Thyroid hormone induces ossification and terminal maturation in a preserved OA cartilage biomimetic model.

OBJECTIVE: To characterize aspects of triiodothyronine (T3) induced chondrocyte terminal maturation within the molecular osteoarthritis pathophysiology using the previously established T3 human ex vivo osteochondral explant model. DESIGNS: RNA-sequencing was performed on explant cartilage obtained from OA patients (n = 8), that was cultured ex vivo with or without T3 (10 ng/ml), and main findings were validated using RT-qPCR in an independent sample set (n = 22). Enrichment analysis was used for functional clustering and comparisons with available OA patient RNA-sequencing and GWAS datasets were used to establish relevance for OA pathophysiology by linking to OA patient genomic profiles. RESULTS: Besides the upregulation of known hypertrophic genes EPAS1 and ANKH, T3 treatment resulted in differential expression of 247 genes with main pathways linked to extracellular matrix and ossification. CCDC80, CDON, ANKH and ATOH8 were among the genes found to consistently mark early, ongoing and terminal maturational OA processes in patients. Furthermore, among the 37 OA risk genes that were significantly affected in cartilage by T3 were COL12A1, TNC, SPARC and PAPPA. CONCLUSIONS: RNA-sequencing results show that metabolic activation and recuperation of growth plate morphology are induced by T3 in OA chondrocytes, indicating terminal maturation is accelerated. The molecular mechanisms involved in hypertrophy were linked to all stages of OA pathophysiology and will be used to validate disease models for drug testing.

NMR detection in biofluid extracts at sub-µM concentrations via para-H2 induced hyperpolarization.

NMR spectroscopy is one of the most powerful techniques to simultaneously obtain qualitative and quantitative information in chemical analysis. Despite its versatility, the applications of NMR in the study of biofluids are often limited by the insensitivity of the technique, further aggravated by the poor signal dispersion in the (1)H spectra. Recent advances in para-H2 induced hyperpolarization have proven to address both these limitations for specific classes of compounds. Herein, this approach is for the first time applied for quantitative determination in biofluid extracts. We demonstrate that a combination of solid phase extraction, para-hydrogen induced hyperpolarization and selective NMR detection quickly reveals a doping substance, nikethamide, at sub-µM concentrations in urine. We suggest that this method can be further optimized for the detection of different analytes in various biofluids, anticipating a wider application of hyperpolarized NMR in metabolomics and pharmacokinetics studies in the near future.

CHIVA strategy in chronic venous disease treatment: instructions for users.

Along the years, scientific clinical data have been collected concerning the possible saphenous flow restoration without any ablation and according with the CHIVA strategy. Moreover, in 2013 a Cochrane review highlighted the smaller recurrence risk following a CHIVA strategy rather than a saphenous stripping. Nevertheless, the saphenous sparing strategy surely remains a not-so-worldwide-spread and accepted therapeutic option, also because considered not so immediate and easy to perform. Aim of this paper is to provide an easily accessible guide to an everyday use of a saphenous sparing strategy for chronic venous disease, highlighting how even apparently too complicated reflux patterns classifications can be fastly and successfully managed and exploited for a hemodynamic correction.

A Preliminary microRNA Analysis of Non Syndromic Thoracic Aortic Aneurysms.

The development of thoracic aortic aneurysms (TAAs) involves a multifactorial process resulting in alterations of the structure and composition of the extracellular matrix (ECM). Recently, modifications in microRNA (miRNA) expression were implicated in the pathogenesis of TAA. This study presents a preliminary miRNA microarray analysis conducted on pooled ascending aorta RNAs obtained from non familial non syndromic TAA patients (five males and five females) compared to matched control pools. Ninety-nine differentially expressed miRNAs with >1.5-fold-up- or down-regulation in TAAs compared to controls were identified, 16.0% of which were similarly regulated in the two sexes. Genes putatively targeted by differentially expressed miRNAs belonged preferentially to focal adhesion and adherens junction pathways. The results indicate an altered regulation of miRNA-mediated gene expression in the cellular interactions of aneurysmal aortic wall.

The reproducibility of colour Doppler in chronic cerebrospinal venous insufficiency associated with multiple sclerosis.

AIM: Chronic cerebrospinal venous insufficiency (CCSVI) is a syndrome described in multiple sclerosis (MS) patients, characterized by stenosis of the main extracranial veins with hampered cerebral venous outflow. In the original description echo-colour Doppler demonstrated to be an ideal non invasive tool for screening CCSVI patients, but the reproducibility was not assessed. Aim of this study is to assess the variability coefficient between trained and in not trained echo-colour Doppler operators. METHODS: Thirty-six (36) subjects, matched for age and gender, were subset in 3 groups (group A, 12 healthy controls, HC; group B, 12 multiple sclerosis patients, MS; group C, 12 patients with other neurological disease, OND) underwent echo-colour Doppler screening for CCSVI according to an original protocol previously described. The inter observer variability rate was assessed by comparing respectively trained vs not trained operators, and trained vs trained operators, by using the same echo-colour Doppler equipment. In addition, by scanning 15 subjects after one month from the first session, intra observer coefficient was also assessed in trained operator. RESULTS: The inter observer variability rate between trained and not trained echo-colour Doppler operators, were not completely satisfactory (K coefficient 0.47 95% CI 0.27-0.68). To the contrary the inter observer agreement between trained operators was much more reliable (K coefficient 0.80 95% CI 0.59-1.01). Finally, the intra observer variability rate in trained operators was 0.93, (95% CI 0.80-1.06) confirming a highly satisfactory agreement. CONCLUSION: Echo-colour Doppler is a powerful, non-invasive and reproducible tool for screening CCSVI-MS but it needs special training.

1H, 13C and 15N NMR assignments of Duck HBV primer loop of the encapsidation signal epsilon.

The replication of the hepatitis B virus is initiated by binding of the viral reverse transcriptase protein complex to the apical stem loop of the epsilon element to place it next to the primer loop, from which a four nucleotide DNA primer is subsequently synthesized. Here, we present the (1)H/(13)C/(15)N NMR assignments of the bases and sugars of the 37 residues primer loop of Duck HBV epsilon (BMRB-entry 15786).

1H, 13C and 15N NMR assignments of Duck HBV apical stem loop of the epsilon encapsidation signal.

The replication of Hepatitis B virus is initiated by binding of its reverse transcriptase to the apical stem loop and primer loop of epsilon. Here, we present the (1)H/(13)C/(15)N NMR assignments of the bases and sugars of the 29 residues apical stem loop of Duck HBV epsilon.

Selective down-regulation of [(125)I]Y0-alpha-conotoxin MII binding in rat mesostriatal dopamine pathway following continuous infusion of nicotine.

Prolonged exposure to nicotine, as occurs in smokers, results in up-regulation of all the neuronal nicotinic acetylcholine receptor subtypes studied so far, the only differences residing in the extent and time course of the up-regulation. alpha6beta2*-Nicotinic acetylcholine receptors are selectively enriched in the mesostriatal dopaminergic system and may play a crucial role in nicotine dependence. Here we show that chronic nicotine treatment (3mg/kg/day for two weeks, via s.c. osmotic minipumps) caused a significant decrease (36% on average) in the binding of [(125)I]Y(0)-alpha-conotoxin MII (a selective ligand for alpha6beta2*-nicotinic acetylcholine receptors in this system) to all the five regions of the rat dopaminergic pathway analyzed in this study. After one week of withdrawal, binding was still lower than control in striatal terminal regions (namely the caudate putamen and the accumbens shell and core). In somatodendritic regions (the ventral tegmental area and the substantia nigra) the decrease was significant at the end of the treatment and recovered within one day of withdrawal. This effect was not due to displacement of [(125)I]Y(0)-alpha-conotoxin MII binding by residual nicotine. In fact the binding was not changed by 565 ng/g nicotine (obtained with a single injection of nicotine), a concentration much higher than that found in the brain of rats chronically treated with nicotine (240 ng/g). In addition, consistent with previous studies reporting an up-regulation of other subtypes of nicotinic acetylcholine receptors, we found that nicotine exposure significantly increased (40% on average) the binding of [(125)I]epibatidine (a non-selective agonist at most neuronal heteromeric nicotinic acetylcholine receptors) in three up to five regions containing only alpha-conotoxin MII-insensitive [(125)I]epibatidine binding sites, namely the primary motor, somatosensory and auditory cortices. In conclusion, this work is the first to demonstrate that alpha6beta2*-nicotinic acetylcholine receptors, unique within the nicotinic acetylcholine receptor family, are down-regulated following chronic nicotine treatment in rat dopaminergic mesostriatal pathway, a finding that may shed new light in the complex mechanisms of nicotine dependence.

Nicotine self-administration and withdrawal: modulation of anxiety in the social interaction test in rats.

RATIONALE: Most smokers report smoking has an anxiolytic effect, which may contribute to nicotine dependence. OBJECTIVE: To examine effects in the social interaction test (SI) of anxiety after 4 weeks' self-administered nicotine (15 infusions of 0.03 mg/kg, totalling 0.45 mg/kg per day), and after 24 and 72 h of withdrawal. The effect of exposure to the operant chamber on withdrawal responses was also examined. METHODS: Animals were trained to self-administer saline or nicotine and after 4 weeks they were tested in SI after their daily self-administration session. Animals were retested after 24 and 72 h withdrawal, when they were either taken directly from the home cage or were tested 5 min after a 30-min exposure to the operant chamber. RESULTS: Compared with the saline control group, the animals that had been self-administering nicotine for 4 weeks showed decreased social interaction with no decrease in locomotor activity, indicating a significant anxiogenic effect of the nicotine infusions. There was no change in social interaction after 24 and 72 h withdrawal from chronic nicotine, regardless of whether or not the rats were exposed to the operant chamber just prior to being tested. CONCLUSIONS: Nicotine self-administration is not maintained because of its anxiolytic effect, but despite, or because of, its anxiogenic effect. There was no evidence of an anxiogenic response after either 24 or 72 h of withdrawal and thus increased anxiety on withdrawal from nicotine does not seem to contribute to nicotine self-administration.

The Mad1-Sin3B interaction involves a novel helical fold.

Sin3A or Sin3B are components of a corepressor complex that mediates repression by transcription factors such as the helix-loop-helix proteins Mad and Mxi. Members of the Mad/Mxi family of repressors play important roles in the transition between proliferation and differentiation by down-regulating the expression of genes that are activated by the proto-oncogene product Myc. Here, we report the solution structure of the second paired amphipathic helix (PAH) domain (PAH2) of Sin3B in complex with a peptide comprising the N-terminal region of Mad1. This complex exhibits a novel interaction fold for which we propose the name 'wedged helical bundle'. Four alpha-helices of PAH2 form a hydrophobic cleft that accommodates an amphipathic Mad1 alpha-helix. Our data further show that, upon binding Mad1, secondary structure elements of PAH2 are stabilized. The PAH2-Mad1 structure provides the basis for determining the principles of protein interaction and selectivity involving PAH domains.

A novel experiment for the quantitative measurement of CSA(1H(N))/CSA(15N) cross-correlated relaxation in 15N-labeled proteins.

An experiment is presented which allows for the quantitative measurement of the relaxation interference between the 1H(N) CSA and 15N CSA interactions in 15N labeled proteins. A constant-time buildup scheme is used to measure the differential relaxation rate, eta, between double-quantum (DQ) and zero-quantum (ZQ) 1H(N)-15N coherences. The CSA/CSA experiment was recorded at three different Bo field strengths. The CSA(1H(N))/CSA(15N) cross-correlation rate was obtained from the linear fit of the measured rate, eta, versus Bo2 for 77 residues of the EH2 domain from mouse Eps15.

The EH1 domain of Eps15 is structurally classified as a member of the S100 subclass of EF-hand-containing proteins.

The Eps15 homology (EH) domain is a protein-protein interaction module that binds to proteins containing the asparagine-proline-phenylalanine (NPF) or tryptophan/phenylalanine-tryptophan (W/FW) motif. EH domain-containing proteins serve important roles in signaling and processes connected to transport, protein sorting, and organization of subcellular structure. Here, we report the solution structure of the apo form of the EH1 domain of mouse Eps15, as determined by high-resolution multidimensional heteronuclear NMR spectroscopy. The polypeptide folds into six alpha-helices and a short antiparallel beta-sheet. Additionally, it contains a long, structured, topologically unique C-terminal loop. Helices 2-5 form two EF-hand motifs. Structural similarity and Ca(2+) binding properties lead to classification of the EH1 domain as a member of the S100 subclass of EF-hand-containing proteins, albeit with a unique set of interhelical angles. Binding studies using an eight-residue NPF-containing peptide derived from RAB, the cellular cofactor of the HIV Rev protein, show a hydrophobic peptide-binding pocket formed by conserved tryptophan and leucine residues.

Neural substrate of nicotine addiction as defined by functional brain maps of gene expression.

The distributed neural networks involved in the intravenous self-administration of nicotine and cocaine, and in a model of relapse of nicotine-taking after abstinence, were compared in Wistar rats. Post-mortem brain maps of c-fos-related antigens expression showed specific activation in prefrontal cortex, anterior cingulate and nucleus accumbens for both drugs, but of the anterior cingulate cortex only during relapse, suggesting that a subset of the neural network involved in drug self-administration is activated during relapse.

Heteronuclear NMR studies of the combined Src homology domains 2 and 3 of pp60 c-Src: effects of phosphopeptide binding.

The results of heteronuclear NMR studies on the combined Src homology domains 2 and 3 (SH3-SH2) of pp60 c-Src are presented. Resonance assignments were obtained using heteronuclear triple-resonance experiments in conjunction with 15N-separated nuclear Overhauser effect spectroscopy (NOESY) data. A modified three-dimensional 13CO-15N-1H spectral correlation experiment [(HACA)CO(CA)-NH] with improved sensitivity is presented that provided additional sequential information and resolved several ambiguities. Chemical shifts and sequential- and medium-range NOE cross peaks indicate that the structures of both the SH3 and SH2 portions of the polypeptide are very similar to those of the isolated SH3 and SH2 domains. Binding of a high-affinity phosphopeptide, EPQpYEEIPIYL, induces large chemical shift changes at several locations in the SH2 domain. Comparison with known results for peptide binding to SH2 domains shows that the residues displaying the largest effects are all involved in peptide binding or undergo significant conformational changes upon binding. However, subtle changes of both 1H and 15N chemical shifts are observed for residues within the SH3 domain and the connecting linker region, indicating possible cross-domain communication.

Common neural substrates for the addictive properties of nicotine and cocaine.

Regional brain activation was assessed by mapping of Fos-related protein expression in rats trained to self-administration of intravenous nicotine and cocaine. Both drugs produced specific overlapping patterns of activation in the shell and the core of the nucleus accumbens, medial prefrontal cortex, and medial caudate areas, but not in the amygdala. Thus, the reinforcing properties of cocaine and nicotine map on selected structures of the terminal fields of the mesocorticolimbic dopamine system, supporting the idea that common substrates for these addictive drugs exist.

A 15n-filtered 2D 1H TOCSY experiment for assignment of aromatic ring resonances and selective identification of tyrosine ring resonances in proteins: Description and application to Photoactive Yellow Protein.

A new method to selectively detect the ring resonances of the aromatic residues in 15N-labelled proteins is presented. The experiment consists of a 2D 1H TOCSY sequence withremoval of the amide signals via 15N-filtering. Experiments are acquired in the absence andpresence of water inversion; combining the two spectra allows selective observation of thetyrosine ring resonances and enables the identification of their delta andepsilon ring protons. The experiment is demonstrated on a 15N-labelled sample of Photoactive Yellow Protein and isshown to give good selectivity for tyrosine ring resonances under a wide range oftemperatures and pH values.