BUSCADOR RECOLECTA

Despite the great advances in macroautophagy/autophagy research in the last years, the in vivo role of the different members of the four mammalian orthologs of yeast Atg4 protease (ATG4A-D) remain unclear. To gain further insights into the functional relevance of Atg4 orthologs, we have generated mutant mice deficient in Atg4c. These mice are viable and fertile, and do not display any obvious abnormalities, indicating that they are able to develop the autophagic response required during the early neonatal period. However, they show tissue-specific autophagy alterations, including reduced autophagic flux in diaphragm and show decreased breathing and locomotor activity after fasting. In addition, atg4c-/- mice show reduced number of circulating T and B lymphocytes, which is associated with accumulation of apoptotic cells in the spleen and an increased susceptibility to develop chemically-induced fibrosarcomas. Moreover, through the analysis of cells and mice simultaneously deficient for ATG4C and ATG4D proteases we also reveal a role for ATG4C in mATG8 proteins delipidation. ATG4 (autophagy related 4 cysteine peptidase); ATG4A (autophagy related 4A cysteine peptidase); ATG4B (autophagy related 4B cysteine peptidase); ATG4C (autophagy related 4C cysteine peptidase); ATG4D (autophagy related 4D cysteine peptidase); Atg8 (autophagy related 8); GABARAP (GABA type A receptor-associated protein); GABARAPL1(GABA type A receptor-associated protein like 1); GABARAPL2 (GABA type A receptor-associated protein like 2); MAP1LC3A/LC3A (microtubule associated protein 1 light chain 3 alpha); MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta); mATG8 (mammalian Atg8); PE (phosphatidylethanolamine); PS (phosphatydylserine); SQSTM1/p62 (sequestosome 1)., This work was supported by grants from Ministerio Ciencia eInnovación (Spain) (PID2021-127534OB-I00), the South-Eastern 1315 Norway Regional Health Authority (2021088 to N.E.) and Instituto de Salud Carlos III (RTICC Spain). Jesús Prieto-Lloret is funded by Programa Estrategico IBGM, Escalera de Excelencia, ref. CCVC8485, Consejería de Educación, Junta de Castilla y León (Spain). Funding for open Access Charge: Roche Farma”, as the aricle will be published via Open access and the OA costs will be funded by Roche Farma., Peer reviewed

Table S1. Reagents used in this work. Table S2: Patient Cohort characteristics. Figure S1. PLD2 expression in OC patients and patient survival in OC public databases. (A) PLD2 expression in the OC patient databases GSE12172, GSE9891, GSE63885 and GSE2109 indicating OC subtype. (B) PLD2 expression in the OC patient databases GSE18520, GSE40595, and GSE38666, indicating OC subtype. (C) Kaplan-Meier plots showing overall survival (OS) of patients with high (red) or low (green) PLD2 expression levels in three OC databases with survival data: GSE13876 (advanced HGSOC); GSE23554 (advanced serous epithelial OC); and GSE31245 (92% serous, 2% endometroid, 6% clear cell). Data were analyzed with the log-rank test, and the associated P-values are shown in the graphs. (D) Kaplan-Meier plots showing overall survival (OS) of patients with high (red) or low (green) risk. PLD2 expression for each group is shown on the right of each graph. (E) Kaplan-Meier plots generated with Kaplan-Meier Plotter showing PFS (left column), PPS (middle column) and OS (right column) by splitting patients according to PLD2 expression in all OC patients (top row) or only HGSOC patients (bottom row). Expression levels are shown as log2 transformed values from the R2 database. Data were analyzed using Student’s t-test. *, P <0.05; **, P < 0.01; ***, P < 0.001. Figure S2. HIF1a levels in OC cell lines in response to hypoxia. (A) Representative images of HIF-1a protein levels by immunofluorescence in SKOV3, OVCAR8 and ES-2 cells under normoxia and hypoxia or in the presence of the HIF-hydroxylase inhibitor DMOG. (B) Western blot showing HIF-1a and alpha-tubulin protein levels in SKOV3, OVCAR8 and ES-2 cells under normoxia and hypoxia or in the presence of the HIF-hydroxylase inhibitor DMOG. A minimum of three independent experiments were performed. Figure S3. Differential analyses of chromatin accessibility in OC cells. (A) Volcano plot showing differential analyses of chromatin accessibility between SKOV3 cells carrying Ev and plasmid overexpressing PLD2 in normoxia. (B) Heatmaps and average profiles plotting normalized ATAC-seq signal in SKOV3 cells carrying Ev and overexpressing PLD2 for the differentially accessible regions (DARs) in (A). (C) Gene Ontology term enrichment analyses of biological processes for the genes associated with DARs in SKOV3 cells carrying EV in normoxia versus hypoxia. (D) Gene Ontology term enrichment analyses of biological processes for the genes associated with DARs in SKOV3 cells carrying Ev versus overexpressing PLD2 in normoxia. Figure S4. Motif and footprint analyses of transcription factor binding in OC cells. (A-B) Motif enrichment analyses of the increased and decreased ATAC peaks in OC cells carrying Ev in hypoxia vs. normoxia (A) and +/- PLD2 expression in normoxia (B). The three motifs with the lowest p values are shown in each case. (C) Venn diagrams plotting the overlap between TFs with increased binding in hypoxia and expressing PLD2 in normoxia (top) or the overlap between TFs with increased binding in hypoxia and expressing shPLD2 in hypoxia (bottom). (D) Distribution of fold changes in the ATAC peaks containing the motifs FOS::JUN in Ev normoxia vs. Ev hypoxia (top) or in Ev hypoxia vs. shPLD2 hypoxia. (E) Aggregate footprint signal of the peaks containing the motifs in (D). Figure S5. Clustering of differentially accessible regions in OC cells. (A) Heatmaps plotting normalized ATAC-seq signal at peaks associated with stemness genes in SKOV3 cells carrying Ev or plasmid expressing PLD2 in normoxia and carrying Ev or plasmid expressing shPLD2 in hypoxia, for the differentially accessible regions (DARs) clustered using k-means method in 4 clusters. (B) Tracks with ATAC-seq in SKOV3 cells carrying Ev or expressing PLD2 in normoxia and carrying Ev or shPLD2 in hypoxia, at the JAG1 locus. Figure S6. Expression of stemness and hypoxia genes correlated with PLD2 in OC patients. Heatmaps showing the expression z-scores of stemness-associated genes or hypoxia-response genes whose expression correlated with PLD2 in GSE40595 and GSE38666 OC patient databases. Figure S7. Hypoxia induces CSCs in ovarian cancer cells. (A) Top, Representative images of tumorspheres formed by SKOV3, OVCAR8 and ES-2 cells in normoxia or hypoxia. Bottom, quantification of the number and size of tumorspheres. Scale bars: 250 μm. (B) Percentage of holoclones formed by SKOV3, OVCAR8 and ES-2 cells in normoxia or hypoxia. At least 200 individual clones were analyzed. (C) Analysis of the expression of NANOG, SOX2, CD44 and EPCAM stemness-associated genes by RT-qPCR in SKOV3, OVCAR8 and ES-2 cells in normoxia or hypoxia. The mRNA expression was calculated as 2-∆Ct relative to the ACTB gene. (D) Percentage of CD133 positive cells measured by FACS in SKOV3, OVCAR8 and ES-2 cells in normoxia and hypoxia The average and SD of three independent experiments are shown in all cases. A minimum of three independent experiments were performed and the data were compared using Student’s t tests. Asterisks indicate statistical significance with respect to normoxia. *p < 0.05; **p < 0.01; ***p < 0.001. 13 Figure S8. Relative protein quantification of PLD2 normalized to alpha-tubulin from the western blots in Figure 4B. Figure S9. PLD2 expression and clone analyses. (A) Western blot showing PLD2 and alpha-tubulin protein levels in SKOV3, OVCAR8 and ES-2 OC cells carrying Ev, a plasmid expressing shPLD2 or plasmids expressing shPLD2 and PLD2. (B) Percentage of paraclones, meroclones and holoclones formed by SKOV3, OVCAR8 and ES-2 cells carrying Ev or plasmids expressing PLD2, shPLD2 or both in hypoxia or normoxia. At least 200 individual clones were analyzed. The average of three independent experiments is shown. A dotted line represents the percentage of holoclones in Ev carrying cells as a reference. Data were compared using Student’s t tests. Asterisks indicate statistical significance with respect to Ev carrying cells. *p< 0.05. (C) Left, determination of PLD2 protein levels by immunofluorescence in tumorspheres formed by OC cells carrying Ev and expressing PLD2 or shPLD2. Right, quantification of the percentage of cells with PLD2 expression in tumorspheres. Scale bars: 100 μm. Figure S10. Analyses of the EMT in OC cells in response to hypoxia and/or PLD2 expression. (A) Heatmaps showing the expression z-scores of epithelial-to-mesenchymal transition-associated genes obtained from TaqMan Arrays. Genes are sorted according to decreasing z-scores in the Ev-carrying cells under normoxia. (B) Heatmaps showing the z-scores of EMT genes expression levels in SKOV3 cells carrying EV or plasmid overexpressing PLD2 under normoxia conditions and carrying EV or plasmid expressing shPLD2 under hypoxia condition. Hierarchical clustering of the samples is shown. (C) Expression levels of SNAI1, VIM, CDH1 and CDH2 EMT-associated genes in cells carrying Ev or plasmids expressing PLD2 under normoxia or shPLD2 under hypoxia conditions. (D) Left, representative images of the Boyden chamber migration assays in SKOV3 and OVCAR8 cells carrying Ev or plasmids expressing PLD2 or shPLD2 under normoxic or hypoxic conditions. Right, quantification of the Boyden chamber migration assays. A minimum of three independent experiments were performed and the data were analyzed using Student’s t-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001., Supplementary Material 1: Essential role of PLD2 in hypoxia-induced stemness and therapy resistance in ovarian tumors., Funding: Consejo Superior de Investigaciones Cientificas (CSIC)., Peer reviewed

Experimental studies and clinical trials of nanoparticles for treating diseases are increasing continuously. However, the reach to the market does not correlate with these efforts due to the enormous cost, several years of development, and off-target effects like cardiotoxicity. Multicellular organisms such as the Caenorhabditis elegans (C. elegans) can bridge the gap between in vitro and vertebrate testing as they can provide extensive information on systemic toxicity and specific harmful effects through facile experimentation following 3R EU directives on animal use. Since the nematodes’ pharynx shares similarities with the human heart, we assessed the general and pharyngeal effects of drugs and polypyrrole nanoparticles (Ppy NPs) using C. elegans. The evaluation of FDA-approved drugs, such as Propranolol and Racepinephrine reproduced the arrhythmic behavior reported in humans and supported the use of this small animal model. Consequently, Ppy NPs were evaluated due to their research interest in cardiac arrhythmia treatments. The NPs’ biocompatibility was confirmed by assessing survival, growth and development, reproduction, and transgenerational toxicity in C. elegans. Interestingly, the NPs increased the pharyngeal pumping rate of C. elegans in two slow-pumping mutant strains, JD21 and DA464. Moreover, the NPs increased the pumping rate over time, which sustained up to a day post-excretion. By measuring pharyngeal calcium levels, we found that the impact of Ppy NPs on the pumping rate could be mediated through calcium signaling. Thus, evaluating arrhythmic effects in C. elegans offers a simple system to test drugs and nanoparticles, as elucidated through Ppy NPs., Peer reviewed

(A) An asynchronous culture of GAL-SIC1ΔNT GLN3-9MYC (YMF4471) was grown at 30 °C in medium lacking galactose. After the addition of nocodazole, culture was synchronised in G2-M phase for 1 generation time. Cells were then transferred to fresh medium containing galactose to allow overexpression of SIC1ΔNT. Cells were maintained as well as in nocodazole. Cell extract were made over the course of 2 h to examine Gln3 mobility. Raw data for blots can be found in Supporting information (S1 Raw Images). (B) TUB1-GFP cdc15-2 (YMF3976) cells were grown in YPD and arrested in late anaphase by raising the temperature to 37 °C before the addition of rapamycin to half of the culture. Subsequently, to allow progression through the cell cycle, cells were released in the absence (−) or presence (+) of rapamycin. Samples were taken at the indicated times. Using fluorescence microscopy, the proportion of cells with anaphase spindles in the absence (i) or presence (ii) of rapamycin was investigated. Examples of TUB1-GFP cdc15-2 cells at 120 min after the release at 24 °C are shown in the absence (iii) or presence (iv) of rapamycin. Scale bars indicate 5 μm. (C) cdc15-2 cells (CC2274) were grown in parallel with strains for Fig 2A, but instead or releasing cells at the permissive temperature of 24 °C after the addition of rapamycin like in Fig 2A, cells were maintained at the restrictive temperature of 37 °C in the presence of rapamycin (i). Samples were taken at the indicated times to determine DNA content by flow cytometry analysis (ii) and cell morphology at the end of the experiment (iii). Scale bars indicate 5 μm. (D) cdc14-1 cdc15-2 cells (CC6441) were grown in YPD and arrested in late anaphase by shifting the temperature to 37 °C before the addition of rapamycin to half of the culture (ii). Then, cells were released at 24 °C in the absence (i) or presence (ii) of rapamycin. Samples were taken at the specified times to determine DNA content by FACS analysis. Using light microscopy, we studied cell morphology in the absence (iii) or presence (iv) of rapamycin at the 120 min time point after the release from late anaphase arrest. Scale bars indicate 5 μm. (E) INN1-GFP cdc15-2 (YMF3162) cells were grown as in A. Samples were taken at the indicated times. The proportion of cells with total Inn1-GFP signal in the absence (i) or presence (ii) of rapamycin was determined using fluorescence microscopy. The percentage of cells with either medial rings or spots of Inn1-GFP was calculated too. Examples of cells expressing Inn1-GFP 30 min after the release are shown in the absence (iii) or presence (iv) of rapamycin. Scale bars indicate 5 μm. (F) 3GFP-RAS2 cdc14-1 cells (CC6296) were grown as described in A. Cells are shown at 120 min after the release in the absence (i) or the presence (ii) of rapamycin. Red arrows denote cells where examination of each z-level at the bud neck showed divided cytoplasm and new buds are marked with white asterisks. Scale bars indicate 5 μm. Underlying data for all the graphs can be found in S1 Data file. FACS graphs can be found in the supplementary FACS file (S1 File)., journal.pbio.3002263.s001.pdf, Peer reviewed

Coordinators: Natividad Benito (GEIO-SEIMC), Juan Carlos Martínez-Pastor (SECOT), Jesús Saavedra-Lozano (SEIP).-- Methods: Eighteen clinical questions were formulated under the three major headings of Diagnostics, Therapeutics and Follow-up. The coordinators assigned each clinical question to a subgroup of panellists. For each clinical question, all significant scientific literature was reviewed and summarised in comprehensive tables following the PICO system (P– Populations/People/Patient/Problem; I– Intervention(s); C-Comparison; O– Outcome). The criteria used to evaluate the strength of the recommendation and the quality of the evidence are summarised in Table 1. The coordinators wrote the first draft based on the sections submitted by each subgroup of panellists. The draft was reviewed by all members of the panel of experts, controversial issues were debated, and a final version was prepared. Before its final approval, the document was posted on the SEIMC intranet and left open for suggestions and comments from members. All authors have approved the contents of the document and the final recommendations. Possible conflicts of interest associated with all members of the panel of experts are listed at the end of the document., Infectious arthritis is an infection of one or more joints caused by bacteria (including mycobacteria), fungi and viruses. Only a small number of viruses directly infect the joint, mainly as part of a self-limiting systemic infection. While the term septic arthritis (SA) usually refers to bacterial or fungal arthritis, bacteria are by far the most common agents of these infections. The annual incidence of SA is between 2 and 7 cases per 100,000 inhabitants in most Western European series. 2–7 Higher rates have been observed in other areas, such as Australasia. 8–10 Even though SA remains relatively rare in the general population, the overall incidence is increasing, due to the rising rate among older patients, who have more underlying co-morbidities and joint disorders, undergo more invasive procedures with increased use of immunosuppressive treatments., Peer reviewed

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See the supplementary material for further details on the electronic structure and quantum dynamical methods, electronic diabatic Hamiltonians, and characterization of the normal modes used in the simulations., Peer reviewed

Microglial cells are recognized as very dynamic brain cells, screening the environment and sensitive to signals from all other cell types in health and disease. Apolipoprotein D (ApoD), a lipid-binding protein of the Lipocalin family, is required for nervous system optimal function and proper development and maintenance of key neural structures. ApoD has a cell and state-dependent expression in the healthy nervous system, and increases its expression upon aging, damage or neurodegeneration. An extensive overlap exists between processes where ApoD is involved and those where microglia have an active role. However, no study has analyzed the role of ApoD in microglial responses. In this work, we test the hypothesis that ApoD, as an extracellular signal, participates in the intercellular crosstalk sensed by microglia and impacts their responses upon physiological aging or damaging conditions. We find that a significant proportion of ApoD-dependent aging transcriptome are microglia-specific genes, and show that lack of ApoD in vivo dysregulates microglial density in mouse hippocampus in an age-dependent manner. Murine BV2 and primary microglia do not express ApoD, but it can be internalized and targeted to lysosomes, where unlike other cell types it is transiently present. Cytokine secretion profiles and myelin phagocytosis reveal that ApoD has both long-term pre-conditioning effects on microglia as well as acute effects on these microglial immune functions, without significant modification of cell survival. ApoD-triggered cytokine signatures are stimuli (paraquat vs. Aβ oligomers) and sex-dependent. Acute exposure to ApoD induces microglia to switch from their resting state to a secretory and less phagocytic phenotype, while long-term absence of ApoD leads to attenuated cytokine induction and increased myelin uptake, supporting a role for ApoD as priming or immune training factor. This knowledge should help to advance our understanding of the complex responses of microglia during aging and neurodegeneration, where signals received along our lifespan are combined with damage-triggered acute signals, conditioning both beneficial roles and limitations of microglial functions., Peer reviewed

[Introduction]: The Unfolded Protein Response, a mechanism triggered by the cell in response to Endoplasmic reticulum stress, is linked to inflammatory responses. Our aim was to identify novel Unfolded Protein Response-mechanisms that might be involved in triggering or perpetuating the inflammatory response carried out by the Intestinal Epithelial Cells in the context of Inflammatory Bowel Disease., [Methods]: We analyzed the transcriptional profile of human Intestinal Epithelial Cell lines treated with an Endoplasmic Reticulum stress inducer (thapsigargin) and/or proinflammatory stimuli. Several genes were further analyzed in colonic biopsies from Ulcerative Colitis patients and healthy controls. Lastly, we generated Caco-2 cells lacking HMGCS2 by CRISPR Cas-9 and analyzed the functional implications of its absence in Intestinal Epithelial Cells., [Results]: Exposure to a TLR ligand after thapsigargin treatment resulted in a powerful synergistic modulation of gene expression, which led us to identify new genes and pathways that could be involved in inflammatory responses linked to the Unfolded Protein Response. Key differentially expressed genes in the array also exhibited transcriptional alterations in colonic biopsies from active Ulcerative Colitis patients, including NKG2D ligands and the enzyme HMGCS2. Moreover, functional studies showed altered metabolic responses and epithelial barrier integrity in HMGCS2 deficient cell lines., [Conclusion]: We have identified new genes and pathways that are regulated by the Unfolded Protein Response in the context of Inflammatory Bowel Disease including HMGCS2, a gene involved in the metabolism of Short Chain Fatty Acids that may have an important role in intestinal inflammation linked to Endoplasmic Reticulum stress and the resolution of the epithelial damage., Peer reviewed

S1 Fig. Workflow for the manual and automatic text-mining searches. A starting list of 6034 mammal viruses was used to obtain, which were then reviewed using both manual and PubmedKB-based automated strategies. The resulting virus-receptor pairs were combined with known databases and manually curated (see text for full description). M, manual strategy; PKB, PubmedKB strategy., S2 Fig. Roles of known receptors according to viral family. Only families with at least 10 known virus-host interactions are represented. Families of enveloped and non-enveloped viruses are shown, and within each group, families are sorted by the fraction of known receptors that are sufficient for viral entry (main plus alternative receptors)., S3 Fig. Research effort versus the known number of host proteins used as receptors for different viral families. Data points correspond to individual viruses. Those corresponding to the indicated family are shown in color (blue for non-enveloped viruses; yellow for enveloped viruses), and grey points correspond to all other viruses. The colored and grey dashed lines show the GLM prediction obtained specifically for the family and all viruses, respectively. Only families with at least 5 viral species in the dataset were considered., S1 Table. Database of virus receptors generated in this study. The following information is provided: the viral species, number of PubMed records and Genbank sequences available for each virus, viral family, presence of an envelope, number of host species, receptor symbol, receptor nature, functional role of the receptor, corresponding gene symbol, original publication PMID, year of discovery, and whether the virus-receptor interaction was reported in previous reviews and databases., S2 Table. Scores obtained from the GBM. The gene symbol, assigned score (probability of being a receptor), and whether the corresponding protein is a known receptor are indicated., S3 Table. Relevant features identified by the GBM. Gain represents the relative contribution of each variable to the model prediction. Cover indicates the relative number of observations that are related to a given variable., Peer reviewed