Cytogenetic characterization and evaluation of c-MYC gene amplification in PG100, a new Brazilian gastric cancer cell line

Gastric cancer is the fourth most frequent type of cancer and the second cause of cancer mortality worldwide. The genetic alterations described so far for gastric carcinomas include amplifications and mutations of the c-ERBB2, KRAS, MET, TP53, and c-MYC genes. Chromosomal instability described for gastric cancer includes gains and losses of whole chromosomes or parts of them and these events might lead to oncogene overexpression, showing the need for a better understanding of the cytogenetic aspects of this neoplasia. Very few gastric carcinoma cell lines have been isolated. The establishment and characterization of the biological properties of gastric cancer cell lines is a powerful tool to gather information about the evolution of this malignancy, and also to test new therapeutic approaches. The present study characterized cytogenetically PG100, the first commercially available gastric cancer cell line derived from a Brazilian patient who had a gastric adenocarcinoma, using GTG banding and fluorescent in situ hybridization to determine MYC amplification. Twenty metaphases were karyotyped; 19 (95%) of them presented chromosome 8 trisomy, where the MYC gene is located, and 17 (85%) presented a deletion in the 17p region, where the TP53 is located. These are common findings for gastric carcinomas, validating PG100 as an experimental model for this neoplasia. Eighty-six percent of 200 cells analyzed by fluorescent in situ hybridization presented MYC overexpression. Less frequent findings, such as 5p deletions and trisomy 16, open new perspectives for the study of this tumor.

Estudo das reações de oxidação de etileno glicol e glicerol sobre nanopartículas intermetálicas de PtSb/C e PdSb/C

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

COMT Met (158) modulates facial emotion recognition in bipolar I disorder mood episodes

Background: One of the many cognitive deficits reported in bipolar disorder (BD) patients is facial emotion recognition (FER), which has recently been associated with dopaminergic catabolism. Catechol-O-methyltransferase (COMT) is one of the main enzymes involved in the metabolic degradation of dopamine (DA) in the prefrontal cortex (PFC). The COMT gene polymorphism rs4680 (Val(158)Met) Met allele is associated with decreased activity of this enzyme in healthy controls. The objective of this study was to evaluate the influence of Val(158)Met on FER during manic and depressive episodes in BD patients and in healthy controls. Materials and methods: 64 BD type I patients (39 in manic and 25 in depressive episodes) and 75 healthy controls were genotyped for COMT rs4680 and assessed for FER using the Ekman 60 Faces (EK60) and Emotion Hexagon (Hx) tests. Results: Bipolar manic patients carrying the Met allele recognized fewer surprised faces, while depressed patients with the Met allele recognized fewer "angry" and "happy" faces. Healthy homozygous subjects with the Met allele had higher FER scores on the Hx total score, as well as on "disgust" and "angry" faces than other genotypes. Conclusion: This is the first study suggesting that COMT rs4680 modulates FER differently during BD episodes and in healthy controls. This provides evidence that PFC DA is part of the neurobiological mechanisms of social cognition. Further studies on other COMT polymorphisms that include euthymic BD patients are warranted. ClinicalTrials.gov Identifier: NCT00969. (C) 2011 Elsevier B.V. All rights reserved.

I sarcofagi tra III e IV secolo d. C.: problemi di iconologia

Cosa e’ stato fatto e il fine della ricerca in questi tre anni si è esaminato il materiale edito sui sarcofagi del periodo in esame, sui culti funerari, i problemi religiosi ed artistici. Per trovare confronti validi si sono resi necessari alcuni viaggi sia in Italia che all’estero. Lo scopo della ricerca è stato quello di “leggere” i messaggi insiti nelle figurazioni delle casse dei sarcofagi, per comprendere meglio la scelta dei committenti verso determinati temi e la ricezione di questi ultimi da parte del pubblico. La tomba, infatti, è l’ultima traccia che l’uomo lascia di sé ed è quindi importante cercare di determinare l’impatto psicologico del monumento funerario sul proprietario, che spesso lo acquistava quando ancora era in vita, e sui familiari in visita alla tomba durante la celebrazione dei riti per i defunti. Nell’ultimo anno, infine, si è provveduto a scrivere la tesi suddivindendo i capitoli e i pezzi in base all’argomento delle figurazioni (mitologici, di virtù, etc.). I capitoli introduttivi Nel primo capitolo di introduzione si è cercato di dare un affresco per sommi capi del periodo storico in esame da Caracalla a Teodosio e della Chiesa di III e IV secolo, mettendo in luce la profonda crisi che gravava sull’Impero e le varie correnti che frammentavano il cristianesimo. In particolare alcune dispute, come quella riguardante i lapsi, sarà alla base di ipotesi interpretative riguardanti la raffigurazione del rifiuto dei tre giovani Ebrei davanti a Nabuchodonosor. Nel capitolo seguente vengono esaminati i riti funerari e il ruolo dei sarcofagi in tali contesti, evidenziando le diverse situazioni emotive degli osservatori dei pezzi e, quindi, l’importanza dei temi trattati nelle casse. I capitolo Questo capitolo tratta dei sarcofagi mitologici. Dopo una breve introduzione, dove viene spiegata l’entrata in uso dei pezzi in questione, si passa alla discussione dei temi, suddivisi in paragrafi. La prima classe di materiali è qualla con la “caduta di Fetonte” la cui interpretazione sembra chiara: una tragedia familiare. Il compianto sul corpo di un ragazzo morto anzi tempo, al quale partecipa tutto il cosmo. L’afflizione dei familiari è immane e sembra priva di una possibile consolazione. L’unico richiamo a riprendere a vivere è solo quello del dovere (Mercurio che richiama Helios ai propri impegni). La seconda classe è data dalle scene di rapimento divino visto come consolazione e speranza in un aldilà felice, come quelle di Proserpina e Ila. Nella trasposizione della storia di Ila è interessante anche notare il fatto che le ninfe rapitrici del giovane – defunto abbiano le sembianze delle parenti già morte e di un fanciullo, probabilmente la madre, la nonna e un fratello deceduto anzi tempo. La terza classe presenta il tema del distacco e dell’esaltazione delle virtù del defunto nelle vesti dei cacciatori mitici Mealeagro, Ippolito e Adone tutti giovani, forti e coraggiosi. In questi sarcofagi ancor più che negli altri il motivo della giovinezza negata a causa della morte è fondamentale per sottolineare ancora di più la disperazione e il dolore dei genitori rimasti in vita. Nella seguente categoria le virtù del defunto sono ancora il tema dominante, ma in chiave diversa: in questo caso l’eroe è Ercole, intento ad affrontare le sue fatiche. L’interpretazione è la virtù del defunto che lo ha portato a vincere tutte le difficoltà incontrate durante la propria vita, come dimostrerebbe anche l’immagine del semidio rappresentato in età diverse da un’impresa all’altra. Vi è poi la categoria del sonno e della morte, con i miti di Endimione, Arianna e Rea Silvia, analizzato anche sotto un punto di vista psicologico di aiuto per il superamento del dolore per la perdita di un figlio, un marito, o, ancora, della sposa. Accanto ai sarcofagi con immagini di carattere narrativo, vi sono numerosi rilievi con personaggi mitici, che non raccontano una storia, ma si limitano a descrivere situazioni e stati d’animo di felicità. Tali figurazioni si possono dividere in due grandi gruppi: quelle con cortei marini e quelle con il tiaso dionisiaco, facendo dell’amore il tema specifico dei rilievi. Il fatto che quello del tiaso marino abbia avuto così tanta fortuna, forse, per la numerosa letteratura che metteva in relazione l’Aldilà con l’acqua: l’Isola dei Beati oltre l’Oceano, viaggi per mare verso il mondo dei morti. Certo in questo tipo di sarcofagi non vi sono esplicitate queste credenze, ma sembrano più che altro esaltare le gioie della vita. Forse il tutto può essere spiegato con la coesistenza, nei familiari del defunto, della memoria retrospettiva e della proiezione fiduciosa nel futuro. Sostanzialmente era un modo per evocare situazioni gioiose e di godimento sensibile, riferendole ai morti in chiave beneaugurale. Per quanto rigurda il tiaso di Bacco, la sua fortuna è stata dettata dal fatto che il suo culto è sempre stato molto attivo. Bacco era dio della festa, dell’estasi, del vino, era il grande liberatore, partecipe della crescita e della fioritura, il grande forestiero , che faceva saltare gli ordinamenti prestabiliti, i confini della città con la campagna e le convenzioni sociali. Era il dio della follia, al quale le menadi si abbandonavano nella danza rituale, che aggrediva le belve, amante della natura, ma che penetra nella città, sconvolgendola. Del suo seguito facevano parte esseri ibridi, a metà tra l’ordine e la bestialità e animali feroci ammansiti dal vino. I suoi nemici hanno avuto destini orribili di indicibile crudeltà, ma chi si è affidato anima e corpo a lui ha avuto gioia, voluttà, allegria e pienezza di vita. Pur essendo un valente combattente, ha caratteri languidi e a tratti femminei, con forme floride e capelli lunghi, ebbro e inebriante. Col passare del tempo la conquista indiana di alessandro si intrecciò al mito bacchico e, a lungo andare, tutti i caratteri oscuri e minacciosi della divinità scomparirono del tutto. Un esame sistematico dei temi iconografici riconducibili al mito di Bacco non è per nulla facile, in quanto l’oggetto principale delle raffigurazioni è per lo più il tiaso o come corteo festoso, o come gruppi di figure danzanti e musicanti, o, ancora, intento nella vendemmia. Ciò che interessava agli scultori era l’atmosfera gioiosa del tiaso, al punto che anche un episodio importante, come abbiamo visto, del ritrovamento di Arianna si pèerde completamente dentro al corteo, affollatissimo di personaggi. Questo perché, come si è detto anche al riguardo dei corte marini, per creare immagini il più possibile fitte e gravide di possibilità associative. Altro contesto iconografico dionisiaco è quello degli amori con Arianna. Sui sarcofagi l’amore della coppia divina è raffigurato come una forma di stupore e rapimento alla vista dell’altro, un amore fatto di sguardi, come quello del marito sulla tomba della consorte. Un altro tema , che esalta l’amore, è senza ombra di dubbio quello di Achille e Pentesilea, allegoria dell’amore coniugale. Altra classe è qualla con il mito di Enomao, che celebra il coraggio virile e l’attitudine alla vittoria del defunto e, se è presente la scena di matrimonio con Ippodamia, l’amore verso la sposa. Infine vi sono i sarcofagi delle Muse: esaltazione della cultura e della saggezza del morto. II capitolo Accanto ad i grandi ambiti mitologici dei due tiasi del capitolo precedente era la natura a lanciare un messaggio di vita prospera e pacifica. Gli aspetti iconografici diq uesto tema sono due: le stagioni e la vita in un ambiente bucolico. Nonostante la varietà iconografica del soggetto, l’idea di fondo rimane invariata: le stagioni portano ai morti i loro doni affinchè possano goderne tutto l’anno per l’eternità. Per quanto riguarda le immagini bucoliche sono ispirate alla vita dei pastori di ovini, ma ovviamente non a quella reale: i committenti di tali sarcofagi non avevano mai vissuto con i pastori, né pensavano di farlo i loro parenti. Le immagini realistiche di contadini, pastori, pescatori, tutte figure di infimo livello sociale, avevano assunto tratti idilliaci sotto l’influsso della poesia ellenistica. Tutte queste visioni di felicità mancano di riferimenti concreti sia temporali che geografici. Qui non vi sono protagonisti e situazioni dialogiche con l’osservatore esterno, attraverso la ritrattistica. I defunti se appaiono sono all’interno di un tondo al centro della cassa. Nei contesti bucolici, che andranno via, via, prendendo sempre più piede nel III secolo, come in quelli filosofici, spariscono del tutto le scene di lutto e di cordoglio. Le immagini dovevano essere un invito a godersi la vita, ma dovevano anche dire qualcosa del defunto. In una visione retrospettiva, forse, si potrebbero intendere come una dichiarazione che il morto, in vita, non si era fatto mancare nulla. Nel caso opposto, poteve invece essere un augurio ad un’esistenza felice nell’aldilà. III capitolo Qui vengono trattati i sarcofagi con l’esaltazione e l’autorappresentazione del defunto e delle sue virtù in contesti demitizzati. Tra i valori ricorrenti, esaltati nei sarcofagi vi è l’amore coniugale espresso dal tema della dextrarum iunctio, simbolo del matrimonio, la cultura, il potere, la saggezza, il coraggio, esplicitato dalle cacce ad animali feroci, il valore guerriero e la giustizia, dati soprattutto dalle scene di battaglia e di giudizio sui vinti. IV capitolo In questo capitolo si è provato a dare una nuova chiave di lettura ai sarcofagi imperiali di S. Elena e di Costantina. Nel primo caso si tratterebbe della vittoria eterna sul male, mentre nel secondo era un augurio di vita felice nell’aldilà in comunione con Dio e la resurrezione nel giorno del giudizio. V capitolo Il capitolo tratta le mediae voces, quei pezzi che non trovano una facile collocazione in ambito religioso poiché presentano temi neutri o ambivalenti, come quelli del buon pastore, di Prometeo, dell’orante. VI capitolo Qui trovano spazio i sarcofagi cristiani, dove sono scolpite varie scene tratte dalle Sacre Scritture. Anche in questo caso si andati al di là della semplice analisi stilistica per cercare di leggere il messaggio contenuto dalle sculture. Si sono scoperte preghiere, speranze e polemiche con le correnti cristiane considerate eretiche o “traditrici” della vera fede, contro la tradizionale interpretazione che voleva le figurazioni essenzialmente didascaliche. VII capitolo Qui vengono esposte le conclusioni da un punto di vista sociologico e psicologico.

Targeted alpha-radionuclide therapy of functionally critically located gliomas with 213Bi-DOTA-[Thi8,Met(O2)11]-substance P: a pilot trial

Functionally critically located gliomas represent a challenging subgroup of intrinsic brain neoplasms. Standard therapeutic recommendations often cannot be applied, because radical treatment and preservation of neurological function are contrary goals. The successful targeting of gliomas with locally injected beta radiation-emitting (90)Y-DOTAGA-substance P has been shown previously. However, in critically located tumours, the mean tissue range of 5 mm of (90)Y may seriously damage adjacent brain areas. In contrast, the alpha radiation-emitting radionuclide (213)Bi with a mean tissue range of 81 microm may have a more favourable toxicity profile. Therefore, we evaluated locally injected (213)Bi-DOTA-substance P in patients with critically located gliomas as the primary therapeutic modality.

Comparison of C-11 methionine and C-11 choline for PET imaging of brain metastases: a prospective pilot study

The aim of the study was the comparison of C-11 methionine (MET) and C-11 choline (CHO) in the positron emission tomography (PET) imaging of brain metastases in correlation to the histopathology findings in stereotactic biopsy.

Role of hepatitis C virus genotype 3 in liver fibrosis progression - a systematic review and meta-analysis

The progression of liver fibrosis in chronic hepatitis C has long been considered to be independent from viral genotypes. However, recent studies suggest an association between Hepatitis C virus (HCV) genotype 3 and accelerated liver disease progression. We completed a systematic review and meta-analysis of studies evaluating the association between HCV genotypes and fibrosis progression. PubMed, Embase and ISI Web of Knowledge databases were searched for cohort, cross-sectional and case-control studies on treatment-naïve HCV-infected adults in which liver fibrosis progression rate (FPR) was assessed by the ratio of fibrosis stage in one single biopsy to the duration of infection (single-biopsy studies) or from the change in fibrosis stage between two biopsies (paired biopsies studies). A random effect model was used to derive FPR among different HCV genotypes. Eight single-biopsy studies (3182 patients, mean/median duration of infection ranging from 9 to 21 years) and eight paired biopsies studies (mean interval between biopsies 2-12 years) met the selection criteria. The odds ratio for the association of genotype 3 with accelerated fibrosis progression was 1.52 (95% CI 1.12-2.07, P = 0.007) in single-biopsy studies and 1.37 (95% CI 0.87-2.17, P = 0.17) in paired biopsy studies. In conclusion, viral genotype 3 was associated with faster fibrosis progression in single-biopsy studies. This observation may have important consequences on the clinical management of genotype 3-infected patients. The association was not significant in paired biopsies studies, although the latter may be limited by important indication bias, short observation time and small sample size.

Met-thodology

Met-regulated expression signature defines a subset of human hepatocellular carcinomas with poor prognosis and aggressive phenotype. Kaposi-Novak P, Lee JS, Gomez-Quiroz L, Coulouarn C, Factor VM, Thorgeirsson SS. Identification of specific gene expression signatures characteristic of oncogenic pathways is an important step toward molecular classification of human malignancies. Aberrant activation of the Met signaling pathway is frequently associated with tumour progression and metastasis. In this study, we defined the Met-dependent gene expression signature using global gene expression profiling of WT and Met-deficient primary mouse hepatocytes. Newly identified transcriptional targets of the Met pathway included genes involved in the regulation of oxidative stress responses as well as cell motility, cytoskeletal organization, and angiogenesis. To assess the importance of a Met-regulated gene expression signature, a comparative functional genomic approach was applied to 242 human hepatocellular carcinomas (HCCs) and 7 metastatic liver lesions. Cluster analysis revealed that a subset of human HCCs and all liver metastases shared the Met-induced expression signature. Furthermore, the presence of the Met signature showed significant correlation with increased vascular invasion rate and microvessel density as well as with decreased mean survival time of HCC patients. We conclude that the genetically defined gene expression signatures in combination with comparative functional genomics constitute an attractive paradigm for defining both the function of oncogenic pathways and the clinically relevant subgroups of human cancers. [Abstract reproduced by permission of J Clin Invest 2006;116:1582-1595].

C-Port Flex-A-assisted automated anastomosis for high-flow extracranial-intracranial bypass surgery in patients with symptomatic carotid artery occlusion: a feasibility study. Clinical article

OBJECT: Preliminary experience with the C-Port Flex-A Anastomosis System (Cardica, Inc.) to enable rapid automated anastomosis has been reported in coronary artery bypass surgery. The goal of the current study was to define the feasibility and safety of this method for high-flow extracranial-intracranial (EC-IC) bypass surgery in a clinical series. METHODS: In a prospective study design, patients with symptomatic carotid artery (CA) occlusion were selected for C-Port-assisted high-flow EC-IC bypass surgery if they met the following criteria: 1) transient or moderate permanent symptoms of focal ischemia; 2) CA occlusion; 3) hemodynamic instability; and 4) had provided informed consent. Bypasses were done using a radial artery graft that was proximally anastomosed to the superficial temporal artery trunk, the cervical external, or common CA. All distal cerebral anastomoses were performed on M2 branches using the C-Port Flex-A system. RESULTS: Within 6 months, 10 patients were enrolled in the study. The distal automated anastomosis could be accomplished in all patients; the median temporary occlusion time was 16.6+/-3.4 minutes. Intraoperative digital subtraction angiography (DSA) confirmed good bypass function in 9 patients, and in 1 the anastomosis was classified as fair. There was 1 major perioperative complication that consisted of the creation of a pseudoaneurysm due to a hardware problem. In all but 1 case the bypass was shown to be patent on DSA after 7 days; furthermore, in 1 patient a late occlusion developed due to vasospasm after a sylvian hemorrhage. One-week follow-up DSA revealed transient asymptomatic extracranial spasm of the donor artery and the radial artery graft in 1 case. Two patients developed a limited zone of infarction on CT scanning during the follow-up course. CONCLUSIONS: In patients with symptomatic CA occlusion, C-Port Flex-A-assisted high-flow EC-IC bypass surgery is a technically feasible procedure. The system needs further modification to achieve a faster and safer anastomosis to enable a conclusive comparison with standard and laser-assisted methods for high-flow bypass surgery.

Molecular mechanisms of Ca$\sp{2+}$ signal transduction from cardiac troponin C to troponin I

$\rm Ca\sp{2+}$-dependent exposure of an N-terminal hydrophobic region in troponin C (TnC) is thought to be important for the regulation of contraction in striated muscle. To study these conformational changes in cardiac troponin (cTnC), the $\varepsilon$C and $\varepsilon$H chemical shifts for all 10 Met residues in cTnC were sequence-specific assigned on NMR spectra using a combination of two dimensional NMR techniques and site-directed mutagenesis. The assigned methyl-Met chemical shifts were used as structural markers to monitor conformational changes induced by $\rm Ca\sp{2+}.$ The results showed that binding of $\rm Ca\sp{2+}$ to the regulatory site in the N-domain induced large changes in the $\varepsilon$H and $\varepsilon$C chemical shifts of Met 45, Met 80, Met 81 in the predicted N-terminal hydrophobic region, but had no effect on the chemical shifts of Met residues located in the C-domain. These results suggest that the $\rm Ca\sp{2+}$-dependent functions of cTnC are mainly through N-terminal domain of cTnC.^ To further define the molecular mechanism by which TnC regulates muscle contraction, single Cys residues were engineered at positions 45, 81, 84 or 85 in the N-terminal hydrophobic region of cTnC to provide sites for attachment of specific blocking groups. Blocking groups were coupled to these Cys residues in cTnC mutants and the covalent adducts were tested for activity in TnC-extracted myofibrils. Covalent modification of cTnC(C45) had no effect on maximal myofibril ATPase activity. Greatly decreased myofibril ATPase activity resulted when the peptide or biotin was conjugated to residue 81 in cTnC(C81), while less inhibition resulted from covalent modification of cTnC(C84) or cTnC(C85). The results suggest that limited sites of the N-terminal hydrophobic region in cTnC are important for transducing the $\rm Ca\sp{2+}$ signal to troponin I (TnI) and are sensitive to modification, while other regions are less important or can adapt to steric hindrances introduced by bulky blocking groups.^ Although the exposed TnI interaction site in the N-terminal hydrophobic region of TnC is crucial for function of TnC, other regions in the N-domain of TnC may also participate in transducing the $\rm Ca\sp{2+}$ signal and conferring the maximal activation of actomyosin ATPase. The interactions between the B-/C-helices of cTnC and cTnI were characterized using a combination of site-directed mutagenesis, fluorescence and covalent modification. The results suggest that the $\rm Ca\sp{2+}$-dependent interactions of the B-/C-helices of cTnC with TnI may be required for the maximal activation of muscle contraction. ^

STABILITY AND FOLDING OF MUTANT FORMS OF ISO-1 CYTOCHROME-C FROM SACCHAROMYCES CEREVISIAE

Partially functional forms of iso-1-cytochrome c from Saccharomyces cerevisiae were obtained by replacements of the evolutionarily conserved proline 71 with valine, isoleucine and threonine (Ernst et.al.,1985). Pro-71 lies at the juncture of two short helical regions and is believed to be important for proper local polypeptide chain folding within the iso-1-cytochrome c structure.^ To study folding in the absence of intermolecular disulfide dimer formation the free sulfhydryl group of Cys-102 was modified in both wild type and mutant proteins with an alkylating reagent, methyl methanethiosulfonate. Spectral analysis of the wild type and mutant proteins shows that the native-like functional (or partially functional) folded structure of cytochrome c is retained in the chemically modified derivatives. The replacement of Pro-71 with valine, isoleucine or threonine reduces the intensity of the 696 nm absorbance band which is an indicator of the Met-80 ligation to the heme. Thermal stability and guanidine hydrochloride unfolding studies of the mutant proteins shows a destabilization of the protein as a result of mutation. The degree of destabilization depends on the chemical nature of the substituent amino acid in the mutant protiens.^ Kinetics of folding/unfolding reactions of the proteins were monitored by fluorescence changes using stopped flow mixing to obtain guanidine hydrochloride concentration jumps ending below, within, and above the transition zone. The replacement of Pro-71 alters the rate on one of the fastest phases, $\tau\sb3$, while the two other phases, $\tau\sb1$ & $\tau\sb2$, remain the same.^ Slow refolding kinetic studies indicate that replacement of Pro-71 does not completely eliminate the absorbance or fluorescence detected slow phases leading to the conclusion that Pro-71 is not involved in the generation of the slow phases in the folding kinetics of iso-1-cytochrome c.^ The alkaline conformational change involving the disappearance of the 696 nm absorbance band occurs with increasing pH in the alkaline pH region (Davis et al., 1974). The apparent pK of this conformational change in mutant proteins is shifted as much as two pH units compared to wild type. The equilibrium and kinetic data of alkaline transition for the wild type follows a simple mechanism proposed by Davis et al., (1974) for horse heart cytochrome c. A more complex mechanism is proposed for the behavior of the mutant proteins. ^

Efficacy of motivational enhancement therapy on alcohol use disorders in patients with chronic hepatitis C: a randomized controlled trial

AIMS: To determine the efficacy of motivational enhancement therapy (MET) on alcohol use in patients with the hepatitis C virus (HCV) and an alcohol use disorder (AUD). DESIGN: Randomized, single-blind, controlled trial comparing MET to a control education condition with 6-month follow-up. SETTING: Patients were recruited from hepatitis clinics at the Minneapolis, Minnesota and Portland, Oregon Veterans Affairs Health Care Systems, USA. PARTICIPANTS AND INTERVENTION: Patients with HCV, an AUD and continued alcohol use (n = 139) were randomized to receive either MET (n = 70) or a control education condition (n = 69) over 3 months. MEASUREMENTS: Data were self-reported percentage of days abstinent from alcohol and number of standard alcohol drinks per week 6 months after randomization. FINDINGS: At baseline, subjects in MET had 34.98% days abstinent, which increased to 73.15% at 6 months compared to 34.63 and 59.49% for the control condition. Multi-level models examined changes in alcohol consumption between MET and control groups. Results showed a significant increase in percentage of days abstinent overall (F(1120.4)  = 28.04, P < 0.001) and a significant group × time effect (F(1119.9)  = 5.23, P = 0.024) with the MET group showing a greater increase in percentage of days abstinent at 6 months compared with the education control condition. There were no significant differences between groups for drinks per week. The effect size of the MET intervention was moderate (0.45) for percentage of days abstinent. CONCLUSION: Motivational enhancement therapy (MET) appears to increase the percentage of days abstinent in patients with chronic hepatitis C, alcohol use disorders and ongoing alcohol use. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

The association between diabetes and hepatitis C: A systematic review of epidemiological evidence

Background. Several studies have proposed a link between type 2 Diabetes mellitus (DM2) and Hepatitis C infection (HCV) with conflicting results. Since DM2 and HCV have high prevalence, establishing a link between the two may guide further studies aimed at DM2 prevention. A systematic review was conducted to estimate the magnitude and direction of association between DM2 and HCV. Temporality was assessed from cohort studies and case-control studies where such information was available. ^ Methods. MEDLINE searches were conducted for studies that provided risk estimates and fulfill criteria regarding the definition of exposure (HCV) and outcomes (DM2). HCV was defined in terms of method of diagnosis, laboratory technique and method of data collection; DM2 was defined in terms of the classification [World Health Organization (WHO) and American Diabetes Association (ADA)] 1-3 used for diagnosis, laboratory technique and method of data collection. Standardized searches and data abstraction for construction of tables was performed. Unadjusted or adjusted measures of association for individual studies were obtained or calculated from the full text of the studies. Template designed by Dr. David Ramsey. ^ Results. Forty-six studies out of one hundred and nine potentially eligible articles finally met the inclusion and exclusion criteria and were classified separately based on the study design as cross-sectional (twenty four), case-control (fifteen) or cohort studies (seven). The cohort studies showed a three-fold high (confidence interval 1.66–6.29) occurrence of DM2 in individuals with HCV compared to those who were unexposed to HCV and cross sectional studies had a summary odds ratio of 2.53 (1.96, 3.25). In case control studies, the summary odds ratio for studies done in subjects with DM2 was 3.61 (1.93, 6.74); in HCV, it was 2.30 (1.56, 3.38); and all fifteen studies, together, yielded an odds ratio of 2.60 (1.82, 3.73). ^ Conclusion. The above results support the hypothesis that there is an association between DM and HCV. The temporal relationship evident from cohort studies and proposed pathogenic mechanisms also suggest that HCV predisposes patients to development of DM2. Further cohort or prospective studies are needed, however, to determine whether treatment of HCV infections prevents development of DM2.^

NMR structural analysis of cardiac troponin C: Monitoring conformational changes induced by binding calcium, troponin I, and calcium -sensitizing drugs

Contraction of cardiac muscle is regulated through the Ca2+ dependent protein-protein interactions of the troponin complex (Tn). The critical role cardiac troponin C (cTnC) plays as the Ca2+ receptor in this complex makes it an attractive target for positive inotropic compounds. In this study, the ten Met methyl groups in cTnC, [98% 13C ϵ]-Met cTnC, are used as structural markers to monitor conformational changes in cTnC and identify sites of interaction between cTnC and cardiac troponin I (cTnI) responsible for the Ca2+ dependent interactions. In addition the structural consequences that a number of Ca2+-sensitizing compounds have on free cTnC and the cTnC·cTnI complex were characterized. Using heteronuclear NMR experiments and monitoring chemical shift changes in the ten Met methyl 1H-13C correlations in 3Ca2+ cTnC when bound to cTnI revealed an anti-parallel arrangement for the two proteins such that the N-domain of cTnI interacts with the C-domain of cTnC. The large chemical shifts in Mets-81, -120, and -157 identified points of contact between the proteins that include the C-domain hydrophobic surface in cTnC and the A, B, and D helical interface located in the regulatory N-domain of cTnC. TnI association [cTnI(33–80), cTnI(86–211), or cTnI(33–211)] was found also to dramatically reduce flexibility in the D/E central linker of cTnC as monitored by line broadening in the Met 1H- 13C correlations of cTnC induced by a nitroxide spin label, MTSSL, covalently attached to cTnC at Cys 84. TnI association resulted in an extended cTnC that is unlike the compact structure observed for free cTnC. The Met 1H-13C correlations also allowed the binding characteristics of bepridil, TFP, levosimendan, and EMD 57033 to the apo, 2Ca2+, and Ca2+ saturated forms of cTnC to be determined. In addition, the location of drug binding on the 3Ca2+cTnC·cTnI complex was identified for bepridil and TFP. Use of a novel spin-labeled phenothiazine, and detection of isotope filtered NOEs, allowed identification of drug binding sites in the shallow hydrophobic cup in the C-terminal domain, and on two hydrophobic surfaces on N-regulatory domain in free 3Ca2+ cTnC. In contrast, only one N-domain drug binding site exists in 3Ca2+ cTnC·cTnI complex. The methyl groups of Met 45, 60 and 80, which are grouped in a hydrophobic patch near site II in cTnC, showed the greatest change upon titration with bepridil or TFP, suggesting that this is a critical site of drug binding in both free cTnC and when associated with cTnI. The strongest NOEs were seen for Met-60 and -80, which are located on helices C and D, respectively, of Ca2+ binding site II. These results support the conclusion that the small hydrophobic patch which includes Met-45, -60, and -80 constitutes a drug binding site, and that binding drugs to this site will lead to an increase in Ca2+ binding affinity of site II while preserving maximal cTnC activity. Thus, the subregion in cTnC makes a likely target against which to design new and selective Ca2+-sensitizing compounds. ^