Further, subgroup analysis was performed based on Treg definition markers and disease activity to identify potential sources of heterogeneity. Forty-seven studies involving a total of 4373 participants were included in the meta-analysis. The Treg/PBMC and Treg/CD4+ T cell ratios were significantly lower in AS patients than those in healthy controls (HCs). A subgroup analysis indicated that patients defined by CD4+CD25+/high, CD4+CD25+CD127low/−, and CD4+CD25+FOXP3+ had much lower Treg/PBMC and Treg/CD4+ T cell ratios than HCs. Active AS patients also had a substantially lower proportion of Tregs/PBMCs and Treg/CD4+ T cells than HCs. The proportion of Tregs among both PBMCs and CD4+ T cells was significantly decreased in AS patients. Treg definition markers and disease activity may influence the proportion of Tregs measured among the PBMC and CD4+ T cell populations. Further study of the correlation between AS disease activity and the proportion of Tregs in peripheral blood is needed to determine the physiological role of this association. This study implies that loss of Tregs may play a role in the pathogenesis of AS and helps clarify the contradictory Treg results in AS patients. This trial is registered with PROSPERO (CRD42019147064).Studies on the number and proportion of regulatory T cells (Tregs) in ankylosing spondylitis (AS) patients have been controversial, which has led to a disagreement regarding the role of Tregs in the pathogenesis of AS. To clarify this debate, we conducted a meta-analysis to verify the reported changes in Tregs during AS. We systematically searched the PubMed, Foreign Medical Retrieval System (FMRS), and China National Knowledge Infrastructure (CNKI) web of knowledge databases for eligible articles. A meta-analysis of studies that examined the proportion and number of Tregs among peripheral blood mononuclear cells (PBMCs) and CD4 In detail, we found significant differences in the proportion of Tregs between AS patients and healthy controls when Tregs were defined as “CD4+CD25+” cells  and as “CD4+CD25high” cells .We hypothesized that the primary reason for the unexpected results might be that the definitions of Tregs were inconsistent. Thus, we performed a subgroup analysis based on Treg definitions to explore the potential sources of heterogeneity . First, low/−” to define Tregs showed that such cell numbers decreased in AS patients  with statistical heterogeneity . More specifically, we found significant differences in the proportion of Tregs between AS patients and healthy controls when Tregs were defined as “CD4+CD25+CD127low/−” cells  and as “CD4+CD25high CD127low/−” cells .Second, the other 14 groups that used “CD127+” cells. Pooled analysis of all 13 trials showed that such cell numbers decreased in AS patients  with statistical heterogeneity . More specifically, twelve studies used “CD4+CD25+FOXP3+” to define Tregs, and pooled analysis showed that such cell numbers decreased in AS patients  with statistical heterogeneity . One study used “CD3+CD4+FOXP3+” to define Tregs, so no further analysis was conducted on this study.Finally, we analyzed 13 groups in which Tregs were defined as “FOXP3p ≤ 0.001], regardless of the Tregs definitions used. The I2 values also showed very high heterogeneity . Thus, we performed a subgroup analysis based on the Treg definitions to explore the potential sources of heterogeneity in active AS patients. In detail, we found significant differences in the proportion of Tregs between active AS patients and healthy controls when Tregs were defined as “CD25-positive” cells  with statistical heterogeneity . More specifically, Tregs were identified as “CD4+CD25+” cells , but no significant difference was apparent when Tregs were defined as “CD4+CD25high” cells .To explore the correlation between AS disease activity and the number of Tregs in peripheral blood, we further contrasted the results for active AS patients and HCs . We havelow/−” cells, pooled analysis of all 36 trials revealed a significant difference  with statistical heterogeneity . More specifically, Tregs were identified as “CD4+CD25+CD127low/−” cells  and as “CD4+CD25highCD127low/−” cells . Finally, the other eight groups that used “CD4+CD25+FOXP3+” to define Tregs showed that such cell numbers decreased in active AS patients  with statistical heterogeneity .When Tregs were defined as “CD127p ≤ 0.001] and the Ankylosing Spondylitis Disease Activity Score (ASDAS) .We also consider that the evaluation criteria of the AS active period are different, which may be another source of heterogeneity. So, we conducted a subgroup analysis based on the different evaluation criteria of the AS activity period. We found significant differences in the proportion of Tregs between active AS patients and healthy controls when the evaluation criteria of AS active period were based on the Bath Ankylosing Spondylitis Activity Index (BASDAI) [−0.793, , + T cells in AS patients and HCs. We initially compared the proportion of Tregs in AS patients and healthy controls regardless of the Treg definition used. In the overall analysis, high heterogeneity  was observed between the studies, and a random-effects model was used in the meta-analysis. We discovered that the percentages of Tregs in AS were significantly lower than those in controls , p ≤ 0.001)  . We also≤ 0.001) .+CD25+CD127low/−” cells , p=0.006) with statistical heterogeneity  and Tregs were identified as “CD4+CD25+FOXP3+” cells , p ≤ 0.001) with statistical heterogeneity .Tregs were identified as “CD4+ T cells. We found significant differences in the proportion of Tregs between active AS patients and healthy controls when Tregs were defined as “CD4+CD25+CD127low/−” cells , p ≤ 0.001) with statistical heterogeneity , but no significant difference was apparent when Tregs were defined as “CD4+CD25positive” cells , p=0.428). There was only one study that defined Tregs as “CD4+CD25+FOXP3+”, so no further analysis was conducted for this study. Both ASDAS and BASDAI can be used as a measure of AS disease activity. So, we also conducted a subgroup analysis based on the different evaluation criteria of the AS activity period (p=0.002) and as “ASDAS” cells , p=0.005) and as “FOXP3+” cells , p=0.005).To explore the correlation between AS disease activity and the number of Tregs in peripheral blood, we further contrasted results for active AS patients and HCs . Similary period . We foun+ T cells in patients with AS showed no obvious publication bias by Egger's test  and , respectively)  .+ T cells. Single studies were successively eliminated without any substantial change in the results. This indicated that the results of the meta-analysis were relatively stable  [+ T cell subsets express the highest levels of CD25 (CD4+CD25 high) and exhibit in vitro immunosuppressive properties [Tregs were originally described as expressing the peripheral CD4n (CD25)  and may n (CD25) , 68. Furoperties . Furtheroperties , which ioperties . Classicoperties . FOXP3, operties , 19, 69.operties . Tregs ioperties .Although studies have confirmed that ASDAS and BASDAI are highly correlated, they are good measures of AS disease activity , 72. ButAlthough we also conducted a subgroup analysis of the active AS period according to the different criteria of the activity period, the actual disease evaluation scores are different; some may have very high scores, while some may just meet the activity period judgment criteria. In addition, not only in the study of active AS but also in all studies, the disease state of each patient is unlikely to be at the same level, which may be the main source of heterogeneity.In conclusion, our meta-analysis also indicates to a certain extent that the absence of Tregs may play an important role in the pathogenesis of AS. This is also consistent with the performance of Tregs in other immune diseases . This wi


P-values (Welch's two-sample t-test), and q-values  from all pairwise comparisons among experimental groups. These statistical data are intended to serve as means of identification for lipid species whose levels were altered by the disease, and which could be used as biomarkers of symptom emergence and disease progression. Because of few studies on the subject, the association between modulation of lipid biosynthetic pathways and disease progression in grapevine has remained poorly understood. The analysis of the data described here has already provided new perspectives regarding the pathogenesis of ESCA leaf symptom formation. Reanalysis of these data would undoubtedly unravel some physiological roles played by lipids in the adaptation of vine plants to stressful conditions.The dataset presented in this paper comprises the masses of 208 lipid species and other compounds of lipid metabolism, and their levels in leaves of vines with brown wood streaking and grapevine leaf stripe, two symptomatic expressions of Esca complex disease (ESCA). Healthy, asymptomatic and symptomatic leaves were collected from the cultivar Malvasia Fina grown in a vineyard. The lipidome of these leaves was characterized using a platform consisting of an Ultrahigh Performance Liquid Chromatography and a Gas chromatography equipment coupled to a Q-Exactive Hybrid Quadrupole-Orbitrap high resolution/accurate Mass Spectrometer interfaced with a heated electrospray ionization source. The analysis permitted the detection of 158 molecular species of known identity and 50 species of unknown structural identity. The area counts of these molecular species is reported in the dataset, along with fold changes (log2-ratio),  Additional analyses such as principal component, hierarchical clustering, and random forest could be performed to explore whether different classification methods nominate different candidate predictors of the presence of the disease.A direct use of these data would be for identification of metabolite markers associated with delay appearance of ESCA-foliar symptoms in grapevine. Fold change and •This type of data is likely to be extremely beneficial to researchers working on finding molecular markers that could assist in the current diagnostic approaches for ESCA. The identified biomarkers could be used by investors for the development and commercialization of a non-invasive biochemical method for early esca diagnosis by winegrowers.•So far, no characterization has been made of global lipid changes that occur following infection of vines by pathogens. The lipidome data generated from the leaves of grapevine and reported in this paper could be used to determine whether and how reprogramming of lipid metabolism exert a role on the etiopathogenesis of ESCA and other vine diseases.•These lipidomic data can also be analyzed together with other published grapevine-related OMIC data in order to discover new insights into the host's mechanisms of resistance, or to elucidate key issues in grapevine cell biology such as evolution and crop enhancement.•Through pathway enrichment or correlation network analyses, data reported here could yield important information about the metabolic pathways associated with reduced foliar symptom expression that might consent to engineer resistant cultivars.1. There were six biological replicates for each of the leaf group, for a total of 24 samples coded PD-UDTD-00001 to PD-UDTD-00024 (Table S1).The data provided in the MENDELEY repository is related to the levels of lipid species in four experimental leaf groups  as described in Fig. 1m/z) in Table S1; each compound is assigned a unique ID (001 to 208). Chromatographic peaks corresponding to lipid species were quantified as area-under-the-curve detector ion counts and area counts were normalized to the sample weight to obtain the raw data in Table S1. The proportions of samples with successful quantification (detection rates) are also shown in the table. For each compound, the chromatographic condition used for quantification is listed i.e. LC/MS Pos Early, LC/MS Pos Late, LC/MS Polar, LC/MS Neg or GC/MS. Empirical names (LIPID NAME) and chemical structures (SMILES = Simplified Molecular-Input Line-Entry System) were generated for each given accurate mass. Compounds with the same mass and retention time that could not be integrated independently (i.e. isobars) are denoted by adding a number sign after the isobaric species name and a number within brackets after its abbreviation. Peaks that had reproducible retention time, mass, and MS/MS spectra, but could not be associated with a chemical library are given a numerical designation starting with X (e.g. X–23440); these are labeled “UNNAMED” in the column “TYPE”. The designation enables these compounds to be documented in future experiments and to be identified by future acquisition of a matching purified standard or by classical structural analyses.In total, 208 lipid species and other compounds of lipid metabolism were successfully detected in all 24 replicate samples and are listed along with their retention indexes and mass-to-charge ratios  classes, including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, and prenol lipids . CompounTable S2, fold changes (log2 ratios) and P-values (Welch's two-sample t-tests) were applied to screen for potential differences between the means. An estimate of the false discovery rate  is also provided to take into account the multiple comparisons. The following colors are used for highlighting differences between the groups: white = P > 0.10 for unaffected levels, dark green = P ≤ 0.05 for decreased levels, light green = P ≤ 0.10 for decreased levels, red = P ≤ 0.05 for increased levels, and light red = P ≤ 0.10 for increased levels. Box-and-whisker plots for 208 molecular species are sorted by biochemical pathway in Figure S1; 29 of the plots are also present in Table S3 for a general definition of colors and further descriptions of codes used in the dataset. For ease of presentation, abbreviations are provided for all molecular species names in the tables and charts.Scaled imputed data used in statistical analyses are reported 22.12.1.12 in 1995 with V. vinifera L. ‘Malvasia Fina’ grafted on 19-17-Castel rootstock. Malvasia Fina, also known as Boal, is a white grape cultivar grown historically in Portugal. Initially, the vineyard had 1247 vines organized in 22 rows. With time, some vines have been uprooted for studies and some have died; missing vines have not been replanted. The distance between the rows is 1.80 m for 1.12 m spacing between vines in the row. Different training systems are used for canopy management. Based on observations of leaf symptoms during the last decade, ESCA is the main disease in the vineyard Samples used for lipidomic analyses were collected in a vineyard naturally infected by ESCA-associated pathogens. The vineyard, called Quinta de Nossa Senhora de Lourdes , is in the city of Vila Real in Portugal. In the immediate surroundings of the vineyard are three other vineyards with different cultivars  separated from each other by roads, and a cherry field (south). The vineyard was planted on an area of 2700 m2/s). Precipitation was limited with 880.7 mm average rainfall and the vineyard had no irrigation system. The soil (Anthrosol) was uniform across the experimental plot and was characterized by 62% sand, 25% silt, and 13% clay (pH 4.2). Pruning, fertilization and plant protection measures were undertaken according to Plant care guidelines developed by Universidade de Trás-os-Montes e Alto Douro.In the sampling year, vines were grown in the field at an average annual air temperature of 13.97  °C with 75% relative humidity and a 16-h photoperiod (1350 μmol/m2.1.2The sampling protocol was developed using four rows of vines (a block of 243 vines) facing north and trained to a bilateral cordon according to the royal-type trellis system. An inspection period of six continuous years gave sufficient time to identify all the vines showing internal and external signs of ESCA in the study block. Visual inspection of all individual vines was conducted yearly after grape cluster harvesting, following published recommendations Affected vines were characterized as having diverse shapes and colors of wood and leaf deterioration and necrosis, and were grouped into I, II, III, and IV. Group I comprised of healthy unaffected control vines (CTL) that had minor or non-necrotic wood tissues and did not express foliar symptoms in any of the inspection year. Group II comprised of wood-symptomatic and foliar-asymptomatic vines (ASY) that expressed tiger stripe-like foliar symptoms no more than three times during the 6-year survey period. These vines exhibited clear signs of brown wood streaking, which is a wood deterioration characteristic of ESCA 2.1.3For each of the experimental groups, ten vines were identified. Approx. 10 leaves facing and opposite a grape bunch were collected from the central portion of each vine, immediately frozen with liquid nitrogen and stored at −80  °C. A similar set of samples was kept at normal temperature and dried in an oven at 85  °C to a constant weight for water content determination. Once the water content was known, samples from six vines  were selected for lipidomic analyses. Prior to these analyses, samples were freeze-dried and pulverized using a blender .2.22.2.1All organic solvents and reagents of analytical grade (≥ 98%) were purchased from Sigma-Aldrich  or Panreac Química SLU . Chemicals for lipid extraction included methanol, chloroform, potassium chloride, hexane, 14% boron trifluoride, and anhydrous sodium sulfate. Standard mixtures of fatty acid methyl esters (FAME37), alkanes mixtures, LC-MS grade acetonitrile and methanol were also purchased from Sigma-Aldrich, as well as ammonium bicarbonate, ammonium formate, ammonium hydroxide, perfluoropentanoic acid and formic acid. Water was obtained from a Millipore Ultra-high purity water dispenser . Isotopically labelled compounds  were purchased from C/D/N Isotopes  and Santa Cruz Biotechnology .2.2.2g for 5 min (25  °C), the top organic phase was collected and divided into four fractions. The fractions were transferred to a speed vacuum concentrator  to remove the organic solvent and the resulting residues frozen at −80  °C. In the second extraction step, 50 mg sample was added with a mixture of methanol, chloroform, water, and potassium chloride as described in Lipids were extracted using a modification of the protocol proposed by 2.2.3The fractions were removed from the freezer and stored under a nitrogen stream at room temperature for ca. 15 h before being reconstituted in solvents compatible to each of the five methods used for chromatographic separations.Fraction 1 was reconstituted in 50 µL 0.1% formic acid in water and analyzed by UPLC using acidic positive ion conditions chromatographically optimized for the elution of hydrophilic compounds (LC/MS Pos early). The extract was gradient-eluted from a C18 column  at 40  °C and 0.35 mL/min using (A) 0.1% formic acid and 0.05% perfluoropentanoic acid in water and (B) 0.1% formic acid and 0.05% perfluoropentanoic acid in methanol .Fraction 2 was also reconstituted in 50 µL 0.1% formic acid in water and analyzed by UPLC using acidic positive ion conditions, but chromatographically optimized for the elution of hydrophobic compounds (LC/MS Pos late). The extract was gradient-eluted from the aforementioned C18 column at an overall higher organic content using (A) 0.01% formic acid in water and (B) 0.05% perfluoropentanoic acid in 60/40 methanol/acetonitrile; and the same gradient profile as for Fraction 1.Fraction 3 was reconstituted in 50 µL of 6.5 mM ammonium bicarbonate in water (pH 8.0) and analyzed by UPLC using basic negative ion optimized conditions (LC/MS Neg). The basic extract was gradient-eluted from a separate base-dedicated C18 column using (A) 6.5 mM ammonium bicarbonate in water (pH 8) and (B) 6.5 mM ammonium bicarbonate in 95/5 methanol/water (same gradient profile as above).Fraction 4 was reconstituted in 50 µL ammonium formate (pH 10.8) in water and analyzed via negative ionization following elution at 0.35 mL/min from a Hydrophilic Interaction Liquid Chromatographic (HILIC) column  using a gradient consisting of (A) 10 mM ammonium formate in 15% water, 5% methanol, 80% acetonitrile; pH 10.8 and (B) 10 mM ammonium formate in 50% water, 50% acetonitrile; pH 10.6 . The pH of all solvents was adjusted by adding ammonium hydroxide, and the conditions were optimized for targeting polar compounds (LC/MS Polar).Fraction 5 was reconstituted in 1 mL hexane. Following vortexing for 3 min, the upper phase was analyzed by GC, with elution on a DB-225MS column . Separation conditions targeted free fatty acids (GC/MS) and were as follows: split ratio of 5:1; inlet temperature of 270  °C; carrier gas, helium ; oven temperature, 200  °C ; ion source temperature, 230  °C; interface temperature, 270  °C; ionization voltage, 70 eV.Reversed phase separation of molecular species was achieved by Ultrahigh Performance Liquid Chromatography using an ACQUITY UPLC System . Capillary gas chromatography was with a Shimadzu GC-2010 Plus . The machines were coupled to a Q-Exactive Hybrid Quadrupole-Orbitrap high resolution/accurate mass spectrometer (HRaMS) interfaced with a heated electrospray ionization (HESI-II) source . In all cases, an aliquot of 5 μL of extract fraction was loaded in the chromatograph using an automatic injector and analyzed using MS.2.2.4Samples were analyzed in positive and negative ion modes depending on the fractions. The MS interface capillary was maintained at 350  °C and the corona discharge current was set at 5 μA. The spray voltages for the positive and negative ion injections were 4.50 and 3.75 kV, respectively. Nebulization was with nitrogen, with a sheath gas flow of 40 (arbitrary units) and an auxiliary gas flow of 5 (arbitrary units) for both positive and negative injections. The nebulizer temperature was set at 400  °C.n) modes, and over the period between 1 and 15 min within UPLC gradients and GC. The scan range varied slightly depending on the fraction analyzed, but covers approximately 70–1000 m/z. The resolution for all scans was set at 35,000 (measured at 200 m/z) and approximately 9 scans were performed per second, which restricted the loading time to 100 ms. MS/MS normalized collision energy was set to 40, activation Q 0.25, and activation time 30 ms, with a 3 m/z isolation window. MS/MS scans were collected using dynamic exclusion with an exclusion time of 3.5 s. The system was calibrated internally as needed to maintain a mass error < 5 ppm for all internal standards (IS) monitored.The mass spectra were recorded alternately between full-scan (MS) and all-ion fragmentation-scan  of six recovery standards in methanol , which permitted the monitoring of extraction efficiency. The percent recovery was ≥ 95% for each standard.All reconstituted fractions were spiked with a series of isotopically labeled IS at fixed concentrations before injection into the MS. The following ISs were used to assess instrument performance and to serve as retention time (RT) markers for chromatographic alignment: d7-glucose, d3-methionine, d3-leucine, d8-phenylalanine, d5-tryptophan, Cl-phenylalanine, Br-phenylalanine, d15-octanoic acid, d19-decanoic acid, d27-tetradecanoic acid, d35-octadecanoic acid for UPLC and alkanes mixtures C8-C40 (even) for GC.Technical replicate samples generated by combining a small portion of each of the 24 experimental samples were also analyzed. Water blanks and solvent blanks were used to provide a baseline reference signal and segregate external contamination sources, respectively. Experimental samples were randomized across the platform run, with technical replicates and blanks spaced evenly among the injections.Instrument variability was determined by calculating the median relative standard deviation (RSD) for the ISs. Overall process coefficient of variation was determined by calculating the median RSD for all endogenous lipids present in technical replicate samples. Median RSD for instrument and process variability were 7% and 8% (< 10%) respectively, indicating that all aspects of the analysis process were operating within specifications.2.2.6m/z) within a matrix along with their respective intensities for each sample.Data acquisition was performed using Xcalibur QuanBrowser 3.0 and Lab Solution 5.71 for UPLC and GC, respectively. Various user-defined peak threshold values were included for baseline correction, chemical noise subtraction, chromatogram alignment, peak detection, and integration. This included a signal-to-noise ratio > 5, peak area > 50,000 (LC) and 100 (GC), peak width = 0.2 min. After this preprocessing step, raw data were extracted and a list of peaks passing above threshold criteria was obtained. These peaks were organized by mass-to-charge ratio . Using retention indexes (RI), LIMS was first used to perform a RT correction of the output matrix based on RT markers placed throughout the chromatographic time window. Then, the resulting MS/MS data were searched against the LIMS library of purified standards and routinely detected unknown entities by comparing their specific Compound identifications were based on three criteria: (i) RI within a narrow window of the proposed identification, typically 75 RI units of the proposed identification or approximately 5 s, (ii) accurate mass match to the library of +/− 10 ppm, (iii) MS/MS forward and reverse scores above 80%. Identifications were automatically approved if all the above criteria were met Hundreds of molecular species were successfully detected in all samples. Compounds that were detected in at least 33% of all replicates for the same test group were included in subsequent analyses. Peaks were quantified as area-under-the-curve detector ion counts and normalized to the sample weight. The raw area count for each compound was rescaled through the division of each sample value by the median value for this specific compound to obtain the scaled imputed data.2.2.8t-tests were used to compare the means of the different leaf groups. Before these analyses, scaled imputed data were log10-transformed to normalize distributions. A P-value correction was performed by estimating the false discovery rate − which was obtained by the q-value −, thereby accounting for multiple comparisons. For a better visualization of the data, box-and-whisker plots of means were constructed for each molecular species.Statistical analyses consisted of a series of pairwise comparisons among experimental groups  using the Excel add-in application “Statistical Analysis Tool” (http://prime.psc.riken.jp/compms/others/main.html#Statistics). Following imputation of missing values with the minimum observed value for each compound, fold changes were calculated as log2 ratio of the scaled imputed mean of an experimental group and that of another experimental group. Welch's two-sample Piebiep Goufo: Conceptualization, methodology, validation, formal analysis, investigation, data curation, writing– original draft preparation, writing– review and editing, visualization. Isabel Cortez Conceptualization, methodology, validation, resources, writing– review and editing, supervision, project administration, funding acquisition.This article does not contain any studies involving animals or humans performed by any of the authorsThe authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.


As a fundament in many biologically relevant processes, endocytosis in its different guises has been arousing interest for decades and still does so. This is true for the actual transport and its initiation alike. In clathrin-mediated endocytosis, a comparatively well understood endocytic pathway, a set of adaptor proteins bind specific lipids in the plasma membrane, subsequently assemble and thus form a crucial bridge from clathrin to actin for the ongoing process. These adaptor proteins are highly interesting themselves and the subject of this manuscript. Using many of the instruments that are available now in the mass spectrometry toolbox, we added some facets to the picture of how these minimal assemblies may look, how they form, and what influences the structure. Especially, lipids in the adaptor protein complexes result in reduced charging of a normal sized complex due to their specific binding position. The results further support our structural model of a double ring structure with interfacial lipids.  •IM-MS and SID reveals that reduced charge states are a result of interfacial lipids.•ANTH subunits in the outer ring interact barely with each other and stabilize ENTH interaction.•Additional intermediates confirm our recent double ring model. Instead, binding of six PIP2 ligands proved sufficient to stabilize the ENTH homo-hexamer . EmployiProteins can easily impose sample preparation- and data interpretation-related obstacles on structural analysis by conventional methods as X-ray crystallography, EM or NMR due to heterogeneity or lack of stability. Protein structural studies utilizing MS methods tend to circumvent such difficulties because of inherently low sample consumption, high mass accuracy and sensitivity, the theoretical absence of an upper size limit and, above all, the option to measure several simultaneously present association- or conformational states. This is partially true for indirect approaches like surface labelling, backbone amide hydrogen exchange or cross-linking  and a ma2Protein production and purification: Human ENTH and the yeast ENTH1, ENTH2, and ANTH were produced recombinantly in E. coli BL21 DE3 (Novagen) from the pETM30 vector as GST fusion proteins with an N-terminal His-tag and a TEV cleavage site between His-GST tag and the protein of interest. Upon cell harvesting, purifications by NiNTA and TEV cleavage followed. For detailed descriptions see Ref. [Sample preparation for native MS: Typically, purified proteins were buffer exchanged to 300 mM ammonium acetate  and 1 mM DL-dithiothreitol , pH 8.0, via centrifugal filter units  at 13,000×g and 4 °C. Complexes were assembled after buffer exchange by mixing with final concentrations of 10 μM ENTH, 10 μM ANTH and 60 μM PIP2 . Native Orbitrap MS: For Orbitrap measurements, yeast proteins were used at a mixing ratio of 0.7 ENTH1: 1 ANTH: 3.5 PIP2 and exchanged into ammonium acetate after complex assembly [−9 mbar in the ultra high vacuum region of the Orbitrap mass analyzer. Source fragmentation parameter and collision energy in the HCD cell, both to force desolvation of the protein complex ions, were 100 V and 50 V, respectively. Transients were recorded for 64 ms per scan, averaging over several minutes to acquire the spectra presented. Native QToF1 MS: Yeast proteins were used at a mixing ratio of 0.7 ENTH1: 1 ANTH: 3.5 PIP2  and buffer exchanged into ammonium acetate, either before or after complex assembly [−2 mbar xenon was used as collision gas. Collision voltage was 75 V. Capillary and sample cone were at 1350 V and 150 V, respectively. Native ion mobility MS: Proteins were prepared as described and introduced into a commercially available travelling wave ion mobility mass spectrometer  by nano-electrospray using home-made gold-coated borosilicate emitters. The instrument is equipped with a 32 k quadrupole allowing transmission of high mass ions and with a pusher frequency of 1900 s−1. All measurements were done in positive ion mode. The highest values used for measuring yeast ENTH:ANTH:PIP2 complexes and still considered native were 2.3 kV, 50 V, and 0.5 V capillary, sample cone, and extraction cone voltage, respectively. Trap and transfer collision energies were 15 V and 5 V, trap DC bias and helium DC bias 45 V and 25 V, respectively, if not stated otherwise. Backing pressure was set to 8 mbar and 2.5 mbar nitrogen (60 ml/min IMS gas flow) was used for ion mobility separation. For ion separation, wave velocities of 650 m/s and wave heights of 40 V were used. Wave velocities in trap and transfer cell were 300 m/s and 64 m/s with wave heights of 6.0 V and 2.9 V, respectively. Collision gas was argon with 4.6 × 10−2 mbar and 4.4 × 10−2 mbar in trap and transfer cell, respectively. Settings for measurements of human PIP2-containing ENTH hexamers were 1.3 kV capillary voltage, 25 V sampling cone, 4 V extraction cone voltage. Trap collision energy, trap DC bias, helium DC and transfer collision energy were set to 25 V, 45 V, 25 V, and 5 V, respectively. Backing pressure was 8.5 mbar and 3.1 mbar nitrogen (90 ml/min IMS gas flow) was used for ion mobility separation. Argon was used as collision gas with 3.1 × 10−2 mbar in the trap and 3.2 × 10−2 mbar in the transfer cell. Travelling waves with heights of 25 V and velocities of 300 m/s were used for ion mobility separation. Wave velocities in trap and transfer cell were 300 m/s and 66 m/s with wave heights of 6.0 V and 2.0 V, respectively. Alcohol dehydrogenase from S. cerevisiae (#A7011), concanavalin A from C. ensiformis (#C2010), glutamate dehydrogenase from bovine liver (#G7882), pyruvate kinase from rabbit muscle (#P9136), and bovine serum albumin (#P7656), all from Sigma-Aldrich, were used as ion mobility calibrants employing a logarithmic fit procedure [2 and the according underestimation, CCS was scaled by the experimentally derived factor of 1.14 [Surface-induced dissociation MS: Samples were prepared and sprayed as described above and analyzed in positive ion mode on a Synapt G2™ HDMS instrument with an additional SID cell located upstream of the ion mobility separation cell [i.e. without a collision with the surface, were acquired with 1.6 kV capillary voltage and 80 V sampling cone voltage. Trap and transfer cell collision energies were both set to 10 V. 2.5 mbar nitrogen was used for ion mobility separation. Travelling waves with heights of 20 V and velocities of 300 m/s were used in the IMS cell. Measurements in SID mode were performed with 1.8 kV capillary voltage and 50 V sampling cone voltage. Trap and transfer collision energies were set to 10 V and 4 V, respectively. 2.2 mbar nitrogen was used for ion mobility separation of SID products in the IMS cell. The potential difference between the trap DC bias and the surface of the SID cell defined the SID collision energy. It was ramped from 30 V upwards. Due to the low signal intensity in MS/MS mode, spectra with broader precursor selection windows were acquired. For that purpose, the MS profile was set to the range of 10,000–15,000 m/z for 75% of the dwell time.see Ref. . Sample assembly . The insrocedure  and measrocedure  with the of 1.14 . Surfaceion cell . The SIDion cell . Spectra33.1As outlined above, PIP2 binding to individual fungal ENTH or ANTH does not change the monomeric state of either protein. However, as soon as both proteins and PIP2 are present, ligand binding induces formation of a rather low-abundant di-hexamer, which quickly grows into the prominent di-octamer. Formation of these complexes proceeds in a highly cooperative manner as no other intermediate species accumulated    predict W1.8868)   for ENTH1-ANTH complexes favored transmission of di-octamers carrying a comparable number of ligands as before (TWCCSN2) for all relevant ENTH-ANTH complexes could thus be calculated  species alike, may result from the Synapt having decreasing transmission at higher s before A and wass before . The sigs before B. The colculated .Fig. 2HiTWCCSN2 values of reference proteins [TWCCSN2 on charge - the larger the ions, the lesser the influence of actual charge on their TWCCSN2. The TWCCSN2 of the yeast ENTH-ANTH complexes, on the other hand increased slightly but significantly with charge , we observed as dominant species the di-octamer complex with 22–31 ligands and a previously described aggregate , a dimer4The preferred charge and ligand-binding states of the yeast ENTH-ANTH di-octamers are a special case: As outlined, theory predicts 45 + as dominant species. Although, membrane proteins may be expected to charge up less in presence of lipids, at least when well resolved they usually match the prediction. The membrane-associated complexes described here almost reached this value (43+) on an Orbitrap instrument as opposed to QToF or Synapt measurements. It is tempting to deduce these differences to peculiarities of the concerned instruments or spray conditions; however, charge states inversely correlate with lower ligand binding; the extremes being 23 PIP2 at 43+ (Orbitrap) and up to 31 PIP2 and 34+ . This also holds on spectrum level. In The simplest explanation for this finding is neutralization of positively charged residues by the negatively charged phospholipid. Charged residues include arginine and lysine residues, which are abundant close to the previously known canonical ligand-binding sites. Neither for the monomers nor the human ENTH hexamer, however, was the PIP2 induced charge reduction observed suggesting that it is specific for the higher order oligomers. This in turn implies that the PIP2-induced charge reduction is due to surplus ligands residing at ANTH-ENTH interfaces. Thus, surficial arginine and lysine residues between outer and inner ring subunits are good candidates. Although of highly speculative nature, this may prompt future modelling attempts. Such surficial lipids cannot be bound as tightly as the CID resistant ones and could be regarded as gradually increasing membranous environment. Upon release of membrane proteins from membrane environments, lipids are gradually stripped off   than the precursor (≈31), only a small fraction of the released SID-ANTH subunits had a bound lipid (To conclude, our results further confirm the proposed assembly model with a ring-shaped ENTH core surrounding a central cavity. PIP2 lipids mostly locate to the interface of the ENTH and ANTH subunits, which is in line with little PIP2 carried along by SID-ejected ANTH subunits, PIP2-induced charge reduction exclusively in ENTH-ANTH complexes and high lipid content in complexes missing ANTH subunits. The additional complex stoichiometries are supportive of the fungal complexes also assembling around an ENTH core, which is however not stable at room temperature in absence of ANTH . SID fur


This study aimed to summarize earlier studies on the effects of RS consumption on the serum levels of inflammatory biomarkers.A comprehensive search was done in the electronic databases that published from 1988 up to May 2019. Two reviewers independently performed screening, data extraction, and risk-of-bias assessment. We used from the effect size, as estimated by the mean difference to perform the fixed method meta-analysis.P = 0.001) levels. However, no significant changes were found in C-reactive protein concentration . Moreover, the changes in interleukin 6 concentration was dependent on study quality and intervention duration.Overall, 13 studies with 14 effect sizes met the inclusion criteria and were included in the final analysis. Sample size of these studies ranged from 15 to 75 and intervention duration ranged from 4 to 14 weeks. Meta-analysis revealed that higher consumption of resistant starch caused a significant reduction in the interleukin 6  and tumor necrosis factor alpha (weighted mean difference = − 2.19 pg/mL; 95% CI: − 3.49, − 0.9 pg/mL; The current meta-analysis indicated that RS intake can improve some inflammatory biomarkers. More research, with a large sample sizes and accurate design is recommended. Low grade systematic inflammation is involved in the development and progression of several metabolic conditions , 2. InflAccording to previous studies, diet can play a role in reducing inflammation , 9. For Resistant starch (RS) is a part of starch in the diet that is not digested and absorbed in the small intestine and is fermented in the colon to produce short chain fatty acids (SCFA) . It is cStudies on the effect of resistant starch on inflammatory markers are conflicting. Some studies showed a significant decrease in inflammatory markers after RS consumption compared to placebo , 18, butAlthough studies have been conducted on the effect of resistant starch on inflammatory markers, as far as we know there is no study that summarizes the findings of previous studies. Therefore, the purpose of this study was to provide a systematic review and meta-analysis of extant literature about the effect of RS consumption on several inflammatory biomarkers.clinicaltrials.gov, Scopus, EMBASE, Cochrane Library and World Health Organization International Clinical Trial Registry Platform). Also, the reference list of related articles was hand searched for additional relevant studies. Combinations of the terms “Resistant starch” OR “resistant maltodextrin” OR “resistant dextrin” OR “indigestible dextrin” OR OR “indigestible starch” OR “high amylose starch” OR “slowly digestible starch” AND inflammation OR inflammatory OR “inflammatory factors” OR “C reactive protein” OR “C-reactive protein”OR “high-sensitivity C-reactive protein” OR hs-CRP OR interleukin-6 OR “interleukin 6” OR IL-6 OR “tumor necrosis factor-” OR “tumor necrosis factor” OR TNF-α were searched. We included only studies with the randomized method and measurement of ≥1 of the primary outcomes. Other type of the studies  and studies that resistant starch was used in combination with other dietary components, supplements or drugs were not included in our study.The reporting of this review is aligned with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) . Systemaκ statistic were calculated to check concordance between reviewers [Data were extracted independently by the 2 investigators (MR and MV) according to eligibility criteria Table . The pereviewers . In the For evaluation of the studies quality, we used from the Jadad Scale and the Downs and Black assessment tools , 24. Thi2 index was used for evaluation of heterogeneity. In the classification of I2 index, lower heterogeneity defined as a I2 < 30%, moderate if I2 = 30–75%, and high if I2 > 75% [We used from the effect size, as estimated by the mean difference to perform the fixed method meta-analysis. Weighted mean difference (WMD) with 95% CI was used for pooling data to determine effect sizes. For each measurement, a random effects meta-analysis was performed. II2 > 75% . To findP < 0.05.Funnel plots were used to visually inspect for the presence of publication bias. Moreover, we used from Begg’s rank correlation and Egger’s linear regression tests for evaluation of publication bias. All analyzes were performed using Stata software and results was regarded significant at Flow diagram of study selection is shown in Fig. The publications included in the meta-analysis are described in Table . According to the JADAD core, eight studies had high quality [Findings from assessing the quality of RCTs are shown in Supplementary Table 1 quality , 32, 34  quality , 33, 35. quality , 32, 34, quality , 33, 35,P = 0.61), with a significant between-study heterogeneity   . Due to the high between-study heterogeneity, we stratified studies based on study quality (> 19 vs. ≤19) and duration of follow up (> 8 weeks vs. ≤8 weeks). The subgroup analysis did not provide additional information. Sensitivity analysis revealed that no individual study had a great effect on the overall results.Seven RCTs with eight effect sizes had reported the effect of resistant starch intake on TNF-α levels , 31, 32.I2 = 93.9, P < 0.001)  and that duration of follow up ≤8 weeks . Sensitivity analysis revealed that no individual study had a great effect on the overall results.Pooling effect sizes from seven studies , 28–33, 01) Fig. . SubgrouP = 0.115; Begg’s test P = 0.24)    by intestinal bacteria. Butyrate is the main SCFA that is produced from the fermentation of RS and acts as an anti-inflammatory agent through interference in various inflammatory pathways –13. The Moreover, consuming RS through the weight loss, especially in obese and overweight people, can help to reduce inflammation. Excess body weight can increase the expression of inflammatory biomarkers such as TNF-a . The exaSome trials showed that RS can improve the levels of inflammatory markers –21, whilThe strengths of this study include identifying randomized trials with a rigorous search strategy and subgroup analysis of based study quality, and duration of intervention. Also, the results from the pooled effect size increased statistical power and are more convincing compared to a single study, considering the intra- and inter-individual variations as well as the small sample size of each eligible study.Our studies has few limitations. Firstly, most of the studies that included in the final analysis had small sample size, which may bring a small study effect. Secondly, the samples were from various diseases and healthy individuals. Finally, the inter-study heterogeneity was high.In conclusion, the current study pooled results from 13 RCTs about the effects of RS consumption on inflammatory mediators. The results of our study showed that RS could have anti-inflammatory effects. Anyway, additional studies must be carried out that include well-designed protocols, and larger sample sizes to illustrate the beneficial effects of RS consumption on inflammation.Additional file 1 Supplementary Table 1-A. Jadad Quality Assessment Scores1.Supplementary Table 2. Downs Quality Assessment Scores1.


Oesophageal cancer is a common and often fatal cancer that has two main histological subtypes: oesophageal squamous cell carcinoma and oesophageal adenocarcinoma. Updated statistics on the incidence and mortality of oesophageal cancer, and on the disability-adjusted life-years (DALYs) caused by the disease, can assist policy makers in allocating resources for prevention, treatment, and care of oesophageal cancer. We report the latest estimates of these statistics for 195 countries and territories between 1990 and 2017, by age, sex, and Socio-demographic Index (SDI), using data from the Global Burden of Diseases, Injuries, and Risk Factors Study 2017 (GBD).We used data from vital registration systems, vital registration-samples, verbal autopsy records, and cancer registries, combined with relevant modelling, to estimate the mortality, incidence, and burden of oesophageal cancer from 1990 to 2017. Mortality-to-incidence ratios (MIRs) were estimated and fed into a Cause of Death Ensemble model (CODEm) including risk factors. MIRs were used for mortality and non-fatal modelling. Estimates of DALYs attributable to the main risk factors of oesophageal cancer available in GBD were also calculated. The proportion of oesophageal squamous cell carcinoma to all oesophageal cancers was extracted by use of publicly available data, and its variation was examined against SDI, the Healthcare Access and Quality (HAQ) Index, and available risk factors in GBD that are specific for oesophageal squamous cell carcinoma  and for oesophageal adenocarcinoma .There were 473 000  new cases of oesophageal cancer and 436 000 (425 000–448 000) deaths due to oesophageal cancer in 2017. Age-standardised incidence was 5·9 (5·7–6·1) per 100 000 population and age-standardised mortality was 5·5 (5·3–5·6) per 100 000. Oesophageal cancer caused 9·78 million (9·53–10·03) DALYs, with an age-standardised rate of 120 (117–123) per 100 000 population. Between 1990 and 2017, age-standardised incidence decreased by 22·0% (18·6–25·2), mortality decreased by 29·0% (25·8–32·0), and DALYs decreased by 33·4% (30·4–36·1) globally. However, as a result of population growth and ageing, the total number of new cases increased by 52·3% (45·9–58·9), from 310 000 (300 000–322 000) to 473 000 (459 000–485 000); the number of deaths increased by 40·0% (34·1–46·3), from 311 000 (301 000–323 000) to 436 000 (425 000–448 000); and total DALYs increased by 27·4% (22·1–33·1), from 7·68 million (7·42–7·97) to 9·78 million (9·53–10·03). At the national level, China had the highest number of incident cases (235 000 [223 000–246 000]), deaths (213 000 [203 000–223 000]), and DALYs (4·46 million [4·25–4·69]) in 2017. The highest national-level age-standardised incidence rates in 2017 were observed in Malawi (23·0 [19·4–26·5] per 100 000 population) and Mongolia (18·5 [16·4–20·8] per 100 000). In 2017, age-standardised incidence was 2·7 times higher, mortality 2·9 times higher, and DALYs 3·0 times higher in males than in females. In 2017, a substantial proportion of oesophageal cancer DALYs were attributable to known risk factors: tobacco smoking (39·0% [35·5–42·2]), alcohol consumption (33·8% [27·3–39·9]), high BMI (19·5% [6·3–36·0]), a diet low in fruits (19·1% [4·2–34·6]), and use of chewing tobacco (7·5% [5·2–9·6]). Countries with a low SDI and HAQ Index and high levels of indoor air pollution had a higher proportion of oesophageal squamous cell carcinoma to all oesophageal cancer cases than did countries with a high SDI and HAQ Index and with low levels of indoor air pollution.Despite reductions in age-standardised incidence and mortality rates, oesophageal cancer remains a major cause of cancer mortality and burden across the world. Oesophageal cancer is a highly fatal disease, requiring increased primary prevention efforts and, possibly, screening in some high-risk areas. Substantial variation exists in age-standardised incidence rates across regions and countries, for reasons that are unclear.Bill & Melinda Gates Foundation. Evidence before this studyOesophageal cancer is one of the leading causes of cancer mortality worldwide. Previous publications have reported on various health metrics of oesophageal cancer, but to our knowledge, no study has provided detailed estimates of incidence, mortality, disability-adjusted life-years (DALYs), and DALYs attributable to major risk factors for 195 countries and territories around the world. Furthermore, previous studies have not investigated the country-level correlation between the proportion of new oesophageal squamous cell carcinoma to all oesophageal cancer cases and potential risk factors for oesophageal squamous cell carcinoma, such as indoor air pollution and inadequate access to improved water sources.Added value of this studyWe used data from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2017 to provide the most up-to-date estimates on a wide range of health measures related to oesophageal cancer at global, regional, and country-specific levels for 195 countries and territories, and by sex, age group, and Socio-demographic Index (SDI). We present estimated numbers and age-standardised incidence rates for oesophageal cancer in 2017, as well as trends from 1990 to 2017. We also describe DALYs attributed to several major risk factors for oesophageal cancer. Furthermore, for the first time, we present the correlation of the country-level proportion of new oesophageal squamous cell carcinoma to all oesophageal cancer cases with several potential risk factors for oesophageal squamous cell carcinoma, including indoor air pollution, inadequate access to improved water sources, and SDI. These correlations can guide further investigations into the causes of oesophageal squamous cell carcinoma.Implications of all the available evidenceOesophageal cancer remains a major cause of cancer mortality and disease burden across the world, requiring substantial preventive measures and possibly screening by use of upper gastrointestinal endoscopy in certain high-risk countries. The highest burden of oesophageal cancer (in terms of the number of DALYs) is in east Asia, particularly China, where screening has been shown to be effective in increasing survival rates. Other high-incidence regions are in central Asia and sub-Saharan Africa. The main reasons for these high rates are unknown. Further research into the causes of oesophageal cancer in these high-risk areas is warranted. Our findings are consistent with previous studies that suggest indoor air pollution, inadequate access to improved water sources, and low socioeconomic status are associated with increased risk of oesophageal squamous cell carcinoma. Several epidemiological studies are ongoing by different research groups, and we expect to see new findings over the next few years.Oesophageal cancer is one of the leading causes of cancer mortality worldwide.3Previous publications have reported on various health metrics of oesophageal cancer, including its incidence, mortality, and DALY rates, as well as its risk factors.There are two main histological subtypes of oesophageal cancer: oesophageal adenocarcinoma  and oesophageal squamous cell carcinoma . Although several high-risk regions for oesophageal squamous cell carcinoma are adjacent to regions with medium or low risk, such a pattern has not been observed for oesophageal adenocarcinoma. Globally, more than 85% of all incident oesophageal cancer cases are oesophageal squamous cell carcinoma.In this analysis of data from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2017, we present estimates of the number of incident cases and deaths, as well as age-standardised incidence, mortality, and DALY rates for oesophageal cancer from 1990 to 2017, by age and sex, for 195 countries and territories. Furthermore, we provide age-standardised DALYs by Socio-demographic Index (SDI). Finally, we provide estimates of the proportion of DALYs attributable to several major oesophageal cancer risk factors, including tobacco smoking and chewing, alcohol consumption, low intake of fruits, and high body-mass index (BMI). Through this comprehensive evaluation of global oesophageal cancer measures, we hope to provide additional information for policy makers, funding agencies, and researchers, so they can develop strategies to help prevent and treat oesophageal cancer in high-risk locations, determine allocation of scarce health resources, and guide the direction of future research on oesophageal cancer.This study is part of GBD 2017, which includes estimates for 195 countries and territories, 21 regions, and seven super-regions. GBD 2017 presented estimates for 359 diseases and injuries; 282 causes of death; and 84 behavioural, environmental, occupational, and metabolic risk factors. The methodology used to collect data for this research has been discussed previously.Data came from vital registration systems (19 323 site-years), vital registration-samples (793 site-years), verbal autopsy records (419 site-years), and cancer registries (5497 site-years).Some locations had high-quality incidence and mortality cancer registry data, whereas data were sparse in other locations.15The final mortality estimates were obtained from CoDCorrect,Following this process, each sequela-specific prevalence rate was multiplied by a sequela-specific disability weight to estimate the sequelae-specific years lived with disability (YLDs). DALYs were calculated as the sum years of life lost (YLLs) due to premature death and YLDs.SDI was used in this study to determine the relationship of a country's socioeconomic development status with age-standardised incidence and mortality rates for oesophageal cancer. In GBD 2017, SDI was revised to better reflect the development status of each country.We calculated and reported the percentage of DALYs due to oesophageal cancer that were attributable to smoking, alcohol use, high BMI, a diet low in fruits, and chewing tobacco . Details about the definitions of these risk factors and their relative risk for oesophageal cancer are described elsewhere.Publicly available data for the two main histological subtypes of oesophageal cancer  were obtained from Cancer Incidence in Five Continents (Vol XI).The funder of the study had no role in study design, data collection, data analysis, data interpretation, or the writing of the report. All authors had full access to the data in the study and had final responsibility for the decision to submit for publication.In 2017, there were 473 000 (95% UI 459 000–485 000) new cases of oesophageal cancer and 436 000 (425 000–448 000) deaths due to oesophageal cancer. Age-standardised incidence was 5·9 (5·7–6·1) per 100 000 population and the age-standardised mortality rate was 5·5 (5·3–5·6) per 100 000 population. 9·78 million (9·53–10·03) DALYs were due to oesophageal cancer, with an age-standardised rate of 119·9 (116·9–123·0) per 100 000 population .Between 1990 and 2017, global age-standardised incidence decreased by 22·0% (95% UI 18·6–25·2), mortality decreased by 29·0% (25·8–32·0), and DALYs decreased by 33·4% , southern sub-Saharan Africa (10·0 [9·5–10·4] per 100 000 population), eastern sub-Saharan Africa (7·8 [7·2–8·6] per 100 000 population), central sub-Saharan Africa (7·3 [6·2–8·5] per 100 000 population), and central Asia . Similarly, in contrast to the high age-standardised incidence rate seen in east sub-Saharan Africa (7·8 [7·2–8·6] per 100 000 population in 2017), the age-adjusted incidence rate in western sub-Saharan Africa was low (4·0 [3·5–4·7] per 100 000 population in 2017).Between 1990 and 2017, the regional age-standardised incidence, mortality, and DALY rates for oesophageal cancer decreased in all GBD regions except for high-income North America for age-standardised incidence rates (7·4% [95% UI 3·9–11·1]), and western sub-Saharan Africa for all three measures , deaths (213 000 [203 000–223 000]), and DALYs (4·46 million [4·25–4·69]) in 2017, comprising nearly half of new cases, deaths, and DALYs globally that year. The highest estimated national-level age-standardised incidence, mortality, and DALY rates in 2017 were observed in certain countries in sub-Saharan Africa , central Asia , and east Asia  attributable to tobacco smoking, 33·8% (27·3–39·9) to alcohol consumption, 19·5% (6·3–36·0) to high BMI, 19·1% (4·2–34·6) to a diet low in fruits, and 7·5% (5·2–9·6) to use of chewing tobacco . The impClear patterns emerged by generating graphs of the proportion of oesophageal squamous cell carcinoma to oesophageal adenocarcinoma cases and to all oesophageal cancer cases versus SDI, HAQ Index, and household air pollution, which were obtained by linking to data from Cancer Incidence in Five Continents Vol XI; . At the The results of GBD 2017 show that the large variations in age-standardised incidence, mortality, and DALY rates across countries and territories are still a main epidemiological feature of oesophageal cancer , indicating the importance of finer delineation of hotspots to specify at-risk populations. GBD estimates show a decrease in age-adjusted incidence rates in most parts of the world, particularly in regions where oesophageal squamous cell carcinoma is the dominant histological subtype , but an increase in these rates in regions where oesophageal adenocarcinoma is the major subtype , emphasising the importance of collecting data to differentiate these two subtypes. Further investigation is needed of the causes of the high age-standardised incidence rates seen in some parts of the world, particularly in high-risk areas of sub-Saharan Africa, Mongolia, China, and Iran. The observed global decrease in age-standardised incidence rate from 1990 to 2017 is perhaps due to improvements in several social and environmental factors, such as the SDI.Our findings add to the literature by providing the most up-to-date data on a wide range of oesophageal cancer health metrics, including age-standardised rates of DALYs for 195 countries and territories, DALYs attributable to several major risk factors of oesophageal cancer, and trends of these measures from 1990 to 2017. Although age-standardised incidence and mortality rates of oesophageal cancer have decreased at the global level over the past three decades, absolute numbers of new oesophageal cancer cases and deaths and DALYs attributable to oesophageal cancer have increased as a result of population growth and ageing. These trends over time indicate that oesophageal cancer still causes a substantial disease burden across the world, and the overall burden could continue to rise. Our results corroborate findings from previous studies but use different sources of data and estimation methods. The country-level positive correlations between the proportion of oesophageal squamous cell carcinoma to all oesophageal cancer cases and high indoor pollution, low access to improved water sources, low SDI, and low HAQ Index are novel and support the hypothesis that these factors could contribute to the risk of oesophageal squamous cell carcinoma. Gastro-oesophageal reflux disease, an established risk factor for oesophageal adenocarcinoma , showed a U-shaped relationship with the proportion of oesophageal squamous cell carcinoma to all oesophageal cancer cases. No clear explanation exists for this unexpected finding, although we can suggest some hypotheses. This U-shaped relationship was primarily due to aberrations seen in eastern Europe, an area that is close to geographical areas of Asia where oesophageal squamous cell carcinoma is prevalent, and adjacent to western Europe, where gastro-oesophageal reflux disease and oesophageal adenocarcinoma are highly prevalent. This geographical proximity, combined with spatial modelling, might have affected the estimates. Another possibility is a potential association between gastro-oesophageal reflux disease prevalence and one or more oesophageal squamous cell carcinoma risk factors in these regions.The estimated numbers of new cases of oesophageal cancer based on GBD estimates were lower than those estimated with the 2018 Global Cancer Observatory (GLOBOCAN).Some countries in east and central Asia, as well as in eastern, central, and southern sub-Saharan Africa, had high age-standardised incidence, mortality, and DALY rates over the study period. These findings are consistent with previous literature. Since at least the early 1970s, vast areas of Asia, extending from China and Mongolia to the Caspian Sea, have been known to have high rates of oesophageal cancer.Although GBD estimates show major variations in age-standardised incidence, mortality, and DALYs across regions and countries, there might be finer variations within countries not captured by these data. A prominent epidemiological feature of oesophageal squamous cell carcinoma, which constitutes the majority of oesophageal cancer cases worldwide, is that, for unknown reasons, rates of this disease can change sharply over relatively short distances. Therefore, countries marked as medium risk or low risk might have areas with a very high risk of oesophageal squamous cell carcinoma. For example, although Iran is not marked as a high-risk country for oesophageal cancer, Golestan Province in the northern part of Iran, bordering Turkmenistan, has some of the highest rates of oesophageal squamous cell carcinoma in the world.Although there was a global decrease in age-standardised incidence rates of oesophageal cancer between 1990 and 2017, the age-standardised rates increased in several countries, particularly in high-income countries. This is likely to be because the rates of oesophageal adenocarcinoma  have increased in high-income countries  since the 1990s.This study found tobacco smoking, tobacco chewing, alcohol consumption, low intake of fruit, and high BMI to be important oesophageal cancer risk factors, accounting for most of the burden. Of these factors, high BMI is an established risk factor for oesophageal adenocarcinoma but has no proven association with oesophageal squamous cell carcinoma risk. Given the poor survival rate of patients with oesophageal cancer, major efforts to reduce these risk factors should be undertaken, particularly given that many oesophageal cancer risk factors  are also important risk factors for other major chronic diseases, such as cardiovascular diseases and other cancers.Although there were some exceptions, we found an overall inverse association between the age-standardised incidence of oesophageal cancer and SDI at the country level. These findings are consistent with previous epidemiological studies that have shown an inverse association between oesophageal squamous cell carcinoma and socioeconomic status.36The poor prognosis of oesophageal cancer, indicated by the high MIR, might also call for screening efforts in the general population or in high-risk groups. Early detection of oesophageal cancer is also appealing because premalignant lesions and early cancers can now be treated with methods that are less invasive than surgery, such as endoscopic mucosal resection and endoscopic submucosal dissection, photodynamic therapy, argon plasma coagulation, radiofrequency ablation, and cryotherapy.Primary prevention of oesophageal cancer with chemopreventive measures has been the subject of many observational studies and randomised trials.Presenting data from 195 countries and territories over three decades, this study is, to our knowledge, the most comprehensive and up-to-date analysis of the global burden, trends, and risk factors of oesophageal cancer. Linking data to Cancer Incidence in Five Continents provided, we believe for the first time, an opportunity to investigate associations with several potential risk factors of oesophageal squamous cell carcinoma at the country level. The data and findings, however, have several limitations. The two main histological subtypes of oesophageal cancer, oesophageal squamous cell carcinoma and oesophageal adenocarcinoma, have distinct risk factors, incidence trends, and geographical distributions,In conclusion, despite reductions in age-standardised incidence and mortality rates, oesophageal cancer remains a major cause of cancer mortality and burden across the world. The bulk of the burden of oesophageal cancer comes from east Asia, particularly from China. Areas with the highest age-standardised incidence, mortality, and DALY rates are located in east and central Asia and eastern, central, and southern sub-Saharan Africa. Although the exact reasons for these high rates are unclear, many epidemiological studies are ongoing, and we expect to see new findings over the next few years. In the meantime, intensive reduction of major known and potential oesophageal squamous cell carcinoma risk factors  and oesophageal adenocarcinoma (primarily obesity and tobacco use) are strongly recommended. Where possible, it would be useful to collect country-level data on other risk factors such as consumption of opium and hot drinks, and water pipes . It would also be advisable to collect data on the histology of oesophageal cancer, and to treat oesophageal squamous cell carcinoma and oesophageal adenocarcinoma as two distinct entities for future analyses. High mortality-to-incidence ratios highlight the importance of primary and secondary prevention. Screening might be advised only in certain geographical areas with a very high risk of oesophageal squamous cell carcinoma and high prevalence of squamous dysplasia. Further development of less invasive screening methods should be highly encouraged.


Scientific Reports 10.1038/s41598-019-49782-x, published online 18 September 2019Correction to: The original version of this Article contained errors.Affiliation 7 was a duplicate of Affiliation 5, and it has now been removed. The original Affiliation 8 is now listed as Affiliation 7.In addition, in the Abstract,“As planned pregnancy was the strongest factor, healthcare professionals should ensure that childbearing women taking antiepileptics are informed of the importance of planned pregnancy.”now reads:“As unplanned pregnancy was the strongest factor, healthcare professionals should ensure that childbearing women taking antiepileptics are informed of the importance of planned pregnancy.”Moreover, in Table “Planned pregnancy, n (%) Unplanned pregnancy”now reads:“Planned pregnancy, n (%)”Finally, in the Discussion“In the present study, a planned pregnancy was the strongest factor associated with folic acid intake”now reads:“In the present study, a planned pregnancy was the strongest factor associated with adequate folic acid intake”


The quality and nutritional value of dietary proteins are determined by the quantity, digestibility and bioavailability of essential amino acids (EAA), which play a critical role in human growth, longevity and metabolic health. Plant-source protein is often deficient in one or more EAAs  and, in its natural form, is less digestible than animal-source protein. Nevertheless, dietary intake of plant-source protein has been promoted because of its potential health benefits, lower cost of production and lower environmental impact compared to animal-source protein. Implementation of dietary strategies that improve both human and planetary health are of critical importance and subject to growing interest from researchers and consumers. Therefore, in this review we analyse current plant protein intake patterns and discuss possible countermeasures that can enhance plant protein nutrition, examples include: (1) combining different plant proteins with complementary EAA profiles; (2) identification and commercial cultivation of new and novel high-quality plant proteins; (3) industrial and domestic processing methods; and (4) genome-editing techniques. All 20 thletes) . In devethletes) ,7, wherethletes) . Over the last 60 years, the per capita global protein consumption has increased , but rapPlant crops are the primary harvesters of solar energy acting as a cost and energy-efficient nutrient-rich sink—and a valuable source of protein, carbohydrate, essential vitamins and minerals . Hence, −1·d−1 plant protein of 83 g·capita−1·d−1 total protein) [−1·d−1 (64 g·capita−1·d−1 in 1967 to 82 g·capita−1·d−1 in 2017) and the percent contribution of animal-sourced protein (ASP) by 9 percentage points [Hundreds of plant species have been identified and commercially cultivated for human consumption, however, most of the world’s population only depend on ~20 plant crops-grouped into a few broad categories  ,18. Suppprotein) , but varIn humans, the nutritional value of dietary protein is determined by its ability to meet protein and AA requirements for growth, maintenance and basic physiological needs ,2,3,4,5.−1·d−1 (~132 mg N·kg−1·d−1) is recommended for N-balance , with the estimated average requirement (EAR) of protein  set at 0.66 g·kg−1·d−1 [−1·d−1) [−1·d−1) have been (re)calculated and (re)quantified via segmented regression and the indicator amino acid oxidation (IAAO) technique [For adolescents and adults, a RDA of ~0.8 g·kgkg−1·d−1 ,20. But,[−1·d−1) ,20,21,22[−1·d−1) ,21,22,23In addition to the protein RDA, the quality of the protein must be considered as it is inextricably linked to RDA and protein adequacy. Protein quality is based on the ability of the protein to deliver the individual EAAs in sufficient amounts to meet requirements. Indeed, several studies have reported differences between isonitrogenous PSP and ASP, showing that PSP is utilised with a lower efficiency, implying that PSP is lower-quality than ASP . −1·d−1) the DIAAS represent the fraction of the minimum daily requirement of the first LEAA consumed. Thus, 0.66 g·kg−1 of protein with a DIAAS of 1.0 will, in theory, supply 100% of the minimum daily requirements of the first LEAA and all other EAAs [Quantitative assessment of dietary protein quality was devised to reflect postprandial bioavailability of the EAAs in relation to requirements, determined by the content and profile of EAA and the true ileal digestibility of the protein. Although several methods exist to evaluate protein quality, the digestible indispensable amino acid score (DIAAS) has been adopted by the World Health Organisation (WHO)/Food and Agriculture Organisation (FAO)/United Nations University (UNU) ,25. The her EAAs ,25. If tIn the main cereal crops, Lys is the first LEAA, and the sulphur-containing amino acids (SAA: Met and Cys) are usually the first LEAA in the legume crops, although there are exceptions where Trp  or Lys (black-eyed peas) is the first LEAA , but it −1·d−1). However, in most instances fold increases in PSP intake would not be necessary to meet requirements as normally multiple different dietary PSPs and ASP are consumed [Protein RDA is based on high-quality protein intake  ,25, but consumed . Neverthconsumed ,20,21,22−1 of protein , the caloric equivalent for soybeans would be higher than poultry meat despite having similar DIAAS  [−1), but score markedly better than low-quality, low-density plant foods . In most instances, consuming greater amounts of PSP to compensate for inferior protein quality is not always feasible and may negatively impact health . Therefore, it is pertinent that we review dietary strategies that enhance the quality of PSP [As the DIAAS is calculated per gram of protein, comparative evaluation of the DIAAS between different protein sources is conducted on an isonitrogenous basis . Consequal·kg−1) . Animal-y of PSP ,29,30,31As the DIAAS is expressed in standardised units , different protein sources can be compounded at discrete time intervals . In this approach, two complementary protein sources (with different LEAAs) can be combined to affect the DIAAS, greater than either individual protein. For example, 100 g serving of rice (Oryza sativa) and 100g of lentils (Lens culinaris) would be classified, individually, as low-quality protein sources  . WhereasCereal and legume crops are generally complementary . HoweverThe RDA for protein is normally expressed per diem, which does not consider the timing (i.e. the time between eating occasion (EO)) or distribution (i.e. the number of EO and amount of protein per EO) of protein consumption throughout the day. For PSP, it should be noted that the LEAA Lys (and Thr) are unique among the EAAs as it appears that they can be conserved, indicating that they do not need to be co-ingested with the other EAAs at each EO  . Evidenc−1) and distribution of protein throughout the day  for muscle protein synthesis—yielding per diem equivalents higher than the current RDA  [In addition to providing nitrogen, some other AAs also activate signalling pathways that regulate metabolism  ,2,4,23. g−1·d−1) ,23,40,41In theory, postprandial deficiencies in Lys (and Thr) in PSP can be drawn from body-pools, but postprandial deficiencies in other EAAs  , would lIf available and consumed in appropriate amounts and/or combinations, it appears that adequate levels of protein/AA nutrition can be attained from PSP. However, whether optimal levels of protein/AA nutrition can be achieved exclusively from PSP, especially for clinical and athletic populations with higher MDs, is questionable—as, in its natural form, higher intakes from PSP may not be practicable and may negatively affect health . vs. 90–95%)  , due to Pisum sativum L.) in water increases protein digestibility (75% up to 80%) [Indeed, there are several domestic processing methods that have been shown to improve PSP digestibility  . For exa to 80%) . Industr to 80%) ,46. Howe to 80%) ,46. In p to 80%) . For exa to 80%) .The ease by which the peptides and AA can be liberated from the microstructure influences digestibility and nutritional value. For PSP, complete elimination of the microstructure and the NPC appears to enhance digestibility and protein quality ,46,47. SPSP is usually deficient in one or more EAAs   as they There are inherent difficulties in classical genetic and breeding approaches used to increase the nutritional value PSP, due to the tightly regulated Lys metabolic pathway and their deleterious effects crop yields and plant growth . Thus, oThere is the potential that GM plants can significantly improve PSP quality and nutritional value, however, the biosafety and bioefficacy have yet to be confirmed in humans. Moreover, even though GM crops are only granted regulatory approval after strict assessment , like all other GM crops, the opportunity to improve PSP quality via GM will be conditional on public acceptance ,52. To a2·g Lys−1vs. 0.16 kg CO2·g Lys−1), and larger disparities exist between different PSPs  and ASPs , than between ASP and PSP (e.g. wheat vs. poultry meat)  describes plant-based diets as a ‘major opportunity for mitigating and adapting to climate change’, which includes a policy recommendation to reduce meat consumption . Consequry meat) .2, B12, D3, heme-iron, iodine, calcium, essential fatty acids and zinc). Thus, from a nutritional perspective, there is justification for modest increases in animal food intake in developing regions, particularly those with higher MD [SDGs aim to meet the needs of the present, without compromising the ability of future generations to meet their own needs . Animal-igher MD . Additio−1·d−1 may be a simple and effective strategy to offset protein/AA deficiencies in PSP, however, in practice, this strategy may not always be practicable or efficacious—particularly for those who rely on a limited variety of PSPs by necessity, not by choice. In developed regions, increasing intake of PSP should be little concern as dietary strategies such as protein complementation, supplementation and fortification of PSP with EAA can be implemented to help meet protein/AA requirements. This is fortunate as increased pressure to find sustainable food sources has led to a trend away from animal- to plant-source foods and plant-based diets. At present, it appears that there is ample room to improve sustainable intensification of animal-source food production, whilst simultaneously increasing in the intrinsic nutritional value of plant-source foods via GEd or processing methods. This dual approach will favourably impact global nutrition and the health of both current and future generations, but remains a significant scientific and technological challenge.In this brief review, the nutritional value of PSP related to human protein/AA requirements is highlighted and discussed. Increasing the RDA >1.0 g·kg


Antimicrobial resistance represents a significant world-wide health threat that is looming. To meet this challenge, new classes of antimicrobial agents and the redesign of existing ones will be required. This review summarizes some of the studies that have been carried out in my own laboratories involving membrane-disrupting agents. A major discovery that we made, using a Triton X-100 as a prototypical membrane-disrupting molecule and cholesterol-rich liposomes as model systems, was that membrane disruption can occur by two distinct processes, depending on the state of aggregation of the attacking agent. Specifically, we found that monomers induced leakage, while attack by aggregates resulted in a catastrophic rupture of the membrane. This discovery led us to design of a series of derivatives of the clinically important antifungal agent, Amphotericin B, where we demonstrated the feasibility of separating antifungal from hemolytic activity by decreasing the molecule’s tendency to aggregate, i.e., by controlling its monomer concentration. Using an entirely different approach , we found that by covalently attaching one or more facial amphiphiles (“floats”) to Amphotericin B, its aggregate forms were much less active in lysing red blood cells while maintaining high antifungal activity. The possibility of applying such “monomer control” and “taming” strategies to other membrane-disrupting antimicrobial agents is briefly discussed. Acinetobacter, Candida auris, Clostridioides difficle, Carbapenem-resistant Enterobacteriaceae, and drug-resistant Neisseria gonorrhoeae. The CDC also considers the following 11 bacteria as “serious threats”: drug-resistant Campylobacter, drug-resistant Candida, ESBL-producing Enterobacteriaceae, Vancomycin-resistant Enterococci (VRE), multidrug-resistant Pseudomonas aeruginosa, drug-resistant nontyphoidal Salmonella, drug-resistant Salmonella serotype Typhi, drug-resistant Shigella, Methicillin-resistant Staphylococcus aureus (MRSA), drug-resistant Streptococcus pneumoniae and drug-resistant Tuberculosis. The two bacteria that the CDC views as “concerning threats” are Erythromycin-resistant Group A Streptococcus and Clindamycin-resistant Group B Streptococcus.According to a 2019 Antimicrobial Threats Report from the Centers of Disease Control and Prevention (CDC), more than 2.8 million antimicrobial-resistant infections occur each year in the United States, resulting in more than 35,000 deaths . T. T23]. TThe value of Amphotericin B as an antifungal drug derives from its modest selectivity in destroying fungal cells over mammalian cells. This selectivity appears to be due to a higher affinity that Amphotericin B has towards ergosterol (the sterol found in fungi) over cholesterol   17,22].,22.17,22The significant toxicity associated with Amphotericin B is, in fact, a common problem that exists for virtually all membrane-disrupting antimicrobial agents. To judge the toxicity of an agent to mammalian cells, it is common practice to first measure its hemolytic activity in vitro, i.e., its ability to release hemoglobin from red blood cells. In an enlightening study by Bolard and coworkers, it was reported that while aggregates of Amphotericin B exhibit both hemolytic and antifungal activity, its monomers exhibit only antifungal activity . The basPrior to Bolard’s publication, we initiated a study that was aimed at gaining fundamental insight into how membrane-disrupting agents, in general, act on lipid bilayers . In that1a–c  unit, i.e., conjugates 1a–c  27]..1a–c  in which its amino group was protected in the form of an Fmoc-carbamate, and its carboxylic acid moiety was activated by reaction with N,N,N′,N′-tetramethyl-O-uranium tetrafluoroborate [3 with a family of α,ω-diamines that were monoacylated with cholic acid, followed by deprotection with piperidine afforded the desired conjugates, 2a–d [To prepare these conjugates, we first synthesized a derivative of Amphotericin B , in place of Amphotericin B, favors binding to lipid membranes such that the choloyl moieties lie close to the membrane’s surface [It is noteworthy that in related studies we obtained evidence via a parallax analysis that an analog of  surface .T, m, and P as the total, the monomeric and the aggregate concentrations of Amphotericin B, respectively, and if ε, εm and εp are defined as the apparent molar absorptivity, the molar absorptivity of the monomer and the molar absorptivity of the aggregate components, respectively, then it can be shown that ε = εp + (εm − εp)m/T. Thus, when concentrations Amphotericin B and its conjugates are in excess of their critical aggregation concentrations, m is a constant value and ε is expected to be inversely proportional to T. Thus, by plotting the apparent molar absorptivity at 409 nm as a function of the reciprocal of the analytical concentration of Amphotericin B or a conjugate, one can estimate its critical aggregation concentration from the intercept of two straight lines. Based on this method, the critical aggregation concentration for 2a–d and 4 were all estimated to be ca. 1 µM, which was the same as Amphotericin B, itself [To define the critical aggregation of Amphotericin B and its conjugates, we devised a simple approach that takes advantage of a well-defined absorption band for heptaene macrolide monomers, which lies at 409 nm . Upon ag, itself ,29.2a–d and 4, we determined their minimum inhibitory concentrations (MIC) with respect to C. albicans, C glabrata, C. neoformans, and C. gatti [2a,b, exhibited broad-spectrum antifungal activities that compared favorably with Amphotericin B. Those analogs having longer spacers, 2c, d, exhibited high antifungal activity against some but not all of the fungi that were tested. The conjugate bearing two choloyl moieties, 4, also showed broad spectrum antifungal activities, which compared favorably with Amphotericin B.To evaluate the antifungal properties of C. gatti ,29. As s2a that was required for 50% hemolysis was 465 µM. For 2b–d, negligible hemolysis was found at concentrations as high as 400 µM. With conjugate 4, 50% hemolysis required a concentration of 375 µM. Thus, in all cases, these tamed derivatives of Amphotericin B were two or more orders of magnitude less hemolytic than the native drug.To judge the toxicity of these membrane-disrupting antimicrobial agents towards mammalian cells, we evaluated their hemolytic activity using sheep red blood cells and determined the concentrations required for 50% hemolysis . In the 2a was moderately toxic (ca. 40% cell viability), and 2b was modestly toxic (ca. 70% cell viability). At this same 25 µg/mL, 2c and 2d showed close to negligible toxicity (>90% cell viability). In the case of conjugate 4, no evidence of cytotoxicity was observed with a concentration as high as 100 µM (100% cell viability) [To further evaluate the toxicity of these agents, we also examined their effect on HEK293 T cells. At a concentration of 25 µg/mL, Amphotericin B was highly toxic (<5% cell viability), ability) .In this review, I have discussed our fundamental studies with Triton X-100 that have led to a discovery of two distinct pathways of membrane disruption, i.e., mild leakage induced by monomers and catastrophic membrane rupture by aggregates. I have also described our design and synthesis of derivatives of the clinically important antifungal agent, Amphotericin B, which has allowed for the separation of the antimicrobial activity from the hemolytic activity by controlling their monomer concentration. A taming concept that I have discussed, in which facial amphiphiles are covalently attached to Amphotericin B to reduce its membrane rupturing activity is an entirely distinct approach for enhancing its cellular selectivity.The generality of these two very different strategies (monomer control and taming) for improving the selectivity of other membrane-disrupting agents remains to be established. In the following, I briefly discuss, what I believe are, two broad classes of molecules whose therapeutic potential might be enhanced by either or both of these strategies.Antimicrobial Peptides. Antimicrobial peptides (AMPs) represent a broad class of membrane-disrupting molecules that contain, typically 12 to 50 amino acids [no acids ,33,34,35Closely related to antimicrobial peptides are, what have been termed, “cell-penetrating peptides” (CPPs) ,37. As pQuaternary Ammonium Compounds. Because quaternary ammonium compounds are highly toxic to both mammalian cells and Gram-negative bacteria, their use as antimicrobial agents has been limited as disinfectants [fectants . I beliefectants . Specififectants ,43.


Trifluoroethanol and difluoroethanol units are important motifs in bioactive molecules, but the methods to direct incorporate these units are limited. Herein, we report two organosilicon reagents for the transfer of trifluoroethanol and difluoroethanol units into molecules. Through intramolecular C-Si bond activation by alkoxyl radicals, these reagents were applied in allylation, alkylation and alkenylation reactions, enabling efficient synthesis of various tri(di)fluoromethyl group substituted alcohols. The broad applicability and general utility of the approach are highlighted by late-stage introduction of these fluoroalkyl groups to complex molecules, and the synthesis of antitumor agent Z and its difluoromethyl analog Z′. Methods for direct incorporation of tri- and di-fluoroethanol units in molecules are relatively limited. Here, the authors report two organosilicon reagents which are applied to allylation, alkylation and alkenylation reactions as tri- and di-fluoroethanol transfer reagents. Fluoroalkylsilicon reagents, such as TMSCF3 (Rupert–Prakash reagent), TMSCF2H, and others are widely used reagents in the synthesis of organofluorine compounds3. Among various fluorine-containing molecules, the secondary fluoroalkyl alcohols are of particular importance; monoamine oxidase A inhibitor Befloxatone4 and antitumor agent Z5 are examples of bioactive molecules containing trifluoroethanol motif  it is hard to control the regioselectivity when there are more than one aldehyde sites in the same molecule. Moreover, the design and synthesis of pharmacuticals call for strategies to incorporate important structural motifs at late-stage, because this will aviod de novo synthesis12.Fluorine incorporation has been a routine strategy for the design of new drugs and materials, because it can often improve the chemical, physical, and/or biological properties of organic moleculestif Fig. . Anionictif Fig. 6–8. Howeols Fig. . Actuallols Fig.  possesse1a and 2a. Through intramolecular C–Si bond activation by alkoxyl radicals19, these developed β-fluorinated organosilicon reagents were successfully applied in radical allylation, alkylation, and alkenylation reactions, enabling efficient synthesis of a variety of fluoroalkyl group substituted alcohols .Herein, we report two fluoroalkylsilicon reagents ols Fig. . The bro1a and 2a were easily synthesized in three steps  can be synthesized directly from the reaction of Ph3SiLi and (CF3CO)2O in one step23. The difluoroacetylsilane 6 was easily prepared in 79% yield through the hydrolysis of enol silyl ether 4 under acidic conditions. Reduction of compound 6 delivered difluoromethyl containing reagent 2a in 88% yield.The fluoroalkyl group transfer reagents 1a in hand, we investigated the substitution reaction with allylic sulfone 7a as model substrate. Firstly, we tried anionic activation strategy which has been widely used in the C–Si bond cleavage for the fluoroalkyl transfer reactions8. Common activators such as TBAF, CsF, KF, t-BuOK were tried, but no substitution product 8a or 9a was observed, albeit full conversion of compound 1a was observed , extreme ionization energy (17.418 eV), see ref 1a, pp 5–8). Therefore, we envisioned that in-situ generated carbon radical from alkoxyl radicals should not prefer β-F elimination. Consequently, they could be trapped by radical acceptors to generate trifluoroethanol transfer products . The attempt to use Smith’s condition19 to achieve the reaction between 1a and 7a failed to afford any amount of product 8a or 9a, highlighting the influence of CF3 group on the reactivity of reagent 1a.With this idea in mind, we investigated a variety of conditions which could generate alkoxyl radicals 2·4H2O are efficient catalysts for the transformation. When TBAF was used to quench the reaction, the silyl ether was converted to alcohol in one pot, and compound 9a was isolated in 62% yield. It was found that a variety of allylic sulfones34 bearing different groups could be used as the radical acceptors, affording the α-CF3-substituted homoallylic alcohols 9a–9ap in 40–83% yields. This reaction can be scaled up, and a yield of 65% was obtained for compound 9c (1.25 g isolated), and the sulfone 7c was recovered in 61% yield after silica gel chromatography. It is worth noting that the current allylation protocol can tolerate many functional groups. The Csp2–F, Csp2–Cl, Csp2–Br, Csp2–I bond can be kept after the reaction, affording compounds 9d–9i, 9u, and 9v in 56–83% yields. Moreover, allylic bromide can also be tolerated . These functional groups are well-known convertible motifs under transition metal-mediated/catalyzed reactions. Electron donating OMe group, electron withdrawing groups, such as NO2, CN can also be tolerated, affording compounds 9k–9m in 68–80% yields. Electron-rich heterocyclic groups, such as furyl and thienyl are also be tolerated . Naphthyl-substituted product was also successfully made via the Mn-catalyzed substitution reaction, affording alcohol 9p in 72% yield. A variety of alkyl ethers and benzylic alcohol-derived esters are tolerated under the current oxidation conditions, and alcohols 9q–9w were obtained in 56–73% yields. Not only ester-substituted and ketone-substituted allylic sulfones, but also amide, sulfone, phenyl, and alkyl groups-substituted allylic sulfones can be applied in the current substitution process, affording corresponding α-CF3-substituted homoallylic alcohols . Moreover, this substitution process is compatible with many base-sensitive functionality, such as primary alcohol , alkyl aldehyde , alkyl ketone . It is worthy to note that the above-mentioned aldehyde and ketone-containing products are challenging to be synthesized by methods based on these functional groups3. In addition, the current radical allylic substitution can be applied in the functionalization of complex molecules, such as the derivatives from biologically active naturally occuring epiandrosterone, cholesterol, testosterone, diosgenin, vitamin E, estrone, and (8α)-estradiol, affording corresponding alcohols 9ai–9ap in 46–75% yields.We next explored the scope of the allylic substitution reaction Fig. . Both Mn10 were chosen to test the possibility38. To our delight, under Mn(II)/TBPB conditions, various acryl amides can be converted to corresponding trifluoromethylated alcohols 11 in 63–91% yield  is isopolar and isosteric to an SH or OH group, and can also behave as a hydrogen donor through hydrogen bonding47. Therefore, difluoromethylated compounds are strong candidate for drugs. There are examples showing that the CF2H-containing compounds exhibit higher bioactivity than their CF3-containing counterparts49. Encouraged by the above success of radical C–Si bond activation to access trifluoromethylated allylic, alkyl, and alkenyl alcohols, we extended the strategy to directly transfer 2,2-difluoroethanol to organic molecules with reagent 2a . Therefore, radical process might be involved in current substitution reaction. Our investigation revealed that Mn(OAc)3·2H2O is able to mediate the reaction without external oxidant, but Mn(OAc)2·4H2O cannot mediate the reaction without TBPB  intermediate53. Carbon radical III would be generated through Brook rearrangement, and then undergo further reaction to generate product IV. Mn(III) catalyst is likely to be regenerated by the oxidation of Mn(II) by TBPB. The alcohol product V would be generated after the desilylation step. It is worthy to note that the reaction pathways in the allylation, alkylation, and alkenylation reactions are probably different  the reaction partners in the above three kinds of reactions are electron-deficient olefins; (2) more electron-deficient substrate afforded higher yield ; (3) the carbon radical generated from trifluoroethanol under γ-ray irradiation could add to highly electrophilic hexafluoro-2-butyne27.The radical inhibition experiments revealed that addition of 1 equiv. of TEMPO or BHT can completely inhibit the allylation reaction, albeit more than 60% of compound ses Fig. . In addiZ and its difluoromethyl analog Z′. Further application of the radical C–Si bond activation of organosilicon reagents are underway in our lab.In conclusion, we have developed two fluorinated organosilicon reagents, which were used in direct transfer of trifluoroethanol and difluoroethanol units into organic molecules. A radical C–Si bond activation strategy was developed to solve the problem of β-fluorine elimination in anionic activation methods. Upon intramolecular activation of C–Si bond by alkoxyl radicals, the β-fluoro carbon radicals were generated and participated in efficient allylation, alkylation, and alkenylation reactions, enabling efficient synthesis of numerous fluoroalkyl alcohols. The broad applicability and general utility of the approach are highlighted by late-stage introduction of fluoroalkyl groups to complex molecules and the synthesis of antitumor agent 2 atmosphere, to a dried 10 mL Schlenk tube equipped with a magnetic stir bar containing Mn(OAc)2·4H2O  was added DCM , 1a , 7a , and TBPB  sequentially. The tube was sealed, and the resulting mixture was kept stirring at 70 °C in a heating block for 18 h. The mixture was then cooled to 5 °C with ice bath, TBAF  was added and the resulting mixture was stirred at 5 °C for 0.5 h. The reaction mixture was quenched with water (2 mL), extracted with DCM (3 × 10 mL) and organic phase was combined and washed with brine, dried over Na2SO4, concentrated under reduced pressure. The crude product was purified with column chromatography on silica gel (200–300 mesh) and PE/EA  as eluent to afford 40.0 mg of compound 9a as a colorless oil (62% yield).Under NSupplementary informationPeer Review File