Protein-templated reactions using DNA-antibody conjugates

DNA-templated chemical reactions have found wide applications in drug discovery, programmed multistep synthesis, nucleic acid detection, and targeted drug delivery. The control of these reactions has, however, been limited to nucleic acid hybridization as a means to direct the proximity between reactants. In this work a system capable of translating protein-protein binding events into a DNA-templated reaction which leads to the covalent formation of a product is introduced. Protein-templated reactions by employing two DNA-antibody conjugates that are both able to recognize the same target protein and to colocalize a pair of reactant DNA strands able to undergo a click reaction are achieved. Two individual systems, each responsive to human serum albumin (HSA) and human IgG, are engineered and it is demonstrated that, while no reaction occurs in the absence of proteins, both protein-templated reactions can occur simultaneously in the same solution without any inter-system crosstalk

Atypical development of the executive attention network in children with chromosome 22q11.2 deletion syndrome

Impairment in the executive control of attention has been found in youth with chromosome 22q11.2 deletion syndrome (22q11.2DS). However, how this impairment is modified by other factors, particularly age, is unknown. Forty-six typically developing and 53 children with 22q11.2DS were tested with the attention networks task (ANT) in this cross-sectional study. We used logarithmic transform and linear modeling to assess age effects on the executive index of the ANT. Mixed modeling accounted for between subject variability, age, handedness, catecholamine-O-transferase (COMT; codon 158) genotype, and gender on performance for all experimental conditions (cue × flanker) and their two-level interactions. Children with 22q11.2DS showed a relative, age-dependent executive index impairment but not orienting or alerting network index impairments. In factorial analysis, age was a major predictor of overall performance. There was a significant effect of the 22q11.2DS on overall performance. Of note, children with 22q11.2DS are specifically vulnerable to incongruent flanker interference, especially at younger ages. We did not find an overall effect of COMT genotype or handedness. Children with 22q11.2DS demonstrated age-related impairment in the executive control of attention. Future investigation will likely reveal that there are different developmental trajectories of executive attentional function likely related to the development of schizophrenia in 22q11.2DS

Grey-matter texture abnormalities and reduced hippocampal volume are distinguishing features of schizophrenia

Neurodevelopmental processes are widely believed to underlie schizophrenia. Analysis of brain texture from conventional magnetic resonance imaging (MRI) can detect disturbance in brain cytoarchitecture. We tested the hypothesis that patients with schizophrenia manifest quantitative differences in brain texture that, alongside discrete volumetric changes, may serve as an endophenotypic biomarker. Texture analysis (TA) of grey matter distribution and voxel-based morphometry (VBM) of regional brain volumes were applied to MRI scans of 27 patients with schizophrenia and 24 controls. Texture parameters (uniformity and entropy) were also used as covariates in VBM analyses to test for correspondence with regional brain volume. Linear discriminant analysis tested if texture and volumetric data predicted diagnostic group membership (schizophrenia or control). We found that uniformity and entropy of grey matter differed significantly between individuals with schizophrenia and controls at the fine spatial scale (filter width below 2 mm). Within the schizophrenia group, these texture parameters correlated with volumes of the left hippocampus, right amygdala and cerebellum. The best predictor of diagnostic group membership was the combination of fine texture heterogeneity and left hippocampal size. This study highlights the presence of distributed grey-matter abnormalities in schizophrenia, and their relation to focal structural abnormality of the hippocampus. The conjunction of these features has potential as a neuroimaging endophenotype of schizophrenia

A Comparison of Red Fluorescent Proteins to Model DNA Vaccine Expression by Whole Animal In Vivo Imaging

DNA vaccines can be manufactured cheaply, easily and rapidly and have performed well in pre-clinical animal studies. However, clinical trials have so far been disappointing, failing to evoke a strong immune response, possibly due to poor antigen expression. To improve antigen expression, improved technology to monitor DNA vaccine transfection efficiency is required. In the current study, we compared plasmid encoded tdTomato, mCherry, Katushka, tdKatushka2 and luciferase as reporter proteins for whole animal in vivo imaging. The intramuscular, subcutaneous and tattooing routes were compared and electroporation was used to enhance expression. We observed that overall, fluorescent proteins were not a good tool to assess expression from DNA plasmids, with a highly heterogeneous response between animals. Of the proteins used, intramuscular delivery of DNA encoding either tdTomato or luciferase gave the clearest signal, with some Katushka and tdKatushka2 signal observed. Subcutaneous delivery was weakly visible and nothing was observed following DNA tattooing. DNA encoding haemagglutinin was used to determine whether immune responses mirrored visible expression levels. A protective immune response against H1N1 influenza was induced by all routes, even after a single dose of DNA, though qualitative differences were observed, with tattooing leading to high antibody responses and subcutaneous DNA leading to high CD8 responses. We conclude that of the reporter proteins used, expression from DNA plasmids can best be assessed using tdTomato or luciferase. But, the disconnect between visible expression level and immunogenicity suggests that in vivo whole animal imaging of fluorescent proteins has limited utility for predicting DNA vaccine efficacy

A tale worth telling: the impact of the diagnosis experience on disclosure of genetic disorders: Impact of the diagnosis experience on disclosure

Research suggests children with genetic disorders exhibit greater coping skills when they are aware of their condition and its heritability. While the experiences parents have at diagnosis may influence their decision to disclose the diagnosis to their children, there is little research into this communication. The aim of the current study was to examine the relationship between the diagnosis experience and the disclosure experience for parents of children with intellectual disabilities; with a child affected by 22q11.2 deletion syndrome (22q11DS) compared to a group of parents with children affected with other genetic diagnoses, with a similar age of diagnosis (e.g., Fragile X syndrome) and a group where diagnosis generally occurs early (i.e., Down syndrome)

SNPs and real-time quantitative PCR method for constitutional allelic copy number determination, the VPREB1 marker case

<p>Abstract</p> <p>Background</p> <p>22q11.2 microdeletion is responsible for the DiGeorge Syndrome, characterized by heart defects, psychiatric disorders, endocrine and immune alterations and a 1 in 4000 live birth prevalence. Real-time quantitative PCR (qPCR) approaches for allelic copy number determination have recently been investigated in 22q11.2 microdeletions detection. The qPCR method was performed for 22q11.2 microdeletions detection as a first-level screening approach in a genetically unknown series of patients with congenital heart defects. A technical issue related to the <it>VPREB1 </it>qPCR marker was pointed out.</p> <p>Methods</p> <p>A set of 100 unrelated Italian patients with congenital heart defects were tested for 22q11.2 microdeletions by a qPCR method using six different markers. Fluorescence In Situ Hybridization technique (FISH) was used for confirmation.</p> <p>Results</p> <p>qPCR identified six patients harbouring the 22q11.2 microdeletion, confirmed by FISH. The <it>VPREB1 </it>gene marker presented with a pattern consistent with hemideletion in one 3 Mb deleted patient, suggestive for a long distal deletion, and in additional five non-deleted patients. The long distal 22q11.2 deletion was not confirmed by Comparative Genomic Hybridization. Indeed, the <it>VPREB1 </it>gene marker generated false positive results in association with the rs1320 G/A SNP, a polymorphism localized within the <it>VPREB1 </it>marker reverse primer sequence. Patients heterozygous for rs1320 SNP, showed a qPCR profile consistent with the presence of a hemideletion.</p> <p>Conclusions</p> <p>Though the qPCR technique showed advantages as a screening approach in terms of cost and time, the <it>VPREB1 </it>marker case revealed that single nucleotide polymorphisms can interfere with qPCR data generating erroneous allelic copy number interpretations.</p

Suicidal tendencies and attitude towards freedom to choose suicide among Lithuanian schoolchildren: results from three cross-sectional studies in 1994, 1998, and 2002

BACKGROUND: Suicidal behaviour is increasingly becoming a phenomenon associated with young people and an important public health issue in Lithuania. However, there are very few studies evaluating impact of young peoples' attitudes towards suicide to their suicidal behaviour. A better understanding of the relations among the variables associated with suicidal ideation and threats in the normal population of adolescents may eventually result in a better understanding of the more serious forms of adolescent suicidal behaviour. The aim of the present study was to evaluate prevalence of suicidal tendencies among Lithuanian schoolchildren and to estimate its association with an attitude towards suicide in 1994 – 2002. METHODS: Three country representative samples of schoolchildren, aged 11, 13 and 15, were surveyed in 1994 (n = 5428), 1998 (n = 4513), and 2002 (n = 5645) anonymously in conformity with the methodology of the World Health Organization Cross – National study on Health Behaviour in School-aged Children (HBSC). RESULTS: About one third of respondents reported about suicidal ideation, plans or attempts to commit suicide. In the study period of eight years, the percentage of adolescents who reported sometime suicidal ideation decreased but the percentage of adolescents who declared serious suicidal behaviour remained on the same high level (8.1%, 9.8% and 8.4% correspondingly in 1994, 1998 and 2002). Moreover, the number of suicidal attempts changed from 1.0% in 1994 to 1.8% in the year 1998 and to 1,7% in the year 2002. The schoolchildren's attitude towards suicide became more agreeable: 36.6%, 41.9% and 62.5% of respondents, correspondingly in 1994, 1998 and 2002, answered that they agree with a person's freedom to make a choice between life and suicide. A multiple logistic regression analysis with low level of suicidality and high level of suicidality versus non suicidal behaviour as dependent variables for gender, age, year of the survey and attitude towards freedom to choose suicide as independent variables approved a significant association between studied covariates over the entire study period. CONCLUSION: Suicidal tendencies are quite frequent among Lithuanian adolescents. An increasing number of schoolchildren are expressing an agreeable attitude towards suicide. The approving attitude towards suicide among adolescents correlates with suicidal ideation and behaviour

Search for copy number variants in chromosomes 15q11-q13 and 22q11.2 in obsessive compulsive disorder

<p>Abstract</p> <p>Background</p> <p>Obsessive-compulsive disorder (OCD) is a clinically and etiologically heterogeneous syndrome. The high frequency of obsessive-compulsive symptoms reported in subjects with the 22q11.2 deletion syndrome (DiGeorge/velocardiofacial syndrome) or Prader-Willi syndrome (15q11-13 deletion of the paternally derived chromosome), suggests that gene dosage effects in these chromosomal regions could increase risk for OCD. Therefore, the aim of this study was to search for microrearrangements in these two regions in OCD patients.</p> <p>Methods</p> <p>We screened the 15q11-13 and 22q11.2 chromosomal regions for genomic imbalances in 236 patients with OCD using multiplex ligation-dependent probe amplification (MLPA).</p> <p>Results</p> <p>No deletions or duplications involving 15q11-13 or 22q11.2 were identified in our patients.</p> <p>Conclusions</p> <p>Our results suggest that deletions/duplications of chromosomes 15q11-13 and 22q11.2 are rare in OCD. Despite the negative findings in these two regions, the search for copy number variants in OCD using genome-wide array-based methods is a highly promising approach to identify genes of etiologic importance in the development of OCD.</p

Topical Gene Electrotransfer to the Epidermis of Hairless Guinea Pig by Non-invasive Multielectrode Array

Topical gene delivery to the epidermis has the potential to be an effective therapy for skin disorders, cutaneous cancers, vaccinations and systemic metabolic diseases. Previously, we reported on a non-invasive multielectrode array (MEA) that efficiently delivered plasmid DNA and enhanced expression to the skin of several animal models by in vivo gene electrotransfer. Here, we characterized plasmid DNA delivery with the MEA in a hairless guinea pig model, which has a similar histology and structure to human skin. Significant elevation of gene expression up to 4 logs was achieved with intradermal DNA administration followed by topical non-invasive skin gene electrotransfer. This delivery produced gene expression in the skin of hairless guinea pig up to 12 to 15 days. Gene expression was observed exclusively in the epidermis. Skin gene electrotransfer with the MEA resulted in only minimal and mild skin changes. A low level of human Factor IX was detected in the plasma of hairless guinea pig after geneelectrotransfer with the MEA, although a significant increase of Factor IX was obtained in the skin of animals. These results suggest geneelectrotransfer with the MEA can be a safe, efficient, non-invasive skin delivery method for skin disorders, vaccinations and potential systemic diseases where low levels of gene products are sufficient