Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Challenges in microbial ecology: building predictive understanding of community function and dynamics.

The importance of microbial communities (MCs) cannot be overstated. MCs underpin the biogeochemical cycles of the earth's soil, oceans and the atmosphere, and perform ecosystem functions that impact plants, animals and humans. Yet our ability to predict and manage the function of these highly complex, dynamically changing communities is limited. Building predictive models that link MC composition to function is a key emerging challenge in microbial ecology. Here, we argue that addressing this challenge requires close coordination of experimental data collection and method development with mathematical model building. We discuss specific examples where model-experiment integration has already resulted in important insights into MC function and structure. We also highlight key research questions that still demand better integration of experiments and models. We argue that such integration is needed to achieve significant progress in our understanding of MC dynamics and function, and we make specific practical suggestions as to how this could be achieved

Advancements in kidney organoids and tubuloids to study (dys)function

The rising prevalence of kidney diseases urges the need for novel therapies. Kidney organoids and tubuloids are advanced in vitro models and have recently been described as promising tools to study kidney (patho)physiology. Recent developments have shown their application in disease modeling, drug screening, and nephrotoxicity. These applications rely on their ability to mimic (dys)function in vitro including endocrine activity and drug, electrolyte, and water transport. This review provides an overview of these emerging kidney models and focuses on the most recent developments that utilize their functional capabilities. In addition, we cover current limitations and provide future perspectives for this rapidly evolving field, including what these functional properties mean for translational and personalized medicine now and in the future.</p

EFFECT OF CHEMICAL TREATMENT ON THE AROMATIC CARBON CONTENT AND PARTICLE

The present experimental work was carried out to examine the physicochemical treatability of a high-strength olive oil mill wastewater (OMW). Firstly, the wastewater was subjected to environmental characterization and particle size distribution-based COD-TOC-UV254-UV280 analyses. The following treatment schemes were selected for the study: Coagulation-flocculation in the presence of anionic and commercial polymers at varying pH and coagulant/polymer dosages Fenton treatment at different pH's and Fe(II): H2O2 concentrations as well as electrocoagulation using stainless steel electrodes at different electrolyte concentrations and current densities. Results of the study have indicated that none of the investigated physicochemical treatment methods was capable of removing the organic carbon content of the wastewater by more than 30% in terms of COD and 20% in terms of TOC that is at least partially attributable to the high, soluble organic carbon content of the wastewater. Alternative treatment processes and/or combinations have to be explored for effective treatment of OMW effluent

Assessment of organic carbon removal by particle size distribution analysis

Particle size distribution (PSD)-based fractionation, an alternative methodology in wastewater characterization, was employed to gather detailed information on the organic matter content of a raw olive mill wastewater (OMW) and to investigate the changes brought about in this characteristic after application of certain chemical treatment alternatives, namely, lime precipitation-coagulation, Fenton oxidation, and electrocoagulation. PSD-based analysis of the untreated OMW demonstrated that more than two-thirds of its organic carbon content, measured as chemical oxygen demand (COD) (69%), total organic carbon (TOC) (74%), as well as antioxidant activity (AOA) (74%) caused by the polyphenolic carbon fraction, was at the soluble range (<2 nm). Treatability experiments, with maximum overall removal efficiencies below 50% even under optimized conditions, indicated that none of the applied chemical pretreatment methods was sufficiently effective in removing the organics from the investigated OMW. Nonetheless, PSD-based fractionation of the pretreated samples provided an insight about the treatment alternatives and the size fractions where they performed better. Electrocoagulation was capable of removing particulate and colloidal matter and provided a relatively higher overall performance with 23, 20, and 34% decreases in COD, TOC, and polyphenol contents of the OMW, respectively. Fenton process, on the other hand, showed a lower overall performance in terms of COD and TOC removal (17 and 15%, respectively), yet it was significantly effective on the soluble fraction, and thus more successful in removing the polyphenols mostly originating from the soluble range, with an overall efficiency of 42%. Based on these observations, PSD analysis was suggested as a useful tool to perform detailed wastewater characterization, as well as to differentiate between specific impacts of different pretreatment processes and help choose between alternatives, especially when a particular pollutant is targeted. Copyright 2009, Mary Ann Liebert, Inc

Effect of chemical treatment on the aromatic carbon content and particle size distribution-based organic matter profile of Olive mill wastewaters

The present experimental work was carried out to examine the physicochemical treatability of a high-strength olive oil mill wastewater (OMW). Firstly, the wastewater was subjected to environmental characterization and particle size distribution-based COD-TOC-UV254-UV280 analyses. The following treatment schemes were selected for the study: Coagulation-flocculation in the presence of anionic and commercial polymers at varying pH and coagulant/polymer dosages; Fenton treatment at different pH's and Fe(II): H2O2 concentrations as well as electrocoagulation using stainless steel electrodes at different electrolyte concentrations and current densities. Results of the study have indicated that none of the investigated physicochemical treatment methods was capable of removing the organic carbon content of the wastewater by more than 30% in terms of COD and 20% in terms of TOC that is at least partially attributable to the high, soluble organic carbon content of the wastewater. Alternative treatment processes and/or combinations have to be explored for effective treatment of OMW effluent. © by PSP

WASTE-WATER MANAGEMENT FOR ISTANBUL - BASIS FOR TREATMENT AND DISPOSAL

A number of studies have so far been conducted to assess the magnitude of pollution generated by domestic and industrial sources in the Istanbul Metropolitan Area. They indicate that a management scheme for wastewater treatment and disposal should involve a total discharge of 15.4 m3 s-1 with a potential pollution load of 330 tons BOD5 day-1 for 1990. A scheme of this magnitude inevitably requires careful evaluation of receiving water characteristics, both from an oceanographic and a quality standpoint

engineering

The paper evaluates the scientific merit of doctoral studies on environmental sciences and technology, involving completed doctoral studies in Turkey between 2000-2007, as a case study. The investigation revealed 446 scientific papers derived from 170 completed doctoral studies. This level represented 22% of the total number of publications at departments with graduate programs in this field. 42 of the completed studies (25%) did not produce published papers other than the compulsory doctoral thesis. These publications received 2766 citations, corresponding to 6.2 citations per publication; 65 of 170 doctoral studies completed (38%) had no record of any citation, indicating that aside from 42 doctoral theses which did not produce any publications, results of 22 doctoral studies were published but received no citations. Impact factors of selected journals for publication varied within a wide range of 0.429 to 3.894 with an average value of 1.65, based on 2007 impact factor records