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

Business models and consumers' value proposition for PEDs value generation by PEDs: best practices case study book

Executive Summary: The EU's SET Plan has proposed the development of Positive Energy Districts (PEDs) to achieve the transition towards a more sustainable energy system through the adoption of renewable energy technologies and energy efficiency measures. PEDs are envisioned to be neighbourhoods that utilise such technologies and create an environment that enables citizens to lead environmentally‐friendly lifestyles. At their core, PEDs create value across three sustainability dimensions: environmental, social, and economic. PEDs and PED‐like projects can be developed in a variety of ways and are a product of the physical, social, and economic characteristics of the environment. Innovative business models – the configurations in which value is created, delivered, and captured – are integral to leveraging these contextual characteristics in order to achieve the goals of PEDs. This study aims to identify best practice cases and categorise them into archetypes of business models operating in PEDs in order to summarise the opportunities available in PED creation. The authors synthesise existing literature in the area of sustainable business models to develop a conceptual framework that analyses the extent to which the value dimensions of business models address the economic, social, and environmental dimensions of sustainability, with a focus on PEDs. This allows the building blocks of the original business model canvas to be modified and extended. The authors use a qualitative analytical approach, to a set of selected European cases, through the lens of the conceptual framework to identify business model archetypes of PEDs and PED‐like projects

Deleterious Effects of Cold Air Inhalation on Coronary Physiological Indices in Patients With Obstructive Coronary Artery Disease

Background Cold air inhalation during exercise increases cardiac mortality, but the pathophysiology is unclear. During cold and exercise, dual‐sensor intracoronary wires measured coronary microvascular resistance (MVR) and blood flow velocity (CBF), and cardiac magnetic resonance measured subendocardial perfusion. Methods and Results Forty‐two patients (62±9 years) undergoing cardiac catheterization, 32 with obstructive coronary stenoses and 10 without, performed either (1) 5 minutes of cold air inhalation (5°F) or (2) two 5‐minute supine‐cycling periods: 1 at room temperature and 1 during cold air inhalation (5°F) (randomized order). We compared rest and peak stress MVR, CBF, and subendocardial perfusion measurements. In patients with unobstructed coronary arteries (n=10), cold air inhalation at rest decreased MVR by 6% (P=0.41), increasing CBF by 20% (P<0.01). However, in patients with obstructive stenoses (n=10), cold air inhalation at rest increased MVR by 17% (P<0.01), reducing CBF by 3% (P=0.85). Consequently, in patients with obstructive stenoses undergoing the cardiac magnetic resonance protocol (n=10), cold air inhalation reduced subendocardial perfusion (P<0.05). Only patients with obstructive stenoses performed this protocol (n=12). Cycling at room temperature decreased MVR by 29% (P<0.001) and increased CBF by 61% (P<0.001). However, cold air inhalation during cycling blunted these adaptations in MVR (P=0.12) and CBF (P<0.05), an effect attributable to defective early diastolic CBF acceleration (P<0.05) and associated with greater ST‐segment depression (P<0.05). Conclusions In patients with obstructive coronary stenoses, cold air inhalation causes deleterious changes in MVR and CBF. These diminish or abolish the normal adaptations during exertion that ordinarily match myocardial blood supply to demand

Gene expression profile of circulating tumor cells in breast cancer by RT-qPCR

<p>Abstract</p> <p>Background</p> <p>Circulating tumor cells (CTCs) have been associated with prognosis especially in breast cancer and have been proposed as a liquid biopsy for repeated follow up examinations. Molecular characterization of CTCs is difficult to address since they are very rare and the amount of available sample is very limited.</p> <p>Methods</p> <p>We quantified by RT-qPCR <it>CK-19, MAGE-A3, HER-2, TWIST1, hTERT α+β+</it>, and <it>mammaglobin </it>gene transcripts in immunomagnetically positively selected CTCs from 92 breast cancer patients, and 28 healthy individuals. We also compared our results with the CellSearch system in 33 of these patients with early breast cancer.</p> <p>Results</p> <p>RT-qPCR is highly sensitive and specific and can detect the expression of each individual gene at the one cell level. None of the genes tested was detected in the group of healthy donors. In 66 operable breast cancer patients, <it>CK-19 </it>was detected in 42.4%, <it>HER-2 </it>in 13.6%, <it>MAGE-A3 </it>in 21.2%, <it>hMAM </it>in 13.6%, <it>TWIST-1 </it>in 42.4%, and <it>hTERT α+β+ </it>in 10.2%. In 26 patients with verified metastasis, <it>CK-19 </it>was detected in 53.8%, <it>HER-2 </it>in 19.2%, <it>MAGE-A3 </it>in 15.4%, <it>hMAM </it>in 30.8%, <it>TWIST-1 </it>in 38.5% and <it>hTERT </it>α⁺β⁺in 19.2%. Our preliminary data on the comparison between RT-qPCR and CellSearch in 33 early breast cancer patients showed that RT-qPCR gives more positive results in respect to CellSearch.</p> <p>Conclusions</p> <p>Molecular characterization of CTCs has revealed a remarkable heterogeneity of gene expression between breast cancer patients. In a small percentage of patients, CTCs were positive for all six genes tested, while in some patients only one of these genes was expressed. The clinical significance of these findings in early breast cancer remains to be elucidated when the clinical outcome for these patients is known.</p

Trioctahedral entities in palygorskite: Near-infrared evidence for sepiolite-palygorskite polysomatism

The mixed dioctahedral-trioctahedral character of Mg-rich palygorskite has been previously described by the formula yMg5 Si8 O20(OH)2(OH2)4(1–y)[xMg2Fe2(1–x)Mg2 Al2] Si8 O20(OH)2(OH2)4, where y is the trioctahedral fraction of this two-chain ribbon mineral with an experimentally determined upper limit of y 0.5 and x is the FeIII content in the M2 sites of the dioctahedral component. Ideal trioctahedral (y ¼ 1) palygorskite is elusive, although sepiolite Mg8Si12O30(OH)4(OH2)4 with a similar composition, three-chain ribbon structure and distinct XRD pattern is common. A set of 22 samples identified by XRD as palygorskite and with variable composition (0 , x , 0.7, 0 , y , 0.5) were studied to extrapolate the structure of an ideal trioctahedral (y ¼ 1) palygorskite and to compare this structure to sepiolite. Near-infrared spectroscopy was used to study the influence of octahedral composition on the structure of the TOT ribbons, H2O in the tunnels and surface silanols of palygorskite, as well as their response to loss of zeolitic H2O. All spectroscopic evidence suggests that palygorskite consists of discrete dioctahedral and trioctahedral entities. The dioctahedral entities have variable structure determined solely by x=FeIII/(Al+FeIII) and their content is proportional to (1–y). In contrast, the trioctahedral entities have fixed octahedral composition or ribbon structure and are spectroscopically identical to sepiolite. The value of d200 in palygorskite follows the regression d200 (A°)= 6.362 + 0.129 x(1–y) + 0.305y, R2 = 0.96, σ = 0.013A°. When extrapolated to y = 1,d200 is identical to sepiolite. Based on this analysis, we propose that palygorskite samples with non-zero trioctahedral character should be considered as members of a polysomatic series of sepiolite and (dioctahedral) palygorskite described by the new formula y'Mg8 Si12 O30(OH)4(OH2)4.(1–y')[x'Mg2Fe2(1–x')Mg2Al2]Si8O20(OH)2(OH2)4, with 0 < x'= x < 0.7 and 0 < y' = y/(2–y) < 0.33

Real-time RT–PCR detection of disseminated tumour cells in bone marrow has superior prognostic significance in comparison with circulating tumour cells in patients with breast cancer

This study assessed the ability of real-time reverse transcription–polymerase chain reaction (RT–PCR) analysis to detect disseminated epithelial cells (DEC) in peripheral blood (PB) and bone marrow (BM) of patients with breast cancer (BC). Detection of DEC in BM is an obvious choice in BC, but blood sampling is more convenient. The aim of this study was to evaluate whether the detection of DEC in either PB or BM predicts overall survival (OS). Peripheral blood and BM samples were collected from 148 patients with primary (stage M0, n=116/78%) and metastatic (stage M+, n=32/21%) BC before the initiation of any local or systemic treatment. Peripheral blood of healthy volunteers and BM of patients with a nonmalignant breast lesion or a haematological malignancy served as the control group. Disseminated epithelial cells was detected by measuring relative gene expression (RGE) for cytokeratin-19 (CK-19) and mammaglobin (MAM), using a quantitative RT–PCR detection method. The mean follow-up time was 786 days (+/− 487). Kaplan–Meier analysis was used for predicting OS. By taking the 95 percentile of the RGE of CK-19 (BM: 26.3 and PB: 58.7) of the control group as cutoff, elevated CK-19 expression was detected in 42 (28%) BM samples and in 22 (15%) PB samples. Mammaglobin expression was elevated in 20% (both PB and BM) of the patients with BC. There was a 68% (CK-19) and 75% (MAM) concordance between PB and BM samples when classifying the results as either positive or negative. Patients with an elevated CK-19 or MAM expression in the BM had a worse prognosis than patients without elevated expression levels (OS: log-rank test, P=0.0045 (CK-19) and P=0.025 (MAM)). For PB survival analysis, no statistical significant difference was observed between patients with or without elevated CK-19 or MAM expression (OS: log-rank test, P=0.551 (CK-19) and P=0.329 (MAM)). Separate analyses of the M0 and M+ patients revealed a marked difference in OS according to the BM CK-19 or MAM status in the M+ patient group, but in the M0 group, only MAM expression was a prognostic marker for OS. Disseminated epithelial cells, measured as elevated CK-19 or MAM mRNA expression, could be detected in both PB and BM of patients with BC. Only the presence of DEC in BM was highly predictive for OS. The occurrence of DEC in the BM is probably less time-dependent and may act as a filter for circulating BC cells. The use of either larger volumes of PB or performing an enrichment step for circulating tumour in blood cells might improve these results