Analog-sensitive inhibition was used to define phospho-sites regulated by Sch9, PKA and Ypk1. A considerable number of sites affected by Ypk1 inhibition fell into RRxS/T-contexts and overlapped with PKA sites. Neutral trehalase is activated through phosphorylation by both PKA and Ypk1. Signaling through Ypk1 is therefore more closely related to PKA than previously appreciated. HighlightsAcute inhibition of growth-regulatory AGC-kinases Sch9, PKA and Ypk1.Phospho-proteomic profiling of 6373 phsopho-sites.Ypk1 regulates phospho-sites in RRxS/T-context and functionally overlaps with PKA.Ypk1 and PKA activate trehalase activity of Nth1. S. cerevisiae. To understand how these kinases co-ordinately regulate cellular functions we compared the phospho-proteome of exponentially growing cells without and with acute chemical inhibition of PKA, Sch9 and Ypk1. Sites hypo-phosphorylated upon PKA and Sch9 inhibition were preferentially located in RRxS/T-motifs suggesting that many are directly phosphorylated by these enzymes. Interestingly, when inhibiting Ypk1 we not only detected several hypo-phosphorylated sites in the previously reported RxRxxS/T-, but also in an RRxS/T-motif. Validation experiments revealed that neutral trehalase Nth1, a known PKA target, is additionally phosphorylated and activated downstream of Ypk1. Signaling through Ypk1 is therefore more closely related to PKA- and Sch9-signaling than previously appreciated and may perform functions previously only attributed to the latter kinases.Protein phosphorylation cascades play a central role in the regulation of cell growth and protein kinases PKA, Sch9 and Ypk1 take center stage in regulating this process in  Cell growth is dynamic and highly regulated by signaling pathways that are conserved across evolution. To accomplish this regulation, eukaryotes have developed intricate means to assess growth conditions and to rapidly communicate this information to the processes controlling the accumulation of mass, the modification of cellular volume and of membrane surface area. Although many signal transduction pathways are involved in this regulation, those employing AGC-family kinases  are prominent.S. cerevisiae TORC1 primarily responds to changes in carbon and nitrogen availability and regulates ribosome biogenesis, cell cycle progression and stress responses via the AGC-kinase Sch9, like S6K downstream of mammalian TORC1 (The target of rapamycin complexes 1 and 2 (TORC1 and TORC2) are central sensors of environmental conditions and regulators of cell growth 3Tpk1, Tpk2, and Tpk3 are the partially redundant paralogs of the catalytic subunit of PKA. When cells are starved of carbon, cAMP levels are low and, therefore, PKA is kept inactive by its regulatory subunit Bcy1. Glucose addition induces activation of the adenylate cyclase Cyr1/Cdc35 via the small GTPase Ras1/2 and the G protein-coupled receptor Gpr1. The subsequent increase in cAMP levels triggers the dissociation of Bcy1 from the Tpks allowing them to phosphorylate their substrates , 8. Addi10YPK2 produces no obvious phenotype suggesting that it only plays a minor role in cell growth control  treated escribed . In briem urea, 50 mm ammonium bicarbonate, and 5 mm EDTA, then lysed by bead beating. For each biological replicate, 3 mg protein were reduced using 5 mm TCEP, alkylated in 10 mm iodoacetamide, and digested overnight with trypsin (1:125 w/w). Reverse phase chromatography was used to purify samples before phospho-peptide enrichment, which was performed with titanium dioxide resin (1.25 mg resin for each sample) (Cell pellets were suspended in a buffer consisting of 8  sample) . IsolateThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  partner https://downloads.yeastgenome.org/sequence/S288C_reference/orf_protein/orf_trans_all.fasta.gz; 6713 entries) from 2015–01-13 was used to match the spectra. Finally, a peptide and modification site FDR of 0.01 was applied to filter peptide spectrum matches (PSMs). Contaminants and decoys were subsequently removed from the results.Mass spectrometry data were processed with the MaxQuant software (v. 1.6.0.16), which uses the Andomeda MS/MS spectrum search engine , 33. Thesupplemental Table S1 and S2 respectively) were imported into the R statistical environment to perform missing value imputation and intensity normalization. Only peptides containing at least one phosphorylation were retained, together with all possible positions of the phosphorylation and their respective scores. All intensity values were log2 transformed .The MaxQuant result tables PDPYGINDKFFDLEK and one Sch9 and one PKA value are missing. In the sibling peptide (see below) HSS(ph)PDPYGINDK with clearly higher MS1 intensities all values are present . This shows that even if all 3 replicates values are missing, one cannot conclude absence of the corresponding peptide, but these missing values are most likely artifacts inherent to MS1 based quantitation. Phospho-peptides with no missing values in 3 replicates have stronger average MS1 signal intensities compared with peptides with missing values . If only one value out of 3 replicate values is missing, the 2 remaining positive replicate values can be used to estimate or impute the missing values (see below). However, if 2 or 3 values are missing, imputation becomes more error prone and can easily lead to false positives, when searching for peptides of differential abundance. Therefore, we only retained peptides with maximally one missing value per 3 replicates. 67.3% of all phospho-peptides fall in this category .2-transformed intensity value P,S,iI of peptide P in replicate i of sample S byP,S,i is the mean of the 2 non-missing values of peptide P in sample S and 𝒩 is a Gaussian error term with mean μ and standard deviation σ. In order to estimate this error term, we first need the notion of sibling peptides. Two peptides are called siblings if the sequence of one peptide is a N/C-terminal extension of the sequence of the other one (i.e. they differ by a missed cleavage) and if they carry the same modifications. It is unlikely that the sample condition has a direct influence on the trypsin cleavage, and it can be expected that two sibling peptides have similar expression profiles.We imputed a missing logP with a missing value in replicate i in sample S, we searched for sibling peptides P' of P with no missing values in the 3 replicates of sample S. Then we calculated the difference between the P',S,iI and αP',S,i and used all these differences to estimate μ and σ. The result shown in Fsupplemental Fig. S1 reveals that the missing values are on average slightly, but significantly lower than the average of the 2 non-missing values , a small bias that we took into account in the imputation The imputation parameters μ and σ are then estimated using the sibling peptides present in the MaxQuant results: for each peptide normalizeBetweenArrays R-function was used to equalize the log2-intensity distributions in all 30 LC-MS runs. The lmFit function was used to remove eventual batch effects present in the 10 samples. The empirical Bayes (eBayes) method was the applied to compare the 1NM-PP1 treated to the control (DMSO) samples for each strain and to calculate fold-changes and p values adjusted for multiple testing (FDR adjustment) . This method performs a correction of the standard deviation of each peptide by the overall empirical standard deviation.After missing value imputation, the R package limma  was usedp value of less than 0.05 the sequence from −5 to +5 amino acids from each of the phospho-sites localized on the peptide was extracted. In case of ambiguity between isoforms with different phospho-localizations, the peptide with the best localization score was chosen. If a phospho-site was represented by several peptides, the sequence surrounding the site was listed only once. This was performed individually for each strain for hyper- and hypo-phosphorylated peptides and the union of both.For each phospho-peptide that exhibited a change upon treatment with an adjusted Sequence logos were generated with pLogo using all quantified phospho-peptides as a background .et al., 2015 were based on sites in supplemental Table S2 of that study and generated in an analogous manner  were treated with ATP-analog (1NM-PP1) or mock (DMSO). Three replicates per strain were used for shotgun and targeted proteomics and six replicates for the trehalase assay. Independent starter cultures were used for each strain to generate replicates. The number of replicates used is the highest number deemed justified considering cost and the ability to process samples in parallel. No replication was used in Western blotting as the results were verified by Parallel Reaction Monitoring (PRM) as an alternative approach.In the label-free phospho-proteomics, targeted proteomics and the trehalase activity assay, the stated number of replicate cultures of analog-sensitive strains or wt-strain (aswt) was employed in all experiments. For Western blotting, a strain not carrying a FLAG-tag and another in which five serines of Nth1 had been mutated to alanine were used as additional controls.A strain which was not expected to be affected by 1NM-PP1 addition . Using this approach an observed false positive rate of 0.4% was calculated based on the results from the aswt strain.For label-free phospho-proteomics, after data processing as described above, the empirical Bayes (eBayes) method of the limma R package  was thent-tests for each strain.For targeted proteomics and the trehalase activity assay, 1NM-PP1-treated samples were compared with mock-treated samples using unpaired, two-sided Student's e.g. phospho-sites), a trade-off exists between the quality with which each point is measured and the number of points representing each set. To achieve meaningful set-sizes we did not apply further localization score thresholds when analyzing ensembles of phospho-sites (e.g. for generating Venn-diagrams or sequence-logos), however, we ensured that sites are based on phospho-isoforms with the highest localization score. As stated in the RESULTS-section, when performing analyses on the level of individual phospho-sites we required the presence of sequence motifs as additional criteria.When analyzing sets of data points, rather than individual data points -IMAC beads  were used for phospho-peptide enrichment, essentially following the manufacturer's instructions.Samples for targeted proteomics by PRM were prepared from 250 μg of protein in a similar manner as for label-free phospho-proteomics, with the exception that a mixture of magnetic TiOversus 0.1% formic acid in water. MS-scans were acquired at 60 000 resolution, with an ion target of 4 × 105 and maximum injection time of 118 ms. Targeted MS/MS employing higher energy collisional dissociation on 9 selected precursors representing Nth1 phospho-peptides and targeted single ion monitoring on two precursors were each acquired at a resolution of 60,000 with an ion target of 4 × 105 and maximum injection time of 200 ms.Data acquisition was performed on an Orbitrap Fusion Tribrid mass spectrometer coupled to an nLC1000 uHPLC without use of internal standard peptides (Tier 3). Peptides were separated on a 25 cm EASY-spray column (Thermo Scientific) with a 30 min gradient from 3 to 25% acetonitrile, 0.1% formic acid For data analysis in Skyline 4.2.0.19072 we employed spectral libraries from previously generated data-dependent acquisition and filtered for seven b- or y-ions from ion 3 to last ion .t test.The extracted total areas were normalized to the total ion current area of the corresponding sample and an intensity-weighted mean was calculated for cases in which phospho-sites were represented by several precursors. The significance of changes between DMSO- and 1NM-PP1 treated samples was evaluated using a two-sided Student's PRM data are accessible at ProteomeXchange via identifier PXD015760.Yeast strains were inoculated in YPD (pH 5.6) and incubated at 30 °C and 120 rpm overnight, diluted to OD = 0.1 the next morning and again to 0.0002 in the evening with incubation under the same conditions. Once an OD of about 0.8 was reached cultures were diluted to OD 0.08 and further in a 10x dilution series in the same medium.m 1NM-PP1 or an equal volume of DMSO and incubated at 30 °C for 2 days.For spot assays 3 μl of these cultures were spotted onto YPD agar plates containing 500 n2 and 0.8–1.0 circularity. Overlapping colonies or colonies at the edge of plates were manually excluded. The difference of colony sizes of analog-sensitive strains compared with wtas was evaluated using the Mann-Whitney-Wilcoxon test.Yeast strains were inoculated in YPD (pH 5.6) and incubated at 30 °C and 120 rpm overnight, diluted to OD = 0.1, grown to an OD of about 0.8 and diluted to an OD of 0.00008. Of the diluted culture, 100 μl were spread out with glass beads on three YPD-plates per strain and incubated at 30 °C. After 2 days, images were taken and colony sizes quantified using ImageJ (1.52p) : After sWt and analog-sensitive  versions of GST-tagged Sch9 and Ypk1 were expressed in BY4741 from uracil-selectable plasmids employing galactose-inducible promoters: Strains were inoculated in 5 ml SC-Ura + 2% glucose and expanded in 200 ml SC-Ura + 2% raffinose at 30 °C. Protein expression was induced by addition of galactose to 2%. After 3 h, cells were spun down and washed once with cold PBS before snap freezing and storage at - 80 °C.m CHAPS, Complete Protease Inhibitor Mixture, 1 mm PMSF) and lysed using a French press . Lysates were spun down for 20 min at 20,000 × g and 4 °C. The supernatant was incubated with 2 ml glutathione Sepharose 4B beads  for 2 h under gentle rotation at 4 °C. Beads were washed once with lysis buffer and an additional three times with lysis buffer containing 350 mm NaCl. Proteins were eluted in lysis buffer containing 50 mm reduced glutathione (pH 8.0), dialyzed against 25 mm HEPES pH 7.5, 150 mm KCl, 2.5 mm CHAPS, 5 mm MgCl2, 50% glycerol, adjusted to 60 pmol/μl according to Bradford assay and stored at −80 °C.Pellets were resuspended in 10 ml/g lysis buffer  plasmid (a kind gift from the Hahn-lab) . Cell wem imidazole.Ni-NTA beads  were washed twice in lysis buffer, before incubation with lysate for 1 h at 4 °C. The beads were washed three times with lysis buffer and proteins eluted with lysis buffer containing 500 m32P-ATP in PBS with 20% glycerol, 0.5% Tween-20, 4 mm MgCl2, 10 mm DTT, 2 mg/ml heparin, and Complete Protease Inhibitor Mixture w/o EDTA. After 5, 10, 17, 25, 40, 60 min at room temperature, an aliquot was mixed with 0.5 volumes 0.5 m EDTA with bromphenol blue.Kinase assays were based on the method by Asensio and Garcia with some modifications : Reactiom orthophosphoric acid and once with acetone and dried. An imager screen was exposed to the filter paper for 1 h and the screen read out using a Typhoon FLA 9500 imager .Of the stopped reactions 5 μl were spotted onto P81-paper, which were air-dried, washed five times 10 min with 200 ml 75 mm 1NM-PP1 or the corresponding volume of DMSO for 15 min and harvested by TCA-precipitation as described for phospho-proteomics sample preparation.To site-specifically validate changes in Nth1 phosphorylation by Western blotting, yeast cultures were treated with 500 nm Tris buffer containing 6 m urea and 1% SDS. Cleared lysate corresponding to 1.3 mg protein was diluted 1:10 in IP-buffer  and phosphatase- (Thermo Scientific) inhibitors and mixed with 25 μl Epoxy-beads (Thermo Scientific) that had been coupled to anti-FLAG antibody  in 0.1 m sodium phosphate buffer containing 1 m ammonium sulfate at 37 °C overnight, and washed four times with IP-buffer.Yeast pellets were lysed by bead beating (5 × 45s at 6500 rpm) in 25 mThe lysate was incubated with the beads at 4 °C for 4 h, then washed four times with IP-buffer. Proteins were eluted in 15 μl Laemmli-buffer at 65 °C for 10 min, then in further 15 μl Laemmli-buffer at 95 °C for 5 min.The eluates from both steps were combined and resolved on a 7.5% SDS-PAGE gel. Proteins were transferred to a nitrocellulose membrane using an iBlot2 system, blocked in 5% BSA in PBS for 1 h. The membrane was incubated with primary antibodies (1:1000 mouse anti-FLAG (Merck) and 1:100 rabbit anti-Nth1-S83p, a kind gift from the Thevelein lab ) in 5% Baswt, aspka and asypk1, as well as one sample of an nth1Δ-strain as a control, were inoculated in YPD (pH 5.6) and incubated at 30 °C overnight. Cultures were diluted to an OD of 0.05 and incubated for about 10 h before further dilution to OD 0.001 in YPD. The cultures were grown to an OD of ∼0.7 and, where necessary, cultures were diluted during this time to arrive at a similar OD for all cultures. Subsequently, 100 ml of each culture was centrifuged for 10 min at 2000 × g, pellets resuspended in 10 ml YPD and distributed to two 5 ml cultures in 50 ml Erlenmeyer flasks. The cultures were left to recover at 30 °C with shaking at 120 rpm for 25 min before treatment with 500 nm 1NM-PP1 (from a 250 μm stock in DMSO) or the corresponding volume of DMSO. After 15 min of shaking at 30 °C 0.5 ml of each culture were harvested into 0.5 ml of 0.2 m tricine (pH 7.0) with 0.1% Triton-X 100 and 1× phosphatase inhibitor, 200 nm Okadaic acid  and 1 EDTA-free protease inhibitor tablet (Roche) per 30 ml, immediately frozen in liquid nitrogen and stored at −80 °C.Measurement of trehalase specific activity was performed as previously described with modifications . Six repg and the pellet washed in 1 ml of cold 0.2 m tricine pH 7.0 . Pellets were resuspended in 1 ml of cold 0.2 m tricine (pH 7.0) and transferred into the wells of a deep-well 96-well plate on ice. The plate was spun for 5 min at maximum speed and 4 °C, supernatants removed, and pellets resuspended in 320 μl 62.5 mm tricine pH 7.0, 250 μm CaCl2 containing protease- and phosphatase-inhibitors. A well which did not contain a yeast pellet was used as blank. The plate and 0.5 m trehalose were equilibrated to 30 °C for 5 min before 80 μl of 0.5 m trehalose were added to each sample and incubation at 30 °C. After 10, 20, 30, and 40 min a 25 μl aliquot of each sample was transferred into PCR-stripes and heated to 98 °C for at least 3 min.Samples were thawed at 30 °C in a thermomixer at 1400 rpm, centrifuged for 1 min at 12,000 × The samples were cooled to room temperature and cell debris spun down briefly. The amount of glucose in the supernatants was determined using a Glucose-Oxidase/Peroxidase kit (Merck) using 5 μl input in a 150 μl reaction volume.versus time after trehalose addition with y-intercept of zero was determined as a measure for trehalase activity.The amount of glucose generated  was determined against a glucose standard and the blank value subtracted from each sample. The slope of the curve of trehalose degraded m urea in 25 mm Tris (pH 6.8). The trehalase activity per protein content of the aspka and asypk1 strains were each compared with that of the aswt strain using a two-sided, unpaired Student's t test.This activity was normalized to the protein level determined by BCA assay (Thermo Scientific) after spinning the remaining samples and bead beating of the resulting pellets in 400 μl 1% SDS, 6 E. coli (1 μm according to Bradford assay) was mixed with 0.5 μm GST-Ypk1 purified from S. cerevisiae or bovine PKA  and 300 μm ATP in 25 mm HEPES pH 7.5, 150 mm KCl, 2.5 mm CHAPS, 5 mm MgCl2, 1 mm DTT. After 10 min at 30 °C, 12 μm 6xHis-Bmh1 purified from E. coli and 30 mm trehalose were added. Samples were incubated at 30 °C and aliquots taken after 5, 10, 20, 30, 40 min, which were heated to 95 °C.Recombinant Nth1 purified from The amount of glucose produced was measured as described above using 5 μl of a 1:100 dilution of the samples as input.wt kinases were replaced with alleles encoding the corresponding analog-sensitive enzymes  were replaced with analog-sensitive alleles. The YPK1 paralog YPK2, which is considered to play a minor role compared with YPK1  versus the isogenic background (referred to as aswt) by assessing their growth on plates with and without ATP-analog . Given the central role of all three kinases in growth regulation, we expected visible effects on colony size upon the inhibition of any of them. A slight reduction of colony size was observed for the assch9 and aspka+assch9 strains even on plates without 1NM-PP1, indicating that these strains were mildly hypomorphic. The significance of this effect (p < 10−10) was verified independently by measuring the sizes of individual colonies when the strains were spread out on YPD-plates .We characterized these analog-sensitive strains . Inhibition of Sch9 alone also exhibited a visible reduction in growth. Interestingly, a small number of regular-sized colonies formed under these conditions, indicating that a limited number of cells adapted or became insensitive to Sch9-inhibition. This is consistent with a previous observation in our lab of an sch9Δ-strain rapidly generating adapted clones  and this finding further suggested that the compensation for PKA function is mainly through Sch9, which is consistent with previous findings .In contrast, the presence of 1NM-PP1 only mildly compromised colony size in case of the t viable , 8, 16. versus DMSO were compared by label-free, quantitative shotgun proteomics.For phospho-proteome analyses, analog-sensitive strains treated with 1NM-PP1 At a false-discovery rate (FDR) of 0.01, 7575 peptides, 4628 of which were phosphorylated (61%), were identified from phospho-peptide enriched samples from the respective strains. These phospho-peptides corresponded to 6373 phospho-sites (at site-decoy fraction: 0.01), mapping to 1430 proteins.supplemental Table S3). An overview of the data as volcano plots is shown in Fig. 2A. It is apparent that most phospho-peptides are unaffected by kinase inhibition. At a multiple-testing corrected p value cut-off (pAdj) of 0.05, 439 phospho-peptides were found to be altered in their abundance upon 1NM-PP1 treatment in the Ypk1-, 128 in the Sch9-, 118 in the PKA- and 293 in the PKA+Sch9 data set . For comparison, 12 phospho-peptides were classified as changing when aswt cells were treated with the ATP-analog, corresponding to a 0.4% false-positive rate.Out of the 4628 identified phospho-peptides, 3113 had no more than one missing value in three replicates of any sample and were thus considered for further analysis . Although the aspka strain was localized near aswt, the asypk1 strain on one hand and assch9 and aspka+assch9 strains on the other formed separate clusters.Principal component analysis of these data revealed that replicate samples clustered, and that separation of samples was mainly explained by genotype rather than by treatment . This again suggests that the gatekeeper mutation in Sch9 reduces the catalytic activity of this kinase and renders the strains hypomorphic. In contrast, we did not observe impaired colony formation of the asypk1 strain. This indicates that the extent of reduced phosphorylation of Ypk1 targets in this hypomorphic strain is not enough to lead to an observable proliferation phenotype.With respect to the in vitro kinase assay compared with the respective wt versions .We argued that the observed hypomorphic phenotypes were because of reduction of kinase activity by the gatekeeper mutations. Indeed, GST-tagged analog sensitive Ypk1 and Sch9 purified from yeast displayed a reduced activity in an Adj < 0.05) hypo- or hyper-phosphorylated upon kinase inhibition is shown in Fig. 2B. This global overview suggests a similar pattern of changes in the PKA+Sch9, Sch9 and PKA data sets, albeit for some phospho-peptides changes are only apparent in the PKA+Sch9 data set. In contrast, the Ypk1 data set contains some phospho-peptides which change in the same direction as in one or more of the other data sets, but the majority appears to change uniquely in this sample.A heatmap depicting the intensity fold changes of peptides significantly (pAdj < 0.05) altered phospho-peptides observed for each strain tested .To further assess the commonalities and differences between the inhibition of these kinases we determined the overlap among significantly (pi.e. hyper- or hypo-phosphorylated) in one of the data sets was also significant in the other (50 out of 117 and 128 respectively). This may indicate a strong overlap of, or cross-talk between, the signaling pathways. In the former case it can be envisioned that for some shared targets the inhibition of one of the inputs is compensated by the other. An argument in favor of this proposition is the finding that approximately twice as many significant phospho-peptides (293) were identified in the PKA+Sch9 data set compared with the individual PKA and Sch9 data sets. Furthermore, 112 of these were unique to the PKA+Sch9 data set, consistent with the notion that wild type PKA or Sch9 masks the loss of the other for many targets. On the other hand, 80% of the Sch9- and 66% of the PKA-targets were significantly altered also in the PKA+Sch9 data set, arguing that the high number of sites unique to PKA+Sch9 is not merely because of failure to detect true positives repeatedly over data sets.We first asked whether our data would recapitulate the previously reported interaction between PKA and Sch9 signaling in the form of shared targets . Indeed,We next addressed the overlap between PKA/Sch9- and Ypk1-targets, which we expected to be low as TORC2-signaling is less connected to PKA- and Sch9-signaling than the latter two to each other. Consistent with this idea, we found 297 phospho-peptides uniquely affected by Ypk1 inhibition. Surprisingly, 142 phospho-peptides (32%) were shared with at least one other data set, especially with PKA+Sch9 (27%).Bsupplemental Fig. S4) and hyper-phosphorylated  phospho-peptides, in that targets unique to the Ypk1- and PKA+Sch9-data sets constituted the largest groups whereas a strong prevalence of phospho-peptides shared between sets was also apparent.The results were qualitatively similar when focusing only on hypo- (p < 0.05) among proteins with regulated phospho-sites is given in supplemental Table S4. Among the Biological Process GO terms, “endocytosis.” which was enriched in the Ypk1-data set, is a major downstream process regulated by this kinase   (A full list of gene ontology (GO) terms significantly enriched  . It shouFig. S5) . Beyond Fig. S5) .“Regulation of transcription involved in meiotic cell cycle” and “Response to abiotic stimulus” were both enriched in the PKA-, whereas no significant terms were found for the Sch9- and PKA+Sch9-data sets.supplemental Table S4). The fact that both homologs Gsy1 and Gsy2 were detected based on distinct peptides lends validity to the finding that the regulation of storage carbohydrate metabolism is an important function of PKA and Sch9 signaling . Additionally, the neutral trehalase Nth1 was found to be affected by PKA and Ypk1 inhibition (see below).Similar gene ontologies related to transferase/glucosyltransferase activity were detected for the PKA, Sch9 and PKA+Sch9 data set and the proteins associated with these GO-terms in the three data sets strongly overlapped and included subunits of the trehalose 6-phosphate/phosphatase complex (Tsl1 and Tps3), glycogen phosphorylase Gph1 and glycogen synthases Gsy1 and Gsy2 , which argues that the sequence specificity of Ypk1 was not altered because of the gatekeeper mutation.For hypo-phosphorylated sites in the Ypk1-data set in contrast, arginine was more strongly enriched in the -3 position and further enrichment of arginines N-terminal of the phosphorylated residue, at -5 and -2, could be observed. The sequence context determined for Ypk1 fitted the reported RxRxxS/T-motif because of the enrichment of arginine in the -5 and -3 position , howeverIt is noteworthy that a slight enrichment of arginine in the -3 position of sites hyper-phosphorylated upon kinase inhibition was observable. As the change in abundance was opposite to what was expected for direct targets, it is possible that this residue forms part of the recognition sequence of a kinase that is itself repressed by the analog-sensitive kinases studied.In each case, the PKA+Sch9 motifs constituted a more pronounced version of the PKA and Sch9 motifs, therefore indicating that Sch9- and PKA-signaling converge either through shared targets or cross-talk.A–S5Csupplemental Tables S5.To estimate if our approach was suitable to identify direct AGC-kinase targets we asked how many of their previously known target-sites were identified and exhibited significant down-regulation upon kinase inhibition in our data set. Ypk1, Sch9 and PKA target sites derived from the literature are listed in bona fide kinase target sites were detected (i.e. identified and quantified) by mass spectrometry, most of these sites were significantly hypo-phosphorylated upon inhibition of the respective kinase:Although only 14% to 19% of bona fide PKA-sites were detected and three of them were down-regulated in the PKA  and six in the PKA+Sch9  data set  harbor more than one regulated phospho-site   –58. We t to S5C) , 59.supplemental Table S3).Notable observations included the detection of two regulated RxxS-sites on the previously reported Sch9-target Stb3 in the Sch9 and PKA+Sch9 data sets . We alsoi.e. without a basic residue in the -2 position, but with a proline at +1 . Interestingly, four regulated phospho-sites were detected on the trehalose synthase subunit Tsl1 in all four data sets.Similarly, several of the regulated RxxS/T-sites on multi-site proteins in the PKA data set were found in relaxed motifs, Finally, three RxxS phospho-sites on Bcy1 were significantly affected in the PKA and PKA+Sch9 data sets. Phospho-peptides containing either S83 or S145 exhibited an ∼45–75% reduced abundance upon kinase inhibition in both data sets, whereas phospho-peptides with T129 showed an up to 2-fold increase in the PKA data set.As Bcy1 is the common regulatory subunit of all three catalytic PKA-subunits , it is conceivable that these sites form part of a feedback loop regulating PKA activity. Indeed, S145 has previously been reported as a PKA target as part of its negative feedback mechanism . The samWe did not detect any peptides on phospho-diesterase Pde1, which has been reported as another component of cAMP-signaling that is a target PKA . InsteadThe reason for the clustering of phosphorylation-sites on AGC-kinase targets is not well understood in most cases, but it may allow generating switch-like responses similarly to other kinase targets . In any adj < 0.05) hypo-phosphorylated in at least one of the data sets. Because this is consistent with previously reported kinase consensus motifs (RRxS/T for Sch9 and PKA and RxRxxS/T for Ypk1)  , 59, we A).Like the data on all hypo-phosphorylated sites, the vast majority of RxxS/T-sites in the PKA or Sch9 data set were also part of the PKA+Sch9 set. Not all of these sites overlapped between the PKA and Sch9 samples, indicating that they may be targets of only one of the two kinases or that inhibition of one kinase targeting a site is compensated by the second kinase A.Although 45 RxxS/T-sites were uniquely affected by Ypk1-inhibition, 36 sites were also hypo-phosphorylated in at least one other data set. As the Ypk1 consensus motif is distinct from the PKA/Sch9 motif, we asked if this overlap mainly comprised sites within RRxS/T- or RxRxxS/T-motifs.B and 5C). As expected, the majority of RxRxxS/T-sites (20 of 24) were unique to the Ypk1-set. Consequently, sites with an RxRxxS/T-motif do not explain the overlap of RxxS/T-sites between the Ypk1 and the other three data sets.We therefore evaluated the overlap of significantly hypo-phosphorylated RxRxxS/T and RRxS/T sites between data sets B and 5C.C). Among these 31 sites only four were simultaneously in an RxRxxS/T-motif (i.e. RxRRxS). Assuming these sites are indeed direct Ypk1 targets, the previously defined Ypk1 consensus motif of RxRxxS/T may need to be reconsidered.On the other hand, 31 RRxS/T-sites were hypo-phosphorylated in the Ypk1 data set, of which 24 overlapped with at least one of the other sets C. Among supplemental Fig. S7).We next asked if the RRxS/T-sites that are potential Ypk1 targets had any other sequence characteristics close to the phospho-site, but no obvious amino acid enrichments at other positions within five residues from the phospho-site were observed  of neutral trehalase Nth1 with potential phosphorylation at S83 or T87 (or a mix of both), down-regulated with a fold-change (FC) of 0.6 (p = 0.007), 0.5 (p = 0.009) and 0.3 (p = 4 × 10−5) on Ypk1, PKA and PKA+Sch9 inhibition respectively. A second phospho-peptide of Nth1 mapping to S60 (58TMSVFDNVSPFKK70 + phos) exhibited significant down-regulation upon Ypk1 , PKA  and PKA+Sch9  inhibition.Our phospho-proteomics assay detected a phospho-peptide .We evaluated the phosphorylation of this site on endogenously 3xFLAG-tagged Nth1 after immuno-affinity purification by Western blotting with an antibody against phosphorylated S83 (S83p) . Indeed,Fig. 7These findings validate the effect of Ypk1 on phosphorylation of a site located within an RRxS-motif and therefore not assumed to be a direct Ypk1 target based on its previously reported consensus motif .It has been previously reported that the PKA-dependent phosphorylation of the N-terminal tail of Nth1 stimulates the enzyme's trehalase activity , 25, 28.aswt, aspka and asypk1 strains treated for 15 min with 1NM-PP1 with mock-treated controls and assessed trehalase activity by a method similar to the one described by De Virgilio, 1991  indicating that Nth1 is the only trehalase active under these conditions. Note that acidic trehalase is not expected to be active at the neutral pH used for this assay. We observed that trehalase activity was strongly reduced in the aspka, and somewhat also in the asypk1 strain, even in the absence of 1NM-PP1, indicating the hypomorphic nature of these strains . Importantly, a significant reduction of trehalase activity in both analog-sensitive strains was observed upon 1NM-PP1 addition relative to mock treated controls . This indicates that, as for PKA, Nth1-activity was positively regulated in a Ypk1-dependent manner.We employed a Finally, we tested if Ypk1 can activate Nth1 in a direct manner. To this end, we incubated Nth1 with GST-Ypk1 and ATP before measuring trehalase activity. Indeed, similarly to PKA, GST-Ypk1 strongly induced trehalase activity . This inIt can therefore be envisioned that stresses inactivating Ypk1 contribute to trehalose accumulation. Vice versa, return to favorable growth conditions and re-activation of Ypk1 may contribute to trehalose degradation by Nth1. Lastly, basal Ypk1 activity appears to contribute to basal Nth1 activity in exponentially growing cells.S. cerevisiae, PKA , Sch9 and Ypk1. For this purpose, we employed analog-sensitive versions of these kinases in combination with phospho-proteomics. A ypk1Δ ypk2Δ double- and tpk1Δ tpk2Δ tpk3Δ triple-deletion renders cells inviable and deletion of SCH9 leads to severe phenotypic effects, demonstrating the important roles that these kinases perform  and was defined by the Thorner lab based on nine peptides . This repeptides . It is isequence , the secsequence , 72. ParAfter the initial definition of the Ypk1 consensus motif  12 furthIt should also be noted that the assays performed in our study targeted Ypk1 during conditions of basal activity rather than in contexts where it is highly activated. It may be speculated that full activation of Ypk1 leads to a change in its substrate preference. This idea is particularly intriguing as distinct phosphorylation steps by Pkh1/2 and TORC2 may lead to partial and full activation of Ypk1 , 21.bona fide Sch9 target sites are generally in an RRxS/T-motif  by PKA has been previously reported , 28, 64.Few Ypk1 targets are currently known. Regulation of trehalose metabolism may therefore be a so far overlooked TORC2-dependent stress response. If this regulation is indeed via direct phosphorylation of the N-terminal tail of Nth1 by Ypk1, remains to be determined. The fact that two of the presumed regulatory sites, S20 and S60, reside within an RxRxxS/T-sequence, the classical Ypk1 target motif, argues in favor of this possibility, regardless of if Ypk1 can also phosphorylate RRxS/T-sites.Based on the finding that trehalose prevents the aggregation of partially unfolded proteins, but also interferes with their re-folding, it has been proposed that rapid activation of trehalase in heat-shock recovery is important to restore protein function . We specFurther phospho-sites on trehalose-6-P synthase/phosphatase subunits Tsl1, Tps2 and Tps3 were affected by Ypk1 inhibition and may provide further ways for TORC2-signaling to modulate trehalose metabolism.The TORC2-dependent regulation of endocytosis in contrast is well-established, however mechanistically not well understood . Our datFinally, we reported phospho-sites affected by the inhibition of AGC-kinases on proteins that had not been previously linked to signaling through these kinases. Based on the close match of sequence motifs derived from these sites and the reported consensus motifs of the kinases it is likely that we detected several direct kinase targets, setting the stage for future focused studies.DDA data have been deposited to the ProteomeXchange Consortium via the PRIDEpartner repository with the data set identifier PXD015668 and 10.6019/PXD015668. Spectra were additionally deposited to MS-Viewer and are accessible using key 99yh6tladc.Supplemental Table S1 is available at https://doi.org/10.6084/m9.figshare.11474685.https://panoramaweb.org/wMKBjd.url and are accessible at ProteomeXchange via identifier PXD015760.PRM data have been deposited at Panorama Public:


Given the increasing number of osteoporotic fractures of the distal femur, screw augmentation with bone cement is an option to enhance implant anchorage. However, in implant removal or revision surgeries, the cement cannot be removed from the distal femur without an extended surgical procedure. Therefore, the aims of this study were to investigate (1) whether cement augmentation has any influence on screw removal and removal torque, and (2) whether the implantation of a femoral component of a knee arthroplasty and its initial interface stability are affected by the remaining screws/cement.Eight pairs of fresh-frozen human female cadaveric distal femurs  with a simulated AO/OTA 33 A3 fracture were randomized in paired fashion to two groups and fixed with a distal femoral locking plate using cannulated perforated locking screws. Screw augmentation with bone cement was performed in one of the groups, while the other group received no screw augmentation. Following biomechanical testing until failure (results published separately), the screws were removed and the removal torque was measured. A femoral component of a knee arthroplasty was then implanted, and pull-out tests were performed after cement curing. Interference from broken screws/cement was assessed, and the maximum pull-out force was measured.p = 0.65) screw groups. However, there were significantly more broken screws in in the augmented screw group . There was no significant difference in the pull-out force of the femoral component between the augmented  and nonaugmented  screw groups.The mean screw removal torque was not significantly different between the augmented  and nonaugmented (4.6 Nm, SD 1.3, The screw removal torque during implant removal surgery does not significantly differ between augmented and nonaugmented screws. In the augmented screw group, significantly more screws failed. To overcome this, the use of solid screws in holes B, C, and G can be considered. Additionally, it is possible to implant a femoral component for knee arthroplasty that retains the initial anchorage and does not suffer from interference with broken screws and/or residual cement. 5 Only 6% of all femoral fractures occur at the distal femur, so they are relatively rare . HoweverSupracondylar fractures are the most common type in the elderly due to attenuated bone quality, especially within the metaphyseal part of the distal femur , 4. The Figure Therefore, the aims of this study were to investigate whether (1) screw removal is possible following augmentation, (2) the screw removal torque is affected by the augmentation, and (3) there is interface instability in cases when knee arthroplasty is required after screw augmentation.We hypothesized that augmentation significantly affects (1) the screw removal and (2) the interface stability.All human specimens in this study were prepared and used in accordance with the “Gesetz über das Leichen-, Bestattungs- und Friedhofswesen (Bestattungsgesetz) vom 04.02.2005, Abschnitt II, § 9 ,” which allows donor bodies to be dissected for scientific and/or educational purposes.Eight pairs of fresh-frozen human female cadaveric distal femora aged 86 years on average (range 81–92 years) were used in this study.Bone mineral density (BMD) was measured by means of high-resolution peripheral quantitative computed tomography  operated at 60 kVp and 900 µA, with 750 projections, an acquisition time of 200 ms, and a resolution of 123 µm. Following an established routine, BMD was evaluated within a cancellous region of interest. Selection was based on the largest condyle diameter with a thickness of 180 slices. A semiautomated segmentation procedure for the cancellous bone was applied .All specimens underwent an investigation beforehand to check the feasibility of cement augmentation for enhancement of implant anchorage in osteoporotic distal femoral fractures .In brief, the distal 7 cm of each femur was cut parallel to the joint line and plated in a simulated AO/OTA 33 A3 fracture model using a locking compression plate for the distal femur  made of titanium alloy. Distal fixation was performed with monocortically placed , cannulated and perforated 5 mm self-tapping locking screws (DePuy Synthes) tightened with a 4 Nm torque limiter.The specimens from each pair were then randomly split into two study groups with equal numbers of right and left distal femora. No screw augmentation was performed in group I. One milliliter of PMMA-based bone cement  was injected through each of the distal screws of the specimens in group II. Cement curing was enhanced by storing all the augmented specimens for 6 h at room temperature.Biomechanical testing was performed on a servohydraulic testing machine  equipped with a 4 kN load cell. All specimens underwent progressively increasing cyclic sinusoidal axial loading at 2 Hz until construct failure . WhereasPhoto and X-ray documentation of the construct failure modes was performed after biomechanical testing. The failure mode significantly differed between the groups; screw cut-out was observed in all nonaugmented specimens, whereas screw and plate breakage occurred in all augmented specimens . FollowiFollowing implant removal, the HR-pQCT scanning of each specimen was repeated to check the bony integrity of the condyles Fig. . A balanPull-out testing of the femoral component was performed on an electromechanical testing machine  with a 10 kN load cell. Each specimen was fixed to the machine base by clamping its PMMA block Fig. , left. Tt test was used to check for significant differences between the study groups. Fisher’s exact test was applied to detect significant differences between the groups with regard to the numbers of broken screws. Pearson correlation analysis was performed to screen the correlation between the pull-out force and BMD. The significance level was set at p = 0.05 for all statistical tests.Statistical analysis was performed using SPSS software package . The Shapiro–Wilk test was applied to screen and prove the normality of the data distribution related to the bone quality and biomechanical testing parameters. The paired-samples A summary of the results is presented in Table p < 0.001). All screws broke directly at the transition from the screw head to the screw shaft, i.e., directly behind the plate. In the augmented screw group, 7 broken screws were located in hole B (the nearest to the fracture line), 6 broken screws were in hole C, and 4 broken screws were in hole G. In the nonaugmented screw group, 5 broken screws were located in hole G, 3 broken screws were in hole C, and 1 broken screw was in hole B.All broken screws were located in the three most proximal plate holes near the fracture line. Nine of the 24 proximal screws were broken in the nonaugmented screw group, versus 17 in the augmented screw group (p = 0.65).The average peak torque for screw removal was 4.97 Nm (standard deviation (SD) 0.8 Nm) in the augmented screw group and 4.86 Nm (SD 1.0 Nm) in the nonaugmented screw group; the difference between the groups was not significantly different (p = 0.94). There was also no significant correlation between the pull-out force and BMD (p = 0.15).The average pull-out force was 2625 N (SD 603 N) in the augmented screw group and 2653 N (SD 542 N) in the nonaugmented screw group; the difference between the groups was not significant . In both groups, all broken screws were located close to the fracture gap. In addition, all screws broke at the head–shaft transition. Hence, it is assumed that the cannulated screws were overstrained at this position due to plate bending. A possible option would be to use standard noncannulated screws at these vulnerable positions. In our study, there was no significant difference between the augmented and nonaugmented screw groups in terms of average maximum screw removal torque. This finding agrees with previous investigations of solid screws that reported no significant difference between augmented and nonaugmented pedicle screws in relation to their average maximum torque during screw removal , 13. In Another concern is the interference with remaining screws and cement during revision surgeries with implantation of femoral components of knee prostheses. We could not find any evidence of interference with the remaining screws and cement in this investigation. Since only the proximal screws were broken, the implantation of a normal bicondylar femoral component could be performed without removing the broken screws. The cement clouds also did not interfere with the osteotomies preformed for femoral component implantation. Previous studies have demonstrated that the use of small cement volumes  leads to significantly improved implant anchorage , 10, 16.Nevertheless, there is a scenario for revision where augmented plate osteosynthesis does appear to be problematic, namely in case of infection in the region of the distal femur. In such a case, it is not easy to completely remove both the implant and cement. That is why we consider augmented plate osteosynthesis in the region of the distal femur to be performed only as a rescue procedure, providing the surgeon with a treatment option in difficult cases.This study has some limitations inherent to all investigations that use a small number of cadaveric specimens. Because of the limited availability of osteoporotic human cadaveric bones, and for ethical reasons, we tried to keep the number of specimens as low as possible. The selected fracture type—supracondylar fracture—represents just one type of clinically occurring distal femoral fracture. However, supracondylar femoral fracture is the predominant form of osteoporotic fracture at the distal femur. In a study of 283 osteoporotic distal femoral fractures by Myers et al., 76% were supracondylar fractures (5% simple and 19% complex intraarticular) . AdditioThe implant removal torque does not significantly differ between augmented and nonaugmented screws. Significantly more screws failed by breakage in the augmented screw group. Solid screws could be used in holes B, C, and G of the locked distal femoral plate to overcome this issue. Additionally, it is possible to implant a femoral component for knee arthroplasty while retaining the initial anchorage and without interference with broken screws and/or residual cement. However, in case of infection, a complicated surgical procedure would be needed to remove the cement.


EIS measurements were target specific and the sensor response was linearly increased with step wise increase in target analyte (Anti-CCP-ab) concentrations. The developed sensor showed a linear range for the addition of Anti-CCP-ab between 1 IU mL−1 → 800 IU mL−1 in phosphate buffered saline (PBS) and Human serum (HS), respectively. The sensor showed a limit of detection of 0.60 IU mL−1 and 0.82 IU mL−1 in the PBS and HS, respectively. The develop bio-electrode showed a good reproducibility (relative standard deviation (RSD), 1.52%), selectivity and stability (1.5% lost at the end of 20th day) with an acceptable recovery rate (98.0% → 101.18%) and % RSD’s for the detection of Anti-CCP-ab in spiked HS samples.Rheumatoid arthritis (RA) is a chronic autoimmune disease that produces a progressive inflammatory response that leads to severe pain, swelling, and stiffness in the joints of hands and feet, followed by irreversible damage of the joints. The authors developed a miniaturized, label-free electrochemical impedimetric immunosensor for the sensitive and direct detection of arthritis Anti-CCP-ab biomarker. An interdigitated-chain-shaped microelectrode array (ICE) was fabricated by taking the advantage of microelectromechanical systems. The fabricated ICE was modified with a self-assembled monolayer (SAM) of Mercaptohexanoic acid (MHA) for immobilization of the synthetic peptide bio-receptor (B-CCP). The B-CCP was attached onto the surface of SAM modified ICE through a strong avidin-biotin bio-recognition system. The modified ICE surface with the SAM and bio-molecules  was morphologically and electrochemically characterized. The change in the sensor signal upon analyte binding on the electrode surface was probed through the electrochemical impedance spectroscopy (EIS) property of charge-transfer resistance (R Nearly 1% of the world’s population is affected by RA, a chronic autoimmune and degenerative disease ,3. The eThe fabrication of electrochemical antibody detection biosensors, for the quantitative analysis and monitoring of disease biomarkers, is presently an emerging field of research activity. Recently, a few electrochemical biosensors for the sensitive detection of antibodies for SARS-CoV-2 spike protein , Zika vid = ~10−15 M), is rapid, unaffected by pH, temperature, organic solvents and was used to attach biomolecules to electrode surface ·3H2O), potassium ferricyanide (K3[Fe(CN)6]), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-Hydroxysuccinimide (NHS), Avidin from egg white , Bovine serum albumin (BSA), and human serum were purchased from Sigma-Aldrich (USA). Anti-Interferon-γ-antibody (Anti-IFN-γ-ab), Anti-insulin-antibody (Anti-insulin-ab), IgM-Rheumatoid factor (IgM-RF), Anti-C-reactive-protein-antibody (Anti-CRP-ab), Human-Immunoglobulin-G-antibody (Human-IgG-ab) were procured from Abcam . Phosphate buffered saline  and 1 × PBS containing 0.05% Tween 20 were procured from Tech and Innovation . All solutions were prepared using deionized water  from Purescience . All other chemicals were of analytical reagent grade and are used without any further purification.Biotin-labelled cyclic citrullinated peptide (Biotin-CCP) (4166 IU/mL) (Peptide sequence: Biotin-HQCHQEST-Cit-GRSRGRCGRSGS-COOH) with a disulfide bond between the Cyc3 and Cyc16 residues was procured from Peptron Inc. . Anti-cyclic citrullinated peptide antibody (Anti-CCP-ab)  with an operating accelerated voltage of 15 kV and energy dispersive X-ray.X-ray photoelectron spectroscopy (XPS) elemental surface analysis was carried out using a PHI 5000 Versa Probe (Ulvac-PHI) spectrometer (Japan) with monochromator A1 Kα (1486.6 eV). Survey scan was first recorded and then region scans were measured for the S(2p) and C(1s) photoelectron binding energy regions. A 50 eV band pass energy, 1 eV step size and 200 μm × 200 μm X-ray spot size were used for measuring survey scan (range = 1200 to −5 eV). For C1s and S2p region scans a pass energy of 20 eV and 0.1 eV step size were used. All the region scans were fitted by a standard Gaussian curve fit with Shirley background subtraction . Atomic 6]3− in 1 × PBS  (pH 7.4), at a sweep rate of 50 mV s−1 within a potential range of −0.2 V to +0.5 V vs. Ag/AgCl external RE. The electrochemical impedance spectroscopy (EIS) measurements were carried out in 5 mM [Fe(CN)6]3−/4− (1:1) in 1 × PBS, at room temperature (RT) (24 °C) using a two-electrode setup. The spectra were measured by selecting an alternating current perturbation voltage with a root mean square value of 0.05 V in a frequency range of 0.1 Hz–1 MHz and three points were collected for a decade of frequency. The measured impedance spectra were fitted by using a suitable equivalent circuit model consisting of solution resistance (Rs), electrode interfacial capacitance (CPE) and charge transfer resistance (Rct). The fitting of the EIS spectra was carried out by ZView-a non-linear curve fitting software from Scribner Associates Inc. .Cyclic voltammetry (CV) was carried out in a three-electrode configuration  consisting of a working electrode (WE) (bare and modified ICEs), a counter electrode (CE) (Platinum coil), and a reference electrode (RE) (Ag/AgCl). The cyclic voltammograms (CVs) were carried out in 5 mM 3−/4− (1:1) in 1 × PBS. The electrode interfacial property (charge-transfer resistance: Rct) was obtained by fitting the measured EIS spectra to an equivalent-circuit model  as shown in i) displays a small semicircle with Rct of 15640 Ω, which is characteristic for a bare gold electrode with no diffusional limiting process at lower frequencies, which is an additional benefit of the developed ICE microelectrodes. The diameter of the semicircle was substantially increased with a Rct of 978,900 Ω, due of the surface modification of ICE with mercaptohexanoic acid (ii), suggesting that the alkanethiol film created an electrical hindrance for the electrons transfer between the [Fe(CN)6]3−/4− redox probe and the electrode surface. The semicircle diameter was significantly decreased with a Rct of 86,520 Ω (iii), after the EDC-NHS carbodiimide coupling reaction with the terminal –COOH of MHA-SAM, suggesting that formation of succinimide ester on the SAM modified electrode surface facilitating the fast electron transfer between the electrode and the interface. The semicircle diameter is slightly increased  with the linking of avidin on the SAM surface, due to the development of electron-blocking layer on the ICE surface by successful immobilization of avidin (iv). The ICE/MHA/EDC-NHS/B-CCP impedance spectrum shows an increase in diameter of semicircle with a Rct of 197,400 Ω (v) due to the formation of electron transfer obstruction layer on the electrode surface by the immobilization of B-CCP synthetic peptide, which is a highly non-conductive biological material. The rinsing of the electrode with 1 × PBS buffer containing 0.05% Tween-20 and 0.5% BSA for 2 min has again increased  the diameter of the semicircle (vi), due to the binding of BSA on ICE surface to prevent non-specific binding of the Anti-CCP-ab onto the ICE surface. vii shows a great increase in diameter of the semicircle  when incubated with the Anti-CCP-ab for 10 min, suggesting the binding of analyte with the target and results in decrease in electron transfer rate by formation of ferrocyanide transport obstruction layer between the electrode surface and the electrolyte. The measured impedance spectra are consistent with the CV results  in 1 × PBS to measure the variations in electrode behavior introduced at major stages of electrode modification . The electrochemical response was drastically changed and the anodic and the cathodic peak currents were intensely altered, after functionalization of the ICE with MHA-SAM when compared to bare ICE (ii). The effect is due to the slow electron transfer kinetics induced by the formation of a high degree of the insulation layer developed by the SAM functionalization. The CV of the ICE/MHA/EDC-NHS shows regeneration of the redox peak for the [Fe(CN)6]3−/4− (iii). The effect is due to the formation of succinimide ester on the electrode surface which results in fast-electron transfer kinetics between the electrode and electrolyte (iv). The CV of ICE/MHA/EDC-NHS/B-CCP electrode, ICE/MHA/EDC-NHS/B-CCP/BSA electrode and ICE/MHA/EDC-NHS/B-CCP/BSA/Anti-CCP-ab shows a reduced electrochemical response in compared to the ICE/MHA/EDC-NHS electrode, due to the immobilization of biologically active synthetic peptide (B-CCP), BSA and Anti-CCP-ab binding on the corresponding electrodes, which results in very low electron transfer kinetics and a greatly decreased in the ferricyanide response on the electrode surface (v-vii).CV of the bare and modified ICEs were recorded in a solution of 5 mM  (1:1) redox probe prepared in 1× PBS. As shown in ct values linearly increased with increasing Anti-CCP-ab concentration. The ΔRct was also proportionate to the linear increase in Anti-CCP-Ab concentrations on the electrode over a range of 1 IU mL−1 → 800 IU mL−1 (ct = 0.5060 + 0.00313*Canti-ccp-ab(PBS) with a correlation coefficient of 0.9986 . The LOD was calculated by using an expression (3*SE/slope)  electrolyte solution. In comparison to the blank electrode, HS (anti-CCP-ab) added electrode impedance was increased with linear increase in concentration of analyte  with a correlation coefficient of 0.9983 (−1 (S/N = 3). The impedance signal measured with HS samples are dependent on concentration of Anti-CCP-ab. The calibration curve constructed for determination of Anti-CCP-ab in HS is used for analysis of real samples.In order to construct a good linear calibration curve and to avoid the matrix effects a dilute human serum solution , Anti-insulin-ab (200 pg mL−1), IgM-RF (50 IU mL−1), Anti-CRP-ab (200 pg mL−1), Human-IgG-ab (100 pg mL−1), Hemoglobin (15 mg mL−1), Anti-CCP-ab (10 IU mL−1). The concentration of the interferents selected is two folds greater than the expected concentration levels of the healthy individuals. These interferents prepared in 1 × PBS are added to the ICE/MHA/EDC-NHS/B-CCP/BSA bioelectrode separately and electrode response is obtained in a frequency range of 0.1 Hz–1 MHz. The interference of the ICE/MHA/EDC-NHS/B-CCP/BSA bioelectrode is studied by measuring the impedance signal in the presence and absence of different proteins co-exist in the HS. The interfering proteins selected for the study are Anti-IFN-γ-ab  on three independently prepared ICE/MHA/EDC-NHS/B-CCP/BSA bio-electrodes. The RSD calculated for the three measurements was 1.52%, suggesting that the fabricated immunosensor shows an acceptable reproducibility. The reproducibility for the three different ICE/MHA/EDC-NHS/B-CCP/BSA bioelectrode (R0) was also verified in 1 × PBS, and the results demonstrated a RSD of 1.02%. A very good reproducibility and stability is endorsed due to a robust Avidin-Biotin interaction system and covalent immobilization of avidin molecules on the gold surface, which created a bio-compatible substrate and prevents the removal of bio-receptors from the modified ICE surface. moreover, the presented impedimetric interdigitated microelectrode array sensor shows several advantages compared to the other reported methods for CCP detection. The developed assay (a) is label-free, (b) mass production of ICE at low cost, (c) no Warburg diffusion at lower frequencies, (d) requirement of low sample volumes (10 µL), (e) low-cost sample preparation, (f) a low assay time and (e) direct signal readout in the form of an electrical signal.Reproducibility is an important factor for the potential application of the immunosensor in clinical diagnostics. Thus, the reproducibility of the fabricated ICE/MHA/EDC-NHS/B-CCP/BSA bioelectrode was verified by sensing Anti-CCP-ab (10 IU mL−1) into 1% v/v PBS diluted HS samples. The HS samples spiked with bio-analyte were loaded onto the developed ICE/MHA/EDC-NHS/B-CCP/BSA bio-electrode. Three different measurements for each concentration was obtained and the average of three measurements was calculated and the recovery rates with the %RSD was tabulated. In −1. The calculated recoveries are 98.0% (n = 3), 99.9% (n = 3), 99.5% (n = 3), 101.18% (n = 3) and 99.80 (n = 3), respectively. The results suggest that the biosensor shows a good recovery rate for the five samples with no significant variation between the spiked and the found Anti-CCP-ab concentrations. Thus, the ICE/MHA/EDC-NHS/B-CCP/BSA bioelectrode fabricated can be potentially used for the accurate quantification of the Anti-CCP-ab’s in the real samples.To validate the practical application of the developed ICE/MHA/EDC-NHS/B-CCP/BSA bioelectrode, Anti-CCP-ab was quantified in spiked HS samples. The analysis was carried out by spiking five different concentrations of Anti-CCP-ab . Additionally, the microscale design of the sensor (3.5 × 14 mm) enabled the ICE to easily assemble with a portable-potentiostat making the bio-electrode suitable for POC testing. The fabricated bioelectrode is economical and feasible for mass production and the detection has not required any fluorophores. It sensor can applied for detection of arthritis AAb (Anti-CCP-ab) in individuals at early stages of disease development.Miniaturized electrochemical impedance immunosensor based on Interdigitated microelectrode array for selective detection of Anti-CCP-ab was developed through a simple Biotin-Avidin recognition system. The avidin is attached on the ICE surface by modification of electrode with MHA-SAM and the functionalization of the ICE with SAM is characterized through AFM, EDX, CV, XPS, EIS. SAM provided a bio-compatible environment to attach and preserve the bio-activity of the B-CCP. The Avidin-Biotin recognition system provided significant stability to the CCP bound onto the electrode surface. The sensor is successfully validated as an impedimetric biosensor for the quantification of Anti-CCP-Ab in PBS and HS. The biosensor showed a linear range of 1 IU mL


The presence of retained products of conception could not be ruled out. Therefore, to further confirm the condition, the patient underwent an emergency hysterectomy. Gross examination showed an enlarged and boggy uterus with numerous dilated and ecstatic thrombosed blood vessels in the implantation site of the endomyometrium. Histologic and immunohistochemical examination of the implantation site revealed the presence of persistently patent uteroplacental arteries with variable degrees of thrombosis adjacent to normally involuted vessels. Hence, a diagnosis of placental site vessel subinvolution (VSI) was established in this case. We also reviewed the related literature to illustrate the informative histologic findings of subinvolution. Preparing the ground for diagnosing subinvolution is important as this process defines that the cause of delayed postpartum uterine bleeding is idiopathic, rather than iatrogenic.Subinvolution of the implantation site is a significant contributor to delayed postpartum hemorrhage (PPH). There is immense literature documenting the histologic features, development, and involution of the uteroplacental site; however, practice-oriented literature on subinvolution of the implantation site is sparse. In the present study, we briefly review the physiologic characteristics associated with the normal development and involution of uteroplacental arteries and the proposed pathophysiologic attributes of subinvolution. Furthermore, we engage in a comparison of the condition with preeclampsia. Herein, we report a case of postpartum vaginal bleeding that persisted for two weeks following cesarean delivery. The bleeding was nonresponsive to conservative treatment. Sonography performed revealed that a heterogeneous intrauterine vascular mass measuring 14.6 × 9.2 × 10.4 cm Postpartum hemorrhage (PPH) is a major cause of maternal deaths worldwide due to its association with hemorrhagic shock and the tendency to develop disseminated intravascular coagulation ,2. The cCauses of primary PPHPrimary PPH is prevalent during the first 24 hours post-completion of the third stage of labor. The leading causes of primary PPH are as follows: acquired coagulopathy, uterine atony, trauma associated with the lower genital tract, congenital coagulopathy, and retained products of conception -14.Causes of secondary PPHSecondary PPH usually occurs between 24 hours and six weeks post-parturition. Secondary PPH has received sparse attention, probably due to its low incidence of complications, which account for only 1% of all pregnancies, and its regular association with maternal morbidity rather than mortality ,16. HoweA major clinical area that has garnered interest as far as PPH is concerned is vessel subinvolution (VSI) of the placental implantation site . There h3; total protein level: 5.5 g/dL; albumin level: 3.1 g/dL; globulin: 2.4 g/dL; and albumin:globulin ratio: 1.3. Serum glutamic-oxaloacetic transaminase (SGOT) level was 18 U/L, alkaline phosphatase level was 62 IU/L, and serum glutamic-pyruvic transaminase (SGPT) level was 10 U/L. The clinical findings also showed that the patient had a γ-glutamyl transferase level of 10 U/L, total bilirubin level of 0.3 mg/dL, conjugated bilirubin level of 0.2 mg/dL, and an unconjugated bilirubin level of 0.1 mg/dL. The patient was resuscitated with fluids and conservatively treated with four units of packed red blood cells and tranexamic acid (one amp intravenously twice per day and stat). However, bleeding persisted and the hemoglobin levels progressively decreased.A 38-year-old woman (P3L3) was brought to the hospital’s emergency department due to vaginal bleeding accompanied by clinical symptoms, such as chills and lightheadedness, which had persisted for 14 days post-cesarean delivery at the end of her pregnancy, which was described as uneventful. At the time of her admission, she complained of irregular abdominal pain. Additionally, she reported frequent profuse vaginal bleeding, during which 10 pads were soaked over a 10-hour period. Physical examination revealed that her abdomen was moderately tender; bloody vaginal discharge was also noted. The patient was afebrile and had tachycardia (blood pressure: 80/50 mmHg). The initial blood test revealed leukocytosis, with a WBC count of 12,000/μL and a hemoglobin level of 7.8 g/dL. Other laboratory findings were as follows: serum β-human chorionic gonadotropin (β-HCG): 2.75 mIU/mL; prothrombin time: 15.72 seconds ; activated partial thromboplastin time: 26.3 seconds ; bleeding time: 2.25 minutes; clotting time: 5.5 minutes; platelet count: 280,000 cells/mmSonography, performed using the Voluson S7 ultrasound unit , revealed that a heterogeneous mass measuring 14.6 × 9.2 × 10.4 cm was present, distending the endometrial cavity. Substantial vascularity was observed around the anterior endometrium and was more localized in the myometrial vessel along the left anterior myometrium. The findings further showed the formation of venous varix and the indication of the subinvolution of the placental site. Despite these results, the presence of retained products of conception could not be ruled out. Therefore, to further confirm the condition, the patient underwent an emergency hysterectomy.Further assessment of the pathologic gross examination of the uterus revealed a considerable amount of thrombi in the endometrial cavity. Microscopic examination of the myometrium beneath the placental implantation site revealed the presence of persistently patent uteroplacental arteries adjacent to normally involuted vessels Figure .Clusters of large ectatic and patent blood vessels with luminal red blood cells and loose fibrin were found in the endomyometrium Figures , 2B, 2C.Numerous constant patent uteroplacental arteries with different degrees of thrombosis located next to normally involuted vessels were also observed Figures , 3B.Persistent extravillous trophoblast (EVT) cells found within the subinvoluted vessel wall can be observed in Figures The immunohistochemical analysis determined the presence of residual trophoblasts within the abnormal vessel walls and interstitium. In the subinvoluted vessels, EVTs replaced the normal endothelium Figure , revealiHistochemical staining for elastin provides a significant benefit for describing the structure of the vessel wall. PAS staining was used for this purpose and revealed disrupted histomorphology of persistently patent vessels Figures , 6C, 6D.The patient was followed up with serial beta-HCG monitoring and CBC assessment. She recovered fully and was found to be asymptomatic on follow-up.Physiology of involutionComplex anatomic and physiologic changes occur in the uterine vessels at the time of pregnancy and during the postpartum period . These cPost-delivery, the physiologic mechanism of uteroplacental arterial involution is crucial for eliminating the remodeled changes in the vessel and for preventing major blood loss. Both the vascular and interstitial EVTs in the implantation site start to be replaced with maternal endothelial cells . SeveralEtiology of subinvolutionThe normal remodeling of the vessels during the third trimester is either delayed or insufficient in the subinvolution of the placental implantation site; the uterus fails to return to the nonpregnant state , resultiInadequate remodeling of the spiral arteries by EVTs is seen in patients with preeclampsia Figure ; in subiAnother crucial piece of information about the mechanism of subinvolution is based on EVT apoptosis, i.e., EVTs seem to lose Bcl-2 expression in preeclampsia, whereas increased Bcl-2 expression has been described in subinvolution ,9,15. PeThe molecular mechanisms include altered expression of the growth factors associated with the uterine scarring process and the upregulation of proangiogenic and antiangiogenic factors as well as receptors at the interface between the fetal and maternal tissues ,6. InvesVSI can also be enhanced by the occurrence of active acute inflammation, which can be enhanced via proinflammatory factor production and local tissue coagulopathy . This acDiagnosis of subinvolutionThe Pathologist’s Perspective of a Case of PPHSubinvolution can be recognized in severe cases by the characteristic clinical features and histologic findings in postpartum endometrial curettage samples from the placental site and hysterectomy specimens. However, ruling out the presence of chorionic villi, endometritis, placenta accreta, and gestational trophoblastic disease in this clinical setting is not sufficient.In placental site subinvolution, the uterus is grossly enlarged and boggy. Multiple microscopic sections of the placental implantation site should be acquired to find the cause of hemorrhage and rule out other causes of secondary PPH . PatholoThe histologic findings in subinvolution probably reflect the nature of this process along a biologic continuum of changes, somewhere between appropriate involution and its complete absence. The extent of subinvolution varies from case to case; the diagnostic findings may be quantitatively limited in any given specimen ,11. In cAn infrequent intravascular collection of obliterated fibrous chorionic villi can be identified; however, it should be properly investigated as to whether it accounts for the hemorrhage. Vascular subinvolution may be detected in the presence of rare degenerating intraluminal/intravascular chorionic villi, which is a valuable observation when the amount of villous tissue present does not account for the hemorrhage ,15. TherSonographic DiagnosisIn establishing the sonographic diagnosis of the subinvolution of the placental site, it is necessary to visualize low-resistance vessels that are found along the inner third of the myometrium. In particular, gray-scale imaging is useful in revealing hypoechoic tortuous vessels found in the myometrium.Another useful technique is pulsed-wave Doppler sonography, which can be used to confirm the vascular characteristics of an elevated optimum systolic velocity using a low-resistance waveform. Using this approach, the presence of increased areas of vascularity is an indication of the placental implantation site of either the second or third-trimester sonography . It is iIn addition, the products of retained conception may mimic the above-mentioned findings and, therefore, should be documented as part of the differential diagnosis if echogenic tissue is observed in the endometrial cavity.ManagementThe therapeutic management of secondary PPH is similar to that of primary PPH. The success of treatment requires coordination and collaboration of multidisciplinary care teams in order to achieve fast hemodynamic stabilization in the patient, replace the volume of the depleted blood, and prevent the occurrence of coagulopathy .It is mandatory to perform a speculum examination of the lower genital tract and cervix to eliminate possible lacerations. Furthermore, it is compulsory to perform uterine ultrasonography to exclude the presence of retained placental tissue . VaginalThe symptomatic subinvolution of the placental site is a health condition of concern with bleeding, the management of which has remained a topic of debate. Some of the suggested techniques that can be used to manage conduction are vaginal packing, the use of uterotonic drugs, and blood and plasma unit transfusion. There are also more aggressive interventions that are considered standard therapies, such as uterine vessel ligation  and hystClinical features and associationsDelayed PPH should be considered as an indication of subinvolution. Other signs of the condition are frequent bleeding that occurs between one week to several months post-parturition ,16. The Placental site VSI is a rare form of secondary PPH that has always been underdiagnosed by clinicians. An immune-related cause involving miscommunication of the maternal and fetal tissues has been proposed regarding its occurrence. In the present study, the diagnosis was confirmed by the presence of endovascular EVTs and histology of the dilated as well as clustered myometrial arteries that were partly occluded by old and new thrombi. Nevertheless, confirmation was primarily obtained after conducting an emergency hysterectomy.Furthermore, we considered that this kind of secondary PPH has an idiopathic cause and used the available data from a previous study to show that no predictive factors  related to it are known. It is crucial to identify the severity of secondary PPH promptly to implement treatment via targeted therapy and in order to preserve fertility.


Dietary counselling favorably modified body mass, body fat, glucose regulation, and fasting concentrations of triglyceride and very low-density lipoprotein cholesterol (main effects of counselling: p < 0.05); SGLT2 inhibition did not influence any of these adaptations (counselling × medication interactions: p > 0.05). However, SGLT2 inhibition when combined with dietary counselling led to greater loss of fat-free mass (counselling × medication interaction: p = 0.047) and attenuated the rise in high-density lipoprotein cholesterol (counselling × medication interaction: p = 0.028). In light of these data and the health implications of decreased fat-free mass, we recommend careful consideration before implementing SGLT2 inhibition as an adjunct to dietary counselling for weight loss in sedentary adults with obesity.Sedentary obesity is associated with increased risk of many cardio-metabolic diseases, including type 2 diabetes. Weight loss is therefore a desirable goal for sedentary adults with obesity. Weight loss is also a well-documented side effect of sodium glucose co-transporter 2 (SGLT2) inhibition, a pharmaceutical strategy for diabetes treatment. We hypothesized that, compared with placebo, SGLT2 inhibition as an adjunct to out-patient dietary counselling for weight loss would lead to more favorable modification of body mass and composition, and greater improvement in glucose regulation and lipid profile. Using a randomized, double-blind, repeated measures parallel design, 50 sedentary men and women (body mass index: 33.4 ± 4.7 kg/m Two thirds of adults within the United States of America are overweight; approximately half of those who are overweight satisfy the criteria for obesity ,2. RecenPerhaps as a result of the increasing prevalence of type 2 diabetes, during the previous decade there has been a surge in the development of new pharmacological therapies. Among these new therapies is a class of medications that work via sodium glucose co-transporter 2 (SGLT2) inhibition. Therapeutic benefits of SGLT2 inhibition include short-term and sustained favorable modification of glycosylated hemoglobin (HBA1c), and fasting blood glucose ,10,11,12Weight loss is a desirable goal for sedentary adults with obesity for many health-related reasons, including decreasing the risk of developing type 2 diabetes ,20. DietConsidering the influence of SGLT2 inhibition on the absorption of dietary carbohydrates, a secondary aim was to explore the effects of dapagliflozin and dietary counselling on dietary preferences and hunger and appetite prior to and following acute feeding. In recent studies, the influence of short-term (2 weeks) SGLT2 inhibition on dietary preferences  and appetite did not attain statistical significance . In the A double-blind, repeated measures parallel design was utilized. Stratified random assignment was used to allocate participants to treatment. The trial was conducted in accordance with the guidelines for Good Clinical Practice and was registered at ClinicalTrials.gov (Identifier: NCT03180489). All procedures were reviewed and approved by the Institutional Review Board at Colorado State University in accordance with the principles set out in the Declaration of Helsinki. All participants provided written informed consent before commencing involvement in the study.2, sedentary, as defined as less than or equal to three low-intensity physical activity sessions per week, of less than or equal to 30 min per session over the previous year, negative pregnancy test for women of childbearing potential, no known metabolic diseases, and willingness to modestly reduce daily caloric intake for twelve weeks. Exclusion criteria consisted of evidence of any significant disease that may interfere with study objectives, habitual use of herbal preparations or prescription drugs other than birth control and/or medications to treat depression, current enrollment in another clinical study, habitual or recent use of tobacco in the last two years, history of hypersensitivity reaction to SGLT2 inhibition, renal impairment (based on estimated glomerular filtration rate), abnormal liver function, bilirubin >2.0 mg/dL (34.2 μmol/L), diagnosis of hepatitis B or C, history of bladder cancer, recent cardiovascular events, or considered unsuitable for participation by our medical monitor.Sedentary, but otherwise healthy, men and women volunteered and participated. Inclusion criteria included: provision of informed consent, aged between 18–65 years, body mass index (BMI) greater than 27.5 kg/mFollowing screening, all research participants began 12 weeks of dietary counselling for weight loss, combined with daily ingestion of either a placebo or a SGLT2 inhibitor. Prior to and following 12 weeks of dietary counselling, participants were assessed for body mass and composition, resting metabolic rate, blood glucose regulation, plasma lipid profile, and hunger and appetite.Screening included review of medical history, physical examination, venous blood sampling, and analysis for standard markers of kidney and liver function , and completion of an exercise stress test  with 12-lead electrocardiogram and blood pressure assessment. Following participant enrollment, as detailed below, primary outcome variables were measured over several separate visits to the laboratory before and after dietary counselling.2peak) was determined via indirect calorimetry  during incremental stationary cycle ergometer exercise to volitional fatigue, as previously described [Body composition was assessed using dual energy X-ray absorptiometry , as previously described . In addiescribed ,25.2 and carbon dioxide production (VCO2) were averaged each minute for 30 min using a custom built ventilated hood indirect calorimetry system  that utilized a respiratory mass spectrometer  and an ultrasonic flow sensor . The system was calibrated daily with precision mixed gases . Energy expenditure was calculated using the Weir formula [2 of 0.93 [Following a 12-h fast, and 24-h abstention from exercise and caffeine, participants reported to the laboratory for measurement of resting metabolic rate (RMR) and respiratory exchange ratio (RER), and completion of an oral glucose tolerance test (OGTT). RMR was measured over 45 min, as previously described ,27. The  formula . In our  of 0.93 .Immediately following the measurement of RMR, participants ingested 75 g of glucose diluted in 300 mL water; venous blood was sampled prior to ingestion and systematically over the next two hours for the determination of circulating concentrations of glucose  and insulin (enzyme-linked immunosorbent assay (ELISA): Crystal Chem, Inc., Elk Grove Village, IL, USA) in a manner similar to that previously described ,30. HomeOn a separate day, participants underwent testing for assessment of hunger and fullness. Following a 12-h fast and 24-h abstention from exercise and caffeine, participants ingested a small mixed macronutrient liquid meal . Sixty minutes after the liquid preload, participants selected food from an all-you-can-eat breakfast buffet. Participants were provided 20 min to eat. The selected food choices (packaging included) were recorded and weighed before and after participant consumption. Remaining uneaten food was reweighed and participants’ energy and macronutrient intakes were determined using the Food Intake Analysis System software . Participants remained at the facility for the next three hours for follow up satiety measurements.At baseline, 0 and 60 min after liquid pre-load consumption, and 0, 60, 120, and 180 min after breakfast, participants completed a hunger and satiety survey. Surveys consisted of five questions: (1) How strong is your desire to eat right now? (2) How hungry do you feel right now? (3) How full do you feel right now? (4) How much do you think you could eat right now? (5) How thirsty do you feel right now? Participants answered these questions by marking a vertical line on a 100 mm visual analog scale, as previously described .Participants completed a three-day diet log (two weekdays and one weekend day) before initiation of the 12-week nutrition education and counselling intervention. Diet logs included all food and drink consumed (ingredients and amounts), location of meal, time of day, and hunger level before meal. Commercially available software  was used to determine the caloric and micro- and macronutrient make up of each item consumed. Self-reported dietary analyses are often inaccurate in quantifying energy and macronutrient intake ,34, thus® developed by the Kendall Reagan Nutrition Center at Colorado State University) was initiated, which consisted of weekly, one-on-one, 30-min meetings providing nutrition education/counselling for the study participants. Counselling focused on modest caloric restriction with an initial target of RMR multiplied by an activity factor of 1.2 and then, as needed, lowered over time toward an energy intake closer to the measured baseline RMR. This dietary reduction was designed to reduce each participant’s weight by approximately 0.5 kg per week [A 12-week outpatient dietary intervention program .Baseline (pre-counselling) differences between placebo and SGLT2 inhibition groups were compared using independent Student t-tests. Consistent with the experimental design , the influence of SGLT2 inhibition on the physiological responses to dietary counselling was examined using two-way (group: placebo vs. SGLT2 inhibition × time: before vs. after dietary counselling) analysis of variance (ANOVA). The influence of SGLT2 inhibition and dietary counselling on the dynamic responses to oral glucose ingestion were examined using three-way ANOVA (placebo vs. SGLT2 inhibition × before vs. after dietary counselling × time points during the OGTT). Pairwise multiple comparison procedures were performed using the Holm–Sidak method. Relations of interest were described using Pearson product-moment correlations. The level of statistical significance was set at 2 in 36/50 participants; the remainder of the participants ranged between 27.5 and 29.9 kg/m2. There were no baseline differences in any of the primary variables between the placebo and SGLT2 inhibitor groups . The dose of dapagliflozin began as 5 mg/day for the first 14 days and was increased to 10 mg/day for all participants for the remainder of the study. Baseline daily dietary intake data, calculated from three-day diet diary records, are also presented in The progress of all participants throughout the trial (from screening to completion) is presented in p = 0.015); this difference appeared to be mediated primarily by a greater carbohydrate ingestion (p = 0.012). Average daily ingestion of fat and protein was not different between placebo and SGLT2 inhibition (both p > 0.09).Average daily dietary intake was greater in the placebo group ; SGLT2 inhibition did not influence this response . Similarly, RER was also decreased (main effect p = 0.002) and the response unaffected by SGLT2 inhibition . There were no group differences for either of these dependent variables (main effect of group: both p > 0.228).Following 12 weeks of dietary counselling, RMR was decreased (main effect of time: p = 0.751), was unchanged (p = 0.174) over 12 weeks, and SGLT2 inhibition did not influence the overall response . Circulating glucose concentration during the OGTT is presented in p = 0.004), but there was no group difference (main effect p = 0.458) or influence of SGLT2 inhibition (interaction p = 0.406). When expressed as area under the curve,  glucose was lower during the week 12 OGTT (main effect p < 0.001), but there was no group difference (main effect p = 0.797) or influence of SGLT2 inhibition (interaction p = 0.653).Fasting glucose was not different between groups (p = 0.251), was unchanged (p = 0.056) over 12 weeks, and SGLT2 inhibition did not influence the overall response . Circulating insulin concentration during the OGTT is presented in p = 0.032), and compared with week 0, insulin was lower during the week 12 OGTT (main effect p < 0.001); there was no influence of SGLT2 inhibition (interaction p = 0.382). When expressed as area under the curve,  insulin was lower during the week 12 OGTT (main effect p < 0.001), but there was no group difference (main effect p = 0.435) or influence of SGLT2 inhibition (interaction p = 0.357). HOMA-IR was not different between groups (p = 0.30), was unchanged (p = 0.561) over 12 weeks, and SGLT2 inhibition did not influence the overall response .Fasting insulin was not different between groups (p = 0.406), was unchanged (p = 0.13) over 12 weeks, and SGLT2 inhibition did not influence the overall response (interaction p = 0.388). Circulating glucagon concentration during the OGTT is presented in p = 0.023) but was unchanged over 12 weeks (main effect p < 0.582); SGLT2 inhibition did not influence this response (interaction p = 0.276). When expressed as area under the curve,  glucagon was similar between groups (main effect p = 0.344), unchanged over time (main effect p = 0.731) and the overall response was not influenced by SGLT2 inhibition (interaction p = 0.236).Fasting glucagon was not different between groups (p = 0.175), did not change over 12 weeks (p = 0.059), and SGLT2 inhibition did not influence the overall response (interaction p = 0.146). During the OGTT the ratio was higher in the placebo group (main effect p = 0.022) but was unchanged over 12 weeks (main effect p = 0.288); SGLT2 inhibition did not influence this response (interaction p = 0.369).The ratio of fasting insulin to fasting glucagon  was not different between groups (p ≥ 0.05). Total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and the ratio of total to high-density lipoprotein cholesterol were unchanged following dietary counselling. However, there were interactions with time and placebo vs. SGLT2 inhibition for high-density lipoprotein cholesterol, and the ratio of total to high-density lipoprotein cholesterol, driven by favorable modification with placebo and moderate unfavorable modification with SGLT2 inhibition.Circulating cholesterol and triglyceride concentrations are presented in Energy intake at the breakfast buffet and responses to the hunger and satiety questionnaire are presented in p > 0.05).Dietary counselling decreased sensations of hunger and satiety (questions 3 and 4) when assessed one hour after ingestion of the liquid primer meal. None of the other responses to the hunger and satiety questions at any given time were influenced by dietary counselling or SGLT2 inhibition . This magnitude of weight loss is comparable to other studies of dietary counseling focusing on modest caloric restriction ,36. SimiWhile diet-induced weight loss is often desirable, changes in body composition are usually of greater clinical relevance. In this regard, in our study, when SGLT2 inhibition was administered as an adjunct to dietary counselling, there was a greater decrease in fat-free mass . Our observation is consistent with several previous studies that also report loss of fat-free mass with SGLT2 inhibition ,38,39. NIn addition to weight loss and changes in body composition, we studied the influence of dietary counselling on glucose regulation. As expected, and consistent with other weight loss studies, glucose regulation (reflected by the lower areas under the glucose and insulin response curves during an OGTT) was improved. Unexpectedly, SGLT2 inhibition provided no additional benefit. One potential explanation for this observation pertains to glucagon secretion. SGLT2 inhibition has been suggested to promote endogenous glucose production in response to increased pancreatic secretion of glucagon ,42, thusWe hypothesized that some of the benefits associated with weight loss, such as the favorable modification of the lipid profile, would be further enhanced with SGLT2 inhibition. While some of the lipid profile parameters  were favorably modified, contrary to our hypothesis, SGLT2 inhibition provided no additional benefit, and in some instances evoked a potentially unfavorable response such as a very modest decrease in high-density lipoprotein cholesterol. This observation is not without precedent; there have been previous reports of modest decreases in high-density lipoprotein cholesterol following 8-weeks of significant carbohydrate restriction (akin to SGLT2 inhibition) before increasing at 6 and 12 months . The expstatistically significant effects were observed [An additional aim of our study was to explore the effects of SGLT2 inhibition and dietary counselling on dietary preferences and hunger and appetite prior to and following acute feeding. Our rationale was based on the influence of SGLT2 inhibition on the absorption of dietary carbohydrates. In light of the increased glucosuria, the SGLT2 inhibited state is unique in that dietary carbohydrate restriction is not required; patients receiving SGLT2 inhibitors may ingest but are unable to fully utilize carbohydrates. Essentially, the SGLT2 inhibited state is akin to a modest carbohydrate-restricted mimetic. Noteworthy, the SGLT2 inhibited state has also been discussed in the context of a ketogenic diet ,50 and oobserved . In the observed , we obseobserved . Our datOur study and design have several potential limitations that warrant additional consideration. The purpose of the study was to determine the efficacy of supplementing dietary counselling for weight loss with SGLT2 inhibition, and the hypothesis was developed from the perspective of how SGLT2 inhibition might influence physiological adaptation to weight loss. However, it is possible that our question was misplaced, and instead we should have asked how weight loss influences SGLT2 inhibition. It is feasible that the responses to a pharmaceutical prescribed to decrease macronutrient (carbohydrate) absorption may become redundant when co-administered with a prescription for caloric restriction. To fully address this question, a four-square approach would have been necessary: placebo with/without dietary counselling vs. SGLT2 inhibition with/without dietary counselling. Unfortunately, this design was beyond the scope of our resources.The total weight loss mediated by dietary counselling was not influenced by SGLT2 inhibition. In addition to decreased dietary intake (or absorption), weight loss is also mediated by increased physical activity and habitual exercise. Although the decrease in resting energy expenditure was similar between the placebo and SGLT2 inhibition treatments, it may have been useful to have assessed the other components of total daily energy expenditure: the thermic effects of feeding and physical activity. At this time, we are unaware of any evidence to indicate SGLT2 inhibition has appreciable effects on either of these other components. Further, although two previous studies have already described dapagliflozin-mediated glucosuria (up to 80 mg/day) in healthy, diabetes-free adults ,14, the n = 50) might be considered small, thus it is plausible that with greater statistical degrees of freedom, we may have identified additional interactions between SGLT2 inhibition and dietary counselling. However, with the exception of two of the lipid profile parameters . Further, even with a relatively small sample size, we were able to identify statistical interactions between SGLT2 inhibition and dietary counselling pertaining to fat-free mass and high-density lipoprotein cholesterol. Thus, we suggest that from the dependent variables quantified in the current study, with the exception of two lipid profile parameters, only two dependent variables were subject to SGLT2 inhibition and dietary counselling interaction.Finally, our total sample size (Twelve weeks of out-patient dietary counseling was associated with weight loss, fat loss, decreased resting metabolic rate, increased reliance on fat oxidation at rest, improved glucose regulation, and decreased circulating concentrations of triglyceride, and very low-density lipoprotein cholesterol; SGLT2 inhibition did not modify any of these responses. However, SGLT2 inhibition was associated with a greater decrease in fat-free mass, and a small decrease in plasma high-density lipoprotein cholesterol. In light of the health implications of the modest decreases in fat-free mass and circulating high-density lipoprotein cholesterol, we recommend careful consideration before implementing SGLT2 inhibition as an adjunct to dietary counselling for weight loss in sedentary adults with obesity.