Comparative analysis of the minimal information for studies of extracellular vesicles guidelines: Advancements and implications for extracellular vesicle research.

In 2014, the International Society for Extracellular Vesicles (ISEV) introduced the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines to establish standards for extracellular vesicle (EV) research. These guidelines aimed to enhance reliability and reproducibility, addressing the expanding field of EV science. EVs, membrane-bound particles released by cells, play crucial roles in intercellular communication and are potential biomarkers for various conditions. Over the years, the EV landscape witnessed a surge in publications, emphasizing their roles in cancer and immune modulation. In response, the MISEV guidelines underwent evolution, leading to the MISEV2018 update. This version, generated through community outreach, provided a comprehensive framework for EV research methodologies, emphasizing separation, characterization, reporting standards, and community engagement. The MISEV2018 guidelines reflected responsiveness to feedback, acknowledging the evolving EV research landscape. The guidelines served as a testament to the commitment of the scientific community to rigorous standards and the collective discernment of experts. The present article compares previous MISEV guidelines with its 2023 counterpart, highlighting advancements, changes, and impacts on EV research standardization. The 2023 guidelines build upon the 2018 principles, offering new recommendations for emerging areas. This comparative exploration contributes to understanding the transformative journey in EV research, emphasizing MISEV's pivotal role and the scientific community's adaptability to challenges.

Immune evasion on the nanoscale: Small extracellular vesicles in pancreatic ductal adenocarcinoma immunity.

Pancreatic ductal adenocarcinoma (PDAC) is a type of cancer alarmingly expanding in our modern societies that is still proving to be very challenging to counteract. This disease constitutes a quintessential example of the multiple interactions existing between the tumour and its surrounding microenvironment. In particular, PDAC is characterized by a very immunosuppressive environment that favours cancer growth and makes this cancer type very resistant to immunotherapy. The primary tumour releases many factors that influence both the microenvironment and the immune landscape. Extracellular vesicles (EVs), recently identified as indispensable entities ensuring cell-to-cell communication in both physiological and pathological processes, seem to play a pivotal function in ensuring the delivery of these factors to the tumour-surrounding tissues. In this review, we summarize the present understanding on the crosstalk among tumour cells and the cellular immune microenvironment emphasizing the pro-malignant role played by extracellular vesicles. We also discuss how a greater knowledge of the roles of EVs in tumour immune escape could be translated into clinical applications.

Proteomic profiling of small extracellular vesicles derived from mouse pancreatic cancer and stellate cells: Role in pancreatic cancer.

Small extracellular vesicles (sEVs) are cell-derived vesicles evolving as important elements involved in all stages of cancers. sEVs bear unique protein signatures that may serve as biomarkers. Pancreatic cancer (PC) records a very poor survival rate owing to its late diagnosis and several cancer cell-derived proteins have been reported as candidate biomarkers. However, given the pivotal role played by stellate cells (PSCs, which produce the collagenous stroma in PC), it is essential to also assess PSC-sEV cargo in biomarker discovery. Thus, this study aimed to isolate and characterise sEVs from mouse PC cells and PSCs cultured alone or as co-cultures and performed proteomic profiling and pathway analysis. Proteomics confirmed the enrichment of specific markers in the sEVs compared to their cells of origin as well as the proteins that are known to express in each of the culture types. Most importantly, for the first time it was revealed that PSC-sEVs are enriched in proteins (including G6PI, PGAM1, ENO1, ENO3, and LDHA) that mediate pathways related to development of diabetes, such as glucose metabolism and gluconeogenesis revealing a potential role of PSCs in pancreatic cancer-related diabetes (PCRD). PCRD is now considered a harbinger of PC and further research will enable to identify the role of these components in PCRD and may develop as novel candidate biomarkers of PC.

Synergistic Anticancer Activity of Plumbagin and Xanthohumol Combination on Pancreatic Cancer Models.

Among diverse cancers, pancreatic cancer is one of the most aggressive types due to inadequate diagnostic options and treatments available. Therefore, there is a necessity to use combination chemotherapy options to overcome the chemoresistance of pancreatic cancer cells. Plumbagin and xanthohumol, natural compounds isolated from the Plumbaginaceae family and Humulus lupulus, respectively, have been used to treat various cancers. In this study, we investigated the anticancer effects of a combination of plumbagin and xanthohumol on pancreatic cancer models, as well as the underlying mechanism. We have screened in vitro numerous plant-derived extracts and compounds and tested in vivo the most effective combination, plumbagin and xanthohumol, using a transgenic model of pancreatic cancer KPC (KrasLSL.G12D/+; p53R172H/+; PdxCretg/+). A significant synergistic anticancer activity of plumbagin and xanthohumol combinations on different pancreatic cancer cell lines was found. The combination treatment of plumbagin and xanthohumol influences the levels of B-cell lymphoma (BCL2), which are known to be associated with apoptosis in both cell lysates and tissues. More importantly, the survival of a transgenic mouse model of pancreatic cancer KPC treated with a combination of plumbagin and xanthohumol was significantly increased, and the effect on BCL2 levels has been confirmed. These results provide a foundation for a potential new treatment for pancreatic cancer based on plumbagin and xanthohumol combinations.

Pharmacohistory of Cannabis Use-A New Possibility in Future Drug Development for Gastrointestinal Diseases.

Humans have employed cannabis for multiple uses including medicine, recreation, food, and fibre. The various components such as roots, flowers, seeds, and leaves have been utilized to alleviate pain, inflammation, anxiety, and gastrointestinal disorders like nausea, vomiting, diarrhoea, and inflammatory bowel diseases (IBDs). It has occupied a significant space in ethnomedicines across cultures and religions. Despite multi-dimensional uses, the global prohibition of cannabis by the USA through the introduction of the Marijuana Tax Act in 1937 led to prejudice about the perceived risks of cannabis, overshadowing its medicinal potential. Nevertheless, the discovery of tetrahydrocannabinol (THC), the primary psychoactive compound in cannabis, and the endocannabinoid system renewed scientific interest in understanding the role of cannabis in modulating different conditions, including gastrointestinal disorders. Preparations combining cannabidiol and THC have shown promise in mitigating gut symptoms through anti-inflammatory and motility-enhancing effects. This review revisits the ethnomedicinal use of cannabis in gastrointestinal diseases and emphasizes the need for further research to determine optimal dosages, formulations, and safety profiles of cannabis-based medicines. It also underscores the future potential of cannabinoid-based therapies by leveraging the role of the expanded endocannabinoid system, an endocannabinoidome, in the modulation of gastrointestinal ailments.

Sex-divergent expression of cytochrome P450 and SIRTUIN 1-7 proteins in toxicity evaluation of a benzimidazole-derived epigenetic modulator in mice.

Efforts in precision medicine to combat aberrant epigenome have led to the development of epigenetic targeting drugs. We have previously reported the capability of the BZD9L1 epigenetic modulator to impede colorectal tumour growth in vitro and in vivo through sirtuin (SIRT) inhibition. Although most benzimidazole derivatives are commonly less toxic, their effects on SIRTs and cytochrome P450 (CYP) regulations have not been explored alongside toxicity assessments. SIRTs are histone deacetylases that are crucial in maintaining metabolic homeostasis, whereas CYP is essential in drug metabolism. This study aims to determine the toxicology profile of BZD9L1 through oral acute and repeated dose toxicity evaluations, along with molecular analyses of SIRT, CYP and relevant toxicity markers through western blot and quantitative polymerase chain reaction (qPCR). BZD9L1 demonstrated no sign of acute toxicity at the limit dose (2000 mg/kg). The 28-day toxicity study highlighted the tolerability of repeated dose administration without adverse effects. BZD9L1 showed a sex-divergent regulation of hepatic SIRT1-7, CYP2A5 and CYP2D proteins. Furthermore, BZD9L1 did not induce the expression of organ injury proteins or alter the gene expression of cellular function indicators in mouse liver and kidneys, hence demonstrating, at least in part, the safety of BZD9L1 in short-term evaluations. The present study cautions for personalised strategies when employing benzimidazole-derived epigenetic therapeutics.

Modulatory role of the endocannabinoidome in the pathophysiology of the gastrointestinal tract.

Originating from Eastern Asia, the plant Cannabis sativa has been used for centuries as a medicinal treatment. The unwanted psychotropic effects of one of its major components, Δ9-tetrahydrocannabinol, discouraged its therapeutic employment until, recently, the discovery of cannabinoids receptors and their endogenous ligands endocannabinoids reignited the interest. The endocannabinoid system has lately been found to play an important role in the maintenance of human health, both centrally and peripherally. However, the initial idea of the endocannabinoid system structure has been quickly understood to be too simplistic and, as new receptors, mediators, and enzymes have been discovered to participate in a complex relationship, the new, more comprehensive term "expanded endocannabinoid system" or "endocannabinoidome", has taken over. The discovery of other endocannabinoid-like receptors, such as the G protein-coupled receptor 119 and G protein-coupled receptor 55, has opened the way to the development of potential therapeutic targets for the treatment of various metabolic disorders. In addition, recent findings have also provided evidence suggesting the potential therapeutic link between the endocannabinoidome and various inflammatory-based gut diseases, such as inflammatory bowel disease and cancer. This review will provide an introduction to the endocannabinoidome, focusing on its modulatory role in the gastrointestinal tract and on the interest generated by the link between gut microbiota, the endocannabinoid system and metabolic diseases such as inflammatory bowel disease, type-2 diabetes and obesity. In addition, we will look at the potential novel aspects and benefits of drugs targeting the endocannabinoid system.

Precision Medicine Approaches with Metabolomics and Artificial Intelligence.

Recent technological innovations in the field of mass spectrometry have supported the use of metabolomics analysis for precision medicine. This growth has been allowed also by the application of algorithms to data analysis, including multivariate and machine learning methods, which are fundamental to managing large number of variables and samples. In the present review, we reported and discussed the application of artificial intelligence (AI) strategies for metabolomics data analysis. Particularly, we focused on widely used non-linear machine learning classifiers, such as ANN, random forest, and support vector machine (SVM) algorithms. A discussion of recent studies and research focused on disease classification, biomarker identification and early diagnosis is presented. Challenges in the implementation of metabolomics-AI systems, limitations thereof and recent tools were also discussed.

Pharmacological and structure-activity relationship studies of oleoyl-lysophosphatidylinositol synthetic mimetics.

Metabolic diseases, such as obesity and type 2 diabetes, are relentlessly spreading worldwide. The beginning of the 21st century has seen the introduction of mechanistically novel types of drugs, aimed primarily at keeping these pathologies under control. In particular, an important family of therapeutics exploits the beneficial physiology of the gut-derived glucagon-like peptide-1 (GLP-1), with important clinical benefits, from glycaemic control to cardioprotection. Nonetheless, these protein-based drugs act systemically as exogenous GLP-1 mimetics and are not exempt from side effects. The food-derived lipid oleoyl-lysophosphatidylinositol (LPI) is a potent GPR119-dependent GLP-1 secreting agent. Here we present a structure-activity relationship (SAR) study of a synthetic library of oleoyl-LPI mimetics capable to induce the physiological release of GLP-1 from gastrointestinal enteroendocrine cells (EECs). The best lead compounds have shown potent and efficient release of GLP-1 in vitro from human and murine cells, and in vivo in diabetic db/db mice. We have also generated a molecular model of oleoyl-LPI, as well as its best performing analogues, interacting with the orthosteric site of GPR119, laying foundational evidence for their pharmacological activity.

Does the SARS-CoV-2 Spike Protein Receptor Binding Domain Interact Effectively with the DPP4 (CD26) Receptor? A Molecular Docking Study.

ACE2 has been established as the main receptor for SARS-CoV-2. Since other human coronaviruses are known to use co-receptors for viral cell entry, it has been suggested that DPP4 (CD26) could be a potential additional binding target or co-receptor, supported by early molecular docking simulation studies. However, recent biophysical studies have shown this interaction to be very weak. We have conducted detailed molecular docking simulations to predict the potential binding interactions between the receptor binding domain (RBD) of the spike protein of SARS-CoV-2 and DPP4 and compare them with the interactions observed in the experimentally determined structure of the complex of MERS-CoV with DPP4. Whilst the overall binding mode of the RBD of SARS-CoV-2 to DPP4 is predicted to be similar to that observed in the MERS-CoV-DPP4 complex, including a number of equivalent interactions, important differences in the amino acid sequences of SARS-CoV-2 and MERS-CoV result in substantially weakened interactions with DPP4. This is shown to arise from differences in the predicted proximity, nature and secondary structure at the binding interface on the RBD of SARS-CoV-2. These findings do not support DPP4 being a significant receptor for SARS-CoV-2.

Inositol Polyphosphate-Based Compounds as Inhibitors of Phosphoinositide 3-Kinase-Dependent Signaling.

Signaling pathways regulated by the phosphoinositide 3-kinase (PI3K) enzymes have a well-established role in cancer development and progression. Over the past 30 years, the therapeutic potential of targeting this pathway has been well recognized, and this has led to the development of a multitude of drugs, some of which have progressed into clinical trials, with few of them currently approved for use in specific cancer settings. While many inhibitors compete with ATP, hence preventing the catalytic activity of the kinases directly, a deep understanding of the mechanisms of PI3K-dependent activation of its downstream effectors led to the development of additional strategies to prevent the initiation of this signaling pathway. This review summarizes previously published studies that led to the identification of inositol polyphosphates as promising parent molecules to design novel inhibitors of PI3K-dependent signals. We focus our attention on the inhibition of protein-membrane interactions mediated by binding of pleckstrin homology domains and phosphoinositides that we proposed 20 years ago as a novel therapeutic strategy.

Large-Scale Plasma Analysis Revealed New Mechanisms and Molecules Associated with the Host Response to SARS-CoV-2.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread to nearly every continent, registering over 1,250,000 deaths worldwide. The effects of SARS-CoV-2 on host targets remains largely limited, hampering our understanding of Coronavirus Disease 2019 (COVID-19) pathogenesis and the development of therapeutic strategies. The present study used a comprehensive untargeted metabolomic and lipidomic approach to capture the host response to SARS-CoV-2 infection. We found that several circulating lipids acted as potential biomarkers, such as phosphatidylcholine 14:0_22:6 (area under the curve (AUC) = 0.96), phosphatidylcholine 16:1_22:6 (AUC = 0.97), and phosphatidylethanolamine 18:1_20:4 (AUC = 0.94). Furthermore, triglycerides and free fatty acids, especially arachidonic acid (AUC = 0.99) and oleic acid (AUC = 0.98), were well correlated to the severity of the disease. An untargeted analysis of non-critical COVID-19 patients identified a strong alteration of lipids and a perturbation of phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, aminoacyl-tRNA degradation, arachidonic acid metabolism, and the tricarboxylic acid (TCA) cycle. The severity of the disease was characterized by the activation of gluconeogenesis and the metabolism of porphyrins, which play a crucial role in the progress of the infection. In addition, our study provided further evidence for considering phospholipase A2 (PLA2) activity as a potential key factor in the pathogenesis of COVID-19 and a possible therapeutic target. To date, the present study provides the largest untargeted metabolomics and lipidomics analysis of plasma from COVID-19 patients and control groups, identifying new mechanisms associated with the host response to COVID-19, potential plasma biomarkers, and therapeutic targets.

Signalling Properties of Inositol Polyphosphates.

Several studies have identified specific signalling functions for inositol polyphosphates (IPs) in different cell types and have led to the accumulation of new information regarding their cellular roles as well as new insights into their cellular production. These studies have revealed that interaction of IPs with several proteins is critical for stabilization of protein complexes and for modulation of enzymatic activity. This has not only revealed their importance in regulation of several cellular processes but it has also highlighted the possibility of new pharmacological interventions in multiple diseases, including cancer. In this review, we describe some of the intracellular roles of IPs and we discuss the pharmacological opportunities that modulation of IPs levels can provide.

Oncogenic and Non-Malignant Pancreatic Exosome Cargo Reveal Distinct Expression of Oncogenic and Prognostic Factors Involved in Tumor Invasion and Metastasis.

Exosomes are small extracellular membrane vesicles important in intercellular communication, with their oncogenic cargo attributed to tumor progression and pre-metastatic niche formation. To gain an insight into key differences in oncogenic composition of exosomes, human non-malignant epithelial and pancreatic cancer cell models and purified and characterized resultant exosome populations are utilized. Proteomic analysis reveals the selective enrichment of known exosome markers and signaling proteins in comparison to parental cells. Importantly, valuable insights into oncogenic exosomes (362 unique proteins in comparison to non-malignant exosomes) of key metastatic regulatory factors and signaling molecules fundamental to pancreatic cancer progression (KRAS, CD44, EGFR) are provided. It is reported that oncogenic exosomes contain factors known to regulate the pre-metastatic niche (S100A4, F3, ITGß5, ANXA1), clinically-relevant proteins which correlate with poor prognosis (CLDN1, MUC1) as well as protein networks involved in various cancer hallmarks including proliferation (CLU, CAV1), invasion (PODXL, ITGA3), metastasis (LAMP1, ST14) and immune surveillance escape (B2M). The presence of these factors in oncogenic exosomes offers an understanding of select differences in exosome composition during tumorigenesis, potential components as prognostic and diagnostic biomarkers in pancreatic cancer, and highlights the role of exosomes in mediating crosstalk between tumor and stromal cells.

PLC-gamma-1 phosphorylation status is prognostic of metastatic risk in patients with early-stage Luminal-A and -B breast cancer subtypes.

BACKGROUND: Phospholipase Cγ1 (PLCγ1) is highly expressed in human tumours. Our previous studies reported that both stable and inducible PLCγ1 down-regulation can inhibit formation of breast-cancer-derived experimental lung metastasis. Further, high expression of PLCγ1 and its constitutively activated forms (i.e., PLCγ1-pY1253, PLCγ1-pY783) is associated with worse clinical outcome in terms of incidence of distant metastases, but not of local relapse in T1-T2, N0 breast cancer patients. METHODS: In the present retrospective study, we analysed the prognostic role of PLCγ1 in early breast cancer patients stratified according to the St. Gallen criteria and to their menopausal status. PLCγ1-pY1253 and PLCγ1-pY783 protein expression levels were determined by immunohistochemistry on tissue microarrays, and were correlated with patients' clinical data, using univariate and multivariate statistical analyses. RESULTS: In our series, the prognostic value of PLCγ1 overexpression was restricted to Luminal type tumours. From multivariate analyses, pY1253-PLCγ1High was an independent prognostic factor only in postmenopausal patients with Luminal-B tumours (hazard ratio [HR], 2.4; 95% confidence interval [CI], 1.1-5.3; P = 0.034). Conversely, PLCγ1-pY783High was a remarkably strong risk factor (HR, 20.1; 95% CI, 2.2-178.4; P = 0.003) for pre/perimenopausal patients with Luminal-A tumours. CONCLUSIONS: PLCγ1 overexpression is a strong predictive surrogate marker of development of metastases in early Luminal-A and -B breast cancer patients, being able to discriminate patients with high and low risk of metastases. Therefore, targeting the PLCγ1 pathway can be considered of potential benefit for prevention of metastatic disease.

Blood-brain barrier disturbances in diabetes-associated dementia: Therapeutic potential for cannabinoids.

Type-2 diabetes (T2D) increases the risk of dementia by ˜5-fold, however the mechanisms by which T2D increases dementia risk remain unclear. Evidence suggests that the heightened inflammation and oxidative stress in T2D may lead to disruption of the blood-brain barrier (BBB), which precedes premature cognitive decline. Studies show that vascular-targeted anti-inflammatory treatments protect the BBB by attenuating neuroinflammation, and in some studies attenuate cognitive decline. Yet, this potential pathway is understudied in T2D-associated cognitive impairment. In recent years, therapeutic potential of cannabinoids has gained much interest. The two major cannabinoids, cannabidiol and tetrahydrocannabinol, exert anti-inflammatory and vascular protective effects, however few studies report their potential for reversing BBB dysfunction, particularly in T2D. Therefore, in this review, we summarize the current findings on the role of BBB dysfunction in T2D-associated dementia and consider the potential therapeutic use of cannabinoids as a protectant of cerebrovascular BBB protection.

Oleoyl-lysophosphatidylinositol enhances glucagon-like peptide-1 secretion from enteroendocrine L-cells through GPR119.

The gastrointestinal tract is increasingly viewed as critical in controlling glucose metabolism, because of its role in secreting multiple glucoregulatory hormones, such as glucagon like peptide-1 (GLP-1). Here we investigate the molecular pathways behind the GLP-1- and insulin-secreting capabilities of a novel GPR119 agonist, Oleoyl-lysophosphatidylinositol (Oleoyl-LPI). Oleoyl-LPI is the only LPI species able to potently stimulate the release of GLP-1 in vitro, from murine and human L-cells, and ex-vivo from murine colonic primary cell preparations. Here we show that Oleoyl-LPI mediates GLP-1 secretion through GPR119 as this activity is ablated in cells lacking GPR119 and in colonic primary cell preparation from GPR119-/- mice. Similarly, Oleoyl-LPI-mediated insulin secretion is impaired in islets isolated from GPR119-/- mice. On the other hand, GLP-1 secretion is not impaired in cells lacking GPR55 in vitro or in colonic primary cell preparation from GPR55-/- mice. We therefore conclude that GPR119 is the Oleoyl-LPI receptor, upstream of ERK1/2 and cAMP/PKA/CREB pathways, where primarily ERK1/2 is required for GLP-1 secretion, while CREB activation appears dispensable.

CD31 signals confer immune privilege to the vascular endothelium.

Constitutive resistance to cell death induced by inflammatory stimuli activating the extrinsic pathway of apoptosis is a key feature of vascular endothelial cells (ECs). Although this property is central to the maintenance of the endothelial barrier during inflammation, the molecular mechanisms of EC protection from cell-extrinsic, proapoptotic stimuli have not been investigated. We show that the Ig-family member CD31, which is expressed by endothelial but not epithelial cells, is necessary to prevent EC death induced by TNF-α and cytotoxic T lymphocytes in vitro. Combined quantitative RT-PCR array and biochemical analysis show that, upon the engagement of the TNF receptor with TNF-α on ECs, CD31 becomes activated and, in turn, counteracts the proapoptotic transcriptional program induced by TNF-α via activation of the Erk/Akt pathway. Specifically, Akt activation by CD31 signals prevents the localization of the forkhead transcription factor FoxO3 to the nucleus, thus inhibiting transcription of the proapoptotic genes CD95/Fas and caspase 7 and de-repressing the expression of the antiapoptotic gene cFlar. Both CD31 intracellular immunoreceptor tyrosine-based inhibition motifs are required for its prosurvival function. In vivo, CD31 gene transfer is sufficient to recapitulate the cytoprotective mechanisms in CD31(-) pancreatic ß cells, which become resistant to immune-mediated rejection when grafted in fully allogeneic recipients.

Pancreatic cancer: Current research and future directions.

Despite the survival rate advancements in different types of cancer in the last 40 years, the perspective for pancreatic cancer patients has seen no substantial changes. Indeed, the five year survival rate remains around 5%. Nevertheless, in the last decade we have witnessed an increased interest in pancreatic cancer biology and this has produced a substantial increment in our knowledge on pancreatic cancer progression. The big challenge is now to translate this knowledge in better outcomes for patients. The aim of this review is to describe the latest discoveries and advancements in pancreatic cancer research and to discuss future directions.

Defining the Anti-Cancer Activity of Tricarbonyl Rhenium Complexes: Induction of G2/M Cell Cycle Arrest and Blockade of Aurora-A Kinase Phosphorylation.

Rhenium and ruthenium complexes containing N-heterocylic carbene (NHC) ligands and conjugated to indomethacin were prepared. The anticancer properties were probed against pancreatic cell lines, revealing a remarkable activity of the rhenium fragment as anticancer agent. The ruthenium complexes were found to be inactive against the same pancreatic cancer cell lines, either alone or in conjugation with indomethacin. An in-depth biological study revealed the origin of the anticancer properties of the rhenium tricarbonyl fragment, of which a complete elucidation had yet to be achieved. It was found that the rhenium complexes induce cell cycle arrest at the G2/M phase by inhibiting the phosphorylation of Aurora-A kinase. A preliminary study on the structure-activity relationship on a large family of these complexes revealed that the anticancer properties are mainly associated with the lability of the ancillary ligand, with inert complexes showing limited to no anticancer properties.