Quantitative analysis of energy transfer between fluorescent proteins in CFP-GBP-YFP and its response to Ca2+

This article reports the full characterisation of the optical properties of a biosynthesised protein consisting of fused cyan fluorescent protein, glucose binding protein and yellow fluorescent protein. The cyan and yellow fluorescent proteins act as donors and acceptors for intramolecular fluorescence resonance energy transfer. Absorption, fluorescence, excitation and fluorescence decays of the compound protein were measured and compared with those of free fluorescent proteins. Signatures of energy transfer were identified in the spectral intensities and fluorescence decays. A model describing the fluorescence properties including energy transfer in terms of rate equations is presented and all relevant parameters are extracted from the measurements. The compound protein changes conformation on binding with calcium ions. This is reflected in a change of energy transfer efficiency between the fluorescent proteins. We track the conformational change and the kinetics of the calcium binding reaction from fluorescence intensity and decay measurements and interpret the results in light of the rate equation model. This visualisation of change in protein conformation has the potential to serve as an analytical tool in the study of protein structure changes in real time, in the development of biosensor proteins and in characterizing protein-drug interactions

Prognostic Role of RASSF1A, SOX17 and Wif-1 Promoter Methylation Status in Cell-Free DNA of Advanced Gastric Cancer Patients

Epigenetic modification of several genes is a key component in the development of gastric cancer. The methylation status of RASSF1A, SOX17 and Wif-1 genes was evaluated in the cell free circulating DNA of 70 patients with advanced gastric cancer, using methylation-specific PCR. Patients with higher cell-free DNA concentration seem to have lower PFS, than patients with lower cell-free DNA concentration (p = 0.001). RASSF1A was the tumor suppressor gene, most frequently methylated in metastatic gastric cancer patients, followed by SOX17 and Wif-1 (74.3%, 60.0% and 47.1%, respectively). Patients having the SOX17 promoter methylated, had lower progression free survival and overall survival, than unmethylated ones (p < 0.001). Patients having the Wif-1 promoter methylated, had lower progression free survival and overall survival, than unmethylated ones (p = 0.001). Patients having the RASSF1A promoter methylated, had lower progression free survival and overall survival, than unmethylated ones (p = 0.004). Promoter methylation of the examined genes was significantly associated with a decrease in progression free survival and overall survival, comparing to that of patients without methylation. Simultaneous methylation of the above genes was associated with even worse progression free survival and overall survival. The methylation of RASSF1A, SOX-17 and Wif-1 and genes, is a frequent epigenetic event in patients with advanced gastric cancer

Circulating cell-free DNA in breast cancer: size profiling, levels, and methylation patterns lead to prognostic and predictive classifiers

Blood circulating cell-free DNA (ccfDNA) is a suggested biosource of valuable clinical information for cancer, meeting the need for a minimally-invasive advancement in the route of precision medicine. In this paper, we evaluated the prognostic and predictive potential of ccfDNA parameters in early and advanced breast cancer. Groups consisted of 150 and 16 breast cancer patients under adjuvant and neoadjuvant therapy respectively, 34 patients with metastatic disease and 35 healthy volunteers. Direct quantification of ccfDNA in plasma revealed elevated concentrations correlated to the incidence of death, shorter PFS, and non-response to pharmacotherapy in the metastatic but not in the other groups. The methylation status of a panel of cancer-related genes chosen based on previous expression and epigenetic data (KLK10, SOX17, WNT5A, MSH2, GATA3) was assessed by quantitative methylation-specific PCR. All but the GATA3 gene was more frequently methylated in all the patient groups than in healthy individuals (all p < 0.05). The methylation of WNT5A was statistically significantly correlated to greater tumor size and poor prognosis characteristics and in advanced stage disease with shorter OS. In the metastatic group, also SOX17 methylation was significantly correlated to the incidence of death, shorter PFS, and OS. KLK10 methylation was significantly correlated to unfavorable clinicopathological characteristics and relapse, whereas in the adjuvant group to shorter DFI. Methylation of at least 3 or 4 genes was significantly correlated to shorter OS and no pharmacotherapy response, respectively. Classification analysis by a fully automated, machine learning software produced a single-parametric linear model using ccfDNA plasma concentration values, with great discriminating power to predict response to chemotherapy (AUC 0.803, 95% CI [0.606, 1.000]) in the metastatic group. Two more multi-parametric signatures were produced for the metastatic group, predicting survival and disease outcome. Finally, a multiple logistic regression model was constructed, discriminating between patient groups and healthy individuals. Overall, ccfDNA emerged as a highly potent predictive classifier in metastatic breast cancer. Upon prospective clinical evaluation, all the signatures produced could aid accurate prognosis. © 2019, Springer Nature Limited

Complex Immune Dysregulation in COVID-19 Patients with Severe Respiratory Failure

Proper management of COVID-19 mandates better understanding of disease pathogenesis. Giamarellos-Bourboulis et al. describe two main features preceding severe respiratory failure associated with COVID-19: the first is macrophage activation syndrome; the second is defective antigen-presentation driven by interleukin-6. An IL-6 blocker partially rescues immune dysregulation in vitro and in patients. © 2020 Elsevier Inc. Proper management of COVID-19 mandates better understanding of disease pathogenesis. The sudden clinical deterioration 7–8 days after initial symptom onset suggests that severe respiratory failure (SRF) in COVID-19 is driven by a unique pattern of immune dysfunction. We studied immune responses of 54 COVID-19 patients, 28 of whom had SRF. All patients with SRF displayed either macrophage activation syndrome (MAS) or very low human leukocyte antigen D related (HLA-DR) expression accompanied by profound depletion of CD4 lymphocytes, CD19 lymphocytes, and natural killer (NK) cells. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) production by circulating monocytes was sustained, a pattern distinct from bacterial sepsis or influenza. SARS-CoV-2 patient plasma inhibited HLA-DR expression, and this was partially restored by the IL-6 blocker Tocilizumab; off-label Tocilizumab treatment of patients was accompanied by increase in circulating lymphocytes. Thus, the unique pattern of immune dysregulation in severe COVID-19 is characterized by IL-6-mediated low HLA-DR expression and lymphopenia, associated with sustained cytokine production and hyper-inflammation. © 2020 Elsevier Inc