Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

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Analysis of intraoperative transfusions of red blood cell concentrates in adults

BACKGROUND: Transfusion of red blood cell (RBC) concentrates is the most common allogeneic transplantation. The aim of the study was to analyse the indications for RBC transfusions, compared to the estimated intraoperative blood loss and the actual requirements for blood transfusion.METHODS: We retrospectively analysed the files of 250 adult patients who were transfused over the year 2006, during various general, oncologic, trauma, vascular, plastic and thoracic surgical procedures. Preoperative screening was done in a hospital laboratory, whereas postoperative haemoglobin concentration and haematocrit were assessed at the bedside using a co-oximeter.RESULTS: The majority of RBC transfusions were started at relatively high haemoglobin concentrations (mean 5.6 mmol L-1), contrary to the current guidelines. A high correlation coefficient (r=0.82) was found between the estimated blood loss and the volume of RBCs transfused; therefore we concluded that the observed blood loss was the main factor in transfusion decisions.CONCLUSIONS: Despite enormous progress in transfusion science, the current practice in our institution is still far from ideal; RBCs are frequently transfused too early and without a real indication.BACKGROUND: Transfusion of red blood cell (RBC) concentrates is the most common allogeneic transplantation. The aim of the study was to analyse the indications for RBC transfusions, compared to the estimated intraoperative blood loss and the actual requirements for blood transfusion.METHODS: We retrospectively analysed the files of 250 adult patients who were transfused over the year 2006, during various general, oncologic, trauma, vascular, plastic and thoracic surgical procedures. Preoperative screening was done in a hospital laboratory, whereas postoperative haemoglobin concentration and haematocrit were assessed at the bedside using a co-oximeter.RESULTS: The majority of RBC transfusions were started at relatively high haemoglobin concentrations (mean 5.6 mmol L-1), contrary to the current guidelines. A high correlation coefficient (r=0.82) was found between the estimated blood loss and the volume of RBCs transfused; therefore we concluded that the observed blood loss was the main factor in transfusion decisions.CONCLUSIONS: Despite enormous progress in transfusion science, the current practice in our institution is still far from ideal; RBCs are frequently transfused too early and without a real indication

The pyrazole derivative of usnic acid inhibits the proliferation of pancreatic cancer cells in vitro and in vivo

Abstract Background Pancreatic cancer is one of the leading causes of cancer death in Western societies. Its late diagnosis and resistance to chemotherapies result in a high mortality rate; thus, the development of more effective therapies for the treatment of pancreatic cancer is strongly warranted. Usnic acid (UA) is a secondary metabolite of lichens that shows modest antiproliferative activity toward cancer cells. Recently, we reported the synthesis of a UA pyrazole derivative, named 5, which was more active than the parent compound toward cervical cancer cells. Here, its anticancer potential has been evaluated in detail in other cancer cells, particularly pancreatic cancer cells. Methods The impact of UA and derivative 5 on cell viability, morphology, cell cycle, and death was assessed using the MTT test, electron microscopy, flow cytometry, and immunoblotting, respectively. The calcium ions level was detected fluorometrically. In vivo, the anticancer activity of 5 was evaluated in a murine xenograft model. Results Derivative 5 inhibited the viability of different cancer cells. Noncancerous cells were less sensitive. It induced the release of calcium ions from the endoplasmic reticulum (ER) and ER stress, which was manifested by cell vacuolization. It was accompanied by G0/G1 cell cycle arrest and cell death of pancreatic cancer cells. When applied to nude mice with xenografted pancreatic cancer cells, 5 inhibited tumor growth, with no signs of kidney or liver toxicity. Conclusions UA derivative 5 is superior to UA inhibiting the growth and proliferation of pancreatic cancer cells. ER stress exaggeration is a mechanism underlying the activity of derivative 5