The Acidic Amino-Terminal Region of Varicella-Zoster Virus Open Reading Frame 4 Protein Is Required for Transactivation and Can Functionally Replace the Corresponding Region of Herpes Simplex Virus ICP27

AbstractBoth varicella-zoster virus open reading frame 4 (ORF4) protein and its herpes simplex virus type 1 homolog ICP27 have highly acidic amino-terminal regions and cysteine-rich carboxy-terminal regions. To investigate the functional domains of these proteins, mutants were constructed and their transregulatory functions were tested in transient expression assays using two reporter plasmids, pTK-CAT-SV40A and pTK-CAT-synA, containing the same promoter sequences but different mRNA processing signals. ORF4 transactivates both pTK-CAT-SV40A and pTK-CAT-synA, while ICP27 transrepresses pTKCAT-SV40A and transactivates pTK-CAT-synA. Deletion of the ORF4 amino-terminal region abolished most of the transactivating activity for pTK-CAT-synA but retained most of the transactivating activity for pTK-CAT-SV40A. Construction of chimeric ORF4-ICP27 molecules indicated that the ORF4 amino-terminal region was able to replace the corresponding region of ICP27 which is required for both transrepression of pTK-CAT-SV40A and transactivation of pTK-CAT-synA. Similarly, the ICP27 amino-terminal region was able to partially replace the corresponding region of ORF4 which is required for transactivation of pTK-CAT-synA. Thus, while ORF4 and ICP27 have different properties in transient expression assays, the aminoterminal regions of ORF4 and ICP27 are functionally homologous to each other and are important in regulating gene expression

Recognition of the latency-associated immediate early protein IE63 of varicella-zoster virus by human memory T-lymphocytes

peer reviewedVaricella-zoster virus (VZV) is a human alpha herpesvirus that establishes latency in sensory ganglia. Latency is characterized by the abundant expression of the immediate early protein 63 (IE63), whereas other viral proteins have not yet been detected during the quiescent phase of VZV infection. The IE63 protein is a component of the virion and is expressed very early in the infectious cycle. The IE63 protein is also expressed in skin during episodes of varicella and herpes zoster. We have evaluated the cell-mediated immune response against IE63 in naturally immune adults with a history of chickenpox, by T lymphoproliferation and cytotoxicity assays. Among donors who had T cell proliferation to unfractionated VZV Ags from infected cell extract, 59% had T cell recognition of purified IE63. The CTL response to IE63 was equivalent to CTL recognition of IE62, the major tegument component of VZV whose immunogenicity has been previously described. IgG Abs against IE63 were detected in serum from healthy immune adults. These results indicate that IE63 is an important target of immunity to VZV. T cell recognition of IE63 is likely to be involved in controlling VZV reactivation from latency

Transfer of heroin profiling method from conventional GC-MS to Fast-GC-MS and UHPLC-MS/MS

Peer reviewe

Innovative methodology to transfer conventional GC-MS heroin profiling to UHPLC-MS/MS

Nowadays, in forensic laboratories, heroin profiling is frequently carried out by gas chromatography coupled with mass spectrometry (GC-MS). This analytical technique is well established, provides good sensitivity and reproducibility, and allows the use of large databases. Despite those benefits, recently introduced analytical techniques, such as ultra-high-pressure liquid chromatography (UHPLC), could offer better chromatographic performance, which needs to be considered to increase the analysis throughput for heroin profiling. With the latter, chromatographic conditions were optimized through commercial modeling software and two atmospheric pressure ionization sources were evaluated. Data obtained from UHPLC-MS/MS were thus transferred, thanks to mathematical models to mimic GC-MS data. A calibration and a validation set of representative heroin samples were selected among the database to establish a transfer methodology and assess the models' abilities to transfer using principal component analysis and hierarchical classification analysis. These abilities were evaluated by computing the frequency of successful classification of UHPLC-MS/MS data among GC-MS database. Seven mathematical models were tested to adjust UHPLC-MS/MS data to GC-MS data. A simplified mathematical model was finally selected and offered a frequency of successful transfer equal to 95%. Figur

COMBINATION OF INDEPENDENT COMPONENT ANALYSIS, DESIGN OF EXPERIMENTS AND DESIGN SPACE FOR A NOVEL METHODOLOGY TO DEVELOP CHROMATOGRAPHIC METHODS

As defined by ICH [1] and FDA, Quality by Design (QbD) stands for “a systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management”. A risk–based QbD–compliant approach is proposed for the robust development of analytical methods. This methodology based on Design of Experiments (DoE) to study the experimental domain models the retention times at the beginning, the apex and the end of each peak corresponding to the compounds of a mixture and uses the separation criterion (S) rather than the resolution (RS) as a Critical Quality Attribute. Stepwise multiple linear regressions are used to create the models. The estimated error is propagated from the modelled responses to the separation criterion (S) using Monte Carlo simulations in order to estimate the predictive distribution of the separation criterion (S) over the whole experimental domain. This allows finding ranges of operating conditions that will guarantee a satisfactory quality of the method in its future use. These ranges define the Design Space (DS) of the method. In chromatographic terms, the chromatograms processed at operating conditions within the DS will assuredly show high quality, with well separated peaks and short run time, for instance. This Design Space can thus be defined as the subspace, necessarily encompassed in the experimental domain (i.e. the knowledge space), within which the probability for the criterion to be higher than an advisedly selected threshold is higher than a minimum quality level. Precisely, the DS is defined as “the multidimensional combination and interaction of input variables (e.g., material attributes) and process parameters that have been demonstrated to provide assurance of quality” [1]. Therefore, this DS defines a region of operating conditions that provide prediction of assurance of quality rather than only quality as obtained with traditional mean response surface optimisation strategies. For instance, in the liquid chromatography there is a great difference in e.g. predicting a resolution (RS) higher than 1.5 vs. predicting that the probability for RS to be higher than 1.5 (i.e. P(RS> 1.5)) is high. The presentation of this global methodology will be illustrated for the robust optimisation and DS definition of several liquid chromatographic methods dedicated to the separation of different mixtures: pharmaceutical formulations, API and impurities/degradation products, plant extracts, separation of enantiomers, … References [1] International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use, Topic Q8(R2): Pharmaceutical development, Geneva, 2009

Incidence of rotavirus gastroenteritis by age in African, Asian and European children: Relevance for timing of rotavirus vaccination

© 2016 The Author(s). Published with license by Taylor & Francis. © GSK Biologicals SA.Variability in rotavirus gastroenteritis (RVGE) epidemiology can influence the optimal vaccination schedule. We evaluated regional trends in the age of RVGE episodes in low- to middle- versus high-income countries in three continents. We undertook a post-hoc analysis based on efficacy trials of a human rotavirus vaccine (HRV; Rotarix™, GSK Vaccines), in which 1348, 1641, and 5250 healthy infants received a placebo in Europe (NCT00140686), Africa (NCT00241644), and Asia (NCT00197210, NCT00329745). Incidence of any/severe RVGE by age at onset was evaluated by active surveillance over the first two years of life. Severity of RVGE episodes was assessed using the Vesikari-scale. The incidence of any RVGE in Africa was higher than in Europe during the first year of life (≤2.78% vs. ≤2.03% per month), but much lower during the second one (≤0.86% versus ≤2.00% per month). The incidence of severe RVGE in Africa was slightly lower than in Europe during the first year of life. Nevertheless, temporal profiles for the incidence of severe RVGE in Africa and Europe during the first (≤1.00% and ≤1.23% per month) and second (≤0.53% and ≤1.13% per month) years of life were similar to those of any RVGE. Any/severe RVGE incidences peaked at younger ages in Africa vs. Europe. In high-income Asian regions, severe RVGE incidence (≤0.31% per month) remained low during the study. The burden of any RVGE was higher earlier in life in children from low- to middle- compared with high-income countries. Differing rotavirus vaccine schedules are likely warranted to maximize protection in different settings

Clinical Outcomes of Percutaneous Coronary Intervention for Bifurcation Lesions According to Medina Classification

Background: Coronary bifurcation lesions (CBLs) are frequently encountered in clinical practice and are associated with worse outcomes after percutaneous coronary intervention. However, there are limited data around the prognostic impact of different CBL distributions. / Methods and Results: All CBL percutaneous coronary intervention procedures from the prospective e‐Ultimaster (Prospective, Single‐Arm, Multi Centre Observations Ultimaster Des Registry) multicenter international registry were analyzed according to CBL distribution as defined by the Medina classification. Cox proportional hazards models were used to compare the hazard ratio (HR) of the primary outcome, 1‐year target lesion failure (composite of cardiac death, target vessel–related myocardial infarction, and clinically driven target lesion revascularization), and its individual components between Medina subtypes using Medina 1.0.0 as the reference category. A total of 4003 CBL procedures were included. The most prevalent Medina subtypes were 1.1.1 (35.5%) and 1.1.0 (26.8%), whereas the least prevalent was 0.0.1 (3.5%). Overall, there were no significant differences in patient and procedural characteristics among Medina subtypes. Only Medina 1.1.1 and 0.0.1 subtypes were associated with increased target lesion failure (HR, 2.6 [95% CI, 1.3–5.5] and HR, 4.0 [95% CI, 1.6–9.0], respectively) at 1 year, compared with Medina 1.0.0, prompted by clinically driven target lesion revascularization (HR, 3.1 [95% CI, 1.1–8.6] and HR, 4.6 [95% CI, 1.3–16.0], respectively) as well as cardiac death in Medina 0.0.1 (HR, 4.7 [95% CI, 1.0–21.6]). No differences in secondary outcomes were observed between Medina subtypes. / Conclusions: In a large multicenter registry analysis of coronary bifurcation percutaneous coronary intervention procedures, we demonstrate prognostic differences in 1‐year outcomes between different CBL distributions, with Medina 1.1.1 and 0.0.1 subtypes associated with an increased risk of target lesion failure