Analysis insights for three FRET pairs of chemically unlinked two-molecule FRET cytometry.

Förster resonance energy transfer (FRET) is the direct energy exchange between two-component fluorescent molecules. FRET methods utilize chemically linked molecules or unlinked fluorescent molecules such as fluoresscent protein-protein interactions. FRET is therefore a powerful indicator of molecular proximity, but standardized determination of FRET efficiency is challenged when investigating natural (chemically unlinked) interactions. In this paper, we have examined the interactions of tumor necrosis factor receptor-1 (TNFR1) molecules expressed as recombinant C-terminal fusion proteins of cyan, yellow, or red fluorescent protein (-CFP, -YFP, or -RFP) to evaluate two-molecule chemically unlinked FRET by flow cytometry. We demonstrate three independent FRET pairs of TNFR1 CFP→YFP (FRET-1), YFP→RFP (FRET-2) and CFP→RFP (FRET-3), by comparing TNFR1+TNFR1 with non-interacting TNFR1+CD27 proteins, on both LSR-II and Fortessa X-20 cytometers. We describe genuine FRET activities reflecting TNFR1 homotypic interactions. The FRET events can be visualized during sample acquisition via the use of "spiked" FRET donor cells, together with TNFR1+TNFR1 co-transfected cells, as FRET channel mean fluorescence intensity (MFI) overlays. FRET events can also be indicated by comparing concatenated files of cells expressing either FRET positive events (TNFR1+TNFR1) or FRET negative events (TNFR1+CD27) to generate single-cell scatter plots showing loss of FRET donor brightness. Robust determination of FRET efficiency is then confirmed at the single-cell level by applying matrix calculations based on the measurements of FRET, using donor, acceptor, and FRET fluorescent intensities (I), detector channel emission coefficient (S), fluorescent protein extinction coefficients (ε) and the α factor. In this TNFR1-based system the mean CFP→YFP FRET-1 efficiency is 0.43 (LSR-II) and 0.41 (Fortessa X-20), the mean YFP→RFP FRET-2 efficiency is 0.30 (LSR-II) and 0.29 (Fortessa X-20), and the mean CFP→RFP FRET-3 efficiency is 0.56 (LSR-II) and 0.54 (Fortessa X-20). This study also embraces multi-dimensional clustering using t-SNE, Fit-SNE, UMAP, Tri-Map and PaCMAP to further demonstrate FRET. These approaches establish a robust system for standardized detection of chemically unlinked TNFR1 homotypic interactions with three individual FRET pairs.

A multi-colour confocal microscopy method for identifying and enumerating macrophage subtypes and adherent cells in the stromal vascular fraction of human adipose.

This study examines leukocytes present in lymphoedema (LE) adipose tissue (AT) by multi-colour confocal microscopy. LE AT, collected by liposuction surgery, was digested with collagenase to separate adipocytes from other tissue cells, comprising blood and lymphatic endothelial cells, fibroblasts, and all vessel- and tissue-resident leukocytes - the stromal vascular fraction (SVF). SVF cells were activated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, adding Brefeldin-A to prevent cytokine secretion during the final 4 hours. Cells were incubated with CD11b-FITC and CD40-APC (M1 MØ)' or CD206-APC (M2 MØ) specific antibodies, fixed, permeabilised, then incubated with either (1) anti-TNF-PE, (2) anti- IL-1ß-PE, (3) anti-IL-6-PE, (4) anti-IL-4-PE, (5) anti-TGFß-PE or (6) isotype-IgG-PE (control), and stained with Hoechst 33342, preserved in permanent mounting media and examined by confocal microscopy. The FITC, PE and APC fluorescence channels were set to achieve minimal cross-channel emission using single-colour controls and voltages set for optimal detection by thresholding on isotype-IgG stained activated cells. Finally, transmission and z-stack images were captured. Cells were analysed as regions of interest (ROI) based on Hoechst-33342 then enumerated as FITC+, FITC+APC+ or FITC+APC+PE+ using an ImageJ script and exported into Excel. This permitted the examination of >9000 SVF cells individually, per LE sample. This method allows for the analysis of a high number of heterogeneous cells defined into any subtype or combination by the investigators' choice of surface and intracellular expression profiles. Fibroblasts, or other cytokine producing cells, can also be analysed by using other antibodies, and the cell count data can be correlated with any clinical or laboratory data.

A panel consisting of three novel circulating lncRNAs, is it a predictive tool for gastric cancer?

Early detection is vital for prolonging 5-year survival for patients with gastric cancer (GC). Numerous studies indicate that circulating long non-coding RNAs (lncRNAs) can be used to diagnose malignant tumours. This study aimed to investigate the capacity of novel lncRNAs for diagnosing GC. A lncRNA microarray assay was used to screen differentially expressed lncRNAs between plasma of patients with GC and healthy controls. Plasma samples from 100 patients with healthy controls were used to construct a multiple-gene panel. An additional 50 pairs of GC patients with healthy controls were used to evaluate the diagnostic accuracy of the panel. Expression levels of lncRNAs were quantified through real-time polymerase chain reaction. The receiver operating characteristic curve and area under curve (AUC) were used to estimate the diagnostic capacity. We identified three lncRNAs, CTC-501O10.1, AC100830.4 and RP11-210K20.5 that were up-regulated in the plasma of GC patients with AUCs 0.724, 0.730 and 0.737, respectively (P < .01). Based on the logistic regression model, the combined AUC of the three lncRNAs was 0.764. The AUC of the panel was 0.700 in the validation cohort. These findings indicate that plasma lncRNAs can serve as potential biomarkers for detection of GC.

Association between alcohol consumption and the risk of gastric cancer: a meta-analysis of prospective cohort studies.

Alcohol consumption is inconsistently associated with the risk of gastric cancer morbidity and mortality. The aim of this study was to systematically evaluate the association between alcohol consumption on gastric cancer risk. The PubMed, Embase, and Cochrane Library databases were searched from inception through April 2017. Prospective cohort studies evaluating the association between alcohol consumption and risk of gastric cancer which report its effect estimates with 95% confidence intervals (CIs) were included. The results summary was performed using the random-effect model. Twenty-two cohort studies involving 22,545 cases of gastric cancer and 5,820,431 participants were identified and included in our data analysis. Overall, drinking had little or no effect on gastric cancer as compared with non-drinkers. Furthermore, light and moderate alcohol consumption had no significant effect on gastric cancer risk when compared with non-drinkers. However, heavy alcohol consumption was associated with a greater risk of gastric cancer when compared with non-drinkers. The findings of the subgroup analyses indicated that light alcohol consumption was associated with a lower risk of gastric cancer in women, while heavy alcohol consumption was associated with an increased risk of gastric cancer regardless of country, gender, whether the study reported gastric cancer incidence, or whether the study adjusted for body mass index, educational attainment, or physical activity. The findings of this study suggest that light alcohol consumption might play a protective effect on gastric cancer in women, while heavy alcohol consumption is associated with a significantly increased risk of gastric cancer in all subgroups.

Efficacy and safety of intraperitoneal chemotherapy in patients with advanced gastric cancer: a cumulative meta-analysis of randomized controlled trials.

Even when a curative gastrectomy is conducted, the majority of advanced gastric cancer patients with invasion die due to peritoneal recurrence. We performed electronic searches to identify randomized controlled trials published through April 2017 evaluating the effect of intraperitoneal chemotherapy (IPC) on survival rates. We included 23 trials reporting data on 2,767 patients with advanced gastric cancer. Overall, we noted that patients who received IPC had a significantly increased 1-year survival rate, and the treatment effect of IPC on 1-year survival was most prominent in studies conducted in Japan or those with a mean age of less than 60 years. IPC was also associated with an increased incidence of 2-year survival rate, but it was not seen to have this effect in studies conducted in China or Australia or with a mean age greater than 60 years. Similarly, IPC associated with a significantly increased 3-year survival rate, but this difference was not detected in studies conducted in Austria or with a mean age greater than 60 years. IPC has no significant effect on the 5-year survival rate. Finally, IPC was associated with a lower risk of recurrence in patients with advanced gastric cancer. The findings of this study suggest that gastric cancer patients who receive IPC associate with increased 1-year, 2-year, and 3-year survival rates, but this does not extend out to a 5-year survival rate. IPC is also shown to play a protective role against the risk of recurrence in patients with advanced gastric cancer.