A resolutive therapy for muscular dystrophies, a heterogeneous group of genetic diseases leading to muscular degeneration and in the severe forms to death, is still lacking. Since inflammation and defects in nitric oxide generation are recognized key pathogenic events in muscular dystrophy, we have analysed the effects of a derivative of ibuprofen, NCX 320, belonging to the class of cyclooxygenase inhibiting nitric oxide donator (CINOD), in the α-sarcoglycan null mice, a severe mouse model of dystrophy. NCX 320 was administered daily in the diet for 8months starting 1month from weaning. Muscle functional recovery was evaluated by free wheel and treadmill tests at 8months. Serum creatine kinase activity, as well as the number of diaphragm inflammatory infiltrates and necrotic fibres, was measured as indexes of skeletal muscle damage. Muscle regeneration was evaluated in diaphragm and tibialis anterior muscles, measuring the numbers of centronucleated fibres and of myogenic precursor cells. NCX 320 mitigated muscle damage, reducing significantly serum creatine kinase activity, the number of necrotic fibres and inflammatory infiltrates. Moreover, NCX 320 stimulated muscle regeneration increasing significantly the number of myogenic precursor cells and regenerating fibres. All these effects concurred in inducing a significant improvement of muscle function, as assessed by both free wheel and treadmill tests. These results describe the properties of a new compound incorporating nitric oxide donation together with anti-inflammatory properties, showing that it is effective in slowing muscle dystrophy progression long term. Of importance, this new compound deserves specific attention for its potential in the therapy of muscular dystrophy given that ibuprofen is well tolerated in paediatric patients and with a profile of safety that makes it suitable for chronic treatment such as the one required in muscular dystrophies.

The SPIRE (Systematic Protein Investigative Research Environment) provides web-based experiment-specific mass spectrometry (MS) proteomics analysis (https://www.proteinspire.org). Its emphasis is on usability and integration of the best analytic tools. SPIRE provides an easy to use web-interface and generates results in both interactive and simple data formats. In contrast to run-based approaches, SPIRE conducts the analysis based on the experimental design. It employs novel methods to generate false discovery rates and local false discovery rates (FDR, LFDR) and integrates the best and complementary open-source search and data analysis methods. The SPIRE approach of integrating X!Tandem, OMSSA and SpectraST can produce an increase in protein IDs (52-88%) over current combinations of scoring and single search engines while also providing accurate multi-faceted error estimation. One of SPIRE's primary assets is combining the results with data on protein function, pathways and protein expression from model organisms. We demonstrate some of SPIRE's capabilities by analyzing mitochondrial proteins from the wild type and 3 mutants of C. elegans. SPIRE also connects results to publically available proteomics data through its Model Organism Protein Expression Database (MOPED). SPIRE can also provide analysis and annotation for user supplied protein ID and expression data.

Acidic mammalian chitinase (AMCase) is produced in the lung during allergic inflammation and asthma, and inhibition of enzymatic activity has been considered as a therapeutic strategy. However, most chitinase inhibitors are nonselective, additionally inhibiting chitotriosidase activity. Here, we describe bisdionin F, a competitive AMCase inhibitor with 20-fold selectivity for AMCase over chitotriosidase, designed by utilizing the AMCase crystal structure and dicaffeine scaffold. In a murine model of allergic inflammation, bisdionin F-treatment attenuated chitinase activity and alleviated the primary features of allergic inflammation including eosinophilia. However, selective AMCase inhibition by bisdionin F also caused dramatic and unexpected neutrophilia in the lungs. This class of inhibitor will be a powerful tool to dissect the functions of mammalian chitinases in disease and represents a synthetically accessible scaffold to optimize inhibitory properties in terms of airway inflammation.

GC 5' splice sites (5'ss) are present in ∼1% of human introns, but factors promoting their efficient selection are poorly understood. Here, we describe a case of X-linked agammaglobulinemia resulting from a GC 5'ss activated by a mutation in BTK intron 3. This GC 5'ss was intrinsically weak, yet it was selected in >90% primary transcripts in the presence of a strong and intact natural GT counterpart. We show that efficient selection of this GC 5'ss required a high density of GAA/CAA-containing splicing enhancers in the exonized segment and was promoted by SR proteins 9G8, Tra2β and SC35. The GC 5'ss was efficiently inhibited by splice-switching oligonucleotides targeting either the GC 5'ss itself or the enhancer. Comprehensive analysis of natural GC-AG introns and previously reported pathogenic GC 5'ss showed that their efficient activation was facilitated by higher densities of splicing enhancers and lower densities of silencers than their GT 5'ss equivalents. Removal of the GC-AG introns was promoted to a minor extent by the splice-site strength of adjacent exons and inhibited by flanking Alu repeats, with the first downstream Alus located on average at a longer distance from the GC 5'ss than other transposable elements. These results provide new insights into the splicing code that governs selection of noncanonical splice sites.

The specific partnering of synaptically connected neurons is central to nervous system function. Proper wiring requires the interchange of signals between a postmitotic neuron and its environment, a distinct pattern of transcription in the nucleus, and deployment of guidance and adhesion cues to the cell surface. To identify genes involved in neurite targeting by retinal ganglion cells (GCs), their presynaptic partners in the retina, and their postsynaptic targets in the optic tectum, we undertook a forward genetic screen for mutations disrupting visual responses in zebrafish. This rapid primary screen was subsequently refined by immunohistochemical labeling of retinal and tectal neurites to detect patterning errors. From this unbiased screen, the notorious (noto) mutant exhibited the most specific phenotypes: intact retinal and tectal differentiation but multiple neurite targeting defects in the retinal inner plexiform layer (IPL) and tectal neuropil. Positional cloning and morpholino phenocopy revealed that the mutation disrupts Topoisomerase IIβ (Top2b), a broadly distributed nuclear protein involved in chromatin modifications during postmitotic differentiation. Top2b-DNA interactions are known to regulate transcription of developmentally important genes, including axon guidance factors and cell adhesion molecules, but a specific role in local synaptic targeting has not been previously described. The neurite targeting defects among GC axons are largely restricted to crossovers between sublaminae of a specific layer, SFGS, and were shown by mosaic analysis to be autonomous to the GC axons. The noto mutant provides the first example of the importance of an epigenetic regulator, Top2b, in the intricate series of events that lead to a properly wired visual system.

Plexin receptors regulate cell adhesion, migration, and guidance. The Rho GTPase binding domain (RBD) of plexin-A1 and -B1 can bind GTPases, including Rnd1. By contrast, plexin-C1 and -D1 reportedly bind Rnd2 but associate with Rnd1 only weakly. The structural basis of this differential Rnd1 GTPase binding to plexin RBDs remains unclear. Here, we solved the structure of the plexin-A2 RBD in complex with Rnd1 and the structures of the plexin-C1 and plexin-D1 RBDs alone, also compared with the previously determined plexin-B1 RBD.Rnd1 complex structure. The plexin-A2 RBD·Rnd1 complex is a heterodimer, whereas plexin-B1 and -A2 RBDs homodimerize at high concentration in solution, consistent with a proposed model for plexin activation. Plexin-C1 and -D1 RBDs are monomeric, consistent with major residue changes in the homodimerization loop. In plexin-A2 and -B1, the RBD β3-β4 loop adjusts its conformation to allow Rnd1 binding, whereas minimal structural changes occur in Rnd1. The plexin-C1 and -D1 RBDs lack several key non-polar residues at the corresponding GTPase binding surface and do not significantly interact with Rnd1. Isothermal titration calorimetry measurements on plexin-C1 and -D1 mutants reveal that the introduction of non-polar residues in this loop generates affinity for Rnd1. Structure and sequence comparisons suggest a similar mode of Rnd1 binding to the RBDs, whereas mutagenesis suggests that the interface with the highly homologous Rnd2 GTPase is different in detail. Our results confirm, from a structural perspective, that Rnd1 does not play a role in the activation of plexin-C1 and -D1. Plexin functions appear to be regulated by subfamily-specific mechanisms, some of which involve different Rho family GTPases.

Diverse stimuli can feed into the MAPK/ERK cascade; this includes receptor tyrosine kinases, G protein-coupled receptors, integrins, and scavenger receptors (LDL receptor-related protein (LRP)). Here, we investigated the consequence of concomitant occupancy of the receptor tyrosine kinases (by EGF, basic FGF, VEGF, etc.) and of LRP family members (by LDL or lactoferrin). The simultaneous stimulation of a receptor tyrosine kinase by its cognate ligand and of LRP-1 (by lactoferrin or LDL) resulted in sustained activation of ERK, which was redirected to the cytoplasm. Accordingly, elevated levels of active cytosolic ERK were translated into accelerated adhesion to vitronectin. The sustained ERK response was seen in several cell types, but it was absent in cells deficient in LRP-1 (but not in cells lacking the LDL receptor). This response was also contingent on the presence of urokinase (uPA) and its receptor (uPAR), because it was absent in uPA(-/-) and uPAR(-/-) fibroblasts. Combined stimulation of the EGF receptor and of LRP-1 delayed nuclear accumulation of phosphorylated ERK. This shift in favor of cytosolic accumulation of phospho-ERK was accounted for by enhanced proteasomal degradation of dual specificity phosphatases DUSP1 and DUSP6, which precluded dephosphorylation of cytosolic ERK. These observations demonstrate that the ERK cascade can act as a coincidence detector to decode the simultaneous engagement of a receptor tyrosine kinase and of LRP-1 and as a signal integrator that encodes this information in a spatially and temporally distinct biological signal. In addition, the findings provide an explanation of why chronic elevation of LRP-1 ligands (e.g. PAI-1) can predispose to cancer.

The multiple transferable resistance (mTR) pump from Neisseria gonorrhoeae MtrCDE multidrug pump is assembled from the inner and outer membrane proteins MtrD and MtrE and the periplasmic membrane fusion protein MtrC. Previously we established that while there is a weak interaction of MtrD and MtrE, MtrC binds with relatively high affinity to both MtrD and MtrE. MtrD conferred antibiotic resistance only when it was expressed with MtrE and MtrC, suggesting that these proteins form a functional tripartite complex in which MtrC bridges MtrD and MtrE. Furthermore, we demonstrated that MtrC interacts with an intraprotomer groove on the surface of MtrE, inducing channel opening. However, a second groove is apparent at the interface of the MtrE subunits, which might also be capable of engaging MtrC. We have now established that MtrC can be cross-linked to cysteines placed in this interprotomer groove and that mutation of residues in the groove impair the ability of the pump to confer antibiotic resistance by locking MtrE in the closed channel conformation. Moreover, MtrE K390C forms an intermolecular disulfide bond with MtrC E149C locking MtrE in the open channel conformation, suggesting that a functional salt bridge forms between these residues during the transition from closed to open channel conformations. MtrC forms dimers that assemble into hexamers, and electron microscopy studies of single particles revealed that these hexamers are arranged into ring-like structures with an internal aperture sufficiently large to accommodate the MtrE trimer. Cross-linking of single cysteine mutants of MtrC to stabilize the dimer interface in the presence of MtrE, trapped an MtrC-MtrE complex with a molecular mass consistent with a stoichiometry of 3:6 (MtrE(3)MtrC(6)), suggesting that dimers of MtrC interact with MtrE, presumably by binding to the two grooves. As both MtrE and MtrD are trimeric, our studies suggest that the functional pump is assembled with a stoichiometry of 3:6:3.

Streptococcus pneumoniae (pneumococcus) is a frequent colonizer of the nasopharynx and one of the leading causative agents of otitis media, pneumonia, and meningitis. The current literature asserts that S. pneumoniae is transmitted person to person via respiratory droplets; however, environmental surfaces (fomites) have been linked to the spread of other respiratory pathogens. Desiccation tolerance has been to shown to be essential for long-term survival on dry surfaces. This study investigated the survival and infectivity of S. pneumoniae following desiccation under ambient conditions. We recovered viable bacteria after all desiccation periods tested, ranging from 1 h to 4 weeks. Experiments conducted under nutrient limitation indicate that desiccation is a condition separate from starvation. Desiccation of an acapsular mutant and 15 different clinical isolates shows that S. pneumoniae desiccation tolerance is independent of the polysaccharide capsule and is a species-wide phenomenon, respectively. Experiments demonstrating that nondesiccated and desiccated S. pneumoniae strains colonize the nasopharynx at comparable levels, combined with their ability to survive long-term desiccation, suggest that fomites may serve as alternate sources of pneumococcal infection.

The intrinsic immunity factor CD317 (BST-2/HM1.24/tetherin) imposes a barrier to HIV-1 release at the cell surface that can be overcome by the viral protein Vpu. Expression of Vpu results in a reduction of CD317 surface levels; however, the mechanism of this Vpu activity and its contribution to the virological antagonism are incompletely understood. Here, we characterized the influence of Vpu on major CD317 trafficking pathways using quantitative antibody-based endocytosis and recycling assays as well as a microinjection/microscopy-based kinetic de novo expression approach. We report that HIV-1 Vpu inhibited both the anterograde transport of newly synthesized CD317 and the recycling of CD317 to the cell surface, while the kinetics of CD317 endocytosis remained unaffected. Vpu trapped trafficking CD317 molecules at the trans-Golgi network, where the two molecules colocalized. The subversion of both CD317 transport pathways was dependent on the highly conserved diserine S52/S56 motif of Vpu; however, it did not require recruitment of the diserine motif interactor and substrate adaptor of the SCF-E3 ubiquitin ligase complex, β-TrCP. Treatment of cells with the malaria drug primaquine resulted in a CD317 trafficking defect that mirrored that induced by Vpu. Importantly, primaquine could functionally replace Vpu as a CD317 antagonist and rescue HIV-1 particle release.

To assess the effect of diacerein on insulin secretion and metabolic control in drug-naïve patients with type 2 diabetes.

To evaluate the long-term intervention effects of oral insulin on the development of type 1 diabetes and to assess the rate of progression to type 1 diabetes before and after oral insulin treatment was stopped in the Diabetes Prevention Trial-Type 1 (DPT-1).

Inherited retinal degeneration results from many different mutations in either photoreceptor-specific or nonphotoreceptor-specific genes. However, nearly all mutations lead to a common blinding phenotype that initiates with rod cell death, followed by loss of cones. In most retinal degenerations, other retinal neuron cell types survive for long periods after blindness from photoreceptor loss. One strategy to restore light responsiveness to a retina rendered blind by photoreceptor degeneration is to express light-regulated ion channels or transporters in surviving retinal neurons. Recent experiments in rodents have restored light-sensitivity by expressing melanopsin or microbial opsins either broadly throughout the retina or selectively in the inner segments of surviving cones or in bipolar cells. Here, we present an approach whereby a genetically and chemically engineered light-gated ionotropic glutamate receptor (LiGluR) is expressed selectively in retinal ganglion cells (RGCs), the longest-surviving cells in retinal blinding diseases. When expressed in the RGCs of a well-established model of retinal degeneration, the rd1 mouse, LiGluR restores light sensitivity to the RGCs, reinstates light responsiveness to the primary visual cortex, and restores both the pupillary reflex and a natural light-avoidance behavior.

The secondary metabolites of higher plants include diverse chemicals, such as alkaloids, isoprenoids and phenolic compounds (phenylpropanoids and flavonoids). Although these compounds are widely used in human health and nutrition, at present they are mainly obtained by extraction from plants and extraction yields are low because most of these metabolites accumulate at low levels in plant cells. Recent advances in synthetic biology and metabolic engineering have enabled tailored production of plant secondary metabolites in microorganisms, but these methods often require the addition of expensive substrates. Here we develop an Escherichia coli fermentation system that yields plant alkaloids from simple carbon sources, using selected enzymes to construct a tailor-made biosynthetic pathway. In this system, engineered cells cultured in growth medium without additional substrates produce the plant benzylisoquinoline alkaloid, (S)-reticuline (yield, 46.0 mg l(-1) culture medium). The fermentation platform described here offers opportunities for low-cost production of many diverse alkaloids.

MicroRNAs are tiny non-coding small endogenous RNAs that regulate gene expression by translational repression, mRNA cleavage and mRNA inhibition. The aim of this study was to investigate the hypermethylation of miR-34b/c and miR-148a in colorectal cancer, and correlate this data to clinicopathological features. We also aimed to evaluate the hypermethylation of miR-34b/c in faeces specimens as a novel non-invasive faecal-DNA-based screening marker.

Nucleophosmin (NPM1) gene mutations resulting in cytoplasmic delocalization of Nucleophosmin (NPMc+) are the most common genetic alteration in acute myeloid leukemia (AML). Here, we attempted to prepare monoclonal antibodies (mAbs) against NPM1 mutation A (NPM-mA) and investigated the mAbs' clinical utility in immunohistochemical detection of NPMc+AML. The pET-32a-NPM-mA vector with the whole open reading frame of the NPM-mA gene was constructed. E.coli BL21 transformed with the vector were induced to express the NPM-mA recombinant protein. BALB/c mice were immunized with the recombinant NPM-mA. Positive clones were selected by indirect ELISA and the mAbs were obtained. Immunohistochemistry was performed to detect the NPMc+ in bone marrow smears from 10 AML patients with NPM-mA. The results showed that the pET-32a-NPM-mA vector was successfully constructed and the NPM-mA recombinant protein was used to immunize the mice. Two positive clones (2G3 and 3F9) were selected. The mAbs against NPM-mA were raised, but did cross-react with wild type NPM1. The mAbs can be used to detect the cytoplasmic dislocation of NPM1 in all AMLs carrying NPM-mA. Our results show that anti-NPM-mA mAbs were produced. Though they would cross-react with wild type NPM1, the mAbs may still have potential in the detection of NPMc+AMLs.

The Hepatitis C Virus Genotype 2 subtype 2c (HCV-2c) is detected as a low prevalence subtype in many countries, except in Southern Europe and Western Africa. The current epidemiology of HCV in Argentina, a low-prevalence country, shows the expected low prevalence for this subtype. However, this subtype is the most prevalent in the central province of Córdoba. Cruz del Eje (CdE), a small rural city of this province, shows a prevalence for HCV infections of 5%, being 90% of the samples classified as HCV-2c. In other locations of Córdoba Province (OLC) with lower prevalence for HCV, HCV-2c was recorded in about 50% of the samples. The phylogenetic analysis of samples from Córdoba Province consistently conformed a monophyletic group with HCV-2c sequences from all the countries where HCV-2c has been sequenced. The phylogeographic analysis showed an overall association between geographical traits and phylogeny, being these associations significant (α = 0.05) for Italy, France, Argentina (places other than Córdoba), Martinique, CdE and OLC. The coalescence analysis for samples from CdE, OLC and France yielded a Time for the Most Common Recent Ancestor of about 140 years, whereas its demographic reconstruction showed a "lag" phase in the viral population until 1880 and then an exponential growth until 1940. These results were also obtained when each geographical area was analyzed separately, suggesting that HCV-2c came into Córdoba province during the migration process, mainly from Europe, which is compatible with the history of Argentina of the early 20th century. This also suggests that the spread of HCV-2c occurred in Europe and South America almost simultaneously, possibly as a result of the advances in medicine technology of the first half of the 20th century.

Olfactory dysfunction in MS patients is reported in the literature. MRI of the olfactory bulb (OB) is discussed as a promising new testing method for measuring olfactory function (OF). Aim of this study was to explore reasons for and optimize the detection of olfactory dysfunction in MS patients with MRI.

Microarray is a powerful tool for genome-wide gene expression analysis. In microarray expression data, often mean and variance have certain relationships. We present a non-parametric mean-variance smoothing method (NPMVS) to analyze differentially expressed genes. In this method, a nonlinear smoothing curve is fitted to estimate the relationship between mean and variance. Inference is then made upon shrinkage estimation of posterior means assuming variances are known. Different methods have been applied to simulated datasets, in which a variety of mean and variance relationships were imposed. The simulation study showed that NPMVS outperformed the other two popular shrinkage estimation methods in some mean-variance relationships; and NPMVS was competitive with the two methods in other relationships. A real biological dataset, in which a cold stress transcription factor gene, CBF2, was overexpressed, has also been analyzed with the three methods. Gene ontology and cis-element analysis showed that NPMVS identified more cold and stress responsive genes than the other two methods did. The good performance of NPMVS is mainly due to its shrinkage estimation for both means and variances. In addition, NPMVS exploits a non-parametric regression between mean and variance, instead of assuming a specific parametric relationship between mean and variance. The source code written in R is available from the authors on request.

Neuronal activity enhances the elaboration of newborn neurons as they integrate into the synaptic circuitry of the adult brain. The role microRNAs play in the transduction of neuronal activity into growth and synapse formation is largely unknown. MicroRNAs can influence the expression of hundreds of genes and thus could regulate gene assemblies during processes like activity-dependent integration. Here, we developed viral-based methods for the in vivo detection and manipulation of the activity-dependent microRNA, miR-132, in the mouse hippocampus. We find, using lentiviral and retroviral reporters of miR-132 activity, that miR-132 is expressed at the right place and right time to influence the integration of newborn neurons. Retroviral knockdown of miR-132 using a specific 'sponge' containing multiple target sequences impaired the integration of newborn neurons into the excitatory synaptic circuitry of the adult brain. To assess potential miR-132 targets, we used a whole-genome microarray in PC12 cells, which have been used as a model of neuronal differentiation. miR-132 knockdown in PC12 cells resulted in the increased expression of hundreds of genes. Functional grouping indicated that genes involved in inflammatory/immune signaling were the most enriched class of genes induced by miR-132 knockdown. The correlation of miR-132 knockdown to increased proinflammatory molecular expression may indicate a mechanistic link whereby miR-132 functions as an endogenous mediator of activity-dependent integration in vivo.