Project description:Intermediate filaments are major components of the cytoskeleton. Desmin and synemin, cytoplasmic intermediate filament proteins and A-type lamins, nuclear intermediate filament proteins, play key roles in skeletal and cardiac muscle. Desmin, encoded by the DES gene (OMIM *125660) and A-type lamins by the LMNA gene (OMIM *150330), have been involved in striated muscle disorders. Diseases include desmin-related myopathy and cardiomyopathy (desminopathy), which can be manifested with dilated, restrictive, hypertrophic, arrhythmogenic, or even left ventricular non-compaction cardiomyopathy, Emery-Dreifuss Muscular Dystrophy (EDMD2 and EDMD3, due to LMNA mutations), LMNA-related congenital Muscular Dystrophy (L-CMD) and LMNA-linked dilated cardiomyopathy with conduction system defects (CMD1A). Recently, mutations in synemin (SYNM gene, OMIM *606087) have been linked to cardiomyopathy. This review will summarize clinical and molecular aspects of desmin-, lamin- and synemin-related striated muscle disorders with focus on LMNA and DES-associated clinical entities and will suggest pathogenetic hypotheses based on the interplay of desmin and lamin A/C. In healthy muscle, such interplay is responsible for the involvement of this network in mechanosignaling, nuclear positioning and mitochondrial homeostasis, while in disease it is disturbed, leading to myocyte death and activation of inflammation and the associated secretome alterations.
Project description:Blood banks around the world store blood components for several weeks ensuring its availability for transfusion medicine. Red blood cells (RBCs) are known to undergo compositional changes during storage, which may impact the cells' function and eventually the recipients' health. We extracted the RBC's cytoplasmic membrane (RBCcm) to study the effect of storage on the membranes' molecular structure and bending rigidity by a combination of X-ray diffraction (XRD), X-ray diffuse scattering (XDS) and coarse grained Molecular Dynamics (MD) simulations. Blood was stored in commercial blood bags for 2 and 5 weeks, respectively and compared to freshly drawn blood. Using mass spectrometry, we measured an increase of fatty acids together with a slight shift towards shorter tail lengths. We observe an increased fraction (6%) of liquid ordered (lo) domains in the RBCcms with storage time, and an increased lipid packing in these domains, leading to an increased membrane thickness and membrane order. The size of both, lo and liquid disordered (ld) lipid domains was found to decrease with increased storage time by up to 25%. XDS experiments reveal a storage dependent increase in the RBCcm's bending modulus κ by a factor of 2.8, from 1.9 kBT to 5.3 kBT. MD simulations were conducted in the absence of proteins. The results show that the membrane composition has a small contribution to the increased bending rigidity and suggests additional protein-driven mechanisms.
Project description:The mature red blood cell (RBC) lacks a nucleus and organelles characteristic of most cells, but it is elegantly structured to perform the essential function of delivering oxygen and removing carbon dioxide from all other cells while enduring the shear stress imposed by navigating small vessels and sinusoids. Over the past several decades, the efforts of biochemists, cell and molecular biologists, and hematologists have provided an appreciation of the complexity of RBC membrane structure, while studies of the RBC membrane disorders have offered valuable insights into structure-function relationships. Within the last decade, advances in genetic testing and its increased availability have made it possible to substantially build upon this foundational knowledge. Although disorders of the RBC membrane due to altered structural organization or altered transport function are heterogeneous, they often present with common clinical findings of hemolytic anemia. However, they may require substantially different management depending on the underlying pathophysiology. Accurate diagnosis is essential to avoid emergence of complications or inappropriate interventions. We propose an algorithm for laboratory evaluation of patients presenting with symptoms and signs of hemolytic anemia with a focus on RBC membrane disorders. Here, we review the genotypic and phenotypic variability of the RBC membrane disorders in order to raise the index of suspicion and highlight the need for correct and timely diagnosis.
Project description:Zika virus (ZIKV) encodes a precursor protein (also called polyprotein) of about 3424 amino acids that is processed by proteases to generate 10 mature proteins and a small peptide. In the present study, we characterized the chemical features, suborganelle distribution and potential function of each protein using Flag-tagged protein expression system. Western blot analysis revealed the molecular weight of the proteins and the polymerization of E, NS1, and NS3 proteins. In addition, we performed multi-labeled fluorescent immunocytochemistry and subcellular fractionation to determine the subcellular localization of these proteins in host cells. We found that 1) the capsid protein colocalizes with 3 different cellular organelles: nucleoli, Golgi apparatus, and lipid droplet; NS2b and NS4a are associated with the Golgi apparatus; 2) the capsid and NS1proteins distribute in both cytoplasm and nucleus, NS5 is a nuclear protein; 3) NS3 protein colocalizes with tubulin and affects Lamin A; 4) Envelope, PrM, and NS2a proteins co-localize with the endoplasmic reticulum; 5) NS1 is associated with autophagosomes and NS4b is related to early endosome; 6) NS5 forms punctate structures in the nucleus that associate with splicing compartments shown by SC35, leading to reduction of SC35 protein level and trafficking of SC35 from the nucleus to the cytoplasm. These data suggest that ZIKV generates 10 functional viral proteins that exhibit distinctive subcellular distribution in host cells.
Project description:Through bioinformatics analyses of a human gene expression database representing 105 different tissues and cell types, we identified 687 skin-associated genes that are selectively and highly expressed in human skin. Over 50 of these represent uncharacterized genes not previously associated with skin and include a subset that encode novel secreted and plasma membrane proteins. The high levels of skin-associated expression for eight of these novel therapeutic target genes were confirmed by semi-quantitative real time PCR, western blot and immunohistochemical analyses of normal skin and skin-derived cell lines. Four of these are expressed specifically by epidermal keratinocytes; two that encode G-protein-coupled receptors (GPR87 and GPR115), and two that encode secreted proteins (WFDC5 and SERPINB7). Further analyses using cytokine-activated and terminally differentiated human primary keratinocytes or a panel of common inflammatory, autoimmune or malignant skin diseases revealed distinct patterns of regulation as well as disease associations that point to important roles in cutaneous homeostasis and disease. Some of these novel uncharacterized skin genes may represent potential biomarkers or drug targets for the development of future diagnostics or therapeutics.
Project description:To study the role of the exonuclease Xrn1 in translational control, we performed ribosome profiling and RNA-seq in Xrn1-depleted cells. By using an auxin-inducible degron, we were able to study immediate effects of Xrn1 depletion in translational control. Therefore, we could overcome experimental limitations associated to stable deletion mutants.
Project description:Haemophilus parasuis is a swine pathogen of significant industry concern, but little is known about how the organism causes disease. A related human pathogen, Haemophilus influenzae, has been better studied, and many of its virulence factors have been identified. Two of these, outer membrane proteins P2 and P5, are known to have important virulence properties. The goals of this study were to identify, analyze, and compare the genetic relatedness of orthologous genes encoding P2 and P5 proteins in a diverse group of 35 H. parasuis strains. Genes encoding P2 and P5 proteins were detected in all H. parasuis strains evaluated. The predicted amino acid sequences for both P2 and P5 proteins exhibit considerable heterogeneity, particularly in regions corresponding to predicted extracellular loops. Twenty-five variants of P2 and 17 variants of P5 were identified. The P2 proteins of seven strains were predicted to contain a highly conserved additional extracellular loop compared to the remaining strains and to H. influenzae P2. Antigenic-site predictions coincided with predicted extracellular loop regions of both P2 and P5. Neighbor-joining trees constructed using P2 and P5 sequences predicted divergent evolutionary histories distinct from those predicted by a multilocus sequence typing phylogeny based on partial sequencing of seven housekeeping genes. Real-time reverse transcription-PCR indicated that both genes are expressed in all of the strains.
Project description:BackgroundExchange transfusion is a mainstay in the treatment of sickle cell anemia. Transfusion recipients with sickle cell disease (SCD) can be transfused over 10 units per therapy, an intervention that replaces circulating sickle red blood cells (RBCs) with donor RBCs. Storage of RBCs makes the intervention logistically feasible. The average storage duration for units transfused at the Duke University Medical Center is approximately 2 weeks, a time window that should anticipate the accumulation of irreversible storage lesion to the RBCs. However, no metabolomics study has been performed to date to investigate the impact of exchange transfusion on recipients' plasma and RBC phenotypes.Study design and methodsPlasma and RBCs were collected from patients with sickle cell anemia before transfusion and within 5 hours after exchange transfusion with up to 11 units, prior to metabolomics analyses.ResultsExchange transfusion significantly decreased plasma levels of markers of systemic hypoxemia like lactate, succinate, sphingosine 1-phosphate, and 2-hydroxyglutarate. These metabolites accumulated in transfused RBCs, suggesting that RBCs may act as scavenger/reservoirs. Transfused RBCs displayed higher glycolysis, total adenylate pools, and 2,3-diphosphoglycerate, consistent with increased capacity to deliver oxygen. Plasma levels of acyl-carnitines and amino acids decreased, while fatty acids and potentially harmful phthalates increased upon exchange transfusion.ConclusionMetabolic phenotypes confirm the benefits of the transfusion therapy in transfusion recipients with SCD and the reversibility of some of the metabolic storage lesion upon transfusion in vivo in 2-week-old RBCs. However, results also suggest that potentially harmful plasticizers are transfused.