Acid Peptide Research Articles

Abstract 3738: A novel mechanism of STAT3 activation by genotoxic chemotherapy

Abstract CARP-1, a perinuclear phospho-protein, is a biphasic regulator of cell survival and apoptosis signaling induced by genotoxic drugs. We have previously found that apoptosis induced by genotoxic drugs involved elevated levels of CARP-1. Exposure to genotoxic drugs also induced CARP-1 interaction with NF-κB kinase subunit γ (aka, NEMO) to promote NF-κB activation and cell survival. To further elucidate chemotherapy-activated, CARP-1-dependent cell survival mechanisms, we UV cross-linked protein extracts from Adriamycin-treated HeLa cervical cancer cells with a CARP-1 (614-638) peptide, and conducted liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the peptide-bound protein complexes. This experiment revealed STAT3 interaction with CARP-1 (614-638) peptide. Our mutagenesis and co-IP-WB experiments revealed CARP-1 (550-650) directly interacts with a 40 amino acid peptide located in the STAT3 DNA binding domain. Overexpression of mutant STAT3 with in-frame-deletion of CARP-1/CCAR1-binding epitope (CE) peptide (Gst-STAT3 (ΔCE) mutant), but not Gst-STAT3 (WT), failed to translocate to nucleus in Adriamycin-treated cells. Disruption of STAT3-CARP-1 interaction or expression of STAT3 (ΔCE) mutant abolished Adriamycin-induced STAT3 Y705 and S727 phosphorylation. Collectively our data demonstrate that CARP-1 interaction with STAT3 regulates genotoxic chemotherapy-activated STAT3 and its nuclear translocation to promote cancer cell survival and growth. Citation Format: Jaganathan Venkatesh, Magesh Muthu, Nuwan C. Acharige, Mary K. Pflum, Arun K. Rishi. A novel mechanism of STAT3 activation by genotoxic chemotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3738.

Abstract 3538: Endogenous HiBiT-tagging of PAX3-FOXO1 identifies potent suppressors of PAX3-FOXO1 protein levels by high-throughput screening

Abstract Background: Oncogenic fusion genes are attractive therapeutic targets due to their tumor-specific expression and driver roles in cancers. PAX3-FOXO1 (P3F) is the dominant oncogenic driver of fusion-positive rhabdomyosarcoma (FP-RMS) with no targeted therapy. We developed methods to directly measure endogenous P3F protein levels amenable to high-throughput drug screens to identify suppressors of P3F. Methods: HiBiT tag, an 11 amino acid peptide of the small fragment of NanoLuc luciferase, was inserted into the endogenous P3F using CRISPR-Cas9 in FP-RMS cell lines RH4 and SCMC. Western analysis was used for HiBiT tag validation and confirmation of P3F suppression. RNA-seq and ChIP-seq were used to assess transcriptomics and DNA binding of HiBiT-tagged P3F (P3F-HiBiT) respectively. High-throughput drug screen using Nano-Glo luciferase assay was performed using the Mechanism Interrogation PlatE (MIPE 5.0) drug library, which included 2,480 drugs with known mechanisms of action. CellTiter-Glo was used to monitor cell viability. We identified drugs that suppressed P3F by Nano-Glo without acute cytotoxicity by CellTiter-Glo at an early 24-hour timepoint. Mouse xenograft model of FP-RMS was used to investigate in vivo efficacy of top hits. Results: We validated HiBiT tagging of P3F and not the wild-type FOXO1 by Western analysis. We showed that the HiBiT tag did not change the function of P3F by transducing human fibroblasts with P3F-HiBiT versus unmodified P3F. Gene Set Enrichment Analysis (GSEA) of RNA-seq showed that P3F-HiBiT activated the same downstream target genes as unmodified P3F. ChIP-seq using HiBiT antibody in HiBiT-tagged FP-RMS cell lines RH4 and SCMC matched the genomic locations from ChIP-seq with P3F antibody in parental RH4 and SCMC. Using a cutoff of Area Under the Curve (AUC) of CellTiter-Glo - AUC of Nano-Glo > 90, in both RH4 and SCMC, identified 182 compounds. Filtering for drugs with ≥ 3 hits for the same target identified 14 drug classes that suppressed P3F protein level including HDAC inhibitors (3), mTOR inhibitors (4), CDK inhibitors (8), and BRD4 inhibitors (3). One top hit was the CDK inhibitor TG02 (Zotiraciclib), currently in human trials. TG02 suppressed P3F protein levels by Nano-Glo and Western analysis. We confirmed induction of apoptosis by PARP cleavage in a panel of FP-RMS cell lines. GSEA analysis of RNA-seq after treatment with TG02 showed marked suppression of P3F target gene sets. TG02 also significantly delayed tumor progression of established tumors in a mouse xenograft model of FP-RMS without weight loss. Conclusion and Future Directions:By HiBiT tagging the fusion oncogene P3F, we identified 182 compounds that suppress P3F levels of which TG02 was a top hit that also showed in vivo efficacy. Drug combination studies are currently underway to identify synergistic suppressors of P3F protein levels that can be translated into clinical trials. Citation Format: Yong Yean Kim, Robert G. Hawley, Mehal Churiwal, Teresa S. Hawley, Christine N. Evans, Raj Chari, David Milewski, Ranuka Sinniah, Young K. Song, Hsien-Chao Chou, Xinyu Wen, Ying Pang, Jing Wu, Craig J. Thomas, Jun S. Wei, Michele Ceribelli, Javed Khan. Endogenous HiBiT-tagging of PAX3-FOXO1 identifies potent suppressors of PAX3-FOXO1 protein levels by high-throughput screening. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3538.

Abstract 3071: Pituitary adenylate cyclase activating polypeptide stimulates NSCLC growth by increasing HER4 tyrosine phosphorylation in a neuregulin-1 dependent manner

Abstract Pituitary adenylate cyclase activating polypeptide [PACAP] is a 27 amino acid peptide which stimulates the growth of numerous cancers including non-small cell lung cancer (NSCLC). PACAP binds with high affinity to the type B G protein-coupled receptor [GPCR] PAC1 (Moody et al., Peptides 2021; 137: 170480). PAC1 activation causes phosphatidyl inositol [PI] turnover stimulating matrix metalloprotease [MMP]. By ELISA, PACAP increased secretion of neuregulin [NRG1] from NSCLC cells. NRG1 binds with high affinity to HER4. Here the ability of PAC1 to regulate transactivation of HER4 was investigated. By RT-PCR and Western blot, PAC1, NRG1 and HER4 but not HER3 or NRG2 were detected in NCI-H522 and H661 cells. Adding PACAP-27 (0.1 μM) or NRG1 (0.01 μg/ml) to NCI-H522 or H661 cells increased P-Tyr1284-HER4 3-fold after 5 min. Using immunoprecipitation techniques, adding PACAP TO NSCLC cells increased formation of HER4 homodimers and HER4-EGFR as well as HER4-HER2 heterodimers. The increase in P-HER4 caused by PACAP-27 was impaired by PACAP(6-38) [PAC1 antagonist], HER4 siRNA, NRG1 antibody [Ab] or ibrutinib [TKI]. The PAC1 regulation of HER4 transactivation is impaired by GM6001 [MMP inhibitor]. The clonal growth of NSCLC cells was stimulated by PACAP-27 or NRG1 but inhibited by PACAP(6-38) or ibrutinib. The results indicate that PACAP-27 stimulates the growth of NSCLC due to release of NRG1 which activates receptor tyrosine kinases such as HER4. Citation Format: Terry W. Moody, Irene Ramos Alvarez, Robert T. Jensen. Pituitary adenylate cyclase activating polypeptide stimulates NSCLC growth by increasing HER4 tyrosine phosphorylation in a neuregulin-1 dependent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3071.

PD04-06 IDENTIFYING NOVEL REGULATORS OF MACROPHAGE TUMOR CELL METABOLIC INTERACTIONS IN PROSTATE CANCER

You have accessJournal of UrologyCME1 Apr 2023PD04-06 IDENTIFYING NOVEL REGULATORS OF MACROPHAGE TUMOR CELL METABOLIC INTERACTIONS IN PROSTATE CANCER Asmaa Elkenawi, Ryan Putney, Amparo Serna, Jasreman Dhillon, and Kosj Yamoah Asmaa ElkenawiAsmaa Elkenawi More articles by this author , Ryan PutneyRyan Putney More articles by this author , Amparo SernaAmparo Serna More articles by this author , Jasreman DhillonJasreman Dhillon More articles by this author , and Kosj YamoahKosj Yamoah More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003228.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: African American men (AA) are more than twice as likely to die of prostate cancer (PCa) compared to European American men (EA). Among many contributing factors, unraveling the metabolic derangements in PCa of AA holds a promise in reducing health disparity. METHODS: We examined publicly available prostate cancer datasets for race dependent differential gene expression. We prospectively examined the differential gene expression of PCa from clinically matched AA and EA in the VANDAAM clinical trial. The VANDAAM study is a validation study of DecipherTM genomic testing in 240 men with localized PCa. We combined computational and experimental approaches to explore the molecular mechanisms of top candidate genes including these involved in metabolism. We then developed a novel metabolomic workflow that profiles patient- specific nutrient consumption to predict anti-cancer efficacy and validate novel biomarkers. RESULTS: We identified aminopeptidase N (ANPEP) as one of the top candidate genes to be overexpressed in AA. Aminopeptidase N is involved in endocytosis, cholesterol and amino acid transport and peptide hydrolysis. Unbiased computational analyses of the VANDAAM revealed that ANPEP correlates with signatures of cholesterol transport, estrogen and androgen receptor (AR) signaling. Based on our recent study demonstrating dominance of these signatures in macrophage rich PCa, we reasoned that ANPEP expression may be driven in part by high macrophage infiltration in AA. Thus, we compared immune cell repertoire in patients with high ANPEP and low ANPEP by deconvoluting immune cell content using the in silico approach, CIBERSORT. These analyses illustrated that only AA patients with high ANPEP expression significantly accumulated high content of M1 inflammatory macrophages. Immune phenotyping of prostate tumors demonstrated that ANPEP indeed represents a marker of M1 inflammatory macrophages. To unravel role of ANPEP in regulating cholesterol and amino acid transport, we developed a Liquid chromatography-high resolution mass spectrometry (LC-HRMS) -based approach to measure cholesterol and amino acids in explants derived from AA and EA prostate cancer patients. This innovative approach enables us to perform mechanistic studies by determining metabolic dependency on specific amino acids as a function ANPEP or macrophage infiltration. CONCLUSIONS: ANPEP represents a macrophage related protein in PCa with a potential role in cholesterol, amino acid transport and / or androgen signaling. Source of Funding: Prostate Cancer Foundation (AE), Urology Care Foundation (AE), Prostate Cancer Foundation (KY) and Department of Defense award CDMRP-PC181013 (KY) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e144 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Asmaa Elkenawi More articles by this author Ryan Putney More articles by this author Amparo Serna More articles by this author Jasreman Dhillon More articles by this author Kosj Yamoah More articles by this author Expand All Advertisement PDF downloadLoading ...

Development of a Nomogram for Predicting Mortality Risk in Sepsis Patients During Hospitalization: A Retrospective Study.

We attempted to establish a model for predicting the mortality risk of sepsis patients during hospitalization. Data on patients with sepsis were collected from a clinical record mining database, who were hospitalized at the Affiliated Dongyang Hospital of Wenzhou Medical University between January 2013 and August 2022. These included patients were divided into modeling and validation groups. In the modeling group, the independent risk factors of death during hospitalization were determined using univariate and multi-variate regression analyses. After stepwise regression analysis (both directions), a nomogram was drawn. The discrimination ability of the model was evaluated using the area under the curve (AUC) of the receiver operating characteristic (ROC) curve, and the GiViTI calibration chart assessed the model calibration. The Decline Curve Analysis (DCA) was performed to evaluate the clinical effectiveness of the prediction model. Among the validation group, the logistic regression model was compared to the models established by the SOFA scoring system, random forest method, and stacking method. A total of 1740 subjects were included in this study, 1218 in the modeling population and 522 in the validation population. The results revealed that serum cholinesterase, total bilirubin, respiratory failure, lactic acid, creatinine, and pro-brain natriuretic peptide were the independent risk factors of death. The AUC values in the modeling group and validation group were 0.847 and 0.826. The P values of calibration charts in the two population sets were 0.838 and 0.771. The DCA curves were above the two extreme curves. Moreover, the AUC values of the models established by the SOFA scoring system, random forest method, and stacking method in the validation group were 0.777, 0.827, and 0.832, respectively. The nomogram model established by combining multiple risk factors could effectively predict the mortality risk of sepsis patients during hospitalization.

Comprehensive metabolomic and lipidomic alterations in response to heat stress during seed germination and seedling growth of Arabidopsis.

Temperature affects seed germination and seedling growth, which is a critical and complex stage in plant life cycle. However, comprehensive metabolic basis on temperature implicating seed germination and seedling growth remains less known. Here, we applied the high-throughput untargeted metabolomic and advanced shotgun lipidomic approaches to profile the Arabidopsis 182 metabolites and 149 lipids under moderate (22°C, 28°C) and extreme high (34°C, 40°C) temperatures. Our results showed that a typical feature of the metabolism related to organic acids/derivates and amines was obviously enriched at the moderate temperature, which was implicated in many cellular responses towards tricarboxylic acid cycle (TCA), carbohydrates and amino acids metabolism, peptide biosynthesis, phenylpropanoid biosynthesis and indole 3-acetate (IAA) biosynthetic pathway. Whereas, under extreme high temperatures, there was no seed germination, but 148 out of total 182 metabolites were highly enriched, involving in the galactose metabolism, fatty acid degradation, tryptophan/phenylalanine metabolism, and shikimic acid-mediated pathways especially including alkaloids metabolism and glucosinolate/flavone/flavonol biosynthesis. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) also exhibited the gradually increased tendency from moderate temperatures to extreme high temperatures; whereas phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylglycerol (PG), monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol (SQDG) were contrary to decrease. Another typical feature of the distinguished metabolites between 22°C and 28°C, the TCA, disaccharides, nucleotides, polypeptides, SQDG and the biosynthesis of fatty acids and glucobrassicin-mediated IAA were obviously decreased at 28°C, while amino acids, trisaccharides, PE, PC, PA, PS, MGDG, DGDG and diacylglycerol (DAG) preferred to enrich at 28°C, which characterized the alteration of metabolites and lipids during fast seedling growth. Taking together, our results provided the comprehensive metabolites phenotyping, revealed the characteristics of metabolites necessary for seed germination and/or seedling growth under different temperatures, and provided insights into the different metabolic regulation of metabolites and lipid homeostasis for seed germination and seedling growth.

Bufalin-loaded vitamin E succinate-grafted chitosan oligosaccharide/RGD-conjugated TPGS mixed micelles inhibit intraperitoneal metastasis of ovarian cancer

BackgroundIntraperitoneal metastasis is one of the major causes of the high mortality rate of ovarian cancer. Bufalin (BU) is an effective component of the traditional Chinese medicine Chansu that exerts antitumor effects, including metastasis inhibition. In our previous studies, we found that BU inhibited the migration and invasion of ovarian cancer cells. However, the application of BU is limited due to its insolubility, toxicity and imprecise targeting. The aim of this study was to use vitamin E succinate (VES)-grafted chitosan oligosaccharide (CSO)/arginine-glycine-aspartic acid peptide (RGD)-conjugated d-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) mixed micelles (VeC/T-RGD MMs) to deliver BU to ovarian cancer cells to inhibit intraperitoneal metastasis. Moreover, the toxicity of BU was reduced by coating it with the mixed micelles to increase its biocompatibility for practical applications.ResultsThe BU-loaded VeC/T-RGD MMs (BU@MMs) had an average diameter of 161 ± 1.4 nm, a zeta potential of 4.49 ± 1.54 mV and a loading efficiency of 2.54%. The results showed that these micelles inhibited cell proliferation, induced apoptosis, and reduced the migration and invasion of A2780 and SKOV3 cells. Further studies indicated that BU@MMs enhanced the levels of e-cadherin and decreased the expression levels of N-cadherin, vimentin and Snail in vitro. In addition, the mixed micelles effectively enhanced the anticancer effect and inhibited intraperitoneal metastasis in intraperitoneal metastatic models. The BU@MMs exhibited fewer toxic side effects than BU, indicating better biocompatibility and biosafety for in vivo applications.ConclusionsOur studies show that BU@MMs are a potential multifunctional nano-drug delivery system that can effectively inhibit the intraperitoneal metastasis of ovarian cancer.

Analytical Method Development and Validation for the Spectrophotometric Estimation of Hipuuric Acid Prodrug (Methenamine Hippurate)

Hippuric acid, pronounced as n-benzoyl glycine, is utilized in the study of nutrition, small bioactive molecules, amino acid derivatives, peptide synthesis, chemical synthesis, cell biology, and chemical biology. It can be found in urine and is produced when glycine and benzoic acid combine. Treatment of urinary tract infections (UTIs) is challenging due to the development of resistant bacterial strains brought on by repeated use of antibiotics and chemotherapy. Methenamine hippurate as a pharmaceutical dosage form hasn’t been the subject of any published research, hence a UV-visible spectrophotometric analytical method development has to be created. Hippuric acid (10 g/mL) solution was made, and when it was scanned at 200–400 nm with distilled water as the green solvent, it was discovered that the maximum absorbance was at 228 nm. The linearity concentration range was found with an R2 = 0.998 correlation coeffi cient. The recovery experiment, which was conducted at three levels of 80, 100, and 120%, was used to evaluate the procedure’s accuracy. The recovery rate was discovered to be between 90 and 120%. The % RSD < 2 it proves that the method is Precise. The results confi rmed the approach’s high capacity to detect Hippuric acid and were consistent with industry standards. LoD and LoQ were discovered to be 0.169 and 0.048, respectively. This approach results in hipuuric acid in formulations

Novel neuroendocrine role of γ-aminobutyric acid and gastrin-releasing peptide in the host response to influenza infection

Gastrin-releasing peptide (GRP), an evolutionarily conserved neuropeptide, significantly contributes to influenza-induced lethality and inflammation in rodent models. Because GRP is produced by pulmonary neuroendocrine cells (PNECs) in response to γ-aminobutyric acid (GABA), we hypothesized that influenza infection promotes GABA release from PNECs that activate GABAB receptors on PNECs to secrete GRP. Oxidative stress was increased in the lungs of influenza A/PR/8/34 (PR8)-infected mice, as well as serum glutamate decarboxylase 1, the enzyme that converts L-glutamic acid into GABA. The therapeutic administration of saclofen, a GABAB receptor antagonist, protected PR8-infected mice, reduced lung proinflammatory gene expression of C-C chemokine receptor type 2 (Ccr2), cluster of differentiation 68 (Cd68), and Toll like receptor 4 (Tlr4) and decreased the levels of GRP and high-mobility group box 1 (HMGB1) in sera. Conversely, baclofen, a GABAB receptor agonist, significantly increased the lethality and inflammatory responses. The GRP antagonist, NSC77427, as well as the GABAB antagonist, saclofen, blunted the PR8-induced monocyte infiltration into the lung. Together, these data provide the first report of neuroregulatory control of influenza-induced disease.

Plasma bioactive adrenomedullin predicts outcome after acute stroke in early rehabilitation

An early and reliable prediction of outcomes after stroke is important for early effective stroke management and the adequate optimal planning of post-stroke rehabilitation and long-term care. Bioactive adrenomedullin (bio-ADM) is a 52-amino acid peptide that is an important peptide hormone in nervous system diseases. The aim of this study was to investigate the prognostic value of bio-ADM on outcomes after rehabilitation in patients with stroke. A total of 557 consecutive patients with a primary diagnosis of ischemic or hemorrhagic stroke (age 69.6–12.9 years, male 51.3%, ischemic stroke 72.5%), who were admitted to an in-patient early rehabilitation center directly after discharge from acute stroke hospital care, were enrolled in this prospective observational study. Plasma concentrations of bio-ADM were determined by using a chemiluminescence immunoassay (functional assay sensitivity 8 pg/ml). The early rehabilitation barthel index (ERBI) was used for the neurological assessment of the patients. The plasma bio-ADM level was analyzed in association with 6-month all-cause mortality as well as a composite outcome of all-cause mortality, unscheduled re-hospitalization, or transfer to a long-term care facility in a vegetative or minimally conscious state. Bio-ADM levels significantly increased in patients with ischemic stroke who died compared to surviving patients (40.4 pg/ml vs. 23.8 pg/ml, p < 0.001) or in those with composite outcomes compared to those with no events (36.9 pg/ml vs. 23.5 pg/ml, p < 0.001). Six-month all-cause mortality was higher in all patients with bio-ADM levels > 70 pg/ml (HR 4.83 [CI 2.28–10.2]). Patients with bio-ADM levels > 70 pg/ml also had higher rates of 6-month composite outcomes (HR 3.82 [CI 2.08–7.01]). Bio-ADM was an independent predictor of all-cause mortality and 6-month composite outcomes after adjusting for age, gender, and ERBI (adjusted OR 1.5; 95% CI 1.0–2.1; p = 0.047 and adjusted OR 1.48; 95% CI 1.1–2.0; p = 0.01, respectively). Bio-ADM may be a suitable novel biomarker to assess the outcomes of patients in rehabilitation after acute stroke. Elevated bio-ADM concentrations may have prognostic value for fatal and nonfatal events in patients with ischemic stroke during early rehabilitation.

Pmu1a, a novel spider toxin with dual inhibitory activity at pain targets hNaV 1.7 and hCaV 3 voltage-gated channels.

Venom-derived peptides targeting ion channels involved in pain are regarded as a promising alternative to current, and often ineffective, chronic pain treatments. Many peptide toxins are known to specifically and potently block established therapeutic targets, among which the voltage-gated sodium and calcium channels are major contributors. Here, we report on the discovery and characterization of a novel spider toxin isolated from the crude venom of Pterinochilus murinus that shows inhibitory activity at both hNaV 1.7 and hCaV 3.2 channels, two therapeutic targets implicated in pain pathways. Bioassay-guided HPLC fractionation revealed a 36-amino acid peptide with three disulfide bridges named μ/ω-theraphotoxin-Pmu1a (Pmu1a). Following isolation and characterization, the toxin was chemically synthesized, and its biological activity was further assessed using electrophysiology, revealing Pmu1a to be a toxin that potently blocks both hNaV 1.7 and hCaV 3. NMR structure determination of Pmu1a shows an inhibitor cystine knot (ICK) fold that is characteristic of many spider peptides. Combined, these data show the potential of Pmu1a as a basis for the design of compounds with dual activity at the therapeutically relevant hCaV 3.2 and hNaV 1.7 voltage-gated channels.

Spider venom-derived peptide JZTX-14 prevents migration and invasion of breast cancer cells via inhibition of sodium channels.

Nav1.5 channel is crucial for the proliferation and migration of breast cancer cells. In this study, we investigated the anticancer effect of JZTX-14, a natural peptide considered an effective antagonist of Nav1.5. First, we successfully isolated and purified the 31amino acid peptide JZTX-14 containing three pairs of disulfide bonds from spider venom and synthesised JZTX-14 by solid phase synthesis. We then predicted their physiochemical properties and structures in the peptide database. Further, we investigated the effects of natural and synthetic JZTX-14 on the proliferation and migration of MDA-MB-231 breast cancer cells via modulation of sodium current through the Nav1.5 channel. The results showed that both synthetic and natural JZTX-14 inhibited Nav1.5 currents, indicating the successful synthesis of JZTX-14. However, JZTX-14 did not affect MDA-MB-231 cell proliferation but inhibited its migration. Transcriptome analysis revealed that JZTX-14 downregulated S100A4 and FBXO2 and upregulated SERPINB2 in MDA-MB-231 cells. Western blot analysis demonstrated an increased level of the epithelial marker, E-cadherin, and decreased levels of the mesenchymal markers, N-cadherin and vimentin, and matrix metalloproteinase (MMP2), indicating the possible underlying mechanism of the inhibition of MDA-MB-231 cell migration by JZTX-14. This study provides a new target for inhibiting breast cancer metastasis and identifies a potent natural peptide for treating Triple-negative breast cancer.

Dietary Protection against Cognitive Impairment, Neuroinflammation and Oxidative Stress in Alzheimer’s Disease Animal Models of Lipopolysaccharide-Induced Inflammation

Alzheimer's disease (AD) is a rapidly growing epidemic with a heavy social and economic burden. Evidence suggests that systemic inflammation, dysregulation of the immune response and the resulting neuroinflammation and neurodegeneration play a significant role in AD pathogenesis. Currently, given that there is no fully convincing cure for AD, the interest in lifestyle factors (such as diet), which potentially delay onset and reduce the severity of symptoms, is increasing. This review is aimed at summarizing the effects of dietary supplementation on cognitive decline, neuroinflammation and oxidative stress in AD-like animal models with a focus on neuroinflammation induced by lipopolysaccharide (LPS) injection, which mimics systemic inflammation in animals. The compounds reviewed include curcumin, krill oil, chicoric acid, plasmalogens, lycopene, tryptophan-related dipeptides, hesperetin and selenium peptides. Despite the heterogeneity of these compounds, there is a strong consensus on their counteracting action on LPS-induced cognitive deficits and neuroinflammatory responses in rodents by modulating cell-signaling processes, such as the NF-κB pathway. Overall, dietary interventions could represent an important resource to oppose AD due to their influence in neuroprotection and immune regulation.

In vitro construction of lung cancer organoids by 3D bioprinting for drug evaluation

The construction of tumor model plays a crucial role in preclinical drug evaluation. However, traditional two-dimensional (2D) monolayer cell models are unable to accurately predict drug activity in vivo, and animal models have species differences and ethical issues. Herein, a droplet-based 3D bioprinting strategy is established to construct lung cancer organoid arrays for drug evaluation. The bioink system is based on sodium alginate (SA, used to crosslink with Ca2+), hyaluronic acid (HA, used to improve gel viscoelasticity and printability), and arginine-glycine-aspartic acid peptide (RGD, used to improve cell adhesion). Hundreds of organoids with 3D multicellular spherical structures are produced in batches after post-printing culture. Compared with 2D monolayer culture models, 3D organoids show higher cell activity and functional expression of P-CK, ProSP-C, MUC1 and Caveolin-1, as well as higher anticancer drug resistance and IC50. This strategy will provide insights into the field of organoid modeling, high-throughput and personalized drug screening.

Effective Inhibition of Thyroid Antigen Presentation Using Retro-Inverso Peptides in Experimental Autoimmune Thyroiditis: A Pathway Toward Immune Therapies of Thyroid Autoimmunity.

Background: Autoimmune thyroid diseases (AITD) represent the most common autoimmune diseases. However, current therapies focus on relieving the symptoms instead of curing AITD, and new therapies to reverse the autoimmune attack on the thyroid are needed. HLA-DRβ1-Arg74 is the key HLA class II allele that triggers AITD by presenting pathogenic thyroglobulin (Tg) peptides that activate thyroid self-reactive T cells. We hypothesized that blocking the presentation of Tg peptides to T cells within the HLA-DRβ1-Arg74 peptide binding cleft could reverse the autoimmune response to the thyroid in AITD. Methods: The approach we used to block Tg peptide presentation within HLA-DRβ1-Arg74 is to design retro-inverso D-amino acid (RID) peptides that have high affinity to the HLA-DRβ1-Arg74 peptide binding pocket. Results: By using computational approaches and molecular dynamics simulations, we designed two RID peptides, RT-15 and VT-15, that blocked peptide binding to recombinant HLA-DRβ1-Arg74 molecule, as well as T cell activation in vitro. Furthermore, RT-15 and VT-15 blocked in vivo T cell activation by thyroglobulin in humanized NOD-DR3 mice induced with experimental autoimmune thyroiditis. Conclusions: In summary, we discovered two RID peptides that block thyroglobulin peptide binding to HLA-DRβ1-Arg74 and their presentation to T cells in AITD. These findings set the stage for a personalized medicine therapeutic approach for AITD patients who carry the DRβ1-Arg74 allele. This antigen-specific therapeutic strategy can potentially be extended to other autoimmune diseases.

Identification of a First-in-Class Small-Molecule Inhibitor of the EIF4E-RBM38 Complex That Enhances Wild-type TP53 Protein Translation for Tumor Growth Suppression.

EIF4E, an mRNA cap-binding protein, is necessary for cap-dependent translation. Overexpression of EIF4E is known to promote cancer development by preferentially translating a group of oncogenic mRNAs. Thus, 4EGI-1, a disruptor of EIF4E-EIF4G1 interaction, was developed to inhibit oncoprotein expression for cancer therapy. Interestingly, RBM38, an RNA-binding protein, interacts with EIF4E on TP53 mRNA, prevents EIF4E from binding to TP53 mRNA cap, and inhibits TP53 expression. Thus, Pep8, an eight amino acid peptide derived from RBM38, was developed to disrupt the EIF4E-RBM38 complex, leading to increased TP53 expression and decreased tumor cell growth. Herein, we have developed a first-in-class small-molecule compound 094, which interacts with EIF4E via the same pocket as does Pep8, dissociates RBM38 from EIF4E, and enhances TP53 translation in RBM38- and EIF4E-dependent manners. Structure-activity relationship studies identified that both the fluorobenzene and ethyl benzamide are necessary for compound 094 to interact with EIF4E. Furthermore, we showed that compound 094 is capable of suppressing three-dimensional tumor spheroid growth in RBM38- and TP53-dependent manners. In addition, we found that compound 094 cooperates with the chemotherapeutic agent doxorubicin and EIF4E inhibitor 4EGI-1 to suppress tumor cell growth. Collectively, we showed that two distinct approaches can be used together to target EIF4E for cancer therapy by enhancing wild-type TP53 expression (094) and by suppressing oncoprotein expression (4EGI-1).

A Significant Difference in Core PDZ Interactivity of SARS-CoV, SARS-CoV2 and MERS-CoV Protein E Peptide PDZ Motifs In Vitro.

Small structural E protein of coronaviruses uses its C-terminal PDZ motif to compromise the cellular PDZ interactome. In this work we compared core PDZ interactivity of small (seven amino acids) peptide PDZ motifs, originating from the envelope proteins of recently transmitted coronaviruses SARS-CoV, SARS-CoV2, and MERS-CoV. As the interaction targets we used 23 domains of the largest PDZ proteins MUPP1/MPDZ and PATJ/INAD. Results revealed exceptional affinity and interaction promiscuity of MERS-CoV PDZ motif in vitro, suggesting an increased probability of potential PDZ targets in vivo. We hypothesize that together with its known ability to enter the cells from both apical and basolateral sites, this might further contribute to its elevated disruption of cellular PDZ pathways and higher virulence.

Diversity of HLA-A2-Restricted and Immunodominant Epitope Repertoire of Human T-Lymphotropic Virus Type 1 (HTLV-1) Tax Protein: Novel Insights among N-Terminal, Central and C-Terminal Regions

The present study sought to search for the immunodominance related to the N-terminal, Central and C-terminal regions of HTLV-1 Tax using novel, cutting-edge peptide microarray analysis. In addition, in silico predictions were performed to verify the presence of nine amino acid peptides present along Tax restricted to the human leukocyte antigen (HLA)-A2.02*01 haplotype, as well as to verify the ability to induce pro-inflammatory and regulatory cytokines, such as IFN-γ and IL-4, respectively. Our results indicated abundant dose-dependent reactivity for HLA-A*02:01 in all regions (N-terminal, Central and C-terminal), but with specific hotspots. Furthermore, the results of fold-change over the Tax11-19 reactivity obtained at lower concentrations of HLA-A*02:01 reveal that peptides from the three regions contain sequences that react 100 times more than Tax11-19. On the other hand, Tax11-19 has similar or superior HLA-A*02:01 reactivity at higher concentrations of this haplotype. The in silico analysis showed a higher frequency of IFN-γ-inducing peptides in the N-terminal portion, while the C-terminal portion showed a higher frequency of IL-4 inducers. Taken together, these results shed light on the search for new Tax immunodominant epitopes, in addition to the canonic Tax11-19, for the rational design of immunomodulatory strategies for HTLV-1 chronic diseases.

Formulated chitosan-sodium tripolyphosphate nanoparticles for co-encapsulation of ellagic acid and anti-inflammatory peptide: characterization, stability and anti-inflammatory activity.

Chitosan (CS) and tripolyphosphate (TPP) can be combined in the development of a material with synergistic properties and promising potential for the conservation of food products. In this study, ellagic acid (EA) and anti-inflammatory peptide (FPL)-loaded CS nanoparticles (FPL/EA NPs) were prepared using the ionic gelation method and optimal preparation conditions were obtained through a single factor design. The synthesized nanoparticles (NPs) were characterized using a scanning electron microscope (SEM), Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and differential scanning calorimetry (DSC). Nanoparticles were spherical, with an average size of 308.33 ± 4.61 nm, a polydispersity index (PDI) of 0.254, a zeta potential of +31.7 ± 0.08 mV, and a high encapsulation capacity (22.16 ± 0.79%). An in vitro release study showed that EA/FPL had a sustainable release from FPL/EA NPs. The stability of the FPL/EA NPs was evaluated for 90 days at 0, 25, and 37 °C. Significant anti-inflammatory activity of FPL/EA NPs was verified by nitric oxide (NO) and tumor necrosis factor-α (TNF-α) reduction. These characteristics support the use of CS nanoparticles to encapsulate EA and FPL and improve their bioactivity in food products. © 2023 Society of Chemical Industry.

Identification, Measurement, and Assessment of the Clinical Utility of Human Pancreatic Polypeptide by Liquid Chromatography-Tandem Mass Spectrometry.

Human pancreatic polypeptide (HPP) is a 36 amino acid peptide hormone that plays a role in the bidirectional communication between the digestive system and the brain. HPP measurements are used to assess vagal nerve function following sham feeding and to detect gastroenteropancreatic-neuroendocrine tumors. These tests have historically been conducted by radioimmunoassays, but liquid chromatography-tandem mass spectrometry (LC-MS/MS) has several advantages such as improved specificity and elimination of radioactive molecules. Here, we present our LC-MS/MS method. Initially, samples were immunopurified and subjected to LC-high resolution accurate mass tandem mass spectrometry (HRAM-MS/MS) to identify circulating forms of the peptide in human plasma. We identified 23 forms of HPP, including several glycosylated forms. The most abundant peptides then were used for targeted LC-MS/MS measurements. LC-MS/MS performance for precision, accuracy, linearity, recovery, limit of detection, and carryover met our acceptance criteria based on CLIA regulations. Additionally, we observed the expected physiological rise in HPP in response to sham feeding. Our results indicate that HPP measurement by LC-MS/MS produces clinically equivalent results to our established immunoassay when several peptides are monitored, making it a suitable replacement. The measurement of peptide fragments, including modified species, might have additional clinical value.