A robust system for RNA interference in the chicken using a modified microRNA operon

AbstractRNA interference (RNAi) provides an effective method to silence gene expression and investigate gene function. However, RNAi tools for the chicken embryo have largely been adapted from vectors designed for mammalian cells. Here we present plasmid and retroviral RNAi vectors specifically designed for optimal gene silencing in chicken cells. The vectors use a chicken U6 promoter to express RNAs modelled on microRNA30, which are embedded within chicken microRNA operon sequences to ensure optimal Drosha and Dicer processing of transcripts. The chicken U6 promoter works significantly better than promoters of mammalian origin and in combination with a microRNA operon expression cassette (MOEC), achieves up to 90% silencing of target genes. By using a MOEC, we show that it is also possible to simultaneously silence two genes with a single vector. The vectors express either RFP or GFP markers, allowing simple in vivo tracking of vector delivery. Using these plasmids, we demonstrate effective silencing of Pax3, Pax6, Nkx2.1, Nkx2.2, Notch1 and Shh in discrete regions of the chicken embryonic nervous system. The efficiency and ease of use of this RNAi system paves the way for large-scale genetic screens in the chicken embryo

The Sensor Technology and Rehabilitative Timing (START) Protocol: A Randomized Controlled Trial for the Rehabilitation of Mild Traumatic Brain Injury

BACKGROUND: Clinical practice for rehabilitation after mild traumatic brain injury (mTBI) is variable, and guidance on when to initiate physical therapy is lacking. Wearable sensor technology may aid clinical assessment, performance monitoring, and exercise adherence, potentially improving rehabilitation outcomes during unsupervised home exercise programs. OBJECTIVE: The objectives of this study were to: (1) determine whether initiating rehabilitation earlier than typical will improve outcomes after mTBI, and (2) examine whether using wearable sensors during a home-exercise program will improve outcomes in participants with mTBI. DESIGN: This was a randomized controlled trial. SETTING: This study will take place within an academic hospital setting at Oregon Health & Science University and Veterans Affairs Portland Health Care System, and in the home environment. PARTICIPANTS: This study will include 160 individuals with mTBI. INTERVENTION: The early intervention group (n = 80) will receive one-on-one physical therapy 8 times over 6 weeks and complete daily home exercises. The standard care group (n = 80) will complete the same intervention after a 6- to 8-week wait period. One-half of each group will receive wearable sensors for therapist monitoring of patient adherence and quality of movements during their home exercise program. MEASUREMENTS: The primary outcome measure will be the Dizziness Handicap Inventory score. Secondary outcome measures will include symptomatology, static and dynamic postural control, central sensorimotor integration posturography, and vestibular-ocular-motor function. LIMITATIONS: Potential limitations include variable onset of care, a wide range of ages, possible low adherence and/or withdrawal from the study in the standard of care group, and low Dizziness Handicap Inventory scores effecting ceiling for change after rehabilitation. CONCLUSIONS: If initiating rehabilitation earlier improves primary and secondary outcomes post-mTBI, this could help shape current clinical care guidelines for rehabilitation. Additionally, using wearable sensors to monitor performance and adherence may improve home exercise outcomes

Identifying the Rules of Engagement Enabling Leukocyte Rolling, Activation, and Adhesion

The LFA-1 integrin plays a pivotal role in sustained leukocyte adhesion to the endothelial surface, which is a precondition for leukocyte recruitment into inflammation sites. Strong correlative evidence implicates LFA-1 clustering as being essential for sustained adhesion, and it may also facilitate rebinding events with its ligand ICAM-1. We cannot challenge those hypotheses directly because it is infeasible to measure either process during leukocyte adhesion following rolling. The alternative approach undertaken was to challenge the hypothesized mechanisms by experimenting on validated, working counterparts: simulations in which diffusible, LFA1 objects on the surfaces of quasi-autonomous leukocytes interact with simulated, diffusible, ICAM1 objects on endothelial surfaces during simulated adhesion following rolling. We used object-oriented, agent-based methods to build and execute multi-level, multi-attribute analogues of leukocytes and endothelial surfaces. Validation was achieved across different experimental conditions, in vitro, ex vivo, and in vivo, at both the individual cell and population levels. Because those mechanisms exhibit all of the characteristics of biological mechanisms, they can stand as a concrete, working theory about detailed events occurring at the leukocyte–surface interface during leukocyte rolling and adhesion experiments. We challenged mechanistic hypotheses by conducting experiments in which the consequences of multiple mechanistic events were tracked. We quantified rebinding events between individual components under different conditions, and the role of LFA1 clustering in sustaining leukocyte–surface adhesion and in improving adhesion efficiency. Early during simulations ICAM1 rebinding (to LFA1) but not LFA1 rebinding (to ICAM1) was enhanced by clustering. Later, clustering caused both types of rebinding events to increase. We discovered that clustering was not necessary to achieve adhesion as long as LFA1 and ICAM1 object densities were above a critical level. Importantly, at low densities LFA1 clustering enabled improved efficiency: adhesion exhibited measurable, cell level positive cooperativity

Inhibition of NF-kB 1 (NF-kBp50) by RNA interference in chicken macrophage HD11 cell line challenged with Salmonellaenteritidis

The NF-kB pathway plays an important role in regulating the immunity response in animals. In this study, small interfering RNAs (siRNA) were used to specifically inhibit NF-kB 1 expression and to elucidate the role of NF-kB in the signal transduction pathway of the Salmonella challenge in the chicken HD11 cell line. The cells were transfected with either NF-kB 1 siRNA, glyceraldehyde 3-phosphate dehydrogenase siRNA (positive control) or the negative control siRNA for 24 h, followed by Salmonella enteritidis (SE) challenge or non-challenge for 1 h and 4 h. Eight candidate genes related to the signal pathway of SE challenge were selected to examine the effect of NF-kB 1 inhibition on their expressions by mRNA quantification. The results showed that, with a 36% inhibition of NF-kB 1 expression, gene expression of both Toll-like receptor (TLR) 4 and interleukin (IL)-6 was consistently and significantly increased at both 1 h and 4 h following SE challenge, whereas the gene expression of MyD88 and IL-1β was increased at 1 h and 4 h, respectively. These findings suggest a likely inhibitory regulation by NF-kB 1, and could lay the foundation for studying the gene network of the innate immune response of SE infection in chickens

Biomarkers of acute lung injury: worth their salt?

The validation of biomarkers has become a key goal of translational biomedical research. The purpose of this article is to discuss the role of biomarkers in the management of acute lung injury (ALI) and related research. Biomarkers should be sensitive and specific indicators of clinically important processes and should change in a relevant timeframe to affect recruitment to trials or clinical management. We do not believe that they necessarily need to reflect pathogenic processes. We critically examined current strategies used to identify biomarkers and which, owing to expedience, have been dominated by reanalysis of blood derived markers from large multicenter Phase 3 studies. Combining new and existing validated biomarkers with physiological and other data may add predictive power and facilitate the development of important aids to research and therapy

Tensile and fatigue strength of hydrogen-treated Ti-6Al-4V alloy

Tensile, fatigue and fractographic data on Ti-6Al-4V microstructures attained through a series of post-β-annealing treatments which used hydrogen as a temporary alloying element are presented. Hydrogen-alloying treatments break up the continuous grain boundary α and colony structure, and produce a homogeneous microstructure consisting of refined α-grains in a matrix of discontinuous β. These changes in microstructural morphology result in significant increases of the yield strength (974 to 1119 MPa), ultimate strength (1025 to 1152 MPa) and high cycle fatigue strength (643 to 669 MPa) compared to respective values for lamellar microstructures (902, 994, 497 MPa). The strengths are also significantly greater than the strengths of equiaxed microstructures (914, 1000, 590 MPa). The strengths of hydrogen-alloy treated samples are therefore superior to strengths attainable via other thermal cycling techniques.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44703/1/10853_2004_Article_BF00576523.pd

Effectiveness of waste minimisation projects in reducing water demand by UK industry

There is growing interest in managing water demand in the UK. A series of waste minimization clubs have been set up within the country and this paper identifies the effectiveness of these clubs in reducing the demand for water within industry. Membership of these clubs is voluntary and the only incentive for industry to reduce water consumption, and consequently the production of effluent, is the almost immediate financial saving made by the company, often achieved by accounting for the water consumption and loss within site from the point of input from the water supplier to output in the form of effluent. On average, companies are able to reduce water consumption by up to 30 percent. If the entire industrial sector within the UK were to achieve this degree of savings, it is possible that approximately 1300Ml/d could be saved