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The aim of the study was to find a practical and inexpensive method for freezing goose semen for use in routine inseminations under farm conditions. Two basic freezing protocols [(1) dynamic, programmable freezing and (2) static, nitrogen vapour method] were evaluated with varying concentrations of dimethylformamide (DMF) plus additional osmoprotectants such as betaine, trehalose, and sucrose, using cryovials as containers. Altogether eight different treatments were compared. sperm viability before freezing and after thawing was examined by in vitro tests and, in the case of the simplest effective method, also by in vivo fertility test. There were no significant differences in sperm survival either in the dynamic (48-50%) or in the static protocol (43-46%), except for the treatment where the lowest DMF concentration was used without any osmoprotectant in the dynamic protocol (42.6%). The addition of osmoprotectants did not improve thawed sperm viability in any case. Fertility with frozen/thawed sperm using the simplest method was 58.5%, while that obtained with fresh, diluted semen was 66.9%. The study proved that the simple freezing of gander semen in nitrogen vapour with 9% DMF in cryovials could produce acceptable fertility. The newly elaborated method can be successfully used for routine inseminations by small- and large-scale goose breeders.
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Criopreservação/métodos , Preservação do Sêmen/veterinária , Sêmen/fisiologia , Animais , Congelamento , Gansos , Masculino , Nitrogênio/análise , Análise do Sêmen/veterinária , Preservação do Sêmen/métodosRESUMO
Hungary has a large number of thermal baths and spa facilities which attract hundreds of thousands of tourists annually. Until recently, however, the free-living amoebae were not of public health concern. Genotyping of Acanthamoeba species, potential agents of keratitis and granulomatous encephalitis, was carried out in 20 Hungarian public baths for the first time to assess the incidence and molecular diversity of the genus in the country. Our results show that 6.7% of the samples were positive for Acanthamoeba. Of these positive samples, 6.5 and 7% was from sterilized and unsterilized pools, respectively. The 18S rRNA gene investigation of the nine Acanthamoeba strains found reveals that seven belong to the hazardous T4 genotype. The remaining two samples were of the T15 type. All the strains kept growing at 36 °C. Our results underline the need to develop a control system for free-living amoebae and supervise the disinfection of Hungarian public baths.
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Acanthamoeba/classificação , Água Doce/parasitologia , Filogenia , Logradouros Públicos , RNA Ribossômico 18S/genética , Acanthamoeba/genética , Acanthamoeba/isolamento & purificação , Amebíase/epidemiologia , Amebíase/parasitologia , Banhos , Monitoramento Epidemiológico , Genes de RNAr , Genótipo , Humanos , Hungria/epidemiologia , IncidênciaRESUMO
Poisoning caused by coumarin-type anticoagulant rodenticides (ARs) stands as the predominant method for controlling rodents globally. ARs, through secondary poisoning, pose a significant threat to predators due to their lethal and sublethal effects. We examined the concentration of accumulated ARs in liver samples of mostly road-killed steppe polecats (Mustela eversmanii) and European polecats (M. putorius) collected throughout Hungary between 2005 and 2021. The steppe polecat samples were found mainly from Eastern Hungary, while European polecats from Western Hungary. We measured the concentration of six residues by HPLC-FLD. Our analysis revealed the presence of one first-generation and four second-generation ARs in 53% of the steppe polecat (36) and 39% of the European polecat (26) samples. In 17 samples we detected the presence of at least two AR compounds. Although we did not find significant variance in AR accumulation between the two species, steppe polecats displayed greater prevalence and maximum concentration of ARs, whereas European polecat samples exhibited a more diverse accumulation of these compounds. Brodifacoum and bromadiolone were the most prevalent ARs; the highest concentrations were 0.57 mg/kg and 0.33 mg/kg, respectively. The accumulation of ARs was positively correlated with human population density and negatively correlated with the extent of the more natural habitats in both species. To the best of our knowledge, this is the first study to demonstrate anticoagulant rodenticide exposure in steppe polecats globally, and for European polecats in Central European region. Although the extent of AR accumulation in European polecat in Hungary appears comparatively lower than in many other European countries, the issue of secondary poisoning remains a serious problem as these ARs intrude into food webs. Reduced and more prudent usage of pesticides would provide several benefits for wildlife, included humans. However, we advocate a prioritization of ecosystem services through the complete prohibition of the toxicants.
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Anticoagulantes , Monitoramento Ambiental , Rodenticidas , Animais , Anticoagulantes/análise , Hungria , Mustelidae , Poluentes Ambientais/análiseRESUMO
BACKGROUND: Single cell genomics has revolutionized microbial sequencing, but complete coverage of genomes in complex microbiomes is imperfect due to enormous variation in organismal abundance and amplification bias. Empirical methods that complement rapidly improving bioinformatic tools will improve characterization of microbiomes and facilitate better genome coverage for low abundance microbes. METHODS: We describe a new approach to sequencing individual species from microbiomes that combines antibody phage display against intact bacteria with fluorescence activated cell sorting (FACS). Single chain (scFv) antibodies are selected using phage display against a bacteria or microbial community, resulting in species-specific antibodies that can be used in FACS for relative quantification of an organism in a community, as well as enrichment or depletion prior to genome sequencing. RESULTS: We selected antibodies against Lactobacillus acidophilus and demonstrate a FACS-based approach for identification and enrichment of the organism from both laboratory-cultured and commercially derived bacterial mixtures. The ability to selectively enrich for L. acidophilus when it is present at a very low abundance (<0.2%) leads to complete (>99.8%) de novo genome coverage whereas the standard single-cell sequencing approach is incomplete (<68%). We show that specific antibodies can be selected against L. acidophilus when the monoculture is used as antigen as well as when a community of 10 closely related species is used demonstrating that in principal antibodies can be generated against individual organisms within microbial communities. CONCLUSIONS: The approach presented here demonstrates that phage-selected antibodies against bacteria enable identification, enrichment of rare species, and depletion of abundant organisms making it tractable to virtually any microbe or microbial community. Combining antibody specificity with FACS provides a new approach for characterizing and manipulating microbial communities prior to genome sequencing.
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Anticorpos Antibacterianos/metabolismo , Carga Bacteriana/métodos , Citometria de Fluxo/métodos , Lactobacillus acidophilus/isolamento & purificação , Microbiota , Análise de Sequência de DNA/métodos , Anticorpos de Cadeia Única/metabolismo , Anticorpos Antibacterianos/imunologia , Anticorpos Antibacterianos/isolamento & purificação , Técnicas de Visualização da Superfície Celular , Lactobacillus acidophilus/genética , Lactobacillus acidophilus/imunologia , Dados de Sequência Molecular , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/isolamento & purificaçãoRESUMO
This article aims to examine the relationship between job crafting activities and employees' readiness to change. Confirmatory factor analysis and hierarchical regression analysis were conducted on a representative sample of 500 employees. Sampling was carried out in a European country in a period strongly affected by COVID-19 to isolate the five dimensions of job crafting and their separate effects on employees' readiness to change. The findings show that the five dimensions of job crafting can be distinguished from each other and that they have differential effects on employees' readiness to change. Extending task crafting shows a positive relationship with employees' readiness to change while reducing task crafting showed no significant relationship. Surprisingly extending and reducing relationship crafting showed no significant relationship with readiness to change. Cognitive crafting was found to be significantly positively related to the dependent variable. This research contributes to the development of job crafting theory by providing empirical support that job crafting can be associated with readiness to change but that this relationship may vary across its dimensions. The results may also provide important conclusions for change leaders and HR professionals.
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This study investigates how compatibility and perceived enjoyment affect the link between intention to use and actual technology use in Romanian preschool education, building on earlier studies. METHODS: 300 participants were invited to participate in this research from 15 Romanian counties. 182 preschool teachers' questionnaires were utilized for data analysis after the return and screening of responses. A valid and accurate scale evaluating preschool teachers' behavior towards technology adoption was included in the questionnaire, along with self-reported demographic data, professional identification, and other information. Data was analyzed using SPSS V.16. RESULTS: (1) Intention to use, compatibility, perceived enjoyment, and actual use were positively associated. (2) The effect of compatibility and perceived enjoyment on the link between intention to use and actual technology use was carried out in the following way: Intention to use â Compatibility with technology â Perceived enjoyment â Actual use. We hypothesize that intention to use affects compatibility, compatibility affects perceived enjoyment, and, lastly, perceived enjoyment affects actual use. For a more robust validation of results, we have also modelled this relationship with the Radial Basis Function (RBF) neural network. CONCLUSION: Compatibility and perceived enjoyment partially mediate the relationship between intention to use and actual technology use in class by Romanian preschool teachers. According to the theory of planned behavior, this study brought to light the intricacy of the relationship between preschool teachers' intention to utilize technology in the classroom and their actual usage of it. Limitations and implications are discussed.
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Filamentous phage display has been extensively used to select proteins with binding properties of specific interest. Although many different display platforms using filamentous phage have been described, no comprehensive comparison of their abilities to display similar proteins has been conducted. This is particularly important for the display of cytoplasmic proteins, which are often poorly displayed with standard filamentous phage vectors. In this article, we have analyzed the ability of filamentous phage to display a stable form of green fluorescent protein and modified variants in nine different display vectors, a number of which have been previously proposed as being suitable for cytoplasmic protein display. Correct folding and display were assessed by phagemid particle fluorescence, and with anti-GFP antibodies. The poor correlation between phagemid particle fluorescence and recognition of GFP by antibodies, indicates that proteins may fold correctly without being accessible for display. The best vector used a twin arginine transporter leader to transport the displayed protein to the periplasm, and a coil-coil arrangement to link the displayed protein to g3p. This vector was able to display less robust forms of GFP, including ones with inserted epitopes, as well as fluorescent proteins of the Azami green series. It was also functional in mock selection experiments.
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Corantes Fluorescentes/análise , Vetores Genéticos , Proteínas de Fluorescência Verde/análise , Inovirus/genética , Citoplasma/química , Proteínas de Fluorescência Verde/genética , Plasmídeos/químicaRESUMO
According to Sustainable Development Goal 4.2 (SDG 4.2), Equal Access to Quality Pre-primary Education, governments throughout the world are working to ensure that all children have access to high-quality early childhood development, care, and pre-primary education by 2030. In order to organize available evidence into a coherent framework, the current scoping review represents an exploratory synthesis addressing the broad question of what qualitative and inclusive Early Childhood Education and Care strategies are currently being established globally to achieve SDG4 targets. The goal of this scoping review in this respect, was to map the available research and offer an overview of micro-, meso-, and macro-level perspectives on evidence-based interventions and strategies, for the promotion of SDG4 globally. A layered model of early childhood education that is both inclusive and egalitarian education emerged, starting with the micro level: child, family and community, mezo level: nursery, and kindergarten and macro level: national policies and SDG 4.2 Agenda for 2030. The mezzo level connects the micro and macro levels, being the most solicited level of implementing inclusive and qualitative ECEC strategies. Thus, starting with putting a real emphasis on children rights, creating a qualitative and inclusive culture with a holistic understanding of child development, then investing in teacher preparation and instilling a strong belief and positive attitudes toward equity in early childhood services, developing inclusive educational policies with an authentic community support offered by all stakeholders, then adapting curriculum and assessment methods to all early childhood educational contexts and lastly piloting and up-scaling good practices, and investing in infrastructure, facilities and innovative educational services, SDG4.2 targets could transparently and efficiently be attained by 2030, with all the setbacks arisen from the pandemic context. The data provide light on a vast topic range, including human rights and values, policy actions, and ideologies. The micro-level themes emphasized the importance of fostering equitable and inclusive environments for children., as well as instructional approaches that encourage positive attitudes toward diversity and instructors' levels of experience in dealing with diversity. We also discovered the significance of creating chances that promote socialization, connection development, and a sense of belonging. Meso-level principles emphasized the relevance of schooling in a child's holistic development and skill acquisition. Mainstream availability for all children, national curriculum regulations, teacher preparation for inclusive early childhood education, excellent funding and governance, evaluation and monitoring, and research on inclusive early childhood education comprise the macro level. As a concept and an approach, inclusive and qualitative education necessitates the preparedness of all relevant educational components to participate. Providing inclusive education in the early years requires setting the foundation for subsequent levels of schooling. The active engagement of a young kid should be directed by developmentally and individually suitable curricula. Access to and participation in age-appropriate general curricula becomes critical in identifying and providing specialized support services. Inclusive programming does not imply that the educational programs will necessarily be of good quality. Efficiency and wellbeing are synonymous with equity. Equitable education investment benefits everyone in society, not just the most marginalized. Investing in education will help communities achieve all of the Sustainable Development Goals related to education.
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Predicting preschool teachers' intention to adopt qualitative and inclusive early childhood intentional behaviors represents an important research field. The objective of this research is first to develop and validate a scale to assess the integrative-qualitative intentional behavior (IQIB-ECEC) of preschool teachers in order to achieve SDG4.2's objective of ensuring that all children have access to high-quality pre-primary education and then to systemically analyze the relationship between variables with Network Analysis. The theory of planned behavior (TPB) describes key individual beliefs (attitudes, subjective norms, and perceived behavior control) that affect people's intentions to engage in a certain conduct and has previously been used with success in evaluating people's intentions to adopt a certain behavior. This research represents one of the first Romanian attempts to use the theory of planned behavior to study the Integrative-Qualitative Intentional Behavior in Romanian Preschool Education and systemically analyze results with Network Analysis approach. This study used a randomized 300 Romanian preschool teachers enrolled in a National Training Program entitled Qualitative and Inclusive Early Childhood Education managed by the Romanian Educational Ministry. Data were collected via an online questionnaire. The scale validation followed a confirmatory factor analysis (CFA). The fitting of the IQIB-ECEC 19-item scale showed that all coefficients CFI (0.942), TLI (0.920), SRMR (0.0452), and RMSEA (0.0451) bring strong evidence in the favor of the statistical validity of the scale. The final IQIB-ECEC 19 items and 8 factors scale obtained a Cronbach's alpha of 0.77. The systemic Network Analysis approach was used in interpreting data. The centrality of the network model was further investigated and the clustering coefficients index were calculated. According to the results, perceived power/control beliefs and behavioral intention were detected as the most important dimensions, whereas behavioral beliefs were less important. These findings were discussed in terms of their theoretical and practical significance.
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BACKGROUND: In order to carry out experimental gene annotation, DNA encoding open reading frames (ORFs) derived from real genes (termed "genic") in the correct frame is required. When genes are correctly assigned, isolation of genic DNA for functional annotation can be carried out by PCR. However, not all genes are correctly assigned, and even when correctly assigned, gene products are often incorrectly folded when expressed in heterologous hosts. This is a problem that can sometimes be overcome by the expression of protein fragments encoding domains, rather than full-length proteins. One possible method to isolate DNA encoding such domains would to "filter" complex DNA (cDNA libraries, genomic and metagenomic DNA) for gene fragments that confer a selectable phenotype relying on correct folding, with all such domains present in a complex DNA sample, termed the "domainome". RESULTS: In this paper we discuss the preparation of diverse genic ORF libraries from randomly fragmented genomic DNA using ß-lactamase to filter out the open reading frames. By cloning DNA fragments between leader sequences and the mature ß-lactamase gene, colonies can be selected for resistance to ampicillin, conferred by correct folding of the lactamase gene. Our experiments demonstrate that the majority of surviving colonies contain genic open reading frames, suggesting that ß-lactamase is acting as a selectable folding reporter. Furthermore, different leaders (Sec, TAT and SRP), normally translocating different protein classes, filter different genic fragment subsets, indicating that their use increases the fraction of the "domainone" that is accessible. CONCLUSIONS: The availability of ORF libraries, obtained with the filtering method described here, combined with screening methods such as phage display and protein-protein interaction studies, or with protein structure determination projects, can lead to the identification and structural determination of functional genic ORFs. ORF libraries represent, moreover, a useful tool to proceed towards high-throughput functional annotation of newly sequenced genomes.
Assuntos
Clostridium thermocellum/genética , Genômica/métodos , Fases de Leitura Aberta , DNA Bacteriano , Biblioteca Gênica , Anotação de Sequência Molecular , Análise de Sequência de DNA , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
Enhanced consensus green protein variant 123 (eCGP123) is an extremely thermostable green fluorescent protein (GFP) that exhibits useful negative reversible photoswitching properties. eCGP123 was derived by the application of both a consensus engineering approach and a recursive evolutionary process. Diffraction-quality crystals of recombinant eCGP123 were obtained by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant. The eCGP123 crystal diffracted X-rays to 2.10 Å resolution. The data were indexed in space group P1, with unit-cell parameters a = 74.63, b = 75.38, c = 84.51 Å, α = 90.96, ß = 89.92, γ = 104.03°. The Matthews coefficient (V(M) = 2.26 Å(3) Da(-1)) and a solvent content of 46% indicated that the asymmetric unit contained eight eCGP123 molecules.
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Proteínas de Fluorescência Verde/química , Processos Fotoquímicos , Sequência de Aminoácidos , Cristalização , Cristalografia por Raios X , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/isolamento & purificação , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Alinhamento de SequênciaRESUMO
The coincidence of systemic autoimmune diseases and pregnancy may modify the outcome of the disease and the pregnancy due to the background immunologic and hormonal processes. The great majority of patients with autoimmune diseases are young females in their reproductive years, willing to have babies. Consequently, we have to prepare for this special situation. Our concept on childbearing in autoimmune women has changed within the last 30 years. Earlier, systemic lupus erythematosus flared in about 50% of patients during pregnancy, but the flare rate has significantly decreased recently. This improvement can be attributed to increased attention to low diseases activity at the time of conception, which might reduce to the half of the risk for flare. Tight control of patients and appropriate use of corticosteroids also contribute to the better results. The adequate use of anti-thrombotic agents resulted in a significant amelioration of pregnancy outcome in antiphospholipid syndrome. The earlier use of methotrexate and the introduction of tumor necrosis factor-alpha inhibitors in the treatment of rheumatoid arthritis have changed the natural characteristics of the disease. The increase in remission rate indirectly has beneficial effect on the number of planned and carried out pregnancies. Authors review the connection between systemic autoimmune disorders and pregnancy as well as the possibilities of medical treatment of such diseases during pregnancy.
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Doenças Autoimunes/tratamento farmacológico , Imunossupressores/administração & dosagem , Complicações na Gravidez/tratamento farmacológico , Complicações na Gravidez/imunologia , Corticosteroides/administração & dosagem , Anti-Inflamatórios/administração & dosagem , Antimaláricos/administração & dosagem , Síndrome Antifosfolipídica/tratamento farmacológico , Síndrome Antifosfolipídica/imunologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/imunologia , Doenças Autoimunes/imunologia , Azatioprina/administração & dosagem , Ciclofosfamida/administração & dosagem , Ciclofosfamida/efeitos adversos , Ciclosporina/administração & dosagem , Esquema de Medicação , Feminino , Humanos , Imunoglobulinas Intravenosas/administração & dosagem , Imunossupressores/efeitos adversos , Isoxazóis/administração & dosagem , Leflunomida , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/imunologia , Metotrexato/administração & dosagem , Metotrexato/efeitos adversos , Gravidez , Resultado da Gravidez , Indução de Remissão , Síndrome de Sjogren/tratamento farmacológico , Síndrome de Sjogren/imunologia , Sulfassalazina/administração & dosagem , Teratogênicos , Fator de Necrose Tumoral alfa/administração & dosagemRESUMO
The C1858T allele of the PTPN22 gene has been reported to confer risk for RA; but in some reports, the effect was restricted to RF- and/or anti-CCP-seropositive patients. Hungarian RA patients and matched controls were genotyped. The 1858T allele showed an increased prevalence in RA patients compared to controls. The 1858T allele represents a risk factor in the whole RA population (P = 0.001); an association was found both in RF-seropositive (P = 0.001) and anti-CCP-seropositive patients (P = 0.001), and in subjects with the combination of these factors (P = 0.002). In TT homozygotes, the estimated susceptibility to RA was more than double (OR = 5.04) of that seen in TC heterozygotes (OR = 1.89); the same gene dosage effect was observed in all seropositive RA subgroups. Our data show that the Hungarian RA patients belong to the populations in which the 1858T allele represents a susceptibility factor both in the RF- and/or anti-CCP-seropositive subjects, and the association exhibit a gene dosage dependency.
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Artrite Reumatoide/enzimologia , Artrite Reumatoide/genética , Predisposição Genética para Doença/genética , Polimorfismo Genético/genética , Proteína Tirosina Fosfatase não Receptora Tipo 22/genética , Adulto , Distribuição por Idade , Idoso , Artrite Reumatoide/etnologia , Análise Mutacional de DNA , Feminino , Dosagem de Genes/genética , Frequência do Gene/genética , Marcadores Genéticos/genética , Testes Genéticos , Genótipo , Homozigoto , Humanos , Hungria/etnologia , Ativação Linfocitária/genética , Masculino , Pessoa de Meia-Idade , Fator Reumatoide/genética , Distribuição por Sexo , Linfócitos T/enzimologiaRESUMO
OBJECTIVE: The prevalence of hyperostosis frontalis interna (HFI) was examined in different periods of the Carpathian Basin from 4900 BCE to 17th century AD. The study seeks to evaluate temporal changes in HFI and the possible impact of lifestyle on it. MATERIALS: The studied material consisted of 4668 crania from Hungary and Serbia. METHODS: The crania were analyzed employing macroscopic and endoscopic examination. RESULTS: In historic periods, sex and age played a pivotal role in HFI development. Among predominantly pastoralist populations of the 5th-8th and 10th centuries, prevalence of HFI was considerably higher than in the medieval populations of the 9th-17th centuries. CONCLUSIONS: In addition to age and sex, other factors could be implicated in HFI development. The physiological effects of the pastoralist lifestyle and diet on insulin regulation could explain the increased risk of developing HFI in the 5th-8th and 10th-century populations. SIGNIFICANCE: The study provides the first comprehensive dataset of HFI from different archaeological periods from the Carpathian Basin. It has implications for lifestyle and risk of HFI development in past populations. LIMITATIONS: The archaeological periods are not equally represented. SUGGESTIONS FOR FURTHER RESEARCH: In order to better understand the etiology of HFI, lifestyle factors can be used to elucidate the risk of developing HFI in ancient populations.
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Osso Frontal/patologia , Hiperostose Frontal Interna/história , Estilo de Vida , Arqueologia/história , Arqueologia/métodos , Fósseis/história , História do Século XVI , História do Século XVII , Humanos , Hungria , Paleopatologia/métodos , Prevalência , Risco , SérviaRESUMO
In the use of non-antibody proteins as affinity reagents, diversity has generally been derived from oligonucleotide-encoded random amino acids. Although specific binders of high-affinity have been selected from such libraries, random oligonucleotides often encode stop codons and amino acid combinations that affect protein folding. Recently it has been shown that specific antibody binding loops grafted into heterologous proteins can confer the specific antibody binding activity to the created chimeric protein. In this paper, we examine the use of such antibody binding loops as diversity elements. We first show that we are able to graft a lysozyme-binding antibody loop into green fluorescent protein (GFP), creating a fluorescent protein with lysozyme-binding activity. Subsequently we have developed a PCR method to harvest random binding loops from antibodies and insert them at predefined sites in any protein, using GFP as an example. The majority of such GFP chimeras remain fluorescent, indicating that binding loops do not disrupt folding. This method can be adapted to the creation of other nucleic acid libraries where diversity is flanked by regions of relative sequence conservation, and its availability sets the stage for the use of antibody loop libraries as diversity elements for selection experiments.
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Regiões Determinantes de Complementaridade/genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Reação em Cadeia da Polimerase/métodos , Proteínas Recombinantes de Fusão/imunologia , Diversidade de Anticorpos , Sequência de Bases , Clonagem Molecular/métodos , Ensaio de Imunoadsorção Enzimática , Biblioteca Gênica , Proteínas de Fluorescência Verde/análise , Humanos , Dados de Sequência Molecular , Muramidase/imunologia , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Análise de Sequência de DNARESUMO
UNLABELLED: Rheumatoid arthritis (RA) is a chronic, progressive polyarthritis leading to substantial disability. Standardised data on consequences of disease progression are needed for clinical assessments and also for cost-effectiveness models. AIM: To analyse the impact of disease progression on health status, disease specific quality of life and costs in Hungary. METHODS: A cross-sectional survey was performed between April and August, 2004, involving consecutive RA patients of 6 hospital based rheumatology outpatient centres. Self-completed questionnaires were used to assess functional (HAQ) and health status (EQ-5D), quality of life (RAQoL). Disease activity (DAS) and costs were also surveyed, statistical analysis was performed. RESULTS: 255 patients were involved [mean age 55.5 +/- 12.3 years; disease duration 9.0 +/- 9.3 years; HAQ 1.38 +/- 0.76; EQ-5D 0.46 +/- 0.33; RAQoL 16.2 +/- 8.1; DAS 5.09 +/- 1.42; costs 1,043,163 (+/- 844,750) HUF/patient/year, conversion 1 Euro = 250 HUF]. Correlation was significant between the parameters ( p < 0.01): EQ-5D index = 1.014 - 0.25 x HAQ-0.041 x DAS; HAQ = 0.314 + 0.065 x RAQoL. Analysis by disease severity levels (HAQ groups 0.5 difference) revealed that health status worsens (mean EQ-5D: 0.784; 0.576; 0.504; 0.367; 0.211; 0.022) and costs increase (mean 628,280; 888,187; 953,759; 1,291,218; 1,346,112; 1,371,674 HUF/patient/year) with disease progression. Minimally important worsening of functional ability (0.25 HAQ increase) corresponds to -0.0705 EQ-5D and +1.884 RAQoL change. Lower health status difference (EQ-5D -0.05725) was calculated in patients with lower disease activity (DAS < 5.1). CONCLUSIONS: Correlation between disease progression, health status, quality of life and costs does not differ significantly from international results. The amount of costs is much lower in all disease severity levels than in developed European countries. Our study serves baseline data for health economic analysis in RA in Hungary.
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Artrite Reumatoide , Custos de Cuidados de Saúde , Nível de Saúde , Qualidade de Vida , Adulto , Idoso , Artrite Reumatoide/economia , Artrite Reumatoide/patologia , Artrite Reumatoide/fisiopatologia , Estudos Transversais , Progressão da Doença , Feminino , Humanos , Hungria , Masculino , Pessoa de Meia-Idade , Inquéritos e QuestionáriosRESUMO
This article describes a set of standard control experiments for the authentication of new protein variants isolated through library selection and site-directed mutagenesis. These controls are specifically designed to rule out artifacts derived from 'double transformants' -- i.e. cells transformed with, or infected by, two different plasmids simultaneously. These seem to have been the source of past artifacts and, as demonstrated here, are far more common than generally recognized. By following standard protocols for cloning, plasmid isolation, subcloning, in combination with functional assays, the presence of such artifacts can be ruled out. This protocol needs to be applied for any new variant isolated from heterogeneous gene repertoires, and in particular for variants isolated by selection for either enzymatic activity, or binding.
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Artefatos , Evolução Molecular Direcionada/métodos , Técnicas Microbiológicas/métodos , Transformação Bacteriana , Evolução Molecular Direcionada/normas , Escherichia coli/genética , Técnicas Microbiológicas/normas , Plasmídeos/genética , Transformação Bacteriana/genéticaRESUMO
Consensus engineering has been used to increase the stability of a number of different proteins, either by creating consensus proteins from scratch or by modifying existing proteins so that their sequences more closely match a consensus sequence. In this paper we describe the first application of consensus engineering to the ab initio creation of a novel fluorescent protein. This was based on the alignment of 31 fluorescent proteins with >62% homology to monomeric Azami green (mAG) protein, and used the sequence of mAG to guide amino acid selection at positions of ambiguity. This consensus green protein is extremely well expressed, monomeric and fluorescent with red shifted absorption and emission characteristics compared to mAG. Although slightly less stable than mAG, it is better expressed and brighter under the excitation conditions typically used in single molecule fluorescence spectroscopy or confocal microscopy. This study illustrates the power of consensus engineering to create stable proteins using the subtle information embedded in the alignment of similar proteins and shows that the benefits of this approach may extend beyond stability.
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Sequência Consenso , Proteínas de Fluorescência Verde/química , Engenharia de Proteínas , Sequência de Aminoácidos , Proteínas de Fluorescência Verde/isolamento & purificação , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de AminoácidosRESUMO
Autoimmune diseases generally belong to the rare diseases, however, some of them are frequent in the population. In the present work the authors analyse whether can any increase be observed in the number of patients suffering from autoimmune diseases and whether do the frequency of certain autoimmune disorders increase. Due mainly to epigenetic factors the incidence of autoimmune diseases are increasing, therefore there are more patients recognised with particular disorders. On the other hand the incidence is increased by improving diagnostic possibilities, by the use of more specific and sensitive classification criteria and more sophisticated laboratory tests, resulted in the recognition of milder and atypical disease variants as well. The prevalence is also increasing in consequence of novel immune suppressive therapeutic possibilities and the consequent improvement of survival in the most of these diseases. Besides, more and more diseases have been revealed to have autoimmune background, and lot of new autoimmune syndromes, diseases have been characterised recently. This increases the number of the known autoimmune rheumatic disorders with a consequent increase in the number of autoimmune patients. Assigned to the increasing number of variable chronic autoimmune disorders, and the increasing number of disabled patients with such diseases increasing medical and social attention has to be focused on.