Designing lentiviral gene vectors

Lentiviral gene vectors are an important tool in gene therapy and basic biomedical research. They are transducing viral particles, normally replication defective, which are generated using the packaging machinery of lentiviruses. These vectors are used to deliver the encapsidated payload genes to the nuclei of the target cells, offering stable transgene expression in many settings in vitro and in vivo. Successful generation of high-titre lentiviral vectors capable of efficiently expressing transgenes over long period of time is governed by a number of vector design rules, some of which are common to all gene vectors while others are specific to lentiviral vectors. Construction of lentiviral vectors with the cargo genes driven by tissue-specific promoters is a particular challenge. This review focuses both on the guiding principles and the technical know-how of the lentiviral gene vector design.Published versio

Presión, temperatura y tiempo de procesamiento para mejorar la extracción de aceite de Camelina sativa mediante pretratamiento texturizado de descompresión instantánea controlada (DIC)

Instant Controlled Pressure Drop (DIC) was evaluated as a texturing pre-treatment for the extraction of Camelina sativa (L.) oil. DIC was coupled to Accelerated Solvent Extraction (ASE), Pressing and Dynamic Maceration (DM). DIC optimization was performed by studying the effects of pressure, temperature and processing time on oil yield. DIC + ASE obtained seed-oil yields of 615.9±0.5 against 555.5±0.5 g oil/kg-ddb for untextured seeds (RM). Via pressing, oil yields were 490.9±0.5 and 444.7±0.5 g oil/kg-ddb for textured and untextured seeds, respectively. Through coupling DIC (P: 0.63 MPa and t: 105 s) to the pressing extraction (60 s) of seeds along with 2h of DM of meals, it was possible to reach 605.8 g oil/kg ddb of oil yield. The same results were not obtained for RM seeds, where after 24 h of DM extraction, the oil yield was 554.7 g oil/kg ddb. DIC allowed for an increase in Camelina oil yields, reduced extraction time and valorized pressing meals.La tecnología de Descompresión Instantánea Controlada (DIC) fue evaluada como un pretratamiento para la extracción de aceite de Camelina sativa (L.). El pretratamiento DIC fue acoplado a la Extracción Acelerada de Disolventes (ASE), al Prensado y a la Maceración Dinámica (DM). La optimización de DIC fue llevada a cabo a través del estudio de los efectos de presión, temperatura y tiempo de proceso en el rendimiento del aceite. ASE + DIC permitió alcanzar rendimientos de 615,9±0,5 comparado con 555,5±0,5 g aceite/kg-ddb (base seca) en el caso de las semillas sin texturización (RM). En el caso del prensado, los rendimientos fueron de 490,9±0,5 y 444,7±0,5 g aceite/kg-ddb para las semillas con y sin texturización, respectivamente. Al acoplar el tratamiento DIC (P: 0.63 MPa y t: 105 s) + la extracción por prensado de las semillas (60 s) + 2h de DM de las harinas, fue posible alcanzar un rendimiento de 606,7 g aceite/ kg ddb. No así para las semillas sin tratamiento, en las que posterior a 24 h de extracción por DM, el rendimiento fue de 554,7 g oil/kg ddb. La texturización DIC permitió incrementar los rendimientos del aceite de Camelina, reducir los tiempos de extracción y valorizar las harinas del prensado

Bis(7-meth­oxy-1-methyl-4,9-dihydro-3H-β-carbolinium) tetra­chloridozincate

In the title compound, (C13H15N2O)2[ZnCl4], also known as di(harmalinium) tetra­chloridozincate, the ZnII atom is in a distorted tetrahedral coordination of the chlorido ligands. In the cation, the meth­oxy and methyl groups are both coplanar with with rings to which they are attached [maximum deviations of 0.232 (4) and 0.259 (4) Å, respectively]. In the crystal, the alkaloid cations and metal complex anions inter­act by way of N—H⋯Cl hydrogen bonds involving each Cl atom, resulting in a network structure

The fetal mouse is a sensitive genotoxicity model that exposes lentiviral-associated mutagenesis resulting in liver oncogenesis

This article is available open access through the publisher’s website at the link below. Copyright @ 2013 The American Society of Gene & Cell Therapy.Genotoxicity models are extremely important to assess retroviral vector biosafety before gene therapy. We have developed an in utero model that demonstrates that hepatocellular carcinoma (HCC) development is restricted to mice receiving nonprimate (np) lentiviral vectors (LV) and does not occur when a primate (p) LV is used regardless of woodchuck post-translation regulatory element (WPRE) mutations to prevent truncated X gene expression. Analysis of 839 npLV and 244 pLV integrations in the liver genomes of vector-treated mice revealed clear differences between vector insertions in gene dense regions and highly expressed genes, suggestive of vector preference for insertion or clonal outgrowth. In npLV-associated clonal tumors, 56% of insertions occurred in oncogenes or genes associated with oncogenesis or tumor suppression and surprisingly, most genes examined (11/12) had reduced expression as compared with control livers and tumors. Two examples of vector-inserted genes were the Park 7 oncogene and Uvrag tumor suppressor gene. Both these genes and their known interactive partners had differential expression profiles. Interactive partners were assigned to networks specific to liver disease and HCC via ingenuity pathway analysis. The fetal mouse model not only exposes the genotoxic potential of vectors intended for gene therapy but can also reveal genes associated with liver oncogenesis.Imperial College London, the Wellcome Trust, and Brunel University

Musculoskeletal pain is associated with a long-term increased risk of cancer and cardiovascular-related mortality

Objectives. To test the hypothesis that individuals with regional and widespread pain disorders have an increased risk of mortality

Clinical and biochemical prediction of early fatal outcome following hip fracture in the elderly

Hip fracture, a moderate musculoskeletal trauma, is associated with a high postoperative mortality. Most patients are elderly, with comorbid conditions and often with heart disease. The objective of this study was to find out if clinical parameters and analyses of specific muscle enzymes could predict three month postoperative mortality. A total of 302 patients above 75 years of age with hip fracture were consecutively enrolled. Baseline information on age, sex and comorbidity assessed with the American Society of Anesthesiologists (ASA) score was obtained before surgery. Creatine kinase (CK), myocardium-specific creatine kinase (CK-MB) and troponin T (TnT) were analysed from venous blood, collected the day before surgery (−1) and postoperatively, within 24 hours (0) and on days one (+1) and four (+4). The overall three month mortality was 19.5%. Multivariate analyses showed that age, male sex and comorbidity (ASA) correlated with mortality (p = 0.027, p = 0.002, p < 0.001, respectively). Surgery induced a two- to threefold increase of CK and CK-MB but without any correlation with mortality. However, high TnT levels >0.04 μg/l correlated significantly with death (days −1, +1 and +4, p = 0.003, p = 0.005 and p = 0.003, respectively). Multivariate analyses, adjusted for age, sex and ASA category, confirmed this correlation (day +4, p = 0.008). Thus, in elderly patients with comorbidities undergoing hip fracture surgery information on sex, age, ASA category and postoperative laboratory analyses on TnT provide the clinicians with useful information on patients at risk of fatal outcome

Chronic widespread bodily pain is increased among individuals with history of fracture:findings from UK Biobank

Acknowledgments This work was supported by grants from the Medical Research Council, British Heart Foundation, Arthritis Research UK, National Osteoporosis Society, International Osteoporosis Foundation, NIHR Southampton Biomedical Research Centre, University of Southampton and University Hospital Southampton NHS Foundation Trust, and NIHR Musculoskeletal Biomedical Research Unit, University of Oxford. This research has been conducted using the UK Biobank Resource. Compliance with ethical standards.Peer reviewedPublisher PD

Central motor control failure in fibromyalgia: a surface electromyography study

<p>Abstract</p> <p>Background</p> <p>Fibromyalgia (FM) is characterised by diffuse musculoskeletal pain and stiffness at multiple sites, tender points in characteristic locations, and the frequent presence of symptoms such as fatigue. The aim of this study was to assess whether the myoelectrical manifestations of fatigue in patients affected by FM are central or peripheral in origin.</p> <p>Methods</p> <p>Eight female patients aged 55.6 ± 13.6 years (FM group) and eight healthy female volunteers aged 50.3 ± 9.3 years (MCG) were studied by means of non-invasive surface electromyography (s-EMG) involving a linear array of 16 electrodes placed on the skin overlying the biceps brachii muscle, with muscle fatigue being evoked by means of voluntary and involuntary (electrically elicited) contractions. Maximal voluntary contractions (MVCs), motor unit action potential conduction velocity distributions (mean ± SD and skewness), and the mean power frequency of the spectrum (MNF) were estimated in order to assess whether there were any significant differences between the two groups and contraction types.</p> <p>Results</p> <p>The motor pattern of recruitment during voluntary contractions was altered in the FM patients, who also showed fewer myoelectrical manifestations of fatigue (normalised conduction velocity rate of changes: -0.074 ± 0.052%/s in FM vs -0.196 ± 0.133%/s in MCG; normalised MNF rate of changes: -0.29 ± 0.16%/s in FM vs -0.66 ± 0.34%/s in MCG). Mean conduction velocity distribution and skewnesses values were higher (p < 0.01) in the FM group. There were no between-group differences in the results obtained from the electrically elicited contractions.</p> <p>Conclusion</p> <p>The apparent paradox of fewer myoelectrical manifestations of fatigue in FM is the electrophysiological expression of muscle remodelling in terms of the prevalence of slow conducting fatigue-resistant type I fibres. As the only between-group differences concerned voluntary contractions, they are probably more related to central motor control failure than muscle membrane alterations, which suggests pathological muscle fibre remodelling related to altered suprasegmental control.</p