A New Large-DNA-Fragment Delivery System Based on Integrase Activity from an Integrative and Conjugative Element.

During the past few decades, numerous plasmid vectors have been developed for cloning, gene expression analysis, and genetic engineering. Cloning procedures typically rely on PCR amplification, DNA fragment restriction digestion, recovery, and ligation, but increasingly, procedures are being developed to assemble large synthetic DNAs. In this study, we developed a new gene delivery system using the integrase activity of an integrative and conjugative element (ICE). The advantage of the integrase-based delivery is that it can stably introduce a large DNA fragment (at least 75 kb) into one or more specific sites (the gene for glycine-accepting tRNA) on a target chromosome. Integrase recombination activity in Escherichia coli is kept low by using a synthetic hybrid promoter, which, however, is unleashed in the final target host, forcing the integration of the construct. Upon integration, the system is again silenced. Two variants with different genetic features were produced, one in the form of a cloning vector in E. coli and the other as a mini-transposable element by which large DNA constructs assembled in E. coli can be tagged with the integrase gene. We confirmed that the system could successfully introduce cosmid and bacterial artificial chromosome (BAC) DNAs from E. coli into the chromosome of Pseudomonas putida in a site-specific manner. The integrase delivery system works in concert with existing vector systems and could thus be a powerful tool for synthetic constructions of new metabolic pathways in a variety of host bacteria.

Is the consumption-income ratio stationary? Evidence from linear and nonlinear panel unit root tests for OECD and non-OECD countries

This paper applies recently developed heterogeneous nonlinear and linear panel unit root tests that account for cross-sectional dependence to 24 OECD and 33 non-OECD countries’ consumption-income ratios over the period 1951–2003. We apply a recently developed methodology that facilitates the use of panel tests to identify which individual cross-sectional units are stationary and which are nonstationary. This extends evidence provided in the recent literature to consider both linear and nonlinear adjustment in panel unit root tests, to address the issue of cross-sectional dependence, and to substantially expand both time-series and cross sectional dimensions of the data analysed. We find that the majority (65%) of the series are nonstationary with slightly fewer OECD countries’ (61%) series exhibiting a unit root than non-OECD countries (68%).

The use of the likelihood ratio for evaluative and investigative purposes in comparative forensic handwriting examination

This paper extends previous research and discussion on the use of multivariate continuous data, which are about to become more prevalent in forensic science. As an illustrative example, attention is drawn here on the area of comparative handwriting examinations. Multivariate continuous data can be obtained in this field by analysing the contour shape of loop characters through Fourier analysis. This methodology, based on existing research in this area, allows one describe in detail the morphology of character contours throughout a set of variables. This paper uses data collected from female and male writers to conduct a comparative analysis of likelihood ratio based evidence assessment procedures in both, evaluative and investigative proceedings. While the use of likelihood ratios in the former situation is now rather well established (typically, in order to discriminate between propositions of authorship of a given individual versus another, unknown individual), focus on the investigative setting still remains rather beyond considerations in practice. This paper seeks to highlight that investigative settings, too, can represent an area of application for which the likelihood ratio can offer a logical support. As an example, the inference of gender of the writer of an incriminated handwritten text is forwarded, analysed and discussed in this paper. The more general viewpoint according to which likelihood ratio analyses can be helpful for investigative proceedings is supported here through various simulations. These offer a characterisation of the robustness of the proposed likelihood ratio methodology.

Gender Inequality and the Sex Ratio in Three Emerging Economies

The aim of this paper is to study inequality and deprivations as reflected in the human sex ratio (commonly defined as the number of males per 100 females). The particular focus is on three emerging economies, viz., Russia, India and China. The paper compares and contrasts the experiences of these countries and discusses policy issues. It is noted that while the feminist perspective on the issues surrounding the sex ratio is important, it would be wrong to view these issues always or exclusively through the prism of that perspective . It is also suggested that India and China probably have better prospects of sustained economic growth in the foreseeable future than does Russia.

Fractional Integration of the Price-Dividend Ratio in a Present-Value Model of Stock Prices

We re-examine the dynamics of returns and dividend growth within the present-value framework of stock prices. We find that the finite sample order of integration of returns is approximately equal to the order of integration of the first-differenced price-dividend ratio. As such, the traditional return forecasting regressions based on the price-dividend ratio are invalid. Moreover, the nonstationary long memory behaviour of the price-dividend ratio induces antipersistence in returns. This suggests that expected returns should be modelled as an AFIRMA process and we show this improves the forecast ability of the present-value model in-sample and out-of-sample.

Meta-analysis identifies 13 new loci associated with waist-hip ratio and reveals sexual dimorphism in the genetic basis of fat distribution.

Waist-hip ratio (WHR) is a measure of body fat distribution and a predictor of metabolic consequences independent of overall adiposity. WHR is heritable, but few genetic variants influencing this trait have been identified. We conducted a meta-analysis of 32 genome-wide association studies for WHR adjusted for body mass index (comprising up to 77,167 participants), following up 16 loci in an additional 29 studies (comprising up to 113,636 subjects). We identified 13 new loci in or near RSPO3, VEGFA, TBX15-WARS2, NFE2L3, GRB14, DNM3-PIGC, ITPR2-SSPN, LY86, HOXC13, ADAMTS9, ZNRF3-KREMEN1, NISCH-STAB1 and CPEB4 (P = 1.9 × 10⁻⁹ to P = 1.8 × 10⁻⁴⁰) and the known signal at LYPLAL1. Seven of these loci exhibited marked sexual dimorphism, all with a stronger effect on WHR in women than men (P for sex difference = 1.9 × 10⁻³ to P = 1.2 × 10⁻&supl;³). These findings provide evidence for multiple loci that modulate body fat distribution independent of overall adiposity and reveal strong gene-by-sex interactions.

Impact of the C2/C6 ratio of high-molecular-weight hydroxyethyl starch on pharmacokinetics and blood coagulation in pigs

Rapport de synthèse : Contexte: l'hydroxyéthylamidon (HEA) est largement utilisé comme expanseur volémique en anesthésiologie et réanimation. Cependant, cette classe de produits perturbe le system de la coagulation. Des améliorations restent possibles dans le choix de la combinaison optimale de poids moléculaire, de degré de substitution en radicaux éthyle et de localisation de ces radicaux sur le squelette glucidique des polymères, afin d'optimiser leur efficacité et leur tolérance. L'HEA de poids moléculaire élevé et faiblement substitué n'affecte pas plus la coagulation sanguine que de l'HEA de bas poids moléculaire faiblement substitué. Nous examinons in vivo l'effet d'un abaissement du rapport C2/C6 sur les caractéristiques pharmacocinétiques et l'impact sur la coagulation sanguine d'un HEA de haut poids moléculaire faiblement substitué. Matériels et méthode: nous comparons dans une étude prospective, randomisée et parallèle l'HEA 650/0.42/2.8 avec l'HEA 650/0.42/5.6 auprès de 30 cochons. Avant, pendant et jusqu'à 630 minutes après une perfusion de 30 ml/kg d'HEA, des échantillons sanguins ont été collectés pour mesurer les concentrations d'HEA, les tests de coagulation plasmatique classiques et la coagulation sanguine par thrombélastographie (TEG®, Haemoscope Corporation, Niles, II, U.S.). Les paramètres pharmacocinétiques ont été estimés en adaptant un modèle à deux compartiments. Résultats: la constante d'élimination est de 0.009 ± 0.001 (min-1) pour l'HEA 650/0.42/2.8 et 0.007 ± 0.001 (min-1) pour l'HEA 650/0.42/5.6 (p<0.001); la surface sous la courbe de concentration est de 1374 ± 340 min*g/L pour l'HEA 650/0.42/2.8 et 1697 ± 411 min*g/L pour l'HEA 650/0.42/5.6 (p=0.026). Les concentrations mesurées d'HEA ne montrent pas de différence entre l'HEA 650/0.42/2.8 et l'HEA 650/0.42/5.6. Les deux solutions d'HEA affectent de façon identique la coagulation sanguine: l'index de coagulation thrombélastographique diminue pareillement à ta fin de la perfusion d'HEA 650/0.42/2.8 et d'HEA 650/0.42/5.6 (p=0.29). De même, le temps de thromboplastine partielle activée et le temps de prothrombine augmentent de manière similaire pour l'HEA 650/0.42/2.8 et l'HEA 650/0.42/5.6 (p=0.83). Conclusion: la réduction du rapport C2/C6 de l'HEA de poids moléculaire élevé et faiblement substitué aboutit à une élimination légèrement accélérée d'HEA. Cependant, elle ne modifie pas l'effet perturbateur sur la coagulation.

Particulate formulations for the delivery of poly(I:C) as vaccine adjuvant.

Current research and development of antigens for vaccination often center on purified recombinant proteins, viral subunits, synthetic oligopeptides or oligosaccharides, most of them suffering from being poorly immunogenic and subject to degradation. Hence, they call for efficient delivery systems and potent immunostimulants, jointly denoted as adjuvants. Particulate delivery systems like emulsions, liposomes, nanoparticles and microspheres may provide protection from degradation and facilitate the co-formulation of both the antigen and the immunostimulant. Synthetic double-stranded (ds) RNA, such as polyriboinosinic acid-polyribocytidylic acid, poly(I:C), is a mimic of viral dsRNA and, as such, a promising immunostimulant candidate for vaccines directed against intracellular pathogens. Poly(I:C) signaling is primarily dependent on Toll-like receptor 3 (TLR3), and on melanoma differentiation-associated gene-5 (MDA-5), and strongly drives cell-mediated immunity and a potent type I interferon response. However, stability and toxicity issues so far prevented the clinical application of dsRNAs as they undergo rapid enzymatic degradation and bear the potential to trigger undue immune stimulation as well as autoimmune disorders. This review addresses these concerns and suggests strategies to improve the safety and efficacy of immunostimulatory dsRNA formulations. The focus is on technological means required to lower the necessary dosage of poly(I:C), to target surface-modified microspheres passively or actively to antigen-presenting cells (APCs), to control their interaction with non-professional phagocytes and to modulate the resulting cytokine secretion profile.

Sex-dependent selection on an autosomal melanic female ornament promotes the evolution of sex ratio bias.

Sex-dependent selection often leads to spectacularly different phenotypes in males and females. In species in which sexual dimorphism is not complete, it is unclear which benefits females and males derive from displaying a trait that is typical of the other sex. In barn owls (Tyto alba), females exhibit on average larger black eumelanic spots than males but members of the two sexes display this trait in the same range of possible values. In a 12-year study, we show that selection exerted on spot size directly or on genetically correlated traits strongly favoured females with large spots and weakly favoured males with small spots. Intense directional selection on females caused an increase in spot diameter in the population over the study period. This increase is due to a change in the autosomal genes underlying the expression of eumelanic spots but not of sex-linked genes. Female-like males produced more daughters than sons, while male-like females produced more sons than daughters when mated to a small-spotted male. These sex ratio biases appear adaptive because sons of male-like females and daughters of female-like males had above-average survival. This demonstrates that selection exerted against individuals displaying a trait that is typical of the other sex promoted the evolution of specific life history strategies that enhance their fitness. This may explain why in many organisms sexual dimorphism is often not complete.

Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery.

BACKGROUND: Gene transfer to nociceptive neurons of the dorsal root ganglia (DRG) is a promising approach to dissect mechanisms of pain in rodents and is a potential therapeutic strategy for the treatment of persistent pain disorders such as neuropathic pain. A number of studies have demonstrated transduction of DRG neurons using herpes simplex virus, adenovirus and more recently, adeno-associated virus (AAV). Recombinant AAV are currently the gene transfer vehicles of choice for the nervous system and have several advantages over other vectors, including stable and safe gene expression. We have explored the capacity of recombinant AAV serotype 6 (rAAV2/6) to deliver genes to DRG neurons and characterized the transduction of nociceptors through five different routes of administration in mice. RESULTS: Direct injection of rAAV2/6 expressing green fluorescent protein (eGFP) into the sciatic nerve resulted in transduction of up to 30% eGFP-positive cells of L4 DRG neurons in a dose dependent manner. More than 90% of transduced cells were small and medium sized neurons (< 700 microm 2), predominantly colocalized with markers of nociceptive neurons, and had eGFP-positive central terminal fibers in the superficial lamina of the spinal cord dorsal horn. The efficiency and profile of transduction was independent of mouse genetic background. Intrathecal administration of rAAV2/6 gave the highest level of transduction (approximately 60%) and had a similar size profile and colocalization with nociceptive neurons. Intrathecal administration also transduced DRG neurons at cervical and thoracic levels and resulted in comparable levels of transduction in a mouse model for neuropathic pain. Subcutaneous and intramuscular delivery resulted in low levels of transduction in the L4 DRG. Likewise, delivery via tail vein injection resulted in relatively few eGFP-positive cells within the DRG, however, this transduction was observed at all vertebral levels and corresponded to large non-nociceptive cell types. CONCLUSION: We have found that rAAV2/6 is an efficient vector to deliver transgenes to nociceptive neurons in mice. Furthermore, the characterization of the transduction profile may facilitate gene transfer studies to dissect mechanisms behind neuropathic pain.

Colloidal systems for the delivery of cyclosporin A to the anterior segment of the eye.

Due to the eye's specific anatomical and physiological conformation, the treatment of eye diseases is a real challenge for pharmaceutical therapy. The presence of efficient protective barriers (i.e., the conjunctival and corneal membranes) and protective mechanisms (i.e., blinking and nasolachrymal drainage) makes this organ particularly impervious to local drug therapy. To overcome these issues, numerous strategies have been envisioned using pharmaceutical technology. Many formulations currently on the market or still under development are emulsions or colloidal systems intended to enhance precorneal residence time and corneal penetration, causing a consequent increase in drug bioavailability after instillation. After a review of some recent developments in the field of cyclosporin A formulations for the eye, a novel micellar formulation of cyclosporine A based on a diblock methoxy-poly(ethylene glycol)-hexysubstituted poly(lactides) (MPEG-hexPLA) is described.

Polyanalgesic Consensus Conference--2012: recommendations on trialing for intrathecal (intraspinal) drug delivery: report of an interdisciplinary expert panel.

INTRODUCTION: Trialing for intrathecal pump placement is an essential part of the decision-making process in placing a permanent device. In both the United States and the international community, the proper method for trialing is ill defined. METHODS: The Polyanalgesic Consensus Conference (PACC) is a group of well-published experienced practitioners who meet to update the state of care for intrathecal therapies on the basis of current knowledge in the literature and clinical experience. Anexhaustive search is performed to create a base of information that the panel considers when making recommendations for best clinical practices. This literature, coupled with clinical experience, is the basis for recommendations and for identification of gaps in the base of knowledge regarding trialing for intrathecal pump placement. RESULTS: The panel has made recommendations for the proper methods of trialing for long-term intrathecal drug delivery. CONCLUSION: The use of intrathecal drug delivery is an important part of the treatment algorithm for moderate to severe chronic pain. It has become common practice to perform a temporary neuroaxial infusion before permanent device implantation. On the basis of current knowledge, the PACC has developed recommendations to improve care. The need to update these recommendations will be very important as new literature is published.

Efficient gene delivery and selective transduction of astrocytes in the mammalian brain using viral vectors.

Astrocytes are now considered as key players in brain information processing because of their newly discovered roles in synapse formation and plasticity, energy metabolism and blood flow regulation. However, our understanding of astrocyte function is still fragmented compared to other brain cell types. A better appreciation of the biology of astrocytes requires the development of tools to generate animal models in which astrocyte-specific proteins and pathways can be manipulated. In addition, it is becoming increasingly evident that astrocytes are also important players in many neurological disorders. Targeted modulation of protein expression in astrocytes would be critical for the development of new therapeutic strategies. Gene transfer is valuable to target a subpopulation of cells and explore their function in experimental models. In particular, viral-mediated gene transfer provides a rapid, highly flexible and cost-effective, in vivo paradigm to study the impact of genes of interest during central nervous system development or in adult animals. We will review the different strategies that led to the recent development of efficient viral vectors that can be successfully used to selectively transduce astrocytes in the mammalian brain.

Delivery of Adaptations in Bristol

Adaptació de l'habitatge a Bristol (UK. Ponència del "2º Espacio de Encuentro :Rehabilitación y Adaptación Funcional de la Vivienda" (San6 Sebastià, 9 Juny 2010)