.008). In obese diabetic patients, the serum level of TNF-p=0.008). In obese diabetic patients, the serum level of TNF-α) is a cytokine that is released by adipocytes and inflammatory cells in response to chronic inflammation. Type 2 diabetes mellitus (T2DM) is believed to be associated with low-grade chronic inflammation. The current study aims to investigate the involvement of leptin and TNF-r = 0.361, p=0.008). In obese diabetic patients, the serum level of TNF-r = 0.361, p=0.008). In obese diabetic patients, the serum level of TNF- Our study showed a significantly higher level of TNF-α is associated with concurrent obesity and T2DM and correlates with HbA1c. This suggests that TNF-α needs further investigation to explore if it has a role in monitoring the effectiveness of management in individuals with obesity and T2DM.α) is a cytokine that is released by adipocytes and inflammatory cells in response to chronic inflammation. Type 2 diabetes mellitus (T2DM) is believed to be associated with low-grade chronic inflammation. The current study aims to investigate the involvement of leptin and TNF-α) is a cytokine that is released by adipocytes and inflammatory cells in response to chronic inflammation. Type 2 diabetes mellitus (T2DM) is believed to be associated with low-grade chronic inflammation. The current study aims to investigate the involvement of leptin and TNF- TNF- Among the most fundamental systems required for survival are metabolic and immune systems, and there is a strong integration between regulation of metabolism and the immune responses. Chronic inflammation due to abnormal productions of cytokines and activation of inflammatory signaling pathways is closely associated with metabolic disorders such as obesity, insulin resistance, and T2DM . Obesityα. It has been reported that TNF-α reduces the expression of insulin-regulated glucose transporter type 4 (GLUT4) which is located mainly in adipocytes and skeletal and cardiac muscles [α by inducing serine phosphorylation of insulin receptor substrate-1 can act as an inhibitor of peripheral insulin action which leads to insulin resistance [α which showed a positive correlation with insulin resistance [α plasma level is related to the amount of visceral fat and is not instantly affected in poorly controlled diabetic patients by acute lowering of blood glucose level [The first proinflammatory cytokine recognized for its involvement in pathogenesis of insulin resistance and T2DM was TNF-sistance , 6. In ose level .Research about the role of TNF-alpha in obesity and diabetes is not settled yet and is still a subject of active research work. Prior studies showed conflicting results about the correlation of obesity with TNF-alpha level. A recent systematic review and meta-analysis investigated the association between TNF-alpha and type 2 diabetes mellitus and concluded that there is considerable heterogeneity between studies and further work is needed . Additio2.In this study, we aim to look into the correlation between TNF-alpha, insulin resistance, and HBA1c level in addition to the association between TNF-alpha, obesity, and diabetes. Specifically, we will investigate the effect of obesity and diabetes separately on the level of TNF-alpha, and we aim to recruit participants with BMI higher than 30 kg/mIn this cross-sectional study, we recruited healthy employees and patients with T2DM from the primary care clinics at King Khalid University Hospital, Riyadh, Saudi Arabia. The control group was evaluated by detailed history, clinical examination, and investigations. Patients with complications of T2DM such as nephropathy, neuropathy, and cardiovascular incidents were excluded from the study. All T2DM patients included in the study were receiving oral hypoglycemic agents, and 18 (24%) patients were on lipid lowering agents. Any patient with pregnancy and using glucocorticoids or oral contraceptive pills was excluded.Body composition was measured using the body composition analyzer . The following measurements were taken for all subjects: body mass index (BMI), percentage of body fat (BF%), lean body mass, and waist-hip ratio (WHR). Before those measurements were taken, the subjects were advised to fast for 10 hours and allowed to rest for 30 minutes. Palms and soles were cleaned with electrolytes tissue, and information about subjects' height, sex, and age was fed to the machine. The subject was asked to stand with barefoot on the platform of the machine.Fasting venous blood samples were analyzed for blood glucose, glycosylated hemoglobin (HbA1c), basal insulin, leptin, and tumor necrosis factor alpha (TNF). HbA1c was measured by the Helena Glyco-Tek Affinity Column method .α immunoassays were performed by the quantitative standard sandwich ELISA technique using monoclonal antibody specific for these parameters with kits supplied by R&D Systems . The indices of basal insulin resistance and beta-cell function were assessed using the homeostasis model assessment (HOMA-IR and HOMA-B) in which HOMA-IR (mmol/L × μIU/mL) = fasting glucose (mmol/L) × fasting insulin (μIU/mL)/22.5 and HOMA-B = fasting insulin (μIU/mL × 20)/(fasting glucose (mmol/L) − 3.5).Insulin, leptin, and TNF-a and Shapiro–Wilk tests were used to see whether data follow normal distribution or not. Those parameters which were not following normal distribution were analyzed by nonparametric Mann–Whitney U test. For continuous data with normal distribution, Student's t-test was used. Correlation between TNF-α, HbA1c, and markers of insulin resistance was determined by simple regression analysis.The data were analyzed by the computer software program Statistical Package for Social Sciences . Descriptive characteristics of the study patients were expressed as mean ± SD (standard deviation). Kolmogorov–SmirnovThe study was approved by the ethical committee of institutional review board of College of Medicine, and it was conducted at the Department of Physiology, College of Medicine, King Saud University, Riyadh. All participants signed a consent form after the study protocol was explained to them, and it was made clear to all subjects that they can withdraw at any time if they want to do so.p=0.005). Patients with T2DM have a higher value of waist to hip ratio when compared to healthy controls . TNF-α level was significantly higher in T2DM patients than in controls , while the difference was not significant for leptin   and sixty-three were healthy control subjects . Clinical and demographic characteristics were compared between controls and T2DM patients . Age ranp=0.331) .α levels in obese diabetic patients were significantly higher than in nonobese diabetic patients (p < 0.018). The obese diabetic patients have significant higher serum TNF-α levels than the obese nondiabetic group (p < 0.001) as shown in Serum TNF-α correlated positively with HbA1c  and HOMA-IR , indicating a significant relationship with glycemic control and insulin resistance (In patients with T2DM, the level of TNF-sistance .α level was significantly higher in T2DM patients compared to healthy subjects while the difference between the two groups was not significant for leptin. The level of TNF-α has a strong positive correlation with HbA1c and was positively associated with insulin resistance. These findings suggest that TNF-α plays an important role in the pathogenesis of T2DM via mechanisms related to insulin peripheral action independent of leptin. Our patients have higher insulin resistance indices and a lower beta-cell function than control subjects.Our study demonstrated that the serum TNF-α on adipose tissue and increased peripheral glucose uptake after neutralizing TNF-α in obese rats indicate its important role in development of insulin resistance and diabetes as a consequence of obesity [α contributes to insulin resistance and T2DM that is associated with obesity [α levels in obese diabetic patients compared to nonobese diabetic patients. Additionally, we reported a higher TNF-α level in obese diabetic patients in comparison with obese nondiabetic healthy subjects.The catabolic effect of TNF- obesity . Previou obesity –13; howe obesity . In our α levels showed a significant positive correlation with insulin resistance. On the other hand, leptin level was not higher in our patients than in control subjects and its correlation with insulin resistance is weak. Owecki and coworkers performed a linear regression test for BMI and leptin to find the threshold of BMI at which serum leptin level starts increasing, and they reported a sudden increase in leptin levels at a BMI of 24.6 kg/m2 which indicates that some metabolic changes occur before reaching obesity levels [The majority of diabetic patients in the current study have BMI above 30, and their elevated TNF-α, the higher their HbA1c value, which means that TNF-α level can be used to predict glycemic control in obese diabetic patients. Mirza et al. showed that diabetes was strongly associated with elevated levels of TNF-α, which was most significantly elevated in the group of patients with HbA1c values higher than 6.5% [α level. Since HbA1c is not a direct measure of glycemia, there is a chance that its level might change due to factors unrelated to blood glucose levels such as rate of glycation and turnover of erythrocytes [α, were strongly associated with increased risk to occurrence of T2DM [α level decreases with good glycemic control would confirm this hypothesis. Interestingly, a previous study showed a significant reduction in TNF-α serum level in obese T2DM patients following 4 weeks of treatment with diet and exercise [α level and HbA1c value after regular exercise in healthy women [α and HbA1c might be useful clinically to follow up the level of glycemic control in patients with T2DM after medical treatment or changed lifestyle or in cases where HbA1c cannot be trusted due to defect in glycation or severe anemia.We found that in patients with T2DM, the higher the level of TNF-han 6.5% . In our hrocytes . Recentlexercise . Tsukui hy women . TheoretLimitations. This is a cross-sectional study with a limited number of patients. A longitudinal study with categorization of patients based on HbA1c and BMI would help in confirming our findings. Follow-up studies after good glycemic control to measure the improvement in beta-cell function and its association with decreased TNF-α level are our future plan.α in diabetic patients correlates with the level of insulin resistance and the glucose metabolism parameters such as HbA1c. The increased TNF-α level is related to the combined effect of obesity and diabetes while increased leptin level is caused by obesity unescorted by diabetes. Our findings suggest that further research should investigate the role of TNF-α as a tool to monitor the effectiveness of diabetes management, particularly in individuals with obesity.Independent of leptin level, serum level of TNF-


To maximize photosynthetic efficiency, plants have evolved a capacity by which leaf area scales allometrically with leaf mass through interactions with the environment. However, our understanding of genetic control of this allometric relationship remains limited.Phaseolus vulgaris) in two different environments. The ontogenetic model identified this QTL plus a few other QTLs that determine developmental trajectories of leaf allometry, whose expression is contingent heavily upon the environment.We integrated allometric scaling laws expressed at static and ontogenetic levels into genetic mapping to identify the quantitative trait loci (QTLs) that mediate how leaf area scales with leaf mass and how such leaf allometry, under the control of these QTLs, varies as a response to environment change. A major QTL detected by the static model constantly affects the allometric growth of leaf area vs. leaf mass for the common bean (Our results gain new insight into the genetic mechanisms of how plants program their leaf morphogenesis to adapt to environmental perturbations. Leaves play a central role in maintaining plants’ survival, growth and reproduction through the interception and transformation of solar energy into chemical energy to produce photosynthates –3. Plantcotton Golgi-related) -mediated pectin methylesterification that determines how carbon is allocated into leaf area and leaf mass through modulating the expansion and positioning of leaf cells in Arabidopsis thaliana. A model named Arabidopsis leaf area growth model was designed to simulate the plant growth process, the emphasis is on the effect of variation in C partitioning between leaf area growth and thickening [The relationship between leaf mass and leaf area has been studied in several fields. As to the molecular mechanisms, Weraduwage et al.  detectedickening . Althougickening , 14, our2) and leaf dry weight (g) [Several theories have been established through mathematical equations to describe the allometric relationship of two different traits, such as leaf area (cmight (g) . An alloor,α is the prefactor whose unit is cm2/gβ and β is the scaling exponent describing how leaf area (A) scales with leaf mass (M) [β < 1.0, = 1.0, or > 1.0, respectively [where mass (M) , 16. Leaectively , 17–20.β < 1). This phenomenon can be explained by the “diminishing return” hypothesis  [β = 1.0) [β > 1, which can be explained by the “network supply constraint” hypothesis  [The larger leaves have higher dry mass investment for per leaf area unit than the smaller ones to provide sufficient mechanical stability to adapt to unfavorable environments, as shown by the scaling exponent of leaf area vs. dry mass less than one (i.e. g light) , 19. Howβ = 1.0) , 18 and β = 1.0) . In sometraints) . Allomettraints) , 23. If Wu et al.  pioneerePhaseolus vulgaris L.) is a legume that is widely grown in tropical and subtropical countries, where it represents an important source of proteins, carbohydrates, dietary fiber, vitamins, minerals, phytonutrients and antioxidants [The common bean  than those in Palmira (b = 0.88); however, leaves were larger and heavier in the latter than in the former. In summary, these plots indicated that allometric scaling of at least the first trifoliate changes with development and that this change depends on the environment. The plots in Fig. Plots of leaf area against leaf mass show that these characters follow the allometric scaling law in the parental and RIL genotypes of the common bean Fig. . AccordiThe static model was used to map those QTLs that control leaf allometric scaling in the RIL family. The significant QTL regions and their peak QTLs detected by static model for each time point were shown in plot of Table An overlapping QTL region on chromosome 7 was detected at both sites at times 2–4, and a major QTL located in this region LSA7.3 ) between markers DIM_7–7 and DIM_7–8 was chosen for further analysis Table , 2. Thisβ ≈ 1.04) over developmental time at Popayan, but it decreases (β = 1.17–0.97) as plants develop at Palmira. Under the control of LOA7.2 (LSA7.3), after the middle stage of plant development, leaves at Popayan tend to invest more energy to surface area growth relative to mass accumulation, with a greater extent than those at Palmira. Taken together with results from Fig. The effect of LOA7.2 (LSA7.3) is basically silent during the early stages of leaf development, and appears to be activated quickly after the leaf reaches maturity, but its effect decreases during the late stages of development Fig. β ≈ .04 over β = 1.01)  eq.  at PopayThe two parents display different patterns of ontogenetic allometry. In Palmira, Jamapa’s slope of leaf area growth over leaf mass growth is greater than that of Calima from early to middle stages of growth Fig. . This diWe derived a dynamic allometry QTL mapping model based on the likelihood function (6), which integrates the ontogenetic allometric eq.  and funcWe further characterized the genotype-dependent mode of action of locus (LOA7.2 LSA7.3), LOA6.1 and LOA9.1) and their environmental dependencies as observed by the changes observed from Palmira to Popayan. The allometric parameter values and genetic effects of the same QTL were different between two sites. The absolute values of environmental effects of each parameter at Palmira were greater than that at Popayan  that affects growth parameters of leaf area and leaf mass for the common bean grown in two different environments by two models. The major QTL region detected by static and ontogeny models at both sites in our study was almost overlapping with the region found in the former study. The peak QTL LOA7.2 (LSA7.3) of this region located between DiM_7–7–DiM_7–8, the same as LeafG1 and QTL LOA6.1 located at DiM_6–15 on chromosome was also very close to LeafG1 (Position: DiM_6–15–Bng088) detected by Jiang et al. The QTLs LOA7.2 (LSA7.3) /LeafG1 and LOA6.1/LeafG2 exerted pleiotropic effects on different traits and displayed environmental pleiotropy on the same trait between different environments [We implemented static and ontogenetic allometry into a genetic mapping setting, which enables the characterization of the genetic origin of allometry. Specific QTLs mapped by this strategy could facilitate our understanding of how covariation between different traits drives the phenotypic evolution of species. We have found specific sets of QTLs for the static allometry and ontogenetic allometry of leaf area vs. leaf mass in the common bean. The genetic effects and environmental effects of the QTLs were different between two sites, implying the pleiotropic effects of the QTLs , 66 RFLP-based markers, three soybean-derived SNP markers, and two phenotypic marker loci for this RILs population. The two parents and RILs were genotyped for 513 molecular markers located on 11 linkage groups each covering a common bean chromosome [A recombinant inbred line (RIL) population was generated from the cross between the Mesoamerican bean cultivar Jamapa and the Andean cultivar Calima of romosome .− 2 d− 1; day length, 15.6 h; and growing season, from 11 Nov 2011 to Jan 2012. In Popayan, the mean air temperature ranges from 13.7 to 25.5 °C; solar radiation, 11.8 MJ m− 2 d− 1; day length, 12.1 h; and growing season, from 23 Mar 2012 to Jun 2012. A randomized complete block row-column design with three replicates (six for each parent) was employed at each site. Each RIL plot had between 30 and 50 plants [− 3 g resolution. We obtained data on leaf area and leaf mass of 173 lines at five time points from each site.The mapping population (including both parents) was planted at two sites, Palmira and Popayan in southwestern Colombia during 2011–2012. These two sites have different temperature regimes . In Palm0 plants . One plan recombinant inbred lines (RILs) in which there are two alternative homozygous genotypes at each marker. We are interested in the allometric covariation of leaf area and leaf mass, which can be described by a power eq. , respectively. Consider a QTL with two genotypes QQ (coded as 1) and qq (coded as 2). Let μy1 and μz1 denote the genotypic values of leaf area and leaf mass for genotype AA, and μy2 and μz2 denote the genotypic value of leaf area and leaf mass for genotype aa, respectively. If this QTL affects leaf allometry, we can establish the following relationships from equation (1b):Suppose there is a mapping population of ower eq.  for eachb = logα. The genetic effects of this QTL on leaf mass and leaf area are calculated as az = μz1 − μz2 and ay = μy1 − μy2 = βaz, respectively. The intercept of leaf area regressed on leaf mass is estimated as by letting Using this QTL’s information, we formulate a bivariate likelihood for two leaf traits based on a mixture model, expressed asωj|i is the conditional probability of QTL genotype j (j = 1 for QQ or 2 for qq), conditional on the marker interval that harbors the QTL, fj is the bivariate density function of leaf area and leaf mass for QTL genotype j, and Φ represents the unknown parameters that describe the location of the QTL and its genetic effects on leaf allometry and residual (co)variances. The conditional probability is expressed in terms of the recombination fraction between the marker interval and QTL [fj is assumed to be a normal density function expressed aswhere  and QTL . fj. The significant QTL was estimated by assuming a QTL at every 2 cM position over the linkage map. After the parameters are estimated, we need to perform hypothesis tests. First, whether there exists a significant QTL can be tested by formulating the following hypotheses:The expectation-maximization (EM) algorithm was implemented to estimate the parameters 1: At least one of the equalities in the H0 does not hold.H0 and H1 is compared against the genome-wide critical threshold determined from permutation tests. Second, we can test whether β is significantly different from a specific value, e.g. 1 or 3/4. The allometry theory can be used to interpret the biological meaning of these parameters [The log-likelihood ratio calculated from the Hrameters .A) is scaled with leaf mass (M) over developmental time (t). Previous studies noted that as leaves grow, increases in surface area and mass are not synchronous [d into the power equation [Ontogenetic allometry states that leaf area  and Zi(t) denote the observed values of leaf area and leaf mass at time t  for RIL i , respectively. By statistical reasoning, we found a goodness-of-fit of eq. (Yi(t) and Zi(t) data for individual RILs QQ and qq. Let Yi = (Yi(1), …, Yi(T)) and Zi = (Zi(1), …, Zi(T)). The bivariate functional mapping is formulated asTo map how a QTL affects the ontogenetic allometry of leaf area vs. leaf mass, we implemented Zhao et al.’s  bivariatωj|i was defined as above, and fj is a bivariate longitudinal normal distribution function for QTL genotype j (j = 1 for QQ and 2 for qq). The genotype-dependent mean vector of fj is expressed aswhere Based on the allometry law expressed by the power eq. , we modeμjz(t) by a growth equation, such as logistic equation [Based on the principle of functional mapping, we model the genotype-dependent growth of leaf mass equation . Thus, wAj, Bj, Rj) are the asymptotic growth, initial growth and relative growth rate of leaf mass for genotype j over time, respectively. Therefore, by substituting eqs. . Such modeling has incorporated the allometric scaling law and growth law, which makes QTL mapping of biological relevance and robustness.where parameters (ing eqs.  and 9)  Aj, Bj, Σ) of fj is a symmetric matrix containing three longitudinal (co)variance matrices within and between leaf area and leaf mass. Zhao et al. [Σ. The bi-SAD (1) model requires the following parameters: antedependence parameters (ρy and ρz) and innovation variances (ρyz). Based on these parameters, Zhao et al. [Φ = variance matrix , and/or its ontogenetic allometry with leaf area (12).http://blast.ncbi.nlm.nih.gov/).If both null hypotheses are rejected, then this means that the QTL pleiotropically affects leaf area and leaf mass growth. Possible functions of all the QTLs were annotated via BLAST in the “nr” database on website of National Center of Biotechnology Information  test statistics over 11 chromosomes for testing the existence of QTLs governing leaf area vs. leaf mass static allometry at different time points 1–5 for the common bean grown at Palmira (red) and Popayan (blue), with the LR values besides the most significant QTLs. The slash horizontal line denotes the genome-wide critical threshold determined from 1000 permutation tests.Additional file 2. The profile of log-likelihood ratio (LR) test statistics over 11 chromosomes for testing the existence of QTLs governing leaf area vs. leaf mass ontogenetic allometry for the common bean grown at Palmira (red) and Popayan (blue), with the LR values besides the most significant QTLs. The slash horizontal line denotes the genome-wide critical threshold determined from 1000 permutation tests.Additional file 3. The function annotation of all the QTLs, marker information and the genetic and environmental effects.Additional file 4 The ontogenetic allometry fitting (curve) of leaf area and leaf mass data (dots) at 5 tome points for each RIL grown at Palmira (red) and Popayan (blue) by the intercepted power eq. (A(t) = αMβ(t) – d.


A successful MGIA could be applied to preclinical and clinical post-vaccination samples to aid in the selection of novel vaccine candidates at an early stage and provide a relevant measure of immunogenicity and protection. However, assay harmonisation is critical to ensure that comparable information can be extracted from different vaccine studies. As part of the FP7 European Research Infrastructures for Poverty Related Diseases (EURIPRED) consortium, we aimed to optimise the direct MGIA, assess repeatability and reproducibility, and harmonise the assay across different laboratories. We observed an improvement in repeatability with increased cell number and increased mycobacterial input. Furthermore, we determined that co-culturing in static 48-well plates compared with rotating 2 ml tubes resulted in a 23% increase in cell viability and a 500-fold increase in interferon-gamma (IFN-γ) production on average, as well as improved reproducibility between replicates, assay runs and sites. Applying the optimised conditions, we report repeatability to be <5% coefficient of variation (CV), intermediate precision to be <20% CV, and inter-site reproducibility to be <30% CV; levels within acceptable limits for a functional cell-based assay. Using relevant clinical samples, we demonstrated comparable results across two shared sample sets at three sites. Based on these findings, we have established a standardised operating procedure (SOP) for the use of the direct PBMC MGIA in TB vaccine development.A major challenge to tuberculosis (TB) vaccine development is the lack of a validated immune correlate of protection. Mycobacterial growth inhibition assays (MGIAs) represent an unbiased measure of the ability to control mycobacterial growth  •Optimised assay conditions for direct PBMC MGIA determined.•Standardised operating procedure for use in TB vaccine studies established.•Consistency across three sites using clinically-relevant samples demonstrated. BCG, thin vitro   represent an alternative to single biomarkers based on predefined individual immune parameters; instead offering an unbiased measure of the functional ability of post-vaccination whole blood or cell samples to control mycobacterial growth in vitro . A succeesearch) . Once biesearch) .Several MGIAs have been reported in the literature  and optiEuropean Research Infrastructures for Poverty Related Diseases (EURIPRED) was a European Commission-funded Framework Program 7 consortium project that ran from 2013 to 2017. The overall goal was to coordinate and integrate international resources into a single specialised infrastructure to support European TB, HIV, Malaria and Hepatitis B/C studies from early drug, vaccine and microbicide discovery to clinical trials. EURIPRED was comprised of several work packages including resource management and quality assurance, sample selection and characterisation of biological materials, development and evaluation of reference reagents, and dissemination and training. Work package 5, standardisation and harmonisation of assays, aimed to select key immunological assays currently used in vaccine development, conduct harmonisation experiments and define standardised SOPs for dissemination.We focussed on the optimisation and harmonisation of the direct MGIA, based on the methods of 22.1Samples used in the optimisation experiments were obtained from local blood transfusion services as buffy coats. PBMC were processed and cryopreserved at one site and vials distributed to the other three laboratories.NCT02380508) conducted at the Jenner Institute, University of Oxford, which was approved by NRES Committee South Central - Oxford B (REC ref. 15/SC/0022). Volunteers were aged between 18 and 45 years with no evidence of latent M.tb infection. Individuals received a single intradermal dose of 2-8 × 105 CFU of BCG SSI and bloods were taken pre-vaccination at day 0, and days 2, 4, 7, 10, 14, 21, 28 and 84 post-vaccination.The phase of the project in which the assay was applied to clinically relevant samples used either PBMC from healthy Dutch donors enrolled in a BCG vaccination study that was approved with protocol P12.087 , or PBMCAll methods used in the clinical trials described were carried out in accordance with the ethical principles set forth in the Declaration of Helsinki as agreed by the World Medical Association General Assembly (Washington 2002), ICH Good Clinical Practice (GCP) and local regulatory requirements. All volunteers gave informed consent after the nature and possible consequences of the studies had been fully explained.2.26 cells per cryovial, frozen overnight at −80 °C and transferred to liquid nitrogen storage the following day. Cryopreserved PBMC were thawed in a water bath at 37 °C until a small amount of frozen material remained. Samples were gradually added to 10 ml RPMI (containing 10% FBS and 2 mM l-glutamine) using a Pasteur pipette. The cryovial was rinsed using 1 ml of fresh medium and added to the corresponding tube, which was then centrifuged at 1500 rpm for 5 min. Supernatants were poured off and cells resuspended at an approximate concentration of 2–3 × 106 cells per ml of RPMI , and 2 μl/ml of 25 U benzonase  added to each tube. Cells were rested at 37 °C for 2 h with 5% CO2 before counting.PBMC were isolated from buffy coat or heparinised peripheral blood samples using density centrifugation. Cells were counted, resuspended in foetal bovine serum (FBS) and incubated at 4 °C for 30 min. An equal volume of FBS containing 20% dimethylsufoxide was then added and cells aliquoted at a concentration of 5–10 × 102.32.3.1l-glutamine and 25 mM HEPES). For harmonisation experiments, frozen aliquots of a single batch of BCG Pasteur stock  grown in Middlebrook 7H9 medium with 10% OADC and 0.05% tyloxapol were circulated and serially diluted prior to inoculation in a standardised way between sites.In-house BCG Pasteur stocks were grown in Middlebrook 7H9 medium with 10% OADC to mid-log phase then divided into 1 ml aliquots and stored at −80 °C until required. Aliquots were thawed at room temperature immediately prior to inoculation and diluted to the correct concentration  in RPMI (containing 2 mM 2.3.26 PBMC and ~500 CFU BCG Pasteur (unless otherwise specified) in a total volume of 600 μl RPMI  were incubated on a 360° rotator  at 37 °C for 96 h. Where specified, cells were lysed at the end of the culture period by centrifuging tubes at 12000 rpm for 10 min, removing supernatant and adding 500 μl sterile water (or other lysis agent as specified) to the pellet before pulse vortexing and transferring to BACTEC MGIT tubes supplemented with PANTA antibiotics  and OADC enrichment broth . For experiments where cells were not lysed, cultures at the end of the 96 h period were transferred directly to supplemented BACTEC MGIT tubes.The in-tube direct PBMC MGIA was performed as previously described . Briefly2.3.36 PBMC and ~500 CFU BCG Pasteur in a total volume of 600 μl RPMI  per well were incubated at 37 °C for 96 h. At the end of the culture period, cultures were added to 2 ml screw-cap tubes and centrifuged at 12000 rpm for 10 min. During this time, 500 μl sterile water was added to each well to lyse adherent monocytes. Supernatants were removed from the 2 ml screw-cap tubes, and water from the corresponding well added to the pellet. Tubes were pulse vortexed and the lysate transferred to BACTEC MGIT tubes supplemented with PANTA antibiotics and OADC enrichment broth .48 well plates containing 3 × 102.3.410 CFU using stock standard curves of TTP against inoculum volume and CFU. Results are presented as growth ratio which is calculated as log10 CFU of sample/log10 CFU of control.At the end of both protocols, tubes were placed on the BACTEC 960 machine  and incubated at 37 °C until the detection of positivity by fluorescence. On day 0, duplicate direct-to-MGIT viability control tubes were set up by inoculating supplemented BACTEC MGIT tubes with the same volume of mycobacteria as the samples. The time to positivity (TTP) read-out was converted to log2.3.5Steps were taken to ensure consistency across sites as far as possible for the harmonisation experiments, including use of the same batch of BCG Pasteur stock preparation diluted in the same series of dilutions at each site, circulating aliquots of the same cryopreserved PBMC isolated at one site, and exchange of protocols with specific details for each experiment. Operator training was conducted between sites at the start of the project in the standard in-tube protocol.2.4Cell viability at the end of the 96 h culture period was quantified by trypan blue exclusion. A 0.4% solution of trypan blue  was prepared in buffered isotonic salt solution (pH 7.2–7.3) and 0.1 ml of trypan blue stock solution added to 0.1 ml PBMC. A haemocytometer slide was loaded and examined under a microscope. The percentage of viable cells was calculated as [1-] x 100, and corrected for the dilution factor.2.5IFN-γ concentration in MGIA culture supernatants at the end of the 96 h period was determined using the Quantikine® ELISA kit  according to the manufacturer's instructions. Briefly, samples were diluted 1:1 with assay diluent and incubated for 2 h at room temperature before aspirating each well and washing 4 times using wash buffer. 200 μl of human IFN-γ conjugate was added to each well and incubated for 2 h at room temperature followed by 4 washes with wash buffer. Substrate solution (200 μl) was added to each well and incubated for 30 min in the dark at room temperature, followed by 50 μl of stop solution. The optical density (OD) of each well was determined using a microplate reader set to 450 nm. Duplicate readings were averaged and the average zero optical density subtracted. A standard curve was generated to convert OD values to concentration in pg/ml.2.6Data was analysed using GraphPad Prism v.7 and IBM SPSS v.25. Normality of data was determined using a Shapiro-Wilk test. For parametric data with multiple groups, a one-way ANOVA or repeated-measured ANOVA was conducted followed by a Dunn's post-test. For comparisons between two groups of normally-distributed data, a t-test or paired t-test was used. For non-parametric data with multiple groups, a Kruskal-Wallis or Friedman test was conducted followed by a Dunn's post-test. For comparisons between two groups of non-parametric data or small sample size, a Mann Whitney or Wilcoxon matched-pairs signed rank test was conducted. A Spearman's rank correlation was used to determine associations between two different measures. For determination of intra-assay precision, intermediate (inter-assay) precision and inter-site reproducibility, the coefficients of variation (standard deviation/mean x 100) and intra-class correlation coefficients  were calculated using raw TTP values.33.16 cells per culture were inoculated with either 50 or 500 CFU of BCG Pasteur in the in-tube direct PBMC MGIA conducted at three different laboratory sites. On average, intra-assay repeatability between replicate cultures was improved with increased cell input and with increased mycobacterial inoculum. The mean CVs for the replicate cultures under the different assay conditions are shown in Human PBMC from 4 donors at a concentration of either 1, 3 or 5 × 103.2Quantification of BCG at the end of the 96 h co-culture period is a critical step in assessing mycobacterial growth inhibition. Intracellular BCG is released by lysis of host cells before addition of the cultures to BACTEC MGIT tubes using sterile water . We comp3.36 PBMC and 500 CFU of BCG in 2 ml screw-cap tubes with no lysis at the end of the culture period) was applied to relevant clinical samples at each of the three sites. At site 1, samples were available from 3 volunteers at baseline and weeks 4, 8, 12 and 52 post-BCG vaccination. There was a trend towards improved control of mycobacterial growth following BCG vaccination up to week 12, but this was not statistically significant  were co-cultured with ~500 CFU of BCG Pasteur in either sealed rotating screw-cap tubes or in static 48-well plates with CO2. At the end of the 96 h culture period, cell viability was assessed by trypan blue exclusion, and IFN-γ production was measured by ELISA. Cell viability was significantly greater in 48-well plates compared with rotating screw-cap tubes , but no correlation between cell viability and mycobacterial growth in the MGIA . IFN-γ concentrations were significantly higher in the supernatants of co-cultures in 48-well plates compared with those in screw-cap tubes . There was also a significant correlation between IFN-γ concentration in the MGIA supernatant and volunteer PPD status as measured by PPD stimulation index  precision. This process was repeated twice across three different sites to evaluate inter-site reproducibility.Human PBMC from 12 donors were assessed by MGIA with co-cultures in either sealed rotating screw-cap tubes or in static 48-well plates with COp = .001, Wilcoxon matched-pairs signed rank test, p = .04, Wilcoxon matched-pairs signed rank test). In run 2, intra-assay repeatability was significantly improved when using 48-well plates compared with rotating screw-cap tubes at sites 1 and 2  precision between experiments was determined by conducting the direct PBMC MGIA using the same sample set at the same site on two different days (henceforth referred to as ‘runs’). Intermediate precision, as measured by CV, was significantly improved when using 48-well plates compared with rotating screw-cap tubes at site 2 (10 CFU for the in-tube and 48-well plate protocols respectively), resulting in a low signal-to-noise ratio. When tested in an additional sample set with a broader dynamic range (range = 0.66 and 0.59 log10 CFU respectively), a similar pattern of control across the individuals was observed in a direct comparison of the two protocols described here with an ICC of 0.54 (moderate agreement) . However, this sample set was homogeneous with similar levels of growth control across volunteers (range = 0.29 and 0.13 logreement) a–b.3.6p = .02, p = .03 and p = .01 respectively, Wilcoxon matched-pairs signed rank test, Cryopreserved cells were taken from adult volunteers who were either BCG naïve or historically BCG vaccinated and the direct PBMC MGIA performed in duplicate across three different sites. There was no overall difference in MGIA outcome between naïve and historically vaccinated volunteers observed at any of the three sites using either the in-tube a or 48-wp = .0007, p = .01 and p = .0002 for sites 1, 2 and 3 respectively, paired t-test, In a second round of experiments, cryopreserved cells were taken from UK adult volunteers at baseline and 12 weeks post-BCG vaccination and the direct PBMC MGIA performed across three different sites using the optimised 48-well plate protocol only. In order to validate detection of a ‘positive response’, volunteer samples were selected based on a) demonstrating improved control of mycobacterial growth following BCG vaccination as measured at the trial site and b) having sufficient availability of cryopreserved PBMC to conduct a comparison across three different sites. 9 volunteers fulfilled these criteria and this sample set was circulated for MGIA analysis. A significant improvement in mycobacterial growth inhibition following BCG vaccination was observed at all three sites (3.7The detailed optimised Standard Operating Procedure (SOP) for the direct PBMC MGIA, produced as a result of the EURIPRED project, is available online at:http://www.euripred.eu/information-trainings/sops-assay-harmonisation.html4While several MGIAs have been described in the literature for use in mice, cattle and humans  did not impact BCG recovery, likely due to the fact that end-of-culture samples are added to BACTEC MGIT tubes containing Middlebrook 7H9 broth in which cells would naturally lyse, releasing intracellular mycobacteria for quantification. This outcome was consistent across the three sites despite the use of different BCG Pasteur stocks between sites 1 and 2/3, and different batches of the same stock between sites 2 and 3 in this experiment, which accounts for the observed inter-site variation in TTP. As such we selected the ‘no lysis’ condition for the in-tube protocol to avoid introducing unnecessary processing steps and enhance technical simplicity.The finding that intra-assay repeatability is improved by increasing cell concentration or mycobacterial input is consistent with previous work by n = 3) and early post-vaccination time-points at site 3 (weeks 2 and 4). It has previously been shown that the peak of response following primary BCG vaccination in this assay occurs at 4–12 weeks post-vaccination, but may vary from individual to individual making it difficult to reach statistical significance at a given time-point . Total cell viability did not correlate with control of mycobacterial growth suggesting that cells dying over the 96 h incubation period may not be those with effector functions. The consistency in outcomes observed in a direct comparison of both assay methods  compared with the values for murine splenocytes. Murine splenocytes are widely considered more delicate than PBMC, necessitating the use of fresh samples and suggesting that the human MGIA may be more robust. However, other differences between these studies may also have contributed including the use of virulent  methods  supports6 PBMC compared with 3 × 106 PBMC used in the 48-well plate protocol) to align with the standard in-tube protocol described in previous studies , or artificially high due to systematic differences. ICC compares within-sample variability (between replicates within an assay run or between runs) with between-sample variability and therefore arguably offers a more stringent and meaningful measure. ICC can also be applied using a ‘consistency’ rather than ‘absolute’ agreement, which takes into account systematic differences between operators. Thus ICC was also calculated where sample size permitted. Using the criteria described by 6 cells, 500 CFU BCG, 48-well plates with CO2) to two shared sets of clinical trial samples. In the first cohort, which consisted of BCG naïve and historically vaccinated volunteers, we did not observe a difference between the two groups at any of the three sites or using either protocol. That this finding was consistent across all three sites and both protocols suggests that this is due to biological rather than technical factors. These volunteers were vaccinated at various times in the past (up to 40 years ago) and as such we may not expect to detect an effect of vaccination, particularly given that BCG efficacy has been shown to reduce over time in some studies  using 3 × 10al assay . As suchal assay . The optThe following are the supplementary data related to this article.


Although the diagnosis and management of chronic spontaneous urticaria (CSU) is well documented in the literature, some aspects of the disease remain unclear. We aimed to further describe the natural history, prognostic factors, humanistic burden and uptake of traditional and alternative therapies in patients with CSU.This was a prospective, cross-sectional analysis at a single centre. We reviewed patient medical records and conducted a survey in patients with CSU.72 patients participated in the study with a median duration of CSU of 48 months. 30% of patients had symptoms that resolved in under 2 years with these patients trending towards an older age of onset of CSU (48 ± 17 years). 16% of patients had symptoms lasting 10 years or longer with these patients trending towards a younger age of onset (22 ± 16 years). Patients with a relapsing/remitting disease course (31%) and those with co-existing angioedema (57%) trended towards a longer median duration of CSU (96 and 50 months respectively) and were observed to have a higher proportion of patients reporting CSU duration of 10 years or longer . Patients with co-existing autoimmune/thyroid disease (19%) trended towards a shorter median duration of CSU (37 months). 54 patients (75%) reported sleep disturbance and 29 patients (43%) required emergency room visit(s) for symptomatic control. 84% of patients who trialed second generation antihistamines reported a response to treatment, while 73% of patients who trialed omalizumab reported a response to treatment. Patients using alternative medicine such as acupuncture, traditional Chinese medicine and naturopathic medicine had lower reported response rates (20–29%) to treatment.The natural history of CSU may be longer than previously reported with our study finding a median duration of symptoms of nearly 4 years with one-third of patients reporting a relapsing/remitting disease course. Younger age of onset, a relapsing/remitting disease course and angioedema may predict a longer duration of CSU, whereas older age of onset and co-existing autoimmune/thyroid disease may predict a shorter duration of CSU. Reported symptomatic benefit was higher from guidelines based pharmacologic therapy versus various alternative medicines. Chronic spontaneous urticaria (CSU) is defined as the presence of urticaria with or without angioedema that has been continuous or intermittent for at least 6 weeks . It can A recent cross sectional analysis has estimated that CSU affects about 0.23% of the population in the United States, with women more than twice as likely to have the condition relative to men  Most of Several studies have shown that the symptoms of CSU can often have a significant effect on patient well-being, utilization of health care resources and quality of life, particularly sleep and work impairment –13. TherManagement guidelines for CSU have recently been developed and accepted by most jurisdictions. These guidelines recommend initial treatment with second generation antihistamines, which can be used up to four times the recommended dose if there is inadequate response to standard dosing . OmalizuAlthough there are many different forms of complementary and alternative medicine available, their effectiveness in treating CSU is questionable. There has been some studies that looked at acupuncture for global symptom improvement when used in conjunction with pharmacologic therapy however a recently published meta-analysis showed there is overall a low level of evidence for its use . There hThis was a prospective cross-sectional, single centre study that took place at a private allergy/immunology clinic in Vancouver, British Columbia, Canada. Potential participants were identified by diagnostic code (CSU) in the clinic’s electronic medical record from 2013 to 2015. Each file was manually reviewed by the investigators for inclusion in the study (see criteria below). Relevant medical data obtained from the medical chart included patient age, gender, baseline medical history including medical comorbidities, duration and severity of CSU, prior treatments, relevant co-morbid inducible urticarias, prescribed treatments, and laboratory results (if applicable). Data was collated in an electronic spreadsheet with patients identified by a unique participant identification number.Eligible participants who met the inclusion criteria for the study (see below) were sent a package in the mail which included a letter of initial contact outlining the purpose of the study and an informed consent document. The letter of initial contact stated that eligible participants would be contacted in 4–6 weeks for a follow-up telephone survey if they had signed and returned the consent form to the investigator’s clinic and agreed participate in the study. If patients declined participation, no further contact was made. If patients did not respond to the initial package, a follow-up phone call was made to further describe the study and answer any questions. Some eligible participants were also approached at follow-up visits, as opposed to communication through letter mail, if they had a follow-up visit in the clinic within 4–6 weeks. A telephone survey with a prepared oral script took place 4–6 weeks after initial contact to gather participant-reported data  of the patients were female Table . Slightl22 patients (31%) reported a recurrence of their disease after initial remission with a mean length of remission of 21 ± 10 months. Those with disease recurrence had a similar median age of onset of CSU of 42.5 years compared to 44.5 years in those patients denying CSU recurrence. 3 males (23%) and 19 females (32%) reported CSU recurrence, with no statistical difference (p = 0.516). Of those with resolved CSU, 15 patients (27%) reported a disease recurrence, with a total median duration of disease of 96 months (including disease remission time). 5 of these patients (33%) reported a disease duration of 10 years or longer, compared to 4 patients (10%) who had resolution of their CSU and denied a recurrence of disease (p = 0.033) (Table 41 patients (57%) had associated angioedema and of those patients with resolved CSU, 32 patients (57%) had associated angioedema with their median duration of CSU lasting 50 months. 8 patients (25%) had symptoms lasting 10 years or longer compared to only 1 patient (4%) who did not have angioedema (p = 0.036) , stress 38%), heat (35%), and cold (14%). Other triggers reported by patients included foods (11%), nonsteroidal anti-inflammatory drugs (NSAIDs) (11%), exercise (10%), alcohol (7%) and sunlight (3%)  reported co-existing thyroid and/or autoimmune disease, with hypothyroidism (8%) being most common. Other reported autoimmune diseases included rheumatoid arthritis (7%), Crohn’s disease (1%), vitiligo (1%), celiac disease (1%) and HLA-B27 spondyloarthopathy (1%) (Table 54 patients (75%) reported that they had disrupted sleep due to their urticaria and 29 patients 43%) presented to the emergency room for uncontrollable symptoms of their urticaria. Of note, five patients did not identify if they visited an emergency room for their urticaria Table . 47 fema% presentThe most trialed treatment for CSU were second generation antihistamines with 68 patients 94%) reporting their use. Other pharmacologic treatments employed included first generation antihistamines in 51 patients (71%), prednisone in 28 patients (39%), omalizumab in 15 patients (21%), cyclosporine in three patients (4%), montelukast in two patients (3%), intravenous immunoglobulin (IVIG) in one patient (1%) and methotrexate in one patient (1%) (Table % reportiWe stratified our patient population by response to treatment with second generation antihistamines to see if we could observe any differences in patient characteristics in these two groups Table . Of thos9 /L). 27 patients had evaluation for C-reactive protein (CRP) and it was elevated in 8 patients (> 4.8 mg/L). 38 patients had testing for thyroid peroxidase antibodies (anti-TPO) and it was positive in 4 patients (> 35 IU/ml). 23 patients had laboratory testing for immunoglobulin E (IgE), and it was elevated in 11 patients (> 430 μg/ml).Standardized laboratory investigations were not part of this protocol. However, we did observe that of 42 patients that had blood work including complete blood counts, 2 of them had eosinophilia . Patients with recurrence of their CSU also had a higher proportion of patients with disease lasting 10 years or longer 33%), compared to those without disease recurrence (10%) (p = 0.033). This is a significant finding as details on long term prognosis of patients with CSU is limited. The natural history of the disease process may be longer than previously reported and include more of a relapsing/remitting course. Multiple previous studies that evaluated the natural history of CSU do not comment on recurrence rates after the initial remission, and have generally only followed patients for a few years after diagnosis 3%, compa, 7. NeitPatients with angioedema had a median duration of CSU of 4.2 years and also had a larger proportion of patients with CSU lasting 10 years (25%) compared to those who did not have angioedema (4%) p = 0.036). Previous studies have also reported that patients with co-existing angioedema are more likely to have a longer disease course [6. PrevioThe impact on quality of life was observed to be quite high in our patient population, with three-quarters of our patients reporting sleep disturbance due to the symptoms of CSU. This is a higher proportion than what has previously been reported in the literature . This isThere has also been strong links found between CSU and autoimmunity. Another large Israeli population-based study that spanned 17 years and involving thousands of patients diagnosed with CSU found that patients with CSU were observed to have increased odds of co-existing hypothyroidism, hyperthyroidism, and antithyroid antibodies . In addiThe treatment of CSU observed in this study paralleled the recommended published guidelines, with nearly all patients having tried second generation antihistamines followed by first generation antihistamines, prednisone and omalizumab . HoweverAlthough this is a relatively small analysis, we feel that this adds to the growing body of evidence about CSU. Limitations of this study include the small sample size and single centre origin which makes generalizability to the general population difficult. In addition, there is likely a component of selection bias as patients were required to volunteer to complete the survey and participate in the study. This may predispose individuals who have had a more difficult time controlling their symptoms agreeing to participate in the study. In addition, our study population is based on referrals to an allergy specialist referral centre and are therefore also more likely to have severe disease.CSU has been shown to disproportionately affect females with an average onset of disease in the fifth decade and generally similar rates among various ethnicities. We believe that the natural history of CSU may be longer than previously reported with our study showing a median duration of symptoms of 4 years and one-third of patients having a relapsing/remitting course. Patients with a relapsing/remitting disease course and those with co-existing angioedema trended towards a longer median duration of CSU and were observed to have a higher proportion of patients reporting CSU duration of 10 years or longer. Patients with co-existing autoimmune/thyroid disease trended towards a shorter median duration of CSU. There was also a trend of older age of CSU diagnosis being associated with more rapid CSU resolution (within 2 years) whereas those diagnosed with CSU at a younger age trended towards a longer disease course (10 years or greater). There was a relatively high humanistic burden of disease with 75% of patients reporting inadequate symptom control disrupting their sleep as well as significant utilization of health care resources (i.e. emergency room visits) for patients with uncontrolled symptoms, particularly in those with a relapsing/remitting disease course. Reported symptomatic benefit was higher from guideline based pharmacologic therapy versus various alternative medicines with response rates to antihistamines similar amongst different subgroups of patients. CSU can have a significant impact on quality of life if it is inadequately controlled and even with following guideline-based therapy, symptoms can be difficult to abate in a subset of the population.Additional file 1: Figure S1. Study questionnaire provided to patients.


Diaphragm dysfunction is highly prevalent in mechanically ventilated patients. Recent work showed that changes in diaphragm shear modulus (ΔSMdi) assessed using ultrasound shear wave elastography (SWE) are strongly related to changes in Pdi (ΔPdi) in healthy subjects. The aims of this study were to investigate the relationship between ΔSMdi and ΔPdi in mechanically ventilated patients, and whether ΔSMdi is responsive to change in respiratory load when varying the ventilator settings.r) were used to investigate within-individual relationships between variables, and repeated measure correlations (R) were used for determining overall relationships between variables. Linear mixed models were used to compare breathing indices across the conditions of ventilation.A prospective, monocentric study was conducted in a 15-bed ICU. Patients were included if they met the readiness-to-wean criteria. Pdi was continuously monitored using a double-balloon feeding catheter orally introduced. The zone of apposition of the right hemidiaphragm was imaged using a linear transducer . Ultrasound recordings were performed under various pressure support settings and during a spontaneous breathing trial (SBT). A breath-by-breath analysis was performed, allowing the direct comparison between ΔPdi and ΔSMdi. Pearson’s correlation coefficients . Individual correlation displays a significant correlation in 8 patients out of 25 . Changing the condition of ventilation similarly affected ΔPdi and ΔSMdi. Patients in which ΔPdi–ΔSMdi correlation was non-significant had a faster respiratory rate as compared to that of patient with a significant ΔPdi–ΔSMdi relationship (median (Q1–Q3), 25 (18–33) vs. 21 (15–26) breaths.min−1, respectively).Thirty patients were included and 930 respiratory cycles were analyzed. Twenty-five were considered for the analysis. A significant correlation was found between ΔPdi and ΔSMdi (We demonstrate that ultrasound SWE may be a promising surrogate to Pdi in mechanically ventilated patients. Respiratory rate appears to negatively impact SMdi measurement. Technological developments are needed to generalize this method in tachypneic patients.NCT03832231. Acute respiratory failure is a common cause of admission in the intensive care unit (ICU) that can require invasive mechanical ventilation to relieve respiratory muscles work of breathing and ensure satisfactory gas exchange . HoweverThis study followed the STROBE guidelines for observational study . It was 2 > 90% or PaO2/FiO2 ≥ 150 mmHg with a fraction of inspired oxygen (FiO2) ≤ 40%, no or minimal vasopressor, and a positive end-expiratory pressure (PEEP) ≤ 8 cmH2O. Patients who were pregnant, under a legal protection measure, with a contraindication to the insertion of a gastric-esophageal probe , or with known allergies to anesthetizing were not included.Patients older than 18 years old were eligible for inclusion if they had been intubated and ventilated for at least 24 h, and failed a first spontaneous breathing trial (SBT). They could be included if they met predefined readiness-to-wean criteria on daily screening  and wereA flow sensor  connected to a spirometer  was used to continuously measure flow. Esophageal (Pes) and gastric (Pga) pressures were monitored using a double-balloon feeding catheter . The catheter was inserted through the mouth or nostril in the esophagus as demonstrated by the appearance of cardiac artifacts and appropriate negative swings of pressure during inspiration. Both balloons were inflated with 3 to 4 ml of air and connected to separated differential pressure transducers . The correct position of the esophageal balloon was confirmed by a dynamic occlusion test allowing the visualization of a corresponding deflation in esophageal pressure and airway pressure during inspiratory effort . Flow anpleura and peritoneum .The zone of apposition of the right hemidiaphragm was imaged using an ultrafast US scanner  driving a linear transducer array . The probe was placed on the mid-axillary line, vertical to the chest wall, at the 8th–11th intercostal space and the spot was skin marked. US gel was generously applied to optimize acoustic coupling and minimal pressure was applied to the transducer to limit tissue deformation and/or alteration of breathing mechanics. In this location, the diaphragm appears as a three-layered structure just superficial to the liver, consisting of a relatively non-echogenic muscular layer bounded by two echogenic lines corresponding to the diaphragm Patients were in a semi-recumbent position throughout the study. Sedations were not modified during the protocol. The study was carried out as follows: i) recordings under different conditions of pressure support ventilation, ii) recordings during a SBT.+25%), (iii) -25% pressure support with baseline PEEP (PS-25%) and (iv) baseline pressure support and zero end-expiratory pressure (PSZEEP). Each breathing condition was maintained for 10 min with 30-s acquisitions performed at 3 and 9 min within the condition.At baseline, patients were ventilated under pressure support ventilation mode. In each patient, 4 consecutive conditions of ventilation were applied in a randomized order: (i) initial ventilator settings predefined by the attending physician (PS), (ii) + 25% pressure support with baseline PEEP (PS2O). This modality of SBT, part of usual care of our ICU, reflects the work of breathing after extubation [Every patient underwent a planned 30 min SBT during which no assistance was provided from the ventilator . SMdi was reported as the median shear modulus within each ROI.Data were analyzed offline using standardized MATLAB  scripts. A rectangular region of interest (ROI) was drawn manually in the center of the diaphragm on the first frame of each recording. The ROI was replicated on subsequent frames. Diaphragm shear modulus was calculated as SMdi = ρ ⋅Vsmax) was measured before the SBT using a one-way valve allowing exhalation only [max was calculated as the difference between Pdi at functional residual capacity and maximal Pdi during the Mueller maneuver. A video showing US imaging along with the temporal evolution of flow, internal pressures, and SMdi is available in Additional file Changes in Pdi (ΔPdi), Pes (ΔPes), Pga (ΔPga) and SMdi (ΔSMdi) were computed for each respiratory cycle. The cycles were delimited by the deflations of the esophageal pressure signal and not with the flow signal since it could mask the onset of the inspiratory effort, especially when the patient has to overcome intrinsic PEEP , 30. Durion only . Pdimax Results are presented as median (Q1-Q3) for descriptive statistics. We calculated our sample size based on the expected correlation between ΔPdi and ΔSMdi of 0.7 . A requip < 0.05) ΔPdi–ΔSMdi correlation and their counterparts. Analyses were performed in the computing environment R [p < 0.05 for all tests.Pearson’s correlation coefficients were used to investigate within-individual relationships between variables. Repeated measure correlation  was used for determining overall relationships between variables . Paired onment R . Signifi2O and a PEEP level of 5 (5–5) cmH2O. A total of 3878 breathing cycles were recorded and 930 were considered for further analysis (i.e. corresponding to triplicate for each condition of ventilation). All patients tolerated each condition of the protocol.Between February 2019 and February 2020, 1087 patients were admitted within the ICU. 404 patients received invasive mechanical ventilation, 99 fulfilled the inclusion criteria leading to the enrollment of 30 patients. Twenty-five patients were considered for subsequent analysis . Regarding within-subject correlation analysis, ΔSMdi and ΔPdi exhibited a significant correlation in 8 patients and no significant correlation in 17 patients . Figure r = 0.81, p = 0.002) and in a patient with no ΔPdi–ΔSMdi correlation  during the protocol. Patients in which ΔPdi–ΔSMdi correlation was non-significant were older, had a faster respiratory rate, lower ΔPdi, lower PTPdi and lower ΔPga as compared to that of patients with significant ΔPdi–ΔSMdi correlation  failed the SBT. Reasons of failure were acute respiratory distress (5/13), neurologic impairment 4/13) and weaning induced pulmonary edema (4/13). ∆Pdi and PTPdi tended to be higher at the start of the SBT in patients who failed the SBT as compared to their counterpart. No difference was found regarding ∆SMdi between the two groups of patients. Table /13 and wThe present work provides new insights regarding the use of ultrasound shear wave elastography for the assessment of diaphragm function in patients under mechanical ventilation using a breath-by-breath analysis. First, we found that changes in diaphragm shear modulus and changes in transdiaphragmatic pressure were significantly correlated. However, when considering the relationship between diaphragm shear modulus and transdiaphragmatic pressure within individuals, the correlation was significant in only a third of patients and it was absent in the remaining patients. Second, we found no significant difference in diaphragm function and change in diaphragm shear modulus in response to changes in ventilatory conditions. Changes in transdiaphragmatic pressure and in diaphragm shear modulus significantly increased during the SBT. Third, we found no significant difference regarding SBT-induced changes in diaphragm shear modulus between patients who failed or succeeded the spontaneous breathing trial.R2 = 0.94 ± 0.05) [20) was much wider than the one in the present study (0–50 cmH2O with 62% of ΔPdi < 10 cmH2O). Therefore, the moderate relationship between ΔSMdi and ΔPdi observed in our study may be partly explained by the relatively low, albeit more physiological, range over which diaphragm effort was analyzed. Our results support this hypothesis, as patients with a significant ΔPdi–ΔSMdi correlation displayed higher ΔPdi , as compared to our previous work in healthy subjects  . In a re ± 0.05) . Our findi Table . Second,ts Table . Also, Δ0 cmH2O, ) and ii)As expected, removing PS was associated with a significant increase in diaphragm function as assessed using ΔPdi and PTPdi as repeatedly observed , 42, 43.A secondary aim of the present study was to investigate differences in diaphragm function and diaphragm shear modulus according to the outcome of a SBT. As previously reported, we observed that patients who failed the SBT had larger  ΔPdi and ΔPTPdi at the start of the SBT as compared to patients who succeeded  was found between changes in transdiaphragmatic pressure (ΔPdi) and changes in diaphragm shear modulus (ΔSMdi) can be found at the following link: https://figshare.com/s/f53dbea5b18c420a1490.Additional file 2.  Figure S2: Individual correlation coefficients between changes in transdiaphragmatic pressure (ΔPdi) and changes in diaphragm shear modulus (SMdi) can be found at the following link: https://figshare.com/s/c0f06ddf34d5a784ccb5.Additional file 3. Figure S3: Movie clip of temporal changes in esophageal pressure, gastric pressure, transdiaphragmatic pressure, and diaphragm shear modulus during pressure support ventilation in one patient with a breathing frequency of 33 breaths/min in which no correlation  was found between changes in transdiaphragmatic pressure (ΔPdi) and changes in diaphragm shear modulus (ΔSMdi) can be found at the following link: https://figshare.com/s/fb33c7701fb50c35c98d.