Priming by Chemokines Restricts Lateral Mobility of the Adhesion Receptor LFA-1 and Restores Adhesion to ICAM-1 Nano-Aggregates on Human Mature Dendritic Cells

LFA-1 is a leukocyte specific β2 integrin that plays a major role in regulating adhesion and migration of different immune cells. Recent data suggest that LFA-1 on mature dendritic cells (mDCs) may function as a chemokine-inducible anchor during homing of DCs through the afferent lymphatics into the lymph nodes, by transiently switching its molecular conformational state. However, the role of LFA-1 mobility in this process is not yet known, despite that the importance of lateral organization and dynamics for LFA-1-mediated adhesion regulation is broadly recognized. Using single particle tracking approaches we here show that LFA-1 exhibits higher mobility on resting mDCs compared to monocytes. Lymphoid chemokine CCL21 stimulation of the LFA-1 high affinity state on mDCs, led to a significant reduction of mobility and an increase on the fraction of stationary receptors, consistent with re-activation of the receptor. Addition of soluble monomeric ICAM-1 in the presence of CCL21 did not alter the diffusion profile of LFA-1 while soluble ICAM-1 nano-aggregates in the presence of CCL21 further reduced LFA-1 mobility and readily bound to the receptor. Overall, our results emphasize the importance of LFA-1 lateral mobility across the membrane on the regulation of integrin activation and its function as adhesion receptor. Importantly, our data show that chemokines alone are not sufficient to trigger the high affinity state of the integrin based on the strict definition that affinity refers to the adhesion capacity of a single receptor to its ligand in solution. Instead our data indicate that nanoclustering of the receptor, induced by multi-ligand binding, is required to maintain stable cell adhesion once LFA-1 high affinity state is transiently triggered by inside-out signals.Peer ReviewedPostprint (published version

Growth disrupting mutations in epigenetic regulatory molecules are associated with abnormalities of epigenetic aging.

Germline mutations in fundamental epigenetic regulatory molecules including DNA methyltransferase 3 alpha (DNMT3A) are commonly associated with growth disorders, whereas somatic mutations are often associated with malignancy. We profiled genome-wide DNA methylation patterns in DNMT3A c.2312G > A; p.(Arg771Gln) carriers in a large Amish sibship with Tatton-Brown-Rahman syndrome (TBRS), their mosaic father, and 15 TBRS patients with distinct pathogenic de novo DNMT3A variants. This defined widespread DNA hypomethylation at specific genomic sites enriched at locations annotated as genes involved in morphogenesis, development, differentiation, and malignancy predisposition pathways. TBRS patients also displayed highly accelerated DNA methylation aging. These findings were most marked in a carrier of the AML-associated driver mutation p.Arg882Cys. Our studies additionally defined phenotype-related accelerated and decelerated epigenetic aging in two histone methyltransferase disorders: NSD1 Sotos syndrome overgrowth disorder and KMT2D Kabuki syndrome growth impairment. Together, our findings provide fundamental new insights into aberrant epigenetic mechanisms, the role of epigenetic machinery maintenance, and determinants of biological aging in these growth disorders

The psychic costs of migration: evidence from Irish return migrants

Within the economics literature, the 'psychic costs' of migration have been incorporated into theoretical models since Sjaastad (J Polit Econ 70:80 93, 1962). However, the existence of such costs has rarely been investigated in empirical papers. In this paper, we look at the psychic costs of migration by using alcohol problems as an indicator. Rather than comparing immigrants and natives, we look at the native-born in a single country and compare those who have lived away for a period of their lives and those who have not. We use data from the first wave of the Irish Longitudinal Study on Ageing which is a large, nationally representative sample of older Irish adults. We find that men who lived away are more likely to have suffered from alcohol problems than men who stayed. For women, we again see a higher incidence of alcohol problems for short-term migrants. However, long-term female migrants are less likely to have suffered from alcohol problems. For these women, it seems that migration provided psychic benefits, and this is consistent with findings fro

Direct Ubiquitin Independent Recognition and Degradation of a Folded Protein by the Eukaryotic Proteasomes-Origin of Intrinsic Degradation Signals

Eukaryotic 26S proteasomes are structurally organized to recognize, unfold and degrade globular proteins. However, all existing model substrates of the 26S proteasome in addition to ubiquitin or adaptor proteins require unstructured regions in the form of fusion tags for efficient degradation. We report for the first time that purified 26S proteasome can directly recognize and degrade apomyoglobin, a globular protein, in the absence of ubiquitin, extrinsic degradation tags or adaptor proteins. Despite a high affinity interaction, absence of a ligand and presence of only helices/loops that follow the degradation signal, apomyoglobin is degraded slowly by the proteasome. A short floppy F-helix exposed upon ligand removal and in conformational equilibrium with a disordered structure is mandatory for recognition and initiation of degradation. Holomyoglobin, in which the helix is buried, is neither recognized nor degraded. Exposure of the floppy F-helix seems to sensitize the proteasome and primes the substrate for degradation. Using peptide panning and competition experiments we speculate that initial encounters through the floppy helix and additional strong interactions with N-terminal helices anchors apomyoglobin to the proteasome. Stabilizing helical structure in the floppy F-helix slows down degradation. Destabilization of adjacent helices accelerates degradation. Unfolding seems to follow the mechanism of helix unraveling rather than global unfolding. Our findings while confirming the requirement for unstructured regions in degradation offers the following new insights: a) origin and identification of an intrinsic degradation signal in the substrate, b) identification of sequences in the native substrate that are likely to be responsible for direct interactions with the proteasome, and c) identification of critical rate limiting steps like exposure of the intrinsic degron and destabilization of an unfolding intermediate that are presumably catalyzed by the ATPases. Apomyoglobin emerges as a new model substrate to further explore the role of ATPases and protein structure in proteasomal degradatio

Identificación molecular de micobacterias no tuberculosas mediante el análisis de los patrones de restricción, Colombia 1995-2005

Introduction. Nontuberculous mycobacteria can be saprophytic, pathogenic or opportunistic. The most common diseases produced by these microorganisms are the post-surgical infections due to anesthetic procedures, infections associated with catheters, disseminated cutaneous diseases and pulmonary and central nervous system diseases that especially affect HIV patients. Identification of the nontuberculous mycobacteria can take several weeks and even then, differentiation of complex members is not possible.Objective. The PCR-restriction analysis (PRA) technique was evaluated as a method for genotypic identification of nontuberculous mycobacteria isolated of clinical samples located in the culture collection of the Instituto Nacional de Salud (National Institute of Health), Bogotá, Colombia.Materials and methods. Seventy clinical isolates of nontuberculous mycobacteria stored in 50% glycerol at -70°C were identified by phenotypic techniques. The genotypic identification was made using the PCR-restriction analysis (PRA) using the restriction enzymes BstEII and HseIII, the restriction products were visualized on gels of agarose to 3%, and the concordance between the methodologies was evaluated.Results. A matching of 100% was obtained in the identification of Mycobacterium terrae, M. szulgai, M. avium, M. chelonae and M. scrofulaceum, the matching between M. fortuitum species, M. abscessus, M. gordonae and M. intracellulare varied from 44 to 89%; there was no concurrence in the identification of species M. flavescens and M. malmoense. Conclusions. PRA provided a fast, inexpensive and accurate alternative for the identification of nontuberculous mycobacteria that permited the differentiation among species of a complex and determining the subtype of each species sample.Introducción. Las micobacterias no tuberculosas pueden ser saprofitas, patógenas u oportunistas; las enfermedades más comunes producidas por estos microorganismos son las infecciones posquirúrgicas, principalmente por procedimiento estéticos, infecciones asociadas con catéteres, enfermedades cutáneas diseminadas, enfermedades pulmonares y del sistema nervioso central que afectan especialmente a pacientes infectados con el virus de la inmunodeficiencia humana. La identificación fenotípica de las micobacterias no tuberculosas incluye pruebas microbiológicas y bioquímicas, las cuales pueden tomar varias semanas y algunas veces no logran diferenciar entre los miembros de un complejo.Objetivo. El objetivo fue evaluar la metodología de reacción en cadena de la polimerasaanálisis de restricción, como método de identificación genotípica de micobacterias no tuberculosas aisladas de muestras clínicas que pertenecen a la colección del Instituto Nacional de Salud.Materiales y métodos. Se estudiaron 70 aislamientos clínicos de micobacterias no tuberculosas, criopreservados en glicerol al 50% e identificados mediante metodologías fenotípicas. La identificación genotípica se realizó por reacción en cadena de la polimerasa-análisis de restricción y se evaluó la concordancia entre las metodologías.Resultados. Se obtuvo una concordancia del 100% en la identificación de Mycobacterium terrae, M. szulgai, M. avium, M. chelonae y M. scrofulaceum, en las especies M. fortuitum, M. abscessus M. gordonae y M. intracellulare varió de 44% a 89%; no se obtuvo concordancia en la identificación de las especies M. flavescens y M. malmoense.Conclusiones. El análisis de restricción es una alternativa para la identificación de especies de micobacterias no tuberculosas, rápida, económica y segura para la identificación, que permite la diferenciación entre especies de un complejo y la determinación del subtipo de cada especie

Surface Co-Expression of Two Different PfEMP1 Antigens on Single Plasmodium falciparum-Infected Erythrocytes Facilitates Binding to ICAM1 and PECAM1

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigens play a major role in cytoadhesion of infected erythrocytes (IE), antigenic variation, and immunity to malaria. The current consensus on control of variant surface antigen expression is that only one PfEMP1 encoded by one var gene is expressed per cell at a time. We measured var mRNA transcript levels by real-time Q-PCR, analysed var gene transcripts by single-cell FISH and directly compared these with PfEMP1 antigen surface expression and cytoadhesion in three different antibody-selected P. falciparum 3D7 sub-lines using live confocal microscopy, flow cytometry and in vitro adhesion assays. We found that one selected parasite sub-line simultaneously expressed two different var genes as surface antigens, on single IE. Importantly, and of physiological relevance to adhesion and malaria pathogenesis, this parasite sub-line was found to bind both CD31/PECAM1 and CD54/ICAM1 and to adhere twice as efficiently to human endothelial cells, compared to infected cells having only one PfEMP1 variant on the surface. These new results on PfEMP1 antigen expression indicate that a re-evaluation of the molecular mechanisms involved in P. falciparum adhesion and of the accepted paradigm of absolutely mutually exclusive var gene transcription is required

Karyotypic Determinants of Chromosome Instability in Aneuploid Budding Yeast

Recent studies in cancer cells and budding yeast demonstrated that aneuploidy, the state of having abnormal chromosome numbers, correlates with elevated chromosome instability (CIN), i.e. the propensity of gaining and losing chromosomes at a high frequency. Here we have investigated ploidy- and chromosome-specific determinants underlying aneuploidy-induced CIN by observing karyotype dynamics in fully isogenic aneuploid yeast strains with ploidies between 1N and 2N obtained through a random meiotic process. The aneuploid strains exhibited various levels of whole-chromosome instability (i.e. chromosome gains and losses). CIN correlates with cellular ploidy in an unexpected way: cells with a chromosomal content close to the haploid state are significantly more stable than cells displaying an apparent ploidy between 1.5 and 2N. We propose that the capacity for accurate chromosome segregation by the mitotic system does not scale continuously with an increasing number of chromosomes, but may occur via discrete steps each time a full set of chromosomes is added to the genome. On top of such general ploidy-related effect, CIN is also associated with the presence of specific aneuploid chromosomes as well as dosage imbalance between specific chromosome pairs. Our findings potentially help reconcile the divide between gene-centric versus genome-centric theories in cancer evolution