The Impact of HAART on the Respiratory Complications of HIV Infection: Longitudinal Trends in the MACS and WIHS Cohorts

Objective: To review the incidence of respiratory conditions and their effect on mortality in HIV-infected and uninfected individuals prior to and during the era of highly active antiretroviral therapy (HAART). Design: Two large observational cohorts of HIV-infected and HIV-uninfected men (Multicenter AIDS Cohort Study [MACS]) and women (Women's Interagency HIV Study [WIHS]), followed since 1984 and 1994, respectively. Methods: Adjusted odds or hazards ratios for incident respiratory infections or non-infectious respiratory diagnoses, respectively, in HIV-infected compared to HIV-uninfected individuals in both the pre-HAART (MACS only) and HAART eras; and adjusted Cox proportional hazard ratios for mortality in HIV-infected persons with lung disease during the HAART era. Results: Compared to HIV-uninfected participants, HIV-infected individuals had more incident respiratory infections both pre-HAART (MACS, odds ratio [adjusted-OR], 2.4; 95% confidence interval [CI], 2.2-2.7; p<0.001) and after HAART availability (MACS, adjusted-OR, 1.5; 95%CI 1.3-1.7; p<0.001; WIHS adjusted-OR, 2.2; 95%CI 1.8-2.7; p<0.001). Chronic obstructive pulmonary disease was more common in MACS HIV-infected vs. HIV-uninfected participants pre-HAART (hazard ratio [adjusted-HR] 2.9; 95%CI, 1.02-8.4; p = 0.046). After HAART availability, non-infectious lung diseases were not significantly more common in HIV-infected participants in either MACS or WIHS participants. HIV-infected participants in the HAART era with respiratory infections had an increased risk of death compared to those without infections (MACS adjusted-HR, 1.5; 95%CI, 1.3-1.7; p<0.001; WIHS adjusted-HR, 1.9; 95%CI, 1.5-2.4; p<0.001). Conclusion: HIV infection remained a significant risk for infectious respiratory diseases after the introduction of HAART, and infectious respiratory diseases were associated with an increased risk of mortality. © 2013 Gingo et al

The Extrachromosomal EAST Protein of Drosophila Can Associate with Polytene Chromosomes and Regulate Gene Expression

The EAST protein of Drosophila is a component of an expandable extrachromosomal domain of the nucleus. To better understand its function, we studied the dynamics and localization of GFP-tagged EAST. In live larval salivary glands, EAST-GFP is highly mobile and localizes to the extrachromosomal nucleoplasm. When these cells are permeabilized, EAST-GFP rapidly associated with polytene chromosomes. The affinity to chromatin increases and mobility decreases with decreasing salt concentration. Deleting the C-terminal residues 1535 to 2301 of EAST strongly reduces the affinity to polytene chromosomes. The bulk of EAST-GFP co-localizes with heterochromatin and is absent from transcriptionally active chromosomal regions. The predominantly chromosomal localization of EAST-GFP can be detected in non-detergent treated salivary glands of pupae as they undergo apoptosis, however not in earlier stages of development. Consistent with this chromosomal pattern of localization, genetic evidence indicates a role for EAST in the repression of gene expression, since a lethal east mutation is allelic to the viable mutation suppressor of white-spotted. We propose that EAST acts as an ion sensor that modulates gene expression in response to changing intracellular ion concentrations

Hippocampal LTP and contextual learning require surface diffusion of AMPA receptors

Long-term potentiation (LTP) of excitatory synaptic transmission has long been considered a cellular correlate for learning and memory. Early LTP (eLTP, <1 hour) had initially been explained either by presynaptic increases in glutamate release or by direct modification of post-synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) function. Compelling models have more recently proposed that synaptic potentiation can occur by the recruitment of additional post-synaptic AMPARs, sourced either from an intracellular reserve pool by exocytosis or from nearby extra synaptic receptors pre-existing on the neuronal surface. However, the exact mechanism through which synapses can rapidly recruit new AMPARs during eLTP is still unknown. In particular, direct evidence for a pivotal role of AMPAR surface diffusion as a trafficking mechanism in synaptic plasticity is still lacking. Using AMPAR immobilization approaches, we show that interfering with AMPAR surface diffusion dramatically impaired synaptic potentiation of Schaffer collateral/commissural inputs to cornu ammonis area 1 (CA1) in cultured slices, acute slices and in vivo. Our data also identifies distinct contributions of various AMPAR trafficking routes to the temporal profile of synaptic potentiation. In addition, AMPAR immobilization in vivo in the dorsal hippocampus (DH) before fear conditioning, indicated that AMPAR diffusion is important for the early phase of contextual learning. Therefore, our results provide a direct demonstration that the recruitment of new receptors to synapses by surface diffusion is a critical mechanism for the expression of LTP and hippocampal learning. Since AMPAR surface diffusion is dictated by weak Brownian forces that are readily perturbed by protein-protein interactions, we anticipate that this fundamental trafficking mechanism will be a key target for modulating synaptic potentiation and learning

Sustained Oscillations of NF-κB Produce Distinct Genome Scanning and Gene Expression Profiles

NF-κB is a prototypic stress-responsive transcription factor that acts within a complex regulatory network. The signaling dynamics of endogenous NF-κB in single cells remain poorly understood. To examine real time dynamics in living cells, we monitored NF-κB activities at multiple timescales using GFP-p65 knock-in mouse embryonic fibroblasts. Oscillations in NF-κB were sustained in most cells, with several cycles of transient nuclear translocation after TNF-α stimulation. Mathematical modeling suggests that NF-κB oscillations are selected over other non-oscillatory dynamics by fine-tuning the relative strengths of feedback loops like IκBα. The ability of NF-κB to scan and interact with the genome in vivo remained remarkably constant from early to late cycles, as observed by fluorescence recovery after photobleaching (FRAP). Perturbation of long-term NF-κB oscillations interfered with its short-term interaction with chromatin and balanced transcriptional output, as predicted by the mathematical model. We propose that negative feedback loops do not simply terminate signaling, but rather promote oscillations of NF-κB in the nucleus, and these oscillations are functionally advantageous

Interphase Nucleo-Cytoplasmic Shuttling and Localization of SIRT2 during Mitosis

The human NAD+-dependent protein deacetylase SIRT2 resides predominantly in the cytoplasm where it functions as a tubulin deacetylase. Here we report that SIRT2 maintains a largely cytoplasmic localization during interphase by active nuclear export in a Crm1-dependent manner. We identified a functional, leptomycin B-sensitive, nuclear export signal sequence within SIRT2. During the cell cycle, SIRT2 becomes enriched in the nucleus and is associated with mitotic structures, beginning with the centrosome during prophase, the mitotic spindle during metaphase, and the midbody during cytokinesis. Cells overexpressing wild-type or a catalytically inactive SIRT2 exhibit an increase in multinucleated cells. The findings suggest a novel mechanism of regulating SIRT2 function by nucleo-cytoplasmic shuttling, as well as a role for SIRT2 in the nucleus during interphase and throughout mitosis

Can visco-elastic phase separation, macromolecular crowding and colloidal physics explain nuclear organisation?

BACKGROUND: The cell nucleus is highly compartmentalized with well-defined domains, it is not well understood how this nuclear order is maintained. Many scientists are fascinated by the different set of structures observed in the nucleus to attribute functions to them. In order to distinguish functional compartments from non-functional aggregates, I believe is important to investigate the biophysical nature of nuclear organisation. RESULTS: The various nuclear compartments can be divided broadly as chromatin or protein and/or RNA based, and they have very different dynamic properties. The chromatin compartment displays a slow, constrained diffusional motion. On the other hand, the protein/RNA compartment is very dynamic. Physical systems with dynamical asymmetry go to viscoelastic phase separation. This phase separation phenomenon leads to the formation of a long-lived interaction network of slow components (chromatin) scattered within domains rich in fast components (protein/RNA). Moreover, the nucleus is packed with macromolecules in the order of 300 mg/ml. This high concentration of macromolecules produces volume exclusion effects that enhance attractive interactions between macromolecules, known as macromolecular crowding, which favours the formation of compartments. In this paper I hypothesise that nuclear compartmentalization can be explained by viscoelastic phase separation of the dynamically different nuclear components, in combination with macromolecular crowding and the properties of colloidal particles. CONCLUSION: I demonstrate that nuclear structure can satisfy the predictions of this hypothesis. I discuss the functional implications of this phenomenon

Protein Diffusion in Mammalian Cell Cytoplasm

We introduce a new method for mesoscopic modeling of protein diffusion in an entire cell. This method is based on the construction of a three-dimensional digital model cell from confocal microscopy data. The model cell is segmented into the cytoplasm, nucleus, plasma membrane, and nuclear envelope, in which environment protein motion is modeled by fully numerical mesoscopic methods. Finer cellular structures that cannot be resolved with the imaging technique, which significantly affect protein motion, are accounted for in this method by assigning an effective, position-dependent porosity to the cell. This porosity can also be determined by confocal microscopy using the equilibrium distribution of a non-binding fluorescent protein. Distinction can now be made within this method between diffusion in the liquid phase of the cell (cytosol/nucleosol) and the cytoplasm/nucleoplasm. Here we applied the method to analyze fluorescence recovery after photobleach (FRAP) experiments in which the diffusion coefficient of a freely-diffusing model protein was determined for two different cell lines, and to explain the clear difference typically observed between conventional FRAP results and those of fluorescence correlation spectroscopy (FCS). A large difference was found in the FRAP experiments between diffusion in the cytoplasm/nucleoplasm and in the cytosol/nucleosol, for all of which the diffusion coefficients were determined. The cytosol results were found to be in very good agreement with those by FCS

Identification of biomolecule mass transport and binding rate parameters in living cells by inverse modeling

BACKGROUND: Quantification of in-vivo biomolecule mass transport and reaction rate parameters from experimental data obtained by Fluorescence Recovery after Photobleaching (FRAP) is becoming more important. METHODS AND RESULTS: The Osborne-Moré extended version of the Levenberg-Marquardt optimization algorithm was coupled with the experimental data obtained by the Fluorescence Recovery after Photobleaching (FRAP) protocol, and the numerical solution of a set of two partial differential equations governing macromolecule mass transport and reaction in living cells, to inversely estimate optimized values of the molecular diffusion coefficient and binding rate parameters of GFP-tagged glucocorticoid receptor. The results indicate that the FRAP protocol provides enough information to estimate one parameter uniquely using a nonlinear optimization technique. Coupling FRAP experimental data with the inverse modeling strategy, one can also uniquely estimate the individual values of the binding rate coefficients if the molecular diffusion coefficient is known. One can also simultaneously estimate the dissociation rate parameter and molecular diffusion coefficient given the pseudo-association rate parameter is known. However, the protocol provides insufficient information for unique simultaneous estimation of three parameters (diffusion coefficient and binding rate parameters) owing to the high intercorrelation between the molecular diffusion coefficient and pseudo-association rate parameter. Attempts to estimate macromolecule mass transport and binding rate parameters simultaneously from FRAP data result in misleading conclusions regarding concentrations of free macromolecule and bound complex inside the cell, average binding time per vacant site, average time for diffusion of macromolecules from one site to the next, and slow or rapid mobility of biomolecules in cells. CONCLUSION: To obtain unique values for molecular diffusion coefficient and binding rate parameters from FRAP data, we propose conducting two FRAP experiments on the same class of macromolecule and cell. One experiment should be used to measure the molecular diffusion coefficient independently of binding in an effective diffusion regime and the other should be conducted in a reaction dominant or reaction-diffusion regime to quantify binding rate parameters. The method described in this paper is likely to be widely used to estimate in-vivo biomolecule mass transport and binding rate parameters

Structural plasticity of single chromatin fibers revealed by torsional manipulation

Magnetic tweezers are used to study the mechanical response under torsion of single nucleosome arrays reconstituted on tandem repeats of 5S positioning sequences. Regular arrays are extremely resilient and can reversibly accommodate a large amount of supercoiling without much change in length. This behavior is quantitatively described by a molecular model of the chromatin 3-D architecture. In this model, we assume the existence of a dynamic equilibrium between three conformations of the nucleosome, which are determined by the crossing status of the entry/exit DNAs (positive, null or negative). Torsional strain, in displacing that equilibrium, extensively reorganizes the fiber architecture. The model explains a number of long-standing topological questions regarding DNA in chromatin, and may provide the ground to better understand the dynamic binding of most chromatin-associated proteins.Comment: 18 pages, 7 figures, Supplementary information available at http://www.nature.com/nsmb/journal/v13/n5/suppinfo/nsmb1087_S1.htm

Salivary gland branching morphogenesis: a quantitative systems analysis of the Eda/Edar/NFκB paradigm

<p>Abstract</p> <p>Background</p> <p>Ectodysplasin-A appears to be a critical component of branching morphogenesis. Mutations in mouse <it>Eda </it>or human <it>EDA </it>are associated with absent or hypoplastic sweat glands, sebaceous glands, lacrimal glands, salivary glands (SMGs), mammary glands and/or nipples, and mucous glands of the bronchial, esophageal and colonic mucosa. In this study, we utilized <it>Eda</it>^{<it>Ta </it>}(Tabby) mutant mice to investigate how a marked reduction in functional Eda propagates with time through a defined genetic subcircuit and to test the proposition that canonical NFκB signaling is sufficient to account for the differential expression of developmentally regulated genes in the context of <it>Eda </it>polymorphism.</p> <p>Results</p> <p>The quantitative systems analyses do not support the stated hypothesis. For most NFκB-regulated genes, the observed time course of gene expression is nearly unchanged in Tabby (<it>Eda</it>^<it>Ta</it>) as compared to wildtype mice, as is NFκB itself. Importantly, a subset of genes is dramatically differentially expressed in Tabby (<it>Edar</it>, <it>Fgf8</it>, <it>Shh</it>, <it>Egf</it>, <it>Tgfa</it>, <it>Egfr</it>), strongly suggesting the existence of an alternative Eda-mediated transcriptional pathway pivotal for SMG ontogeny. Experimental and <it>in silico </it>investigations have identified C/EBPα as a promising candidate.</p> <p>Conclusion</p> <p>In Tabby SMGs, upregulation of the Egf/Tgfα/Egfr pathway appears to mitigate the potentially severe abnormal phenotype predicted by the downregulation of Fgf8 and Shh. Others have suggested that the buffering of the phenotypic outcome that is coincident with variant Eda signaling could be a common mechanism that permits viable and diverse phenotypes, normal and abnormal. Our results support this proposition. Further, if branching epithelia use variations of a canonical developmental program, our results are likely applicable to understanding the phenotypes of other branching organs affected by <it>Eda </it>(<it>EDA</it>) mutation.</p