Ribonucleic acid (RNA) represents an important target of a wide array of laboratory anal yses. Thus, RNA purification is a critical first preceding step of a number of preparative and analytical methods, important particularly in diagnostics of dozens of viral, bacterial, and parasitic diseases, dia gnosis of inherited disorders, and tumours, as well as in basic research. To provide relevant and reliable results, techniques of molecular biology used for such purposes require pure and intact molecules of purified RNA. Moreover, RNA has to be purified effectively and reproducibly from various heterogeneous materials such as fresh or frozen tissues, cell lines, PCR products or long-term chemically preserved samples. Principally, methods of RNA purification can be divided into three groups. The first group of methods is based on organic phenol:chloroform extraction. The second group encompasses methods of RNA purification by means of its ability to bind specific surfaces in the presence of chaotropic salt, and the third group includes methods exploiting RNA isolation on isopycnic gradients. Although RNA can be isolated from either prokaryotic or eukaryotic organisms, this review is to give out a basic outline of methods available for eukaryotic, with emphasis on mammalian, tissues.

• MeSH
• adsorpce MeSH
• lidé MeSH
• RNA chemie   izolace a purifikace   MeSH
• rozpouštědla chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• RNA MeSH
• rozpouštědla MeSH

BACKGROUND: This study compared the strength of incorporation and biocompatibility of 2 porcine-derived grafts (cross-linked and non-cross-linked) in a rat hernia model. METHODS: A standardized 2 × 4 cm(2) fascial defect was created in 30 Wistar rats and repaired with either a cross-linked or a non-cross-linked graft. The rats were killed 3, 6, and 12 months later. The strength of incorporation, vascularization, cellular invasion, foreign body reaction, and capsule formation were evaluated. RESULTS: Both graft materials showed cellular ingrowth and neovascularization by 3 months postimplantation. The average level of cellularization was significantly higher in the non-cross-linked grafts than in the cross-linked grafts at 6 months (2 vs 1; P = .029). Vascularization was significantly higher in the non-cross-linked grafts than in the cross-linked grafts at 6 months postimplantation (2 vs 1; P = .029) and insignificant at 3 months (2 vs 1.75; P = .311) and 12 months (1 vs 0.67; P = 1). The maximum load and breaking strength of both biomaterials increased during the study period. Overall, the strength of incorporation of the non-cross-linked grafts increased from 3 months (0.75 MPa) to 12 months (3.06 MPa) postimplantation. The strength of incorporation of the cross-linked grafts also increased from 3 months (0.59 MPa) to 12 months (1.58 MPa) postimplantation. CONCLUSIONS: The results of our study suggest that non-cross-linked grafts may be slightly more biocompatible and allow a more rapid and higher degree of cellular penetration and vascularization, resulting in stronger attachment to the tissues.

• Klíčová slova
• biocompatibility, biologics, cross-linking, extracellular matrix, hernia,
• Publikační typ
• časopisecké články MeSH

• MeSH
• biologické modely * MeSH
• gnotobiologické modely * MeSH
• imunita * MeSH
• imunoglobuliny biosyntéza   MeSH
• imunologická tolerance MeSH
• infekce imunologie   MeSH
• přirozená imunita MeSH
• stárnutí MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• imunoglobuliny MeSH

Thyroid hormone system disruption (THSD) is a growing concern in chemical hazard assessment due to its impact on human and environmental health and the scarce methods available for assessing the THSD potential of chemicals. In particular, the general lack of validated in silico and in vitro methods for assessing THS activity is of high concern. This manuscript provides an inventory of test methods relevant to THSD. Building on the Organisation for Economic Co-operation and Development (OECD) Guidance Document 150 and recent international developments, we highlight progress in in silico and in vitro methods, as well as in vivo assays. The provided inventory categorizes available methods according to the levels of the OECD Conceptual Framework, with an assessment of the validation status of each method. At Level 1, 12 in silico models that have been statistically validated and are directly related to THSD have been identified. At Level 2, 67 in vitro methods have been listed including those assessed in key initiatives such as the European Union Network of Laboratories for the Validation of Alternative Methods (EU-NETVAL) validation study to identify potential thyroid disruptors. At Levels 3-5, THSD-sensitive endpoints are being included in existing fish-based OECD Test Guidelines to complement amphibian assays. In total, the inventory counts 108 entries comprising established methods (e.g., OECD Test Guidelines) as well as citable methods that are under further development and in some cases are ready for validation or in the initial stages of validation. This work aims to support the ongoing development of strategies for regulatory hazard assessment, such as integrated approaches to testing and assessment (IATAs), for endocrine disruptors, addressing critical gaps in the current testing landscape for THSD in both human and environmental health contexts.

Endocrine disruption - the potential of chemicals, such as industrial chemicals or pesticides, to disrupt hormonal systems and cause adverse health effects - is of growing concern due to its impact on human and environmental health and the scarce methods available for assessing such hazards. In particular, the limited methods available for assessing disruption of the thyroid hormone system, is of high concern. This manuscript provides an inventory of test methods relevant for the assessment of thyroid hormone system disruption. We highlight progress in different types of methods such as computer simulations, cell-based methods, non-mammalian embryo-based methods and animal methods and include an assessment of the readiness of each method for implementation in chemical evaluations. In total, the inventory counts 108 entries comprising already established methods as well as recent developments. This work aims to support the ongoing development of strategies for evaluating endocrine disruption, addressing critical gaps in the current testing landscape for thyroid hormone system disruption in both human and environmental health contexts.

• Klíčová slova
• One Health, Thyroid hormone system disruption, endocrine disruption, new approach methods,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Animal testing has been prohibited for the safety assessment of cosmetic ingredients or finished products. Thus, alternative non-animal methods, followed by confirmatory clinical studies on human volunteers, should be used as the sole legally acceptable approach within the EU. The safety assessment of cosmetic products requires the involvement of multiple scientific disciplines, including analytical chemistry and biomedicine, as well as in chemico, in vitro and in silico toxicology. Recent data suggest that fragrance components may exert multiple adverse biological effects, e.g. cytotoxicity, skin sensitisation, (photo)genotoxicity, mutagenicity, reprotoxicity and endocrine disruption. Therefore, a pilot study was conducted with selected samples of fragrance-based products, such as deodorant, eau de toilette and eau de parfum, with the aim of integrating results from a number of alternative non-animal methods suitable for the detection of the following toxicological endpoints: cytotoxicity (with 3T3 Balb/c fibroblasts); skin sensitisation potential (in chemico method, DPRA); skin sensitisation potential (LuSens in vitro method, based on human keratinocytes); genotoxicity potential (in vitro Comet assay with 3T3 Balb/c cells); and endocrine disruption (in vitro YES/YAS assay). The presence of twenty-four specific known allergens in the products was determined by using GC-MS/MS. The strategies for estimation of the NOAEL of a mixture of allergens, which were proposed by the Scientific Committee on Consumer Products in their 'Opinion on Tea tree oil' document and by the Norwegian Food Safety Authority in their 'Risk Profile of Tea tree oil' report, were used as models for the NOAEL estimation of the mixtures of allergens that were identified in the individual samples tested in this study.

• Klíčová slova
• 3Rs, Three Rs, allergens, alternative methods to animal testing, cosmetics, cytotoxicity, endocrine disruption, genotoxicity, in vitro methods, mixtures, perfumes, sensitisers, skin sensitisation,
• MeSH
• alergeny toxicita   analýza   MeSH
• kosmetické přípravky * toxicita   MeSH
• lidé MeSH
• parfém * analýza   MeSH
• pilotní projekty MeSH
• plynová chromatografie s hmotnostně spektrometrickou detekcí MeSH
• tandemová hmotnostní spektrometrie MeSH
• tea tree oil * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• alergeny MeSH
• kosmetické přípravky * MeSH
• parfém * MeSH
• tea tree oil * MeSH

OBJECTIVES: Malathion is generally not classified as toxic. However, the toxicity seems to be species-dependent. Local and systemic toxicity data for birds are rare, but a decrease of wild bird densities in areas where malathion was applied was reported. Aim of the study was to extend knowledge on malathion toxicity on cellular and organ level and to evaluate embryotoxicity and genotoxicity for birds using the chick embryo model HET-CAM. METHODS: Skin and eye irritation was determined using reconstructed skin and eye cornea tissues and the chorioallantoic membrane of chick embryo to simulate conjunctiva. Cytotoxicity in 3T3 Balb/c fibroblast culture was determined to estimate acute systemic toxicity. Chick embryo model was further employed to evaluate acute embryotoxicity for birds (mortality and genotoxicity). Data were analysed by means of general linear models. RESULTS: Malathion is not a skin and eye irritant. Cytotoxicity in vitro test provided LD50 value of 616 mg/kg suggesting higher toxic potential than is generally published based on in vivo tests on laboratory rodents. Embryotoxicity studies revealed dose and age dependent mortality of chick embryos. Genotoxicity was identified by means of micronucleus test in erythroid cells isolated from chorioallantois vascular system of chick embryos. CONCLUSIONS: Using in vitro alternative toxicological methods, a higher toxic potential of malathion was demonstrated than is generally declared. An increased health and environmental hazard may occur in areas with intensive agricultural production. The environmental consequences of delayed effects and embryotoxicity for bird populations in areas exposed to organophosphate insecticides, such as malathion, are obvious.

• MeSH
• biologické modely MeSH
• buňky BALB 3T3 cytologie   účinky léků   MeSH
• chorioalantoická membrána cytologie   účinky léků   MeSH
• dráždivé látky toxicita   MeSH
• druhová specificita MeSH
• embryo nesavčí cytologie   účinky léků   MeSH
• insekticidy toxicita   MeSH
• kur domácí MeSH
• kuřecí embryo MeSH
• lineární modely MeSH
• malathion toxicita   MeSH
• mitóza účinky léků   MeSH
• myši MeSH
• rohovka cytologie   účinky léků   MeSH
• techniky in vitro MeSH
• testy akutní toxicity MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• dráždivé látky MeSH
• insekticidy MeSH
• malathion MeSH

The aim of this study was to assess two protocols for their capacities to simultaneously isolate RNA, mtDNA and ncDNA from mammalian cells. We compared the Invitrogen TRIzol-based method and Qiagen DNeasy columns, using the HepG2 cell line and human primary glioblastoma stem cells. Both methods allowed the isolation of all three types of nucleic acids and provided similar yields in mtDNA. However, the yield in ncDNA was more than tenfold higher on columns, as observed for both cell types. Conversely, the TRIzol method proved more reproducible and was the method of choice for isolating RNA from glioblastoma cells, as demonstrated for the housekeeping genes RPLP0 and RPS9.

• Klíčová slova
• RNA, mammalian cells, mitochondria, mitochondrial DNA (mtDNA), nuclear DNA (ncDNA),
• MeSH
• biochemie metody   MeSH
• buněčné jádro metabolismus   MeSH
• buňky Hep G2 MeSH
• glioblastom metabolismus   patologie   MeSH
• lidé MeSH
• messenger RNA izolace a purifikace   MeSH
• mitochondriální DNA izolace a purifikace   MeSH
• nádorové kmenové buňky metabolismus   MeSH
• reagenční diagnostické soupravy MeSH
• RNA izolace a purifikace   MeSH
• savci metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• messenger RNA MeSH
• mitochondriální DNA MeSH
• reagenční diagnostické soupravy MeSH
• RNA MeSH

A computerised set of methods for the risk assessment of infectious diseases has been developed as part of a software package which deals with animal population health and disease analyses and programming (EPIZOO, version 2.6). This set includes methods for calculating the following: general indicators of risk of animal disease. Risk probability assessment of propagation of disease. Risk probability assessment of the introduction of disease through the importation of animals and their products, based on predefined criteria and on non-predefined criteria. Risk comparison of the introduction of one disease agent from several territories. Risk comparison of the introduction of several disease agents from one territory. Risk of disease propagation related to the movement and concentration of the animal population. Risk of disease propagation related to the transfer of animal products. Risk of the probability of negative test results in infected animals. Risk of the probability that at least one imported animal or animal product unit is infected.

• MeSH
• hodnocení rizik MeSH
• infekční nemoci epidemiologie   přenos   MeSH
• obchod MeSH
• pravděpodobnost MeSH
• rizikové faktory MeSH
• software * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

To clarify differences in the incidence and number of nucleoli in the granulopoietic lineage, these nuclear components were studied in human and rabbit granulocyte precursors and granulocytes after visualization by 2 widely employed cytochemical procedures, i.e. a procedure for the demonstration of RNA and the silver reaction for the demonstration of nucleolar silver stainable proteins (SSPs). In early stages of the granulocyte proliferating compartment, substantial differences were not found between specimens in which nucleoli were visualized by both procedures. However, in contrast to specimens stained with the silver reaction, the number of cells without nucleoli was substantially larger in advanced stages of granulocyte development in specimens stained for RNA. The number of nucleoli per cell as expressed by the nucleolar coefficient was generally larger in specimens stained with the silver reaction for nucleolar SSPs. These differences were significant starting with the stage of myelocytes. Moreover, in specimens stained with the silver reaction, most of human mature granulocytes did not contain nucleoli but nucleoli were present in all mature granulocytes of rabbits. Such differences were not observed in specimens stained for RNA in which most granulocytes were without RNA-containing nucleoli. Thus, the evaluation of the presence or absence of nucleoli in specimens depended on the visualization procedure. It is likely that in micronucleoli which are characteristic for terminal differentiation of the granulocytic lineage, RNA-containing structures may be lost or are below the detection limit of the light microscope. In addition, differences in the presence of nucleoli exist apparently between human and rabbit granulocytes in specimens stained for SSPs but not in those stained for RNA.

• MeSH
• barvení stříbrem MeSH
• buněčné jadérko chemie   MeSH
• granulocyty chemie   cytologie   MeSH
• hematopoetické kmenové buňky chemie   cytologie   MeSH
• jaderné proteiny analýza   MeSH
• králíci MeSH
• lidé MeSH
• organizátor jadérka chemie   MeSH
• RNA analýza   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• jaderné proteiny MeSH
• RNA MeSH

Public awareness and discussion about animal experiments and replacement methods has greatly increased in recent years. The term 'the Three Rs', which stands for the Replacement, Reduction and Refinement of animal experiments, is inseparably linked in this context. A common goal within the Three Rs scientific community is to develop predictive non-animal models and to better integrate all available data from in vitro, in silico and omics technologies into regulatory decision-making processes regarding, for example, the toxicity of chemicals, drugs or food ingredients. In addition, it is a general concern to implement (human) non-animal methods in basic research. Toward these efforts, there has been an ever-increasing number of Three Rs centres and platforms established over recent years - not only to develop novel methods, but also to disseminate knowledge and help to implement the Three Rs principles in policies and education. The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes gave a strong impetus to the creation of Three Rs initiatives, in the form of centres and platforms. As the first of a series of papers, this article gives an overview of the European Three Rs centres and platforms, and their historical development. The subsequent articles, to be published over the course of ATLA's 50th Anniversary year, will summarise the current focus and tasks as well as the future and the plans of the Three Rs centres and platforms. The Three Rs centres and platforms are very important points of contact and play an immense role in their respective countries as 'on the ground' facilitators of Directive 2010/63/EU. They are also invaluable for the widespread dissemination of information and for promoting implementation of the Three Rs in general.

• Klíčová slova
• 3R, 3Rs, EU3Rnet, NAM, NAMs, new approach methodologies, non-animal methods, novel approach methodologies,
• MeSH
• alternativy testů na zvířatech MeSH
• experimenty na zvířatech * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Evropa MeSH