Some transition metal complexes of 8-amino- quinoline

Transition metal complexes of 8-aminoquinolin

Design and cloning strategies for constructing shRNA expression vectors

BACKGROUND: Short hairpin RNA (shRNA) encoded within an expression vector has proven an effective means of harnessing the RNA interference (RNAi) pathway in mammalian cells. A survey of the literature revealed that shRNA vector construction can be hindered by high mutation rates and the ensuing sequencing is often problematic. Current options for constructing shRNA vectors include the use of annealed complementary oligonucleotides (74 % of surveyed studies), a PCR approach using hairpin containing primers (22 %) and primer extension of hairpin templates (4 %). RESULTS: We considered primer extension the most attractive method in terms of cost. However, in initial experiments we encountered a mutation frequency of 50 % compared to a reported 20 – 40 % for other strategies. By modifying the technique to be an isothermal reaction using the DNA polymerase Phi29, we reduced the error rate to 10 %, making primer extension the most efficient and cost-effective approach tested. We also found that inclusion of a restriction site in the loop could be exploited for confirming construct integrity by automated sequencing, while maintaining intended gene suppression. CONCLUSION: In this study we detail simple improvements for constructing and sequencing shRNA that overcome current limitations. We also compare the advantages of our solutions against proposed alternatives. Our technical modifications will be of tangible benefit to researchers looking for a more efficient and reliable shRNA construction process

The effects of stem length and core placement on shRNA activity

<p>Abstract</p> <p>Background</p> <p>Expressed short hairpin RNAs (shRNA) used in mammalian RNA interference (RNAi) are often designed around a specific short interfering RNA (siRNA) core. Whilst there are algorithms to aid siRNA design, hairpin-specific characteristics such as stem-length and siRNA core placement within the stem are not well defined.</p> <p>Results</p> <p>Using more than 91 hairpins designed against HIV-1 Tat and Vpu, we investigated the influence of both of these factors on suppressive activity, and found that stem length does not correspond with predictable changes in suppressive activity. We also detected multiple processed products for all stem lengths tested. However, the entire length of the hairpin stem was not equally processed into active products. As such, the placement of the siRNA core at the base terminus was critical for activity.</p> <p>Conclusion</p> <p>We conclude that there is no fixed correlation between stem length and suppressive activity. Instead, core selection and placement likely have a greater influence on the effectiveness of shRNA-based silencing.</p

Development and application of a novel Peptide Nucleic Acid probe for the specific detection of Cronobacter (Enterobacter sakazakii) in powdered infant formula

Cronobacter spp. are causative agents of meningitis, septicemia and necrotizing enterocolitis in neonates and immunocompromised infants. Recently, contaminated powdered infant formula (PIF) has been reported as a source of these infections. In order to minimize the risk of infection, the development of a rapid, sensitive and specific method for the early detection of this bacterium in infant formula is of the utmost importance. Fluorescence in situ hybridization (FISH), a technique that allows direct visualization of whole cells, has been combined with specific peptide nucleic acid (PNA) probes, a new synthetic molecule with a better hybridization performance than DNA probes. In this work, a new FISH method for the detection of Cronobacter spp. using a novel PNA probe is reported. This PNA-FISH method was then adapted for the detection of this bacterium in PIF. The PNA-FISH procedure using the Cronobacter probe proved to be a reliable method for the detection of this pathogen in PIF samples and an alternative to existing molecular methods. It presented high specificity and sensitivity, detected less than 1 CFU per 10g of Cronobacter in infant formula and provided detection in less than 12 hours. Direct visualization of bacterial cells was possible and the method was simple and easy to use, without any special equipment apart from an epifluorescence microscope. The samples can be also analysed by flow cytometry

A comparison of multiple shRNA expression methods for combinatorial RNAi

RNAi gene therapies for HIV-1 will likely need to employ multiple shRNAs to counter resistant strains. We evaluated 3 shRNA co-expression methods to determine their suitability for present use; multiple expression vectors, multiple expression cassettes and single transcripts comprised of several dsRNA units (aka domains) with each being designed to a different target. Though the multiple vector strategy was effective with 2 shRNAs, the increasing number of vectors required is a major shortcoming. With single transcript configurations we only saw adequate activity from 1 of 10 variants tested, the variants being comprised of 2 - 3 different target domains. Whilst single transcript configurations have the most advantages on paper, these configurations can not yet be rapidly and reliably re-configured for new targets. However, our multiple cassette combinations of 2, 3 and 4 (29 bp) shRNAs were all successful, with suitable activity maintained in all positions and net activities comparable to that of the corresponding single shRNAs. We conclude that the multiple cassette strategy is the most suitably developed for present use as it is easy to design, assemble, is directly compatible with pre-existing shRNA and can be easily expanded

A history of Proterozoic terranes in southern South America: From Rodinia to Gondwana

The role played by Paleoproterozoic cratons in southern South America from the Mesoproterozoic to the Early Cambrian is reconsidered here. This period involved protracted continental amalgamation that led to formation of the supercontinent Rodinia, followed by Neoproterozoic continental break-up, with the consequent opening of Clymene and Iapetus oceans, and finally continental re-assembly as Gondwana through complex oblique collisions in the Late Neoproterozoic to Early Cambrian. The evidence for this is based mainly on a combination of precise U-Pb SHRMP dating and radiogenic isotope data for igneous and metamorphic rocks from a large area extending from the Rio de la Plata craton in the east to the Argentine Precordillera in the west and as far north as Arequipa in Peru. Our interpretation of the paleogeographical and geodynamic evolution invokes a hypothetical Paleoproterozoic block (MARA) embracing basement ultimately older than 1.7 Ga in the Western Sierras Pampeanas (Argentina), the Arequipa block (Peru), the Rio Apa block (Brazil), and probably also the Paraguaia block (Bolivia).Centro de Investigaciones Geológica

Burdigalian deposits of the Santa Cruz Formation in the Sierra Baguales, Austral (Magallanes) Basin: Age, depositional environment and vertebrate fossils

Indexación: Web of Science; Scielo.ABSTRACT. A succession of marine and continental strata on the southern flank of Cerro Cono in the Sierra Baguales, northeast of Torres del Paine, can be correlated with stratigraphic units exposed along the southern border of the Lago Argentino region in Santa Cruz Province, Argentina. These include the Estancia 25 de Mayo Formation and the basal part of the Santa Cruz Formation. The lithological correlation is also confirmed by detrital zircon ages (maximum age of 18.23±0.26 Ma) and a rich assemblage of terrestrial vertebrate fossils, biostratigraphically equivalent to a postColhuehuapian, pre-Santacrucian South American Land Mammal Age (SALMA) fauna, suggesting a range of 19 to 17.8 Ma. Similar ages have been obtained from the basal part of the Santa Cruz Formation at Estancia Quién Sabe in southwestern Argentina, supporting the assumption of a regional continuity between these deposits. A measured lithostratigraphic column is presented and the depositional environment is interpreted as a coastal plain with small, meandering rivers and ephemeral floodplain lakes. The sedimentation coincides with intensified uplift of the Patagonian Andes during the ‘Quechua Phase’ of Andean tectonism, which is reflected by a change in paleocurrent directions from northwest to east-northeast. Keywords: Burdigalian, Santa Cruz Formation, Santacrucian SALMA, ‘Notohippidian’ fauna, Meandering rivers.RESUMEN. Una sucesión de estratos marinos y continentales en el flanco meridional del cerro Cono, en la sierra Baguales, al noreste de Torres del Paine, se correlaciona con estratos al sur de la región de lago Argentino en la Provincia de Santa Cruz, República Argentina. Estas unidades incluyen la Formación Estancia 25 de Mayo y la parte basal de la Formación Santa Cruz. La correlación litológica es, además, confirmada por datación de circones detríticos (edad máxima de 18,23±0,26 Ma) y un variado ensamble de vertebrados fósiles terrestres de edad post-Colhuehuapense a pre-Santacrucense en la escala de Edades Mamífero Sudamericanas (EMAS), con un rango temporal de entre 19 a 17,8 Ma. Edades similares han sido reportadas para la parte basal de la Formación Santa Cruz, en estancia Quién Sabe, en el suroeste de Argentina, ratificando la continuidad regional entre estos depósitos. Se presenta una columna estratigráfica y se interpreta el ambiente de depositación como una llanura costera con pequeños ríos sinuosos y lagos efímeros. La edad de sedimentación coincide con el solevantamiento de los Andes Patagónicos durante la 'Fase Quechua', lo que se ve reflejado por un cambio en la dirección de las paleocorrientes desde el noroeste hacia el este-noreste.http://ref.scielo.org/csxwd

Etiology of Experimental Osteoarthritis: Early Events and Potential Clinical Implications

Introduction Osteoarthritis (OA) is the most common form of arthritis and accounts for 50% of all chronic conditions in the elderly. One in two adults reported a chronic musculoskeletal condition in 2005, twice the rate of reported chronic heart or respiratory conditions(2). In addition, persons aged 45 to 64 account for an increasingly greater proportion of total musculoskeletal disease treatment costs and lost wages, a trend that will continue for the next several decades(3). Surgical treatment culminating in total joint replacement (TJR) remains the most effective therapy for late stage OA. Current treatment of pre-surgical OA consists of pain relieving medications (i.e. NSAIDs), physical therapy, and mechanical supports (i.e. braces, canes, and walkers). Despite the wealth of clinical data on OA, there is currently no cure for the disease. Our previous work in developing potential disease-modifying osteoarthritis drugs (DMOADs) had yielded promising results, showing a decrease in OA cartilage lesion areas and histological grades (Figure 1). Interestingly, we noted that animals treated for only the first 3 weeks demonstrated near 6-week levels of OA reduction. These differences in treatment responsiveness necessitate a better characterization of the specific cellular phases of OA throughout the natural disease progression. The current study was undertaken to clarify this progression of early OA events. Methods OA was induced in the right knees of 10-week-old male 129 S6/SvEv (Taconic) mice via DMM surgery. Mice receiving sham surgery with no destabilization were used as negative controls. Both groups were sacrificed at 4, 8, 12, 16, and 20-day intervals in order to evaluate OA progression. Knees were harvested, processed, and sectioned at 6um intervals. Sections were stained for cartilage composition (Safranin-O) and scored for progression and severity of OA by 3 blinded observers using a 0-5 scale (modified Mankin System)(4). Both ‘mean maximal’ scores (highest scores per knee), and ‘mean summed scores (sum of scores per knee) were generated using this scale. All scores were averaged across observers. Cartilage lesion area, subchondral bone area (sclerosis), and apoptosis (TUNEL method) were measured using a histomorphometric analysis package (ImageJ)(5). Conclusions Measurable osteoarthritic changes in articular cartilage and underlying bone following meniscal injury occur far earlier than previously described. Some changes are clearly degenerative (OA grade, stage & lesion area), however, some changes (subchondral bone thickening) could be regarded as compensatory supportive mechanisms. Cell death (apoptosis) is an acute event following relatively minor changes to knee biomechanics. Our results suggest an opportunity for intervention early on in OA before the resulting articular changes become irreversible. Specifically, consideration of anti-apoptosis based therapies could prevent much of the subsequent structural changes in articular cartilage. Future Directions Apoptosis data suggests pursuing an anti-apoptotic therapy strategy in the DMM model of OA Early bone sclerotic events suggest bone tissue as a target for anti-OA therapy. Translationally, preventing or delaying OA due to soft tissue injuries (e.g., sports injuries) may be possible with early medical treatment of OA proximal to the time of injury. References (1) International Bone and Joint Decade 2000-2010 Organization, 1999. (2) National Center for Health Statistics, National Health Interview Survey, 2005. (3) Kurtz, SM, Lau, E, et al. Future Young Patient Demand for Primary and Revision Joint Replacement: National Projections from 2010 to 2030. Clinical Orthopaedics and Related Research, April 2009. (4) Kurtz, SM, Ong, K, et. al. Projections of Primary and Revision Hip and Knee Arthroplasty in the United States from 2005 to 2030. The Journal of Bone and Joint Surgery, 2007;89:780-5. (5) http://rsbweb.nih.gov/ij