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Journal articles on the topic 'AAL2'

Author: Grafiati
Published: 4 June 2021
Last updated: 1 February 2022

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1

Bucha, Tomáš, Juraj Papčo, Ivan Sačkov, Jozef Pajtík, Maroš Sedliak, Ivan Barka, and Ján Feranec. "Woody Above-Ground Biomass Estimation on Abandoned Agriculture Land Using Sentinel-1 and Sentinel-2 Data." Remote Sensing 13, no. 13 (June 25, 2021): 2488. http://dx.doi.org/10.3390/rs13132488.

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Abandoned agricultural land (AAL) is a European problem and phenomenon when agricultural land is gradually overgrown with shrubs and forest. This wood biomass has not yet been systematically inventoried. The aim of this study was to experimentally prove and validate the concept of the satellite-based estimation of woody above-ground biomass (AGB) on AAL in the Western Carpathian region. The analysis is based on Sentinel-1 and -2 satellite data, supported by field research and airborne laser scanning. An improved AGB estimate was achieved using radar and optical multi-temporal data and polarimetric coherence by creating integrated predictive models by multiple regression. Abandonment is represented by two basic AAL classes identified according to overgrowth by shrub formations (AAL1) and tree formations (AAL2). First, an allometric model for AAL1 estimation was derived based on empirical material obtained from blackthorn stands. AAL2 biomass was quantified by different procedures related to (1) mature trees, (2) stumps and (3) young trees. Then, three satellite-based predictive mathematical models for AGB were developed. The best model reached R2 = 0.84 and RMSE = 41.2 t·ha−1 (35.1%), parametrized for an AGB range of 4 to 350 t·ha−1. In addition to 3214 hectares of forest land, we identified 992 hectares of shrub–tree formations on AAL with significantly lower wood AGB than on forest land and with simple shrub composition.
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Adegbesan, S. I., and I. Abdulraheem. "Growth performance, nutrient utilization, haematology and serum biochemistry of African catfish (Clarias gariepinus) broodstock fed varying levels of Aspilia africana leaves-paste." Nigerian Journal of Animal Production 47, no. 1 (December 19, 2020): 129–39. http://dx.doi.org/10.51791/njap.v47i1.197.

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Dietary supplementation with phytobiotics is a promising area in fish nutrition towards promoting the growth and health status of cultured fish. This experiment was conducted to determine the effects of Aspilia africana leaves-paste (AAL) on the growth, haematology and serum biochemistry of Clarias gariepinus broodstock. Experiment was carried out in a 24 net-happa (0.6m x 1.07m x 1.2m) suspended in an earthen pond (30 m x 5 m x 1.2m). Forty-eight (7 months old fish, 24 males (0.80±0.04kg) and 24 females (0.70±0.03kg)) were stocked at two fish per net-happa under four treatments in six replicates in a completely randomized design. Four diets (40% crude protein) were formulated to contained: control (0%); AAL1 (0.5%); AAL2 (1%) and AAL3 (1.5%). Fish were fed ad libitum twice daily for 16 weeks. Data on all parameters were analyzed using ANOVA. The highest mean weight gain, MWG: 3.13±0.15kg and lowest feed conversion ratio, FCR: 1.30± 0.04 were recorded in broodstock fed 1.5% AAL3. The lowest MWG (1.27±0.03kg) and highest FCR: 1.72±0.03) were obtained in broodstock fed control diet. The lowest packed cell volume: 15.83 ± 0.17 % and haemoglobin: 5.25 ± 0.1 (g/dL) were recorded in fish fed 1% and 1.5% AAL. No significant differences in the total protein and creatinine values obtained between fish fed 1% and 1.5% AAL. The study recommended the dietary inclusion of 1.5% A. africana leaves-paste to effectively promote growth and nutrient utilization of cultured C. gariepinus broodstock
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Wang, Zhangxia, and Haibo Ma. "Mechanisms of a Cyclobutane-Fused Lactone Hydrolysis in Alkaline and Acidic Conditions." Molecules 26, no. 12 (June 9, 2021): 3519. http://dx.doi.org/10.3390/molecules26123519.

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Searching for functional polyesters with stability and degradability is important due to their potential applications in biomedical supplies, biomass fuel, and environmental protection. Recently, a cyclobutane-fused lactone (CBL) polymer was experimentally found to have superior stability and controllable degradability through hydrolysis reactions after activation by mechanical force. In order to provide a theoretical basis for developing new functional degradable polyesters, in this work, we performed a detailed quantum chemical study of the alkaline and acidic hydrolysis of CBL using dispersion-corrected density functional theory (DFT-D3) and mixed implicit/explicit solvent models. Various possible hydrolysis mechanisms were found: BAC2 and BAL2 in the alkaline condition and AAC2, AAL2, and AAL1 in the acidic condition. Our calculations indicated that CBL favors the BAC2 and AAC2 mechanisms in alkaline and acidic conditions, respectively. In addition, we found that incorporating explicit water solvent molecules is highly necessary because of their strong hydrogen-bonding with reactant/intermediate/product molecules.
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4

Saito, H. "Bandwidth management for AAL2 traffic." IEEE Transactions on Vehicular Technology 49, no. 4 (July 2000): 1364–77. http://dx.doi.org/10.1109/25.875261.

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5

Saito, Hiroshi. "Performance evaluation of AAL2 voice multiplexer." Performance Evaluation 42, no. 1 (September 2000): 57–83. http://dx.doi.org/10.1016/s0166-5316(99)00085-1.

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6

Moore, Jeffrey A., and John M. Schwab. "Unprecedented observation of lactone hydrolysis by the AAl2 mechanism." Tetrahedron Letters 32, no. 21 (May 1991): 2331–34. http://dx.doi.org/10.1016/s0040-4039(00)79916-3.

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7

Petr, David W., Gopi K. Vaddi, Raghushankar R. Vatte, and Sampath Sreepathi-Komanduri. "Analytic and Simulation Modeling for AAL2 Connection Admission Control." SIMULATION 78, no. 4 (April 2002): 239–48. http://dx.doi.org/10.1177/0037549702078004542.

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8

Ko, Gwangzeen, Sungdon Moon, Aftab Ahmad, and Kiseon Kim. "Performance Analysis of the ATM Adaptation Layer 2 (AAL2)." AEU - International Journal of Electronics and Communications 58, no. 3 (January 2004): 193–99. http://dx.doi.org/10.1078/1434-8411-54100228.

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9

Makké, Rani, Samir Tohmé, Jean-Yves Cochennec, and Sophie Pautonnier. "Performance evaluation of the AAL2 protocol within the UTRAN." Annales Des Télécommunications 58, no. 7-8 (July 2003): 1041–65. http://dx.doi.org/10.1007/bf03001871.

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10

LEE, S. K. "Performance Analysis on VoDSL with Splitting Two Sublayers in AAL2." IEICE Transactions on Communications E88-B, no. 4 (April 1, 2005): 1677–81. http://dx.doi.org/10.1093/ietcom/e88-b.4.1677.

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11

Villasenor-Gonzalez, Luis, Sophia Tsakiridou, Luis Orozco-Barbosa, and Louise Lamont. "Performance analysis of wireless ATM/AAL2 over a burst error channel." Computer Communications 25, no. 1 (January 2002): 1–8. http://dx.doi.org/10.1016/s0140-3664(01)00335-8.

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12

Samhat, A., and T. Chahed. "IP versus AAL2 for transport in the UMTS radio access network." Computer Communications 28, no. 5 (March 2005): 477–84. http://dx.doi.org/10.1016/j.comcom.2004.09.009.

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13

Eneroth, G., G. Fodor, G. Leijonhufvud, A. Racz, and I. Szabo. "Applying ATM/AAL2 as a switching technology in third-generation mobile access networks." IEEE Communications Magazine 37, no. 6 (June 1999): 112–23. http://dx.doi.org/10.1109/35.769285.

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14

Sriram, K., and Yung-Terng Wang. "Voice over ATM using AAL2 and bit dropping: performance and call admission control." IEEE Journal on Selected Areas in Communications 17, no. 1 (1999): 18–28. http://dx.doi.org/10.1109/49.743693.

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15

Lee, H. J., and J. H. Kim. "Analysis of Bandwidth Gain Over Various Timer$_$CU of AAL2 for Voice Traffic Multiplexing." IEEE Transactions on Vehicular Technology 54, no. 4 (July 2005): 1438–46. http://dx.doi.org/10.1109/tvt.2005.851321.

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16

Lorenz, B., J. Lenzi, J. Cmaidalka, R. L. Meng, Y. Y. Sun, Y. Y. Xue, and C. W. Chu. "Superconductivity in the C32 intermetallic compounds AAl2−xSix, with A=Ca and Sr; and 0.6." Physica C: Superconductivity 383, no. 3 (December 2002): 191–96. http://dx.doi.org/10.1016/s0921-4534(02)02056-7.

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17

Sriram, K., T. G. Lyons, and Yung-Terng Wang. "Anomalies due to delay and loss in AAL2 packet voice systems: performance models and methods of mitigation." IEEE Journal on Selected Areas in Communications 17, no. 1 (1999): 4–17. http://dx.doi.org/10.1109/49.743690.

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18

Travis, Laura, Leon J. Worth, Jason Trubiano, Karin Thursky, and Noleen Bennett. "Burden of antibiotic allergy labels in Australian aged care residents: Findings from a national point-prevalence survey." Infection Control & Hospital Epidemiology 41, no. 6 (March 19, 2020): 641–44. http://dx.doi.org/10.1017/ice.2020.53.

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AbstractObjective:To determine the prevalence of antibiotic allergy labels (AALs) in Australian aged care residents and to describe the impact of labels on antibiotic prescribing practices.Design:Point-prevalence survey.Setting:Australian residential aged care facilities.Participants:We surveyed 1,489 residents in 407 aged care facilities.Methods:Standardized data were collected on a single day between June 1 and August 31, 2018, for residents prescribed an antibiotic. An AAL was reported if it was documented in the resident’s health record. Resident-level data were used to calculate overall prevalence, and antibiotic-level data were used to report relative frequency of AALs for individual antibiotics and classes.Results:Among 1,489 residents, 356 (24%) had 1 or more documented AALs. The AALs for penicillin (28.3%), amoxicillin or amoxicillin/clavulanic acid (10.5%), cefalexin (7.2%), and trimethoprim (7.0%) were most commonly reported. The presence of an AAL was associated with significantly less prescribing of penicillins (OR, 0.43; 95% CI, 0.31–0.62; P < .001) and significantly more prescribing of lincosamides (OR, 4.81; P < .001), macrolides (OR, 2.03; P = .007), and tetracyclines (OR, 1.54; P = .033). Of residents with AALs, 7 residents (1.9%) were prescribed an antibiotic that was listed on the allergy section of their health record.Conclusions:A high prevalence of AALs was observed among residents of Australian aged care facilities, comparable to the prevalence of AALs in high-risk hospitalized patients. Significant increases in prescribing of lincosamide, macrolide, and tetracycline agents poses a potential risk to aged populations, and future studies must evaluate the benefits of AAL delabelling programs tailored for aged care settings.
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Earley, Lauriel F., Yasuhiro Kawano, Kei Adachi, Xiao-Xin Sun, Mu-Shui Dai, and Hiroyuki Nakai. "Identification and Characterization of Nuclear and Nucleolar Localization Signals in the Adeno-Associated Virus Serotype 2 Assembly-Activating Protein." Journal of Virology 89, no. 6 (December 31, 2014): 3038–48. http://dx.doi.org/10.1128/jvi.03125-14.

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ABSTRACTAssembly-activating protein (AAP) of adeno-associated virus serotype 2 (AAV2) is a nucleolar-localizing protein that plays a critical role in transporting the viral capsid VP3 protein to the nucleolus for assembly. Here, we identify and characterize AAV2 AAP (AAP2) nuclear (NLS) and nucleolar (NoLS) localization signals near the carboxy-terminal region of AAP2 (amino acid positions 144 to 184) (AAP2144–184). This region contains five basic-amino-acid-rich (BR) clusters, KSKRSRR (AAP2BR1), RRR (AAP2BR2), RFR (AAP2BR3), RSTSSR (AAP2BR4), and RRIK (AAP2BR5), from the amino terminus to the carboxy terminus. We created 30 AAP2BR mutants by arginine/lysine-to-alanine mutagenesis or deletion of AAP2BRs and 8 and 1 green fluorescent protein (GFP)-AAP2BR and β-galactosidase–AAP2BR fusion proteins, respectively, and analyzed their intracellular localization in HeLa cells by immunofluorescence microscopy. The results showed that AAP2144–184has redundant multipartite NLSs and that any combinations of 4 AAP2BRs, but not 3 or less, can constitute a functional NLS-NoLS; AAP2BR1 and AAP2BR2 play the most influential role for nuclear localization, but either one of the two AAP2BRs is dispensable if all 4 of the other AAP2BRs are present, resulting in 3 different, overlapping NLS motifs; and the NoLS is shared redundantly among the five AAP2BRs and functions in a context-dependent manner. AAP2BR mutations not only resulted in aberrant intracellular localization, but also attenuated AAP2 protein expression to various degrees, and both of these abnormalities have a significant negative impact on capsid production. Thus, this study reveals the organization of the intermingling NLSs and NoLSs in AAP2 and provides insights into their functional roles in capsid assembly.IMPORTANCEAdeno-associated virus (AAV) has become a popular and successful vector forin vivogene therapy; however, its biology has yet to be fully understood. In this regard, the recent discovery of the assembly-activating protein (AAP), a nonstructural, nucleolar-localizing AAV protein essential for viral capsid assembly, has provided us a new opportunity to better understand the fundamental processes required for virion formation. Here, we identify clusters of basic amino acids in the carboxy terminus of AAP from AAV serotype 2 (AAV2) that act as nuclear and nucleolar localization signals. We also demonstrate their importance in maintaining AAP expression levels and efficient production of viral capsids. Insights into the functions of AAP can elucidate the requirements and process for AAV capsid assembly, which may lead to improved vector production for use in gene therapy. This study also contributes to the growing body of work on nuclear and nucleolar localization signals.
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Chang, Xiao, Xiaoliang Jia, Kuo Liu, and Hao Hu. "Knowledge-enabled digital twin for smart designing of aircraft assembly line." Assembly Automation 41, no. 4 (June 8, 2021): 441–56. http://dx.doi.org/10.1108/aa-09-2020-0133.

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Purpose The purpose of this paper is to provide a knowledge-enabled digital twin for smart design (KDT-SD) of aircraft assembly line (AAL) to enhance the AAL efficiency, performance and visibility. Modern AALs usually need to have capabilities such as digital-physical interaction and self-evaluation that brings significant challenges to traditional design method for AAL. The digital twin (DT) combining with reusable knowledge, as the key technologies in this framework, is introduced to promote the design process by configuring, understanding and evaluating design scheme. Design/methodology/approach The proposed KDT-SD framework is designed with the introduction of DT and knowledge. First, dynamic design knowledge library (DDK-Lib) is established which could support the various activities of DT in the entire design process. Then, the knowledge-driven digital AAL modeling method is proposed. At last, knowledge-based smart evaluation is used to understand and identify the design flaws, which could further improvement of the design scheme. Findings By means of the KDT-SD framework proposed, it is possible to apply DT to reduce the complexity and discover design flaws in AAL design. Moreover, the knowledge equips DT with the capacities of rapid modeling and smart evaluation that improve design efficiency and quality. Originality/value The proposed KDT-SD framework can provide efficient design of AAL and evaluate the design performance in advance so that the feasibility of design scheme can be improved as much as possible.
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GUARNIERI, FERNANDA, and FRANCISCO GIOVANNI DAVID VIEIRA. "REINVENTANDO O COTIDIANO: ANÁLISE DE PRÁTICAS DE CONSUMO SOB A ÓTICA DE CERTEAU." Revista de Administração de Empresas 60, no. 5 (October 2020): 311–21. http://dx.doi.org/10.1590/s0034-759020200502.

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RESUMO O objetivo deste trabalho é compreender como a frequência às Academias ao Ar Livre (AALs) por parte dos idosos molda suas práticas de consumo e (re)inventa o seu cotidiano. Como suporte teórico, adotamos uma abordagem interdisciplinar entre pós-modernismo e estudos de cultura de consumo com a teoria das práticas cotidianas de Michel de Certeau. A pesquisa é de natureza qualitativa, e as informações foram coletadas com idosos frequentadores de AALs. As análises seguiram a concepção da pesquisa interpretativista para organização e categorização das informações. A frequência à AAL revelou-se como ponto de partida para a (re)invenção do cotidiano dos idosos. Essa (re)invenção do cotidiano ocorre a partir de novas práticas de consumo que emergem por meio dos desdobramentos de uma nova dinâmica na vida deles. Ao final, indicamos nova possibilidade de investigação teórica em estudos de consumo relacionados com práticas, além da relevância apresentada no âmbito social.
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Torda, Adrienne, and Victor Chan. "Antibiotic Allergies – Is De-labeling Based on Clinical History Feasible?" Open Forum Infectious Diseases 4, suppl_1 (2017): S342. http://dx.doi.org/10.1093/ofid/ofx163.816.

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Abstract Background Up to 25% of patients admitted to hospital have an antibiotic allergy label (AAL), most of which are towards penicillin. However, up to 90% of patients who claim to be allergic to penicillin are actually able to tolerate them1. Whilst skin testing is safe and efficacious in de-labeling patients with a penicillin allergy label, it is usually not widely available. Therefore, we investigated the feasibility of de-labeling based solely upon clinical grounds. Quality of allergy documentation and subsequent antibiotic use was also assessed. Methods This was a cross-sectional study assessing all patients admitted to a tertiary referral teaching hospital over a 5-month period in 2016. All newly admitted patients were prospectively screened for the presence of an antibiotic allergy documented in their electronic medical record. Unless unable to participate, patients were interviewed regarding the detailed nature of their antibiotic allergy. Information regarding allergy documentation, medical condition and antibiotic use was obtained from medical records. Results 3855 patients were screened, 553 (14.35%) had an AAL, and 352 were interviewed. There were 426 allergies, 276 (64.8%) towards a penicillin. Only 52% of patients had a convincing history consistent with antibiotic allergy, and 48% of these were mild cutaneous reactions. It was felt that de-labeling and direct re-challenge would be relatively safe in 70% (298/42) of AALs (if the mild cutaneous allergic group were included). In patients who were prescribed antibiotics during study admission, 25.6% (41/160) of antibiotic prescriptions in our cohort were found to be inappropriate in patients with AALs. Conclusion Direct re-challenge based upon clinical grounds appears to be a feasible clinical option in many patients with AALs and would allow de-labeling of these patients. The major barriers continue to be patient acceptance and risk of severe adverse reactions. Our study also found that major improvements could be made in the specific documentation of allergy and also in selection of guideline-recommended alternate antibiotics. 1. Joint Task Force on Practice Parameters. Drug Allergy: An Updated Practice Parameter. Ann Allergy Asthma Immunol. 2010; 105(4): p. 259–273. Disclosures All authors: No reported disclosures.
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Simon, Leah E., and Fred Hamann. "Tracing Metallicity in High Redshift Quasars." Proceedings of the International Astronomical Union 5, S265 (August 2009): 183–84. http://dx.doi.org/10.1017/s1743921310000517.

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AbstractWe present two ongoing studies of gas phase abundances around high redshift quasars. First, we examine broad emission line (BEL) metallicities for 29 quasars with 2.3 < z < 4.6 and far-infrared (far-IR) luminosities (LFIR) from 1013.4 to ≤ 1012.2 L⊙, corresponding to star formation rates (SFRs) of 6740 to ≤ 1360 M⊙ yr−1. Quasar samples sorted by LFIR might represent an evolutionary sequence if SFRs in quasar hosts generally diminish across quasar lifetimes. We create three composite spectra from rest-frame ultra-violet Sloan Digital Sky Survey spectra with increasing far-IR luminosity. We measure the N V(λ1240)/C IV(λ1550) and Si IV(λ1397)+O IV](λ1402)/C IV(λ1550) emission line flux ratios for each composite and find uniformly high (~5-10 times solar) metallicities for the three composites, and no evidence for changes in metal enrichment with changes in ongoing SFR. Second, we present preliminary results from the largest ever survey of high resolution associated absorption line (AAL) region metallicities and physical properties in a sample of high redshift (z > 3) quasars. This includes five quasars with previously known AALs at z > 4 and two well measured z ~3 quasars with unusually rich absorption spectra. We determine well-constrained metallicities of about twice solar for five AAL systems. We find a range of lower limits for AAL metallicities in the z > 4 quasars from 1/100ths solar to 3 times solar. Overall, these results for typically super-solar gas-phase metallicities near quasars are consistent with evolutionary schemes where the major episodes of star formation in the host galaxies occur before the visibly luminous quasar phase. High SFRs (comparable to ULIRGs) in the host galaxies are not clearly linked to younger or chemically less mature quasar environments.
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Peng, Tao, and Binghai Zhou. "Scheduling multiple servers to facilitate just-in-time part-supply in automobile assembly lines." Assembly Automation 38, no. 3 (August 6, 2018): 347–60. http://dx.doi.org/10.1108/aa-08-2017-102.

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Purpose With regard to product variety and cost competition, just-in-time (JIT) part-supply has become a critical issue in automobile assembly lines (AALs). This paper aims to investigate a multiple server scheduling problem (MSSP) encountered in the JIT part-supply process of AALs. Parts are stored in boxes and allotted from the JIT-supermarket to consumptive stations with a multiple server system. The schedule is to dispatch and sequence material boxes on each server for minimizing line-side inventory levels. Design/methodology/approach A mixed integer linear programming (MILP) model is established to formulate the proposed MSSP to pave the way for CPLEX procedure. Considering the high complexity of MSSP, a hybrid ant colony optimization (HACO) approach is developed by integrating basic ant colony optimization (ACO) with local optimizers that comprise of a fast local search and a tailored breadth-first tree search method. Findings Both CPLEX and HACO approach are capable of solving small-scale instances to optimality within reasonable computation time. The proposed HACO has been well enhanced with the embedded fast local search and tailored breadth-first tree search, and it performs robustly in a statistically significant manner when applied to real-world scale instances. Originality/value No stock-outs constraints and weighted line-side inventory level are considered in this paper, and the MSSP is solved satisfactorily to facilitate an efficient JIT part-supply of the AAL. In terms of the algorithm design, a tree search-based local optimizer is embedded into ACO to combine the mechanisms of ACO and problem-specific optimization.
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Poppe, Jean Lucas, and Taffarel De Oliveira Fontela. "ESPAÇOS PÚBLICOS EM PROL DA SAÚDE COLETIVA: INVESTIGAÇÃO, CARACTERIZAÇÃO E PERSPECTIVAS." Vivências 17, no. 33 (June 21, 2021): 37–56. http://dx.doi.org/10.31512/vivencias.v17i33.256.

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O sedentarismo tem sido um dos principais causadores de morte no mundo todo, sendo assim, surgiram ideias para combatê-lo, como, por exemplo, o incremento da infraestrutura de praças e parques públicos para a prática de exercícios físicos. O presente estudo busca conhecer a população que frequenta as praças de São Luiz Gonzaga, identificando os interesses esportivos e os elementos potencializadores e inibidores para a adoção de hábitos saudáveis, apresentando soluções que minimizem os níveis de sedentarismo populacional. O público de interesse foi entrevistado por meio de questionários e os dados obtidos foram analisados estatisticamente. A maioria dos entrevistados busca por melhores condições de saúde. Destes, 81% podem ser considerados ativos de acordo com padrões estabelecidos pela Organização Mundial de Saúde. Caminhada foi a atividade física predominante. Os maiores índice de IMC estão presentes entre os homens, assim como a ocorrência de lesões, sendo o tornozelo e o joelho as principais regiões acometidas. Cerca de 50% dos entrevistados mencionaram utilizar a academia pública ao ar livre (AAL), no entanto, apenas 45% da população relatam conhecer a finalidade dos equipamentos. Então, foi elaborada uma cartilha instrutiva com relação à utilização adequada de cada equipamento da AAL, tal material foi vinculado a um QR code fixado nas placas de identificação das AALs. Portanto, os investimentos nessas praças são válidos, pois incentivam a população a tornar-se fisicamente ativa, porém, ações de Educação em Saúde devem ser pensadas para aumentar o número de frequentadores na praça e promover a adoção de hábitos saudáveis pela população local.
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Alam, Samina, and Craig Meyers. "Adeno-Associated Virus Type 2 Induces Apoptosis in Human Papillomavirus-Infected Cell Lines but Not in Normal Keratinocytes." Journal of Virology 83, no. 19 (July 22, 2009): 10286–92. http://dx.doi.org/10.1128/jvi.00343-09.

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ABSTRACT The results of seroepidemiological studies suggest that infection with adeno-associated virus type 2 (AAV2) is negatively correlated with the incidence of human papillomavirus (HPV)-associated cervical cancer. We studied the potential of AAV2 oncosuppression of HPV and showed that HPV/AAV2 coinfection of cells culminated in apoptotic death, as determined by DNA laddering and caspase-3 cleavage. The induction of apoptosis coincided with AAV2 Rep protein expression; increased S-phase progression; upregulated pRb displaying both hyper- and hypophosphorylated forms; increased levels of p21WAF1, p16INK4, and p27KIP1 proteins; and diminished levels of E7 oncoprotein. In contrast, normal keratinocytes that were infected with AAV2 or transfected with the cloned full-length AAV2 genome failed to express Rep proteins or undergo apoptosis. The failure of AAV2 to productively infect normal keratinocytes could be clinically advantageous. The delineation of the molecular mechanisms underlying the HPV/AAV2 interaction could be harnessed for developing novel AAV2-derived therapeutics for cervical cancer.
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Yang, Grace S., Michael Schmidt, Ziying Yan, Jonathan D. Lindbloom, Thomas C. Harding, Brian A. Donahue, John F. Engelhardt, Robert Kotin, and Beverly L. Davidson. "Virus-Mediated Transduction of Murine Retina with Adeno-Associated Virus: Effects of Viral Capsid and Genome Size." Journal of Virology 76, no. 15 (August 1, 2002): 7651–60. http://dx.doi.org/10.1128/jvi.76.15.7651-7660.2002.

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ABSTRACT Gene therapy vectors based on adeno-associated viruses (AAVs) show promise for the treatment of retinal degenerative diseases. In prior work, subretinal injections of AAV2, AAV5, and AAV2 pseudotyped with AAV5 capsids (AAV2/5) showed variable retinal pigmented epithelium (RPE) and photoreceptor cell transduction, while AAV2/1 predominantly transduced the RPE. To more thoroughly compare the efficiencies of gene transfer of AAV2, AAV3, AAV5, and AAV6, we quantified, using stereological methods, the kinetics and efficiency of AAV transduction to mouse photoreceptor cells. We observed persistent photoreceptor and RPE transduction by AAV5 and AAV2 up to 31 weeks and found that AAV5 transduced a greater volume than AAV2. AAV5 containing full-length or half-length genomes and AAV2/5 transduced comparable numbers of photoreceptor cells with similar rates of onset of expression. Compared to AAV2, AAV5 transduced significantly greater numbers of photoreceptor cells at 5 and 15 weeks after surgery (greater than 1,000 times and up to 400 times more, respectively). Also, there were 30 times more genome copies in eyes injected with AAV2/5 than in eyes injected with AAV2. Comparing AAVs with half-length genomes, AAV5 transduced only four times more photoreceptor cells than AAV2 at 5 weeks and nearly equivalent numbers at 15 weeks. The enhancement of transduction was seen at the DNA level, with 50 times more viral genome copies in retinas injected with AAV having short genomes than in retinas injected with AAV containing full-length ones. Subretinal injection of AAV2/6 showed only RPE transduction at 5 and 15 weeks, while AAV2/3 did not transduce retinal cells. We conclude that varying genome length and AAV capsids may allow for improved expression and/or gene transfer to specific cell types in the retina.
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28

Fragkos, Michalis, Madlaina Breuleux, Nathalie Clément, and Peter Beard. "Recombinant Adeno-Associated Viral Vectors Are Deficient in Provoking a DNA Damage Response." Journal of Virology 82, no. 15 (May 7, 2008): 7379–87. http://dx.doi.org/10.1128/jvi.00358-08.

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ABSTRACT Adeno-associated virus type 2 (AAV2) provokes a DNA damage response that mimics a stalled replication fork. We have previously shown that this response is dependent on ataxia telangiectasia-mutated and Rad3-related kinase and involves recruitment of DNA repair proteins into foci associated with AAV2 DNA. Here, we investigated whether recombinant AAV2 (rAAV2) vectors are able to produce a similar response. Surprisingly, the results show that both single-stranded and double-stranded green fluorescent protein-expressing rAAV2 vectors are defective in producing such a response. We show that the DNA damage signaling initiated by AAV2 was not due to the virus-encoded Rep or viral capsid proteins. UV-inactivated AAV2 induced a response similar to that of untreated AAV2. This type of DNA damage response was not provoked by other DNA molecules, such as single-stranded bacteriophage M13 or plasmid DNAs. Rather, the results indicate that the ability of AAV2 to produce a DNA damage response can be attributed to the presence of cis-acting AAV2 DNA sequences, which are absent in rAAV2 vectors and could function as origins of replication creating stalled replication complexes. This hypothesis was tested by using a single-stranded rAAV2 vector containing the p5 AAV2 sequence that has previously been shown to enhance AAV2 replication. This vector was indeed able to trigger DNA damage signaling. These findings support the conclusion that efficient formation of AAV2 replication complexes is required for this AAV2-induced DNA damage response and provide an explanation for the poor response in rAAV2-infected cells.
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29

Tullis, Gregory E., and Thomas Shenk. "Efficient Replication of Adeno-Associated Virus Type 2 Vectors: a cis-Acting Element outside of the Terminal Repeats and a Minimal Size." Journal of Virology 74, no. 24 (December 15, 2000): 11511–21. http://dx.doi.org/10.1128/jvi.74.24.11511-11521.2000.

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ABSTRACT Recombinant adeno-associated virus type 2 (AAV2) can be produced in adenovirus-infected cells by cotransfecting a plasmid containing the recombinant AAV2 genome, which is generally comprised of the viral terminal repeats flanking a transgene, together with a second plasmid expressing the AAV2 rep and cap genes. However, recombinant viruses generally replicate inefficiently, often producing 100-fold fewer virus particles per cell than can be obtained after transfection with a plasmid containing a wild-type AAV2 genome. We demonstrate that this defect is due, at least in part, to the presence of a positive-acting cis element between nucleotides 194 and 1882 of AAV2. Recombinant AAV2 genomes lacking this region accumulated 14-fold less double-stranded, monomer-length replicative-form DNA than did wild-type AAV2. In addition, we demonstrate that a minimum genome size of 3.5 kb is required for efficient production of single-stranded viral DNA. Relatively small recombinant genomes (2,992 and 3,445 bp) accumulated three- to eightfold less single-stranded DNA per monomer-length replicative-form DNA molecule than wild-type AAV2. In contrast, recombinant AAV2 with larger genomes (3,555 to 4,712 bp) accumulated similar amounts of single-stranded DNA per monomer-length replicative-form DNA compared to wild-type AAV2. Analysis of two recombinant AAV2 genomes less than 3.5 kb in size indicated that they were deficient in the production of the extended form of monomer-length replicative-form DNA, which is thought to be the immediate precursor to single-stranded AAV2 DNA.
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30

Allocca, Mariacarmela, Claudio Mussolino, Maria Garcia-Hoyos, Daniela Sanges, Carolina Iodice, Marco Petrillo, Luk H. Vandenberghe, et al. "Novel Adeno-Associated Virus Serotypes Efficiently Transduce Murine Photoreceptors." Journal of Virology 81, no. 20 (August 15, 2007): 11372–80. http://dx.doi.org/10.1128/jvi.01327-07.

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ABSTRACT Severe inherited retinal diseases, such as retinitis pigmentosa and Leber congenital amaurosis, are caused by mutations in genes preferentially expressed in photoreceptors. While adeno-associated virus (AAV)-mediated gene transfer can correct retinal pigment epithelium (RPE) defects in animal models, approaches for the correction of photoreceptor-specific diseases are less efficient. We evaluated the ability of novel AAV serotypes (AAV2/7, AAV2/8, AAV2/9, AAV2rh.43, AAV2rh.64R1, and AAV2hu.29R) in combination with constitutive or photoreceptor-specific promoters to improve photoreceptor transduction, a limiting step in photoreceptor rescue. Based on a qualitative analysis, all AAV serotypes tested efficiently transduce the RPE as well as rod and cone photoreceptors after subretinal administration in mice. Interestingly, AAV2/9 efficiently transduces Müller cells. To compare photoreceptor transduction from different AAVs and promoters in both a qualitative and quantitative manner, we designed a strategy based on the use of a bicistronic construct expressing both enhanced green fluorescent protein and luciferase. We found that AAV2/8 and AAV2/7 mediate six- to eightfold higher levels of in vivo photoreceptor transduction than AAV2/5, considered so far the most efficient AAV serotype for photoreceptor targeting. In addition, following subretinal administration of AAV, the rhodopsin promoter allows significantly higher levels of photoreceptor expression than the other ubiquitous or photoreceptor-specific promoters tested. Finally, we show that AAV2/7, AAV2/8, and AAV2/9 outperform AAV2/5 following ex vivo transduction of retinal progenitor cells differentiated into photoreceptors. We conclude that AAV2/7 or AAV2/8 and the rhodopsin promoter provide the highest levels of photoreceptor transduction both in and ex vivo and that this may overcome the limitation to therapeutic success observed so far in models of inherited severe photoreceptor diseases.
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31

Hildinger, Markus, Alberto Auricchio, Guangping Gao, Lili Wang, Narendra Chirmule, and James M. Wilson. "Hybrid Vectors Based on Adeno-Associated Virus Serotypes 2 and 5 for Muscle-Directed Gene Transfer." Journal of Virology 75, no. 13 (July 1, 2001): 6199–203. http://dx.doi.org/10.1128/jvi.75.13.6199-6203.2001.

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ABSTRACT Vectors based on hybrids consisting of adeno-associated virus types 2 (ITRs and Rep) and 5 (Cap) were evaluated for muscle-directed gene transfer (called AAV2/5). Evaluation in immune-competent mice revealed greater transduction efficacy with AAV2/5 than with AAV2 and no cross-neutralization between AAV2/5 and AAV2. Interestingly, we saw no immunologic evidence of previous exposure to AAV5 capsids in a large population of healthy human subjects.
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32

Kashiwakura, Yuji, Kenji Tamayose, Kazuhisa Iwabuchi, Yukihiko Hirai, Takashi Shimada, Kunio Matsumoto, Toshikazu Nakamura, Masami Watanabe, Kazuo Oshimi, and Hiroyuki Daida. "Hepatocyte Growth Factor Receptor Is a Coreceptor for Adeno-Associated Virus Type 2 Infection." Journal of Virology 79, no. 1 (January 1, 2005): 609–14. http://dx.doi.org/10.1128/jvi.79.1.609-614.2005.

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ABSTRACT After the first attachment of virus to the cell surface through a primary receptor, efficient entry of virus requires the presence of a coreceptor. For adeno-associated virus type 2 (AAV2) infection, heparan sulfate proteoglycan is supposed as the primary receptor, and αvβ5 integrin and FGFR1 are reported to act as coreceptors. In this study, we were able to demonstrate that hepatocyte growth factor receptor, c-Met, is also a coreceptor for AAV2 infection. AAV2-mediated transgene analyses revealed that c-Met expression significantly up-regulated transgene expression without increasing AAV2 cell binding. Moreover, a viral overlay assay elucidated the physical interaction between AAV2 and the β subunit of c-Met. These data suggest that c-Met plays the role of coreceptor for AAV2 infection by facilitating AAV2 internalization into the cytoplasm.
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33

Halbert, Christine L., Elizabeth A. Rutledge, James M. Allen, David W. Russell, and A. Dusty Miller. "Repeat Transduction in the Mouse Lung by Using Adeno-Associated Virus Vectors with Different Serotypes." Journal of Virology 74, no. 3 (February 1, 2000): 1524–32. http://dx.doi.org/10.1128/jvi.74.3.1524-1532.2000.

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ABSTRACT Vectors derived from adeno-associated virus type 2 (AAV2) promote gene transfer and expression in the lung; however, we have found that while gene expression can persist for at least 8 months in mice, it was reduced dramatically in rabbits over a period of 2 months. The efficiency and persistence of AAV2-mediated gene expression in the human lung have yet to be determined, but it seems likely that readministration will be necessary over the lifetime of an individual. Unfortunately, we have found that transduction by a second administration of an AAV2 vector is blocked, presumably due to neutralizing antibodies generated in response to the primary vector exposure. Here, we have explored the use of AAV2 vectors pseudotyped with capsid proteins from AAV serotypes 2, 3, and 6 for readministration in the mouse lung. We found that an AAV6 vector transduced airway epithelial and alveolar cells in the lung at rates that were at least as high as those of AAV2 pseudotype vectors, while transduction rates mediated by AAV3 were much lower. AAV6 pseudotype vector transduction was unaffected by prior administration of an AAV2 or AAV3 vector, and transduction by an AAV2 pseudotype vector was unaffected by prior AAV6 vector administration, showing that cross-reactive neutralizing antibodies against AAV2 and AAV6 are not generated in mice. Interestingly, while prior administration of an AAV2 vector completely blocked transduction by a second AAV2 pseudotype vector, prior administration of an AAV6 vector only partially inhibited transduction by a second administration of an AAV6 pseudotype vector. Analysis of sera obtained from mice and humans showed that AAV6 is less immunogenic than AAV2, which helps explain this finding. These results support the development of AAV6 vectors for lung gene therapy both alone and in combination with AAV2 vectors.
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34

Schultz, Carolyn J., Meier Hsu, Barbara Miesak, and Gloria M. Coruzzi. "Arabidopsis Mutants Define an in Vivo Role for Isoenzymes of Aspartate Aminotransferase in Plant Nitrogen Assimilation." Genetics 149, no. 2 (June 1, 1998): 491–99. http://dx.doi.org/10.1093/genetics/149.2.491.

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Abstract Arabidopsis contains five isoenzymes of aspartate aminotransferase (AspAT) localized to the cytosol, chloroplast, mitochondria, or peroxisomes. To define the in vivo function of individual isoenzymes, we screened for Arabidopsis mutants deficient in either of the two major isoenzymes, cytosolic AAT2 or chloroplastic AAT3, using a native gel activity assay. In a screen of 8,000 M2 seedlings, three independent mutants deficient in cytosolic AAT2 (aat2) and two independent mutants deficient in chloroplastic AAT3 (aat3) were isolated. Mapping of aat2 and aat3 mutations and the five AspAT genes (ASP1–ASP5) established associations as follows: the mutation affecting aat2 maps with and cosegregates with ASP2, one of two expressed genes for cytosolic AspAT; the mutation affecting aat3 maps to the same location as the ASP5 gene encoding chloroplastic AspAT. Phenotypic analysis of the aat2 and aat3 mutants revealed a dramatic aspartate-related phenotype in one of the mutants deficient in cytosolic AAT2. The aat2-2 mutant displays an 80% reduction in levels of aspartate transported in the phloem of light-grown plants, and a 50% reduction in levels of asparagine transported in dark-adapted plants. These results indicate that cytosolic AAT2 is the major isoenzyme controlling aspartate synthesized for nitrogen transport in the light, and that this aspartate pool is converted to asparagine when plants are dark adapted.
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35

Asokan, Aravind, Julie B. Hamra, Lakshmanan Govindasamy, Mavis Agbandje-McKenna, and Richard J. Samulski. "Adeno-Associated Virus Type 2 Contains an Integrin α5β1 Binding Domain Essential for Viral Cell Entry." Journal of Virology 80, no. 18 (September 15, 2006): 8961–69. http://dx.doi.org/10.1128/jvi.00843-06.

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ABSTRACT Integrins have been implicated as coreceptors in the infectious pathways of several nonenveloped viruses. For example, adenoviruses are known to interact with αV integrins by virtue of a high-affinity arginine-glycine-aspartate (RGD) domain present in the penton bases of the capsids. In the case of adeno-associated virus type 2 (AAV2), which lacks this RGD motif, integrin αVβ5 has been identified as a coreceptor for cellular entry. However, the molecular determinants of AAV2 capsid-integrin interactions and the potential exploitation of alternative integrins as coreceptors by AAV2 have not been established thus far. In this report, we demonstrate that integrin α5β1 serves as an alternative coreceptor for AAV2 infection in human embryonic kidney 293 cells. Such interactions appear to be mediated by a highly conserved domain that contains an asparagine-glycine-arginine (NGR) motif known to bind α5β1 integrin with moderate affinity. The mutation of this domain reduces transduction efficiency by an order of magnitude relative to that of wild-type AAV2 vectors in vitro and in vivo. Further characterization of mutant and wild-type AAV2 capsids through transduction assays in cell lines lacking specific integrins, cell adhesion studies, and cell surface/solid-phase binding assays confirmed the role of the NGR domain in promoting AAV2-integrin interactions. Molecular modeling studies suggest that NGR residues form a surface loop close to the threefold axis of symmetry adjacent to residues previously implicated in binding heparan sulfate, the primary receptor for AAV2. The aforementioned results suggest that the internalization of AAV2 in 293 cells might follow a “click-to-fit” mechanism that involves the cooperative binding of heparan sulfate and α5β1 integrin by the AAV2 capsids.
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36

Rutledge, Elizabeth A., Christine L. Halbert, and David W. Russell. "Infectious Clones and Vectors Derived from Adeno-Associated Virus (AAV) Serotypes Other Than AAV Type 2." Journal of Virology 72, no. 1 (January 1, 1998): 309–19. http://dx.doi.org/10.1128/jvi.72.1.309-319.1998.

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ABSTRACT Adeno-associated viruses (AAVs) are single-stranded dependent parvoviruses being developed as transducing vectors. Although at least five serotypes exist (AAV types 1 to 5 [AAV1 to -5]), only AAV2, AAV3, and AAV4 have been sequenced, and the vectors in use were almost all derived from AAV2. Here we report the cloning and sequencing of a second AAV3 genome and a new AAV serotype designated AAV6 that is related to AAV1. AAV2, AAV3, and AAV6 were 82% identical at the nucleotide sequence level, and AAV4 was 75 to 78% identical to these AAVs. Significant sequence variation was noted in portions of the capsid proteins that presumably are responsible for serotype-specific functions. Vectors produced from AAV3 and AAV6 differed from AAV2 vectors in host range and serologic reactivity. The AAV3 and AAV6 vector serotypes were able to transduce cells in the presence of serum from animals previously exposed to AAV2 vectors. Our results suggest that vectors based on alternative AAV serotypes will have advantages over existing AAV2 vectors, including the transduction of different cell types, and resistance to neutralizing antibodies against AAV2. This could be especially important for gene therapy, as significant immunity against AAV2 exists in human populations and many protocols will likely require multiple vector doses.
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37

Nakazawa, N., S. Harashima, and Y. Oshima. "AAR2, a gene for splicing pre-mRNA of the MATa1 cistron in cell type control of Saccharomyces cerevisiae." Molecular and Cellular Biology 11, no. 11 (November 1991): 5693–700. http://dx.doi.org/10.1128/mcb.11.11.5693.

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We have isolated a class of mutants, aar2, showing the alpha mating type due to a defect in a1-alpha 2 repression but with alpha 2 repression activity from a nonmater strain of Saccharomyces cerevisiae expressing both a and alpha mating-type information in duplicate. Cells of the aar2 mutant and the aar2 disruptant also show a growth defect. A DNA fragment complementing the aar2 mutation contains an open reading frame consisting of 355 amino acid codons. Northern hybridization showed that cells of the aar2 mutant and disruptant contained alpha 1 and alpha 2 transcripts of the MAT alpha gene (or HML alpha in sir3 cells), but their a1 transcript of MATa (or HMRa in sir3 cells) migrated more slowly than that of the wild-type cells on gel electrophoresis and gave a diffused band. Primer extension analysis showed that the aar2 mutant and disruptant have a defect in splicing two short introns of the a1 pre-mRNA but not in splicing pre-mRNA of ACT1. The alpha mating type, but not the slow-growing phenotype, of the aar2 mutant was suppressed by introduction of an intronless MATa1 DNA. Thus, the AAR2 gene is involved in splicing pre-mRNA of the a1 cistron and other genes that are important for cell growth. The AAR2 locus was mapped on chromosome II beside the SSA3 locus, with a 276-bp space, but was not allelic to either PRP5 or PRP6, which are both located on chromosome II and function in splicing pre-mRNA of ACT1.
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38

Halbert, Christine L., James M. Allen, and A. Dusty Miller. "Adeno-Associated Virus Type 6 (AAV6) Vectors Mediate Efficient Transduction of Airway Epithelial Cells in Mouse Lungs Compared to That of AAV2 Vectors." Journal of Virology 75, no. 14 (July 15, 2001): 6615–24. http://dx.doi.org/10.1128/jvi.75.14.6615-6624.2001.

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ABSTRACT Although vectors derived from adeno-associated virus type 2 (AAV2) promote gene transfer and expression in many somatic tissues, studies with animal models and cultured cells show that the apical surface of airway epithelia is resistant to transduction by AAV2 vectors. Approaches to increase transduction rates include increasing the amount of vector and perturbing the integrity of the epithelia. In this study, we explored the use of vectors based on AAV6 to increase transduction rates in airways. AAV vectors were made using combinations ofrep, cap, and packaged genomes from AAV2 or AAV6. The packaged genomes encoded human placental alkaline phosphatase and contained terminal repeat sequences from AAV2 or AAV6. We found that transduction efficiency was primarily dependent on the source of Cap protein, defined here as the vector pseudotype. The AAV6 and AAV2 pseudotype vectors exhibited different tropisms in tissue-cultured cells, and cell transduction by AAV6 vectors was not inhibited by heparin, nor did they compete for entry in a transduction assay, indicating that AAV6 and AAV2 capsid bind different receptors. In vivo analysis of vectors showed that AAV2 pseudotype vectors gave high transduction rates in alveolar cells but much lower rates in the airway epithelium. In contrast, the AAV6 pseudotype vectors exhibited much more efficient transduction of epithelial cells in large and small airways, showing up to 80% transduction in some airways. These results, combined with our previous results showing lower immunogenicity of AAV6 than of AAV2 vectors, indicate that AAV6 vectors may provide significant advantages over AAV2 for gene therapy of lung diseases like cystic fibrosis.
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39

Nakazawa, N., S. Harashima, and Y. Oshima. "AAR2, a gene for splicing pre-mRNA of the MATa1 cistron in cell type control of Saccharomyces cerevisiae." Molecular and Cellular Biology 11, no. 11 (November 1991): 5693–700. http://dx.doi.org/10.1128/mcb.11.11.5693-5700.1991.

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We have isolated a class of mutants, aar2, showing the alpha mating type due to a defect in a1-alpha 2 repression but with alpha 2 repression activity from a nonmater strain of Saccharomyces cerevisiae expressing both a and alpha mating-type information in duplicate. Cells of the aar2 mutant and the aar2 disruptant also show a growth defect. A DNA fragment complementing the aar2 mutation contains an open reading frame consisting of 355 amino acid codons. Northern hybridization showed that cells of the aar2 mutant and disruptant contained alpha 1 and alpha 2 transcripts of the MAT alpha gene (or HML alpha in sir3 cells), but their a1 transcript of MATa (or HMRa in sir3 cells) migrated more slowly than that of the wild-type cells on gel electrophoresis and gave a diffused band. Primer extension analysis showed that the aar2 mutant and disruptant have a defect in splicing two short introns of the a1 pre-mRNA but not in splicing pre-mRNA of ACT1. The alpha mating type, but not the slow-growing phenotype, of the aar2 mutant was suppressed by introduction of an intronless MATa1 DNA. Thus, the AAR2 gene is involved in splicing pre-mRNA of the a1 cistron and other genes that are important for cell growth. The AAR2 locus was mapped on chromosome II beside the SSA3 locus, with a 276-bp space, but was not allelic to either PRP5 or PRP6, which are both located on chromosome II and function in splicing pre-mRNA of ACT1.
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40

Ye, Chaoyang, Jianming Qiu, and David J. Pintel. "Efficient Expression of the Adeno-Associated Virus Type 5 P41 Capsid Gene Promoter in 293 Cells Does Not Require Rep." Journal of Virology 80, no. 13 (July 1, 2006): 6559–67. http://dx.doi.org/10.1128/jvi.00387-06.

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ABSTRACT Efficient expression of the adeno-associated virus type 5 (AAV5) P41 capsid gene promoter required adenovirus E1A and/or E1B; however, in contrast to what was observed for expression of the AAV2 capsid gene promoter (P40), neither adenovirus infection nor the large Rep protein was required. Although both the AAV2 and the AAV5 large Rep proteins efficiently bound the (GAGY)3 Rep-binding element, the AAV5 large Rep protein transactivated transcription of the inducible AAV2 P40 promoter much less well than AAV2 large Rep. Differences in their activation potentials were mapped to the amino-terminal region of the proteins, and the poorly transactivating AAV5 Rep protein could competitively inhibit AAV2 Rep transactivation.
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41

Li, Chengwen, Matthew Hirsch, Aravind Asokan, Brian Zeithaml, Hong Ma, Tal Kafri, and R. Jude Samulski. "Adeno-Associated Virus Type 2 (AAV2) Capsid-Specific Cytotoxic T Lymphocytes Eliminate Only Vector-Transduced Cells Coexpressing the AAV2 Capsid In Vivo." Journal of Virology 81, no. 14 (May 2, 2007): 7540–47. http://dx.doi.org/10.1128/jvi.00529-07.

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ABSTRACT A recent clinical trial has suggested that recombinant adeno-associated virus (rAAV) vector transduction in humans induces a cytotoxic T-lymphocyte (CTL) response against the AAV2 capsid. To directly address the ability of AAV capsid-specific CTLs to eliminate rAAV-transduced cells in vitro and in vivo in mice, we first demonstrated that AAV2 capsid-specific CTLs could be induced by dendritic cells with endogenous AAV2 capsid expression or pulsed with AAV2 vectors. These CTLs were able to kill a cell line stable for capsid expression in vitro and also in a mouse tumor xenograft model in vivo. Parent colon carcinoma (CT26) cells transduced with a large amount of AAV2 vectors in vitro were also destroyed by these CTLs. To determine the effect of CTLs on the elimination of target cells transduced by AAV2 vectors in vivo, we carried out adoptive transfer experiments. CTLs eliminated liver cells with endogenous AAV2 capsid expression but not liver cells transduced by AAV2 vectors, regardless of the reporter genes. Similar results were obtained for rAAV2 transduction in muscle. Our data strongly suggest that AAV vector-transduced cells are rarely eliminated by AAV2 capsid-specific CTLs in vivo, even though the AAV capsid can induce a CTL response. In conclusion, AAV capsid-specific CTLs do not appear to play a role in elimination of rAAV-transduced cells in a mouse model. In addition, our data suggest that the mouse model may not mimic the immune response noted in humans and additional modification to AAV vectors may be required for further study in order to elicit a similar cellular immune response.
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42

Cabanes-Creus, Marti, Claus V. Hallwirth, Adrian Westhaus, Boaz H. Ng, Sophia H. Y. Liao, Erhua Zhu, Renina Gale Navarro, et al. "Restoring the natural tropism of AAV2 vectors for human liver." Science Translational Medicine 12, no. 560 (September 9, 2020): eaba3312. http://dx.doi.org/10.1126/scitranslmed.aba3312.

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Recent clinical successes in gene therapy applications have intensified interest in using adeno-associated viruses (AAVs) as vectors for therapeutic gene delivery. Although prototypical AAV2 shows robust in vitro transduction of human hepatocyte–derived cell lines, it has not translated into an effective vector for liver-directed gene therapy in vivo. This is consistent with observations made in Fah−/−/Rag2−/−/Il2rg−/− (FRG) mice with humanized livers, showing that AAV2 functions poorly in this xenograft model. Here, we derived naturally hepatotropic AAV capsid sequences from primary human liver samples. We demonstrated that capsid mutations, likely acquired as an unintentional consequence of tissue culture propagation, attenuated the intrinsic human hepatic tropism of natural AAV2 and related human liver AAV isolates. These mutations resulted in amino acid changes that increased binding to heparan sulfate proteoglycan (HSPG), which has been regarded as the primary cellular receptor mediating AAV2 infection of human hepatocytes. Propagation of natural AAV variants in vitro showed tissue culture adaptation with resulting loss of tropism for human hepatocytes. In vivo readaptation of the prototypical AAV2 in FRG mice with a humanized liver resulted in restoration of the intrinsic hepatic tropism of AAV2 through decreased binding to HSPG. Our results challenge the notion that high affinity for HSPG is essential for AAV2 entry into human hepatocytes and suggest that natural AAV capsids of human liver origin are likely to be more effective for liver-targeted gene therapy applications than culture-adapted AAV2.
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43

Wang, Lili, Roberto Calcedo, Timothy C. Nichols, Dwight A. Bellinger, Aaron Dillow, Inder M. Verma, and James M. Wilson. "Sustained correction of disease in naive and AAV2-pretreated hemophilia B dogs: AAV2/8-mediated, liver-directed gene therapy." Blood 105, no. 8 (April 15, 2005): 3079–86. http://dx.doi.org/10.1182/blood-2004-10-3867.

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AbstractAdeno-associated virus 8 (AAV8), a new member of the AAV family isolated from nonhuman primates, is an attractive candidate for hepatic gene transfer applications because of 10- to 100-fold improved transduction efficiency in mouse liver models. Additionally, AAV8 has lesser frequency of pre-existing immunity in humans. These properties could solve some of the problems associated with AAV2 vectors. The benefits of AAV8 demonstrated in mouse models, however, have not been confirmed in larger animals. In this study, we evaluate the efficacy and safety of AAV2/8 vector in both naive and AAV2-pretreated hemophilia B dogs. Two naive hemophilia B dogs that received a single intraportal administration of AAV2/8 vector have achieved sustained expression of 10% and 26% of normal levels of canine factor IX (cFIX) for more than a year. In an AAV2-pretreated hemophilia B dog, cFIX expression increased from less than 1% to 16% of normal levels when treated with an AAV2/8 vector, and a high level of expression has lasted for more than 2 years. No significant liver toxicity or cFIX-specific antibodies have been detected in these animals. Studies here have demonstrated the safety and improved efficacy of AAV2/8 vector in large-animal models for liver-directed gene therapy.
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44

Rosenzweig, M., T. Wiehagen, A. M. Brufsky, and R. M. Arnold. "Symptom distress, quality of life and challenges of illness according to race and income in women with metastatic breast cancer." Journal of Clinical Oncology 24, no. 18_suppl (June 20, 2006): 8609. http://dx.doi.org/10.1200/jco.2006.24.18_suppl.8609.

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8609 Background: Response to a diagnosis of metastatic breast cancer (MBC) may vary according to race and income. The aims of this study were: 1) to identify quality of life, symptom distress and challenges of illness during MBC treatment and 2) to determine if these variables differ according to race and income. Methods: The study was a 2×2 prospective design conducted at an urban breast cancer center. Women with MBC were categorized into four groups based on race and income: white low (WL), white high (WH), African American high (AAH) and African American low (AAL). Instruments were 1) Symptom Distress Scale (SDS), (higher scores /worse distress) 2) Functional Assessment of Cancer Therapy (FACT), (higher scores/better QOL) and a 3) semi structured interview assessing patient perspectives of MBC. Interview analysis utilized grounded theory. Results: Preliminary results are for 51 women. Mean age was 58.2 years, with mean 24 months since MBC diagnosis. Quantitative data indicated worse quality of life in AA than white women. (P=0.06), with AALI women exhibiting worse symptom distress (P=0.03) as compared to white women. Qualitative data (n=48) corroborated quantitative data. The most prevalent themes among all sociodemographic groups were of hope (33/48 - 69%), faith (28/48 - 58%) and progressive loss (29/48, 60%). Each racial/economic delineation expressed unique themes: AALI talked about physical (7/7,100%)and social distress (6/7, 86%) as well as uncertainty regarding “whether treatment was worth it” (6/7 - 86%). WLI women verbalized an overall optimism, describing themselves as “lucky” (6/14 - 43%), with minimization of symptoms (10/14 - 71%). WHI women articulated a sense of betrayal at their progressive illness (9/20 - 45%) and fear of physical and economic dependence. Conclusion: Race and economic delineation brings unique symptom experience, quality of life and patient perspective to the metastatic breast cancer experience. These findings will advise tailored intervention. [Table: see text] No significant financial relationships to disclose.
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45

Dienhart, Mary K., and Rosemary A. Stuart. "The Yeast Aac2 Protein Exists in Physical Association with the Cytochromebc1-COX Supercomplex and the TIM23 Machinery." Molecular Biology of the Cell 19, no. 9 (September 2008): 3934–43. http://dx.doi.org/10.1091/mbc.e08-04-0402.

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The ADP/ATP carrier (AAC) proteins play a central role in cellular metabolism as they facilitate the exchange of ADP and ATP across the mitochondrial inner membrane. We present evidence here that in yeast (Saccharomyces cerevisiae) mitochondria the abundant Aac2 isoform exists in physical association with the cytochrome c reductase (cytochrome bc1)-cytochrome c oxidase (COX) supercomplex and its associated TIM23 machinery. Using a His-tagged Aac2 derivative and affinity purification studies, we also demonstrate here that the Aac2 isoform can be affinity-purified with other AAC proteins. Copurification of the Aac2 protein with the TIM23 machinery can occur independently of its association with the fully assembled cytochrome bc1-COX supercomplex. In the absence of the Aac2 protein, the assembly of the cytochrome bc1-COX supercomplex is perturbed, whereby a decrease in the III2-IV2assembly state relative to the III2-IV form is observed. We propose that the association of the Aac2 protein with the cytochrome bc1-COX supercomplex is important for the function of the OXPHOS complexes and for the assembly of the COX complex. The physiological implications of the association of AAC with the cytochrome bc1-COX-TIM23 supercomplex are also discussed.
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46

Chai, Zheng, Xintao Zhang, Amanda Lee Dobbins, Ellie Azure Frost, R. Jude Samulski, and Chengwen Li. "Chimeric Capsid Proteins Impact Transduction Efficiency of Haploid Adeno-Associated Virus Vectors." Viruses 11, no. 12 (December 9, 2019): 1138. http://dx.doi.org/10.3390/v11121138.

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Our previous studies have demonstrated that haploid AAV vectors made from capsids of two different serotypes induced high transduction and prevented serotype-specific antibody binding. In this study, we explored the transduction efficiency of several haploid viruses, which were made from the VP1/VP2 of one serotype and VP3 of another compatible serotype. After systemic injection of 2 × 1010 vg of AAV vectors into mice, the haploid AAV vectors, composed of VP1/VP2 from serotypes 8 or 9, and VP3 from AAV2, displayed a two to seven-fold increase in liver transduction compared with those of parental AAV2 vectors. Furthermore, a chimeric AAV2/8 VP1/VP2 with N-terminus of VP1/VP2 from AAV2 and C-terminus (VP3 domain) from AAV8 was constructed, and produced the haploid vector 28m-2VP3 with AAV2 VP3. The haploid 28m-2VP3 vector showed a five-fold higher transduction than that of the vectors composed solely of AAV2 VPs. Remarkably, the 28m-2VP3 vectors also induced a significant increase in transgene expression compared to the vectors composed of AAV8 VP1/VP2 with AAV2 VP3. The results suggest that the difference in the VP1/VP2 N-terminal region between AAV2 and AAV8 may allow better “communication” between the VP1/VP2 N-terminus of AAV2 with its cognate VP3. Similarly, the haploid vectors, VP1/VP2 from serotypes 8 or 9 and VP3 from AAV3, achieved higher transductions in multiple tissue types beyond typical tropism compared with those of AAV3 vectors. Consistently, higher vector genome copy numbers were detected in these tissues, indicating that an incorporation of non-cognate VP1/VP2 might influence the cellular tropism of the haploid vectors. However, there was no significant difference or even decreased transductions when compared with those of parental AAV8 or AAV9 vectors. In summary, these studies provide insight into current development strategies of AAV vectors that can increase AAV transduction across multiple tissues.
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47

Scallan, Ciaran D., Haiyan Jiang, Tongyao Liu, Susannah Patarroyo-White, Jurg M. Sommer, Shangzhen Zhou, Linda B. Couto, and Glenn F. Pierce. "Human immunoglobulin inhibits liver transduction by AAV vectors at low AAV2 neutralizing titers in SCID mice." Blood 107, no. 5 (March 1, 2006): 1810–17. http://dx.doi.org/10.1182/blood-2005-08-3229.

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Long-term cures of hemophilia B have been achieved using AAV2 delivering the factor IX gene to the liver of adeno-associated virus (AAV)–naive hemophilic animals. However, the clinical success of this approach requires overcoming pre-existing AAV neutralizing antibodies prevalent in humans. To better define the inhibition of neutralizing antibodies on AAV2-mediated liver transduction, we developed an in vivo passive immunity model. SCID mice were first reconstituted to a defined neutralizing titer with pooled plasma-derived human immunoglobulin. AAV2-FIX vectors then were administered to the liver, and the transduction efficiency was measured by plasma FIX levels. Unexpectedly, AAV2 neutralizing titers lower than 1:10 were sufficient to neutralize 4 to 20 × 1012 vg/kg of AAV2 vectors in vivo, a capacity that was underestimated by in vitro neutralizing assays. We also evaluated strategies to evade neutralization, including the use of alternative delivery routes, infusion parameters, empty capsids, and alternative AAV serotypes 6 and 8. The results indicate that low AAV2 neutralizing titers can be inhibitory to the tested human and primate AAV vectors delivered into the circulatory system. Therefore, novel nonprimate AAV vectors or compartmentalized delivery may offer more consistent therapeutic effects in the presence of pre-existing AAV neutralizing antibodies.
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48

Jurvansuu, Jaana, Kenneth Raj, Andrzej Stasiak, and Peter Beard. "Viral Transport of DNA Damage That Mimics a Stalled Replication Fork." Journal of Virology 79, no. 1 (January 1, 2005): 569–80. http://dx.doi.org/10.1128/jvi.79.1.569-580.2005.

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ABSTRACT Adeno-associated virus type 2 (AAV2) infection incites cells to arrest with 4N DNA content or die if the p53 pathway is defective. This arrest depends on AAV2 DNA, which is single stranded with inverted terminal repeats that serve as primers during viral DNA replication. Here, we show that AAV2 DNA triggers damage signaling that resembles the response to an aberrant cellular DNA replication fork. UV treatment of AAV2 enhances the G2 arrest by generating intrastrand DNA cross-links which persist in infected cells, disrupting viral DNA replication and maintaining the viral DNA in the single-stranded form. In cells, such DNA accumulates into nuclear foci with a signaling apparatus that involves DNA polymerase delta, ATR, TopBP1, RPA, and the Rad9/Rad1/Hus1 complex but not ATM or NBS1. Focus formation and damage signaling strictly depend on ATR and Chk1 functions. Activation of the Chk1 effector kinase leads to the virus-induced G2 arrest. AAV2 provides a novel way to study the cellular response to abnormal DNA replication without damaging cellular DNA. By using the AAV2 system, we show that in human cells activation of phosphorylation of Chk1 depends on TopBP1 and that it is a prerequisite for the appearance of DNA damage foci.
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49

Thomas, Clare E., Theresa A. Storm, Zan Huang, and Mark A. Kay. "Rapid Uncoating of Vector Genomes Is the Key toEfficient Liver Transduction with Pseudotyped Adeno-Associated VirusVectors." Journal of Virology 78, no. 6 (March 15, 2004): 3110–22. http://dx.doi.org/10.1128/jvi.78.6.3110-3122.2004.

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ABSTRACT Transduction of the liver with single-stranded adeno-associated virus serotype 2 (AAV2) vectors is inefficient; less than 10% of hepatocytes are permissive for stable transduction, and transgene expression is characterized by a lag phase of up to 6 weeks. AAV2-based vector genomes packaged inside AAV6 or AAV8 capsids can transduce the liver with higher efficiency, but the molecular mechanisms underlying this phenomenon have not been determined. We now show that the primary barrier to transduction of the liver with vectors based on AAV2 capsids is uncoating of vector genomes in the nucleus. The majority of AAV2 genomes persist as encapsidated single-stranded molecules within the nucleus for as long as 6 weeks after vector administration. Double-stranded vector genomes packaged inside AAV2 capsids are at least 50-fold more active than single-stranded counterparts, but these vectors also exhibit a lag phase before maximal gene expression. Vector genomes packaged inside AAV6 or AAV8 capsids do not persist as encapsidated molecules and are more biologically active than vector genomes packaged inside AAV2 capsids. Our data suggest that the rate of uncoating of vector genomes determines the ability of complementary plus and minus single-stranded genomes to anneal together and convert to stable, biologically active double-stranded molecular forms.
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50

Zabner, Joseph, Michael Seiler, Robert Walters, Robert M. Kotin, Wendy Fulgeras, Beverly L. Davidson, and John A. Chiorini. "Adeno-Associated Virus Type 5 (AAV5) but Not AAV2 Binds to the Apical Surfaces of Airway Epithelia and Facilitates Gene Transfer." Journal of Virology 74, no. 8 (April 15, 2000): 3852–58. http://dx.doi.org/10.1128/jvi.74.8.3852-3858.2000.

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ABSTRACT In the genetic disease cystic fibrosis, recombinant adeno-associated virus type 2 (AAV2) is being investigated as a vector to transfer CFTR cDNA to airway epithelia. However, earlier work has shown that the apical surface of human airway epithelia is resistant to infection by AAV2, presumably as a result of a lack of heparan sulfate proteoglycans on the apical surface. This inefficiency can be overcome by increasing the amount of vector or by increasing the incubation time. However, these interventions are not very practical for translation into a therapeutic airway-directed vector. Therefore, we examined the efficiency of other AAV serotypes at infecting human airway epithelia. When applied at low multiplicity of infection to the apical surface of differentiated airway epithelia we found that a recombinant AAV5 bound and mediated gene transfer 50-fold more efficiently than AAV2. Furthermore, in contrast to AAV2, AAV5-mediated gene transfer was not inhibited by soluble heparin. Recombinant AAV5 was also more efficient than AAV2 in transferring β-galactosidase cDNA to murine airway and alveolar epithelia in vivo. These data suggest that AAV5-derived vectors bind and mediate gene transfer to human and murine airway epithelia, and the tropism of AAV5 may be useful to target cells that are not permissive for AAV2.
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