Targeting phosphoinositide signaling in cancer: relevant techniques to study lipids and novel avenues for therapeutic intervention

Phosphoinositides serve as essential players in numerous biological activities and are critical for overall cellular function. Due to their complex chemical structures, localization, and low abundance, current challenges in the phosphoinositide field include the accurate measurement and identification of specific variants, particularly those with acyl chains. Researchers are intensively developing innovative techniques and approaches to address these challenges and advance our understanding of the impact of phosphoinositide signaling on cellular biology. This article provides an overview of recent advances in the study of phosphoinositides, including mass spectrometry, lipid biosensors, and real-time activity assays using fluorometric sensors. These methodologies have proven instrumental for a comprehensive exploration of the cellular distribution and dynamics of phosphoinositides and have shed light on the growing significance of these lipids in human health and various pathological processes, including cancer. To illustrate the importance of phosphoinositide signaling in disease, this perspective also highlights the role of a family of lipid kinases named phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks), which have recently emerged as exciting therapeutic targets for cancer treatment. The ongoing exploration of phosphoinositide signaling not only deepens our understanding of cellular biology but also holds promise for novel interventions in cancer therapy

Mycobacterium caprae Infection in Livestock and Wildlife, Spain

Mycobacterium caprae is a pathogen that can infect animals and humans. To better understand the epidemiology of M. caprae, we spoligotyped 791 animal isolates. Results suggest infection is widespread in Spain, affecting 6 domestic and wild animal species. The epidemiology is driven by infections in caprids, although the organism has emerged in cattle

Biological membranes in EV biogenesis, stability, uptake, and cargo transfer: an ISEV position paper arising from the ISEV membranes and EVs workshop

Paracrine and endocrine roles have increasingly been ascribed to extracellular vesicles (EVs) generated by multicellular organisms. Central to the biogenesis, content, and function of EVs are their delimiting lipid bilayer membranes. To evaluate research progress on membranes and EVs, the International Society for Extracellular Vesicles (ISEV) conducted a workshop in March 2018 in Baltimore, Maryland, USA, bringing together key opinion leaders and hands-on researchers who were selected on the basis of submitted applications. The workshop was accompanied by two scientific surveys and covered four broad topics: EV biogenesis and release; EV uptake and fusion; technologies and strategies used to study EV membranes; and EV transfer and functional assays. In this ISEV position paper, we synthesize the results of the workshop and the related surveys to outline important outstanding questions about EV membranes and describe areas of consensus. The workshop discussions and survey responses reveal that while much progress has been made in the field, there are still several concepts that divide opinion. Good consensus exists in some areas, including particular aspects of EV biogenesis, uptake and downstream signalling. Areas with little to no consensus include EV storage and stability, as well as whether and how EVs fuse with target cells. Further research is needed in these key areas, as a better understanding of membrane biology will contribute substantially towards advancing the field of extracellular vesicles.Fil: Russell, Ashley E.. University Johns Hopkins; Estados UnidosFil: Sneider, Alexandra. University Johns Hopkins; Estados UnidosFil: Witwer, Kenneth W.. University Johns Hopkins; Estados UnidosFil: Bergese, Paolo. Università Degli Studi Di Brescia; ItaliaFil: Bhattacharyya, Suvendra N.. Indian Institute of Chemical Biology; IndiaFil: Cocks, Alexander. Cardiff University; Reino UnidoFil: Cocucci, Emanuele. Ohio State University; Estados UnidosFil: Erdbrügger, Uta. University of Virginia; Estados UnidosFil: Falcon Perez, Juan M.. Ikerbasque Basque Foundation for Science; EspañaFil: Freeman, David W.. National Institute On Aging National Institute for Helth ; Estados UnidosFil: Gallagher, Thomas M.. Loyola University Of Chicago; Estados UnidosFil: Hu, Shuaishuai. Technological University Dublin; IrlandaFil: Huang, Yiyao. University Johns Hopkins; Estados Unidos. Southern Medical University; ChinaFil: Jay, Steven M.. University of Maryland; Estados UnidosFil: Kano, Shin-ichi. The University of Alabama at Birmingham School of Medicine; Estados UnidosFil: Lavieu, Gregory. Institut Curie; FranciaFil: Leszczynska, Aleksandra. University of California at San Diego; Estados UnidosFil: Llorente, Alicia M.. Oslo University Hospital; NoruegaFil: Lu, Quan. Harvard University. Harvard School of Public Health; Estados UnidosFil: Mahairaki, Vasiliki. University Johns Hopkins; Estados UnidosFil: Muth, Dillon C.. University Johns Hopkins; Estados UnidosFil: Noren Hooten, Nicole. National Institute On Aging National Institute for Helth ; Estados UnidosFil: Ostrowski, Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; ArgentinaFil: Prada, Ilaria. Consiglio Nazionale delle Ricerche; ItaliaFil: Sahoo, Susmita. Icahn School of Medicine at Mount Sinai ; Estados UnidosFil: Schøyen, Tine Hiorth. Uit The Arctic University Of Norway; Noruega. University Johns Hopkins; Estados UnidosFil: Sheng, Lifuy. University of Washington. School of Medicine; Estados UnidosFil: Tesch, Deanna. Shaw University; Estados UnidosFil: Van Niel, Guillaume. No especifíca;Fil: Vandenbroucke, Roosmarijn E.. University of Ghent; BélgicaFil: Verweij, Frederik J.. No especifíca;Fil: Villar, Ana V.. Universidad de Cantabria; EspañaFil: Wauben, Marca. University of Utrecht; Países BajosFil: Wehman, Ann M.. Universität Würzburg; AlemaniaFil: Ardavan, Arzhang. Peking University; ; ChinaFil: Carter, David Raul Francisco. Oxford Brookes University; Reino UnidoFil: Vader, Pieter. University Medical Center Utrecht; Países Bajo

Biological properties of extracellular vesicles and their physiological functions

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system

Lymphangioleiomyomatosis biomarkers linked to lung metastatic potential and cell stemness

Lymphangioleiomyomatosis (LAM) is a rare lung-metastasizing neoplasm caused by the proliferation of smooth muscle-like cells that commonly carry loss-of-function mutations in either the tuberous sclerosis complex 1 or 2 (TSC1 or TSC2) genes. While allosteric inhibition of the mechanistic target of rapamycin (mTOR) has shown substantial clinical benefit, complementary therapies are required to improve response and/or to treat specific patients. However, there is a lack of LAM biomarkers that could potentially be used to monitor the disease and to develop other targeted therapies. We hypothesized that the mediators of cancer metastasis to lung, particularly in breast cancer, also play a relevant role in LAM. Analyses across independent breast cancer datasets revealed associations between low TSC1/2 expression, altered mTOR complex 1 (mTORC1) pathway signaling, and metastasis to lung. Subsequently, immunohistochemical analyses of 23 LAM lesions revealed positivity in all cases for the lung metastasis mediators fascin 1 (FSCN1) and inhibitor of DNA binding 1 (ID1). Moreover, assessment of breast cancer stem or luminal progenitor cell biomarkers showed positivity in most LAM tissue for the aldehyde dehydrogenase 1 (ALDH1), integrin-ß3 (ITGB3/CD61), and/or the sex-determining region Y-box 9 (SOX9) proteins. The immunohistochemical analyses also provided evidence of heterogeneity between and within LAM cases. The analysis of Tsc2-deficient cells revealed relative over-expression of FSCN1 and ID1; however, Tsc2-deficient cells did not show higher sensitivity to ID1-based cancer inhibitors. Collectively, the results of this study reveal novel LAM biomarkers linked to breast cancer metastasis to lung and to cell stemness, which in turn might guide the assessment of additional or complementary therapeutic opportunities for LAM

Summary of the ISEV workshop on extracellular vesicles as disease biomarkers, held in Birmingham, UK, during December 2017

This report summarises the presentations and activities of the ISEV Workshop on extracellular vesicle biomarkers held in Birmingham, UK during December 2017. Among the key messages was broad agreement about the importance of biospecimen science. Much greater attention needs to be paid towards the provenance of collected samples. The workshop also highlighted clear gaps in our knowledge about pre-analytical factors that alter extracellular vesicles (EVs). The future utility of certified standards for credentialing of instruments and software, to analyse EV and for tracking the influence of isolation steps on the structure and content of EVs were also discussed. Several example studies were presented, demonstrating the potential utility for EVs in disease diagnosis, prognosis, longitudinal serial testing and stratification of patients. The conclusion of the workshop was that more effort focused on pre-analytical issues and benchmarking of isolation methods is needed to strengthen collaborations and advance more effective biomarkers

Analytical techniques for multiplex analysis of protein biomarkers

Introduction: The importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine. Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect the earliest perturbations indicative of disease, minimizing therapeutic intervention or even enabling disease reversal. Protein biomarkers are of particular interest given their central role in biochemical pathways. Hence, capabilities to analyze multiple protein biomarkers in one assay are highly interesting for biomedical research. Areas covered: We here review multiple methods that are suitable for robust, high throughput, standardized, and affordable analysis of protein biomarkers in a multiplex format. We describe innovative developments in immunoassays, the vanguard of methods in clinical laboratories, and mass spectrometry, increasingly implemented for protein biomarker analysis. Moreover, emerging techniques are discussed with potentially improved protein capture, separation, and detection that will further boost multiplex analyses. Expert commentary: The development of clinically applied multiplex protein biomarker assays is essential as multi-protein signatures provide more comprehensive information about biological systems than single biomarkers, leading to improved insights in mechanisms of disease, diagnostics, and the effect of personalized medicine

Detection of circulating miRNAs : comparative analysis of extracellular vesicle-incorporated miRNAs and cell-free miRNAs in whole plasma of prostate cancer patients

Funding Information: This study was supported by the Norwegian Financial Mechanism 2009–2014 under Project Contract No NFI/R/2014/045. The funding body had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. Publisher Copyright: © 2017 The Author(s).Background: Circulating cell-free miRNAs have emerged as promising minimally-invasive biomarkers for early detection, prognosis and monitoring of cancer. They can exist in the bloodstream incorporated into extracellular vesicles (EVs) and ribonucleoprotein complexes. However, it is still debated if EVs contain biologically meaningful amounts of miRNAs and may provide a better source of miRNA biomarkers than whole plasma. The aim of this study was to systematically compare the diagnostic potential of prostate cancer-associated miRNAs in whole plasma and in plasma EVs. Methods: RNA was isolated from whole plasma and plasma EV samples from a well characterised cohort of 50 patient with prostate cancer (PC) and 22 patients with benign prostatic hyperplasia (BPH). Nine miRNAs known to have a diagnostic potential for PC in cell-free blood were quantified by RT-qPCR and the relative quantities were compared between patients with PC and BPH and between PC patients with Gleason score ≥ 8 and ≤6. Results: Only a small fraction of the total cell-free miRNA was recovered from the plasma EVs, however the EV-incorporated and whole plasma cell-free miRNA profiles were clearly different. Four of the miRNAs analysed showed a diagnostic potential in our patient cohort. MiR-375 could differentiate between PC and BPH patients when analysed in the whole plasma, while miR-200c-3p and miR-21-5p performed better when analysed in plasma EVs. EV-incorporated but not whole plasma Let-7a-5p level could distinguish PC patients with Gleason score ≥ 8 vs ≤6. Conclusions: This study demonstrates that for some miRNA biomarkers EVs provide a more consistent source of RNA than whole plasma, while other miRNAs show better diagnostic performance when tested in the whole plasma.publishersversionPeer reviewe

Diagnostic, prognostic and predictive value of cell-free miRNAs in prostate cancer : A systematic review

Publisher Copyright: © 2016 Endzeliņš et al.Prostate cancer, the second most frequently diagnosed cancer in males worldwide, is estimated to be diagnosed in 1.1 million men per year. Introduction of PSA testing substantially improved early detection of prostate cancer, however it also led to overdiagnosis and subsequent overtreatment of patients with an indolent disease. Treatment outcome and management of prostate cancer could be improved by the development of non-invasive biomarker assays that aid in increasing the sensitivity and specificity of prostate cancer screening, help to distinguish aggressive from indolent disease and guide therapeutic decisions. Prostate cancer cells release miRNAs into the bloodstream, where they exist incorporated into ribonucleoprotein complexes or extracellular vesicles. Later, cell-free miRNAs have been found in various other biofluids. The initial RNA sequencing studies suggested that most of the circulating cell-free miRNAs in healthy individuals are derived from blood cells, while specific disease-associated miRNA signatures may appear in the circulation of patients affected with various diseases, including cancer. This raised a hope that cell-free miRNAs may serve as non-invasive biomarkers for prostate cancer. Indeed, a number of cell-free miRNAs that potentially may serve as diagnostic, prognostic or predictive biomarkers have been discovered in blood or other biofluids of prostate cancer patients and need to be validated in appropriately designed longitudinal studies and clinical trials. In this review, we systematically summarise studies investigating cell-free miRNAs in biofluids of prostate cancer patients and discuss the utility of the identified biomarkers in various clinical scenarios. Furthermore, we discuss the possible mechanisms of miRNA release into biofluids and outline the biological questions and technical challenges that have arisen from these studies.publishersversionPeer reviewe

Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points