Regulation of Virulence of Entamoeba histolytica by the URE3-BP Transcription Factor

It is not understood why only some infections with Entamoeba histolytica result in disease. The calcium-regulated transcription factor upstream regulatory element 3-binding protein (URE3-BP) was initially identified by virtue of its role in regulating the expression of two amebic virulence genes, the Gal/GalNac lectin and ferredoxin. Here we tested whether this transcription factor has a broader role in regulating virulence. A comparison of in vivo to in vitro parasite gene expression demonstrated that 39% of in vivo regulated transcripts contained the URE3 motif recognized by URE3-BP, compared to 23% of all promoters (P < 0.0001). Amebae induced to express a dominant positive mutant form of URE3-BP had an increase in an elongated morphology (30% ± 6% versus 14% ± 5%; P = 0.001), a 2-fold competitive advantage at invading the intestinal epithelium (P = 0.017), and a 3-fold increase in liver abscess size (0.1 ± 0.1 g versus 0.036 ± 0.1 g; P = 0.03). These results support a role for URE3-BP in virulence regulation

Systems analysis of the transcriptional response of human ileocecal epithelial cells to Clostridium difficile toxins and effects on cell cycle control

<p>Abstract</p> <p>Background</p> <p>Toxins A and B (TcdA and TcdB) are <it>Clostridium difficile</it>'s principal virulence factors, yet the pathways by which they lead to inflammation and severe diarrhea remain unclear. Also, the relative role of either toxin during infection and the differences in their effects across cell lines is still poorly understood. To better understand their effects in a susceptible cell line, we analyzed the transciptome-wide gene expression response of human ileocecal epithelial cells (HCT-8) after 2, 6, and 24 hr of toxin exposure.</p> <p>Results</p> <p>We show that toxins elicit very similar changes in the gene expression of HCT-8 cells, with the TcdB response occurring sooner. The high similarity suggests differences between toxins are due to events beyond transcription of a single cell-type and that their relative potencies during infection may depend on differential effects across cell types within the intestine. We next performed an enrichment analysis to determine biological functions associated with changes in transcription. Differentially expressed genes were associated with response to external stimuli and apoptotic mechanisms and, at 24 hr, were predominately associated with cell-cycle control and DNA replication. To validate our systems approach, we subsequently verified a novel G₁/S and known G₂/M cell-cycle block and increased apoptosis as predicted from our enrichment analysis.</p> <p>Conclusions</p> <p>This study shows a successful example of a workflow deriving novel biological insight from transcriptome-wide gene expression. Importantly, we do not find any significant difference between TcdA and TcdB besides potency or kinetics. The role of each toxin in the inhibition of cell growth and proliferation, an important function of cells in the intestinal epithelium, is characterized.</p

A compendium of single extracellular vesicle flow cytometry

Flow cytometry (FCM) offers a multiparametric technology capable of characterizing single extracellular vesicles (EVs). However, most flow cytometers are designed to detect cells, which are larger than EVs. Whereas cells exceed the background noise, signals originating from EVs partly overlap with the background noise, thereby making EVs more difficult to detect than cells. This technical mismatch together with complexity of EV-containing fluids causes limitations and challenges with conducting, interpreting and reproducing EV FCM experiments. To address and overcome these challenges, researchers from the International Society for Extracellular Vesicles (ISEV), International Society for Advancement of Cytometry (ISAC), and the International Society on Thrombosis and Haemostasis (ISTH) joined forces and initiated the EV FCM working group. To improve the interpretation, reporting, and reproducibility of future EV FCM data, the EV FCM working group published an ISEV position manuscript outlining a framework of minimum information that should be reported about an FCM experiment on single EVs (MIFlowCyt-EV). However, the framework contains limited background information. Therefore, the goal of this compendium is to provide the background information necessary to design and conduct reproducible EV FCM experiments. This compendium contains background information on EVs, the interaction between light and EVs, FCM hardware, experimental design and preanalytical procedures, sample preparation, assay controls, instrument data acquisition and calibration, EV characterization, and data reporting. Although this compendium focuses on EVs, many concepts and explanations could also be applied to FCM detection of other particles within the EV size range, such as bacteria, lipoprotein particles, milk fat globules, and viruses

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

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Prognostic role of preoperative circulating systemic inflammatory response markers in primary breast cancer: meta-analysis

Background: Circulating markers of the systemic inflammatory response are prognostic in several cancers, but their role in operable breast cancer is unclear. A systematic review and meta-analysis of the literature was carried out. Methods: A search of electronic databases up to August 2020 identified studies that examined the prognostic value of preoperative circulating markers of the systemic inflammatory response in primary operable breast cancer. A meta-analysis was carried out for each marker with more than three studies, reporting a HR and 95 per cent confidence interval for disease-free survival (DFS), breast cancer-specific survival (BCSS) or overall survival (OS). Results: In total, 57 studies were reviewed and 42 were suitable for meta-analysis. Higher neutrophil-to-lymphocyte ratio (NLR) was associated with worse overall survival (OS) (pooled HR 1.75, 95 per cent c.i. 1.52 to 2.00; P &lt; 0.001), disease-free survival (DFS) (HR 1.67, 1.50 to 1.87; P &lt; 0.001), and breast cancer-specific survival (BCSS) (HR 1.89, 1.35 to 2.63; P &lt; 0.001). This effect was also seen with an arithmetically-derived NLR (dNLR). Higher platelet-to-lymphocyte ratio (PLR) was associated with worse OS (HR 1.29, 1.10 to 1.50; P = 0.001) and DFS (HR 1.58, 1.33 to 1.88; P &lt; 0.001). Higher lymphocyte-to-monocyte ratio (LMR) was associated with improved DFS (HR 0.65, 0.51 to 0.82; P &lt; 0.001), and higher C-reactive protein (CRP) level was associated with worse BCSS (HR 1.22, 1.07 to 1.39; P = 0.002) and OS (HR 1.24, 1.14 to 1.35; P = 0.002). Conclusion: Current evidence suggests a role for preoperative NLR, dNLR, LMR, PLR, and CRP as prognostic markers in primary operable breast cancer. Further work should define their role in clinical practice, particularly reproducible thresholds and molecular subtypes for which these may be of most value

Imaging Flow Cytometry Elucidates Limitations of Microparticle Analysis by Conventional Flow Cytometry

Microparticles (MPs) are submicron vesicles released from cell membranes in response to activation, cell injury, or apoptosis. The clinical importance of MPs has become increasingly recognized, although no standardized method exists for their measurement. Flow cytometry (FCM) is the most commonly used technique, however, because of the small size of MPs, and the limitations of current FCM instrumentation, accurate identification is compromised by this methodology. We decided to investigate whether the use of FCM combined with imaging, such as is possible with the ImagestreamX imaging FC (ISX), would be a more sensitive approach to characterizing MPs. Combining FCM with imaging eliminates some of the limitations demonstrated by conventional FCM, whereas also providing morphological confirmation and the ability to distinguish true single events from aggregates and cell debris. The detection limit of standard nonspecialized FCM is suboptimal when compared to ISX. Evaluating MPs below 0.200 mm and sizing remain a challenge as some MPs remain below the detection limit of ISX. Standardized calibrators, that more closely reflect the physical characteristics of MPs, need further development. (C) 2014 International Society for Advancement of Cytometr