Escherichia Coli-Based Cell-Free Protein Synthesis for Iterative Design of Tandem-Core Virus-Like Particles

Tandem-core hepatitis B core antigen (HBcAg) virus-like particles (VLPs), in which two HBcAg monomers are joined together by a peptide linker, can be used to display two different antigens on the VLP surface. We produced universal influenza vaccine candidates that use this scaffold in an Escherichia coli-based cell-free protein synthesis (CFPS) platform. We then used the CFPS system to rapidly test modifications to the arginine-rich region typically found in wild-type HBcAg, the peptide linkers around the influenza antigen inserts, and the plasmid vector backbone to improve titer and quality. Using a minimal plasmid vector backbone designed for CFPS improved titers by at least 1.4-fold over the original constructs. When the linker lengths for the influenza inserts were more consistent in length and a greater variety of codons for glycine and serine were utilized, titers were further increased to over 70 μg/mL (4.0-fold greater than the original construct) and the presence of lower molecular weight product-related impurities was significantly reduced, although improvements in particle assembly were not seen. Furthermore, any constructs with the C-terminal arginine-rich region removed resulted in asymmetric particles of poor quality. This demonstrates the potential for CFPS as a screening platform for VLPs

A viral CTL escape mutation leading to immunoglobulin-like transcript 4-mediated functional inhibition of myelomonocytic cells

Viral mutational escape can reduce or abrogate recognition by the T cell receptor (TCR) of virus-specific CD8+ T cells. However, very little is known about the impact of cytotoxic T lymphocyte (CTL) epitope mutations on interactions between peptide–major histocompatibility complex (MHC) class I complexes and MHC class I receptors expressed on other cell types. Here, we analyzed a variant of the immunodominant human leukocyte antigen (HLA)-B2705–restricted HIV-1 Gag KK10 epitope (KRWIILGLNK) with an L to M amino acid substitution at position 6 (L6M), which arises as a CTL escape variant after primary infection but is sufficiently immunogenic to elicit a secondary, de novo HIV-1–specific CD8+ T cell response with an alternative TCR repertoire in chronic infection. In addition to altering recognition by HIV-1–specific CD8+ T cells, the HLA-B2705–KK10 L6M complex also exhibits substantially increased binding to the immunoglobulin-like transcript (ILT) receptor 4, an inhibitory MHC class I–specific receptor expressed on myelomonocytic cells. Binding of the B2705–KK10 L6M complex to ILT4 leads to a tolerogenic phenotype of myelomonocytic cells with lower surface expression of dendritic cell (DC) maturation markers and co-stimulatory molecules. These data suggest a link between CTL-driven mutational escape, altered recognition by innate MHC class I receptors on myelomonocytic cells, and functional impairment of DCs, and thus provide important new insight into biological consequences of viral sequence diversificatio

A Common Genetic Variant Risk Score is Associated with Drug-Induced QT Prolongation and Torsade de Pointes Risk: A Pilot Study.

Background -Drug-induced QT interval prolongation, a risk factor for life-threatening ventricular arrhythmias, is a potential side effect of many marketed and withdrawn medications. The contribution of common genetic variants previously associated with baseline QT interval to drug-induced QT prolongation and arrhythmias is not known. Methods -We tested the hypothesis that a weighted combination of common genetic variants contributing to QT interval at baseline, identified through genome-wide association studies, can predict individual response to multiple QT-prolonging drugs. Genetic analysis of 22 subjects was performed in a secondary analysis of a randomized, double-blind, placebo-controlled, cross-over trial of 3 QT-prolonging drugs with 15 time-matched QT and plasma drug concentration measurements. Subjects received single doses of dofetilide, quinidine, ranolazine and placebo. The outcome was the correlation between a genetic QT score comprising 61 common genetic variants and the slope of an individual subject's drug-induced increase in heart rate corrected QT (QTc) vs. drug concentration. Results -The genetic QT score was correlated with drug-induced QTc prolongation. Among white subjects, genetic QT score explained 30% of the variability in response to dofetilide (r = 0.55 [95% CI, 0.09-0.81], P = 0.02), 23% in response to quinidine (r = 0.48 [95% CI, -0.03 to 0.79], P = 0.06) and 27% in response to ranolazine (r = 0.52 [95% CI, 0.05 to 0.80], P = 0.03). Furthermore, the genetic QT score was a significant predictor of drug-induced torsade de pointes in an independent sample of 216 cases compared to 771 controls (r(2) = 12%, P = 1x10(-7)). Conclusions -We demonstrate that a genetic QT score comprising 61 common genetic variants explains a significant proportion of the variability in drug-induced QT prolongation and is a significant predictor of drug-induced torsade de pointes. These findings highlight an opportunity for recent genetic discoveries to improve individualized risk-benefit assessment for pharmacologic therapies. Replication of these findings in larger samples is needed to more precisely estimate variance explained and to establish the individual variants that drive these effects. Clinical Trial Registration - http://clinicaltrials.gov Unique identifier: NCT01873950

New directions in cellular therapy of cancer: a summary of the summit on cellular therapy for cancer

A summit on cellular therapy for cancer discussed and presented advances related to the use of adoptive cellular therapy for melanoma and other cancers. The summit revealed that this field is advancing rapidly. Conventional cellular therapies, such as tumor infiltrating lymphocytes (TIL), are becoming more effective and more available. Gene therapy is becoming an important tool in adoptive cell therapy. Lymphocytes are being engineered to express high affinity T cell receptors (TCRs), chimeric antibody-T cell receptors (CARs) and cytokines. T cell subsets with more naïve and stem cell-like characteristics have been shown in pre-clinical models to be more effective than unselected populations and it is now possible to reprogram T cells and to produce T cells with stem cell characteristics. In the future, combinations of adoptive transfer of T cells and specific vaccination against the cognate antigen can be envisaged to further enhance the effectiveness of these therapies

Penis deformity after intra-urethral liquid paraffin administration in a young male: a case report

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Lung adenocarcinoma originates from retrovirus infection of proliferating type 2 pneumocytes during pulmonary post-natal development or tissue repair

Jaagsiekte sheep retrovirus (JSRV) is a unique oncogenic virus with distinctive biological properties. JSRV is the only virus causing a naturally occurring lung cancer (ovine pulmonary adenocarcinoma, OPA) and possessing a major structural protein that functions as a dominant oncoprotein. Lung cancer is the major cause of death among cancer patients. OPA can be an extremely useful animal model in order to identify the cells originating lung adenocarcinoma and to study the early events of pulmonary carcinogenesis. In this study, we demonstrated that lung adenocarcinoma in sheep originates from infection and transformation of proliferating type 2 pneumocytes (termed here lung alveolar proliferating cells, LAPCs). We excluded that OPA originates from a bronchioalveolar stem cell, or from mature post-mitotic type 2 pneumocytes or from either proliferating or non-proliferating Clara cells. We show that young animals possess abundant LAPCs and are highly susceptible to JSRV infection and transformation. On the contrary, healthy adult sheep, which are normally resistant to experimental OPA induction, exhibit a relatively low number of LAPCs and are resistant to JSRV infection of the respiratory epithelium. Importantly, induction of lung injury increased dramatically the number of LAPCs in adult sheep and rendered these animals fully susceptible to JSRV infection and transformation. Furthermore, we show that JSRV preferentially infects actively dividing cell in vitro. Overall, our study provides unique insights into pulmonary biology and carcinogenesis and suggests that JSRV and its host have reached an evolutionary equilibrium in which productive infection (and transformation) can occur only in cells that are scarce for most of the lifespan of the sheep. Our data also indicate that, at least in this model, inflammation can predispose to retroviral infection and cancer

Lying Behavior, Family Functioning and Adjustment in Early Adolescence

Item does not contain fulltextCommunication between children and parents has been the subject of several studies, examining the effects of, for example, disclosure and secrecy on adolescents' social relationships and adjustment. Less attention has paid to adolescent deception. We developed and tested a new instrument on lying behavior in a sample of 671 parent-adolescent couples. Analyses on the psychometric properties showed that this instrument had one principal component, and high internal consistency, item-total correlations and inter-item correlations. Lying was moderately associated with other indicators of parent-child communication, the quality of the parent-child relationship, and with parenting practices. In addition, frequent lying was moderately related to behavioral problems and emotional problems.10 p

Genome sequencing of the extinct Eurasian wild aurochs, Bos primigenius, illuminates the phylogeography and evolution of cattle

Background Domestication of the now-extinct wild aurochs, Bos primigenius, gave rise to the two major domestic extant cattle taxa, B. taurus and B. indicus. While previous genetic studies have shed some light on the evolutionary relationships between European aurochs and modern cattle, important questions remain unanswered, including the phylogenetic status of aurochs, whether gene flow from aurochs into early domestic populations occurred, and which genomic regions were subject to selection processes during and after domestication. Here, we address these questions using whole-genome sequencing data generated from an approximately 6,750-year-old British aurochs bone and genome sequence data from 81 additional cattle plus genome-wide single nucleotide polymorphism data from a diverse panel of 1,225 modern animals. Results Phylogenomic analyses place the aurochs as a distinct outgroup to the domestic B. taurus lineage, supporting the predominant Near Eastern origin of European cattle. Conversely, traditional British and Irish breeds share more genetic variants with this aurochs specimen than other European populations, supporting localized gene flow from aurochs into the ancestors of modern British and Irish cattle, perhaps through purposeful restocking by early herders in Britain. Finally, the functions of genes showing evidence for positive selection in B. taurus are enriched for neurobiology, growth, metabolism and immunobiology, suggesting that these biological processes have been important in the domestication of cattle. Conclusions This work provides important new information regarding the origins and functional evolution of modern cattle, revealing that the interface between early European domestic populations and wild aurochs was significantly more complex than previously thought

Fanconi anaemia with bilateral diffuse pulmonary arterio venous fistulae: a case report

<p>Abstract</p> <p>Background</p> <p>We report a patient with cytogenetically confirmed Fanconi anaemia with associated diffuse bilateral pulmonary arterio-venous fistulae. This is only the second reported case of diffuse pulmonary arterio-venous fistulae with Fanconi anaemia.</p> <p>Case Presentation</p> <p>A 16 year old Sri Lankan boy, with a cytogenetically confirmed Fanconi anaemia was admitted to University Medical Unit, National Hospital of Sri Lanka for further assessment and treatment. Both central and peripheral cyanosis plus clubbing were noted on examination. The peripheral saturation was persistently low on room air and did not improve with supplementary Oxygen. Contrast echocardiography failed to demonstrate an intra cardiac shunt but showed early crossover of contrast, suggesting the possibility of pulmonary arterio-venous fistulae. Computed tomography pulmonary angiogram was inconclusive. Subsequent right heart catheterisation revealed bilateral diffuse arterio-venous fistulae not amenable for device closure or surgical intervention.</p> <p>Conclusion</p> <p>To our knowledge, this is the second reported patient with diffuse pulmonary arterio-venous fistulae associated with Fanconi anaemia. We report this case to create awareness among clinicians regarding this elusive association. We recommend screening patients with Fanconi anaemia using contrast echocardiography at the time of assessment with transthoracic echocardiogram. Though universal screening may be impossible given the cost constraints, such screening should at least be performed in patients with clinical evidence of desaturation or when a therapeutic option such as haematopoietic stem cell transplantation is considered. Treatment of pulmonary arteriovenous fistulae would improve patient outcome as desaturation by shunting worsens the anaemic symptoms by reducing the oxygen carrying capacity of blood.</p