Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy

Mutations in the gene encoding dystrophin, a protein that maintains muscle integrity and function, cause Duchenne muscular dystrophy (DMD). The deltaE50-MD dog model of DMD harbors a mutation corresponding to a mutational “hotspot” in the human DMD gene. We used adeno-associated viruses to deliver CRISPR gene editing components to four dogs and examined dystrophin protein expression 6 weeks after intramuscular delivery (n = 2) or 8 weeks after systemic delivery (n = 2). After systemic delivery in skeletal muscle, dystrophin was restored to levels ranging from 3 to 90% of normal, depending on muscle type. In cardiac muscle, dystrophin levels in the dog receiving the highest dose reached 92% of normal. The treated dogs also showed improved muscle histology. These large-animal data support the concept that, with further development, gene editing approaches may prove clinically useful for the treatment of DMD

Electrode fabrication and interface optimization for imaging of evoked peripheral nervous system activity with electrical impedance tomography (EIT)

Objective. Non-invasive imaging techniques are undoubtedly the ideal methods for continuous monitoring of neural activity. One such method, fast neural electrical impedance tomography (EIT) has been developed over the past decade in order to image neural action potentials with non-penetrating electrode arrays. Approach. The goal of this study is two-fold. First, we present a detailed fabrication method for silicone-based multiple electrode arrays which can be used for epicortical or neural cuff applications. Secondly, we optimize electrode material coatings in order to achieve the best accuracy in EIT reconstructions. Main results. The testing of nanostructured electrode interface materials consisting of platinum, iridium oxide, and PEDOT:pTS in saline tank experiments demonstrated that the PEDOT:pTS coating used in this study leads to more accurate reconstruction dimensions along with reduced phase separation between recording channels. The PEDOT:pTS electrodes were then used in vivo to successfully image and localize the evoked activity of the recurrent laryngeal fascicle from within the cervical vagus nerve. Significance. These results alongside the simple fabrication method presented here position EIT as an effective method to image neural activity

Assessment of a six gene panel for the molecular detection of circulating tumor cells in the blood of female cancer patients

<p>Abstract</p> <p>Background</p> <p>The presence of circulating tumor cells (CTC) in the peripheral blood of cancer patients has been described for various solid tumors and their clinical relevance has been shown. CTC detection based on the analysis of epithelial antigens might be hampered by the genetic heterogeneity of the primary tumor and loss of epithelial antigens. Therefore, we aimed to identify new gene markers for the PCR-based detection of CTC in female cancer patients.</p> <p>Methods</p> <p>Gene expression of 38 cancer cell lines (breast, ovarian, cervical and endometrial) and of 10 peripheral blood mononuclear cell (PBMC) samples from healthy female donors was measured using microarray technology (Applied Biosystems). Differentially expressed genes were identified using the maxT test and the 50% one-sided trimmed maxT-test. Confirmatory RT-qPCR was performed for 380 gene targets using the AB TaqMan^®Low Density Arrays. Then, 93 gene targets were analyzed using the same RT-qPCR platform in tumor tissues of 126 patients with primary breast, ovarian or endometrial cancer. Finally, blood samples from 26 healthy women and from 125 patients (primary breast, ovarian, cervical, or endometrial cancer, and advanced breast cancer) were analyzed following OncoQuick enrichment and RNA pre-amplification. Likewise, <it>hMAM </it>and <it>EpCAM </it>gene expression was analyzed in the blood of breast and ovarian cancer patients. For each gene, a cut-off threshold value was set at three standard deviations from the mean expression level of the healthy controls to identify potential markers for CTC detection.</p> <p>Results</p> <p>Six genes were over-expressed in blood samples from 81% of patients with advanced and 29% of patients with primary breast cancer. <it>EpCAM </it>gene expression was detected in 19% and 5% of patients, respectively, whereas <it>hMAM </it>gene expression was observed in the advanced group (39%) only. Multimarker analysis using the new six gene panel positively identified 44% of the cervical, 64% of the endometrial and 19% of the ovarian cancer patients.</p> <p>Conclusions</p> <p>The panel of six genes was found superior to <it>EpCAM </it>and <it>hMAM </it>for the detection of circulating tumor cells in the blood of breast cancer, and they may serve as potential markers for CTC derived from endometrial, cervical, and ovarian cancers.</p

The frequency of NPM1 mutations in childhood acute myeloid leukemia

<p>Abstract</p> <p>Background</p> <p>Mutations in the nucleophosmin <it>(NPM1) </it>gene have been solely associated with childhood acute myeloid leukemia (AML). We evaluated the frequency of <it>NPM1 </it>mutations in childhood AML, their relation to clinical and cytogenetic features and the presence of common <it>FLT3 </it>and <it>RAS </it>mutations.</p> <p>Results</p> <p><it>NPM1 </it>mutations were found in 8% of cases. They involved the typical type 'A' mutation and one novel mutation characterized by two individual base pair substitutions, which resulted in 2 amino acid changes (W290) and (S293) in the NPM protein. <it>FLT3</it>/ITD mutations were observed in 12% of the cases and in one <it>NPM1-</it>mutated case bearing also t(8;21) (q22;q22). No common <it>RAS </it>mutations were identified.</p> <p>Conclusions</p> <p>A relatively consistent <it>NPM1 </it>mutation rate was observed, but with variations in types of mutations. The role of different types of <it>NPM1 </it>mutations, either individually or in the presence of other common gene mutations may be essential for childhood AML prognosis.</p

Error determination in the photogrammetric assessment of shoreline changes

The evaluation of error or uncertainty in shoreline change studies is an issue of prime importance for providing an adequate framework for calculated rates of change and to allow the establishment of threshold values above which the rates would be significant. In this note, a practical, easy-to-use method is presented to estimate error involved in the calculation of shoreline changes on aerial photographs, including the three most used types of shoreline indicators: high water line, dune/cliff toe and cliff top. This approach takes into account the specific characteristics of each shoreline proxy, such as relief in the case of the cliff top or tidal oscillations in the case of the high water line. At the same time it includes the error components that are independent from the proxy, basically related to the technical aspects of the process such as photo scanning and georeferencing. A practical example of application of the method is provided for several types of data inputs, based on shoreline changes around the Bay of Cádiz (SW Spain)

Global, regional, and national comparative risk assessment of 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks, 1990-2015: a systematic analysis for the Global Burden of Disease Study 2015

SummaryBackground The Global Burden of Diseases, Injuries, and Risk Factors Study 2015 provides an up-to-date synthesis of the evidence for risk factor exposure and the attributable burden of disease. By providing national and subnational assessments spanning the past 25 years, this study can inform debates on the importance of addressing risks in context. Methods We used the comparative risk assessment framework developed for previous iterations of the Global Burden of Disease Study to estimate attributable deaths, disability-adjusted life-years (DALYs), and trends in exposure by age group, sex, year, and geography for 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks from 1990 to 2015. This study included 388 risk-outcome pairs that met World Cancer Research Fund-defined criteria for convincing or probable evidence. We extracted relative risk and exposure estimates from randomised controlled trials, cohorts, pooled cohorts, household surveys, census data, satellite data, and other sources. We used statistical models to pool data, adjust for bias, and incorporate covariates. We developed a metric that allows comparisons of exposure across risk factors—the summary exposure value. Using the counterfactual scenario of theoretical minimum risk level, we estimated the portion of deaths and DALYs that could be attributed to a given risk. We decomposed trends in attributable burden into contributions from population growth, population age structure, risk exposure, and risk-deleted cause-specific DALY rates. We characterised risk exposure in relation to a Socio-demographic Index (SDI). Findings Between 1990 and 2015, global exposure to unsafe sanitation, household air pollution, childhood underweight, childhood stunting, and smoking each decreased by more than 25%. Global exposure for several occupational risks, high body-mass index (BMI), and drug use increased by more than 25% over the same period. All risks jointly evaluated in 2015 accounted for 57·8% (95% CI 56·6–58·8) of global deaths and 41·2% (39·8–42·8) of DALYs. In 2015, the ten largest contributors to global DALYs among Level 3 risks were high systolic blood pressure (211·8 million [192·7 million to 231·1 million] global DALYs), smoking (148·6 million [134·2 million to 163·1 million]), high fasting plasma glucose (143·1 million [125·1 million to 163·5 million]), high BMI (120·1 million [83·8 million to 158·4 million]), childhood undernutrition (113·3 million [103·9 million to 123·4 million]), ambient particulate matter (103·1 million [90·8 million to 115·1 million]), high total cholesterol (88·7 million [74·6 million to 105·7 million]), household air pollution (85·6 million [66·7 million to 106·1 million]), alcohol use (85·0 million [77·2 million to 93·0 million]), and diets high in sodium (83·0 million [49·3 million to 127·5 million]). From 1990 to 2015, attributable DALYs declined for micronutrient deficiencies, childhood undernutrition, unsafe sanitation and water, and household air pollution; reductions in risk-deleted DALY rates rather than reductions in exposure drove these declines. Rising exposure contributed to notable increases in attributable DALYs from high BMI, high fasting plasma glucose, occupational carcinogens, and drug use. Environmental risks and childhood undernutrition declined steadily with SDI; low physical activity, high BMI, and high fasting plasma glucose increased with SDI. In 119 countries, metabolic risks, such as high BMI and fasting plasma glucose, contributed the most attributable DALYs in 2015. Regionally, smoking still ranked among the leading five risk factors for attributable DALYs in 109 countries; childhood underweight and unsafe sex remained primary drivers of early death and disability in much of sub-Saharan Africa. Interpretation Declines in some key environmental risks have contributed to declines in critical infectious diseases. Some risks appear to be invariant to SDI. Increasing risks, including high BMI, high fasting plasma glucose, drug use, and some occupational exposures, contribute to rising burden from some conditions, but also provide opportunities for intervention. Some highly preventable risks, such as smoking, remain major causes of attributable DALYs, even as exposure is declining. Public policy makers need to pay attention to the risks that are increasingly major contributors to global burden. Funding Bill & Melinda Gates Foundation

Ethics and the choice of animal advocacy campaigns

This paper examines how different ethical positions view various types of animal advocacy campaigns concerning a product made using animals as an input. The ethical positions represent common company, society, and animal advocate viewpoints. We adopt an industrial economics approach, modelling a market with a monopolistic supplier and subject to consumer-oriented, technological, collaborative, and direct action campaigns. We determine whether the ethical positions support or oppose each campaign, and in what conditions. We find that animal welfare and rights goals are simultaneously satisfied by three campaigns: negotiation, targeted direct action, and awareness raising that condemns low welfare standards

The Cysteine Protease α-Clostripain is Not Essential for the Pathogenesis of Clostridium perfringens-Mediated Myonecrosis

Clostridium perfringens is the causative agent of clostridial myonecrosis or gas gangrene and produces many different extracellular toxins and enzymes, including the cysteine protease α-clostripain. Mutation of the α-clostripain structural gene, ccp, alters the turnover of secreted extracellular proteins in C. perfringens, but the role of α-clostripain in disease pathogenesis is not known. We insertionally inactivated the ccp gene C. perfringens strain 13 using TargeTron technology, constructing a strain that was no longer proteolytic on skim milk agar. Quantitative protease assays confirmed the absence of extracellular protease activity, which was restored by complementation with the wild-type ccp gene. The role of α-clostripain in virulence was assessed by analysing the isogenic wild-type, mutant and complemented strains in a mouse myonecrosis model. The results showed that although α-clostripain was the major extracellular protease, mutation of the ccp gene did not alter either the progression or the development of disease. These results do not rule out the possibility that this extracellular enzyme may still have a role in the early stages of the disease process

A Genome-Wide Association Study Identifies rs2000999 as a Strong Genetic Determinant of Circulating Haptoglobin Levels

Haptoglobin is an acute phase inflammatory marker. Its main function is to bind hemoglobin released from erythrocytes to aid its elimination, and thereby haptoglobin prevents the generation of reactive oxygen species in the blood. Haptoglobin levels have been repeatedly associated with a variety of inflammation-linked infectious and non-infectious diseases, including malaria, tuberculosis, human immunodeficiency virus, hepatitis C, diabetes, carotid atherosclerosis, and acute myocardial infarction. However, a comprehensive genetic assessment of the inter-individual variability of circulating haptoglobin levels has not been conducted so far