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31p.-7 fig. 1 graph. abst., Frontotemporal dementia is the second most prevalent type of early-onset dementia and up to 40% of cases are familial forms. One of the genes mutated in patients is CHMP2B, which encodes a protein found in a complex important for maturation of late endosomes, an essential process for recycling membrane proteins through the endolysosomal system. Here, we have generated a CHMP2B-mutated human embryonic stem cell line using genome editing with the purpose to create a human in vitro Frontotemporal dementia disease model. To date, most studies have focused on neuronal alterations; however, we present a new co-culture system in which neurons and astrocytes are independently generated from human embryonic stem cells and combined in co-cultures. With this approach, we have identified alterations in the endolysosomal system of Frontotemporal dementia astrocytes, a higher capacity of astrocytes to uptake and respond to glutamate, and a neuronal network hyperactivity as well as excessive synchronization. Overall, our data indicates that astrocyte alterations precede neuronal impairments and could potentially trigger neuronal network changes, indicating the important and specific role of astrocytes in disease development., This study was supported by funding from the Swedish Society for Medical Research (SSMF, S20-0003), Kockska, Segerfalk and Hardebo foundations to IC; the Swedish Research Council (VR:2018-02695), Swedish Alzheimer and Åhlen foundations to HA; the Swedish Research Council (VR:2016-01789) to AH; and the Agencia Estatal de Investigación, Ministerio de Ciencia e Innovación (RTI2018-097624-BI00) and European Regional Development Fund to DPS.

Supplementary Methods: For the characterization of the L-aptamer AON-D21 Supplementary Results: Characterization of the L-aptamer AON-D21 Supplementary References Supplementary Figure S1: Pharmacokinetics of AON-D21 Supplementary Figure S2: Individual follow up of subcutaneous growth and rechallenge Supplementary Figure S3: Splenic leukocyte subpopulations in mice bearing 393P tumors Supplementary Figure S4: Scatter plots of CD8 T cells in treated 393P tumors Supplementary Figure S5: Tumor-infiltrating leukocyte subpopulations in 393P tumors Supplementary Figure S6: Splenic leukocyte subpopulations in mice bearing LLC tumors Supplementary Figure S7: Tumor-infiltrating leukocyte subpopulations and IL2 in LLC tumors Supplementary Figure S8: Flow cytometry gating strategy Supplementary Table S1: Rate constants and dissociation constant of AON-D21 Supplementary Table S2: mRNA expression of immune-related molecules in 393P tumors

WMV - 9857K, Video of scr-siRNA treated A549 cell growth.

The TNF receptor-associated protein 1 (TRAP1) is a mitochondrial HSP that has been related to drug resistance and protection from apoptosis in colorectal and prostate cancer. Here, the effect of TRAP1 ablation on cell proliferation, survival, apoptosis, and mitochondrial function was determined in non–small cell lung cancer (NSCLC). In addition, the prognostic value of TRAP1 was evaluated in patients with NSCLC. These results demonstrate that TRAP1 knockdown reduces cell growth and clonogenic cell survival. Moreover, TRAP1 downregulation impairs mitochondrial functions such as ATP production and mitochondrial membrane potential as measured by TMRM (tetramethylrhodamine methylester) uptake, but it does not affect mitochondrial density or mitochondrial morphology. The effect of TRAP1 silencing on apoptosis, analyzed by flow cytometry and immunoblot expression (cleaved PARP, caspase-9, and caspase-3) was cell line and context dependent. Finally, the prognostic potential of TRAP1 expression in NSCLC was ascertained via immunohistochemical analysis which revealed that high TRAP1 expression was associated with increased risk of disease recurrence (univariate analysis, P = 0.008; multivariate analysis, HR: 2.554; 95% confidence interval, 1.085–6.012; P = 0.03). In conclusion, these results demonstrate that TRAP1 impacts the viability of NSCLC cells, and that its expression is prognostic in NSCLC.Implications: TRAP1 controls NSCLC proliferation, apoptosis, and mitochondrial function, and its status has prognostic potential in NSCLC. Mol Cancer Res; 12(5); 660–9. ©2014 AACR.

WMV - 9842K, Video of TRAP1-siRNA1 treated A549 cell growth.

Cysteine residues can undergo multiple posttranslational modifications with diverse functional consequences, potentially behaving as tunable sensors. The intermediate filament protein vimentin has important implications in pathophysiology, including cancer progression, infection, and fibrosis, and maintains a close interplay with other cytoskeletal structures, such as actin filaments and microtubules. We previously showed that the single vimentin cysteine, C328, is a key target for oxidants and electrophiles. Here, we demonstrate that structurally diverse cysteine-reactive agents, including electrophilic mediators, oxidants and drug-related compounds, disrupt the vimentin network eliciting morphologically distinct reorganizations. As most of these agents display broad reactivity, we pinpointed the importance of C328 by confirming that local perturbations introduced through mutagenesis provoke structure-dependent vimentin rearrangements. Thus, GFP-vimentin wild type (wt) forms squiggles and short filaments in vimentin-deficient cells, the C328F, C328W, and C328H mutants generate diverse filamentous assemblies, and the C328A and C328D constructs fail to elongate yielding dots. Remarkably, vimentin C328H structures resemble the wt, but are strongly resistant to electrophile-elicited disruption. Therefore, the C328H mutant allows elucidating whether cysteine-dependent vimentin reorganization influences other cellular responses to reactive agents. Electrophiles such as 1,4-dinitro-1H-imidazole and 4-hydroxynonenal induce robust actin stress fibers in cells expressing vimentin wt. Strikingly, under these conditions, vimentin C328H expression blunts electrophile-elicited stress fiber formation, apparently acting upstream of RhoA. Analysis of additional vimentin C328 mutants shows that electrophile-sensitive and assembly-defective vimentin variants permit induction of stress fibers by reactive species, whereas electrophile-resistant filamentous vimentin structures prevent it. Together, our results suggest that vimentin acts as a break for actin stress fibers formation, which would be released by C328-aided disruption, thus allowing full actin remodeling in response to oxidants and electrophiles. These observations postulate C328 as a “sensor” transducing structurally diverse modifications into fine-tuned vimentin network rearrangements, and a gatekeeper for certain electrophiles in the interplay with actin.

Supplementary Figure 2 from Inhibitor of Differentiation-1 as a Novel Prognostic Factor in NSCLC Patients with Adenocarcinoma Histology and Its Potential Contribution to Therapy Resistance

Supplementary Figure 3 from Inhibitor of Differentiation-1 as a Novel Prognostic Factor in NSCLC Patients with Adenocarcinoma Histology and Its Potential Contribution to Therapy Resistance

Supplementary Figures S1-S3 and Supplementary Tables S1-S2.

Purpose: High inhibitor of differentiation-1 (Id1) levels have been found in some tumor types. We aimed to study Id1 levels and their prognostic impact in a large series of stages I to IV non-small cell lung cancer (NSCLC) patients. Experiments in cell lines and cells derived from malignant pleural effusions (MPE) were also carried out.Experimental Design: A total of 346 NSCLC samples (three different cohorts), including 65 matched nonmalignant tissues, were evaluated for Id1 expression by using immunohistochemistry. Additional data from a fourth cohort including 111 patients were obtained for Id1 mRNA expression analysis by using publicly available microarrays. In vitro proliferation assays were conducted to characterize the impact of Id1 on growth and treatment sensitivity.Results: Significantly higher Id1 protein levels were found in tumors compared with normal tissues (P < 0.001) and in squamous carcinomas compared with adenocarcinomas (P < 0.001). In radically treated stages I to III patients and stage IV patients treated with chemotherapy, higher Id1 levels were associated with a shorter disease-free survival and overall survival in adenocarcinoma patients in a log-rank test. A Cox model confirmed the independent prognostic value of Id1 levels for both stages I to III and stage IV patients. In silico analysis confirmed a correlation between higher Id1 mRNA levels and poor prognosis for adenocarcinoma subjects. In vitro Id1 silencing in radio/chemotherapy-resistant adenocarcinoma cells from MPEs restored sensitivity to both therapies.Conclusions: In our series, Id1 levels showed an independent prognostic value in patients with adenocarcinoma, regardless of the stage. Id1 silencing may sensitize adenocarcinoma cells to radiotherapy and chemotherapy. Clin Cancer Res; 17(12); 4155–66. ©2011 AACR.

Supplementary Figure 4 from Inhibitor of Differentiation-1 as a Novel Prognostic Factor in NSCLC Patients with Adenocarcinoma Histology and Its Potential Contribution to Therapy Resistance

Supplementary Figure 1 from Inhibitor of Differentiation-1 as a Novel Prognostic Factor in NSCLC Patients with Adenocarcinoma Histology and Its Potential Contribution to Therapy Resistance

PDF file - 409K

Purpose: We investigated the role of the collagen-binding receptor discoidin domain receptor-1 (DDR1) in the initiation and development of bone metastasis.Experimental Design: We conducted immunohistochemical analyses in a cohort of 83 lung cancer specimens and examined phosphorylation status in a panel of human lung cancer cell lines. Adhesion, chemotaxis, invasiveness, metalloproteolytic, osteoclastogenic, and apoptotic assays were conducted in DDR1-silenced cells. In vivo, metastatic osseous homing and colonization were assessed in a murine model of metastasis.Results: DDR1 was expressed in a panel of human lung cancer cell lines, and high DDR1 levels in human lung tumors were associated with poor survival. Knockdown (shDDR1) cells displayed unaltered growth kinetics in vitro and in vivo. In contrast, shDDR1 cells showed reduced invasiveness in collagen matrices and increased apoptosis in basal conditions and induced apoptosis in vitro. More importantly, conditioned media of DDR1-knockdown cells decreased osteoclastogenic activity in vitro. Consequently, in a model of tumor metastasis to bone, lack of DDR1 showed decreased metastatic activity associated with reduced tumor burden and osteolytic lesions. These effects were consistent with a substantial reduction in the number of cells reaching the bone compartment. Moreover, intratibial injection of shDDR1 cells significantly decreased bone tumor burden, suggesting impaired colonization ability that was highly dependent on the bone microenvironment.Conclusions: Disruption of DDR1 hampers tumor cell survival, leading to impaired early tumor–bone engagement during skeletal homing. Furthermore, inhibition of DDR1 crucially alters bone colonization. We suggest that DDR1 represents a novel therapeutic target involved in bone metastasis. Clin Cancer Res; 18(4); 969–80. ©2012 AACR.

Supplementary Figure 5 from Inhibitor of Differentiation-1 as a Novel Prognostic Factor in NSCLC Patients with Adenocarcinoma Histology and Its Potential Contribution to Therapy Resistance

Abnormal alternative splicing has been associated with cancer. Genome-wide microarrays can be used to detect differential splicing events. In this study, we have developed ExonPointer, an algorithm that uses data from exon and junction probes to identify annotated cassette exons. We used the algorithm to profile differential splicing events in lung adenocarcinoma A549 cells after downregulation of the oncogenic serine/arginine-rich splicing factor 1 (SRSF1). Data were generated using two different microarray platforms. The PCR-based validation rate of the top 20 ranked genes was 60% and 100%. Functional enrichment analyses found a substantial number of splicing events in genes related to RNA metabolism. These analyses also identified genes associated with cancer and developmental and hereditary disorders, as well as biologic processes such as cell division, apoptosis, and proliferation. Most of the top 20 ranked genes were validated in other adenocarcinoma and squamous cell lung cancer cells, with validation rates of 80% to 95% and 70% to 75%, respectively. Moreover, the analysis allowed us to identify four genes, ATP11C, IQCB1, TUBD1, and proline-rich coiled-coil 2C (PRRC2C), with a significantly different pattern of alternative splicing in primary non–small cell lung tumors compared with normal lung tissue. In the case of PRRC2C, SRSF1 downregulation led to the skipping of an exon overexpressed in primary lung tumors. Specific siRNA downregulation of the exon-containing variant significantly reduced cell growth. In conclusion, using a novel analytical tool, we have identified new splicing events regulated by the oncogenic splicing factor SRSF1 in lung cancer. Cancer Res; 74(4); 1105–15. ©2013 AACR.

PDF file - 1020K, Supplementary Figure S1. A cassette event in MAPT gene. Supplementary Figure S2. Validation of the cell model of SRSF1 down-regulation. Supplementary Figure S3. Validation of differential alternative splicing events from the top 20 genes selected by the ExonPointer algorithm using microarray data from the Oryzon platform. Supplementary Figure S4. Validation of differential alternative splicing events in a set of genes selected by the ExonPointer algorithm using microarray data from the GeneSplice platform. Supplementary Figure S5. Ingenuity pathway network related to "Cell Morphology, Developmental Disorder and Hereditary Disorder" and derived from the analysis of differential alternative splicing events after SRSF1 down-regulation. Supplementary Figure S6 Ingenuity pathway network with annotations: "Cell Cycle, Cellular Assembly and Organization and Cellular Movement" and derived from the analysis of differential alternative splicing events after SRSF1 down-regulation. Supplementary Figure S7. Ingenuity pathway network with annotations: "Organismal Development, Cancer, Cell Death and Survival" and derived from the analysis of differential alternative splicing events after SRSF1 down-regulation. Supplementary Figure S8. Growth effect of PRRC2C splice variants in HCC827, H2087 and HCC95 cell growth.

PDF file - 96K, Description of the ExonPointer algorithm.

The metabolism and intercellular transfer of glutathione or its precursors may play an important role in cellular defense against oxidative stress, a common hallmark of neurodegeneration. In the 1990s, several studies in the Neurobiology field led to the widely accepted notion that astrocytes produce large amounts of glutathione that serve to feed neurons with precursors for glutathione synthesis. This assumption has important implications for health and disease since a reduction in this supply from astrocytes could compromise the capacity of neurons to cope with oxidative stress. However, at first glance, this shuttling would imply a large energy expenditure to get to the same point in a nearby cell. Thus, are there additional underlying reasons for this expensive mechanism? Are neurons unable to import and/or synthesize the three non-essential amino acids that are the glutathione building blocks? The rather oxidizing extracellular environment favors the presence of cysteine (Cys) as cystine (Cis), less favorable for neuronal import. Therefore, it has also been proposed that astrocytic GSH efflux could induce a change in the redox status of the extracellular space nearby the neurons, locally lowering the Cis/Cys ratio. This astrocytic glutathione release would also increase their demand for precursors, stimulating Cis uptake, which these cells can import, further impacting the local decline of the Cis/Cys ratio, in turn, contributing to a more reduced extracellular environment and subsequently favoring neuronal Cys import. Here, we revisit the experimental evidence that led to the accepted hypothesis of astrocytes acting as suppliers of neuronal glutathione precursors, considering recent data from the Human Protein Atlas. In addition, we highlight some potential drawbacks of this hypothesis, mainly supported by heterogeneous cellular models. Finally, we outline additional and more cost-efficient possibilities by which astrocytes could support neuronal glutathione levels, including its shuttling in extracellular vesicles.

Type 2 diabetes (T2D) is a complex metabolic disease, which involves maintained hyperglycemia, mainly due to the development of an insulin resistance process. Metformin administration is the most prescribed treatment for diabetic patients. In a previously published study, we demonstrated that Pediococcus acidilactici pA1c® (pA1c) protects from insulin resistance and body weight gain in HFD-induced diabetic mice. The present work aimed to evaluate the possible beneficial impact of a 16-week administration of pA1c, metformin, or the combination of pA1c and metformin in a T2D HFD-induced mice model. We found that the simultaneous administration of both products attenuated hyperglycemia, increased high-intensity insulin-positive areas in the pancreas and HOMA-β, decreased HOMA-IR and also provided more beneficial effects than metformin treatment (regarding HOMA-IR, serum C-peptide level, liver steatosis or hepatic Fasn expression), and pA1c treatment (regarding body weight or hepatic G6pase expression). The three treatments had a significant impact on fecal microbiota and led to differential composition of commensal bacterial populations. In conclusion, our findings suggest that P. acidilactici pA1c® administration improved metformin beneficial effects as a T2D treatment, and it would be a valuable therapeutic strategy to treat T2D. This research was funded by a grant from the Government of Navarre (Ayudas para la realización de proyectos de I+D 2020) (Reference: 0011-1365-2020-000086). M.C.O. was funded by a postdoctoral fellowship “Ayudas para la Recualificación del Sistema Universitario Español para 2021–2023, UPNA, Modalidad Margarita Salas”, funded by the European Union—NextGenerationEU. M.O. was supported by Torres-Quevedo grants from the Spanish Ministry of Science and innovation (Ayudas para contratos Torres Quevedo PTQ2019-010384/AEI/10.13039/501100011033)

Methylation of CpG dinucleotides plays a crucial role in the regulation of gene expression and therefore in the development of different pathologies. Aberrant methylation has been associated to the majority of the diseases, including cancer, neurodegenerative, cardiovascular and autoimmune disorders. Analysis of DNA methylation patterns is crucial to understand the underlying molecular mechanism of these diseases. Moreover, DNA methylation patterns could be used as biomarker for clinical management, such as diagnosis, prognosis and treatment response. Nowadays, a variety of high throughput methods for DNA methylation have been developed to analyze the methylation status of a high number of CpGs at once or even the whole genome. However, identification of specific methylation patterns at specific loci is essential for validation and also as a tool for diagnosis. In this review, we describe the most commonly used approaches to evaluate specific DNA methylation. There are three main groups of techniques that allow the identification of specific regions that are differentially methylated: bisulfite conversion-based methods, restriction enzyme-based approaches, and affinity enrichment-based assays. In the first group, specific restriction enzymes recognize and cleave unmethylated DNA, leaving methylated sequences intact. Bisulfite conversion methods are the most popular approach to distinguish methylated and unmethylated DNA. Unmethylated cytosines are deaminated to uracil by sodium bisulfite treatment, while the methyl cytosines remain unconverted. In the last group, proteins with methylation binding domains or antibodies against methyl cytosines are used to recognize methylated DNA. In this review, we provide the theoretical basis and the framework of each technique as well as the analysis of their strength and the weaknesses.

Prediabetes (PreD), which is associated with impaired glucose tolerance and fasting blood glucose, is a potential risk factor for type 2 diabetes mellitus (T2D). Growing evidence suggests the role of the gastrointestinal microbiota in both PreD and T2D, which opens the possibility for a novel nutritional approach, based on probiotics, for improving glucose regulation and delaying disease progression of PreD to T2D. In this light, the present study aimed to assess the antidiabetic properties of

23 p.-9 fig.-8 tab., Catalytic MATa1 subunits associate into kinetically distinct homo-dimers (MAT III) and homo-tetramers (MAT I) that synthesize S-adenosylmethionine in the adult liver. Pathological reductions in S-adenosylmethionine levels correlate with MAT III accumulation; thus, it is important to know the determinants of dimer–dimer associations. Here, polar interactions (, This research was funded by grants of the Ministerio de Ciencia e Innovación (BMC-2002-00243, BFU2005-00050, BFU2008-00666, BFU2009-08977 to M.Á.P.) and Fondo de Investigaciones Sanitarias of the Instituto de Salud Carlos III (01/1077 and RCMN C03/08 to M.Á.P.).

19 p.-9 fig., The SARS-CoV-2 Spike protein mediates docking of the virus onto cells prior to viral invasion. Several cellular receptors facilitate SARS-CoV-2 Spike docking at the cell surface, of which ACE2 plays a key role in many cell types. The intermediate filament protein vimentin has been reported to be present at the surface of certain cells and act as a co-receptor for several viruses; furthermore, its potential involvement in interactions with Spike proteins has been proposed. Nevertheless, the potential colocalization of vimentin with Spike and its receptors on the cell surface has not been explored. Here we have assessed the binding of Spike protein constructs to several cell types. Incubation of cells with tagged Spike S or Spike S1 subunit led to discrete dotted patterns at the cell surface, which consistently colocalized with endogenous ACE2, but sparsely with a lipid raft marker. Vimentin immunoreactivity mostly appeared as spots or patches unevenly distributed at the surface of diverse cell types. Of note, vimentin could also be detected in extracellular particles and in the cytoplasm underlying areas of compromised plasma membrane. Interestingly, although overall colocalization of vimentin-positive spots with ACE2 or Spike was moderate, a selective enrichment of the three proteins was detected at elongated structures, positive for acetylated tubulin and ARL13B. These structures, consistent with primary cilia, concentrated Spike binding at the top of the cells. Our results suggest that a vimentin-Spike interaction could occur at selective locations of the cell surface, including ciliated structures, which can act as platforms for SARS-CoV-2 docking., This work was supported by grants from Consejo Superior de Investigaciones Científicas, CSIC PTI Global Health (PIE 202020E223/CSIC-COV19-100), RTI2018-097624-B-I00 from Ministerio de Ciencia e Innovación, Micinn (Agencia Estatal de Investigación), Spain, and European Regional Development Fund, EDRF (. Á.V.P. and P.G.J. are the recipients of predoctoral contracts BES-2016-076965 and PRE2019-088194, respectively, from Ministerio de Ciencia e Innovación, Spain.

15 p.-5 fig.-3 tab., Protein modification by lipid derived reactive species or lipoxidation is increased during oxidative stress, a common feature observed in many pathological conditions. Biochemical and functional consequences of lipoxidation include changes in the conformation and assembly of the target proteins, altered recognition of ligands and/or cofactors, changes in the interactions with DNA or protein-protein interactions, modifications in membrane partitioning and binding and/or subcellular localization. These changes may impact, directly or indirectly, signaling pathways involved in the activation of cell defense mechanisms, but when these are overwhelmed they may lead to pathological outcomes. Mass spectrometry provides state of the art approaches for the identification and characterization of lipoxidized proteins/residues and the modifying species. Nevertheless, understanding the complexity of the functional effects of protein lipoxidation requires the use of additional methodologies. Herein, biochemical and biophysical methods used to detect and measure functional effects of protein lipoxidation at different levels of complexity, from in vitro and reconstituted cell-like systems to cells, are reviewed, focusing especially on macromolecular interactions. Knowledge generated through innovative and complementary technologies will contribute to comprehend the role of lipoxidation in pathophysiology and, ultimately, its potential as target for therapeutic intervention., This work was supported by the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement number 675132 (http://cordis.europa.eu/project/rcn/198275_en.html), and by grants from the Spanish Ministerio de Economía y Competitividad (MINECO/FEDER, http://www.mineco.gob.es/portal/site/mineco/idi) SAF2015-68590-R to DPS and BFU2016-75471-C2-1-P to GR, and RETIC Aradyal from ISCIII/FEDER (RD16/0006/0021) to DPS.

23 p.-7 fig., The intermediate filament protein vimentin is involved in essential cellular processes, including cell division and stress responses, as well as in the pathophysiology of cancer, pathogen infection, and autoimmunity. The vimentin network undergoes marked reorganizations in response to oxidative stress, in which modifications of vimentin single cysteine residue, Cys328, play an important role, and is modulated by zinc availability. However, the molecular basis for this regulation is not fully understood. Here, we show that Cys328 displays a low pKa, supporting its reactivity, and is readily alkylated and oxidized in vitro. Moreover, combined oxidation and crosslinking assays and molecular dynamics simulations support that zinc ions interact with Cys328 in its thiolate form, whereas Glu329 and Asp331 stabilize zinc coordination. Vimentin oxidation can induce disulfide crosslinking, implying the close proximity of Cys328 from neighboring dimers in certain vimentin conformations, supported by our computational models. Notably, micromolar zinc concentrations prevent Cys328 alkylation, lipoxidation, and disulfide formation. Moreover, zinc selectively protects vimentin from crosslinking using short-spacer cysteine-reactive but not amine-reactive agents. These effects are not mimicked by magnesium, consistent with a lower number of magnesium ions hosted at the cysteine region, according to molecular dynamics simulations. Importantly, the region surrounding Cys328 is involved in interaction with several drugs targeting vimentin and is conserved in type III intermediate filaments, which include glial fibrillary acidic protein and desmin. Altogether, our results identify this region as a hot spot for zinc binding, which modulates Cys328 reactivity. Moreover, they provide a molecular standpoint for vimentin regulation through the interplay between cysteine modifications and zinc availability., This work was supported by the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie Grant Agreement No. 675132 “Masstrplan”, Grants SAF2015-68590-R and RTI2018-097624-B-I00 from Agencia Estatal de Investigación, MICINN/FEDER, Spain, and Instituto de Salud Carlos III/FEDER, RETIC ARADyAL RD16/0006/0021 to DPS; Grant CTQ2017-88353-R from MICINN to SMS; Grant BES-2015-071588 from MICINN to JGC.

The Spike protein from SARS-CoV-2 mediates docking of the virus onto cells and contributes to viral invasion. Several cellular receptors are involved in SARS-CoV-2 Spike docking at the cell surface, including ACE2 and neuropilin. The intermediate filament protein vimentin has been reported to be present at the surface of certain cells and act as a co-receptor for several viruses; furthermore, its potential involvement in interactions with Spike proteins has been proposed. Here we have explored the binding of Spike protein constructs to several cell types using low-temperature immunofluorescence approaches in live cells, to minimize internalization. Incubation of cells with tagged Spike S or Spike S1 subunit led to discrete dotted patterns at the cell surface, which showed scarce colocalization with a lipid raft marker, but consistent coincidence with ACE2. Under our conditions, vimentin immunoreactivity appeared as spots or patches unevenly distributed at the surface of diverse cell types. Remarkably, several observations including potential antibody internalization and adherence to cells of vimentin-positive structures present in the extracellular medium exposed the complexity of vimentin cell surface immunoreactivity, which requires careful assessment. Notably, overall colocalization of Spike and vimentin signals markedly varied with the cell type and the immunodetection sequence. In turn, vimentin-positive spots moderately colocalized with ACE2; however, a particular enrichment was detected at elongated structures positive for acetylated tubulin, consistent with primary cilia, which also showed Spike binding. Thus, these results suggest that vimentin-ACE2 interaction could occur at selective locations near the cell surface, including ciliated structures, which can act as platforms for SARS-CoV-2 docking., This work was supported by grants CSIC PTI Global Health (PIE 202020E223/CSIC-COV19-100), RTI2018-097624-B-I00 from Micinn (Agencia Estatal de Investigación), Spain and ERDF. Á.V.P. and P.G.J. are the recipients of predoctoral contracts BES-2016-076965 and PRE2019-088194, respectively, from Micinn, Spain.

Background: tobacco is the main risk factor for developing lung cancer. Yet, some heavy smokers do not develop lung cancer at advanced ages while others develop it at young ages. Here, we assess for the first time the genetic background of these clinically relevant extreme phenotypes using whole exome sequencing (WES). Methods: we performed WES of germline DNA from heavy smokers who either developed lung adenocarcinoma at an early age ( extreme cases, n=50) or did not present lung adenocarcinoma or other tumors at an advanced age (extreme controls, n=50). We selected non-synonymous variants located in exonic regions and consensus splice sites of the genes that showed significantly different allelic frequencies between both cohorts. We validated our results in all the additional extreme cases (i.e., heavy smokers who developed lung adenocarcinoma at an early age) available from The Cancer Genome Atlas (TCGA). Results: the mean age for the extreme cases and controls was respectively 49.7 and 77.5 years. Mean tobacco consumption was 43.6 and 56.8 pack-years. We identified 619 significantly different variants between both cohorts, and we validated 108 of these in extreme cases selected from TCGA. Nine validated variants, located in relevant cancer related genes, such as PARP4, HLA-A or NQO1, among others, achieved statistical significance in the False Discovery Rate test. The most significant validated variant (P=4.48x10(-5)) was located in the tumor-suppressor gene ALPK2. Conclusions: we describe genetic variants associated with extreme phenotypes of high and low risk for the development of tobacco-induced lung adenocarcinoma. Our results and our strategy may help to identify high-risk subjects and to develop new therapeutic strategies. This work was supported by the Spanish Society of Medical Oncology; Fundación SEOM and Fundación Salud 2000; and Government of Navarra. LMM research work is supported by Foundation for Applied Medical Research (FIMA), Fundación Científica de la Asociación Espanola Contra el Cáncer, Fundación Ramón Areces, and Fondo de Investigación Sanitaria-Fondo Europeo de Desarrollo Regional 'Una manera de hacer Europa' (PI19/00098).

1 p. (Abtracts of SFRR-International 2021 Virtual Meeting), H2020 grant-675132, “Masstrplan”; RTI2018-097624-B-100 (MCINN, ERDF); RETIC-Aradyal RD16/0006/0021 (ISCIII-ERDF); MICINN BES-2016-076965 (AVP), PRE2019-088194 (PG).

Obesity is a worldwide epidemic characterized by excessive fat accumulation, associated with multiple comorbidities and complications. Emerging evidence points to gut microbiome as a driving force in the pathogenesis of obesity. Vinegar intake, a traditional remedy source of exogenous acetate, has been shown to improve glycemic control and to have anti-obesity effects. New functional foods may be developed by supplementing traditional food with probiotics. B. coagulans is a suitable choice because of its resistance to high temperatures. To analyze the possible synergic effect of Vinegar and B. coagulans against the metabolic alterations induced by a high fat diet (HFD), we fed twelve-week-old C57BL/6 mice with HFD for 5 weeks after 2 weeks of acclimation on a normal diet. Then, food intake, body weight, blood biochemical parameters, histology and liver inflammatory markers were analyzed. Although vinegar drink, either alone or supplemented with B. coagulans, reduced food intake, attenuated body weight gain and enhanced glucose tolerance, only the supplemented drink improved the lipid serum profile and prevented hepatic HFD-induced overexpression of CD36, IL-1&beta, IL-6, LXR and SREBP, thus reducing lipid deposition in the liver. The beneficial properties of the B. coagulans-supplemented vinegar appear to be mediated by a reduction in insulin and leptin circulating levels.

Obesity is a worldwide epidemic characterized by excessive fat accumulation, associated with multiple comorbidities and complications. Emerging evidence points to gut microbiome as a driving force in the pathogenesis of obesity. Vinegar intake, a traditional remedy source of exogenous acetate, has been shown to improve glycemic control and to have anti-obesity effects. New functional foods may be developed by supplementing traditional food with probiotics. B. coagulans is a suitable choice because of its resistance to high temperatures. To analyze the possible synergic effect of Vinegar and B. coagulans against the metabolic alterations induced by a high fat diet (HFD), we fed twelve-week-old C57BL/6 mice with HFD for 5 weeks after 2 weeks of acclimation on a normal diet. Then, food intake, body weight, blood biochemical parameters, histology and liver inflammatory markers were analyzed. Although vinegar drink, either alone or supplemented with B. coagulans, reduced food intake, attenuated body weight gain and enhanced glucose tolerance, only the supplemented drink improved the lipid serum profile and prevented hepatic HFD-induced overexpression of CD36, IL-1β, IL-6, LXR and SREBP, thus reducing lipid deposition in the liver. The beneficial properties of the B. coagulans-supplemented vinegar appear to be mediated by a reduction in insulin and leptin circulating levels.

Enzymes of the methionine and homocysteine metabolism catalyze reactions belonging to the methionine and folate cycles and the transsulfuration pathway. The importance of the metabolites produced through these routes (e.g. S-adenosylmethionine, homocysteine) and their role in e.g. epigenetics or redox mechanisms makes their tight regulation essential for a correct cellular function. Pharmacological or pathophysiological insults induce, among others, changes in activity, oligomerization, protein levels, subcellular localization and expression of these enzymes. The abundance of these proteins in liver has made this organ the preferred system to study their regulation. Nevertheless, knowledge about their putative protein-protein interactions is limited in this and other tissues and cell types. High-throughput methods, including immunoprecipitation, affinity purification coupled to mass spectrometry and yeast two-hybrid have rendered the identification of a number of protein-protein interactions involving these enzymes in several systems. Validation by coimmunoprecipitation and/or pull-down has been made, mainly, after coexpression of bait and prey, but few of the interactions have been confirmed. Additionally, information concerning the role of these interactions in the regulation of this pathway and other cellular processes is scarce. Here, we review the current knowledge on mammalian protein-protein interactions involving methionine adenosyltransferases, S-adenosylhomocysteine hydrolase, betaine homocysteine S-methyltransferases, methionine synthase and cystathionine β-synthase, although references to data obtained in other organisms are also made. Moreover, the verified or putative implication of these interactions in the regulation of methionine and homocysteine metabolism, its interplay with other metabolic pathways and its putative link to pathophysiological processes, such as oncogenesis, is discussed., This work was supported by grants of the Ministerio de Ciencia,Innovación y Universidades (BFU2005-00050, BFU2008-00666,BFU2009-08977 to MAP; BFU2008-04285 to FP; BIO2015-67580-Pand PGC2018-097019-B-I00 to JV), the Instituto de Salud Carlos III-Fondo de Investigación Sanitaria PRB3 (IPT17/0019-ISCIII-SGEFI/ERDF, ProteoRed to JV), Fundació Marató TV3 (122/C/2015 to JV)and La Caixa Banking Foundation (HR17-00247 to JV). The CNIC issupported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovación y Universidades (MCNU) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).

16 p.-5 fig., Intermediate filaments (IFs) play key roles in cell mechanics, signaling and homeostasis. Their assembly and dynamics are finely regulated by posttranslational modifications. The type III IFs, vimentin, desmin, peripherin and glial fibrillary acidic protein (GFAP), are targets for diverse modifications by oxidants and electrophiles, for which their conserved cysteine residue emerges as a hot spot. Pathophysiological examples of these modifications include lipoxidation in cell senescence and rheumatoid arthritis, disulfide formation in cataracts and nitrosation in endothelial shear stress, although some oxidative modifications can also be detected under basal conditions. We previously proposed that cysteine residues of vimentin and GFAP act as sensors for oxidative and electrophilic stress, and as hinges influencing filament assembly. Accumulating evidence indicates that the structurally diverse cysteine modifications, either per se or in combination with other posttranslational modifications,elicit specific functional outcomes inducing distinct assemblies or network rearrangements, including filament stabilization, bundling or fragmentation. Cysteine-deficient mutants are protected from these alterations but show compromised cellular performance in network assembly and expansion, organelle positioning and aggresome formation, revealing the importance of this residue. Therefore, the high susceptibility to modification of the conserved cysteine of type III IFs and its cornerstone position in filament architecture sustains their role in redox sensing and integration of cellular responses. This has deep pathophysiological implications and supports the potential of this residue as a drug target., This work was supported by Grants from Agencia Estatal de Investigación, Ministerio de Ciencia e Innovación (MCI, Spain) and European Regional Development Fund, RTI2018-097624-B-I00,Instituto de Salud Carlos III (Spain) RETIC AraDyal RD16/0006/0021,and CSIC PTI Global Health (PIE202020E223/CSIC-COV19-100). AV-P is supported by the FPI program from MCI, reference BES-2016-076965.

Extracorporeal membrane oxygenation (ECMO) is mechanical circulatory and respiratory assistance and offers an alternative to cardiopulmonary bypass (CPB) for certain thoracic procedures including lung transplantation.

Prediabetes (PreD), which is associated with impaired glucose tolerance and fasting blood glucose, is a potential risk factor for type 2 diabetes mellitus (T2D). Growing evidence suggests the role of the gastrointestinal microbiota in both PreD and T2D, which opens the possibility for a novel nutritional approach, based on probiotics, for improving glucose regulation and delaying disease progression of PreD to T2D. In this light, the present study aimed to assess the antidiabetic properties of Pediococcus acidilactici (pA1c) in a murine model of high-fat diet (HFD)-induced T2D. For that purpose, C57BL/6 mice were given HFD enriched with either probiotic (1 × 1010 CFU/day) or placebo for 12 weeks. We determined body weight, fasting blood glucose, glucose tolerance, HOMA-IR and HOMA-β index, C-peptide, GLP-1, leptin, and lipid profile. We also measured hepatic gene expression (G6P, PEPCK, GCK, IL-1β, and IL-6) and examined pancreatic and intestinal histology (% of GLP-1+ cells, % of goblet cells and villus length). We found that pA1c supplementation significantly attenuated body weight gain, mitigated glucose dysregulation by reducing fasting blood glucose levels, glucose tolerance test, leptin levels, and insulin resistance, increased C-peptide and GLP-1 levels, enhanced pancreatic function, and improved intestinal histology. These findings indicate that pA1c improved HFD-induced T2D derived insulin resistance and intestinal histology, as well as protected from body weight increase. Together, our study proposes that pA1c may be a promising new dietary management strategy to improve metabolic disorders in PreD and T2D. This research was funded by grant from Government of Navarre (Ayudas para la realización de proyectos de I+D 2020) [Reference: 0011-1365-2020-000086]. This study was also funded by grants from Genbioma Aplicaciones S.L. Maria Oneca is supported by Torres-Quevedo grants from the Spanish Ministry of Science and innovation (Ayudas para contratos Torres Quevedo PTQ2019-010384/AEI/10.13039/501100011033).

143 p.-17 fig., SIGNIFICANCE:Transsulfuration allows conversion of methionine into cysteine using homocysteine as an intermediate. This pathway produces S-adenosylmethionine, a key metabolite for cell function, and provides 50% of the cysteine needed for hepatic glutathione synthesis. The route requires the intake of essential nutrients (e.g. methionine, vitamins) and is regulated by their availability. Transsulfuration presents multiple interconnections with epigenetics, ATP and glutathione synthesis, polyol and pentose phosphate pathways and detoxification that rely mostly in the exchange of substrates or products. Major hepatic diseases, rare diseases and sensorineural disorders, among others that concur with oxidative stress, present impaired transsulfuration. Recent advances. In contrast to the classical view, a nuclear branch of the pathway, potentiated under oxidative stress, is emerging. Several transsulfuration proteins regulate gene expression, suggesting moonlighting activities. Additionally, abnormalities in homocysteine metabolism link nutrition and hearing loss., CRITICAL ISSUES:Knowledge about the cross-regulation between pathways is mostly limited to the hepatic availability/removal of substrates and inhibitors. However, advances regarding protein-protein interactions involving oncogenes, identification of several post-translational modifications and putative moonlighting activities expand the potential impact of transsulfuration beyond methylations and homocysteine., FUTURE DIRECTIONS:Increasing the knowledge on transsulfuration outside the liver, understanding the protein-protein interaction networks involving these enzymes, the functional role of their post-translational modifications or the mechanisms controlling their nucleocytoplasmic shuttling may provide further insights into the pathophysiological implications of this pathway, allowing design of new therapeutic interventions., This work was support by grants of the Ministerio de Economía y Competitividad (SAF2012-36519 and SAF2015-68590R to DPS) and Instituto de Salud Carlos III (RETICs RIRAAF RD12/0013/0008 and ARADyAL RD16/0006/0021).