Comment on Lai et al. Dipeptidyl Peptidase 4 Stimulation Induces Adipogenesis-Related Gene Expression of Adipose Stromal Cells. Int. J. Mol. Sci. 2023, 24, 16101.

Adiponectin is a circulating hormone secreted by adipose tissue that exerts, unlike other adipokines such as leptin, anti-inflammatory, anti-atherosclerotic and other protective effects on health. Adiponectin receptor agonists are being tested in clinical trials and are expected to show benefits in many diseases. In a recent article, LW Chen's group used monocyte chemoattractant protein-1 (MCP-1/CCL2) to improve plasma levels of adiponectin, suggesting the involvement of dipeptidyl peptidase 4 (DPP4/CD26) in the mechanism. Here, we discuss the significance of the role of DPP4, favoring the increase in DPP4-positive interstitial progenitor cells, a finding that fits with the greater stemness and persistence of other DPP4/CD26-positive cells.

Gut microbiota DPP4-like enzymes are increased in type-2 diabetes and contribute to incretin inactivation.

BACKGROUND: The gut microbiota controls broad aspects of human metabolism and feeding behavior, but the basis for this control remains largely unclear. Given the key role of human dipeptidyl peptidase 4 (DPP4) in host metabolism, we investigate whether microbiota DPP4-like counterparts perform the same function. RESULTS: We identify novel functional homologs of human DPP4 in several bacterial species inhabiting the human gut, and specific associations between Parabacteroides and Porphyromonas DPP4-like genes and type 2 diabetes (T2D). We also find that the DPP4-like enzyme from the gut symbiont Parabacteroides merdae mimics the proteolytic activity of the human enzyme on peptide YY, neuropeptide Y, gastric inhibitory polypeptide (GIP), and glucagon-like peptide 1 (GLP-1) hormones in vitro. Importantly, administration of E. coli overexpressing the P. merdae DPP4-like enzyme to lipopolysaccharide-treated mice with impaired gut barrier function reduces active GIP and GLP-1 levels, which is attributed to increased DPP4 activity in the portal circulation and the cecal content. Finally, we observe that linagliptin, saxagliptin, sitagliptin, and vildagliptin, antidiabetic drugs with DPP4 inhibitory activity, differentially inhibit the activity of the DPP4-like enzyme from P. merdae. CONCLUSIONS: Our findings confirm that proteolytic enzymes produced by the gut microbiota are likely to contribute to the glucose metabolic dysfunction that underlies T2D by inactivating incretins, which might inspire the development of improved antidiabetic therapies.

Excessive accumulation of epicardial adipose tissue promotes microvascular obstruction formation after myocardial ischemia/reperfusion through modulating macrophages polarization.

BACKGROUND: Owing to its unique location and multifaceted metabolic functions, epicardial adipose tissue (EAT) is gradually emerging as a new metabolic target for coronary artery disease risk stratification. Microvascular obstruction (MVO) has been recognized as an independent risk factor for unfavorable prognosis in acute myocardial infarction patients. However, the concrete role of EAT in the pathogenesis of MVO formation in individuals with ST-segment elevation myocardial infarction (STEMI) remains unclear. The objective of the study is to evaluate the correlation between EAT accumulation and MVO formation measured by cardiac magnetic resonance (CMR) in STEMI patients and clarify the underlying mechanisms involved in this relationship. METHODS: Firstly, we utilized CMR technique to explore the association of EAT distribution and quantity with MVO formation in patients with STEMI. Then we utilized a mouse model with EAT depletion to explore how EAT affected MVO formation under the circumstances of myocardial ischemia/reperfusion (I/R) injury. We further investigated the immunomodulatory effect of EAT on macrophages through co-culture experiments. Finally, we searched for new therapeutic strategies targeting EAT to prevent MVO formation. RESULTS: The increase of left atrioventricular EAT mass index was independently associated with MVO formation. We also found that increased circulating levels of DPP4 and high DPP4 activity seemed to be associated with EAT increase. EAT accumulation acted as a pro-inflammatory mediator boosting the transition of macrophages towards inflammatory phenotype in myocardial I/R injury through secreting inflammatory EVs. Furthermore, our study declared the potential therapeutic effects of GLP-1 receptor agonist and GLP-1/GLP-2 receptor dual agonist for MVO prevention were at least partially ascribed to its impact on EAT modulation. CONCLUSIONS: Our work for the first time demonstrated that excessive accumulation of EAT promoted MVO formation by promoting the polarization state of cardiac macrophages towards an inflammatory phenotype. Furthermore, this study identified a very promising therapeutic strategy, GLP-1/GLP-2 receptor dual agonist, targeting EAT for MVO prevention following myocardial I/R injury.

Molecular basis for receptor recognition and broad host tropism for merbecovirus MjHKU4r-CoV-1.

A novel pangolin-origin MERS-like coronavirus (CoV), MjHKU4r-CoV-1, was recently identified. It is closely related to bat HKU4-CoV, and is infectious in human organs and transgenic mice. MjHKU4r-CoV-1 uses the dipeptidyl peptidase 4 (DPP4 or CD26) receptor for virus entry and has a broad host tropism. However, the molecular mechanism of its receptor binding and determinants of host range are not yet clear. Herein, we determine the structure of the MjHKU4r-CoV-1 spike (S) protein receptor-binding domain (RBD) complexed with human CD26 (hCD26) to reveal the basis for its receptor binding. Measuring binding capacity toward multiple animal receptors for MjHKU4r-CoV-1, mutagenesis analyses, and homology modeling highlight that residue sites 291, 292, 294, 295, 336, and 344 of CD26 are the crucial host range determinants for MjHKU4r-CoV-1. These results broaden our understanding of this potentially high-risk virus and will help us prepare for possible outbreaks in the future.

Fragment-based QSAR study to explore the structural requirements of DPP-4 inhibitors: a stepping stone towards better type 2 diabetes mellitus management.

Dipeptidyl peptidase-4 (DPP-4) inhibitors belong to a prominent group of pharmaceutical agents that are used in the governance of type 2 diabetes mellitus (T2DM). They exert their antidiabetic effects by inhibiting the incretin hormones like glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide which, play a pivotal role in the regulation of blood glucose homoeostasis in our body. DPP-4 inhibitors have emerged as an important class of oral antidiabetic drugs for the treatment of T2DM. Surprisingly, only a few 2D-QSAR studies have been reported on DPP-4 inhibitors. Here, fragment-based QSAR (Laplacian-modified Bayesian modelling and Recursive partitioning (RP) approaches have been utilized on a dataset of 108 DPP-4 inhibitors to achieve a deeper understanding of the association among their molecular structures. The Bayesian analysis demonstrated satisfactory ROC values for the training as well as the test sets. Meanwhile, the RP analysis resulted in decision tree 3 with 2 leaves (Tree 3: 2 leaves). This present study is an effort to get an insight into the pivotal fragments modulating DPP-4 inhibition.

In vitro gastrointestinal stability and intestinal absorption of ACE-1 and DPP4 inhibitory peptides from poultry by-product hydrolysates.

Bioactive peptides derived from food are promising health-promoting ingredients that can be used in functional foods and nutraceutical formulations. In addition to the potency towards the selected therapeutic target, the bioavailability of bioactive peptides is a major factor regarding clinical efficacy. We have previously shown that a low molecular weight peptide fraction (LMWPF) from poultry by-product hydrolysates possesses angiotensin-1-converting enzyme (ACE-1) and dipeptidyl-peptidase 4 (DPP4) inhibitory activities. The present study aimed to investigate the bioavailability of the bioactive peptides in the LMWPF. Prior to the investigation of bioavailability, a dipeptide YA was identified from this fraction as a dual inhibitor of ACE-1 and DPP4. Gastrointestinal (GI) stability and intestinal absorption of the bioactive peptides (i.e., YA as well as two previously reported bioactive dipeptides (VL and IY)) in the LMWPF were evaluated using the INFOGEST static in vitro digestion model and intestinal Caco-2 cell monolayer, respectively. Analysis of peptides after in vitro digestion confirmed that the dipeptides were resistant to the simulated GI conditions. After 4 hours of incubation, the concentration of the peptide from the apical side of the Caco-2 cell monolayer showed a significant decrease. However, the corresponding absorbed peptides were not detected on the basolateral side, suggesting that the peptides were not transported across the intestinal monolayer but rather taken up or metabolized by the Caco2 cells. Furthermore, when analyzing the gene expression of the Caco-2 cells upon peptide stimulation, a down-regulation of peptide transporters, the transcription factor CDX2, and the tight junction protein-1 (TJP1) was observed, suggesting the specific effects of the peptides on the Caco-2 cells. The study demonstrated that bioactive dipeptides found in the LMWPF were stable through in vitro GI digestion; however, the overall bioavailability may be hindered by inadequate uptake across the intestinal barrier.

ADA/CD26 axis increases intra-tumor PD-1+CD28+CD8+ T-cell fitness and affects NSCLC prognosis and response to ICB.

Improving cancer immunotherapy efficacy hinges on identifying key T-cell populations critical for tumor control and response to Immune Checkpoint Blockade (ICB). We have recently reported that while the co-expression of PD-1 and CD28 is associated with impaired functionality in peripheral blood, it significantly enhances T-cell fitness in the tumor site of non-small cell lung cancer (NSCLC) patients. To uncover the underlying mechanisms, we explored the role of CD26, a key player in T-cell activation through its interaction with adenosine deaminase (ADA), a crucial intra/extracellular enzyme able to neutralize local adenosine (ADO). We found that an autocrine ADA/CD26 axis enhances CD8+PD-1+CD28+ T-cell function, particularly within an immunosuppressive environment marked by CD39 expression. Then, we interrogated the TCGA and OAK datasets to gain insight into the prognostic/predictive potential of our findings. We identified a signature predicting overall survival (OS) in LUAD patients and response to atezolizumab in advanced LUAD cases. These findings suggest promising avenues for therapeutic intervention targeting the ADA/CD26 axis.

Unconventional T Cells Influence Clinical Outcome After Allogeneic Hematopoietic Cell Transplantation.

We evaluated the impact of early recovery of mucosal-associated invariant T cells (MAIT) and gamma-delta (γδ) T cells, especially Vδ2+ T cells, on the clinical outcomes of 76 patients who underwent allogeneic hematopoietic cell transplantation (allo-HCT). MAIT cells were identified at day 20-30 post-transplant using flow cytometry and defined as CD3+ TCRVα7.2+CD161+. Two subsets of Vδ2+ T cells were analyzed according to the expression of CD26. The cytotoxicity profile of MAIT and Vδ2+ T cells was analyzed according to the intracellular expression of perforin and granzyme B, and intracellular IFN-γ was evaluated after in vitro activation. CD26+Vδ2+ T cells displayed higher intracellular levels of IFN-γ, whereas CD26- Vδ2+ T were found to be more cytotoxic. Moreover, MAIT cell frequency was correlated with the frequency of Vδ2+ T cells with a better correlation observed with Vδ2+CD26+ than with the Vδ2+CD26- T cell subset. By using the composite endpoint graft-versus-host disease (GvHD)-free, relapse-free survival (GRFS) as the primary endpoint, we found that patients with a higher MAIT cell frequency at day 20-30 after allo-HCT had a significantly increased GRFS and a better overall survival (OS) and disease-free survival (DFS). Moreover, patients with a low CD69 expression by MAIT cells had an increased cumulative incidence of grade 2-4 acute GvHD (aGvHD). These results suggest that MAIT cell reconstitution may provide mitigating effects early after allo-HCT depending on their activation markers and functional status. Patients with a high frequency of Vδ2+CD26+ T cells had a significantly higher GRFS, OS and DFS, but there was no impact on cumulative incidence of grade 2-4 aGVHD, non-relapse mortality and relapse. These results revealed that the impact of Vδ2+ T cells on the success of allo-HCT may vary according to the frequency of the CD26+ subset.

Estrogen-mediated DNMT1 and DNMT3A recruitment by EZH2 silences miR-570-3p that contributes to papillary thyroid malignancy through DPP4.

BACKGROUND: Papillary thyroid carcinoma (PTC) is a common endocrine malignancy. Studies have indicated that estrogen can regulate the expression of miRNAs in numerous malignancies. MiR-570-3p has been shown to have a regulatory function in various cancers. However, studies of the regulatory function of miR-570-3p and a direct link between estrogen (especially estradiol E2) and miR-570-3p in PTC have not been done. METHODS: Expression of miR-570-3p and its downstream target DPP4 in PTC tissues and cells was predicted using bioinformatics and validated by qRT-PCR and western blot assays. We then performed a series of gain-and-loss experiments to assess the functional significance of miR-570-3p/DPP4 axis in PTC progression in vitro and in vivo. Additionally, the methylation of the miR-570-3p promoter region was examined via bioinformatics analysis and MSP. Finally, the effects of E2 on PTC progression and the correlation between DNMT1/DNMT3A and EZH2 were predicted by bioinformatic tools and proved by luciferase reporter, ChIP, and co-IP assays. RESULTS: In PTC tumor tissues and cell lines, there was a lower expression level and a higher methylation level of miR-570-3p compared to normal tissues and cell lines. DPP4 was identified as the downstream target of miR-570-3p. Overexpression of miR-570-3p reduced the proliferative, migratory, and invasive capabilities, and promoted apoptosis, while overexpression of DPP4 reversed these effects in PTC cells. It was also discovered that DNMT1 and DNMT3A increased the CpG methylation level of the miR-570-3p promoter in an EZH2-dependent manner, which led to decreased expression of miR-570-3p. Furthermore, we observed that estrogen (E2) enhanced the methylation of miR-570-3p and suppressed its expression levels, resulting in augmented tumor growth in vivo in PTC. CONCLUSION: Estrogen regulates the EZH2/DNMTs/miR-570-3p/DPP4 signaling pathway to promote PTC progression.

Computational Screening for the Dipeptidyl Peptidase-IV Inhibitory Peptides from Putative Hemp Seed Hydrolyzed Peptidome as a Potential Antidiabetic Agent.

Dipeptidyl peptidase-IV (DPPIV) inhibitory peptides are a class of antihyperglycemic drugs used in the treatment of type 2 diabetes mellitus, a metabolic disorder resulting from reduced levels of the incretin hormone GLP-1. Given that DPPIV degrades incretin, a key regulator of blood sugar levels, various antidiabetic medications that inhibit DPPIV, such as vildagliptin, sitagliptin, and linagliptin, are employed. However, the potential side effects of these drugs remain a matter of debate. Therefore, we aimed to investigate food-derived peptides from Cannabis sativa (hemp) seeds. Our developed bioinformatics pipeline was used to identify the putative hydrolyzed peptidome of three highly abundant proteins: albumin, edestin, and vicilin. These proteins were subjected to in silico digestion by different proteases (trypsin, chymotrypsin, and pepsin) and then screened for DPPIV inhibitory peptides using IDPPIV-SCM. To assess potential adverse effects, several prediction tools, namely, TOXINpred, AllerCatPro, and HemoPred, were employed to evaluate toxicity, allergenicity, and hemolytic effects, respectively. COPID was used to determine the amino acid composition. Molecular docking was performed using GalaxyPepDock and HPEPDOCK, 3D visualizations were conducted using the UCSF Chimera program, and MD simulations were carried out with AMBER20 MD software. Based on the predictive outcomes, FNVDTE from edestin and EAQPST from vicilin emerged as promising candidates for DPPIV inhibitors. We anticipate that our findings may pave the way for the development of alternative DPPIV inhibitors.

Comparative Binding Study of Gliptins to Bacterial DPP4-like Enzymes for the Treatment of Type 2 Diabetes Mellitus (T2DM).

The role of the gut microbiota and its interplay with host metabolic health, particularly in the context of type 2 diabetes mellitus (T2DM) management, is garnering increasing attention. Dipeptidyl peptidase 4 (DPP4) inhibitors, commonly known as gliptins, constitute a class of drugs extensively used in T2DM treatment. However, their potential interactions with gut microbiota remain poorly understood. In this study, we employed computational methodologies to investigate the binding affinities of various gliptins to DPP4-like homologs produced by intestinal bacteria. The 3D structures of DPP4 homologs from gut microbiota species, including Segatella copri, Phocaeicola vulgatus, Bacteroides uniformis, Parabacteroides merdae, and Alistipes sp., were predicted using computational modeling techniques. Subsequently, molecular dynamics simulations were conducted for 200 ns to ensure the stability of the predicted structures. Stable structures were then utilized to predict the binding interactions with known gliptins through molecular docking algorithms. Our results revealed binding similarities of gliptins toward bacterial DPP4 homologs compared to human DPP4. Specifically, certain gliptins exhibited similar binding scores to bacterial DPP4 homologs as they did with human DPP4, suggesting a potential interaction of these drugs with gut microbiota. These findings could help in understanding the interplay between gliptins and gut microbiota DPP4 homologs, considering the intricate relationship between the host metabolism and microbial communities in the gut.

Molecular design of peptide therapeutics via N-terminal modification.

Glucagon-like peptide-1, glucose-dependent insulinotropic polypeptide, and glucagon are three naturally occurring peptide hormones that mediate glucoregulation. Several agonists representing appropriately modified native ligands have been developed to maximize metabolic benefits with reduced side-effects and many have entered the clinic as type 2 diabetes and obesity therapeutics. In this work, we describe strategies for improving the stability of the peptide ligands by making them refractory to dipeptidyl peptidase-4 catalyzed hydrolysis and inactivation. We describe a series of alkylations with variations in size, shape, charge, polarity, and stereochemistry that are able to engender full activity at the receptor(s) while simultaneously resisting enzyme-mediated degradation. Utilizing this strategy, we offer a novel method of modulating receptor activity and fine-tuning pharmacology without a change in peptide sequence.

Mid1 promotes synovitis in rheumatoid arthritis via ubiquitin-dependent post-translational modification.

INTRODUCTION: Current anti-rheumatic drugs are primarily modulating immune cell activation, yet their effectiveness remained suboptimal. Therefore, novel therapeutics targeting alternative mechanisms, such as synovial activation, is urgently needed. OBJECTIVES: To explore the role of Midline-1 (Mid1) in synovial activation. METHODS: NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were used to establish a subcutaneous xenograft model. Wild-type C57BL/6, Mid1-/-, Dpp4-/-, and Mid1-/-Dpp4-/- mice were used to establish a collagen-induced arthritis model. Cell viability, cell cycle, qPCR and western blotting analysis were used to detect MH7A proliferation, dipeptidyl peptidase-4 (DPP4) and Mid1 levels. Co-immunoprecipitation and proteomic analysis identified the candidate protein of Mid1 substrates. Ubiquitination assays were used to determine DPP4 ubiquitination status. RESULTS: An increase in Mid1, an E3 ubiquitin ligase, was observed in human RA synovial tissue by GEO dataset analysis, and this elevation was confirmed in a collagen-induced mouse arthritis model. Notably, deletion of Mid1 in a collagen-induced arthritis model completely protected mice from developing arthritis. Subsequent overexpression and knockdown experiments on MH7A, a human synoviocyte cell line, unveiled a previously unrecognized role of Mid1 in synoviocyte proliferation and migration, the key aspects of synovial activation. Co-immunoprecipitation and proteomic analysis identified DPP4 as the most significant candidate of Mid1 substrates. Mechanistically, Mid1 promoted synoviocyte proliferation and migration by inducing ubiquitin-mediated proteasomal degradation of DPP4. DPP4 deficiency led to increased proliferation, migration, and inflammatory cytokine production in MH7A, while reconstitution of DPP4 significantly abolished Mid1-induced augmentation of cell proliferation and activation. Additionally, double knockout model showed that DPP4 deficiency abolished the protective effect of Mid1 defect on arthritis. CONCLUSION: Overall, our findings suggest that the ubiquitination of DPP4 by Mid1 promotes synovial cell proliferation and invasion, exacerbating synovitis in RA. These results reveal a novel mechanism that controls synovial activation, positioning Mid1 as a promising target for therapeutic intervention in RA.

Identification of DPP-IV inhibitory peptides derived from buffalo colostrum: Mining through bioinformatics, in silico and in vitro approaches.

Bioactive peptides derived from foods provide physiological health benefits beyond nutrition. This study focused on profiling small peptide inhibitors against two key serine proteases, dipeptidyl peptidase-IV (DPP-IV) and prolyl oligopeptidase (POP). DPP-IV is a well-known protein involved in diverse pathways regulating inflammation, renal, cardiovascular physiology, and glucose homeostasis. POP is yet another key target protein for neurodegenerative disorders. The study evaluated peptide libraries of buffalo colostrum whey and fat globule membrane proteins derived from pepsin and pepsin-pancreatin digestion through in silico web tools and structure-based analysis by molecular docking and binding free-energy estimation, followed by in vitro assay for DPP-IV inhibition for the lead peptides. The bioinformatic study indicated 49 peptides presented motifs with DPP-IV inhibition while 5 peptides with sequences for POP inhibition. In the molecular docking interactions study, 22 peptides interacted with active site residues of DPP-IV and 3 peptides with that of POP. The synthesized peptides, SFVSEVPEL and LTFQHNF inhibited DPP-IV in vitro with an IC50 of 193.5 µM and 1.782 mM, respectively. The study revealed the key residues for inhibition of DPP-IV and POP thus affirming the DPP-IV inhibitory potential of milk-derived peptides.

Molecular Mechanistic Insights into Dipeptidyl Peptidase-IV Inhibitory Peptides to Decipher the Structural Basis of Activity.

Dipeptidyl peptidase-IV (DPP-IV) inhibiting peptides have attracted increased attention because of their possible beneficial effects on glycemic homeostasis. However, the structural basis underpinning their activities has not been well understood. This study combined computational and in vitro investigations to explore the structural basis of DPP-IV inhibitory peptides. We first superimposed the Xaa-Pro-type peptide-like structures from several crystal structures of DPP-IV ligand-protein complexes to analyze the recognition interactions of DPP-IV to peptides. Thereafter, a small set of Xaa-Pro-type peptides was designed to explore the effect of key interactions on inhibitory activity. The intramolecular interaction of Xaa-Pro-type peptides at the first and third positions from the N-terminus was pivotal to their inhibitory activities. Residue interactions between DPP-IV and residues of the peptides at the fourth and fifth positions of the N-terminus contributed significantly to the inhibitory effect of Xaa-Pro-type tetrapeptides and pentapeptides. Based on the interaction descriptors, quantitative structure-activity relationship (QSAR) studies with the DPP-IV inhibitory peptides resulted in valid models with high R2 values (0.90 for tripeptides; 0.91 for tetrapeptides and pentapeptides) and Q2 values (0.33 for tripeptides; 0.68 for tetrapeptides and pentapeptides). Taken together, the structural information on DPP-IV and peptides in this study facilitated the development of novel DPP-IV inhibitory peptides.

Combination of spironolactone and DPP-4 inhibitors for treatment of SARS-CoV-2 infection: a literature review.

Coronavirus disease 2019 (COVID-19) is still causing hospitalization and death, and vaccination appears to become less effective with each emerging variant. Spike, non-spike, and other possible unrecognized mutations have reduced the efficacy of recommended therapeutic approaches, including monoclonal antibodies, plasma transfusion, and antivirals. SARS-CoV-2 binds to angiotensin-converting enzyme 2 (ACE2) and probably dipeptidyl peptidase 4 (DPP-4) to initiate the process of endocytosis by employing host proteases such as transmembrane serine protease-2 (TMPRSS-2) and ADAM metallopeptidase domain 17 (ADAM17). Spironolactone reduces the amount of soluble ACE2 and antagonizes TMPRSS-2 and ADAM17. DPP-4 inhibitors play immunomodulatory roles and may block viral entry. The efficacy of treatment with a combination of spironolactone and DPP-4 inhibitors does not appear to be affected by viral mutations. Therefore, the combination of spironolactone and DPP-4 inhibitors might improve the clinical outcome for COVID-19 patients by decreasing the efficiency of SARS-CoV-2 entry into cells and providing better anti-inflammatory, antiproliferative, and antifibrotic effects than those achieved using current therapeutic approaches such as antivirals and monoclonal antibodies.

Enrichment and Quantitation of Dipeptidyl Peptidase IV Inhibitory Peptides in Quinoa upon Systematic Malting.

Food-derived peptides with an inhibitory effect on dipeptidyl peptidase IV (DPP-IV) can be used as an additive treatment for type 2 diabetes. The inhibitory potential of food depends on technological protein hydrolysis and gastrointestinal digestion, as the peptides only act after intestinal resorption. The effect of malting as a hydrolytic step on the availability of these peptides in grains has yet to be investigated. In this study, quinoa was malted under systematic temperature, moisture, and time variations. In the resulting malts, the DPP-IV inhibition reached a maximum of 45.02 (±10.28) %, whereas the highest overall concentration of literature-known inhibitory peptides was 4.07 µmol/L, depending on the malting parameters. After in vitro gastrointestinal digest, the inhibition of most malts, as well as the overall concentration of inhibitory peptides, could be increased significantly. Additionally, the digested malts showed higher values in both the inhibition and the peptide concentration than the unmalted quinoa. Concerning the malting parameters, germination time had the highest impact on the inhibition and the peptide concentration after digest. An analysis of the protein sizes before and after malting gave first hints toward the origin of these peptides, or their precursors, in quinoa.

Discovery of Nine Dipeptidyl Peptidase-4 Inhibitors from Coptis chinensis Using Virtual Screening, Bioactivity Evaluation, and Binding Studies.

The objective of this study was to identify multiple alkaloids in Coptis chinensis that demonstrate inhibitory activity against DPP-4 and systematically evaluate their activity and binding characteristics. A combined strategy that included molecular docking, a DPP-4 inhibition assay, surface plasmon resonance (SPR), and a molecular dynamics simulation technique was employed. The results showed that nine alkaloids in Coptis chinensis directly inhibited DPP-4, with IC50 values of 3.44-53.73 µM. SPR-based binding studies revealed that these alkaloids display rapid binding and dissociation characteristics when interacting with DPP-4, with KD values ranging from 8.11 to 29.97 µM. A molecular dynamics analysis revealed that equilibrium was rapidly reached by nine DPP-4-ligand systems with minimal fluctuations, while binding free energy calculations showed that the ∆Gbind values for the nine test compounds ranged from -31.84 to -16.06 kcal/mol. The most important forces for the binding of these alkaloids with DPP-4 are electrostatic interactions and van der Waals forces. Various important amino acid residues, such as Arg125, His126, Phe357, Arg358, and Tyr547, were involved in the inhibition of DPP-4 by the compounds, revealing a mechanistic basis for the further optimization of these alkaloids as DPP-4 inhibitors. This study confirmed nine alkaloids as direct inhibitors of DPP-4 and characterized their binding features, thereby providing a basis for further research and development on novel DPP-4 inhibitors.

In Vitro Investigation of Therapy-Induced Senescence and Senescence Escape in Breast Cancer Cells Using Novel Flow Cytometry-Based Methods.

Although cellular senescence was originally defined as an irreversible form of cell cycle arrest, in therapy-induced senescence models, the emergence of proliferative senescence-escaped cancer cells has been reported by several groups, challenging the definition of senescence. Indeed, senescence-escaped cancer cells may contribute to resistance to cancer treatment. Here, to study senescence escape and isolate senescence-escaped cells, we developed novel flow cytometry-based methods using the proliferation marker Ki-67 and CellTrace CFSE live-staining. We investigated the role of a novel senescence marker (DPP4/CD26) and a senolytic drug (azithromycin) on the senescence-escaping ability of MCF-7 and MDA-MB-231 breast cancer cells. Our results show that the expression of DPP4/CD26 is significantly increased in both senescent MCF-7 and MDA-MB-231 cells. While not essential for senescence induction, DPP4/CD26 contributed to promoting senescence escape in MCF-7 cells but not in MDA-MB-231 cells. Our results also confirmed the potential senolytic effect of azithromycin in senescent cancer cells. Importantly, the combination of azithromycin and a DPP4 inhibitor (sitagliptin) demonstrated a synergistic effect in senescent MCF-7 cells and reduced the number of senescence-escaped cells. Although further research is needed, our results and novel methods could contribute to the investigation of the mechanisms of senescence escape and the identification of potential therapeutic targets. Indeed, DPP4/CD26 could be a promising marker and a novel target to potentially decrease senescence escape in cancer.

Modulation of Paracellular Permeability in SARS-CoV-2 Blood-to-Brain Transcytosis.

SARS-CoV-2 primarily infects the lungs via the ACE2 receptor but also other organs including the kidneys, the gastrointestinal tract, the heart, and the skin. SARS-CoV-2 also infects the brain, but the hematogenous route of viral entry to the brain is still not fully characterized. Understanding how SARS-CoV-2 traverses the blood-brain barrier (BBB) as well as how it affects the molecular functions of the BBB are unclear. In this study, we investigated the roles of the receptors ACE2 and DPP4 in the SARS-CoV-2 infection of the discrete cellular components of a transwell BBB model comprising HUVECs, astrocytes, and pericytes. Our results demonstrate that direct infection on the BBB model does not modulate paracellular permeability. Also, our results show that SARS-CoV-2 utilizes clathrin and caveolin-mediated endocytosis to traverse the BBB, resulting in the direct infection of the brain side of the BBB model with a minimal endothelial infection. In conclusion, the BBB is susceptible to SARS-CoV-2 infection in multiple ways, including the direct infection of endothelium, astrocytes, and pericytes involving ACE2 and/or DPP4 and the blood-to-brain transcytosis, which is an event that does not require the presence of host receptors.