The C-Terminal Domain of the Arabinosyltransferase Mycobacterium tuberculosis EmbC Is a Lectin-Like Carbohydrate Binding Module

The D-arabinan-containing polymers arabinogalactan (AG) and lipoarabinomannan (LAM) are essential components of the unique cell envelope of the pathogen Mycobacterium tuberculosis. Biosynthesis of AG and LAM involves a series of membrane-embedded arabinofuranosyl (Araf) transferases whose structures are largely uncharacterised, despite the fact that several of them are pharmacological targets of ethambutol, a frontline drug in tuberculosis therapy. Herein, we present the crystal structure of the C-terminal hydrophilic domain of the ethambutol-sensitive Araf transferase M. tuberculosis EmbC, which is essential for LAM synthesis. The structure of the C-terminal domain of EmbC (EmbCCT) encompasses two sub-domains of different folds, of which subdomain II shows distinct similarity to lectin-like carbohydrate-binding modules (CBM). Co-crystallisation with a cell wall-derived di-arabinoside acceptor analogue and structural comparison with ligand-bound CBMs suggest that EmbCCT contains two separate carbohydrate binding sites, associated with subdomains I and II, respectively. Single-residue substitution of conserved tryptophan residues (Trp868, Trp985) at these respective sites inhibited EmbC-catalysed extension of LAM. The same substitutions differentially abrogated binding of di- and penta-arabinofuranoside acceptor analogues to EmbCCT, linking the loss of activity to compromised acceptor substrate binding, indicating the presence of two separate carbohydrate binding sites, and demonstrating that subdomain II indeed functions as a carbohydrate-binding module. This work provides the first step towards unravelling the structure and function of a GT-C-type glycosyltransferase that is essential in M. tuberculosis. Author Summary Top Tuberculosis (TB), an infectious disease caused by the bacillus Mycobacterium tuberculosis, burdens large swaths of the world population. Treatment of active TB typically requires administration of an antibiotic cocktail over several months that includes the drug ethambutol. This front line compound inhibits a set of arabinosyltransferase enzymes, called EmbA, EmbB and EmbC, which are critical for the synthesis of arabinan, a vital polysaccharide in the pathogen's unique cell envelope. How precisely ethambutol inhibits arabinosyltransferase activity is not clear, in part because structural information of its pharmacological targets has been elusive. Here, we report the high-resolution structure of the C-terminal domain of the ethambutol-target EmbC, a 390-amino acid fragment responsible for acceptor substrate recognition. Combining the X-ray crystallographic analysis with structural comparisons, site-directed mutagenesis, activity and ligand binding assays, we identified two regions in the C-terminal domain of EmbC that are capable of binding acceptor substrate mimics and are critical for activity of the full-length enzyme. Our results begin to define structure-function relationships in a family of structurally uncharacterised membrane-embedded glycosyltransferases, which are an important target for tuberculosis therapy

Exploring the Conformational Transitions of Biomolecular Systems Using a Simple Two-State Anisotropic Network Model

Biomolecular conformational transitions are essential to biological functions. Most experimental methods report on the long-lived functional states of biomolecules, but information about the transition pathways between these stable states is generally scarce. Such transitions involve short-lived conformational states that are difficult to detect experimentally. For this reason, computational methods are needed to produce plausible hypothetical transition pathways that can then be probed experimentally. Here we propose a simple and computationally efficient method, called ANMPathway, for constructing a physically reasonable pathway between two endpoints of a conformational transition. We adopt a coarse-grained representation of the protein and construct a two-state potential by combining two elastic network models (ENMs) representative of the experimental structures resolved for the endpoints. The two-state potential has a cusp hypersurface in the configuration space where the energies from both the ENMs are equal. We first search for the minimum energy structure on the cusp hypersurface and then treat it as the transition state. The continuous pathway is subsequently constructed by following the steepest descent energy minimization trajectories starting from the transition state on each side of the cusp hypersurface. Application to several systems of broad biological interest such as adenylate kinase, ATP-driven calcium pump SERCA, leucine transporter and glutamate transporter shows that ANMPathway yields results in good agreement with those from other similar methods and with data obtained from all-atom molecular dynamics simulations, in support of the utility of this simple and efficient approach. Notably the method provides experimentally testable predictions, including the formation of non-native contacts during the transition which we were able to detect in two of the systems we studied. An open-access web server has been created to deliver ANMPathway results. © 2014 Das et al

Raising awareness of bronchiectasis in primary care:overview of diagnosis and management strategies in adults

Bronchiectasis is a chronic lung disease characterised by recurrent infection, inflammation, persistent cough and sputum production. The disease is increasing in prevalence, requiring a greater awareness of the disease across primary and secondary care. Mild and moderate cases of bronchiectasis in adults can often be managed by primary care clinicians. Initial assessments and long-term treatment plans that include both pharmacological and non-pharmacological treatments, however, should be undertaken in collaboration with a secondary care team that includes physiotherapists and specialists in respiratory medicine. Bronchiectasis is often identified in patients with other lung diseases, such as chronic obstructive pulmonary disease, asthma, and in a lesser but not insignificant number of patients with other inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease. Overall goals of therapy are to prevent exacerbations, improve symptoms, improve quality of life and preserve lung function. Prompt treatment of exacerbations with antibiotic therapy is important to limit the impact of exacerbations on quality of life and lung function decline. Patient education and cooperation with health-care providers to implement treatment plans are key to successful disease management. It is important for the primary care provider to work with secondary care providers to develop an individualised treatment plan to optimise care with the goal to delay disease progression. Here, we review the diagnosis and treatment of bronchiectasis with a focus on practical considerations that will be useful to primary care.</p

Rhabdomyoblastic Differentiation in Head and Neck Malignancies Other Than Rhabdomyosarcoma

Rhabdomyosarcoma is a relatively common soft tissue sarcoma that frequently affects children and adolescents and may involve the head and neck. Rhabdomyosarcoma is defined by skeletal muscle differentiation which can be suggested by routine histology and confirmed by immunohistochemistry for the skeletal muscle-specific markers myogenin or myoD1. At the same time, it must be remembered that when it comes to head and neck malignancies, skeletal muscle differentiation is not limited to rhabdomyosarcoma. A lack of awareness of this phenomenon could lead to misdiagnosis and, subsequently, inappropriate therapeutic interventions. This review focuses on malignant neoplasms of the head and neck other than rhabdomyosarcoma that may exhibit rhabdomyoblastic differentiation, with an emphasis on strategies to resolve the diagnostic dilemmas these tumors may present. Axiomatically, no primary central nervous system tumors will be discussed.info:eu-repo/semantics/publishedVersio

On the dynamics of the adenylate energy system: homeorhesis vs homeostasis.

Biochemical energy is the fundamental element that maintains both the adequate turnover of the biomolecular structures and the functional metabolic viability of unicellular organisms. The levels of ATP, ADP and AMP reflect roughly the energetic status of the cell, and a precise ratio relating them was proposed by Atkinson as the adenylate energy charge (AEC). Under growth-phase conditions, cells maintain the AEC within narrow physiological values, despite extremely large fluctuations in the adenine nucleotides concentration. Intensive experimental studies have shown that these AEC values are preserved in a wide variety of organisms, both eukaryotes and prokaryotes. Here, to understand some of the functional elements involved in the cellular energy status, we present a computational model conformed by some key essential parts of the adenylate energy system. Specifically, we have considered (I) the main synthesis process of ATP from ADP, (II) the main catalyzed phosphotransfer reaction for interconversion of ATP, ADP and AMP, (III) the enzymatic hydrolysis of ATP yielding ADP, and (IV) the enzymatic hydrolysis of ATP providing AMP. This leads to a dynamic metabolic model (with the form of a delayed differential system) in which the enzymatic rate equations and all the physiological kinetic parameters have been explicitly considered and experimentally tested in vitro. Our central hypothesis is that cells are characterized by changing energy dynamics (homeorhesis). The results show that the AEC presents stable transitions between steady states and periodic oscillations and, in agreement with experimental data these oscillations range within the narrow AEC window. Furthermore, the model shows sustained oscillations in the Gibbs free energy and in the total nucleotide pool. The present study provides a step forward towards the understanding of the fundamental principles and quantitative laws governing the adenylate energy system, which is a fundamental element for unveiling the dynamics of cellular life

The timing of umbilical cord clamping at birth: physiological considerations

While it is now recognized that umbilical cord clamping (UCC) at birth is not necessarily an innocuous act, there is still much confusion concerning the potential benefits and harms of this common procedure. It is most commonly assumed that delaying UCC will automatically result in a time-dependent net placental-to-infant blood transfusion, irrespective of the infant’s physiological state. Whether or not this occurs, will likely depend on the infant’s physiological state and not on the amount of time that has elapsed between birth and umbilical cord clamping (UCC). However, we believe that this is an overly simplistic view of what can occur during delayed UCC and ignores the benefits associated with maintaining the infant’s venous return and cardiac output during transition. Recent experimental evidence and observations in humans have provided compelling evidence to demonstrate that time is not a major factor influencing placental-to-infant blood transfusion after birth. Indeed, there are many factors that influence blood flow in the umbilical vessels after birth, which depending on the dominating factors could potentially result in infant-to-placental blood transfusion. The most dominant factors that influence umbilical artery and venous blood flows after birth are lung aeration, spontaneous inspirations, crying and uterine contractions. It is still not entirely clear whether gravity differentially alters umbilical artery and venous flows, although the available data suggests that its influence, if present, is minimal. While there is much support for delaying UCC at birth, much of the debate has focused on a time-based approach, which we believe is misguided. While a time-based approach is much easier and convenient for the caregiver, ignoring the infant’s physiology during delayed UCC can potentially be counter-productive for the infant

Littoral cell angioma of the spleen in a patient with previous pulmonary sarcoidosis: a TNF-α related pathogenesis?

<p>Abstract</p> <p>Background</p> <p>Littoral cell angioma (LCA) is a rare vascular tumor of the spleen. Generally thought to be benign, additional cases of LCA with malignant features have been described. Thus, its malignant potential seems to vary and must be considered uncertain. The etiology remains unclear, but an immune dysregulation for the apparent association with malignancies of visceral organs or immune-mediated diseases has been proposed.</p> <p>Case Presentation</p> <p>We report a case of LCA in a 43-year old male patient who presented with a loss of appetite and intermittent upper abdominal pain. Computed tomography showed multiple hypoattenuating splenic lesions which were hyperechogenic on abdominal ultrasound. Lymphoma was presumed and splenectomy was performed. Pathological evaluation revealed LCA.</p> <p>Conclusions</p> <p>LCA is a rare, primary vascular neoplasm of the spleen that might etiologically be associated with immune dysregulation. In addition, it shows a striking association with synchronous or prior malignancies. With about one-third of the reported cases to date being co-existent with malignancies of visceral organs or immune-mediated diseases, this advocates for close follow-ups in all patients diagnosed with LCA. To our knowledge, this report is the first one of LCA associated with previous pulmonary sarcoidosis and hypothesizes a TNF-α related pathogenesis of this splenic tumor.</p

Fin-Tail Coordination during Escape and Predatory Behavior in Larval Zebrafish

Larval zebrafish innately perform a suite of behaviors that are tightly linked to their evolutionary past, notably escape from threatening stimuli and pursuit and capture of prey. These behaviors have been carefully examined in the past, but mostly with regard to the movements of the trunk and tail of the larvae. Here, we employ kinematics analyses to describe the movements of the pectoral fins during escape and predatory behavior. In accord with previous studies, we find roles for the pectoral fins in slow swimming and immediately after striking prey. We find novel roles for the pectoral fins in long-latency, but not in short-latency C-bends. We also observe fin movements that occur during orienting J-turns and S-starts that drive high-velocity predatory strikes. Finally, we find that the use of pectoral fins following a predatory strike is scaled to the velocity of the strike, supporting a role for the fins in braking. The implications of these results for central control of coordinated movements are discussed, and we hope that these results will provide baselines for future analyses of cross-body coordination using mutants, morphants, and transgenic approaches

C/EBPβ-Thr217 Phosphorylation Signaling Contributes to the Development of Lung Injury and Fibrosis in Mice

mice are refractory to Bleomycin-induced lung fibrosis the molecular mechanisms remain unknown. Here we show that blocking the ribosomal S-6 kinase (RSK) phosphorylation of the CCAAT/Enhancer Binding Protein (C/EBP)-β on Thr217 (a RSK phosphoacceptor) with either a single point mutation (Ala217), dominant negative transgene or a blocking peptide containing the mutated phosphoacceptor ameliorates the progression of lung injury and fibrosis induced by Bleomycin in mice. mice with a cell permeant, C/EBPβ peptide that inhibits phosphorylation of C/EBPβ on Thr217 (40 µg instilled intracheally on day-2 and day-6 after the single Bleomycin dose) also blocked the progression of lung injury and fibrosis induced by Bleomycin. Phosphorylation of human C/EBPβ on Thr266 (human homologue phosphoacceptor) was induced in collagen-activated human lung fibroblasts in culture as well as in activated lung fibroblasts in situ in lungs of patients with severe lung fibrosis but not in control lungs, suggesting that this signaling pathway may be also relevant in human lung injury and fibrosis.These data suggest that the RSK-C/EBPβ phosphorylation pathway may contribute to the development of lung injury and fibrosis