Centro Andaluz de Biología Molecular y Medicina Regenerativa

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 thatthere 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 flux through the autophagy pathway (i.e., the completeprocess 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, defined as increasedautophagy 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 field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in manycases, 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 forreviewers 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 multipleassays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagyrelated protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that 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 assays, we hope to encourage technical innovation in the field.

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.

PURPOSE: Age-related macular degeneration (AMD) is a frequent, complex disorder in elderly of European ancestry. Risk profiles and treatment options have changed considerably over the years, which may have affected disease prevalence and outcome. We determined the prevalence of early and late AMD in Europe from 1990 to 2013 using the European Eye Epidemiology (E3) consortium, and made projections for the future. DESIGN: Meta-analysis of prevalence data. PARTICIPANTS: A total of 42 080 individuals 40 years of age and older participating in 14 population-based cohorts from 10 countries in Europe. METHODS: AMD was diagnosed based on fundus photographs using the Rotterdam Classification. Prevalence of early and late AMD was calculated using random-effects meta-analysis stratified for age, birth cohort, gender, geographic region, and time period of the study. Best-corrected visual acuity (BCVA) was compared between late AMD subtypes; geographic atrophy (GA) and choroidal neovascularization (CNV). MAIN OUTCOME MEASURES: Prevalence of early and late AMD, BCVA, and number of AMD cases. RESULTS: Prevalence of early AMD increased from 3.5% (95% confidence interval [CI] 2.1%-5.0%) in those aged 55-59 years to 17.6% (95% CI 13.6%-21.5%) in those aged ≥85 years; for late AMD these figures were 0.1% (95% CI 0.04%-0.3%) and 9.8% (95% CI 6.3%-13.3%), respectively. We observed a decreasing prevalence of late AMD after 2006, which became most prominent after age 70. Prevalences were similar for gender across all age groups except for late AMD in the oldest age category, and a trend was found showing a higher prevalence of CNV in Northern Europe. After 2006, fewer eyes and fewer ≥80-year-old subjects with CNV were visually impaired (P = 0.016). Projections of AMD showed an almost doubling of affected persons despite a decreasing prevalence. By 2040, the number of individuals in Europe with early AMD will range between 14.9 and 21.5 million, and for late AMD between 3.9 and 4.8 million. CONCLUSION: We observed a decreasing prevalence of AMD and an improvement in visual acuity in CNV occuring over the past 2 decades in Europe. Healthier lifestyles and implementation of anti-vascular endothelial growth factor treatment are the most likely explanations. Nevertheless, the numbers of affected subjects will increase considerably in the next 2 decades. AMD continues to remain a significant public health problem among Europeans.

Genomes are transmitted faithfully from dividing cells to their offspring. Changes that occur during DNA repair, chromosome duplication, and transmission or via recombination provide a natural source of genetic variation. They occur at low frequency because of the intrinsic variable nature of genomes, which we refer to as genome instability. However, genome instability can be enhanced by exposure to external genotoxic agents or as the result of cellular pathologies. We review the causes of genome instability as well as how it results in hyper-recombination, genome rearrangements, and chromosome fragmentation and loss, which are mainly mediated by double-strand breaks or single-strand gaps. Such events are primarily associated with defects in DNA replication and the DNA damage response, and show high incidence at repetitive DNA, non-B DNA structures, DNA-protein barriers, and highly transcribed regions. Identifying the causes of genome instability is crucial to understanding genome dynamics during cell proliferation and its role in cancer, aging, and a number of rare genetic diseases.

We describe the isolation and detailed structural characterization of stable toxic oligomers of α-synuclein that have accumulated during the process of amyloid formation. Our approach has allowed us to identify distinct subgroups of oligomers and to probe their molecular architectures by using cryo-electron microscopy (cryoEM) image reconstruction techniques. Although the oligomers exist in a range of sizes, with different extents and nature of β-sheet content and exposed hydrophobicity, they all possess a hollow cylindrical architecture with similarities to certain types of amyloid fibril, suggesting that the accumulation of at least some forms of amyloid oligomers is likely to be a consequence of very slow rates of rearrangement of their β-sheet structures. Our findings reveal the inherent multiplicity of the process of protein misfolding and the key role the β-sheet geometry acquired in the early stages of the self-assembly process plays in dictating the kinetic stability and the pathological nature of individual oligomeric species.

Colorectal cancer (CRC) is the fourth leading cause of the worldwide cancer mortality. Different molecular mechanisms have been attributed to the development and progress of CRC. In this review, we will focus on the mitogen-activated protein kinase (MAPK) cascades downstream of the epidermal growth factor receptor (EGFR), Notch, PI3K/AKT pathway, transforming growth factor-β (TGF-β), and Wnt signaling pathways. Various mutations in the components of these signaling pathways have been linked to the development of CRC. Accordingly, numerous efforts have been carried out to target the signaling pathways to develop novel therapeutic approaches. Herein, we review the signaling pathways involved in the incidence and progression of CRC, and the strategies for the therapy targeting components of signaling pathways in CRC.

Sen1 of S. cerevisiae is a known component of the NRD complex implicated in transcription termination of nonpolyadenylated as well as some polyadenylated RNA polymerase II transcripts. We now show that Sen1 helicase possesses a wider function by restricting the occurrence of RNA:DNA hybrids that may naturally form during transcription, when nascent RNA hybridizes to DNA prior to its packaging into RNA protein complexes. These hybrids displace the nontranscribed strand and create R loop structures. Loss of Sen1 results in transient R loop accumulation and so elicits transcription-associated recombination. SEN1 genetically interacts with DNA repair genes, suggesting that R loop resolution requires proteins involved in homologous recombination. Based on these findings, we propose that R loop formation is a frequent event during transcription and a key function of Sen1 is to prevent their accumulation and associated genome instability.

Mesenchymal stem cells (MSCs) are among the most frequently used cell type for regenerative medicine. A large number of studies have shown the beneficial effects of MSC-based therapies to treat different pathologies, including neurological disorders, cardiac ischemia, diabetes, and bone and cartilage diseases. However, the therapeutic potential of MSCs in cancer is still controversial. While some studies indicate that MSCs may contribute to cancer pathogenesis, emerging data reported the suppressive effects of MSCs on cancer cells. Because of this reality, a sustained effort to understand when MSCs promote or suppress tumor development is needed before planning a MSC-based therapy for cancer. Herein, we provide an overview on the therapeutic application of MSCs for regenerative medicine and the processes that orchestrates tissue repair, with a special emphasis placed on cancer, including central nervous system tumors. Furthermore, we will discuss the current evidence regarding the double-edged sword of MSCs in oncological treatment and the latest advances in MSC-based anti-cancer agent delivery systems.

Co-transcriptional RNA-DNA hybrids (R loops) cause genome instability. To prevent harmful R loop accumulation, cells have evolved specific eukaryotic factors, one being the BRCA2 double-strand break repair protein. As BRCA2 also protects stalled replication forks and is the FANCD1 member of the Fanconi Anemia (FA) pathway, we investigated the FA role in R loop-dependent genome instability. Using human and murine cells defective in FANCD2 or FANCA and primary bone marrow cells from FANCD2 deficient mice, we show that the FA pathway removes R loops, and that many DNA breaks accumulated in FA cells are R loop-dependent. Importantly, FANCD2 foci in untreated and MMC-treated cells are largely R loop dependent, suggesting that the FA functions at R loop-containing sites. We conclude that co-transcriptional R loops and R loop-mediated DNA damage greatly contribute to genome instability and that one major function of the FA pathway is to protect cells from R loops.

DNA-end resection is a highly regulated and critical step in the response and repair of DNA double-strand breaks. In higher eukaryotes, CtIP regulates resection by integrating cellular signals via its posttranslational modifications and protein-protein interactions, including cell-cycle-controlled interaction with BRCA1. The role of BRCA1 in DNA-end resection is not clear. Here, we develop an assay to study DNA resection in higher eukaryotes at high resolution. We demonstrate that the BRCA1-CtIP interaction, albeit not essential for resection, modulates the speed at which this process takes place.

One of the mechanisms involved in chromatin remodelling is so-called 'histone replacement'. An example of such a mechanism is the substitution of canonical H2A histone by the histone variant H2A.Z. The ATP-dependent chromatin remodelling complex SWR1 is responsible for this action in yeast. We have previously proposed the existence of an SWR1-like complex in Arabidopsis by demonstrating genetic and physical interaction of the components SEF, ARP6 and PIE1, which are homologues of the yeast Swc6 and Arp6 proteins and the core ATPase Swr1, respectively. Here we show that histone variant H2A.Z, but not canonical H2A histone, interacts with PIE1. Plants mutated at loci HTA9 and HTA11 (two of the three Arabidopsis H2A.Z-coding genes) displayed developmental abnormalities similar to those found in pie1, sef and arp6 plants, exemplified by an early-flowering phenotype. Comparison of gene expression profiles revealed that 65% of the genes differentially regulated in hta9 hta11 plants were also mis-regulated in pie1 plants. Detailed examination of the expression data indicated that the majority of mis-regulated genes were related to salicylic acid-dependent immunity. RT-PCR and immunoblotting experiments confirmed constitutive expression of systemic acquired resistance (SAR) marker genes in pie1, hta9 hta11 and sef plants. Variations observed at the molecular level resulted in phenotypic alterations such as spontaneous cell death and enhanced resistance to the phytopathogenic bacteria Pseudomonas syringae pv. tomato. Thus, our results support the existence in Arabidopsis of an SWR1-like chromatin remodelling complex that is functionally related to that described in yeast and human, and attribute to this complex a role in maintaining a repressive state of the SAR response.

Genome replication involves dealing with obstacles that can result from DNA damage but also from chromatin alterations, topological stress, tightly bound proteins or non-B DNA structures such as R loops. Experimental evidence reveals that an engaged transcription machinery at the DNA can either enhance such obstacles or be an obstacle itself. Thus, transcription can become a potentially hazardous process promoting localized replication fork hindrance and stress, which would ultimately cause genome instability, a hallmark of cancer cells. Understanding the causes behind transcription-replication conflicts as well as how the cell resolves them to sustain genome integrity is the aim of this review.

THO/TREX connects transcription with genome integrity in yeast, but a role of mammalian THO in these processes is uncertain, which suggests a differential implication of mRNP biogenesis factors in genome integrity in yeast and humans. We show that human THO depletion impairs transcription elongation and mRNA export and increases instability associated with DNA breaks, leading to hyper-recombination and γH2AX and 53BP1 foci accumulation. This is accompanied by replication alteration as determined by DNA combing. Genome instability is R-loop-dependent, as deduced from the ability of the AID enzyme to increase DNA damage and of RNaseH to reduce it, or from the enhancement of R-loop-dependent class-switching caused by THOC1-depletion in CH12 murine cells. Therefore, mammalian THO prevents R-loop formation and has a role in genome dynamics and function consistent with an evolutionary conservation of the functional connection between these mRNP biogenesis factors and genome integrity that had not been anticipated.

A healthy diet together with physical activity could induce weight loss and control the progression of non-alcoholic fatty liver disease (NAFLD). However, the composition of diet has not been clearly established. Macronutrients such as saturated fatty acids (SFA), trans-fats, simple sugars and animal proteins have a harmful effect on the liver. On the other hand, monounsaturated fats (MUFAs), polyunsaturated (PUFAs) omega-3-fats, plant-based proteins and dietary fibres are considered to be beneficial to the liver. The impact of specific micronutrients is less well-known. Nutrients are part of the food we eat. Food makes up our meals, which compose our dietary patterns. Non-alcoholic fatty liver disease patients usually follow Western diets which are rich in soda, frozen junk food, juice, red meat, lard, processed meats, whole fat dairy foods, fatty snack foods, take-away foods, cakes and biscuits and poor in cereals, whole grains, fruit, vegetables, extra virgin olive oil (EVOO) and fish. On the other hand, the Mediterranean diet (MD) is beneficial for NAFLD even when it is iso-caloric or there are no changes in body weight. A new approach, called 'nutritional geometry' considers the importance of integrating nutrition, animals and the environment. The goal of this approach is to combine nutrients and foods in a model to understand how food components interact to regulate the properties of diets affecting health and disease. The use of algorithms developed by artificial intelligence (AI) to create a personalized diet for patients can provide customized nutritional counselling to prevent and treat NAFLD.

Vasculogenic mimicry (VM) is a blood supply system independent of endothelial vessels in tumor cells from different origins. It reflects the plasticity of aggressive tumor cells that express vascular cell markers and line tumor vasculature. The presence of VM is associated with a high tumor grade, short survival, invasion and metastasis. Endothelial cells (ECs) express various members of the cadherin superfamily, in particular vascular endothelial (VE-) cadherin, which is the main adhesion receptor of endothelial adherent junctions. Aberrant extra-vascular expression of VE-cadherin has been observed in certain cancer types associated with VM. In this review we focus on non-endothelial VE-cadherin as a prominent factor involved in the acquisition of tubules-like structures by aggressive tumor cells and we summarize the specific signaling pathways, the association with trans-differentiation and stem-like phenotype and the therapeutic opportunities derived from the in-depth knowledge of the peculiarities of the biology of VE-cadherin and other key components of VM.

Metformin, an oral hypoglycemic agent, has been used for decades to treat type 2 diabetes mellitus. Recent studies indicate that mice treated with metformin live longer and have fewer manifestations of age-related chronic disease. However, the molecular mechanisms underlying this phenotype are unknown. Here, we show that metformin treatment increases the levels of the microRNA-processing protein DICER1 in mice and in humans with diabetes mellitus. Our results indicate that metformin upregulates DICER1 through a post-transcriptional mechanism involving the RNA-binding protein AUF1. Treatment with metformin altered the subcellular localization of AUF1, disrupting its interaction with DICER1 mRNA and rendering DICER1 mRNA stable, allowing DICER1 to accumulate. Consistent with the role of DICER1 in the biogenesis of microRNAs, we found differential patterns of microRNA expression in mice treated with metformin or caloric restriction, two proven life-extending interventions. Interestingly, several microRNAs previously associated with senescence and aging, including miR-20a, miR-34a, miR-130a, miR-106b, miR-125, and let-7c, were found elevated. In agreement with these findings, treatment with metformin decreased cellular senescence in several senescence models in a DICER1-dependent manner. Metformin lowered p16 and p21 protein levels and the abundance of inflammatory cytokines and oncogenes that are hallmarks of the senescence-associated secretory phenotype (SASP). These data lead us to hypothesize that changes in DICER1 levels may be important for organismal aging and to propose that interventions that upregulate DICER1 expression (e.g., metformin) may offer new pharmacotherapeutic approaches for age-related disease.

Mutations in the CRB1 gene are associated with variable phenotypes of severe retinal dystrophies, ranging from leber congenital amaurosis (LCA) to rod-cone dystrophy, also called retinitis pigmentosa (RP). Moreover, retinal dystrophies resulting from CRB1 mutations may be accompanied by specific fundus features: preservation of the para-arteriolar retinal pigment epithelium (PPRPE) and retinal telangiectasia with exudation (also referred to as Coats-like vasculopathy). In this publication, we report seven novel mutations and classify over 150 reported CRB1 sequence variants that were found in more that 240 patients. The data from previous reports were used to analyze a potential correlation between CRB1 variants and the clinical features of respective patients. This meta-analysis suggests that the differential phenotype of patients with CRB1 mutations is due to additional modifying factors rather than particular mutant allele combination.

FACT (facilitates chromatin transcription) is a chromatin-reorganizing complex that swaps nucleosomes around the RNA polymerase during transcription elongation and has a role in replication that is not fully understood yet. Here we show that recombination factors are required for the survival of yeast FACT mutants, consistent with an accumulation of DNA breaks that we detected by Rad52 foci and transcription-dependent hyperrecombination. Breaks also accumulate in FACT-depleted human cells, as shown by γH2AX foci and single-cell electrophoresis. Furthermore, FACT-deficient yeast and human cells show replication impairment, which in yeast we demonstrate by ChIP-chip (chromatin immunoprecipitation [ChIP] coupled with microarray analysis) of Rrm3 to occur genome-wide but preferentially at highly transcribed regions. Strikingly, in yeast FACT mutants, high levels of Rad52 foci are suppressed by RNH1 overexpression; R loops accumulate at high levels, and replication becomes normal when global RNA synthesis is inhibited in FACT-depleted human cells. The results demonstrate a key function of FACT in the resolution of R-loop-mediated transcription-replication conflicts, likely associated with a specific chromatin organization.

Type 1 diabetes (T1D) is a T cell-dependent autoimmune disease that is characterized by the destruction of insulin-producing β cells in the pancreas. The administration to patients of ex vivo-differentiated FoxP3(+) regulatory T (Treg) cells or tolerogenic dendritic cells (DCs) that promote Treg cell differentiation is considered a potential therapy for T1D; however, cell-based therapies cannot be easily translated into clinical practice. We engineered nanoparticles (NPs) to deliver both a tolerogenic molecule, the aryl hydrocarbon receptor (AhR) ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), and the β cell antigen proinsulin (NPITE+Ins) to induce a tolerogenic phenotype in DCs and promote Treg cell generation in vivo. NPITE+Ins administration to 8-week-old nonobese diabetic mice suppressed autoimmune diabetes. NPITE+Ins induced a tolerogenic phenotype in DCs, which was characterized by a decreased ability to activate inflammatory effector T cells and was concomitant with the increased differentiation of FoxP3(+) Treg cells. The induction of a tolerogenic phenotype in DCs by NPs was mediated by the AhR-dependent induction of Socs2, which resulted in inhibition of nuclear factor κB activation and proinflammatory cytokine production (properties of tolerogenic DCs). Together, these data suggest that NPs constitute a potential tool to reestablish tolerance in T1D and potentially other autoimmune disorders.

Elucidating the biology of yeast in its full complexity has major implications for science, medicine and industry. One of the most critical processes determining yeast life and physiology is cel-lular demise. However, the investigation of yeast cell death is a relatively young field, and a widely accepted set of concepts and terms is still missing. Here, we propose unified criteria for the defi-nition of accidental, regulated, and programmed forms of cell death in yeast based on a series of morphological and biochemical criteria. Specifically, we provide consensus guidelines on the differ-ential definition of terms including apoptosis, regulated necrosis, and autophagic cell death, as we refer to additional cell death rou-tines that are relevant for the biology of (at least some species of) yeast. As this area of investigation advances rapidly, changes and extensions to this set of recommendations will be implemented in the years to come. Nonetheless, we strongly encourage the au-thors, reviewers and editors of scientific articles to adopt these collective standards in order to establish an accurate framework for yeast cell death research and, ultimately, to accelerate the pro-gress of this vibrant field of research.