Pyropia pseudolinearis. We prepared water soluble proteins (WSP) containing phycoerythrin, phycocyanin, allophycocyanin, and ribulose 1,5-bisphosphate carboxylase/oxygenase. In vitro analysis showed that the thermolysin hydrolysate of the WSP had high ACE inhibitory activity compared to that of WSP. We then identified 42 peptides in the hydrolysate by high-performance liquid chromatography and mass spectrometry. Among 42 peptides, 23 peptides were found in chloroplast proteins. We then synthesized the uncharacterized peptides ARY, YLR, and LRM and measured the ACE inhibitory activity. LRM showed a low IC50 value (0.15 μmol) compared to ARY and YLR (1.3 and 5.8 μmol). In silico analysis revealed that the LRM sequence was conserved in cpcA from Bangiales and Florideophyceae, indicating that the novel ACE inhibitory peptide LRM was highly conserved in red algae.More than 7000 red algae species have been classified. Although most of them are underused, they are a protein-rich marine resource. The hydrolysates of red algal proteins are good candidates for the inhibition of the angiotensin-I-converting enzyme (ACE). The ACE is one of the key factors for cardiovascular disease, and the inhibition of ACE activity is related to the prevention of high blood pressure. To better understand the relationship between the hydrolysates of red algal proteins and the inhibition of ACE activity, we attempted to identify novel ACE inhibitory peptides from  Shokuiku” (Food and Nutrition Education). The guideline recommends eating well-balanced meals combined with vegetables, fruits, milk products, beans, and fish. In addition, information of the nutritional and bioactive functions of foods based on scientific evidence is required. The importance of such food-derived components has been recognized not only in Japan but also worldwide. One of the examples of functional components is bioactive peptide. Until now, there have been various reports for the functions of bioactive peptides, such as decreases in blood pressure, glucose, cholesterol, and neutral lipids and increases in immune response [Japan’s Ministry of Agriculture, Forestry and Fisheries promotes “response . Among tresponse ,3.Since high blood pressure (hypertension) has no obvious indicating symptoms, it is called a “Silent Killer”. Hypertension is a major risk for cardiovascular diseases and causes serious health problems regarding heart failure, atrial fibrillation, chronic kidney disease, heart valve diseases, aortic syndromes, and dementia ,5. The rNeopyropia sp.) and agar (Gelidium sp. and Gracilaria sp.) are used as industrial foodstuffs, and some species such as Grateloupia asiatica, Gloiopeltis furcata, Chondria crassicaulis, and Nemalion vermiculare are consumed as local food. Therefore, other red algae are also expected to be used effectively as food material and a source of bioactive components. The main proteins in red algae are phycobiliproteins that form phycobilisome in a chloroplast and play a light-harvesting role in photosynthesis [On the other hand, Japanese people have been using seaweed as a food source and industrial material since ancient times. Recently, the health functions of seaweed’s nutritional components have been reported ,18,19,20ynthesis . Phycobiynthesis ,27. APC ynthesis . Regardiynthesis ,34,35,36ynthesis , DPP-IV ynthesis , antioxiynthesis ,39,40,41ynthesis , and antynthesis .50 values of ACE inhibitory peptides prepared by the thermolysin hydrolysis of phycobiliproteins and Rubisco [50 value from in silico analysis. Namely, the in silico approach has the potential to predict the biological activity of peptides derived from red algal protein. However, there were slight gaps in the correlation due to the lack of information on ACE inhibitory peptides from red algae. This indicates that the hydrolysates of red algal phycobiliproteins and Rubisco still contain uncovered ACE inhibitory peptides. Therefore, we used Pyropia pseudolinearis, which is called “Uppurui Nori” in Japan and is eaten in certain local areas, as a sample in this study. We first extracted phycobiliproteins and Rubisco as water-soluble proteins (WSP) and prepared their hydrolysates. We then identified the ACE inhibitory peptides in the hydrolysate and found three novel peptides. Further, we determined IC50 values using synthetic peptides and performed docking simulation between the ACE and three peptides. We also determined the nucleotide sequence of the chloroplast proteins of PE, PC, and APC to reveal the source of the peptides. This study is useful for the evaluation of proteins from red algae as a source of ACE inhibitory peptides.Since red algal phycobiliproteins are commonly composed of many hydrophobic and aromatic amino acid residues (>50%), thermolysin is suitable for the preparation of ACE inhibitory peptides ,35,44,45 Rubisco ,47. The P. pseudolinearis, we first prepared WSP and its thermolysin hydrolysate. The composition of WSP was confirmed by SDS-PAGE and spectral analysis. The components of WSP were mainly found to be 20,000 and 55,000 MW , were not found in the other hydrolysates. The peaks of hydrolysates from dulse and G. asiatica were detected with up to 15% acetonitrile, and the peak from M. japonica was detected with up to 18% acetonitrile. The difference might be due to minor proteins detected on SDS-PAGE. Therefore, we attempted to determine the peptide structures in all fractions.To determine the ACE inhibitory peptides, fractionation was performed by RP-HPLC a. Thirtyjaponica ,36, and asiatica , which sPyropia pulchra was determined [P. pseudolinearis [P. pulchra chloroplast genome and performed in silico thermolysin digestion in it  were composed of α- and β-chains, and the gene coding regions of these chains were continually coded with a short AT-rich spacer. The sequences were deposited in the DNA Data Bank of Japan (DDBJ) as LC599086-LC599088. The phycobiliprotein sequences of P. pseudolinearis were compared to those of other red algae. High identities were obtained in the Bangiophyceae species (>98.8%) and in Florideophyceae species (>88.1%) , which were thought to be a related species  and a distant species (Rhodymeniophycidae) [P. palmata in Japan, indicating that Florideophyceae identities were constant. The data in P. pulchra, and the differences in phycobiliproteins between P. pseudolinearis and P. pulchra were cpcB and apcA. The differences were not involved in the ACE inhibitory peptide sequences, indicating that ACE inhibitory peptides from phycobiliproteins were also conserved in P. pseudolinearis.We employed (>88.1%) . We emplhycidae) . The low50 values, respectively  compared to ARY and YLR. LRM in cpcA and YLR in cpcA, apcA, and apcB were conserved in Bangiales and Florideophyceae. From these results, we identified three novel ACE inhibitory peptides , which were highly conserved in red algae. Several ACE inhibitory peptides derived from nori (AKYSY) and sardines (VY) have been used as functional ingredients in Japan (FOSHU). In the present study, we found that LRM possessed relatively high ACE inhibitory activity. In the future, we would like to investigate the digestibility and absorption of ACE inhibitory peptides derived from red algae.Among the predicted 42 peptides, we focused on the peptides from phycobiliproteins since phycobiliproteins were the major components of the WSP. We previously determined several ACE inhibitory peptides from Japanese dulse and presumed that the peptides LDY, LRY, VYRT, AGGEY, VDHY, LKNPG, and YRD were from phycobiliproteins . To findectively . LRM, wh50 value of Captopril was approximately 17–21 nM [We identified three novel ACE inhibitory peptides . Among them, LRM showed strong ACE inhibitory activity compared to the other two peptides. To know the inhibitory mechanism, the docking simulation between ACE and three peptides was performed by using the HPEPDOCK server. The docking energy scores with ACE were as follows: ARY: –155.685 kcal/mol; YLR: –166.290 kcal/mol; LRM: –140.683 kcal/mol. The results contradicted the in vitro synthetic peptide analysis. The binding of the three peptides in ACE was as follows: ARY was coordinated in the S1′ and S2′ pockets of the ACE, LRM was in the S2 and S1 pockets, and YLR was in the S3 and S2 pockets . Althoug17–21 nM ,54). TheP. pseudolinearis, was collected from the coast of Hakodate, Japan. The sample was stored at –30 °C until use. ACE from rabbit lung was purchased from Sigma-Aldrich Co. . Millex-GV (pore size: 0.22 µm) and Millex-LG (pore size: 0.20 µm) were purchased from MERCK MILLIPORE Ltd. . Synthetic peptides (purity: ≥95%) were purchased from Medical Biological Laboratories Co. . Hyppuryl-L-histidyl-L-leucine (Hip-His-Leu), thermolysin (EC 3.4.24.27) from Bacillus thermoproteolyticus, trifluoroacetic acid (TFA), and all other regents were purchased from Fujifilm Wako Pure Chemical .Uppurui Nori, P. pseudolinearis was as per our previous studies [P. pseudolinearis samples were lyophilized and ground into a fine powder by Wonder Blender WB-1 . The powder was dissolved in 20 v/w distilled water, and protein extraction was performed at 4 °C for 12 h. The WSP was recovered by centrifugation at 4 °C, 15,000× g, for 10 min, and the supernatant was then used as the WSP. The protein hydrolysate of the WSP was prepared by the digestion of protein extract with 1.0 wt. % thermolysin at 70 °C for 3 h. The reaction was stopped by boiling for 10 min. The visible ray absorption spectrum of the WSP was analyzed by a spectrophotometer . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed by Laemmli’s method with a 0.1% SDS-13.75% polyacrylamide slab gel [Preparation of the WSP and the hydrolysate from  studies ,47. Nameslab gel . The gelThe hydrolysate of the WSP was separated by RP-HPLC. The hydrolysate was dissolved (20 mg/mL) in ultra-pure water containing 0.1% TFA and applied to sequential filtration by Millex-GV and Millex-LG. An aliquot of the sample (100 μL) was applied to a Mightysil RP-18GP column (4.6 × 150 mm)  and eluted with a linear gradient of acetonitrile from 1–25% containing 0.1% TFA at flow rate of 1.0 mL/min. Absorbance of the eluent was monitored at 228 nm. The eluted peptide fractions were collected and labeled 1–36. The fractions were used for the ACE inhibitory assay. The amino acid sequences of peptides were determined by Matrix Assisted Laser Desorption / Ionization Time of Flight Tandem Mass Spectrometry (MALDI-TOF/MS/MS) using a 4700 Proteomics Analyzer with DeNovo Explorer software version 3.6 . α-Cyano-4-hydroxycinnamic acid was used as matrix.t test. In this study, the IC50 was defined as an absolute quantity of the peptide to inhibit 50% of 1.0 U ACE.The assay was performed using the same method as described previously . The ACEP. pseudolinearis according to the CTAB method with some modifications [P. pseudolinearis phycobiliproteins were detected using BLAST . The sequences of phycobiliproteins were deposited as DDBJ Accession No. LC599086-LC599088.Chloroplast DNA was extracted from the thalli of ications ,57,58. Thttp://www.uwm.edu.pl/biochemia/index.php/pl/biopep) on 11 November 2020. From the database, 1019 ACE inhibitory peptides were extracted. The peptide sequences from Uppurui Nori were manually annotated. The thermolysin-digestion products of chloroplast proteins were predicted using PeptideCutter (https://web.expasy.org/peptide_cutter/). Docking simulation of the ACE inhibitory peptide with the ACE was performed using the HPEPDOCK server [The information on the reported ACE inhibitory peptide was obtained from the BIOPEP-UWM database  compared with ARY and YLR (1.3 and 5.8 μM). Docking simulation analyses showed that three peptides were coordinated in the active site of the ACE. However, these peptides do not bind to the zinc ion, which may be the reason for their relatively low ACE inhibitory activity. Among these three peptides, LRM was conserved in the primary structure of cpcA from Bangiales and Florideophyceae, indicating that the novel ACE inhibitory peptide LRM can be obtained from many red algae. Therefore, red algae have the potential not only as a protein source but also as an ingredient for supplements and functional foods for human. In a following study, we will seek to investigate how effective any practical applications of the WSP hydrolysate are.In this study, we prepared a water-soluble protein (WSP) hydrolysate from the red alga


Heart failure is a multifactorial disease that affects an estimated 38 million people worldwide. Current pharmacotherapy of heart failure with reduced ejection fraction (HFrEF) includes combination therapy with angiotensin-converting enzyme inhibitors (ACEi) and β-adrenergic receptor blockers (β-AR blockers), a therapy also used as treatment for non-cardiac conditions. Our knowledge of the molecular changes accompanying treatment with ACEi and β-AR blockers is limited. Here, we applied proteomics and phosphoproteomics approaches to profile the global changes in protein abundance and phosphorylation state in cardiac left ventricles consequent to combination therapy of β-AR blocker and ACE inhibitor in HFrEF and control hearts. The phosphorylation changes induced by treatment were profoundly different for failing than for non-failing hearts. HFrEF was characterized by profound downregulation of mitochondrial proteins coupled with derangement of β-adrenergic and pyruvate dehydrogenase signaling. Upon treatment, phosphorylation changes consequent to HFrEF were reversed. In control hearts, treatment mainly led to downregulation of canonical PKA signaling. The observation of divergent signaling outcomes depending on disease state underscores the importance of evaluating drug effects within the context of the specific conditions present in the recipient heart. The incidence of HF continues to escalate as risk factors such as diabetes, obesity and ageing3 remain prevalent. HF can be classified based on whether or not ejection faction (EF) is preserved8. The present study focuses on the case of HF with reduced ejection fraction (HFrEF), a condition associated not only with a structural and mechanical deficit but also with an increased likelihood for the occurrence of life-threatening arrhythmias9.Heart failure (HF) is a multifactorial disease with a complex etiology and prognosis that affects an estimated 38 million people worldwide12. Yet, a fundamental knowledge gap remains regarding the global molecular profile of the failing heart, captured from samples treated under uniform conditions. Different studies have characterized transcriptomic changes in the process of heart failure14, but the conversion of transcriptomic data to protein function is challenging, given the poor correlation that exists between the transcriptome and the proteome of the same organ16. Proteomics studies such as those reported by Lau17 and Kuzmanov19 and colleagues have contributed to our understanding of the global changes in protein expression in animal models presenting cardiac remodeling and dysfunction. However, we still have limited knowledge of the protein- and signaling regulation characterizing a chronic state of heart failure, as well as the molecular changes that result, at the proteomics and phosphoproteomics level, following medical treatment. Current pharmacotherapy of HFrEF often includes a combination of angiotensin-converting enzyme inhibitors (ACEi) and β-adrenergic receptor blockers (β-AR blockers)21. The functional consequences of these drugs are well characterized, and some of their therapeutic effects are known to be extra-cardiac. Importantly, these drugs are also used for conditions in the non-failing heart  or that primarily result from dysfunction in other systems . Yet, though the target receptors are abundantly present in the adult cardiac myocytes, the nature and the extent of changes caused in the molecular landscape of the heart as a result of chronic exposure to these drugs, are poorly characterized.Despite years of research, the molecular underpinnings of HFrEF remain unclear, due in part to the multifactorial nature of the condition. As the heart becomes unable to maintain normal output, a global remodeling of the contractile, metabolic and electrical functions takes place. A number of studies have addressed individual aspects of the phenotype, and in doing so, have advanced the fieldIn this study, we have analyzed the proteome and the phosphoproteome of failing hearts. As an experimental model, HFrEF was induced in murine hearts by partial banding of the transverse aorta . Data were also obtained from animals after two weeks of treatment with enalapril and metoprolol . Herein, we provide a comprehensive characterization of the molecular profile of the failing hearts with and without treatment, and the first characterization of the consequences that these drugs have on non-failing hearts.A detailed description of the experimental protocol can be found in the online supplement. All experiments conformed to the EU directive (2010/63/EU) for animal research and were carried out under a license approved by the Animal Experiments Inspectorate. The study was carried out in compliance with the ARRIVE guidelines.25. Efficacy of the treatment was evaluated in pilot experiments by reduced activity of plasma angiotensin converting enzyme and decreased heart rate in mice receiving treatment (Tr) compared to those receiving saline (Vh). Two weeks after onset of treatment , heart function was re-evaluated by echocardiography and the animals were euthanized. After sacrifice, the cardiac samples were deidentified and experiments and analysis were performed in a blinded-to treatment manner. A detailed description of the methods can be found in the online supplemental materials.For induction of heart failure, C57BL6 male mice were anesthetized, their hearts exposed through lateral thoracotomy and a tight and permanent ligature applied to partially constrict the transverse aorta (TAC). Animals were allowed to recover under observation, and their heart function was examined by echocardiography at the end of eight weeks post-surgery. Control animals (SHAM) underwent the same surgical procedure, though only a loose ligature around the aorta was placed. Both SHAM and TAC groups were then followed for an additional two weeks after implantation of osmotic pumps to deliver either vehicle  or treatment (Tr), the latter a combination of enalapril (ACE inhibitor-administered at the dose of 5 mg/kg/day) and metoprolol succinate (specific and nonvasodilating ß-adrenergic receptor blocker-administered at the dose of 30 mg/kg/day). The chronic administration of the medication have been chosen based on previous animal studies26. For phosphoproteomic analyses, peptides were enriched by titanium dioxide chromatography and analyzed in single-run format. For proteome analyses, peptide samples were pre-fractionated into 10 fractions prior to measurement28. Our experimental strategy enabled us to cover 6,004 cardiac proteins  of our proteomic data classified the heart samples according to their disease status . Our studies sought to obtain a comprehensive perspective of the mechanisms underlying heart failure and the molecular pathways targeted by current treatment strategies. Fourteen and seventeen animals were subjected to the SHAM and the TAC procedures, respectively. A total of twelve SHAM and ten TAC animals completed the experimental protocol and were included in the final proteomic and phosphoproteomic evaluation Fig. A. Echocans Table  and 14,9ns Table . The numns Table A-B. As ens Table C29–31. Ptus Fig. . The extVh mice were compared to those obtained from SHAMVh mice. Quantitative proteomics analysis was based on label-free quantification (LFQ) intensities, which enabled us to evaluate relative abundances of 3,915 proteins for these animals , natriuretic peptide A (Nppa), and angiotensin converting enzyme (Ace)hy” Fig. B. The lahy” Fig. .Vh animals included several proteins involved in cell metabolism, such as fructose biphosphate aldolase B (Aldob), cytochrome b-c1 complex subunit 9 (Uqcr10), and acyl-coenzyme A thioesterase 1 (Acot1). Consistently, functional enrichment analysis revealed that downregulated proteins in the TACVh model clustered around mitochondria and mitochondrial metabolic terms such as “citric acid cycle and respiratory electron transport.” , the inward rectifier K+ channel Kir2.1 (Kcnj11), the gap junction protein Connexin 43 (Gja1), the L-type Ca2+ channel  and the sodium/calcium exchanger NCX (Slc8a1). These data add to a body of evidence indicating electrical remodeling in HFrEF hearts12.Proteins downregulated in the failing hearts of TACt.” Fig. B. In fact.” Fig. C. Taken 39. Such signaling changes can better be evaluated from phosphoproteomics data and, accordingly, are discussed in more details below.The functional enrichment also point to a downregulation of proteins involved in adrenergic signaling Fig. B. This rVh and SHAMVh animals. We obtained quantitative information from 6547 phosphorylation events across heart samples from TACVh and SHAMVh animals , Irs2S556, and Camkk2S495. Proteins containing upregulated phosphorylation sites were functionally clustered in terms associated with metabolic processes  or electrical conduction . When grouped together by functional enrichment analysis, downregulated phosphorylation sites related to cardiac conduction, cell metabolism and adrenergic regulation , reflecting the increased phosphorylation of Hdac2S394, a site that has been associated with cardiac hypertrophy41 and increased phosphorylation of AceS1305, which regulates retention of the enzyme in the plasma membrane42, among other targets , ryanodine receptor sites Ryr2S1402, S2803, S2807 (which modulate RyR2 channel open probability44), and ion channel sites Kcnq1S481 and Scn5aS516, which affect gating of their respective channels46. Of note, in the case of PKA as well as Camk2d, a change in the phosphorylation state of the kinases themselves was noted  as well as reduced phosphorylation of the activation sites of phosphodiesterase 3A, Pde3aS310,S435, which indirectly opposes beta-adrenergic signaling pathways47. Our analysis also identified reduced activity of kinases that affect cell metabolism and in particular Pyruvate Dehydrogenase Kinases . Specifically, we found reduction of substrates directly regulated by Pdhk4 such as Pdha1 at its regulatory sites S232, S293, S300, as well as of Sirt1S14 and Foxo1S467. Taken together, our observations suggest a signaling dysregulation of kinases implicated in β-adrenergic signaling response, as well as in mitochondrial metabolic remodeling.The functional enrichment analysis highlights the biological functions of the proteins with altered phosphorylation sites. However, such analysis does not outline which kinases orchestrate the response. To address this, we used an integrative computational approach termed Kinase Substrate Enrichment Analysists Table . Using tTr vs TACVh). Two weeks of treatment did not lead to significant changes in the cardiac proteome  and cardiac contraction . Therefore, we sought to determine the effects of enalapril/metoprolol on the proteome and phosphoproteome of HFrEF hearts  or TACTr vs TACVh (top). Notice the multiple switches between red and blue across the dataset, indicating that treatment led to a reversal of the phosphorylation state of specific sites. Specific phosphosites that were downregulated as consequence of heart failure and then upregulated in response to treatment include Pdha1S232, S295, Foxo1S467, and Sirt1S14, mainly involved in metabolic remodeling. For receptor regulation we observed reduced phosphorylation of alpha-1-adrenergic receptor (Adra1bS464) and the muscarinic acetylcholine receptor M2 (Chrm2S232) in heart failure, which were reversed by treatment. Moreover, the enalapril-metoprolol combination brought about a switch in the representation of multiple RyR2 phosphorylation sites, some with known functional relevance , of Scn5aS516 and of Gja1S297, S372,S373. Finally, notice the treatment-induced shift in phosphorylation sites on key mediators of intracellular signaling pathways including Camk2dS319,S330,S337, PrkcaS638,S657, Map2k4S255 and Map3k5S965. The switch in phosphorylation state was not limited to the examples illustrated in the heatmap. We evaluated the change in phosphorylation state across all measured phosphorylated peptides and observed that they were inversely correlated with a Pearson correlation coefficient of − 0.6  or placebo (SHAMVh). As in the case of failing hearts, treatment did not significantly change left ventricular protein expression (Table S1305 (regulated by CK2) and phosphorylation on the beta-adrenergic receptor Adrb1S401,S417 (involved in the recruitment of the B-arrestin 2)50. Quantitative analysis of 6,353 phosphorylated peptides identified 168 phosphorylation events that were significantly downregulated, and 288 phosphorylation events that were significantly upregulated, in the treated animals , molecules involved in intracellular calcium homeostasis , and key regulators of intracellular signaling . Reduced phosphorylation levels at these sites reflect a general decrease in phosphorylation of downstream targets of the β-adrenergic signaling pathway, as expected for exposure to metoprolol. Consistent with the latter, functional enrichment analysis of proteins with downregulated phosphorylation sites highlighted terms related to β-adrenergic signaling  and ACE inhibitor  in HFrEF hearts as well as in control hearts.44 and reflect the metabolic flexibility and stress responses of cardiac cells as they remodel consequent to the excessive afterload . Our results also confirm reduction in proteins relevant to electromechanical coupling in HFrEF hearts, such as the gap junction protein Connexin43 (Gja1), which can predispose to arrhythmic events55.Our results showed that the predominant effect on the proteome of the left ventricle of TAC-induced HFrEF is a marked metabolic remodeling, intertwined with changes in proteins relevant to the electrical and mechanical functions of the heart. The metabolic changes parallel similar findings previously reported in heart failure in humans56. Herein, analysis of phosphorylated substrates pointed to reduced activities of protein kinase A (Prkaca) and the Camk2 family. Of the latter, Camk2d is the predominant cardiac isoform and we observed reduced phosphorylation of this kinase at residues 319, 330 and 337. Additionally, also targets directly regulated by Camk2d had reduced phosphorylation levels57, such as PlnT17and Scn5aS516. The reported findings overall suggest that reduced Camk2d signaling cascades, and not exclusively upregulation, may contribute to electrical instability and heart failure.The primary findings in our study come from analysis of the signaling changes evaluated by phosphoproteomic analyses. Previous studies have identified several members of the AGC and Camk superfamilies as maladaptive in heart failureS16 and Tnni3S23,S24 (Troponin I) in HFrEF animals61. PKA is known as the main kinase downstream of β-adrenergic receptor activation, and the downregulation of PKA activity suggested by the phosphoproteomic data support the notion that the cardiac dysfunction in the TAC model is characterized by an uncoupling between β-adrenergic responses and calcium/myofilaments mechanisms.Our observation of reduced activity of protein kinase A was, among others, linked to reduced phosphorylation at PlnS16), consistent with known cardiac effects of the β-AR blocker. Secondary to the reduction in PKA signaling we observed reductions in Camk2 and Akt2 activities. The reduction in Camk2d activity and downstream targets (e.g. PlnT17) are in line with previous findings from our group26 and supports the notion that β-blockade can exert its effects not only by reducing PKA signaling pathways but also by downregulating Camk2d signaling. The treatment also led to a predicted decrease in Akt2 activity; Akt2 is an important regulatory isoform involved in insulin signaling. A cross-talk between the beta-adrenergic and insulin signaling pathways in hearts has been reported63, and here our findings suggest that β-blockade can directly attenuate insulin signaling, affecting cardiac metabolism and glucose uptake.An important new finding resulting from our work relates to the effect that treatment with a β-AR blocker and an ACE inhibitor has on the phosphorylation state of proteins depending on whether they are captured from a failing heart or from a control. Phosphoproteomic analysis of treated SHAM hearts revealed a clear downregulation of canonical targets regulated by PKA signaling 58, which is a direct target of the pyruvate dehydrogenase kinase family and implicated in the regulation of the glucose oxidation66. This finding may suggest a potential restored metabolic flux. We observed that a number of phosphorylation changes consequent to TAC were reversed by combination therapy. This reversal was not confined to specific categories  and the change caused by the same treatment in SHAM animals (SHAMVh vs SHAMTr). The combination treatment examined in this study is commonly used both in patients exhibiting HFrEF and also in those with functionally competent hearts. Our paradoxical observation highlights the importance of evaluating possible drug effects within the context of the specific conditions present in the recipient heart.We made the observation that there is partial correlation between the phosphorylation change induced by HFrEF and the change induced by treatment in SHAM animals. This effect may be attributable, among other things, to the convergence of beta-AR desensitization known to occur in the failing hearts, and the loss of beta-AR signaling expected from the use of a beta-AR blocker (metoprolol in our case). Furthermore, we found a lack of correlation between the change in the phosphoproteome caused by the treatment of failing hearts  to predict the kinases regulated in the cardiac phenotype and by the treatment. On one hand, the computational analysis of the kinases based on the phosphoproteomics data led to the identification of new and well-known dysregulated kinases, but on the other hand it exclusively suggested and not functionally defined the kinase activity. Therefore, additional functional follow-up are required to integrate our results.Given the clear limitations of our experimental model along with inference of the kinase activity, extrapolation from our results to the clinically relevant environment can be only speculative and needs to be done with caution.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.Supplementary Information 8.Supplementary Information 9.Supplementary Information 10.Supplementary Information 11.


Tumor necrosis factor-alpha (TNF-α) signaling regulates phosphorylation of L-plastin, which is involved in forming the nascent sealing zone, a precursor zone for the matured sealing ring. This study aimed to illustrate the molecular mechanisms of L-plastin phosphorylation and the subsequent formation of the nascent sealing zone in osteoclasts treated with TNF-α. Here, we report that anti-TNF-receptor 1, inhibitors of signaling proteins , and siRNA of TRAF-6 attenuated the phosphorylation of LPL and filamentous actin content significantly in the presence of TNF-α. An inhibitor of integrin αvβ3, PKC, or PKA did not inhibit TNF-α-induced L-plastin phosphorylation. Inhibitors of Src and PI3-K and not Rho or Rho-kinase reduced tyrosine phosphorylation of TRAF-6, suggesting that Src and PI3-K regulate TRAF-6 phosphorylation, and Rho and Rho-kinase are downstream of TRAF-6 regulation. Osteoclasts expressing constitutively active or kinase-defective Src proteins were used to determine the role of Src on L-plastin phosphorylation; similarly, the effect of Rho was confirmed by transducing TAT-fused constitutively active (V14) or dominant-negative (N19) Rho proteins into osteoclasts. Pull-down analysis with glutathione S-transferase-fused SH2 and SH3 domains of Src and PI3-K demonstrated coprecipitation of L-plastin and TRAF-6 with the SH3 and SH2 domains of the PI3-K and Src proteins. However, the actual order of the interaction of proteins requires further elucidation; a comprehensive screening should corroborate the initial findings of protein interactions via the SH2/SH3 domains. Ultimately, inhibition of the interaction of proteins with SH2/SH3 could reduce L-plastin phosphorylation and affect NSZ formation and bone resorption in conditions that display osteoclast activation and bone loss. Osteoclasts are bone-resorbing cells, and bone resorption is their sole function. Osteoclasts adhere to the bone matrix to be resorbed. Adhesion of osteoclasts to the bone matrix induces the formation of a sealing zone or sealing ring, which has been deemed an indicator of osteoclast activation for bone resorption. Several studies have shown the involvement of different pathways in the organization of the sealing ring or zone in osteoclasts. However, the actual target molecule(s) that contribute to actin organization requires further elucidation. Many actin-binding proteins (ABPs) have been shown to stabilize and rearrange actin cytoskeletal organization in response to stimuli or during cell migration and adhesion ,3,4,5,6.LPL, also known as fimbrin  or cytos2+ binding sites flanked by EF-hand motifs at the amino-terminal  end; two actin-binding domains (ABD1 and 2), each encompassing two tandem calponin homology (CH) domains at the carboxyl-terminal  end; and the binding of two actin filaments to the spatially close ABDs of plastins, which influences bundling of the actin filaments into tight bundles [Plastins comprise the following: Ca bundles ,12. L-pl bundles ,14. So f bundles ,14,15.Earlier studies, including our own, elucidated the critical role of integrin αvβ3 signaling in osteoclast sealing-zone or -ring formation and function ,23,24,25Most recently, using the TAT-mediated transduction method, we confirmed the role of LPL in NSZ formation. Transduction of TAT-fused full-length LPL peptide significantly increases the number of NSZs and sealing rings. However, transduction of amino-terminal LPL peptides consisting of the serine-5 and -7 amino acids (AAs) reduces the formation of NSZs and sealing rings . FurtherOur next inquiry regarded the possible signaling mechanism that mediates the phosphorylation of LPL. A low concentration of TNF-α was shown to activate osteoclasts and promote actin ring formation in osteoclasts formed in vitro and also extracted from newborn rats . The actSeveral studies have shown the involvement of PKA and PKC in the phosphorylation of LPL ,38,39. LThis study aimed to obtain insight into the molecular mechanisms underlying the phosphorylation of LPL. We used siRNA of TRAF-6 and various inhibitors (Src (PP2), PI3-K (Wortmannin), protein kinase C (PKC) (Go6983 or Staurosporine), protein kinase A (PKA) (H89), and Rho kinase (Y27632), and αv (RGDS peptide)) to determine possible signaling molecules involved in LPL phosphorylation in osteoclasts treated with TNF-α. We found that the TNF-α/TNFR1 signaling pathway involves an Src–PI3K–TRAF-6–Rho/Rho-kinase axis regulating LPL phosphorylation and NSZ formation.C57BL/6 mice (six- to eight-week-old mice) were used for osteoclast preparation. These mice were either bought from Harlan Laboratories or generated in the animal facility of the University of Maryland Dental School. The author followed the guidelines and approval of the Institutional Animal Care and Use Committee in the breeding and maintenance of mice. Marrow cells of long bone were used for the experiments.Antibodies to GAPDH and TNFR1 (TNF-α receptor 1) were purchased from R&D Systems . Secondary antibodies (Cy2- and Cy3-conjugated) were purchased from Jackson Immunoresearch . GAPDH and secondary antibodies for immunoblotting were obtained from GE Healthcare . The antibody to phosphoserine (p-Serine) was bought from Abcam Company . The phospho-SRC pTyr418 antibody was purchased from Thermo Fisher Scientific Company . Mounting solutions for mounting coverslips were bought from Thomas Scientific  or Vector Labs . The exoenzyme C3 transferase enzyme and Rho-GTP binding protein-coupled to GST beads were bought from Cytoskeleton, Inc. . TRAF-6 siRNA and antibodies to L-plastin, Src, TRAF-6, and GAPDH were purchased from Santa Cruz Biotechnology, Inc. . Polyacrylamide gel electrophoresis and protein estimation reagents were bought from Bio-Rad . Rhodamine-phalloidin, Protein A-Sepharose, GST-Sepharose 4B beads, inhibitors to PKC (Gö 6983 and Staurosporine), PKA (H-89), and integrin αv (cyclo RGD peptide aka cRGDfV), and all other chemicals were purchased from Sigma .Osteoclasts were generated in vitro using mouse long bone marrow cells as described previously ,24. AfteAfter removing bone marrow cells, the long mouse bones were washed extensively with PBS and placed in ethanol until use, which kept them free of cells inside and muscle tissue outside. Next, long bones were air-dried in the cell culture biological hood and homogenized with a mini blender. Finally, bone particles were sieved, and bone particles between 60 and 80 µm in size were used for experiments.Before the addition of TNF-α, osteoclasts generated with RANKL and m-CSF-1 were washed extensively with a serum-free alpha-minimum essential medium. Osteoclast cultures were subjected to one of the following inhibitors for 60 min: αv inhibitor , Rho kinase inhibitor , PKA inhibitor , or PKC inhibitor (a pan PKC inhibitor Go6983 (20 nM) or Staurosporine (20 nM)). In addition, some cultures were treated with siRNA of TRAF-6 as described below and previously , or neutTransfection of TRAF-6 siRNA and control or scrambled RNAi  was conducted using the streptolysin O permeabilization method as described previously . After iKinase-defective  and constitutively active Src  were generated essentially based on the pAdEasy-1 system. The virus was propagated as described previously . AdenoviVal14) and dominant-negative (RhoAsn19) (indicated as V14 and N19) were cloned in-frame into a pTAT–HA vector [Constitutively active  membrane . GST-alo5 cells) were cultured on dentine slices and incubated with TNF-α for 3–4 h and 8 h. Immunostaining was done with TRAF-6 and Src antibodies in osteoclasts plated for 3–4 h and 8 h; and TRAF-6 and TNF-R1 antibodies in osteoclasts cultured for 3–4 h. In addition, some cultures were stained with rhodamine-phalloidin as described previously [Osteoclasts (10eviously  to detereviously .We measured the F-actin content in osteoclasts incubated with bone particles (100 µg/mL media) for 3–4 h in the presence of TNF-α (20 ng/mL) and treated as indicated in the figure and above. For each treatment, three to four wells of 24-well culture dishes were used. The cells were fixed and stained with rhodamine-phalloidin. In control experiments, a 10-fold excess of unlabeled phalloidin was used to determine the nonspecific binding. The nonspecific binding was subtracted from the total binding to obtain the specific binding. The cells were washed quickly several times with PBS and extracted with absolute methanol. As previously described, the fluorescence was measured using a spectrofluorometer  .Mature osteoclasts were plated on dentine matrix and allowed to adhere for 2–3 h. Based on previous studies , 3–5 µg/t-test or two-way ANOVA  was used to determine the statistical significance. A p < 0.05 was considered significant.All values were mean ± SEM of three or more experiments performed at different times with different osteoclast preparations. A Student’s LPL phosphorylation and the formation of actin aggregates known as NSZs were observed within 3–4 h of treatment of TNF-α and bone particles . TherefoTRAFs have been implicated in the signaling processes mediated by TNF receptor family members, including TNFR1, TNFR2, CD30, and CD40 . In addiNext, to determine the requirement of TRAF-6 in LPL phosphorylation, we used the RNA interference (RNAi) strategy to reduce TRAF-6 protein levels. We used siRNA-mediated silencing of TRAF-6 at doses of 50 and 100 nM for 36–48 h at 37 °C. A dose-dependent decrease in the level of TRAF-6 was observed at 50 and 100 nM siRNA. The reduction was significant (>80%) at a dose of 100 nM B, lane 1Subsequently, equal amounts of protein lysates were used for immunoprecipitation with an LPL antibody and immunoblotted with a p-serine antibody D in the Confocal microscopy analyses of osteoclasts plated on dentine slices for 3–4 h E demonstWe then assessed the possible TNF-α- and TNFR1-mediated signaling axis using various inhibitors targeting Src, PI3-K, PKC, PKA, Rho, and Rho kinase . OsteoclTo further elucidate the role of Src in LPL phosphorylation, osteoclasts were infected with adenovirus containing constitutively active (CA) and kinase-defective (KD) Src constructs. Immunoblotting analysis with an Src antibody exhibited more CA and KD-Src proteins  correspoTo confirm the role of Rho as an upstream regulator of LPL phosphorylation, osteoclasts were transduced with constitutively active (V14) and dominant-negative (N19) Rho proteins as described previously ,47,48. MThen, we measured the GTP-Rho levels in untransduced (-) and Rho-protein-transduced (V14 and N19) cells , lane 3 Immunoblotting of LPL immunoprecipitates with a p-serine antibody exhibited more LPL phosphorylation in osteoclasts transduced with V14 Rho , lane 3 TRAF-6 and Src knockout mice develop osteopetrosis due to impaired bone resorption by osteoclasts ,36. In aHaving found that Src, PI3-K, TRAF-6, and Rho kinase regulate the phosphorylation of LPL , we thenSubsequently, we confirmed that TRAF-6 did not regulate Src phosphorylation on Y418 using the lysates made from cells treated with siRNA and ScRNAi to TRAF-6. Neither siRNA nor ScRNAi affected the phosphorylation of Src on Y418. Although an increase in Src Y418 phosphorylation was observed in cells transfected with CA-Src E, lane 5Collectively, our data provided evidence that TRAF-6 does not mediate Src phosphorylation. An increase in the phosphorylation of TRAF-6 in osteoclasts expressing CA-Src implied that Src is upstream of TRAF-6 in the signaling pathway, which regulates LPL phosphorylation. Since V14Rho did not affect the phosphorylation of TRAF-6, we suggest it may be a downstream regulator of Src and TRAF-6. Based on these studies , we recoThen we proceeded to determine the binding specificity of TRAF-6 with Src and PI3-K using GST-fused SH2 and SH3 domains of Src, Lck, and PI3-K proteins coupled to Sepharose beads as shown previously . LysatesOsteoclasts cultured on dentine slices in the presence of TNF-α for 3–4 h demonstrated the formation of NSZs. Here, we examined the effect of inhibitors of Src, Rho-kinase (ROK), PI3-K, and SiRNA of TRAF-6, on actin distribution . ConsistAs shown previously , anti-TNL-plastin (LPL) phosphorylation on serine-5 and -7 residues by TNF-alpha signaling regulates the actin-bundling process by LPL and, hence, the assembly of NSZs in mice osteoclasts. We confirmed the role of phosphorylation of Ser-5 and Ser-7 in actin-bundling by using mutated (A5A7) full-length and low-molecular-weight LPL peptides ,30,56. FTRAFs  have been implicated in regulating signals from various TNF-receptor family members, leading to NF-kB activation ,65,66,67TRAF-6 was shown to be a required molecule in the formation of cytoskeletal structures and the resorptive activity of osteoclasts . This pa1MARGSVSDEE10 to inhibit the cellular LPL phosphorylation in osteoclasts [1MARGSVSDEE10) matches the Rho kinase consensus RXS/TX. It also has multiple putative kinase recognition motifs for PKC and PKA (SVSD). Peptide sequences’ RXXS/T or RXS/TX’ is known as the phosphorylation consensus sequence of Rho kinase (ROK or ROCK II) in cardiac troponin T and cardiac troponin 1 [Phosphorylation of LPL was regulated by PKC , PKA, PI3-K, Mst 1 kinase, RSK1, and RSK 2 kinases downstream of the ERK/MAPK pathway ,82,83,84eoclasts ,56. The oponin 1 . PKC hasoponin 1 ,87. PKCαoponin 1 . AlthougPI3-K was first identified as a lipid kinase that can bind viral oncoproteins, including v-Src, v-Ros, and polyomavirus middle T antigen. PI3-K has two  SH2 domains and one SH3 domain . The claThe current study showed that a complex interaction between TNFR1 and signaling proteins regulated the phosphorylation of LPL. The Erk/MAPK pathway was determined as one of the signal-transduction pathways leading to the phosphorylation of LPL ,83. Alth


Traditionally, beers are made with gluten-containing cereals. It is crucial to have rapid analytical methodologies that allow gluten content control of the beers for celiac consumers. We assess the content of gluten in 65 conventional and 41 gluten-free labeled beers commercialized in Europe and compare the results in a subgroup of 71 beers with three ELISA kits. This research allows gathering information on the potential complementary utility of NIR analysis applied to gluten analysis of gluten-free beers in terms of time saving. Results obtained with the ELISA technique identified competitive R5 to be the most sensitive in detecting the prolamins, by eliciting a higher number of beers containing gluten above 20 mg/kg. The gluten content in conventional beers tested increased with the presence of wheat as raw material and with the use of ale-type yeasts. By using competitive R5, 3 out of the 41 gluten-free labeled beers appeared to contain gluten above 20 mg/kg, and conversely, 15 out of 65 of the conventional beers showed a gluten content below this threshold. According to our approaches, NIR did not achieve a suitable correlation with ELISA results, neither for gluten quantification nor for discrimination, and therefore, it cannot be proposed as a complementary technique. Beer represents one of the most frequently consumed beverages worldwide, and brewing is the oldest biotechnological process . In factIn traditional brewing, the maltose obtained from malted barley is used by yeast during fermentation, resulting in alcohol and carbon dioxide production . ProteinConventional beers are unsuitable for people with gluten-related diseases such as celiac disease, which affects 1% of the general population, although this issue has been questioned by several authors . During The main strategies to reduce the presence of gluten in beers are either based on the use of GF cereals and seeds as raw materials  or on enzymatic treatments to process barley-based beers . EnzymesGluten is a complex mixture of protein, prolamins, and glutenins present in a wide range of unprocessed and processed matrices. This fact, in addition to the difficulty of achieving an adequate reference material, especially for hordeins , leads tHence, the aim of this study was to assess the content of gluten in conventional and GF-labeled beers commercialized in Europe by competitive R5 ELISA kit and compare the results in a subgroup of 71 beers with the other two commercially available ELISA kits. Since the NIR technique has complementary proprieties to ELISA, in terms of time and reagent saving, we evaluate the potential correlation between NIR analysis and ELISA results, which may lead to developing a complementary contamination-monitoring sensor for brewing.In total, 160 barley-based beers  sold in Spain but with national and international  origins were specifically selected from November 2019 to February 2020 to analyze descriptor factors such as manufacturer, yeast style, and wheat inclusion as raw material and original extract. Some of them were also found to include other cereals with gluten  or without this protein  as raw materials. Exclusion criteria for the cohort were as follows: flavored beers, mixed with other alcoholic drinks, and toasted beers. Detailed descriptors of this set of beer are provided in Samples were degassed by shaking before the analysis. The gluten content of the overall beer samples was analyzed by commercially available kits: RIDASCREEN Gliadin competitive kit , based on R5 monoclonal antibody. In the case of the 71 beer cohort, other additional tests were used based on R5 antibody  or on the detection of the T-cell stimulatory epitope of α-20 Gliadin (GLUTEN TEC 5171GT(10)03.20, EuroProxima, Arnhem, the Netherlands). All three assays are recommended for hydrolyzed foods and marketed in Spain. RIDASCREEN Gliadin and GLUTEN TEC are competitive enzyme immunoassay (ELISA), while INGEZIM gluten is a direct test. Manufacturers of both competitive kits detailed specific extraction protocols for beers but the direct assays did not. Then, after an internal test (data not shown), the extraction protocol proposed by the manufacturer for tannins or polyphenols was employed in the INGEZIM Gluten Hidrolizado kit.The samples containing gluten at a concentration greater than the dynamic range of the ELISA kits were diluted according to the manufacturer’s recommendations (minimum dilution to be in range was used). The assays were performed in duplicate for each sample, and results were expressed in parts per million of gluten (mg of gluten/kg of sample). Considering that RIDASCREEN Gliadin competitive and GLUTEN TEC kits measure prolamin content, the results were converted into gluten content by multiplication by a factor of two.n = 335) were scanned from 1400 to 2400 nm in Zeutec Spectraanalyser 2.0 (double beam NIR) (Germany) filter-spectrometer. The analysis was carried out by every time selecting 7 NI -filters from 19 included . Each sample was analyzed in duplicate to reduce measuring errors at a temperature of 22 ± 2 °C. Chemometric models of qualitative discrimination and qualitative determination of gluten were prepared from ELISA kits’ data using Application worxG2 software with the multiple linear regression (MLR) model. Due to the big sample number (n = 335), scatter correction was performed in the model, not including the outliers proposed by the software. Three types of outlier were described: (1) chemist, (2) spectroscopy, and (3) the sum of both. The first outlier was calculated for each spectrum included in the calibration and points out the samples that exceed three times the normalized Mahalanobis distance. The second outlier was considered for each spectrum included in the correlation process and indicates all those that exceed 2.5 times the typical error of the estimate. The limit for the sum of both outliers was settled at three times. The outlier amount rejected did not reach 10 percent of the total samples. The representativeness of the model was assessed based on the coefficient of determination (R2). The reliability of the equipment was confirmed by alcohol grade and protein content of 71 beers set .After degassing, the overall set of samples .p < 0.05. Statistical analysis was performed using IBM SPSS Statistics 24.0 .With regard to the comparison of R5 competitive ELISA data in 71 beers by different classification items, the difference in gluten content and frequencies of GC beers were analyzed by Mann–Whitney U and Pearson’s chi-square tests, respectively. Similarly, Pearson’s chi-square test, followed by a post hoc approach (Bonferroni correction), was used to determine differences in frequencies of Codex classification between commercial ELISA kits. Significant differences were considered at The analysis of gluten in 106 beers commercialized in Europe and composed of 41 GF-labeled beers made from gluten-reduced ingredients and 65 conventional beers was carried out by R5 competitive assay . Moreovep = 0.001 and p < 0.001). By contrast, no difference was observed for manufacturer or original extract beer descriptors. The other two ELISA kits also confirmed these significant differences between ale and lager as well as with wheat inclusion or not.For the conventional beers, significant differences were noted with regard to yeasts style  and wheat inclusion using the R5 competitive. Ale- and wheat-containing beers showed greater gluten content than lager and beers without wheat, respectively . These differences were, however, not statistically significant in the subset of GF beers.As far as the conventional beers, 23% of them (15/65) showed a gluten content below 20 mg/kg of gluten by using the R5 competitive assay . This wap < 0.001). This correlates with the low number of beers containing very low gluten (20–100 mg/kg) when gluten was determined by R5 direct (8) or α20gliadin (13) assays, compared to a higher number when R5 competitive was used (27) (p < 0.001). Although the number of beers classified as gluten-containing products by R5 direct was half (7), compared to competitive R5 (17) and α20gliadin (14), the difference did not reach significance.In the cohort of 71 samples, the number of beers with a gluten content below 20 mg/kg was higher when using the R5 direct (35) or the α20gliadin competitive (23) than when using the competitive R5 (6)  . The comn = 335). The obtained correlation coefficient (R2) for the overall beer sample was 0.167. Correlation statistic improved when overall beer set stratification according to gluten content  was carried out, showing R2 values from 0.139 to 0.592. In none of the cases, correlation reached the 0.7 threshold value to be considered a high correlation.2 value than that based on GF and non-GF categories (0.201 vs. 0.185).Two qualitative–discriminatory models were performed. Although similar correlation statistic values were reached for both models, the equation based on a dummy variable with three categories , showed a slightly higher Rn = 71) are defined in 2 values were obtained, with values over the 0.5 threshold to consider moderate correlation .Specific correlation statistics for the well-characterized beer cohort  is a worldwide food chemistry proficiency with specific gluten-testing in beer. The 2019 Fapas® report indicates that RIDASCREEN Gliadin competitive was the most widely used ELISA kit for gluten testing in beers (27 out of 36 laboratories). Although less widely used, other ELISA tests such as RIDASCREEN Gliadin, INGEZIM Gluten Hidrolizado, and Hygiena GlutenTox ELISA Competitive G12 (KIT3012) have been employed. RIDASCREEN Gliadin was rejected from our selection because the competitive kit produced by the same manufacturer was a better option for beer analysis [The selection of those kits was based on several criteria, one of the most important of which is quality assurance. Beer companies rely on laboratories certified by ISO/IEC 17025 for gluten quantification and determination; in the case of our laboratory, it has been certified for gluten analysis in hydrolyzed matrixes since 2014. To archive this accreditation, it is essential to take part in interlaboratory testing. Food Analysis Performance Assessment Scheme  detected more amount of gluten than α20gliadin competitive (GLUTEN TEC) and R5 direct (INGEZIM Gluten Hidrolizado), respectively. R5 competitive kit is, taking into account this comparison, the most restrictive method, and therefore, deviations from the use of the R5 competitive ELISA method may derive into a safety risk to the celiac consumer. Accordingly, specific research about gluten fragment detection indicated that R5 competitive ELISA provided higher prolamin concentration in barley-based beer than the noncompetitive format of the same manufacturer . SimilarHighly variable proteolytic peptides resulting from the brewing processes lead to more appropriate recognition than the requirement of only one epitope as competitive or direct ELISA tests do. A recent review showed that depending on the antibody, contradictory results can be obtained using the same technique (competitive ELISA) for gluten detection of gluten in the same beer samples . In our Since the R5 competitive ELISA was the most restrictive test employed, an in-depth analysis of detected gluten was conducted for 71 beer set by different classification items. Our results in conventional beers reveal greater gluten content in ale than lager beers and in the ones including wheat than those that do not have this cereal as an ingredient. Other researchers have also reported gluten content changes according to beer types. In 2005, Kanerva et al. found remarkable differences between lager and wheat beers using the Tepnel Biokits ELISA assay, based on mAb 401.21 antibody, also called Skerrit antibody . In factRegarding beers labeled as GF, no significant quantitative disparities among beer types were found. Notwithstanding, it is important to highlight that, according to the R5 competitive assay, 3 out of 41 beers labeled as GF  contained more than 30 mg/kg of gluten, and consequently, they were over the threshold stated in the legislation . These aThe use of the PEP enzyme for GF brewing is widespread, both in industrial and craft breweries . Large bAnother worthwhile outcome of the present research was the proportion (15 out of 65) of conventional beer that contained gluten levels below 20 mg/kg of gluten. Further research would be needed to understand the factors that have yielded to those beers so that this could be consistently applied to all batches. In other studies, commercial beer analyzed by R5 competitive , SkerritNowadays, Codex Alimentarius establishes the methodology ELISA R5 for the determination of gluten in foods as a type I method. Fermentation and hydrolysis processes, so common in food preparation, can break down gluten, making it difficult to determine. Thereby, in fermented and hydrolyzed foods, the use of competitive  or similThe time required to perform routine quality control testing of raw material, intermediates, and final products slows down and makes brewing processes more expensive. These ought to obtain analytical data to ensure the suitability of steps performed  . In GF bNIR has been used successfully for the determination of numerous analytical parameters  in cereals and cereal products, milk and dairy products, meat, fish, fruits and vegetables, etc. . A clearNIR has been employed in beers for the determination of other parameters such as pH, alcoholic degree, bitterness, primitive dry extract, and total soluble nitrogen ,41. HoweELISA tests for gluten quantification use a considerable volume of chemical reagents such as fish gelatin, polyvinylpyrrolidone, extraction buffer, or ethanol (at different grades). Additionally, the completion of the assay takes about 75 to 120 min. By contrast, NIR analysis of beer does not entail any chemical reagent and the complete assay for each sample lasts 30 s. In order to answer our hypothesis, we used a filter spectrometer (1200–2600 nm), followed by MLR analysis. This equipment was selected for two main reasons. Firstly, these instruments are simpler and cheaper than monochromators and diode array spectrometers .2R > 0.7), neither when beers were evaluated as a whole nor when samples were stratified by the regulation (EU) No 828/2014 [2 value of 0.167) was a long away from the raw data results described by others measuring wet gluten content  and gliadin content  in whole-wheat grain and flours, respectively [Our first approach for NIR analysis was carried out with 335 beers available on the Spanish market. It is essential to clarify whether a potential correlation occurs between R5 competitive ELISA and NIR results in a large number of beers. The correlation statistics did not achieve the quality goals . In agreement with these results, Albanell et al. reported a convenient NIR calibration for wet gluten content in flour and batter with the exception of concentrations between 0 and 100 mg per 1 Kg food [Further analysis of our results showed that correlation statistics improved with increasing levels of gluten concentration . In fact, similar correlation values were obtained for the evaluation of gluten content as well as GF product discrimination among the three ELISA kits. As with the 335 beer group, the best correlation, defined as RThe lack of a green technique and affordable monitoring of cross-contamination during the brewing process adds to the financial handicap of inconsistent GF labeling of beers. In this study, research presented for the first time shows the potential use of NIR as a complementary technique to ELISA in gluten determination of commercial beers. Our result does not allow us to put forward NIR analysis as a sensor for gluten quantification or GF sample discrimination in beers. For the moment, it is not possible for breweries to use NIR analysis to widen the control throughout the brewing process. However, this work can be considered as a first milestone in the field where future improvement of NIR technique or new chemometric models leads to better alternatives in the gluten analysis.The study also leads us to conclude that the R5 competitive ELISA kit is the most sensitive of the three immunologic methods used here to discriminate low levels of gluten in beer. The analysis performed on conventional beers highlights the significance of lager yeast selection or wheat exclusion on the brewery process to reduce the gluten content of the final product. As for the results on the selected labeled GF beers, we detected some outliers of gluten content that would require attention from the regulatory bodies. On the contrary, some of the conventional beers, not specifically manufactured to be GF, showed levels of gluten below 20 mg/kg. These results are, in all likelihood, pointing at the need for more research in the field of fermented and hydrolyzed foods to understand better all the factors that can yield to produce consistent batches of GF beer within the regulatory threshold, in addition to highlighting the importance of making use of approved validated methods to determine its gluten content with the final aim of protecting celiac consumers.


Poa palustris with seed galls were collected on a bank of river in Teton County, Wyoming, USA. Isolated nematodes were identified by both morphological and molecular methods as Anguina agrostis. This is a first report of A. agrostis in Wyoming and its report on fowl bluegrass.In September 2020, several plants of fowl bluegrass,  Anguina agrostis  Filipjev, 1936 was described from Agrostis capillaris collected in Bavaria, Germany. Many grass species have been included in the list of host plants for this nematode. A. agrostis appeared to be restricted to populations causing characteristic elongate galls and abnormally elongated floral structures in grasses of the genus Agrostis. Analysis of the ITS rRNA gene sequences made by A. agrostis was only found on Agrostis capillaris. Other grasses previously known as hosts for A. agrostis were actual hosts for several other still undescribed species of Anguina. Anguina agrostis is considered as a serious or potentially important nematode pest of bent grass in the Pacific Northwest, USA, and New Zealand   were: L = 820 ± 39 (775-905) µm; W = 13.5±0.8 (12.5-15.0) µm; stylet length = 8.4 ± 0.6 (6.8-8.8) µm; anterior end to median bulb valve =70±2.5 (65-75) µm; a = 60±3.8 (53.7-66.9); b = 5.5 ± 0.5 (5.0-6.5). They are similar with those provided by In September 2020, several plants of fowl bluegrass, mm long)  were colCOI gene). Blastn search showed that the ITS rRNA and COI gene sequences of nematode isolated from Poa palustris were similar in 100% (100% coverage) with corresponding genes of A. agrostis from the USA .DNA was extracted from several live second-stage juveniles. DNA extraction, PCR, and sequencing were performed as described by A. agrostis. This is a first report of A. agrostis in Wyoming, USA and molecularly confirmed report of this species on fowl bluegrass, Poa palustris.Thus, morphological and molecular examination confirmed the species as


A patch antenna sensor with T-shaped slots operating at 2.378 GHz was developed and investigated for wireless ice and frost detection applications. Detection was performed by monitoring the resonant amplitude and resonant frequency of the transmission coefficient between the antenna sensor and a wide band receiver. This sensor was capable of distinguishing between frost, ice, and water with total shifts in resonant frequency of 32 MHz and 36 MHz in the presence of frost and ice, respectively, when compared to the bare sensor. Additionally, the antenna was sensitive to both ice thickness and the surface area covered in ice displaying resonant frequency shifts of 2 MHz and 8 MHz respectively between 80 and 160 μL of ice. By fitting an exponential function to the recorded data, the freezing rate was also extracted. The analysis within this work distinguishes the antenna sensor as a highly accurate and robust method for wireless ice accretion detection and monitoring. This technology has applications in a variety of industries including the energy sector for detection of ice on wind turbines and power lines. Additionally, ice accretion reduces the efficiency of wind turbines regularly by altering their aerodynamic profile and disrupts the energy sector by causing power outages when ice accumulates on power transmission lines5. Ice accretion is also extremely hazardous to the public on roadway infrastructure7. Moreover, ice accumulation in oil pipelines, if unnoticed, can result in the destruction of the pipeline and environmental contamination8. These ice-related hazards exemplify the need for an efficient and accurate system for detecting ice accretion, thickness, and rate of growth.Ice formation and accumulation creates serious hazards and can cause critical damage or malfunction to equipment in various industries. The impact of icing in the wind turbine power generation industry is particularly concerning due to incidents like the recent winter storm event in Texas where ice accretion stopped the operation of the wind turbines resulting in critical power production loss for multiple days13. Recently, sensors operating at microwave frequencies have attracted attention due to their small footprint, ease of fabrication, robust nature, and real-time sensing capabilities20. These advantages have inspired the use of microwave resonators for various sensing applications including ice sensing38. Microwave resonator sensors operate by monitoring the interaction between high concentrations of electric field (E-field) and a material under test, allowing for material property changes to be observed in real time39. In ice detection studies utilizing resonator structures, the microwave sensors were able to detect the accumulation of ice, the thickness of the accreted ice, and the rate of deposition on their surface. However, for real-world implementation on large structures such as wind turbine blades and aircraft wings, transferring the sensed data efficiently can be problematic. In place of the resonator, an antenna sensing structure could overcome this issue by monitoring the S21 parameters between a transmitting sensing antenna and a receiving antenna, thus enabling a wireless ice detection system.Various methods have previously been implemented for ice sensing including piezoelectric sensors, ultrasonic-based methods, and guided wave approaches42. These structures, known as antenna sensors, have previously been utilized to sense a variety of materials in both liquid and solid form, as well as temperature and crack propagation on surfaces51. Here, an antenna sensor was developed for wireless ice accretion sensing. A conventional edge fed patch antenna was modified to operate as an antenna sensor. The microstrip patch antenna was advantageous due to its planar design, directive radiation pattern, satisfactory gain, and resonant profile in its transmission and reflection coefficients. Moreover, the localized high concentration of E-field on the edges of this antenna design increases detection sensitivity. The designed antenna sensor was a T-shaped slotted patch antenna with a rectangular slab element along the width of the patch to enhance sensing capabilities.An antenna can be designed to act as a sensing platform while simultaneously transmitting a signalThis paper is organized as follows: The antenna sensor theory of operation and simulations are described in section II, Section III contains the measurement results for the antenna sensor from three consecutive scenarios of a cooling droplet, a frozen droplet, and a thawing droplet, Section IV presents the conclusions of this technique.r = 6.15 and a thickness of 2.54 mm. The operational frequency of the antenna was designed for 2.45 GHz to take advantage of the available ISM band. Ansys High Frequency Structure Simulator (HFSS) was used for the design and simulation of the antenna sensor. The width, W, and the length, L, of the patch were derived from Eqs. –(4) demonstrated that, for the designed patch, The permittivity and the effective permittivity of the patch are mentclass2pt{minimElectromagnetic simulations were performed in HFSS over the frequency range of 1.5 to 3.5 GHz using 4001 points. The surface copper of the antenna sensor was modeled using finite conductivity boundary conditions and a radiation boundary condition was used for the far-field simulation of the antenna sensor. The integration of the T-shaped slots with the patch antenna was compared against a conventional patch of the same size. As observed in the result of Fig. To simulate ice detection conditions, water and ice were placed between the patch and the slab element to observe their influence on the antenna sensor’s response. Bridging the gap with water resulted in a 340 MHz shift in the resonant frequency of the antenna sensor, while ice shifted the resonant frequency by 14 MHz Fig. b. The suFurther simulations were performed to investigate the E-field concentration on the antenna sensor and to understand the effect of the T-shaped slots and the slab element on the antenna’s sensing performance. The antenna sensor exhibited higher E-field concentration on its surface with the T-shaped slots present and the rectangular slab element did not impact the E-field concentration on the bare antenna sensor Fig. . HoweverThe far-field gain of the antenna sensor was investigated for various simulated conditions in order to validate the sensor’s transmission capability. As observed in the results of Fig. To investigate the dual-antenna system for sensitivity to iced area and ice thickness, simulations were performed in HFSS implementing the antenna sensor and a wideband receiving antenna. The results of the simulations for iced area can be seen in Fig. S11) was measured using a Vector Network Analyzer (VNA) over a frequency range of 1–3 GHz. The operation frequency of the antenna sensor was designed at 2.4–2.5 GHz to exploit the availability of the ISM band. An S11 comparison between the simulated and measured structure was performed , with an operational frequency range from 900 MHz to 12 GHz. The experimental setup can be seen in Fig. Initial experiments were conducted to determine the sensitivity of the antenna sensor to frost and ice accretion. To perform all of the following experiments, a custom experimental setup was created with the ability to cool the patch antenna below the freezing temperature of water using a Peltier cooling stage. The response of the antenna was monitored using a portable Field Fox N9918A Vector Network Analyzer (VNA) and the Two validation experiments were conducted to confirm that the antenna could detect water and ice accretion. First, condensation and subsequently frost were formed on the patch antenna by cooling the device to – 9 °C using the Peltier cooling stage. Water vapor from the surrounding environment condensed on the patch antenna and froze, changing the load impedance of the antenna and ultimately shifting its operational frequency and amplitude Fig. .Figure 8S21 resonant amplitude and resonant frequency of the bare patch antenna was recorded to be − 28.98 dB at 2.375 GHz at room temperature. As the antenna was cooled and condensed water and frost accumulated on the device, the resonant amplitude and resonant frequency shifted to − 32.61 dB at 2.277 GHz and − 28.49 dB at 2.343 GHz, respectively. The resonant amplitude and frequency both decreased during condensation due to the increased loss caused by water accumulating on the surface. The presence of water changed the load conditions on the antenna, which led to changes in the resonant amplitude and frequency. As the condensation froze into frost approximately 400 s into the experiment, the amplitude sharply increased and continued to gradually increase as more frost developed (Fig. The transmitted The Peltier stage was turned off around 1300 s to warm the sensor and thaw the developed frost. During thawing, which began around 1400 s, a sharp drop in resonant frequency and resonant amplitude was observed as the frost melted and became liquid water. The increased water on the sensor resulted in high loss followed by a more gradual return to a steady state. This return was caused by the coalescence of the small, melted frost droplets into larger, discrete droplets. The remaining water did not persist as a thin layer over the entire surface, where no increase in amplitude or frequency would be expected. Regardless, these results clearly demonstrate the antenna sensor’s ability to differentiate frost from liquid water.r = 3.2 and εr = 6.15 respectively, compared to the difference in relative permittivity between air and the substrate, improving signal transmission. Contrasting this, the presence of water degraded signal transmission and reduced the resonant amplitude − 3.63 dB. This occurred because water has a much higher relative permittivity of εr = 81. The resonant frequency will always shift downward in the presence of any additional substance, so long as the material under test has a permittivity higher than air, which increases coupling to the secondary capacitive plate.The total change in resonant amplitude and resonant frequency with respect to the bare antenna due to the presence of frost were 0.49 dB and 0.032 GHz respectively. With condensed water on the antenna the total change in resonant amplitude and resonant frequency were − 3.63 dB and 0.098 GHz respectively. As frost accumulated on the antenna, the resonant amplitude increased 0.49 dB. This was due to the smaller difference in relative permittivity between ice and the substrate, εThe second validation experiment of the antenna sensor involved the freezing of a single droplet of water into ice using the same experimental setup observed in Fig. The resonant amplitude and resonant frequency of the bare antenna, − 28.94 dB at 2.375 GHz, shifted to − 28.24 dB at 2.339 GHz and − 38.29 dB at 2.233 GHz for the single frozen and liquid water droplet, respectively. In the transient graphs, a liquid water droplet was already deposited on the antenna at 0 s. As the antenna was cooled, the droplet froze at 400 s and both the resonant amplitude and resonant frequency increased. This increase in detection parameters due to ice formation was more pronounced compared to the frost experiment due to the increased volume, thickness, and because the ice completely bridged the gap region on the antenna. The signal’s resonant amplitude contained noise in its readout at steady state with ice frozen on the antenna, which was introduced by interference in the environment during detection. This noise indicated that the resonant frequency was more promising to use for stable and robust ice detection, as it would be less affected by interference due to changing environmental conditions. Once the response of the antenna with ice was stable the antenna was allowed to warm up. The ice melted at 700 s and the resonant amplitude and resonant frequency returned to their initial states with minor differences due to additional condensation. This experiment indicated that the antenna can easily detect the presence of ice and water when only a small portion of the antenna is covered.The frost and single droplet experiments successfully validated the antenna sensor for ice detection applications. To explore the sensor’s capabilities further, the sensitivity of the device to iced area and ice thickness was also investigated.To determine the sensitivity of the antenna sensor to the iced area, an experiment was performed using two water droplets of equal volume. First, an 80 µL water droplet at 18 °C was placed on the antenna and then frozen by reducing the stage temperature to − 5 °C. The sensor’s transient response is presented in Fig. Establishing a threshold for ice detection requires the antenna sensor to be capable of detecting changes in the iced surface area coverage. In the above experiment, both the resonant frequency and resonant amplitude shifted as the iced surface area increased. With a single droplet of ice, the time averaged resonant frequency was 2.339 GHz and resonant amplitude was − 28.48 dB. With two droplets of ice the time averaged resonant frequency and resonant amplitude shifted to 2.331 GHz and − 28.50 dB respectively. The time averaged data was used here to eliminate the impact of noise while comparing the response of the sensor. Note that the resonant amplitude experienced a higher level of noise while the resonant frequency remained significantly more stable. As mentioned previously, this was because the resonant amplitude is more susceptible to environmental changes such as movement around the antenna. Although this noise did not preclude the resonant amplitude from being used to threshold detection, the resonant frequency would appear to be a more favorable detection parameter. The total shift in resonant frequency and resonant amplitude due to the increased surface area was 0.023 dB and 8 MHz respectively. The antenna was highly sensitive to the area covered with ice, evidenced by an 8 MHz resonant frequency shift, and can therefore be used to create a threshold of ice coverage before detection is confirmed by other means.2. To avoid leakage before freezing, the mold was attached to the antenna surface using a small piece of double-sided tape.Similarly, a study determining the antenna’s sensitivity to ice thickness was also conducted using two droplets in sequence. This experiment was performed by freezing one 80 µL droplet onto the antenna inside a mold, thereby controlling the surface area covered in ice, and then placing a second 80 µL droplet directly on top of the first. This enabled a study on the impact of ice thickness on the sensor’s transmitted response Fig. . The molThe results of this experiment indicated that ice thickness was also detectable through the shift in resonant frequency and resonant amplitude, although the response was not as pronounced. With a controlled surface area covered in ice, the sensors resonant amplitude and resonant frequency were 2.333 GHz and − 27.92 dB after the first droplet froze, and shifted to 2.329 GHz and − 28.05 dB after the second droplet froze. The total change in resonant amplitude was 0.036 dB and the total change in resonant frequency was 2 MHz due to the increased ice thickness. The sensor was less responsive to ice thickness due to the minimal penetration of the near fields into the ice. In general, the EM near field waves will become weaker moving away from the antenna and will minimally penetrate a material. This effect is amplified as the permittivity of the material under test increases. This expectation was evidenced by the minimal change of the sensor’s resonant amplitude and frequency response, 2.25 dB and 39 MHz, respectively, when the second liquid droplet was added. In contrast, for the surface area experiment Fig. , adding For both the ice thickness and iced area experiments, an exponential curve was fit to the transient resonant amplitude and resonant frequency data, for the first and second droplet freezing curves Table . For eacAccording to the presented results, both the resonant frequency and the resonant amplitude of the antenna sensor displayed high sensitivity to the presence of water, ice, and frost over the sensing region. However, the antenna’s resonant frequency response was more reliable because the resonant frequency was not sensitive to the distance between the sensor antenna and the interrogator antenna, as well as being insensitive to possible obstacles between the two antenna that could interfere with the signal. In contrast, the resonant amplitude would be significantly impacted by the distance between the two antennas. The noise observed within all resonant amplitude results originated from interference in the environment. Overall, the resonant frequency is an excellent decision-making parameter when detecting ice as it is not impacted by environmental factors and was practically noise-free within this controlled environment, while the resonant amplitude could be used as supporting data point to confirm the detection.S21 transmission between the antenna sensor and a wide band receiver could be used to detect ice accretion. However, due to the resonant amplitude being impacted by environmental factors such as distance to the receiver and obstructions in the line-of-sight path, the resonant frequency was the superior decision-making parameter. This proposed ice sensing method is low-cost, real-time, robust, and highly accurate with the additional advantage of wireless readout capability, eliminating the need for long transmission lines on large structures. The detection of ice in both the public and private sectors will create safer environments and protect equipment from being damaged or destroyed. Further development of this sensor will produce an ice detection technique which can be easily implemented into existing structures such as wind turbines for real-time ice sensing.In this work, the use of a patch antenna sensor operating at 2.378 GHz to monitor the presence and accumulation of ice was demonstrated. The antenna sensor was determined to be sensitive not only to the presence of ice, but also the thickness, rate of accretion, and surface area covered. It was determined that both the resonant amplitude and resonant frequency of the Supplementary Information.


Dipeptidyl peptidase-IV (DPP-IV) inhibitors, the enhancers of incretin are used for the treatment of diabetes. The non-glycaemic actions of these drugs  also proved that repurposing of these molecules may be advantageous for other few complicated disorders like cardiovascular diseases, Parkinson's disease, Alzheimer's disease, etc.Curcuma longa.The present study was aimed to investigate the DPP-IV inhibitory potential of Calebin-A, one of the constituents of in silico studies (using Schrödinger Suite) like, Docking analysis, molecular mechanics combined with generalized Born model and solvent accessibility method (MMGBSA) and Induced fit docking (IFD) after validating the protein using Ramachandran plot. Further, the protein-ligand complex was subjected to molecular dynamic simulation studies for 50 nanoseconds. And finally, the results were confirmed through enzyme inhibition study.The phytoconstituent was subjected for various Insilico results revealed possible inhibitory binding interactions in the catalytic pocket  and binding affinity in terms of glide g-score and MMGBSA dG bind values were found to be −6.2 kcal/mol and −98.721 kcal/mol. Further, the inhibitory action towards the enzyme was confirmed by an enzyme inhibition assay, in which it showed dose-dependent inhibition, with maximum % inhibition of 55.9 at 26.3 μM. From molecular dynamic studies (50 nanoseconds), it was understood that Calebin A was found to be stable for about 30 nanoseconds in maintaining inhibitory interactions.in silico and in vitro analysis, the current research emphasizes the consideration of Calebin A to be as a promising or lead compound for the treatment of several ailments where DPP-IV action is culprit.From the   In this context, our research group aimed to find the therapeutic importance of various active constituents of C. longa, one of the important Indian spice plants, via biological and chemical investigations. So far, great attention has been paid to pharmacological research of curcumin, but no significant work has been carried out on other active natural compounds like Calebin A (CA) , hydrogen bonds formed by side chain hydroxyl group of Tyr662, backbone carboxyl group of Glu206 and side chain amino group of Arg358 with 4-hydroxy-3-methoxy phenyl rings of fractions A and B might be responsible in positioning the ligand in the active site of an enzyme to maintain considerable stability. Apart from these, hydrophobic interactions between Tyr666 and phenyl ring of fraction A also contributed for the same.From 15 to 30 ns , despiteFrom 31 to 33 ns , the 4-h3.3.21 subsite followed by hydrophobic contact with Phe357.From 34 to 50 ns, the high conformational flexibility of ligand  caused tInitially, there was an increase in protein RMSD during first 2 ns simulation, which might be due to equilibration of protein-ligand complex with the solvent system. From 2 ns to 50 ns , the RMSIn ligand RMSD, high fluctuation was observed in the range of 2.18–6.58 Å. Initially there was an increase in ligand RMSD having 2.48 at 0.36 ns and then maintained in the range of 2.18–2.88 Å from 10 to 34 ns. After 34 ns, the ligand RMSD was airlifted from 3.0 Å and noticed maximum value of 6.57 Å at 48 ns. The root mean square fluctuations (RMSF) for the amino acid residues of protein Cα, backbone and heavy atoms were observed in the range of 0.52–3.51 Å, 0.53–3.47 Å, and 0.59–4.06 Å respectively . High ro–OOC) (61% of MD trajectory) and another weak bond through a water bridge (20% of MD trajectory). The backbone hydroxyl group of Tyr 662 forms two hydrogen bonds, with methoxy oxygen  and hydroxyl oxygen  of fraction A in a bidentate manner. Another key residue, Arg358 was also noticed to form two hydrogen bonds with hydroxyl group of fraction B, one moderately strong hydrogen bond with hydroxyl group  and a weak bonding through a water bridge (24% of MD trajectory). The phenyl rings of fraction A and fraction B were positioned in the active site through hydrophobic interactions with Tyr666 (46% of MD trajectory) and Phe357 (17% of MD trajectory) respectively. In addition, Ser552 (12% of MD trajectory), Ala654 (11% of MD trajectory), Cys551 , and Glu361  showed weak interactions with CA.The trajectory analysis of MD simulation  conveyed2, 126.18–273.86 Å2 and 180.98–204.9 Å2 respectively.As represented in 4The enzyme DPP-IV belongs to the superfamily of prolyl oligopeptidase and differs from other oligopeptidases in having two glutamate residues (Glu205 and Glu206) in the catalytic pocket of active site that have their own niche role in exhibiting enzymatic activity . GlucagoC. longa were selected. From ancient times, C. longa has its own niche in promoting health. Due to advances in scientific field, many therapeutic actions were proved and many evidences attributed the curcumin to most of the positive actions of C. longa.For the current study, constituents of insilico analysis for the selected receptor. In the current investigation, we have used CA for which except Aβ disaggregating property (in vitro) no other anti-Alzheimer's property has been described.The current research work is a continuation of our repurposing strategy to our earlier research works that involved in targeting DPP-IV inhibitors for Alzheimer's disease ,37,38. C2 extensive subsite , S2 subsite , S1 subsite , S′1 subsite , and S′2 subsite  [The active site of DPP-IV was constructed into different subsites based on substrate and inhibitor binding with respective amino acid residues, they are S His740) . Non-bon His740) .Table 1Ninsilico insights in molecular design of DPP-IV inhibitors [insilico docking and free energy minimization studies to analyze binding interactions of CA with DPP-IV at subsite level. Having molecular surface area of 378.778 Å2, the 4-hydroxy and 3-methoxy groups of fraction A occupied S1 subsite via formation of two hydrogen bonds with Tyr662. However, van der Waals dispersion (hydrophobic) [2 subsite forming two significant hydrogen bonds with side chain and backbone of Glu206. The carbonyl oxygens of both fractions formed three hydrogen bonds with Cys551, Ser552 and Tyr585.The qualitative and quantitative investigations like comparative binding analysis of DPP-IV inhibitors with their inhibitory activities based on X-ray crystal poses , bindinghibitors  and quanhibitors  discloseophobic)  interact2 extensive subsite. It was also reaffirmed by the trajectory analysis of 50 ns MD simulation  with Phe357  and Coulombic interactions with Arg358  , throughmulation   also, both bonded and non-bonded interactions played a key role in stabilising the molecule. The ligand bound amino acids displayed low RMSF  of 0.43–in silico studies is rare, as the usage of in silico technology in the process of drug discovery is emerging now. This might be one of the main reasons for the lack of comparison data among natural DPP-IV inhibitors. Divya et al., reviewed and compiled the data related various natural extracts and compounds that were proved to have DPP-IV inhibition activity through invitro studies [, further supports the current research work [In silico studies on stellasterol isolated from Ganoderma australe highlighted the importance of hydrophobic interactions with Tyr631, Tyr662, Trp659, Tyr666, and Val711 for DPP-IV inhibition which further strengthen the current investigations [In most research studies, related to herbal DPP-IV inhibition activity, extracts were tested for the activity and even if the individual components subjected for the same, progressing them for  studies . As prop studies . Bindingrch work . In siliigations . Other nigations .ex vivo and in vivo methods, direct enzymatic method is an ideal and appropriate method in the initial stages of drug discovery and development process (as many number of compounds can be screened). Due to the limited number of DPP-IV inhibitors in the market, Lin et al., emphasized importance of considering the natural compounds in the drug discovery process and the same can be hastened by implementing in silico and enzyme inhibition methods [, identified the hypoglycemic activity of Brachylaena elliptica is independent of insulin which might be due to its DPP-IV inhibition activity that was proved through invitro study [in vitro methodologies [in vitro assays is measured by statistic parameter called Z'(Z prime) which uses mean and standard deviation of both positive and negative control. Z' is not only a measure of way of doing but also correctness of instrument. Hence, in short we can consider the Z' as statistic measure of overall experimentation. In general, a good Z' value lies between 0.5 and 1. Since the current experiment has got the value of about 0.8, which indicates the quality of overall experimentation. As mentioned earlier, our research mainly focuses on repurposing strategy through DPP-IV inhibitors to target AD, by considering several evidences as facts and support. DPP-IV inhibitors enhance the levels of GLP-1 that can cross Blood brain barrier (BBB) and executes its actions through GLP1R (GLP-1 receptor), present over pyramidal neurons of the hippocampus and purkinje cells of cerebellum [Although DPP-IV inhibitors can be screened by direct enzymatic,  methods . In anotro study . The resdologies . In generebellum . Out of rebellum . GLP-1 wrebellum . Moreoverebellum . By usinrebellum ,58. The rebellum .5Binding mode analysis and results of enzyme inhibition emphasised the consideration of CA for its role as a DPP-IV inhibitor. As role of GLP-1 has extended beyond glycemic actions, DPP-IV inhibitors can be expected to have crucial role in various pathological features. In our previous studies, we have emphasized the importance of these inhibitors in the field of neurodegenerative disorders.insilico methods like molecular docking, binding free energy calculation, induced fit docking and molecular dynamic simulation studies were performed to analyze binding mode of CA with DPP-IV. Having a structurally diverse scaffold from the marketed inhibitors, our studies showed possible interactions of CA with substrate specific recognition site residues like Glu206 (S2 subsite) and Tyr662 (S1 subsite) of “amino hot-spot” and also with Phe357 and Arg358 of S2 extensive subsite, conclusively conveying that CA has the potential to act like class-3 inhibitor according to Nabeno et al. As both in silico and enzyme inhibition studies clearly showed encouraging results for the proposed action, further confirmation through in vivo studies is essential. Furthermore, the rigidification of the CA in the active site to enhance the time of contact with inhibitory interactions with the protein and steps to enhance the bioavailability was directed for future studies to make it clinically viable.In this current investigation, Ministry of AYUSH-A (EMR scheme), Government of India, New Delhi. (Sanction Order: Z.28015/89/2014-HPC (EMR) AYUSH – A).The work was financially supported by None.Nehru sai suresh: Study execution, Literature and experimental data collection, Data Interpretation, Manuscript writing.Srikanth Jupudi: In silico study and statistical analysis.Venkata Ramesh Yasam: Data analysis and Interpretation.Duraiswamy Basawan: Conceptualisation, Supervision, Fund recipient, Manuscript review.