RESUMO
The intracellular distribution of copper in the liver has been investigated in dogs and humans. However, this has not been reported in cats. This study aimed to assess the intracellular copper distribution in liver specimens from cats with a range of hepatic copper concentrations. Twenty-nine frozen liver specimens from cats were included. Each liver specimen was divided into two pieces for overall copper quantification and tissue fractionation. The copper concentrations in liver specimens and liver fractions were measured by flame atomic absorption spectroscopy. Five specimens had copper concentrations < 100 µg/g dry weight, eight had copper concentrations between 100 and 180 µg/g, 14 had copper concentrations between 181 and 700 µg/g, and two had copper concentrations >700 µg/g. Only one specimen had positive copper staining. Regardless of the overall concentrations, copper was mostly found in the cytosolic fraction followed by the nuclear, large granule, and microsomal fractions. Our findings indicate that similarly to other species, intracellular copper is predominantly found in the cytosolic and nuclear fractions in cats. The distribution in cats with copper-loaded conditions, such as primary copper hepatopathy, was not assessed but warrants evaluation.
Assuntos
Cobre/análise , Fígado/química , Espectrofotometria Atômica/veterinária , Animais , Gatos , Fracionamento Celular , Núcleo Celular/química , Citosol/química , Feminino , Humanos , Masculino , Microssomos Hepáticos/químicaRESUMO
Four populations of Rhinella arenarum from aquatic environments with different degrees of disturbance in central Argentina were compared to assess the ability of cytomorphology and cytomorphometry of blood cells as a hematological biomarker. A total of 93 specimens of R. arenarum (adults sexually mature) were captured during the spring. From the analysis of cell, no variations were found in terms of morphology, whereas in nuclear and cell areas and Price-Jones curves, we observed a smaller size in erythrocytes of individuals inhabiting the site most altered, "Villa Dalcar," as well as for leukocytes, lymphocytes, neutrophils, and eosinophils for the same site. This could be caused by presence of different pollutants in the lake. Furthermore, this was confirmed by the high levels of environmental variables (conductivity, total dissolved solids, and salinity) show that Villa Dalcar is the site most affected by human activities.
Assuntos
Núcleo Celular/fisiologia , Eritrócitos/citologia , Animais , Argentina , Bufo arenarum , Núcleo Celular/química , Ecossistema , Saúde Ambiental , LarvaRESUMO
BACKGROUND: Lung cancer seems to have different epidemiological, biomolecular and clinical characteristics in females than in males, with a better prognosis for women. The aim of the study is to determine gender differences in lung adenocarcinoma in terms of androgen (AR), estrogen (ER)α and progesterone (PgR) receptors expression and their impact on outcome. RESULTS: Overall survival was significantly better in ERα and in PgR positive lung adenocarcinoma patients (median survival 45 vs. 19 months).Eight out of 62 patients showed positive expression of nuclear (n) AR and 18 of cytoplasmic (c) AR with a significantly better survival (49 vs. 19 and 45 vs. 19 months, respectively). There was a significant difference in survival between patients with vs. without c-AR expression (30 vs. 17 months). Finally, in the subgroup of women, median survival was greater in positive expression of c-AR than for women with negative c-AR (45 vs. 21 months). MATERIALS AND METHODS: We conducted an analysis on a cohort of 62 patients with advanced NSCLC treated at our institution. We investigated the immunohistochemical expression of n/c AR, ERα and PgR in 62 NSCLC and we correlated it with patients' clinic-pathologic characteristics and with prognosis. CONCLUSIONS: Our results showed that the positive expression of one hormonal receptor could represent a prognostic factor.Furthermore our study suggests that AR should become object of close examination in a larger series of lung adenocarcinoma patients, also for selection of the patients with best prognosis that can perform more chemotherapy lines.
Assuntos
Adenocarcinoma/química , Biomarcadores Tumorais/análise , Carcinoma Pulmonar de Células não Pequenas/química , Receptor alfa de Estrogênio/análise , Disparidades nos Níveis de Saúde , Neoplasias Pulmonares/química , Receptores Androgênicos/análise , Receptores de Progesterona/análise , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adenocarcinoma/terapia , Adenocarcinoma de Pulmão , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Núcleo Celular/química , Citosol/química , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Fatores Sexuais , Fatores de Tempo , Resultado do TratamentoRESUMO
Optogenetics uses light to control and observe the activity of neurons, often using a focused laser beam. As brain tissue is a scattering medium, beams are distorted and spread with propagation through neural tissue, and the beam's degradation has important implications in optogenetic experiments. To address this, we present an analysis of scattering and loss of intensity of focused laser beams at different depths within the brains of zebrafish larvae. Our experimental set-up uses a 488 nm laser and a spatial light modulator to focus a diffraction-limited spot of light within the brain. We use a combination of experimental measurements of back-scattered light in live larvae and computational modelling of the scattering to determine the spatial distribution of light. Modelling is performed using the Monte Carlo method, supported by generalised Lorenz-Mie theory in the single-scattering approximation. Scattering in areas rich in cell bodies is compared to that of regions of neuropil to identify the distinct and dramatic contributions that cell nuclei make to scattering. We demonstrate the feasibility of illuminating individual neurons, even in nucleus-rich areas, at depths beyond 100 µm using a spatial light modulator in combination with a standard laser and microscope optics.
Assuntos
Encéfalo/fisiologia , Luz , Optogenética , Animais , Encéfalo/efeitos da radiação , Núcleo Celular/química , Núcleo Celular/efeitos da radiação , Larva/fisiologia , Método de Monte Carlo , Espalhamento de Radiação , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/fisiologiaRESUMO
Conserved motifs in biological sequences are closely related to their structure and functions. Recently, discriminative motif discovery methods have attracted more and more attention. However, little attention has been devoted to the data imbalance problem, which is one of the main reasons affecting the performance of the discriminative models. In this article, a simulated evolution method is applied to solve the multi-class imbalance problem at the stage of data preprocessing, and at the stage of Hidden Markov Models (HMMs) training, a random under-sampling method is introduced for the imbalance between the positive and negative datasets. It is shown that, in the task of discovering targeting motifs of nine subcellular compartments, the motifs found by our method are more conserved than the methods without considering data imbalance problem and recover the most known targeting motifs from Minimotif Miner and InterPro. Meanwhile, we use the found motifs to predict protein subcellular localization and achieve higher prediction precision and recall for the minority classes.
Assuntos
Algoritmos , Mineração de Dados/estatística & dados numéricos , Proteínas/química , Motivos de Aminoácidos , Animais , Compartimento Celular , Membrana Celular/química , Núcleo Celular/química , Simulação por Computador , Sequência Conservada , Citosol/química , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , Humanos , Cadeias de Markov , Dados de Sequência Molecular , Proteínas/genéticaRESUMO
We have investigated the inhomogeneous interior of confined spherical cavities as capsules containing encapsulated binary hard sphere mixtures for different compositions and cavity wall rigidity. Such a greatly simplified model manifests the effects of macromolecular crowding arising from excluded volume interactions in a tiny cell or a cellular nucleus. By fixing the number of large particles, the level of crowding is adjusted by changing the amount of small hard spheres in the cavity. For a rigid cavity, large spheres tend to pack in liquid-like order apart from the surface to the center of the cavity as the crowding level is increased. Whereas, for a soft cavity, larger spheres tend to blend with small spheres in the peripheral region at near the boundary of the cavity, and are susceptible to be depleted from the interior of the cavity as the cavity becomes more crowded. These results may help future elucidation of the thermodynamic pathways to stabilize the inhomogeneous structure of mixtures confined in cavities, such as the derepression of genome materials around the interior rim of the nucleus in a cancerous cell.
Assuntos
Simulação por Computador , Espaço Intracelular/química , Substâncias Macromoleculares/química , Modelos Biológicos , Núcleo Celular/química , Genoma , Modelos Moleculares , Método de Monte Carlo , TermodinâmicaRESUMO
The intracellular location of nucleic acid intercalators (NAI) in live (not fixed) Saccharomyces cerevisiae cells has been studied using fluorescence microscopy combined with computer pseudospectral image analysis. Three NAI: the anthracycline anticancer drug doxorubicin and the nucleic acid dyes ethidium bromide (E) and 4',6-diamidino-2-phenylindole (DAPI) were used. All three NAI were shown to be localized in nuclei and mitochondria. In contrast to DAPI, which interacted only with DNA, a large fraction of doxorubicin and ethidium bromide apparently bound to mitochondrial membranes. Upon combined application, a competition between these intercalators for binding sites in the nuclear and mitochondrial DNA occurred. It was concluded that this approach may be used in designing new DNA-targeted drugs and in preliminary studies of their interaction with eukaryotic cells.
Assuntos
DNA Fúngico/química , DNA Mitocondrial/química , Doxorrubicina/química , Etídio/química , Processamento de Imagem Assistida por Computador , Indóis/química , Substâncias Intercalantes/química , Núcleo Celular/química , Microscopia de Fluorescência/métodos , Mitocôndrias/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologiaRESUMO
In eukaryotes, the interphase nucleus is organized in morphologically and/or functionally distinct nuclear "compartments". Numerous studies highlight functional relationships between the spatial organization of the nucleus and gene regulation. This raises the question of whether nuclear organization principles exist and, if so, whether they are identical in the animal and plant kingdoms. We addressed this issue through the investigation of the three-dimensional distribution of the centromeres and chromocenters. We investigated five very diverse populations of interphase nuclei at different differentiation stages in their physiological environment, belonging to rabbit embryos at the 8-cell and blastocyst stages, differentiated rabbit mammary epithelial cells during lactation, and differentiated cells of Arabidopsis thaliana plantlets. We developed new tools based on the processing of confocal images and a new statistical approach based on G- and F- distance functions used in spatial statistics. Our original computational scheme takes into account both size and shape variability by comparing, for each nucleus, the observed distribution against a reference distribution estimated by Monte-Carlo sampling over the same nucleus. This implicit normalization allowed similar data processing and extraction of rules in the five differentiated nuclei populations of the three studied biological systems, despite differences in chromosome number, genome organization and heterochromatin content. We showed that centromeres/chromocenters form significantly more regularly spaced patterns than expected under a completely random situation, suggesting that repulsive constraints or spatial inhomogeneities underlay the spatial organization of heterochromatic compartments. The proposed technique should be useful for identifying further spatial features in a wide range of cell types.
Assuntos
Núcleo Celular/química , Centrômero/química , Heterocromatina/química , Imageamento Tridimensional , Modelos Estatísticos , Animais , Arabidopsis/citologia , Embrião de Mamíferos/citologia , Feminino , Glândulas Mamárias Animais/citologia , Microscopia Confocal , Método de Monte Carlo , Proteínas Nucleares/química , CoelhosRESUMO
The nuclear fraction of the ProteoExtract subcellular fractionation kit was assessed using frozen rat liver and heart tissue. Fractionation was evaluated by Western blot using protein markers for various subcellular compartments and followed up with LC/MS/MS analysis of the nuclear fractions. Of the proteins identified, nuclear proteins were in the minority (less than 15%) and there was poor representation of the various nuclear substructures when compared with liver nuclear isolations using a classical density-based centrifugation protocol. The ProteoExtract kit demonstrated poor specificity for the nucleus and offers limited promise for proteomics investigations of the nuclear subproteome in frozen tissue samples.
Assuntos
Fracionamento Celular/métodos , Proteínas Nucleares/análise , Proteoma/análise , Proteômica/métodos , Animais , Núcleo Celular/química , Fígado/citologia , Miocárdio/citologia , Ratos , Sensibilidade e EspecificidadeRESUMO
Many colonial bat species make regional migrations, and the consequent gene flow may eliminate geographic genetic structure resulting from history of colonization. In this study, we verified that history and social organization have detectable impacts on the genetic structure of Miniopterus schreibersii, a cave-dwelling bat with high female philopatry. After studying all known nursing colonies in Portugal, we concluded that there is a significant geographic structure and that the overall pattern is similar for mitochondrial and nuclear DNA. Both pairwise Phi(ST) and F(ST) were significantly correlated with geographical distance, suggesting that isolation by distance is relevant for both mitochondrial and nuclear markers. However, structuring of mitochondrial DNA was much more marked than that of nuclear DNA, a consequence of the strong female philopatry and a bias for male-mediated gene flow. Wintering colonies were more genetically diverse than nursing colonies because the former receive individuals from distinct breeding populations. Haplotype diversity of the northern colonies, the more recent according to population expansion analyses, is only about half of that of the central and southern colonies. This is most likely a consequence of the colonization history of M. schreibersii, which presumably expanded northward from the south of the Iberian Peninsula or North Africa after the last glacial age.
Assuntos
Migração Animal , Quirópteros/genética , DNA Mitocondrial/genética , Genética Populacional , África do Norte , Animais , Sequência de Bases , Núcleo Celular/química , DNA/genética , Feminino , Variação Genética , Masculino , Repetições de Microssatélites/genética , Dados de Sequência Molecular , Dinâmica Populacional , Estações do AnoRESUMO
Considerable advances in microscopy, biophysics, and cell biology have provided a wealth of imaging data describing the functional organization of the cell nucleus. Until recently, cell nuclear architecture has largely been assessed by subjective visual inspection of fluorescently labeled components imaged by the optical microscope. This approach is inadequate to fully quantify spatial associations, especially when the patterns are indistinct, irregular, or highly punctate. Accurate image processing techniques as well as statistical and computational tools are thus necessary to interpret this data if meaningful spatial-function relationships are to be established. Here, we have developed a thresholding algorithm, stable count thresholding (SCT), to segment nuclear compartments in confocal laser scanning microscopy image stacks to facilitate objective and quantitative analysis of the three-dimensional organization of these objects using formal statistical methods. We validate the efficacy and performance of the SCT algorithm using real images of immunofluorescently stained nuclear compartments and fluorescent beads as well as simulated images. In all three cases, the SCT algorithm delivers a segmentation that is far better than standard thresholding methods, and more importantly, is comparable to manual thresholding results. By applying the SCT algorithm and statistical analysis, we quantify the spatial configuration of promyelocytic leukemia nuclear bodies with respect to irregular-shaped SC35 domains. We show that the compartments are closer than expected under a null model for their spatial point distribution, and furthermore that their spatial association varies according to cell state. The methods reported are general and can readily be applied to quantify the spatial interactions of other nuclear compartments.
Assuntos
Forma do Núcleo Celular , Núcleo Celular/ultraestrutura , Microscopia Confocal/métodos , Algoritmos , Linhagem Celular , Núcleo Celular/química , Simulação por Computador , Fibroblastos/citologia , Humanos , Processamento de Imagem Assistida por Computador/métodos , Método de Monte Carlo , Proteínas Nucleares/análise , Proteína da Leucemia Promielocítica , Fatores de Transcrição/análise , Proteínas Supressoras de Tumor/análiseRESUMO
The architecture of the eukaryotic genome is characterized by a high degree of spatial organization. Chromosomes occupy preferred territories correlated to their state of activity and, yet, displace their genes to interact with remote sites in complex patterns requiring the orchestration of a huge number of DNA loci and molecular regulators. Far from random, this organization serves crucial functional purposes, but its governing principles remain elusive. By computer simulations of a statistical mechanics model, we show how architectural patterns spontaneously arise from the physical interaction between soluble binding molecules and chromosomes via collective thermodynamics mechanisms. Chromosomes colocalize, loops and territories form, and find their relative positions as stable thermodynamic states. These are selected by thermodynamic switches, which are regulated by concentrations/affinity of soluble mediators and by number/location of their attachment sites along chromosomes. Our thermodynamic switch model of nuclear architecture, thus, explains on quantitative grounds how well-known cell strategies of upregulation of DNA binding proteins or modification of chromatin structure can dynamically shape the organization of the nucleus.
Assuntos
Núcleo Celular/química , Cromossomos/química , Modelos Biológicos , Sítios de Ligação , Simulação por Computador , DNA/química , Genoma , Modelos Moleculares , Método de Monte Carlo , Conformação de Ácido Nucleico , TermodinâmicaRESUMO
The comet assay is popular for assessments of genotoxicity, but the comparison of results between studies is challenging because of differences in experimental procedures and reports of DNA damage in different units. We investigated the variation of DNA damage in mononuclear blood cells (MNBCs) measured by the comet assay with focus on the variation related to alkaline unwinding and electrophoresis time, number of cells scored, as well as the putative benefits of transforming the primary end points to common units by the use of reference standards and calibration curves. Eight experienced investigators scored pre-made slides of nuclei differently, but each investigator scored constantly over time. Scoring of 200 nuclei per treatment was associated with the lowest residual variation. Alkaline unwinding for 20 or 40 min and electrophoresis for 20 or 30 min yielded different dose-response relationships of cells exposed to gamma-radiation and it was possible to reduce the variation in oxidized purines in MNBCs from humans by adjusting the level of lesions with protocol-specific calibration curves. However, there was a difference in the level of DNA damage measured by different investigators and this variation could not be reduced by use of investigator-specific calibration curves. The mean numbers of lesions per 10(6) bp in MNBCs from seven humans were 0.23 [95% confidence interval (CI): 0.14-0.33] and 0.31 (95% CI: 0.20-0.55) for strand breaks (SBs) and oxidized guanines, respectively. In conclusion, our results indicate that inter-investigator difference in scoring is a strong determinant of DNA damage levels measured by the comet assay.
Assuntos
Ensaio Cometa/estatística & dados numéricos , Ensaio Cometa/normas , Dano ao DNA , DNA/análise , Estresse Oxidativo , Núcleo Celular/química , Núcleo Celular/ultraestrutura , Interpretação Estatística de Dados , Humanos , Leucócitos Mononucleares/química , Leucócitos Mononucleares/ultraestrutura , Variações Dependentes do ObservadorRESUMO
Parafibromin is a protein encoded by the HRPT2 oncosuppressor gene, whose mutation causes the hyperparathyroidism-jaw tumour syndrome, characterized by the occurrence of parathyroid adenoma or carcinoma, fibro-osseous jaw tumours, and renal neoplastic and non-neoplastic abnormalities. Non-morphological techniques, such as Northern and Western blotting and reverse transcriptase-PCR, indicate that parafibromin is ubiquitously expressed, but extensive immunohistochemical studies have not been performed. To increase our knowledge of the distribution and patterns of expression of parafibromin, we examined its expression and location in many different mouse and human organs by immunohistochemistry. There were no substantial differences in parafibromin expression between mouse and human. We found widespread expression of parafibromin, except in connective tissue, smooth muscle, endothelium and some other types of epithelia (colonic, urinary, tubaric, uterine, thyroid). Heterogeneity of positivity intensity and subcellular location (nuclear, nucleocytoplasmic, cytoplasmic) was found between tissues and cell types, suggesting differential functional involvement of parafibromin. Moreover, higher parafibromin expression was found in cell types, such as hepatocytes, cells of the base of gastric glands, renal cortex tubules and the pars intermedia of the hypophysis, which are characterized by different proliferative capacity, thus indicating that the cellular function of parafibromin may not be reduced only to its anti-proliferative effect.
Assuntos
Células/química , Proteínas Supressoras de Tumor/análise , Adulto , Idoso , Animais , Núcleo Celular/química , Citoplasma/química , Feminino , Fibroma/genética , Fibroma/metabolismo , Mucosa Gástrica/química , Hepatócitos/química , Humanos , Hiperparatireoidismo/genética , Hiperparatireoidismo/metabolismo , Imuno-Histoquímica , Neoplasias Maxilomandibulares/genética , Neoplasias Maxilomandibulares/metabolismo , Túbulos Renais/química , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação , Hipófise/química , Especificidade da Espécie , Síndrome , Proteínas Supressoras de Tumor/genéticaRESUMO
This is the first report on the development of an immunohistochemical technique, combined with quantitative image analysis, for the assessment of oxidative stress quantitatively in nuclear DNA in situ, and its application to measure DNA damage in Duchenne muscular dystrophic (DMD) muscles. Three sequential staining procedures for cell nuclei, a cell marker, and a product of oxidative DNA damage, 8-oxoguanine (8-oxoG), were performed. First, the nuclei in muscle sections were stained with Neutral Red followed by the capture of their images with an image analysis system used for absorbance measurements. Second, the same sections were then immunostained for laminin in basement membranes as the cell marker. Next, the sections were treated with 2 N HCl to remove the bound Neutral Red and to denature tissue DNA. Third, the sections were immunostained for 8-oxoG in DNA, using diaminobenzidine (DAB) to reveal the antibody complex. This was followed by capture of the images of the immunostained sections as previously. The absorbances at 451.2 nm of bound Neutral Red and DAB polymer oxides, the final product of 8-oxoG immunostaining, were measured in the same myonuclei in the sections. Analysis of these absorbances permitted indices of the 8-oxoG content, independent of the nuclear densities, to be determined in nuclear DNA in single myofibres and myosatellite cells surrounded by basement membranes. We found that the mean index for the myonuclei in biceps brachii muscles of 2- to 7-year-old patients was 14% higher than that in age-matched normal controls. This finding of the increased oxidative stress in the myonuclei in young DMD muscles agrees with the previous reports of increased oxidative stress in the cytoplasm in the DMD myofibres and myosatellite cells. The present technique for the quantitative assessment of oxidative stress in nuclear DNA in situ is applicable not only in biomedical research but also in the development of effective drugs for degenerative diseases related to oxidative stress.
Assuntos
DNA/química , Distrofina/metabolismo , Guanosina/análogos & derivados , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Estresse Oxidativo , Animais , Núcleo Celular/química , Criança , Pré-Escolar , Distrofina/deficiência , Guanosina/análise , Humanos , Imuno-Histoquímica , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/químicaRESUMO
Diagnostic assays for HER2 in breast cancer provide important prognostic information and independently help guide management by identifying patients who are the most likely to benefit from Herceptin-targeted therapy. The biological events underlying HER2 -driven breast cancer that can be assessed in routine clinical specimens include the evaluation of gene amplification by fluorescence in situ hybridization (FISH), enhanced messenger RNA expression by real-time polymerase chain reaction, and the assessment of protein overexpression at the tumor cell membrane by immunohistochemistry (IHC). Immunohistochemistry and FISH methodologies have the advantage of being morphologically driven, allowing for correlations between HER2 expression and morphologic features. However, each has important advantages and disadvantages, which are discussed in detail. Although immunohistochemistry is familiar and readily accommodated in most surgical pathology laboratories, increasing demands for FISH testing in the clinical setting will require greater familiarity with the technical aspects of FISH assays and their interpretation by the greater laboratory community. In this review, we provide an overview of FISH testing for HER2 in breast cancer, with an emphasis on technical considerations, interpretive guidelines, scoring criteria, and quality control. The development of automated platforms for hybridization, image analysis for signal enumeration, and experience with FISH interpretation should broaden the availability of this technology for clinical diagnostic testing.
Assuntos
Neoplasias da Mama/genética , Hibridização in Situ Fluorescente , Receptor ErbB-2/genética , Neoplasias da Mama/química , Neoplasias da Mama/patologia , Núcleo Celular/química , Citoplasma/química , Feminino , Expressão Gênica , Guias como Assunto , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente/métodos , Controle de Qualidade , Receptor ErbB-2/análiseRESUMO
Qualitative evaluation of protein content in formalin fixed, paraffin-embedded tissues is usually performed by means of cytofluorimetric analysis. On the other hand, several studies underline the opportunity to measure the concentration of nuclear proteins, which is often accomplished by using complex techniques and instrumentation. In the present work, we suggest a new application for the spectrophotometric evaluation of protein content on extracted and isolated nuclei, based on EDTA treatment of specimens and chemical extraction of proteins, followed by direct spectrophotometric measurement at UV wavelengths. We also demonstrate how this parameter correlates with other diagnostic factors, such as the proliferation index (MIB-1) and the DNA content (ploidy) of cells. This method is simple and effective, yet less expensive than other protein quantitation protocols.
Assuntos
Neoplasias da Mama/química , Proteínas Nucleares/análise , Espectrofotometria/métodos , Neoplasias da Mama/patologia , Núcleo Celular/química , Núcleo Celular/patologia , DNA de Neoplasias/análise , DNA de Neoplasias/isolamento & purificação , Feminino , Fixadores/química , Citometria de Fluxo , Formaldeído/química , Humanos , Imuno-Histoquímica , Antígeno Ki-67/análise , Proteínas Nucleares/química , Proteínas Nucleares/isolamento & purificação , Inclusão em Parafina , Ploidias , Fixação de TecidosRESUMO
Nuclear morphometry is a method for quantitative measurement of histopathologic changes in the appearance of stained cell nuclei. Numerous studies have indicated that these assessments may provide clinically relevant information related to the degree of progression and malignant potential of breast neoplasia. Nuclear features are derived from computerized analysis of digitized microscope images, and a quantitative Feulgen stain for DNA was used. Features analyzed included: (1) DNA content; (2) nuclear size and shape; and (3) texture features, describing spatial features of chromatin distribution. In this study replicated measurements are described on a series of 54 breast carcinoma specimens of differing pathologic grades. Duplicate measurements were performed using two serial sections, which were processed and analyzed separately. The value of a single feature measurement, the nuclear area profile, was shown to be the strongest indicator of progression. A quantitative nuclear grade was derived and shown to be strongly correlated with not only the pathologic nuclear grade, but also with tubule formation, mitotic grade, and with the overall histopathologic grade. Analysis of replication precision showed that the standard methods of the histopathology laboratory, if practiced in a uniform manner, are sufficient to ensure reproducibility of these assessments. We argue that nuclear morphometry provides a standardized and reproducible framework for quantitative pathologic assessments.
Assuntos
Neoplasias da Mama/patologia , Núcleo Celular/patologia , Algoritmos , Biomarcadores Tumorais/análise , Neoplasias da Mama/química , Núcleo Celular/química , Cromatina/patologia , DNA de Neoplasias/análise , Feminino , Humanos , Prognóstico , Reprodutibilidade dos Testes , Fatores de RiscoRESUMO
Intracytoplasmic sperm injection (ICSI) has made irrelevant the conventional criteria of concentration, motility and morphology for assessment of sperm quality and so we urgently need new assays by which to gauge sperm 'health'. ICSI may be facilitating the transfer of genetic disorders to future generations by bypassing all the natural hurdles for sperm selection without imposing more pertinent criteria of selection. Sperm DNA quality is vital to the future offspring irrespective of whether the child is conceived naturally, by in vitro fertilization (IVF) or by ICSI. The DNA integrity of sperm can be determined quickly and accurately using a range of techniques that also have strong prognostic power in predicting successful IVF and ICSI outcomes with ejaculated sperm. Moreover, there is a close correlation between testicular nuclear DNA integrity and pregnancy rates in ICSI. Mitochondrial DNA can be measured using long PCR in ejaculated and testicular sperm and is also useful for predicting success in assisted conception. This review discusses how the integrity of both nuclear and mitochondrial affect the choice of sperm for assisted conception.
Assuntos
Núcleo Celular/química , DNA Mitocondrial/análise , DNA/análise , Fertilização in vitro , Espermatozoides/fisiologia , Antioxidantes/administração & dosagem , Dano ao DNA , Epididimo/citologia , Deleção de Genes , Humanos , Masculino , Oligospermia/genética , Espécies Reativas de Oxigênio , Injeções de Esperma Intracitoplásmicas , Espermatozoides/química , Testículo/citologia , Resultado do TratamentoRESUMO
Traditional semen analysis is essential for the diagnosis of male infertility. A number of studies over the past decade have reported that a significant contributing factor to male fertility that is not revealed as part of semen analysis is sperm DNA, specifically its composition and organization. Exogenous and endogenous factors can cause damage to sperm DNA. For example, topoisomerase II activity, which is necessary for sperm DNA packaging, can adversely influence the competence of sperm DNA if the activity of the enzyme is abnormal. Germ cell apoptosis can be induced by oxygen radicals produced from environmental (for example cigarette smoke) or testicular (for example localized ischaemia) sources. Several assays have been developed that are useful for assessing sperm DNA composition and organization. To date, each of these assays has revealed that when sperm DNA has been damaged or packaged improperly there is a concomitant and often significant decline in male fertility.