Bipyridyl- and biphenyl-DNA: A recognition motif based on interstrand aromatic stacking

The synthesis and incorporation into oligonucleotides of C-nucleosides containing the two aromatic, non-hydrogen-bonding nucleobase substitutes biphenyl (I) and bipyridyl (Y) are described. Their homo- and hetero-recognition properties in different sequential arrangements were then investigated via UV-melting curve analysis, gel mobility assays, CD- and NMR spectroscopy. An NMR analysis of a dodecamer duplex containing one biphenyl pair in the center, as well as CD data on duplexes with multiple insertions provide further evidence for the zipper-like interstrand stacking motif that we proposed earlier based on molecular modeling. UV-thermal melting experiments with duplexes containing one to up to seven I- or Y base pairs revealed a constant increase in T(m) in the case of I and a constant decrease for Y. Mixed I/Y base pairs lead to stabilities in between the homoseries. Insertion of alternating I/abasic site- or Y/abasic site pairs strongly decreases the thermal stability of duplexes. Asymmetric distribution of I- or Y residues on either strand of the duplex were also investigated in this context. Duplexes with three natural base pairs at both ends and 50 % of I pairs in the center are still readily formed, while duplexes with blunt ended I pairs tend to aggregate unspecifically. Duplexes with one natural overhang at the end of a I-I base pair tract can both aggregate or form ordered duplexes, depending on the nature of the natural bases in the overhang

Elderly age is not a negative predictive factor for virological response to therapy with pegylated interferon-α and ribavirin in chronic hepatitis C virus patients

BACKGROUND & AIMS: Age is frequently discussed as negative host factor to achieve a sustained virological response (SVR) to antiviral therapy of chronic hepatitis C. However, elderly patients often show advanced fibrosis/cirrhosis as known negative predictive factor. The aim of this study was to assess age as an independent predictive factor during antiviral therapy. METHODS: Overall, 516 hepatitis C patients were treated with pegylated interferon-α and ribavirin, thereof 66 patients ≥60 years. We analysed the impact of host factors (age, gender, fibrosis, haemoglobin, previous hepatitis C treatment) and viral factors (genotype, viral load) on SVR per therapy course by performing a generalized estimating equations (GEE) regression modelling, a matched pair analysis and a classification tree analysis. RESULTS: Overall, SVR per therapy course was 42.9 and 26.1%, respectively, in young and elderly patients with hepatitis C virus (HCV) genotypes 1/4/6. The corresponding figures for HCV genotypes 2/3 were 74.4 and 84%. In the GEE model, age had no significant influence on achieving SVR. In matched pair analysis, SVR was not different in young and elderly patients (54.2 and 55.9% respectively; P = 0.795 in binominal test). In classification tree analysis, age was not a relevant splitting variable. CONCLUSIONS: Age is not a significant predictive factor for achieving SVR, when relevant confounders are taken into account. As life expectancy in Western Europe at age 60 is more than 20 years, it is reasonable to treat chronic hepatitis C in selected elderly patients with relevant fibrosis or cirrhosis but without major concomitant diseases, as SVR improves survival and reduces carcinogenesis.

Robust meta-analytic-predictive priors in clinical trials with historical control information.

Historical information is always relevant for clinical trial design. Additionally, if incorporated in the analysis of a new trial, historical data allow to reduce the number of subjects. This decreases costs and trial duration, facilitates recruitment, and may be more ethical. Yet, under prior-data conflict, a too optimistic use of historical data may be inappropriate. We address this challenge by deriving a Bayesian meta-analytic-predictive prior from historical data, which is then combined with the new data. This prospective approach is equivalent to a meta-analytic-combined analysis of historical and new data if parameters are exchangeable across trials. The prospective Bayesian version requires a good approximation of the meta-analytic-predictive prior, which is not available analytically. We propose two- or three-component mixtures of standard priors, which allow for good approximations and, for the one-parameter exponential family, straightforward posterior calculations. Moreover, since one of the mixture components is usually vague, mixture priors will often be heavy-tailed and therefore robust. Further robustness and a more rapid reaction to prior-data conflicts can be achieved by adding an extra weakly-informative mixture component. Use of historical prior information is particularly attractive for adaptive trials, as the randomization ratio can then be changed in case of prior-data conflict. Both frequentist operating characteristics and posterior summaries for various data scenarios show that these designs have desirable properties. We illustrate the methodology for a phase II proof-of-concept trial with historical controls from four studies. Robust meta-analytic-predictive priors alleviate prior-data conflicts ' they should encourage better and more frequent use of historical data in clinical trials.

The prevalence and predictive value of dipstick urine protein in HIV-positive persons in Europe.

INTRODUCTION Proteinuria (PTU) is an important marker for the development and progression of renal disease, cardiovascular disease and death, but there is limited information about the prevalence and factors associated with confirmed PTU in predominantly white European HIV+ persons, especially in those with an estimated glomerular filtration rate (eGFR) of 60 mL/min/1.73 m(2). PATIENTS AND METHODS Baseline was defined as the first of two consecutive dipstick urine protein (DPU) measurements during prospective follow-up >1/6/2011 (when systematic data collection began). PTU was defined as two consecutive DUP >1+ (>30 mg/dL) >3 months apart; persons with eGFR <60 at either DPU measurement were excluded. Logistic regression investigated factors associated with PTU. RESULTS A total of 1,640 persons were included, participants were mainly white (n=1,517, 92.5%), male (n=1296, 79.0%) and men having sex with men (n=809; 49.3%). Median age at baseline was 45 (IQR 37-52 years), and CD4 was 570 (IQR 406-760/mm(3)). The median baseline date was 2/12 (IQR 11/11-6/12), and median eGFR was 99 (IQR 88-109 mL/min/1.73 m(2)). Sixty-nine persons had PTU (4.2%, 95% CI 3.2-4.7%). Persons with diabetes had increased odds of PTU, as were those with a prior non-AIDS (1) or AIDS event and those with prior exposure to indinavir. Among females, those with a normal eGFR (>90) and those with prior abacavir use had lower odds of PTU (Figure 1). CONCLUSIONS One in 25 persons with eGFR>60 had confirmed proteinuria at baseline. Factors associated with PTU were similar to those associated with CKD. The lack of association with antiretrovirals, particularly tenofovir, may be due to the cross-sectional design of this study, and additional follow-up is required to address progression to PTU in those without PTU at baseline. It may also suggest other markers are needed to capture the deteriorating renal function associated with antiretrovirals may be needed at higher eGFRs. Our findings suggest PTU is an early marker for impaired renal function.

Prognostic and predictive roles of MGMT protein expression and promoter methylation in sporadic pancreatic neuroendocrine neoplasms.

BACKGROUND/AIMS O(6)-methylguanine-methyltransferase (MGMT) is an important enzyme of DNA repair. MGMT promoter methylation is detectable in a subset of pancreatic neuroendocrine neoplasms (pNEN). A subset of pNEN responds to the alkylating agent temozolomide (TMZ). We wanted to correlate MGMT promoter methylation with MGMT protein loss in pNEN, correlate the findings with clinico-pathological data and determine the role of MGMT to predict response to TMZ chemotherapy. METHODS We analysed a well-characterized collective of 141 resected pNEN with median follow-up of 83 months for MGMT protein expression and promoter methylation using methylation-specific PCR (MSP). A second collective of 10 metastasized, pretreated and progressive patients receiving TMZ was used to examine the predictive role of MGMT by determining protein expression and promoter methylation using primer extension-based quantitative PCR. RESULTS In both collectives there was no correlation between MGMT protein expression and promoter methylation. Loss of MGMT protein was associated with an adverse outcome, this prognostic value, however, was not independent from grade and stage in multivariate analysis. Promoter hypermethylation was significantly associated with response to TMZ. CONCLUSION Loss of MGMT protein expression is associated with adverse outcome in a surgical series of pNET. MGMT promoter methylation could be a predictive marker for TMZ chemotherapy in pNEN, but further, favourably prospective studies will be needed to confirm this result and before this observation can influence clinical routine.

PDZ domain-binding motif regulates cardiomyocyte compartment-specific NaV1.5 channel expression and function

BACKGROUND Sodium channel NaV1.5 underlies cardiac excitability and conduction. The last 3 residues of NaV1.5 (Ser-Ile-Val) constitute a PDZ domain-binding motif that interacts with PDZ proteins such as syntrophins and SAP97 at different locations within the cardiomyocyte, thus defining distinct pools of NaV1.5 multiprotein complexes. Here, we explored the in vivo and clinical impact of this motif through characterization of mutant mice and genetic screening of patients. METHODS AND RESULTS To investigate in vivo the regulatory role of this motif, we generated knock-in mice lacking the SIV domain (ΔSIV). ΔSIV mice displayed reduced NaV1.5 expression and sodium current (INa), specifically at the lateral myocyte membrane, whereas NaV1.5 expression and INa at the intercalated disks were unaffected. Optical mapping of ΔSIV hearts revealed that ventricular conduction velocity was preferentially decreased in the transversal direction to myocardial fiber orientation, leading to increased anisotropy of ventricular conduction. Internalization of wild-type and ΔSIV channels was unchanged in HEK293 cells. However, the proteasome inhibitor MG132 rescued ΔSIV INa, suggesting that the SIV motif is important for regulation of NaV1.5 degradation. A missense mutation within the SIV motif (p.V2016M) was identified in a patient with Brugada syndrome. The mutation decreased NaV1.5 cell surface expression and INa when expressed in HEK293 cells. CONCLUSIONS Our results demonstrate the in vivo significance of the PDZ domain-binding motif in the correct expression of NaV1.5 at the lateral cardiomyocyte membrane and underline the functional role of lateral NaV1.5 in ventricular conduction. Furthermore, we reveal a clinical relevance of the SIV motif in cardiac disease.

General distress, hopelessness—suicidal ideation and worrying in adolescence: Concurrent and predictive validity of a symptom-level bifactor model for clinical diagnoses

BACKGROUND: Clinical disorders often share common symptoms and aetiological factors. Bifactor models acknowledge the role of an underlying general distress component and more specific sub-domains of psychopathology which specify the unique components of disorders over and above a general factor. METHODS: A bifactor model jointly calibrated data on subjective distress from The Mood and Feelings Questionnaire and the Revised Children's Manifest Anxiety Scale. The bifactor model encompassed a general distress factor, and specific factors for (a) hopelessness-suicidal ideation, (b) generalised worrying and (c) restlessness-fatigue at age 14 which were related to lifetime clinical diagnoses established by interviews at ages 14 (concurrent validity) and current diagnoses at 17 years (predictive validity) in a British population sample of 1159 adolescents. RESULTS: Diagnostic interviews confirmed the validity of a symptom-level bifactor model. The underlying general distress factor was a powerful but non-specific predictor of affective, anxiety and behaviour disorders. The specific factors for hopelessness-suicidal ideation and generalised worrying contributed to predictive specificity. Hopelessness-suicidal ideation predicted concurrent and future affective disorder; generalised worrying predicted concurrent and future anxiety, specifically concurrent generalised anxiety disorders. Generalised worrying was negatively associated with behaviour disorders. LIMITATIONS: The analyses of gender differences and the prediction of specific disorders was limited due to a low frequency of disorders other than depression. CONCLUSIONS: The bifactor model was able to differentiate concurrent and predict future clinical diagnoses. This can inform the development of targeted as well as non-specific interventions for prevention and treatment of different disorders.

DNA with Hydrophobic Base Substitutes: A Stable, Zipperlike Recognition Motif Based On Interstrand-Stacking Interactions

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Triple-helix formation in the antiparallel binding motif of oligodeoxynucleotides containing N(9)- and N(7)-2-aminopurine deoxynucleosides

Triplex-forming oligodeoxynucleotide 15mers, designed to bind in the antiparallel triple-helical binding motif, containing single substitutions (Z) of the four isomeric alphaN(7)-, betaN(7)-, alphaN(9)- and betaN(9)-2-aminopurine (ap)-deoxyribonucleosides were prepared. Their association with double-stranded DNA targets containing all four natural base pairs (X-Y) opposite the aminopurine residues was determined by quantitative DNase I footprint titration in the absence of monovalent metal cations. The corresponding association constants were found to be in a rather narrow range between 1.0 x 10(6) and 1.3 x 10(8) M(-1). The following relative order in Z x X-Y base-triple stabilities was found: Z = alphaN(7)ap: T-A > A-T> C-G approximately G-C; Z = betaN(7)ap: A-T > C-G > G-C > T-A; Z = alphaN(9)ap: A-T = G-C > T-A > C-G; and Z = betaN(9)ap: G-C > A-T > C-G > T-A

Conformational diversity versus nucleic acid triplex stability, a combinatorial study

The stability of a triple helix formed between a DNA duplex and an incoming oligonucleotide strand strongly depends on the solvent conditions and on intrinsic chemical and conformational factors. Attempts to increase triple helix stability in the past included chemical modification of the backbone, sugar ring, and bases in the third strand. However, the predictive power of such modifications is still rather poor. We therefore developed a method that allows for rapid screening of conformationally diverse third strand oligonucleotides for triplex stability in the parallel pairing motif to a given DNA double helix sequence. Combinatorial libraries of oligonucleotides of the requisite (fixed) base composition and length that vary in their sugar unit (ribose or deoxyribose) at each position were generated. After affinity chromatography against their corresponding immobilized DNA target duplex, utilizing a temperature gradient as the selection criterion, the oligonucleotides forming the most stable triple helices were selected and characterized by physicochemical methods. Thus, a series of oligonucleotides were identified that allowed us to define basic rules for triple helix stability in this conformationally diverse system. It was found that ribocytidines in the third strand increase triplex stability relative to deoxyribocytidines independently of the neighboring bases and position along the strand. However, remarkable sequence-dependent differences in stability were found for (deoxy)thymidines and uridines