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Background and aims: CD4+ T cells constitute central players in inflammatory bowel diseases (IBDs), driving inflammation in the gut mucosa. Current evidence indicates that CCR9 and the integrin α4β7 are necessary and sufficient to imprint colonic homing on CD4+ T cells upon inflammation. Interestingly, dopaminergic signaling has been previously involved in leukocyte homing. Despite dopamine levels are strongly reduced in the inflamed gut mucosa, the role of dopamine in the gut homing of T cells remains unknown. Here, we study how dopaminergic signaling affects T cells upon gut inflammation. Methods: Gut inflammation was induced by transfer of naïve T cells into Rag1-/- mice or by administration of dextran sodium sulfate. T cell migration and differentiation were evaluated by adoptive transfer of congenic lymphocytes followed by flow cytometry analysis. Protein interaction was studied by bioluminescence resonance energy transfer analysis, bimolecular fluorescence complementation, and in situ proximity ligation assays. Results: We show the surface receptor providing colonic tropism to effector CD4+ T cells upon inflammation is not CCR9 but the complex formed by CCR9 and the dopamine receptor D5 (DRD5). Assembly of the heteromeric complex was demonstrated in vitro and in vivo using samples from mouse and human origin. The CCR9:DRD5 heteroreceptor was upregulated in the intestinal mucosa of IBD patients. Signaling assays confirmed that complexes behave differently than individual receptors. Remarkably, the disruption of CCR9:DRD5 assembly attenuated the recruitment of CD4+ T cells into the colonic mucosa. Conclusions: Our findings describe a key homing receptor involved in gut inflammation and introduce a new cell surface module in immune cells: macromolecular complexes formed by G protein-coupled receptors integrating the sensing of multiple molecular cues. Keywords: Chemokine Receptors; Dopaminergic Regulation; G Protein-Coupled Receptors Heteromers; Gut Tropism; Inflammatory Bowel Diseases; Inflammatory Colitis; T Cell Migration.

Alteration of normal ploidy (aneuploidy) can have a number of opposing effects, such as unbalancing protein abundances and inhibiting cell growth but also accelerating genetic diversification and rapid adaptation. The interplay of these detrimental and beneficial effects remains puzzling. Here, to understand how cells develop tolerance to aneuploidy, we subject disomic (i.e. with an extra chromosome copy) strains of yeast to long-term experimental evolution under strong selection, by forcing disomy maintenance and daily population dilution. We characterize mutations, karyotype alterations and gene expression changes, and dissect the associated molecular strategies. Cells with different extra chromosomes accumulated mutations at distinct rates and displayed diverse adaptive events. They tended to evolve towards normal ploidy through chromosomal DNA loss and gene expression changes. We identify genes with recurrent mutations and altered expression in multiple lines, revealing a variant that improves growth under genotoxic stresses. These findings support rapid evolvability of disomic strains that can be used to characterize fitness effects of mutations under different stress conditions., Aneuploidy (abnormal chromosome number) can enable rapid adaptation to stress conditions, but it also entails fitness costs from gene imbalance. Here, the authors experimentally evolve yeast while forcing maintenance of aneuploidy to identify the mechanisms that promote tolerance of aneuploidy.

Familial exudative vitreoretinopathy (FEVR) is a human disease characterized by defective retinal angiogenesis and associated complications that can result in vision loss. Defective Wnt/β-catenin signaling is an established cause of FEVR, whereas other molecular alterations contributing to the disease remain insufficiently understood. Here, we show that integrin-linked kinase (ILK), a mediator of cell-matrix interactions, is indispensable for retinal angiogenesis. Inactivation of the murine Ilk gene in postnatal endothelial cells results in sprouting defects, reduced endothelial proliferation and disruption of the blood-retina barrier, resembling phenotypes seen in established mouse models of FEVR. Retinal vascularization defects are phenocopied by inducible inactivation of the gene for α-parvin (Parva), an interactor of ILK. Screening genomic DNA samples from exudative vitreoretinopathy patients identifies three distinct mutations in human ILK, which compromise the function of the gene product in vitro. Together, our data suggest that defective cell-matrix interactions are linked to Wnt signaling and FEVR., Integrin-linked kinase (ILK) is an important mediator of integrin signaling. Here Park et al. show that mice with endothelial-specific deletion of Ilk develop vascular defects that resemble familial exudative vitreoretinopathy, and identify mutations in ILK in patients with exudative vitreoretinopathy suggesting a potential role in human pathogenesis.

Summary Osteocytes, the most abundant of bone cells, differentiate while they remain buried within the bone matrix. This encasement limits their access to nutrients and likely affects their differentiation, a process that remains poorly defined. Here, we show that restriction in glucose supply promotes the osteocyte transcriptional program while also being associated with increased mitochondrial DNA levels. Glucose deprivation triggered the activation of the AMPK/PGC-1 pathway. AMPK and SIRT1 activators or PGC-1α overexpression are sufficient to enhance osteocyte gene expression in IDG-SW3 cells, murine primary osteoblasts, osteocytes, and organotypic/ex vivo bone cultures. Conversely, osteoblasts and osteocytes deficient in Ppargc1a and b were refractory to the effects of glucose restriction. Finally, conditional ablation of both genes in osteoblasts and osteocytes generate osteopenia and reduce osteocytic gene expression in mice. Altogether, we uncovered a role for PGC-1 in the regulation of osteocyte gene expression., Graphical Abstract, Highlights • Glucose restriction promotes osteocytic gene expression and mitochondrial function • Glucose restriction triggers activation of the AMPK/PGC-1 pathway • Effects of glucose restriction on osteocyte gene expression depend on Ppargc1a/b • Deletion of Ppargc1a/b in osteoblasts and osteocytes leads to osteopenia, Molecular Mechanism of Behavior; Molecular Physiology; Specialized Functions of Cells

The ultimate goal of gene regulation should focus on the protein level. However, as mRNA is an obligate intermediary, and because the amounts of mRNAs and proteins are controlled by their synthesis and degradation rates, the cellular amount of a given protein can be attained following different strategies. By studying omics datasets for six expression variables (mRNA and protein amounts, plus their synthesis and decay rates), we previously demonstrated the existence of common expression strategies (CES) for functionally-related genes in the yeastSaccharomyces cerevisiae. Here we extend that study to two other eukaryotes: the distantly related yeastSchizosaccharomyces pombeand cultured human HeLa cells. We also use genomic datasets from the model prokaryoteEscherichia colias an external reference. We show that CES are also present in all the studied organisms and the differences in them between organisms can be used to establish their phylogenetic relationships. The phenogram based on 6VP has the expected topology for the phylogeny of these four organisms, but shows interesting branch length differences to DNA sequence-based trees.The analysis of the correlations among the six variables supports that most gene expression control occurs in actively growing organisms at the transcription rate level, and that translation plays a minor role in it. We propose that all living cells use CES for the genes acting on the same physiological pathways, especially for those belonging to stable macromolecular complexes, but CES have been modeled by evolution to adapt to the specific life circumstances of each organism. The obtained phenograms may reflect both evolutionary constraints in expression strategies, and lifestyle convergences.

Developmental patterning in Caenorhabditis elegans is known to proceed in a highly stereotypical manner, which raises the question of how developmental robustness is achieved despite the inevitable stochastic noise. We focus here on a population of epidermal cells, the seam cells, which show stem cell-like behaviour and divide symmetrically and asymmetrically over post-embryonic development to generate epidermal and neuronal tissues. We have conducted a mutagenesis screen to identify mutants that introduce phenotypic variability in the normally invariant seam cell population. We report here that a null mutation in the fusogen eff-1 increases seam cell number variability. Using time-lapse microscopy and single molecule fluorescence hybridisation, we find that seam cell division and differentiation patterns are mostly unperturbed in eff-1 mutants, indicating that cell fusion is uncoupled from the cell differentiation programme. Nevertheless, seam cell losses due to the inappropriate differentiation of both daughter cells following division, as well as seam cell gains through symmetric divisions towards the seam cell fate were observed at low frequency. We show that these stochastic errors likely arise through accumulation of defects interrupting the continuity of the seam and changing seam cell shape, highlighting the role of tissue homeostasis in suppressing phenotypic variability during development. Funding: Some C. elegans strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). This work was funded by the European Research Council (ROBUSTNET-639485)

Hyperphosphorylation of protein tau is a hallmark of Alzheimer’s disease (AD). Changes in energy and lipid metabolism have been correlated with the late onset of this neurological disorder. However, it is uncertain if metabolic dysregulation is a consequence of AD or one of the initiating factors of AD pathophysiology. Also, it is unclear whether variations in lipid metabolism regulate the phosphorylation state of tau. Here, we show that in humanized yeast, tau hyperphosphorylation is stimulated by glucose starvation in coincidence with the downregulation of Pho85, the yeast ortholog of CDK5. Changes in inositol phosphate (IP) signaling, which has a central role in energy metabolism, altered tau phosphorylation. Lack of inositol hexakisphosphate kinases Kcs1 and Vip1 (IP6 and IP7 kinases in mammals) increased tau hyperphosphorylation. Similar effects were found by mutation of IPK2 (inositol polyphosphate multikinase), or PLC1, the yeast phospholipase C gene. These effects may be explained by IP-mediated regulation of Pho85. Indeed, this appeared to be the case for plc1, ipk2, and kcs1. However, the effects of Vip1 on tau phosphorylation were independent of the presence of Pho85, suggesting additional mechanisms. Interestingly, kcs1 and vip1 strains, like pho85, displayed dysregulated sphingolipid (SL) metabolism. Moreover, genetic and pharmacological inhibition of SL biosynthesis stimulated the appearance of hyperphosphorylated forms of tau, while increased flux through the pathway reduced its abundance. Finally, we demonstrated that Sit4, the yeast ortholog of human PP2A protein phosphatase, is a downstream effector of SL signaling in mediating the tau phosphorylation state. Altogether, our results add new knowledge on the molecular effectors involved in tauopathies and identify new targets for pharmacological intervention., This research has been supported by the Comisión Interministerial de Ciencia y Tecnología Project (BIO2015-71059-R) from the Spanish Ministry of Science, Innovation and Universities (MICINN/FEDER), the i-LINK program (i-LINK-2016-1109) from the National Spanish Research Council (CSIC), the NIH (grant AI125770 to MDP), and the KU Leuven Research Fund (grant C14/17/063 to JW).

Nervous systems are complex cellular structures that allow animals to interact with their environment, which includes both the external and the internal milieu. The astonishing diversity of nervous system architectures present in all animal clades has prompted the idea that selective forces must have shaped them over evolutionary time. In most cases, neurons seem to coalesce into specific (centralized) structures that function as “central processing units” (CPU): “brains.” Why did neural systems adopt this physical configuration? When did it first happen? What are the physiological, computational, and/or structural advantages of concentrating many neurons in a specific place within the body? Here we examine the concept of nervous system centralization and factors that might have contributed to the evolutionary success of this centralization strategy. In particular, we suggest a putative scenario for the evolution of neural system centralization that incorporates different strands of evidence. This scenario is based on some premises: (1) Receptors originated before neurons (sensors before transmitters) and there were deployed in the first organisms in an asymmetric fashion (deposited randomly in the outer layer); (2) Receptors were segregated in a preferential position in response to an anisotropic environment, (3) Neurons were born in association with this receptors and used to transmit signals distally; (4) Energetics preferentially selected the localization of neurons, and synapsis, close to the receptors (to minimize wire use, for instance); (5) The presence of condensed areas of neurons could have stimulated the proliferation of more receptors in the vicinity, increasing the repertoire of signals processed in an specific body domain (i.e., head) plus contributing to amplify the computational power of the neuronal aggregate; (6) The proliferation of receptors would have induced the proliferation of more neurons in the aggregate, with a further increase in its computational power (hence, diversifying the behavioral repertoire). These last two steps of proliferation and aggregation could have been sustained through a feedback loop, reiterated many times, generating distinct topologies in different lineages. Our main aim in this paper is to examine the brain as both a biological and a physical or computational device.

The fine structure of the infective hexacanths of Echinococcus multilocularis was examined with particular emphasis on the functional ultrastructure of penetration glands and nerve cells directly involved in the mechanism of initial host infection. The oncosphere contains two types of penetration glands, PG1 and PG2, that differ slightly in size and form a large U-shaped bi-nucleated syncytial structure. The arms of each gland at each end of the U, directed towards the hook region, exit into the tegument peripheral layer between the median and lateral hook pairs. The lobate nuclei of PG1 and PG2 contain prominent spherical nucleoli surrounded by several large electron-dense islands of heterochromatin. The syncytial cytoplasm of both types of glands is rich in free ribosomes, polysomes, several mitochondria, and heavy accumulations of discoid secretory granules of moderate to high electron density. The secretory granules, sg1 and sg2, differ in their ultrastructure and electron density; the sg2 are much smaller and more flattened in shape. A common characteristic for sg1 and sg2, evident under high magnification, is their high electron density and discoidal shape, with two bi-concave surfaces. Both sg1 and sg2 are frequently grouped in characteristic parallel stacks, the “rouleau”-shaped assemblages with sometimes six to ten granules. Two nerve cells of neurosecretory type are situated in the central part of the hexacanth, each one in a deep U-shaped invagination between the two penetration glands. The nuclei of nerve cells contain several large heterochromatin islands closely adjacent to their nuclear membranes. Their cytoplasm is characterized by having membrane-bound, dense-cored neurosecretory-like granules not only in nerve cell perikarya but also in the elongated nerve processes frequently adjacent to gland arms and to both somatic or body musculature, including the complex system of hook muscles. The results of the present study, when supported with literature data on oncospheres of other cestode species, allow for a better understanding of the important role and coordinated functions of three structural components, i.e., oncospheral hooks, penetration glands, and nerve cells, in the mechanism of intermediate host infection. Presence or absence of nerve cells in oncospheres of various cestodes is reviewed, and perspectives on the value and application of research on functional morphology of oncospheres are discussed.

VEGFR-2/Notch signalling regulates angiogenesis in part by driving the remodelling of endothelial cell junctions and by inducing cell migration. Here, we show that VEGF-induced polarized cell elongation increases cell perimeter and decreases the relative VE-cadherin concentration at junctions, triggering polarized formation of actin-driven junction-associated intermittent lamellipodia (JAIL) under control of the WASP/WAVE/ARP2/3 complex. JAIL allow formation of new VE-cadherin adhesion sites that are critical for cell migration and monolayer integrity. Whereas at the leading edge of the cell, large JAIL drive cell migration with supportive contraction, lateral junctions show small JAIL that allow relative cell movement. VEGFR-2 activation initiates cell elongation through dephosphorylation of junctional myosin light chain II, which leads to a local loss of tension to induce JAIL-mediated junctional remodelling. These events require both microtubules and polarized Rac activity. Together, we propose a model where polarized JAIL formation drives directed cell migration and junctional remodelling during sprouting angiogenesis., The formation of new blood vessels requires both polarized cell migration and coordinated control of endothelial cell contacts. Here, Cao and colleagues describe at the sub-cellular level the cytoskeletal and cell junction dynamics regulating these processes upon VEGF-induced cell elongation.

Folding and packing of membrane proteins are highly influenced by the lipidic component of the membrane. Here, we explore how the hydrophobic mismatch (the difference between the hydrophobic span of a transmembrane protein region and the hydrophobic thickness of the lipid membrane around the protein) influences transmembrane helix packing in a cellular environment. Using a ToxRED assay in Escherichia coli and a Bimolecular Fluorescent Complementation approach in human-derived cells complemented by atomistic molecular dynamics simulations we analyzed the dimerization of Glycophorin A derived transmembrane segments. We concluded that, biological membranes can accommodate transmembrane homo-dimers with a wide range of hydrophobic lengths. Hydrophobic mismatch and its effects on dimerization are found to be considerably weaker than those previously observed in model membranes, or under in vitro conditions, indicating that biological membranes (particularly eukaryotic membranes) can adapt to structural deformations through compensatory mechanisms that emerge from their complex structure and composition to alleviate membrane stress. Results based on atomistic simulations support this view, as they revealed that Glycophorin A dimers remain stable, despite of poor hydrophobic match, using mechanisms based on dimer tilting or local membrane thickness perturbations. Furthermore, hetero-dimers with large length disparity between their monomers are also tolerated in cells, and the conclusions that one can draw are essentially similar to those found with homo-dimers. However, large differences between transmembrane helices length hinder the monomer/dimer equilibrium, confirming that, the hydrophobic mismatch has, nonetheless, biologically relevant effects on helix packing in vivo. publishedVersion publishedVersion

A preexisting intradermal nevus (IDN) on the chest of a 30-year-old patient changed compared with her baseline total body photography. Clinical examination found a 6-mm shiny pink papule (Fig 1, inset). Clinical differential diagnosis included irritated IDN, basal cell carcinoma (BCC), nodular melanoma, nevoid melanoma, and melanoma arising in a nevus.

The vitamin D receptor (VDR) must be relevant to liver lipid metabolism because VDR deficient mice are protected from hepatosteatosis. Therefore, our objective was to define the role of VDR on the overall lipid metabolism in human hepatocytes. We developed an adenoviral vector for human VDR and performed transcriptomic and metabolomic analyses of cultured human hepatocytes upon VDR activation by vitamin D (VitD). Twenty percent of the VDR responsive genes were related to lipid metabolism, including MOGAT1, LPGAT1, AGPAT2, and DGAT1 (glycerolipid metabolism), CDS1, PCTP, and MAT1A (phospholipid metabolism), and FATP2, SLC6A12, and AQP3 (uptake of fatty acids, betaine, and glycerol, respectively). They were rapidly induced (4&ndash, 6 h) upon VDR activation by 10 nM VitD or 100 &micro, M lithocholic acid (LCA). Most of these genes were also upregulated by VDR/VitD in mouse livers in vivo. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) metabolomics demonstrated intracellular accumulation of triglycerides, with concomitant decreases in diglycerides and phosphatidates, at 8 and 24 h upon VDR activation. Significant alterations in phosphatidylcholines, increases in lyso-phosphatidylcholines and decreases in phosphatidylethanolamines and phosphatidylethanolamine plasmalogens were also observed. In conclusion, active VitD/VDR signaling in hepatocytes triggers an unanticipated coordinated gene response leading to triglyceride synthesis and to important perturbations in glycerolipids and phospholipids.

Chromosome structure is a crucial regulatory factor for a wide range of nuclear processes. Chromosome conformation capture (3C)-based experiments combined with computational modelling are pivotal for unveiling 3D chromosome structure. Here, we introduce TADdyn, a tool that integrates time-course 3C data, restraint-based modelling, and molecular dynamics to simulate the structural rearrangements of genomic loci in a completely data-driven way. We apply TADdyn on in situ Hi-C time-course experiments studying the reprogramming of murine B cells to pluripotent cells, and characterize the structural rearrangements that take place upon changes in the transcriptional state of 21 genomic loci of diverse expression dynamics. By measuring various structural and dynamical properties, we find that during gene activation, the transcription starting site contacts with open and active regions in 3D chromatin domains. We propose that these 3D hubs of open and active chromatin may constitute a general feature to trigger and maintain gene transcription., Regulation of chromosome structure plays essential roles in many nuclear processes. Here, the authors present TADdyn, a tool that integrates time-course 3C data, restraint-based modelling, and molecular dynamics to simulate the structural rearrangements of genomic loci and find that during gene activation, transcription starting sites contact with open chromatin regions into active physical domains.

The number of synapses present in a neuronal circuit is not fixed. Neurons must compensate for changes in connectivity caused by synaptic pruning, learning processes or pathological conditions through the constant adjustment of the baseline level of neurotransmission. Here, we show that cholinergic neurons grown in an autaptic circuit in the absence of glia sense the loss of half of their synaptic contacts triggered by exposure to peptide p4.2, a C-terminal fragment of SPARC. Synaptic elimination is driven by a reorganization of the periodic F-actin cytoskeleton present along neurites, and occurs without altering the density of postsynaptic receptors. Neurons recover baseline neurotransmission through a homeostatic presynaptic response that consists of the coordinated activation of rapid synapse formation and an overall potentiation of presynaptic calcium influx. These results demonstrate that neurons establishing autaptic connections continuously sense and adjust their synaptic output by tweaking the number of functional contacts and neurotransmitter release probability., Cecilia Velasco and Artur Llobet study how autapses respond to synapse elimination. They employ microisland cultures free of glial cells, treat with a SPARC-derived peptide and show that neurons forming autaptic circuits continuously sense and regulate the number of contacts and neurotransmitter release.

The physical microenvironment regulates cell behavior during tissue development and homeostasis. How single cells decode information about their geometrical shape under mechanical stress and physical space constraints within tissues remains largely unknown. Here, using a zebrafish model, we show that the nucleus, the biggest cellular organelle, functions as an elastic deformation gauge that enables cells to measure cell shape deformations. Inner nuclear membrane unfolding upon nucleus stretching provides physical information on cellular shape changes and adaptively activates a calcium-dependent mechanotransduction pathway, controlling actomyosin contractility and migration plasticity. Our data support that the nucleus establishes a functional module for cellular proprioception that enables cells to sense shape variations for adapting cellular behavior to their microenvironment. Funding: V.V. acknowledges support from the ICFOstepstone PhD Programme funded by the European Union’s Horizon 2020 research and innovation program under Marie Skłodowska-Curie grant agreement 665884. F.P. and Q.T.-R. acknowledge grants funded by Ministerio de Ciencia, Innovación y Universidades and Fondo Social Europeo (FSE) (BES2017-080523-SO, PRE2018-084393). M.A.V. acknowledges support from the Spanish Ministry of Science, Education and Universities through grant RTI2018-099718-B-100 and an institutional “Maria de Maeztu Programme” for Units of Excellence in R&D (CEX2018-000792-M) and FEDER funds.S.W. and M.K. acknowledge support from the Spanish Ministry of Economy and Competitiveness through the Severo Ochoa program for Centres of Excellence in R&D (CEX2019-000910-S), from Fundació Privada Cellex, Fundación Mig-Puig, and from Generalitat de Catalunya through the CERCA program and LaserLab (654148). M.K. acknowledges support through Spanish Ministry of Economy and Competitiveness (RYC-2015-17935, EQC2018-005048-P, AEI-010500-2018-228, and PGC2018-097882-A-I00),Generalitat de Catalunya (2017 SGR 1012), the ERC (715243), and the HFSPO (CDA00023/2018). S.W. acknowledges support through the Spanish Ministry of Economy and Competitiveness via MINECO’s Plan Nacional (PGC2018-098532-A-I00). V.R. acknowledges support from the Spanish Ministry of Science and Innovation to the EMBL partnership, the Centro de Excelencia Severo Ochoa, the CERCA Programme/Generalitat de Catalunya, and the MINECO’s Plan Nacional (BFU2017-86296-P)

Background: The role of the T helper 17 (Th17) cell subset in anti-neutrophil cytoplasm antibodies (ANCA) associated vasculitis (AAV) is controversial. We hypothesized that a specific Th17 response to myeloperoxidase (MPO) or proteinase 3 (PR3) is detectable in AAV patients and is different among the disease phases. Methods: We analyzed 43 AAV patients with renal involvement (21 acute and 22 remission patients), and 12 healthy controls. Peripheral blood mononuclear cells (PBMCs) were cultured with PR3/MPO over 48 h. Thereafter, frequencies of MPO/PR3-specific Th17 cells were assessed using an enzyme-linked immunosorbent spot (ELISpot) assay. Supernatant IL-17 concentration was quantified using ELISA. Finally, specific Th17 response after depletion of T regulatory lymphocytes (T-regs) in some remission patients was compared to the non T-reg-depleted response. Results: Specific Th17 cell number was higher in acute patients compared to remission (p = 0.004). Specific Th17 cell number performed well in the disease activity detection (ROC curve area under the curve (AUC) = 0.87, p = 0.0001) with an optimal cut-off of 6 spots/million. Patients above this cut-off showed higher serum creatinine (p = 0.004), C-reactive protein (CRP) (p = 0.001) and ANCA titer (p = 0.032). Supernatant IL-17 concentration was higher in acute patients compared to remission (p = 0.035) and did not normalize to healthy control levels (p = 0.01). Conclusions: A specific Th17 cell response is present in AAV patients. This response is more pronounced in the acute phase, but persists in remission.

Loss of proteostasis and cellular senescence are key hallmarks of aging, but direct cause-effect relationships are not well understood. We show that most yeast cells arrest in G1 before death with low nuclear levels of Cln3, a key G1 cyclin extremely sensitive to chaperone status. Chaperone availability is seriously compromised in aged cells, and the G1 arrest coincides with massive aggregation of a metastable chaperone-activity reporter. Moreover, G1-cyclin overexpression increases lifespan in a chaperone-dependent manner. As a key prediction of a model integrating autocatalytic protein aggregation and a minimal Start network, enforced protein aggregation causes a severe reduction in lifespan, an effect that is greatly alleviated by increased expression of specific chaperones or cyclin Cln3. Overall, our data show that proteostasis breakdown, by compromising chaperone activity and G1-cyclin function, causes an irreversible arrest in G1, configuring a molecular pathway postulating proteostasis decay as a key contributing effector of cell senescence., This work was funded by the Ministry of Economy and Competitiveness of Spain, Consolider-Ingenio 2010, and the European Union (FEDER) to MA. KJ was supported by the EPSRC Centre for Doctoral Training in Cross-Disciplinary Approaches to Non-Equilibrium Systems (CANES, EP/L015854/1). DFM received an FI fellow of Generalitat de Catalunya.

Oxidative stress; Small RNA sequencing; Neurodegeneration Estrés oxidativo; Secuenciación de ARN pequeño; Neurodegeneración Estrès oxidatiu; Seqüenciació d'ARN petit; Neurodegeneració Small non-coding RNAs (sncRNAs), including microRNAs (miRNAs) are important post-transcriptional gene expression regulators relevant in physiological and pathological processes. Here, we combined a high-throughput functional screening (HTFS) platform with a library of antisense oligonucleotides (ASOs) to systematically identify sncRNAs that affect neuronal cell survival in basal conditions and in response to oxidative stress (OS), a major hallmark in neurodegenerative diseases. We considered hits commonly detected by two statistical methods in three biological replicates. Forty-seven ASOs targeting miRNAs (miRNA-ASOs) consistently decreased cell viability under basal conditions. A total of 60 miRNA-ASOs worsened cell viability impairment mediated by OS, with 36.6% commonly affecting cell viability under basal conditions. In addition, 40 miRNA-ASOs significantly protected neuronal cells from OS. In agreement with cell viability impairment, damaging miRNA-ASOs specifically induced increased free radical biogenesis. miRNAs targeted by the detrimental ASOs are enriched in the fraction of miRNAs downregulated by OS, suggesting that the miRNA expression pattern after OS contributes to neuronal damage. The present HTFS highlighted potentially druggable sncRNAs. However, future studies are needed to define the pathways by which the identified ASOs regulate cell survival and OS response and to explore the potential of translating the current findings into clinical applications. This work was supported by the Spanish Ministry of Economy and Competitiveness and FEDER funds (SAF2014-60551-R and SAF2017-88452-R). We acknowledge the support of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership and the Centro de Excelencia Severo Ochoa 2013-2017 (SEV-2012-0208). We acknowledge the support of the Spanish Ministry of Science Innovation and Universities, Maria Maeztu Unit of Excellence Programme. We thank the staff of the Genomics Unit for the preparation of sRNA libraries and sequencing and the staff of the Biomolecular Screening and Protein Technologies Unit for their help in the setting up the high-throughput screening.

© 2019 Moreno et al., The precise coordination of growth and proliferation has a universal prevalence in cell homeostasis. As a prominent property, cell size is modulated by the coordination between these processes in bacterial, yeast, and mammalian cells, but the underlying molecular mechanisms are largely unknown. Here, we show that multifunctional chaperone systems play a concerted and limiting role in cell-cycle entry, specifically driving nuclear accumulation of the G1 Cdk–cyclin complex. Based on these findings, we establish and test a molecular competition model that recapitulates cell-cycle-entry dependence on growth rate. As key predictions at a single-cell level, we show that availability of the Ydj1 chaperone and nuclear accumulation of the G1 cyclin Cln3 are inversely dependent on growth rate and readily respond to changes in protein synthesis and stress conditions that alter protein folding requirements. Thus, chaperone workload would subordinate Start to the biosynthetic machinery and dynamically adjust proliferation to the growth potential of the cell., This work was funded by the Spanish Ministry of Science, Consolider-Ingenio 2010, and the European Union (FEDER) to M Aldea. DF Moreno received an FI fellowship from Generalitat de Catalunya.

We have generated two human induced pluripotent stem cell (iPSC) lines from CD133+ cells isolated from umbilical cord blood (CB) of a female child using non-integrative Sendai virus. Here we describe the complete characterization of these iPSC lines: PRYDi-CB5 and PRYDi-CB40.

Many key cell processes require prior cell uptake of amino acids from the environment, which is facilitated by cell membrane amino acid transporters such as those of the L-type amino acid transporter (LAT) subfamily. Alterations in LAT subfamily amino acid transport are associated with several human diseases, including cancer, aminoacidurias, and neurodegenerative conditions. Therefore, from the perspective of human health, there is considerable interest in obtaining structural information about these transporter proteins. We recently solved the crystal structure of the first LAT transporter, the bacterial alanine-serine-cysteine exchanger of Carnobacterium sp AT7 (BasC). Here, we provide a complete functional characterization of detergent-purified, liposome-reconstituted BasC transporter to allow the extension of the structural insights into mechanistic understanding. BasC is a sodium- and proton-independent small neutral amino acid exchanger whose substrate and inhibitor selectivity are almost identical to those previously described for the human LAT subfamily member Asc-1. Additionally, we show that, like its human counterparts, this transporter has apparent affinity asymmetry for the intra- and extracellular substrate binding sites—a key feature in the physiological role played by these proteins. BasC is an excellent paradigm of human LAT transporters and will contribute to our understanding of the molecular mechanisms underlying substrate recognition and translocation at both sides of the plasma membrane.

Changes in the equilibrium of pro- and anti-apoptotic members of the B-cell lymphoma-2 (Bcl-2) protein family in the mitochondrial outer membrane (MOM) induce structural changes that commit cells to apoptosis. Bcl-2 homology-3 (BH3)-only proteins participate in this process by either activating pro-apoptotic effectors or inhibiting anti-apoptotic components and by promoting MOM permeabilization. The association of BH3-only proteins with MOMs is necessary for the activation and amplification of death signals; however, the nature of this association remains controversial, as these proteins lack a canonical transmembrane sequence. Here we used an in vitro expression system to study the insertion capacity of hydrophobic C-terminal regions of the BH3-only proteins Bik, Bim, Noxa, Bmf, and Puma into microsomal membranes. An Escherichia coli complementation assay was used to validate the results in a cellular context, and peptide insertions were modeled using molecular dynamics simulations. We also found that some of the C-terminal domains were sufficient to direct green fluorescent protein fusion proteins to specific membranes in human cells, but the domains did not activate apoptosis. Thus, the hydrophobic regions in the C termini of BH3-only members associated in distinct ways with various biological membranes, suggesting that a detailed investigation of the entire process of apoptosis should include studying the membranes as a setting for protein-protein and protein-membrane interactions.

SUMMARY Cells adopt distinct signaling pathways to optimize cell locomotion in different physical microenvironments. However, the underlying mechanism that enables cells to sense and respond to physical confinement is unknown. Using microfabricated devices and substrate-printing methods along with FRET-based biosensors, we report that, as cells transition from unconfined to confined spaces, intracellular Ca2+ level is increased, leading to phosphodiesterase 1 (PDE1)-dependent suppression of PKA activity. This Ca2+ elevation requires Piezo1, a stretch-activated cation channel. Moreover, differential regulation of PKA and cell stiffness in unconfined versus confined cells is abrogated by dual, but not individual, inhibition of Piezo1 and myosin II, indicating that these proteins can independently mediate confinement sensing. Signals activated by Piezo1 and myosin II in response to confinement both feed into a signaling circuit that optimizes cell motility. This study provides a mechanism by which confinement-induced signaling enables cells to sense and adapt to different physical microenvironments., In Brief Hung et al. demonstrate that a Piezo1-dependent intracellular calcium increase negatively regulates protein kinase A (PKA) as cells transit from unconfined to confined spaces. The Piezo1/PKA and myosin II signaling modules constitute two confinement-sensing mechanisms. This study provides a paradigm by which signaling enables cells to sense and adapt to different microenvironments.

Many fungal species are dimorphic, exhibiting both unicellular yeast-like and filamentous forms. Schizosaccharomyces japonicus, a member of the fission yeast clade, is one such dimorphic fungus. Here, we first identify fruit extracts as natural, stress-free, starvation-independent inducers of filamentation, which we use to describe the properties of the dimorphic switch. During the yeast-to-hypha transition, the cell evolves from a bipolar to a unipolar system with 10-fold accelerated polarized growth but constant width, vacuoles segregated to the nongrowing half of the cell, and hyper-lengthening of the cell. We demonstrate unusual features of S. japonicus hyphae: these cells lack a Spitzenkörper, a vesicle distribution center at the hyphal tip, but display more rapid cytoskeleton-based transport than the yeast form, with actin cables being essential for the transition. S. japonicus hyphae also remain mononuclear and undergo complete cell divisions, which are highly asymmetric: one daughter cell inherits the vacuole, the other the growing tip. We show that these elongated cells scale their nuclear size, spindle length, and elongation rates, but display altered division size controls. This establishes S. japonicus as a unique system that switches between symmetric and asymmetric modes of growth and division.

How cells communicate to initiate a regenerative response after damage has captivated scientists during the last few decades. It is known that one of the main signals emanating from injured cells is the Reactive Oxygen Species (ROS), which propagate to the surrounding tissue to trigger the replacement of the missing cells. However, the link between ROS production and the activation of regenerative signaling pathways is not yet fully understood. We describe here the non-autonomous ROS sensing mechanism by which living cells launch their regenerative program. To this aim, we used Drosophila imaginal discs as a model system due to its well-characterized regenerative ability after injury or cell death. We genetically-induced cell death and found that the Apoptosis signal-regulating kinase 1 (Ask1) is essential for regenerative growth. Ask1 senses ROS both in dying and living cells, but its activation is selectively attenuated in living cells by Akt1, the core kinase component of the insulin/insulin-like growth factor pathway. Akt1 phosphorylates Ask1 in a secondary site outside the kinase domain, which attenuates its activity. This modulation of Ask1 activity results in moderate levels of JNK signaling in the living tissue, as well as in activation of p38 signaling, both pathways required to turn on the regenerative response. Our findings demonstrate a non-autonomous activation of a ROS sensing mechanism by Ask1 and Akt1 to replace the missing tissue after damage. Collectively, these results provide the basis for understanding the molecular mechanism of communication between dying and living cells that triggers regeneration., Author summary One of the early events that occur after tissue damage consists on the production of Reactive Oxygen Species (ROS), that signal to the surrounding tissue to initiate wound healing and regeneration. Many signaling pathways, such as JNK and p38, respond to oxidative stress and are necessary for regenerative growth. As the link between ROS and regenerative signaling is not well understood, we decided to explore the mechanism that underlies this process. To do that, we genetically induced cell death in specific areas of Drosophila wing imaginal discs and then studied the mechanism that drives living cells to replace the damage zone until it is completely regenerated. We found that the Drosophila Apoptosis signal-regulating kinase 1 (Ask1), a protein that is sensitive to oxidative stress, is a key player in this scenario. This protein acts as an intracellular sensor that upon damage activates the JNK and p38 regenerative signaling pathways. However, high activity of Ask1 can be toxic for the cell. This is controlled by Akt, a kinase downstream the insulin pathway, which attenuates the activity of Ask1 in the living cells that will participate in the regeneration process. In consequence, Ask1 and Akt act synergistically to respond to the stress generated after tissue damage and drive regeneration. Our results provide a first overview within the framework of how insulin signaling inputs could modulate the capacity to overcome tissue damage.

Background: Red blood cell distribution width (RDW) is a risk factor related to adverse outcome in patients with heart failure (HF). Less is known about its role in patients in their first hospitalization for HF. Aims: Our objective was to investigate the prognostic role of RDW in elderly patients hospitalized for acute HF for the first time. Methods: We reviewed all patients aged 65 years or older admitted to a tertiary-care university hospital with a main diagnosis of acute HF during a 2-year period (January 2013 to December 2014). Patients were divided into 2 groups according to admission RDW values (

Oxidative damage is involved in the pathophysiology of age-related ailments, including Alzheimer’s disease (AD). Studies have shown that the brain tissue and also lymphocytes from AD patients present increased oxidative stress compared to healthy controls (HCs). Here, we use lymphoblastoid cell lines (LCLs) from AD patients and HCs to investigate the role of resveratrol (RV) and selenium (Se) in the reduction of reactive oxygen species (ROS) generated after an oxidative injury. We also studied whether these compounds elicited expression changes in genes involved in the antioxidant cell response and other aging-related mechanisms. AD LCLs showed higher ROS levels than those from HCs in response to H2O2 and FeSO4 oxidative insults. RV triggered a protective response against ROS under control and oxidizing conditions, whereas Se exerted antioxidant effects only in AD LCLs under oxidizing conditions. RV increased the expression of genes encoding known antioxidants (catalase, copper chaperone for superoxide dismutase 1, glutathione S-transferase zeta 1) and anti-aging factors (sirtuin 1 and sirtuin 3) in both AD and HC LCLs. Our findings support RV as a candidate for inducing resilience and protection against AD, and reinforce the value of LCLs as a feasible peripheral cell model for understanding the protective mechanisms of nutraceuticals against oxidative stress in aging and AD., This research was funded by Spanish MINECO and FEDER, grant number SAF2016-75508; Catalan Autonomous Government AGAUR, grant number 2017-SGR-106; and the CERCA Programme/Generalitat de Catalunya.

In this study, we describe the correction of single-point mutations in mammalian cells by repair-polypurine reverse Hoogsteen hairpins (repair-PPRHs). These molecules consist of (1) a PPRH hairpin core that binds to a polypyrimidine target sequence in the double-stranded DNA (dsDNA), producing a triplex structure, and (2) an extension sequence homologous to the DNA sequence to be repaired but containing the wild-type nucleotide instead of the mutation and acting as a donor DNA to correct the mutation. We repaired different point mutations in the adenosyl phosphoribosyl transferase (aprt) gene contained in different aprt-deficient Chinese hamster ovary (CHO) cell lines. Because we had previously corrected mutations in the dihydrofolate reductase (dhfr) gene, in this study, we demonstrate the generality of action of the repair-PPRHs. Repaired cells were analyzed by DNA sequencing, mRNA expression, and enzymatic activity to confirm the correction of the mutation. Moreover, whole-genome sequencing analyses did not detect any off-target effect in the repaired genome. We also performed gel-shift assays to show the binding of the repair-PPRH to the target sequence and the formation of a displacement-loop (D-loop) structure that can trigger a homologous recombination event. Overall, we demonstrate that repair-PPRHs achieve the permanent correction of point mutations in the dsDNA at the endogenous level in mammalian cells without off-target activity., Graphical Abstract

Summary Cell reprogramming is thought to be associated with a full metabolic switch from an oxidative- to a glycolytic-based metabolism. However, neither the dynamics nor the factors controlling this metabolic switch are fully understood. By using cellular, biochemical, protein array, metabolomic, and respirometry analyses, we found that c-MYC establishes a robust bivalent energetics program early in cell reprogramming. Cells prone to undergo reprogramming exhibit high mitochondrial membrane potential and display a hybrid metabolism. We conclude that MYC proteins orchestrate a rewiring of somatic cell metabolism early in cell reprogramming, whereby somatic cells acquire the phenotypic plasticity necessary for their transition to pluripotency in response to either intrinsic or external cues., Graphical Abstract, Highlights • Endogenous MYC biological activity is necessary for cell reprogramming • MYC drives mitochondrial fission early in cell reprogramming • MYC establishes a bivalent energetics state necessary for cell reprogramming • MYC polarizes mitochondria, poising cells for reprogramming, Torres and colleagues describe an MYC-dependent remodeling of mitochondrial dynamics and metabolism during the first stage of cell reprogramming. Endogenous MYC activity was found to be necessary for cell reprogramming, likely by establishing a hybrid metabolic state characterized by elevated glycolytic flux and a somatic oxidative metabolic rate. MYC polarized mitochondria by increasing ATPIF1 and labeled cells prone to cell reprogramming.

We review recent evidence showing that cell and tissue dynamics are governed by mesoscale physical principles. These principles can be understood in terms of simple state diagrams in which control variables include force, density, shape, adhesion and self-propulsion. An appropriate combination of these physical quantities gives rise to emergent phenomena such as cell jamming, topological defects and underdamped waves. Mesoscale physical properties of cell assemblies are found to precede and instruct biological functions such as cell division, extrusion, invasion and gradient sensing. These properties are related to properties of biomolecules, but cannot be predicted from biochemical principles. Thus, biological function is governed by emergent mesoscale states that can be predicted by a simple set of physical properties.

The beneficial effects of estrogen on the cardiovascular system have been reported extensively. In fact, the incidence of cardiovascular diseases in women is lower than in age-matched men during their fertile stage of life, a benefit that disappears after menopause. These sex-related differences point to sexual hormones, mainly estrogen, as possible cardiovascular protective factors. The regulation of vascular function by estrogen is mainly related to the maintenance of normal endothelial function and is mediated by both direct and indirect gene transcription through the activity of specific estrogen receptors. Some of these mechanisms are known, but many remain to be elucidated. In recent years, microRNAs have been established as non-coding RNAs that regulate the expression of a high percentage of protein-coding genes in mammals and are related to the correct function of human physiology. Moreover, within the cardiovascular system, miRNAs have been related to physiological and pathological conditions. In this review, we address what is known about the role of estrogen-regulated miRNAs and their emerging involvement in vascular biology.

Single-cell RNA sequencing significantly deepened our insights into complex tissues and latest techniques are capable processing ten-thousands of cells simultaneously. With bigSCale, we provide an analytical framework being scalable to analyze millions of cells, addressing challenges of future large datasets. Unlike previous methods, bigSCale does not constrain data to fit an a priori-defined distribution and instead uses an accurate numerical model of noise. We evaluated the performance of bigSCale using a biological model of aberrant gene expression in patient derived neuronal progenitor cells and simulated datasets, which underlined its speed and accuracy in differential expression analysis. We further applied bigSCale to analyze 1.3 million cells from the mouse developing forebrain. Herein, we identified rare populations, such as Reelin positive Cajal-Retzius neurons, for which we determined a previously not recognized heterogeneity associated to distinct differentiation stages, spatial organization and cellular function. Together, bigSCale presents a perfect solution to address future challenges of large single-cell datasets.Extended AbstractSingle-cell RNA sequencing (scRNAseq) significantly deepened our insights into complex tissues by providing high-resolution phenotypes for individual cells. Recent microfluidic-based methods are scalable to ten-thousands of cells, enabling an unbiased sampling and comprehensive characterization without prior knowledge. Increasing cell numbers, however, generates extremely big datasets, which extends processing time and challenges computing resources. Current scRNAseq analysis tools are not designed to analyze datasets larger than from thousands of cells and often lack sensitivity and specificity to identify marker genes for cell populations or experimental conditions. With bigSCale, we provide an analytical framework for the sensitive detection of population markers and differentially expressed genes, being scalable to analyze millions of single cells. Unlike other methods that use simple or mixture probabilistic models with negative binomial, gamma or Poisson distributions to handle the noise and sparsity of scRNAseq data, bigSCale does not constrain the data to fit an a priori-defined distribution. Instead, bigSCale uses large sample sizes to estimate a highly accurate and comprehensive numerical model of noise and gene expression. The framework further includes modules for differential expression (DE) analysis, cell clustering and population marker identification. Moreover, a directed convolution strategy allows processing of extremely large data sets, while preserving the transcript information from individual cells.We evaluate the performance of bigSCale using a biological model for reduced or elevated gene expression levels. Specifically, we perform scRNAseq of 1,920 patient derived neuronal progenitor cells from Williams-Beuren and 7q11.23 microduplication syndrome patients, harboring a deletion or duplication of 7q11.23, respectively. The affected region contains 28 genes whose transcriptional levels vary in line with their allele frequency. BigSCale detects expression changes with respect to cells from a healthy donor and outperforms other methods for single-cell DE analysis in sensitivity. Simulated data sets, underline the performance of bigSCale in DE analysis as it is faster and more sensitive and specific than other methods. The probabilistic model of cell-distances within bigSCale is further suitable for unsupervised clustering and the identification of cell types and subpopulations. Using bigSCale, we identify all major cell types of the somatosensory cortex and hippocampus analyzing 3,005 cells from adult mouse brains. Remarkably, we increase the number of cell population specific marker genes 4-6-fold compared to the original analysis and, moreover, define markers of higher order cell types. These include CD90 (Thy1), a neuronal surface receptor, potentially suitable for isolating intact neurons from complex brain samples.To test its applicability for large data sets, we apply bigSCale on scRNAseq data from 1.3 million cells derived from the pallium of the mouse developing forebrain (E18, 10x Genomics). Our directed down-sampling strategy accumulates transcript counts from cells with similar transcriptional profiles into index cell transcriptomes, thereby defining cellular clusters with improved resolution. Accordingly, index cell clusters provide a rich resource of marker genes for the main brain cell types and less frequent subpopulations. Our analysis of rare populations includes poorly characterized developmental cell types, such as neuron progenitors from the subventricular zone and neocortical Reelin positive neurons known as Cajal-Retzius (CR) cells. The latter represent a transient population which regulates the laminar formation of the developing neocortex and whose malfunctioning causes major neurodevelopmental disorders like autism or schizophrenia. Most importantly, index cell cluster can be deconvoluted to individual cell level for targeted analysis of populations of interest. Through decomposition of Reelin positive neurons, we determined a previously not recognized heterogeneity among CR cells, which we could associate to distinct differentiation stages as well as spatial and functional differences in the developing mouse brain. Specifically, subtypes of CR cells identified by bigSCale express different compositions of NMDA, AMPA and glycine receptor subunits, pointing to subpopulations with distinct membrane properties. Furthermore, we found Cxcl12, a chemokine secreted by the meninges and regulating the tangential migration of CR cells, to be also expressed in CR cells located in the marginal zone of the neocortex, indicating a self-regulated migration capacity.Together, bigSCale presents a perfect solution for the processing and analysis of scRNAseq data from millions of single cells. Its speed and sensitivity makes it suitable to the address future challenges of large single-cell data sets.

Cells exert, sense, and respond to physical forces through an astounding diversity of mechanisms. Here we review recently developed tools to quantify the forces generated by cells. We first review technologies based on sensors of known or assumed mechanical properties, and discuss their applicability and limitations. We then proceed to draw an analogy between these human-made sensors and force sensing in the cell. As mechanics is increasingly revealed to play a fundamental role in cell function we envisage that tools to quantify physical forces may soon become widely applied in life-sciences laboratories.

Includes supplementary materials for the online appendix. Many eukaryotic genes contain embedded antisense transcripts and repetitive sequences of unknown function. We report that male germline-specific expression of an antisense transcript contained in an intron of C. elegans Target of Rapamycin (TOR, let-363) is associated with (1) accumulation of endo-small interfering RNAs (siRNAs) against an embedded Helitron transposon and (2) activation of an alternative 3′ splice site of TOR. The germline-specific Argonaute proteins PRG-1 and CSR-1, which participate in self/nonself RNA recognition, antagonistically regulate the generation of these endo-siRNAs, TOR mRNA levels, and 3′ splice-site selection. Supply of exogenous double-stranded RNA against the region of sense/antisense overlap reverses changes in TOR expression and splicing and suppresses the progressive multigenerational sterility phenotype of prg-1 mutants. We propose that recognition of a “nonself” intronic transposon by endo-siRNAs/the piRNA system provides physiological regulation of expression and alternative splicing of a host gene that, in turn, contributes to the maintenance of germline function across generations. We thank A. Fire, S. Ahmed, and E. Miska for sharing information and helpful discussions, members of our laboratories for critical feedback, the Caenorhabditis Genetics Center (CGC) for providing strains, Matthew Ragle for collection of sperm samples, Alan Zahler for assistance and critical feedback, and Montserrat Porta de la Riva for technical assistance. S.B.-S. was supported by an EMBO postdoctoral fellowship. A.T.L. is a Taub Fellow, supported by the Taub Foundation. J.C. is a Miguel Servet Junior Investigator. Work in J.V.’s lab is supported by Fundación Botín, Consolider RNAREG and Ministerio de Economía y Competitividad (MINECO). Work in B.L.’s lab is supported by the European Research Council (ERC), MINECO Plan Nacional grants BFU2008-00365 and BFU2011-26206, ERASysBio+ ERANET project EUI2009-04059 GRAPPLE, the EMBO Young Investigator Program, EU Framework 7 project 277899 4DCellFate, the AXA Research Fund, and the EMBL/CRG Systems Biology Program. Support from the CRG Genomics Facility is also acknowledged.

Background: Tracing stable isotopes, such as 13C using various mass spectrometry (MS) methods provides a valuable information necessary for the study of biochemical processes in cells. However, extracting such information requires special care, such as a correction for naturally occurring isotopes, or overlapping mass spectra of various components of the cell culture medium. Developing a method for a correction of overlapping peaks is the primary objective of this study.Results: Our computer program-MIDcor (free at https://github.com/seliv55/mid_correct) written in the R programming language, corrects the raw MS spectra both for the naturally occurring isotopes and for the overlapping of peaks corresponding to various substances. To this end, the mass spectra of unlabeled metabolites measured in two media are necessary: in a minimal medium containing only derivatized metabolites and chemicals for derivatization, and in a complete cell incubated medium. The MIDcor program calculates the difference (D) between the theoretical and experimentally measured spectra of metabolites containing only the naturally occurring isotopes. The result of comparison of D in the two media determines a way of deciphering the true spectra. (1) If D in the complete medium is greater than that in the minimal medium in at least one peak, then unchanged D is subtracted from the raw spectra of the labeled metabolite. (2) If D does not depend on the medium, then the spectrum probably overlaps with a derivatized fragment of the same metabolite, and D is modified proportionally to the metabolite labeling. The program automatically reaches a decision regarding the way of correction. For some metabolites/fragments in the case (2) D was found to decrease when the tested substance was 13C labeled, and this isotopic effect also can be corrected automatically, if the user provides a measured spectrum of the substance in which the 13C labeling is known a priori.Conclusion: Using the developed program improves the reliability of stable isotope tracer data analysis.

Human CMT2-FiPS4F1 cell line was generated from fibroblasts of a patient with Charcot-Marie-Tooth disease harbouring the following mutations in the GDAP1 gene in heterozygosis: p.Q163X/p.T288NfsX3. This patient did not present mutations in the PM22, MPZ or GJB genes. Human reprogramming factors OCT3/4, KLF4, SOX2 and C-MYC were delivered using a non-integrative methodology that involves the use of Sendai virus.

Ribosome assembly requires the concerted expression of hundreds of genes, which are transcribed by all three nuclear RNA polymerases. Transcription elongation involves dynamic interactions between RNA polymerases and chromatin. We performed a synthetic lethal screening in Saccharomyces cerevisiae with a conditional allele of SPT6, which encodes one of the factors that facilitates this process. Some of these synthetic mutants corresponded to factors that facilitate pre-rRNA processing and ribosome biogenesis. We found that the in vivo depletion of one of these factors, Arb1, activated transcription elongation in the set of genes involved directly in ribosome assembly. Under these depletion conditions, Spt6 was physically targeted to the upregulated genes, where it helped maintain their chromatin integrity and the synthesis of properly stable mRNAs. The mRNA profiles of a large set of ribosome biogenesismutants confirmed the existence of a feedback regulatory network among ribosome assembly genes. The transcriptional response in this network depended on both the specific malfunction and the role of the regulated gene. In accordance with our screening, Spt6 positively contributed to the optimal operation of this global network. On the whole, this work uncovers a feedback control of ribosome biogenesis by fine-tuning transcription elongation in ribosome assembly factor-coding genes. Ministerio de Economía y Competitividad BFU2013-48643-C3-1-P, BFU2016-77728-C3-1-P, BFU2013-48643-C3- 3-P, BFU2013-42958-P Junta de Andalucía P12-BIO1938MO, P08-CVI-03508 Comunidad Valenciana 2015/006

The cyclin-dependent kinase inhibitor p27Kip1 (p27) also behaves as a transcriptional repressor. Data showing that the p300/CBP-associated factor (PCAF) acetylates p27 inducing its degradation suggested that PCAF and p27 could collaborate in the regulation of transcription. However, this possibility remained to be explored. We analyzed here the transcriptional programs regulated by PCAF and p27 in the colon cancer cell line HCT116 by chromatin immunoprecipitation sequencing (ChIP-seq). We identified 269 protein-encoding genes that contain both p27 and PCAF binding sites being the majority of these sites different for PCAF and p27. PCAF or p27 knock down revealed that both regulate the expression of these genes, PCAF as an activator and p27 as a repressor. The double knock down of PCAF and p27 strongly reduced their expression indicating that the activating role of PCAF overrides the repressive effect of p27. We also observed that the transcription factor Pax5 interacts with both p27 and PCAF and that the knock down of Pax5 induces the expression of p27/PCAF target genes indicating that it also participates in the transcriptional regulation mediated by p27/PCAF. In summary, we report here a previously unknown mechanism of transcriptional regulation mediated by p27, Pax5 and PCAF., Ministerio de Economía y Competitividad (MINECO) [SAF2012-38078 to O.B. and BFU2015-65311-R to R.A.]; Instituto de Salud Carlos III [RD12/0036/0054 to O.B. and CB16/12/00244 (CIBERONC) to A.B.]; ‘La Caixa’ Foundation 2016-052407 (to R.A.). Funding for open access charge: MInisterio the Economia y Competitividad, Spain La Caixa Foundation. Spain.

Experimental studies on isolated cardiomyocytes from different animal species and human hearts have demonstrated that there are regional differences in the Ca2+ release, Ca2+ decay and sarcomere deformation. Local deformation heterogeneities can occur due to a combination of factors: regional/local differences in Ca2+ release and/or re-uptake, intra-cellular material properties, sarcomere proteins and distribution of the intracellular organelles. To investigate the possible causes of these heterogeneities, we developed a two-dimensional finite-element electromechanical model of a cardiomyocyte that takes into account the experimentally measured local deformation and cytosolic [Ca2+] to locally define the different variables of the constitutive equations describing the electro/mechanical behaviour of the cell. Then, the model was individualised to three different rat cardiac cells. The local [Ca2+] transients were used to define the [Ca2+]-dependent activation functions. The cell-specific local Young’s moduli were estimated by solving an inverse problem, minimizing the error between the measured and simulated local deformations along the longitudinal axis of the cell. We found that heterogeneities in the deformation during contraction were determined mainly by the local elasticity rather than the local amount of Ca2+, while in the relaxation phase deformation was mainly influenced by Ca2+ re-uptake. Our electromechanical model was able to successfully estimate the local elasticity along the longitudinal direction in three different cells. In conclusion, our proposed model seems to be a good approximation to assess the heterogeneous intracellular mechanical properties to help in the understanding of the underlying mechanisms of cardiomyocyte dysfunction. This study was partly supported by grants from Ministerio de Economia y Competitividad (ref. SAF2012-37196, TIN2014-52923-R); the Instituto de Salud Carlos III (ref. PI11/01709, PI14/00226) integrado en el Plan Nacional de I+D+I y cofinanciado por el ISCIII-Subdirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER) “Otra manera de hacer Europa”; the EU FP7 for research, technological development and demonstration under grant agreement VP2HF (n° 611823); The Cerebra Foundation for the Brain Injured Child (Carmarthen, Wales, UK); Obra Social “la Caixa” (Barcelona, Spain); Fundació Mutua Madrileña; Fundació Agrupació Mutua (Spain) and AGAUR 2014 SGR grant n° 928 (Barcelona, Spain). P.G.C. was supported by the Programa de Ayudas Predoctorales de Formación en investigación en Salud (FI12/00362) from the Instituto Carlos III, Spain. P.G.C wants to acknowledge to Boehringer Ingelhiem Fonds for the travel grant to do her research stay at LaBS group in Politecnico di Milano.

During cell migration, the cell edge undergoes periodic protrusion–retraction cycles. Quantitative analyses of the forces at the cell edge that drive these cycles are provided. We show that α-actinin links local contractile units and the global actin flow forces at the cell edge and present a novel model based on these results., During spreading and migration, the leading edges of cells undergo periodic protrusion–retraction cycles. The functional purpose of these cycles is unclear. Here, using submicrometer polydimethylsiloxane pillars as substrates for cell spreading, we show that periodic edge retractions coincide with peak forces produced by local contractile units (CUs) that assemble and disassemble along the cell edge to test matrix rigidity. We find that, whereas actin rearward flow produces a relatively constant force inward, the peak of local contractile forces by CUs scales with rigidity. The cytoskeletal protein α-actinin is shared between these two force-producing systems. It initially localizes to the CUs and subsequently moves inward with the actin flow. Knockdown of α-actinin causes aberrant rigidity sensing, loss of CUs, loss of protrusion–retraction cycles, and, surprisingly, enables the cells to proliferate on soft matrices. We present a model based on these results in which local CUs drive rigidity sensing and adhesion formation.

Summary Induced pluripotent stem cells (iPSCs) are a powerful tool for disease modeling. They are routinely generated from healthy donors and patients from multiple cell types at different developmental stages. However, reprogramming leukemias is an extremely inefficient process. Few studies generated iPSCs from primary chronic myeloid leukemias, but iPSC generation from acute myeloid or lymphoid leukemias (ALL) has not been achieved. We attempted to generate iPSCs from different subtypes of B-ALL to address the developmental impact of leukemic fusion genes. OKSM(L)-expressing mono/polycistronic-, retroviral/lentiviral/episomal-, and Sendai virus vector-based reprogramming strategies failed to render iPSCs in vitro and in vivo. Addition of transcriptomic-epigenetic reprogramming “boosters” also failed to generate iPSCs from B cell blasts and B-ALL lines, and when iPSCs emerged they lacked leukemic fusion genes, demonstrating non-leukemic myeloid origin. Conversely, MLL-AF4-overexpressing hematopoietic stem cells/B progenitors were successfully reprogrammed, indicating that B cell origin and leukemic fusion gene were not reprogramming barriers. Global transcriptome/DNA methylome profiling suggested a developmental/differentiation refractoriness of MLL-rearranged B-ALL to reprogramming into pluripotency., Graphical Abstract, Highlights • Neither primary B-ALL blasts nor leukemic B cell lines can be reprogrammed to iPSCs • Global transcriptome and DNA methylome suggest a developmental refractoriness, Despite the interest in generating iPSCs from human primary acute leukemias for disease modeling, reprogramming leukemias is an extremely inefficient process. In this article, Menéndez, Bueno, and colleagues show that many reprogramming strategies reported to date are not sufficient to generate B-ALL-derived iPSCs. Global transcriptome/DNA methylome profiling suggested a developmental/differentiation refractoriness of B-ALL to reprogramming into pluripotency.

Podeu consultar dades primàries associades a l'article a: http://hdl.handle.net/2445/102243, Background. White adipose tissue (WAT) is a complex, diffuse, multifunctional organ which contains adipocytes, and a large proportion of fat, but also other cell types, active in defense, regeneration and signalling functions. Studies with adipocytes often require their isolation from WAT by breaking up the matrix of collagen fibres; however, it is unclear to what extent adipocyte number in primary cultures correlates with their number in intact WAT, since recovery and viability are often unknown. Experimental Design. Epididymal WAT of four young adult rats was used to isolate adipocytes with collagenase. Careful recording of lipid content of tissue, and all fraction volumes and weights, allowed us to trace the amount of initial WAT fat remaining in the cell preparation. Functionality was estimated by incubation with glucose and measurement of glucose uptake and lactate, glycerol and NEFA excretion rates up to 48 h. Non-adipocyte cells were also recovered and their sizes (and those of adipocytes) were measured. The presence of non-nucleated cells (erythrocytes) was also estimated. Results. Cell numbers and sizes were correlated from all fractions to intact WAT. Tracing the lipid content, the recovery of adipocytes in the final, metabolically active, preparation was in the range of 70 75%. Cells showed even higher metabolic activity in the second than in the first day of incubation. Adipocytes were 7%, erythrocytes 66% and other stromal (nucleated cells) 27% of total WAT cells. However, their overall volumes were 90%, 0.05%, and 0.2% of WAT. Non-fat volume of adipocytes was 1.3% of WAT. Conclusions. The methodology presented here allows for a direct quantitative reference to the original tissue of studies using isolated cells. We have also found that the ``live cell mass'' of adipose tissue is very small: about 13 mL/g for adipocytes and 2 mL/g stromal, plus about 1 mL/g blood (the rats were killed by exsanguination). These data translate (with respect to the actual ``live cytoplasm'' size) into an extremely high metabolic activity, which make WAT an even more significant agent in the control of energy metabolism.

Cell size scales with ploidy in a great range of eukaryotes, but the underlying mechanisms remain unknown. Using various orthogonal single-cell approaches, we show that cell size increases linearly with centromere (CEN) copy number in budding yeast. This effect is due to a G1 delay mediated by increased degradation of Cln3, the most upstream G1 cyclin acting at Start, and specific centromeric signaling proteins, namely Mad3 and Bub3. Mad3 binds both Cln3 and Cdc4, the adaptor component of the Skp1/Cul1/F-box (SCF) complex that targets Cln3 for degradation, these interactions being essential for the CEN-dosage dependent effects on cell size. Our results reveal a pathway that modulates cell size as a function of CEN number, and we speculate that, in cooperation with other CEN-independent mechanisms, it could assist the cell to attain efficient mass/ploidy ratios, Ministry of Economy and Competitiveness of Spain (grant number BFU2016-80234-R to MA and BFU 2013-44189-P to JC). JMM-L and DFM acknowledge fellowhips of the FI program of Generalitat de Catalunya. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Bioelectrical signals and ion channels are central to spatial patterns in cell ensembles, a problem of fundamental interest in positional information and cancer processes. We propose a model for electrically connected cells based on simple biological concepts: i) the membrane potential of a single cell characterizes its electrical state; ii) the long-range electrical coupling of the multicellular ensemble is realized by a network of gap junction channels between neighboring cells; and iii) the spatial distribution of an external biochemical agent can modify the conductances of the ion channels in a cell membrane and the multicellular electrical state. We focus on electrical effects in small multicellular ensembles, ignoring slow diffusional processes. The spatio-temporal patterns obtained for the local map of cell electric potentials illustrate the normalization of regions with abnormal cell electrical states. The effects of intercellular coupling and blocking of specific channels on the electrical patterns are described. These patterns can regulate the electrically-induced redistribution of charged nanoparticles over small regions of a model tissue. The inclusion of bioelectrical signals provides new insights for the modeling of cancer biophysics because collective multicellular states show electrical coupling mechanisms that are not readily deduced from biochemical descriptions at the individual cell level.

Nucleosomes provide additional regulatory mechanisms to transcription and DNA replication by mediating the access of proteins to DNA. During the cell cycle chromatin undergoes several conformational changes, however the functional significance of these changes to cellular processes are largely unexplored. Here, we present the first comprehensive genome-wide study of nucleosome plasticity at single base-pair resolution along the cell cycle in Saccharomyces cerevisiae. We determined nucleosome organization with a specific focus on two regulatory regions: transcription start sites (TSSs) and replication origins (ORIs). During the cell cycle, nucleosomes around TSSs display rearrangements in a cyclic manner. In contrast to gap (G1 and G2) phases, nucleosomes have a fuzzier organization during S and M phases, Moreover, the choreography of nucleosome rearrangements correlate with changes in gene expression during the cell cycle, indicating a strong association between nucleosomes and cell cycle-dependent gene functionality. On the other hand, nucleosomes are more dynamic around ORIs along the cell cycle, albeit with tighter regulation in early firing origins, implying the functional role of nucleosomes on replication origins. Our study provides a dynamic picture of nucleosome organization throughout the cell cycle and highlights the subsequent impact on transcription and replication activity., This work was supported by the Spanish Ministry of Science and Innovation (BIO2012- 32868), Instituto de Salud Carlos III (INB) and the European Research Council (SimDNA project)

Spatial self-organization emerges in distributed systems exhibiting local interactions when nonlinearities and the appropriate propagation of signals are at work. These kinds of phenomena can be modeled with different frameworks, typically cellular automata or reaction-diffusion systems. A different class of dynamical processes involves the correlated movement of agents over space, which can be mediated through chemotactic movement or minimization of cell-cell interaction energy. A classic example of the latter is given by the formation of spatially segregated assemblies when cells display differential adhesion. Here, we consider a new class of dynamical models, involving cell adhesion among two stochastically exchangeable cell states as a minimal model capable of exhibiting well-defined, ordered spatial patterns. Our results suggest that a whole space of pattern-forming rules is hosted by the combination of physical differential adhesion and the value of probabilities modulating cell phenotypic switching, showing that Turing-like patterns can be obtained without resorting to reaction-diffusion processes. If the model is expanded allowing cells to proliferate and die in an environment where diffusible nutrient and toxic waste are at play, different phases are observed, characterized by regularly spaced patterns. The analysis of the parameter space reveals that certain phases reach higher population levels than other modes of organization. A detailed exploration of the mean-field theory is also presented. Finally, we let populations of cells with different adhesion matrices compete for reproduction, showing that, in our model, structural organization can improve the fitness of a given cell population. The implications of these results for ecological and evolutionary models of pattern formation and the emergence of multicellularity are outlined., This work has been supported by the Botín Foundation by Banco Santander through its Santander Universities Global Division, a MINECO fellowship and by the Santa Fe Institute.

Phenothiazine derivatives are non-antibiotics with antimicrobial, fungistatic and fungicidal effects. We exposed to a high energy UV laser beam phenothiazines solutions in water at 20mg/mL concentration to increase antibacterial activity of resulting mixtures. Compared to previous results obtained on bacteria, more research is needed about UV laser irradiated phenothiazines applications on cancer cell cultures to evidence possible anticancerous properties. Evaluation of the safety of the newly obtained photoproducts in view of use on humans is also needed. Due to expensive animal testing in toxicology and pressure from general public and governments to develop alternatives to in vivo testing, in vitro cell-based models are attractive for preliminary testing of new materials. Cytotoxicity screening reported here shows that laser irradiated (4h exposure time length) chlorpromazine and promazine are more efficient against some cell cultures. Interaction of laser irradiated phenothiazines with fabrics show that promethazine and chlorpromazine have improved wetting properties. Correlation of these two groups of properties shows that chlorpromazine appears to be more recommended for applications on tissues using fabrics as transport vectors. The reported results concern stability study of phenothiazines water solutions to know the time limits within which they are stable and may be used. Keywords: Culture cells; Fabrics; Hemolysis; In vitro cytotoxicity; Laser; Phenothiazines.

Reactive lymph nodes (LNs) are sites where pMHC-loaded dendritic cells (DCs) interact with rare cognate T cells, leading to their clonal expansion. While DC interactions with T cell subsets critically shape the ensuing immune response, surprisingly little is known on their spatial orchestration at physiologically T cell low precursor frequencies. Light sheet fluorescence microscopy and one of its implementations, selective plane illumination microscopy (SPIM), is a powerful method to obtain precise spatial information of entire organs of 0.5-10mm diameter, the size range of murine LNs. Yet, its usefulness for immunological research has thus far not been comprehensively explored. Here, we have tested and defined protocols that preserve fluorescent protein function during lymphoid tissue clearing required for SPIM. Reconstructions of SPIM-generated 3D data sets revealed that calibrated numbers of adoptively transferred T cells and DCs are successfully detected at a single cell level within optically cleared murine LNs. Finally, we define parameters to quantify specific interactions between antigen-specific T cells and pMHC-bearing DCs in murine LNs. In sum, our studies describe the successful application of light sheet fluorescence microscopy to immunologically relevant tissues. This work was funded by Swiss National Foundation grant 31003A_135649 (to JVS), Sinergia grant CR23I3_156234 and CRSII3_141918 (to JSh and JVS), EC FP7 Marie Curie RG grant 276702 (to JVS), and a Novartis Research (NRG-2011-0341) grant (to AO). JA was supported by Postdoctoral Fellowship for Research Abroad from Japan Society for the Promotion of Science. JR acknowledges support from the EC FP7 CIG grant HIGH-THROUGHPUT TOMO, and MINECO grant FIS2013-41802-R MESO-IMAGING. JSw and JSh acknowledge support of the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013-2017’, SEV-2012-0208. The SPIM setup was financed by a grant of the Pierre Mercier Foundation.

During the process of reprogramming to induced pluripotent stem (iPS) cells, somatic cells switch from oxidative to glycolytic metabolism, a transition associated with profound mitochondrial reorganization. Neither the importance of mitochondrial remodelling for cell reprogramming, nor the molecular mechanisms controlling this process are well understood. Here, we show that an early wave of mitochondrial fragmentation occurs upon expression of reprogramming factors. Reprogramming-induced mitochondrial fission is associated with a minor decrease in mitochondrial mass but not with mitophagy. The pro-fission factor Drp1 is phosphorylated early in reprogramming, and its knockdown and inhibition impairs both mitochondrial fragmentation and generation of iPS cell colonies. Drp1 phosphorylation depends on Erk activation in early reprogramming, which occurs, at least in part, due to downregulation of the MAP kinase phosphatase Dusp6. Taken together, our data indicate that mitochondrial fission controlled by an Erk-Drp1 axis constitutes an early and necessary step in the reprogramming process to pluripotency., Reprogramming of somatic cells is a stepwise process where cells must overcome several barriers before reaching the pluripotent state. Here the authors show that mitochondrial fission in response to ERK1/2 signalling is an important early step during reprogramming to pluripotency.