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

Frequency of the Q192R and L55M polymorphisms of the human serum paraoxonase gene (PON1) in ten Amazonian Amerindian tribes

Human serum paraoxonase (PON1) is an esterase associated with high density lipoproteins (HDLs) in the plasma and may confer protection against coronary artery disease. Serum PON1 levels and activity vary widely among individuals and populations of different ethnic groups, such variations appearing to be related to two coding region polymorphisms (L55M and Q192R). Several independent studies have indicated that the polymorphism at codon 192 (the R form) is a significant risk factor for cardiovascular disease in some populations, although this association has not been confirmed in other populations. Given the possible associations of these mutations with heart diseases and the fact that little or nothing is known of their prevalence in Amerindian populations, we investigated the variability of both polymorphisms in ten Amazonian Indian tribes and compared the variation found with that of other Asian populations in which both polymorphisms have been investigated. The results show that the LR haplotype is the most frequent and the MR haplotype is absent in all Amerindians and Asian populations. We also found that South America Amerindians present the highest frequency of the PON1192*R allele (considered a significant risk factor for heart diseases in some populations) of all the Amerindian and Asian populations so far studied

Influence of Genetic Ancestry on INDEL Markers of <i>NFKβ1</i>, <i>CASP8</i>, <i>PAR1</i>, <i>IL4</i> and <i>CYP19A1</i> Genes in Leprosy Patients

<div><p>Background</p><p>Leprosy is an insidious infectious disease caused by the obligate intracellular bacteria <i>Mycobacterium leprae</i>, and host genetic factors can modulate the immune response and generate distinct categories of leprosy susceptibility that are also influenced by genetic ancestry.</p><p>Methodology/Principal Findings</p><p>We investigated the possible effects of <i>CYP19A1</i> [rs11575899], <i>NFKβ1</i> [rs28362491], <i>IL1α</i> [rs3783553], <i>CASP8</i> [rs3834129], <i>UGT1A1</i> [rs8175347], <i>PAR1</i> [rs11267092], <i>CYP2E1</i> [INDEL 96pb] and <i>IL4</i> [rs79071878] genes in a group of 141 leprosy patients and 180 healthy individuals. The INDELs were typed by PCR Multiplex in ABI PRISM 3130 and analyzed with GeneMapper ID v3.2. The <i>NFKβ1</i>, <i>CASP8</i>, <i>PAR1</i> and <i>IL4</i> INDELs were associated with leprosy susceptibility, while <i>NFKβ1</i>, <i>CASP8</i>, <i>PAR1</i> and <i>CYP19A1</i> were associated with the MB (Multibacilary) clinical form of leprosy.</p><p>Conclusions/Significance</p><p><i>NFKβ1</i> [rs28362491], <i>CASP8</i> [rs3834129], <i>PAR1</i> [rs11267092] and <i>IL4</i> [rs79071878] genes are potential markers for susceptibility to leprosy development, while the INDELs in <i>NFKβ1</i>, <i>CASP8</i>, <i>PAR1</i> and <i>CYP19A1</i> (rs11575899) are potential markers for the severe clinical form MB. Moreover, all of these markers are influenced by genetic ancestry, and European contribution increases the risk to leprosy development, in other hand an increase in African contribution generates protection against leprosy.</p></div

Assessing Interethnic Admixture Using an X-Linked Insertion-Deletion Multiplex

In this study, a PCR multiplex was optimized, allowing the simultaneous analysis of 13 X-chromosome Insertion/deletion polymorphisms (INDELs). Genetic variation observed in Africans, Europeans, and Native Americans reveals high inter-population variability. The estimated proportions of X-chromosomes in an admixed population from the Brazilian Amazon region show a predominant Amerindian contribution (congruent to 41%), followed by European (congruent to 32%) and African (congruent to 27%) contributions. The proportion of Amerindian contribution based on X-linked data is similar to the expected value based on mtDNA and Y-chromosome information. The accuracy for assessing interethnic admixture, and the high differentiation between African, European, and Native American populations, demonstrates the suitability of this INDEL set to measure ancestry proportions in three-hybrid populations, as it is the case of Latin American populations. Am. J. Hum. Biol. 21:707-709, 2009. (C) 2009 Wiley-Liss, Inc.Fundacao para a Ciencia e a Tecnologia (FCT)[SFRH/BD/30039/2006]Milenio/CNPq (Conselho Nacional de Desenvolvimento Cientifico, e Tecnologico)FINEP (Financiadora de Estudos e Projetos)UFPA (Universidade Federal do Para

Demographic and clinical characteristics of the sample according with clinical form of leprosy.

<p>^a<i>t</i>-Test of Student;</p><p>^bFisher's Exact Test;</p><p>^c Mann-whitney test; The data are show like mean ± standard deviation.</p><p>Demographic and clinical characteristics of the sample according with clinical form of leprosy.</p

Allelic and genotypic distribution between leprosy patients and healthy individuals to markers associated whit susceptibility to leprosy.

<p>^<b>a</b><i>p-value</i> obtained for logistic regression adjusted by age, gender and genetic ancestry;</p><p>^b Adjusted Odds Ratio (OR);</p><p>^c p-value after correction for population structure;</p><p>^d A₁—allele with two tandem repeats A₂—allele with three tandem repeats.</p><p>Allelic and genotypic distribution between leprosy patients and healthy individuals to markers associated whit susceptibility to leprosy.</p

In a comparison of 141 leprosy patients and 180 healthy individuals, events with statistically significant (p<0.05) differences can be categorized into six categories of individuals with African and European genetic ancestry (10%>20%> 30%>40%>50%>60%), the p-values were adjusted by age and gender.

<p>The analysis in individuals with Amerindian ancestry was not statistically significant.</p