Deconvolution analysis for classifying gastric adenocarcinoma patients based on differential scanning calorimetry serum thermograms

Recently, differential scanning calorimetry (DSC) has been acknowledged as a novel tool for diagnosing and monitoring several diseases. This highly sensitive technique has been traditionally used to study thermally induced protein folding/unfolding transitions. In previous research papers, DSC profiles from blood samples of patients were analyzed and they exhibited marked differences in the thermal denaturation profile. Thus, we investigated the use of this novel technology in blood serum samples from 25 healthy subjects and 30 patients with gastric adenocarcinoma (GAC) at different stages of tumor development with a new multiparametric approach. The analysis of the calorimetric profiles of blood serum from GAC patients allowed us to discriminate three stages of cancer development (I to III) from those of healthy individuals. After a multiparametric analysis, a classification of blood serum DSC parameters from patients with GAC is proposed. Certain parameters exhibited significant differences (P < 0.05) and allowed the discrimination of healthy subjects/patients from patients at different tumor stages. The results of this work validate DSC as a novel technique for GAC patient classification and staging, and offer new graphical tools and value ranges for the acquired parameters in order to discriminate healthy from diseased subjects with increased disease burden

Rescuing compound bioactivity in a secondary cell-based screening by using gamma-cyclodextrin as a molecular carrier

In vitro primary screening for identifying bioactive compounds (inhibitors, activators or pharmacological chaperones) against a protein target results in the discovery of lead com- pounds that must be tested in cell-based efficacy secondary screenings. Very often lead com- pounds do not succeed because of an apparent low potency in cell assays, despite an excellent performance in primary screening. Primary and secondary screenings differ significantly accord- ing to the conditions and challenges the compounds must overcome in order to interact with their intended target. Cellular internalization and intracellular metabolism are some of the difficulties the compounds must confront and different strategies can be envisaged for minimizing that prob- lem. Using a novel screening procedure we have identified 15 compounds inhibiting the hepatitis C NS3 protease in an allosteric fashion. After characterizing biophysically the interaction with the target, some of the compounds were not able to inhibit viral replication in cell assays. In order to overcome this obstacle and potentially improve cellular internalization three of these compounds were complexed with gamma-cyclodextrin. Two of them showed a five- and 16-fold activity increase, compared to their activity when delivered as free compounds in solution (while gamma-cyclodextrin did not show antiviral activity by itself ). The most remarkable result came from a third compound that showed no antiviral activity in cell assays when delivered free in solu- tion, but its gamma-cyclodextrin complex exhibited a 50% effective concentration of 5 micromoles. Thus, the antiviral activity of these compounds can be significantly improved, even completely rescued, using gamma-cyclodextrin as carrier molecule

Identification of a Drug Targeting an Intrinsically Disordered Protein Involved in Pancreatic Adenocarcinoma

Intrinsically disordered proteins (IDPs) are prevalent in eukaryotes, performing signaling and regulatory functions. Often associated with human diseases, they constitute drug-development targets. NUPR1 is a multifunctional IDP, over-expressed and involved in pancreatic ductal adenocarcinoma (PDAC) development. By screening 1120 FDA-approved compounds, fifteen candidates were selected, and their interactions with NUPR1 were characterized by experimental and simulation techniques. The protein remained disordered upon binding to all fifteen candidates. These compounds were tested in PDAC-derived cell-based assays, and all induced cell-growth arrest and senescence, reduced cell migration, and decreased chemoresistance, mimicking NUPR1-deficiency. The most effective compound completely arrested tumor development in vivo on xenografted PDAC-derived cells in mice. Besides reporting the discovery of a compound targeting an intact IDP and specifically active against PDAC, our study proves the possibility to target the ''fuzzy'' interface of a protein that remains disordered upon binding to its natural biological partners or to selected drugs

Thermal liquid biopsy (TLB) focused on benign and premalignant pancreatic cyst diagnosis

Background: Current efforts in the identification of new biomarkers are directed towards an accurate differentiation between benign and premalignant cysts. Thermal Liquid Biopsy (TLB) has been previously applied to inflammatory and tumor diseases and could offer an interesting point of view in this type of pathology. Methods: In this work, twenty patients (12 males and 8 females, average ages 62) diagnosed with a pancreatic cyst benign (10) and premalignant (10) cyst lesions were recruited, and biological samples were obtained during the endoscopic ultrasonography procedure. Results: Proteomic content of cyst liquid samples was studied and several common proteins in the different groups were identified. TLB cyst liquid profiles reflected protein content. Also, TLB serum score was able to discriminate between healthy and cysts patients (71% sensitivity and 98% specificity) and between benign and premalignant cysts (75% sensitivity and 67% specificity). Conclusions: TLB analysis of plasmatic serum sample, a quick, simple and non-invasive technique that can be easily implemented, reports valuable information on the observed pancreatic lesion. These preliminary results set the basis for a larger study to refine TLB serum score and move closer to the clinical application of TLB providing useful information to the gastroenterologist during patient diagnosis

Platelets induce free and phospholipid-esterified 12-hydroxyeicosatetraenoic acid generation in colon cancer cells by delivering 12-lipoxygenase

Platelets promote tumor metastasis by inducing promalignant phenotypes in cancer cells and directly contributing to cancer-related throm-botic complications. Platelet-derived extracellular vesicles (EVs) can promote epithelial-mesenchymal transition (EMT) in cancer cells, which confers high-grade malignancy. 12S-hydroxyeicosatetraenoic acid (12-HETE) generated by platelet-type 12-lipoxygenase (12-LOX) is considered a key modulator of cancer metastasis through unknown mechanisms. In plate-lets, 12-HETE can be esterified into plasma membrane phospholipids (PLs), which drive thrombosis. Using cocultures of human platelets and human colon adenocarcinoma cells (line HT29) and LC-MS/MS, we investigated the impact of platelets on cancer cell biosynthesis of 12S-HETE and its esterification into PLs and whether platelet ability to transfer its mo-lecular cargo might play a role. To this aim, we performed coculture experiments with CFSE[5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester]-loaded platelets. HT29 cells did not generate 12S-HETE or express 12-LOX. However, they ac-quired the capacity to produce 12S-HETE mainly esterified in plasmalogen phospholipid forms following the uptake of platelet-derived medium-sized EVs (mEVs) expressing 12-LOX. 12-LOX was detected in plasma mEV of patients with adenomas/ adenocarcinomas, implying their potential to deliver the protein to cancer cells in vivo. In cancer cells exposed to platelets, endogenous but not exogenous 12S-HETE contributed to changes in EMT gene expression, mitigated by three structurally unrelated 12-LOX inhibitors. In conclusion, we showed that platelets induce the generation of primarily esteri-fied 12-HETE in colon cancer cells following mEV-mediated delivery of 12-LOX. The modification of cancer cell phospholipids by 12-HETE may functionally impact cancer cell biology and represent a novel target for anticancer agent development

Thermal Liquid Biopsy (TLB) Focused on Benign and Premalignant Pancreatic Cyst Diagnosis

19 pags., 7 figs., 2 tabs. -- This article belongs to the Special Issue Gastrointestinal Cancers and Personalized MedicineBackground: Current efforts in the identification of new biomarkers are directed towards an accurate differentiation between benign and premalignant cysts. Thermal Liquid Biopsy (TLB) has been previously applied to inflammatory and tumor diseases and could offer an interesting point of view in this type of pathology. Methods: In this work, twenty patients (12 males and 8 females, average ages 62) diagnosed with a pancreatic cyst benign (10) and premalignant (10) cyst lesions were recruited, and biological samples were obtained during the endoscopic ultrasonography procedure. Results: Proteomic content of cyst liquid samples was studied and several common proteins in the different groups were identified. TLB cyst liquid profiles reflected protein content. Also, TLB serum score was able to discriminate between healthy and cysts patients (71% sensitivity and 98% specificity) and between benign and premalignant cysts (75% sensitivity and 67% specificity). Conclusions: TLB analysis of plasmatic serum sample, a quick, simple and non-invasive technique that can be easily implemented, reports valuable information on the observed pancreatic lesion. These preliminary results set the basis for a larger study to refine TLB serum score and move closer to the clinical application of TLB providing useful information to the gastroenterologist during patient diagnosis.This research was funded by the Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) (BFU2016-78232-P to A.V.C.); Projects funded by Instituto de Salud Carlos III and co-funded by European Union (ESF, ”Investing in your future”): “PI15/00663 (FIS project to O.A)”, “PI18/00349 (FIS project to O.A. and Contract to LC)”, “FI19/00146 (PFIS contract for SHD)”, “CPII13/00017 (Miguel Servet Program to OA)”; Diputación General de Aragón (Protein Targets and Bioactive Compounds Group E45_17R to A.V.C. and Digestive Pathology Group B25_17R to O.A.); and the Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd).Peer reviewe

Helicobacter Genotyping and Detection in Peroperative Lavage Fluid in Patients with Perforated Peptic Ulcer

Introduction and Objectives Certain Helicobacter pylori genotypes are associated with peptic ulcer disease; however, little is known about associations between the H. pylori genotype and perforated peptic ulcer (PPU). The primary aim of this study was to evaluate which genotypes are present in patients with PPU and which genotype is dominant in this population. The secondary aim was to study the possibility of determining the H. pylori status in a way other than by biopsy. Materials and Methods Serum samples, gastric tissue biopsies, lavage fluid, and fluid from the nasogastric tube were collec

Role of proton pump inhibitors dosage and duration in Helicobacter pylori eradication treatment: Results from the European Registry on H. pylori management

Background: Management of Helicobacter pylori (H. pylori) infection requires co-treatment with proton pump inhibitors (PPIs) and the use of antibiotics to achieve successful eradication. Aim: To evaluate the role of dosage of PPIs and the duration of therapy in the effectiveness of H. pylori eradication treatments based on the ‘European Registry on Helicobacter pylori management’ (Hp-EuReg). Methods: Hp-EuReg is a multicentre, prospective, non-interventionist, international registry on the routine clinical practice of H. pylori management by European gastroenterologists. All infected adult patients were systematically registered from 2013 to 2022. Results: Overall, 36,579 patients from five countries with more than 1000 patients were analysed. Optimal (≥90%) first-line-modified intention-to-treat effectiveness was achieved with the following treatments: (1) 14-day therapies with clarithromycin-amoxicillin-bismuth and metronidazole-tetracycline-bismuth, both independently of the PPI dose prescribed; (2) All 10-day (except 10-day standard triple therapy) and 14-day therapies with high-dose PPIs; and (3) 10-day quadruple therapies with clarithromycin-amoxicillin-bismuth, metronidazole-tetracycline-bismuth, and clarithromycin-amoxicillin-metronidazole (sequential), all with standard-dose PPIs. In first-line treatment, optimal effectiveness was obtained with high-dose PPIs in all 14-day treatments, in 10- and 14-day bismuth quadruple therapies and in 10-day sequential with standard-dose PPIs. Optimal second-line effectiveness was achieved with (1) metronidazole-tetracycline-bismuth quadruple therapy for 14- and 10&nbsp;days with standard and high-dose PPIs, respectively; and (2) levofloxacin-amoxicillin triple therapy for 14&nbsp;days with high-dose PPIs. None of the 7-day therapies in both treatment lines achieved optimal effectiveness. Conclusions: We recommend, in first-line treatment, the use of high-dose PPIs in 14-day triple therapy and in 10-or 14-day quadruple concomitant therapy in first-line treatment, while standard-dose PPIs would be sufficient in 10-day bismuth quadruple therapies. On the other hand, in second-line treatment, high-dose PPIs would be more beneficial in 14-day triple therapy with levofloxacin and amoxicillin or in 10-day bismuth quadruple therapy either as a three-in-one single capsule or in the traditional scheme

Analysis of Clinical Phenotypes through Machine Learning of First-Line H. pylori Treatment in Europe during the Period 2013–2022: Data from the European Registry on H. pylori Management (Hp-EuReg)

The segmentation of patients into homogeneous groups could help to improve eradication therapy effectiveness. Our aim was to determine the most important treatment strategies used in Europe, to evaluate first-line treatment effectiveness according to year and country. Data collection: All first-line empirical treatments registered at AEGREDCap in the European Registry on Helicobacter pylori management (Hp-EuReg) from June 2013 to November 2022. A Boruta method determined the “most important” variables related to treatment effectiveness. Data clustering was performed through multi-correspondence analysis of the resulting six most important variables for every year in the 2013–2022 period. Based on 35,852 patients, the average overall treatment effectiveness increased from 87% in 2013 to 93% in 2022. The lowest effectiveness (80%) was obtained in 2016 in cluster #3 encompassing Slovenia, Lithuania, Latvia, and Russia, treated with 7-day triple therapy with amoxicillin–clarithromycin (92% of cases). The highest effectiveness (95%) was achieved in 2022, mostly in Spain (81%), with the bismuth–quadruple therapy, including the single-capsule (64%) and the concomitant treatment with clarithromycin–amoxicillin–metronidazole/tinidazole (34%) with 10 (69%) and 14 (32%) days. Cluster analysis allowed for the identification of patients in homogeneous treatment groups assessing the effectiveness of different first-line treatments depending on therapy scheme, adherence, country, and prescription year