RESUMEN
MR fingerprinting (MRF) is a promising method for quantitative characterization of tissues. Often, voxel-wise measurements are made, assuming a single tissue-type per voxel. Alternatively, the Sparsity Promoting Iterative Joint Non-negative least squares Multi-Component MRF method (SPIJN-MRF) facilitates tissue parameter estimation for identified components as well as partial volume segmentations. The aim of this paper was to evaluate the accuracy and repeatability of the SPIJN-MRF parameter estimations and partial volume segmentations. This was done (1) through numerical simulations based on the BrainWeb phantoms and (2) using in vivo acquired MRF data from 5 subjects that were scanned on the same week-day for 8 consecutive weeks. The partial volume segmentations of the SPIJN-MRF method were compared to those obtained by two conventional methods: SPM12 and FSL. SPIJN-MRF showed higher accuracy in simulations in comparison to FSL- and SPM12-based segmentations: Fuzzy Tanimoto Coefficients (FTC) comparing these segmentations and Brainweb references were higher than 0.95 for SPIJN-MRF in all the tissues and between 0.6 and 0.7 for SPM12 and FSL in white and gray matter and between 0.5 and 0.6 in CSF. For the in vivo MRF data, the estimated relaxation times were in line with literature and minimal variation was observed. Furthermore, the coefficient of variation (CoV) for estimated tissue volumes with SPIJN-MRF were 10.5% for the myelin water, 6.0% for the white matter, 5.6% for the gray matter, 4.6% for the CSF and 1.1% for the total brain volume. CoVs for CSF and total brain volume measured on the scanned data for SPIJN-MRF were in line with those obtained with SPM12 and FSL. The CoVs for white and gray matter volumes were distinctively higher for SPIJN-MRF than those measured with SPM12 and FSL. In conclusion, the use of SPIJN-MRF provides accurate and precise tissue relaxation parameter estimations taking into account intrinsic partial volume effects. It facilitates obtaining tissue fraction maps of prevalent tissues including myelin water which can be relevant for evaluating diseases affecting the white matter.
Asunto(s)
Encéfalo , Sustancia Blanca , Humanos , Encéfalo/diagnóstico por imagen , Imagen por Resonancia Magnética/métodos , Sustancia Gris/diagnóstico por imagen , Sustancia Blanca/diagnóstico por imagen , Corteza Cerebral , Fantasmas de Imagen , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
OBJECTIVE: The effect of serum autoantibodies on the brain of systemic lupus erythematosus (SLE) patients remains unclear. We investigated whether serum autoantibodies, individually and assessed in groups, are associated with specific brain-MRI abnormalities or whether these structural changes are associated with other SLE-related or traditional cardiovascular disease risk factors. METHODS: All patients underwent brain 3Tesla-MRI. White matter hyperintensities (WMHs), ischemic lesions, inflammatory-like lesions and cerebral atrophy were scored. Serum autoantibodies analyzed included lupus anticoagulant (LAC), anticardiolipine (aCL) IgG and IgM (first 3 also grouped into antiphospholipid autoantibodies (aPL)), anti-dsDNA, anti-SSA, anti-SSB, anti-RNP, and anti-Sm (the latter 5 grouped into SLE-related autoantibodies). Associations were assessed using logistic regression analysis adjusted for potential confounders. Furthermore, a sensitivity analysis including anti-Beta2 glycoprotein-1 antibodies (anti-ß2GP1) in the aPL group was performed and the potential modification role of the neuropsychiatric clinical status in the model was assessed. RESULTS: 325 patients (mean age 42 years (SD 14), 89% female) were included. The following MRI-brain abnormalities were found: WMHs (71%), lacunar infarcts (21%), gliosis (11%), micro-hemorrhages (5%), large hemorrhages (2%), inflammatory-like lesions (6%) and atrophy (14%). No associations were found between individual or total SLE-related autoantibodies and inflammatory-like lesions. A higher number of positive aPL was associated with lacunar infarcts (OR 1.37 (95%CI 1.02-1.99) and gliosis (OR 2.15 (1.37-3.37)). LAC was associated with lacunar infarcts in white matter (OR 3.38 (1.32-8.68)) and atrophy (OR 2.49 (1.01-6.15)), and aCL IgG with gliosis (OR 2.71 (1.05-7.02)). Among other variables, SLE patients with hypertension presented a higher chance for WMHs (OR 5.61 (2.52-12.48)) and lacunar infarcts in WM (OR 2.52 (1.10-5.74)) and basal ganglia (OR 8.34 (2.19-31.70)), while cumulative SLE-damage was correlated with lacunar infarcts in WM (OR 1.43 (1.07-1.90)), basal ganglia (OR 1.72 (1.18-2.51)) and cerebellum (OR 1.79 (1.33-2.41)). These associations were confirmed in the sensitivity analysis. CONCLUSIONS: Brain abnormalities in SLE represent different underlying pathogenic mechanisms. aPL are associated with ischemic brain changes in SLE, while the presence of SLE-related serum autoantibodies is not related to inflammatory-like lesions. Hypertension and cumulative SLE-damage associate with ischemic MRI-brain changes in SLE, suggesting the importance of accelerated atherosclerosis in this process.
Asunto(s)
Autoanticuerpos/sangre , Encéfalo/patología , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/inmunología , Adulto , Encéfalo/diagnóstico por imagen , Enfermedades Cardiovasculares/etiología , Femenino , Humanos , Modelos Logísticos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Sustancia Blanca/patologíaRESUMEN
AIMS: Type 2 diabetes mellitus is associated with cognitive dysfunction, but the underlying structural brain correlates are uncertain. This study examined the association between cognitive functioning and structural brain abnormalities in people with long-standing Type 2 diabetes. METHODS: Ninety-three people with Type 2 diabetes (age 62.3 ± 5.4 years, diabetes duration 9.7 ± 6.7 years; HbA1c 65 ± 10 mmol/mol, 8.1 ± 1.3%) were included. Cognitive functioning was assessed by a test battery covering the domains memory, processing speed and executive functioning. Brain tissue volumes and white matter hyperintensity volumes were automatically determined on MRI. Linear regression analyses were performed adjusted for age, sex and education. RESULTS: In people with Type 2 diabetes, increased white matter hyperintensity volume was associated with decreased processing speed [regression B coefficient = -0.22 (-0.38 to -0.06), P = 0.009], but not with memory or executive function (P > 0.05). Brain tissue volumes were not significantly related to cognitive functioning (P > 0.05). CONCLUSIONS: In people with long-standing, less strictly controlled Type 2 diabetes, white matter hyperintensities volumes were associated with decreased processing speed. This suggests that cerebral small vessel disease is an underlying disease mechanism of cognitive dysfunction in these individuals.
Asunto(s)
Encéfalo/patología , Cognición/fisiología , Diabetes Mellitus Tipo 2/patología , Diabetes Mellitus Tipo 2/psicología , Anciano , Encéfalo/diagnóstico por imagen , Encéfalo/fisiología , Encefalopatías Metabólicas/diagnóstico , Encefalopatías Metabólicas/etiología , Encefalopatías Metabólicas/patología , Estudios de Casos y Controles , Trastornos del Conocimiento/diagnóstico , Trastornos del Conocimiento/etiología , Trastornos del Conocimiento/patología , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/diagnóstico , Femenino , Hemoglobina Glucada/metabolismo , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Neuroimagen/métodos , Pruebas Neuropsicológicas , Tamaño de los ÓrganosRESUMEN
BACKGROUND AND PURPOSE: The clinical relevance of cortical microinfarcts has recently been established; however, studies on microinfarcts in the deep gray matter are lacking. We examined the risk factors and MR imaging correlates of microinfarcts in the deep gray matter on 7T MR imaging and their relation to cognitive functioning. MATERIALS AND METHODS: Within the Second Manifestations of ARTerial disease-Magnetic Resonance (SMART-MR) study, 213 patients (mean age, 68 [SD, 8] years) had a risk-factor assessment, 7T and 1.5T brain MR imaging, and a cognitive examination. Microinfarcts on 7T MR imaging were defined as lesions of <5 mm. Regression models were used to examine the age-adjusted associations among risk factors, MR imaging markers, and microinfarcts. Cognitive function was summarized as composite and domain-specific z scores. RESULTS: A total of 47 microinfarcts were found in 28 patients (13%), most commonly in the thalamus. Older age, history of stroke, hypertension, and intima-media thickness were associated with microinfarcts. On 1.5T MR imaging, cerebellar infarcts (relative risk = 2.75; 95% CI, 1.4-5.33) and lacunes in the white (relative risk = 3.28; 95% CI, 3.28-6.04) and deep gray matter (relative risk = 3.06; 95% CI, 1.75-5.35) were associated with microinfarcts, and on 7T MR imaging cortical microinfarcts (relative risk = 2.33; 95% CI, 1.32-4.13). Microinfarcts were also associated with poorer global cognitive functioning (mean difference in the global z score between patients with multiple microinfarcts versus none = -0.97; 95% CI, -1.66 to -0.28, P = .006) and across all cognitive domains. CONCLUSIONS: Microinfarcts in the deep gray matter on 7T MR imaging were associated with worse cognitive functioning and risk factors and MR imaging markers of small-vessel and large-vessel disease. Our findings suggest that microinfarcts in the deep gray matter may represent a novel imaging marker of vascular brain injury.
Asunto(s)
Grosor Intima-Media Carotídeo , Sustancia Gris , Anciano , Biomarcadores , Cognición , Sustancia Gris/diagnóstico por imagen , Humanos , Imagen por Resonancia Magnética/métodos , Factores de RiesgoRESUMEN
AIMS/HYPOTHESIS: Type 2 diabetes mellitus is associated with moderate decrements in cognitive functioning, mainly in verbal memory, information-processing speed and executive functions. How this cognitive profile evolves over time is uncertain. The present study aims to provide detailed information on the evolution of cognitive decrements in type 2 diabetes over time. METHODS: Sixty-eight patients with type 2 diabetes and 38 controls matched for age, sex and estimated IQ performed an elaborate neuropsychological examination in 2002-2004 and again in 2006-2008, including 11 tasks covering five cognitive domains. Vascular and metabolic determinants were recorded. Data were analysed with repeated measures analysis of variance, including main effects for group, time and the group x time interaction. RESULTS: Patients with type 2 diabetes showed moderate decrements in information-processing speed (mean difference in z scores [95% CI] -0.37 [-0.69, -0.05]) and attention and executive functions (-0.25 [-0.49, -0.01]) compared with controls at both the baseline and the 4 year follow-up examination. After 4 years both groups showed a decline in abstract reasoning (-0.16 [-0.30, -0.02]) and attention and executive functioning (-0.29 [-0.40, -0.17]), but there was no evidence for accelerated cognitive decline in the patients with type 2 diabetes as compared with controls (all p > 0.05). CONCLUSIONS/INTERPRETATION: In non-demented patients with type 2 diabetes, cognitive decrements are moderate in size and cognitive decline over 4 years is largely within the range of what can be viewed in normal ageing. Apparently, diabetes-related cognitive changes develop slowly over a prolonged period of time.
Asunto(s)
Cognición/fisiología , Diabetes Mellitus Tipo 2/psicología , Anciano , Anciano de 80 o más Años , Índice de Masa Corporal , Femenino , Estudios de Seguimiento , Hemoglobina Glucada/metabolismo , Humanos , Inteligencia , Masculino , Memoria , Persona de Mediana Edad , Países Bajos , Inventario de Personalidad , Pruebas Psicológicas , Valores de Referencia , Accidente Cerebrovascular/epidemiología , Aprendizaje VerbalRESUMEN
Delirium, the clinical expression of acute encephalopathy, is a common neuropsychiatric syndrome that is related to poor outcomes, such as long-term cognitive impairment. Disturbances of functional brain networks are hypothesized to predispose for delirium. The aim of this study in non-delirious elderly individuals was to investigate whether predisposing risk factors for delirium are associated with fMRI network characteristics that have been observed during delirium. As predisposing risk factors, we studied age, alcohol misuse, cognitive impairment, depression, functional impairment, history of transient ischemic attack or stroke, and physical status. In this multicenter study, we included 554 subjects and analyzed resting-state fMRI data from 222 elderly subjects (63% male, age range: 65-85 year) after rigorous motion correction. Functional network characteristics were analyzed and based on the minimum spanning tree (MST). Global functional connectivity strength, network efficiency (MST diameter) and network integration (MST leaf fraction) were analyzed, as these measures were altered during delirium in previous studies. Linear regression analyses were used to investigate the relation between predisposing delirium risk factors and delirium-related fMRI characteristics, adjusted for confounding and multiple testing. Predisposing risk factors for delirium were not associated with delirium-related fMRI network characteristics. Older age within our elderly cohort was related to global functional connectivity strength (ß = 0.182, p < 0.05), but in the opposite direction than hypothesized. Delirium-related functional network impairments can therefore not be considered as the common mechanism for predisposition for delirium.
Asunto(s)
Delirio , Imagen por Resonancia Magnética , Anciano , Anciano de 80 o más Años , Encéfalo/diagnóstico por imagen , Estudios Transversales , Delirio/epidemiología , Delirio/etiología , Femenino , Humanos , Masculino , Factores de RiesgoRESUMEN
Northern blots with poly(A)-selected RNAs were prepared from four primary human breast carcinomas and hybridized with a SIS/PDGF-B gene probe. High expression of a normal-sized 3.7 kb SIS/PDGF-B transcript was observed in three samples. These three carcinomas were all T3 (greater than 5 cm) lesions and were histologically infiltrating ductal carcinomas accompanied by moderate to marked desmoplasia. Furthermore in situ hybridization with photobiotinylated probes was performed and demonstrated that the SIS/PDGF-B expression was localized within the epithelial cells of malignant as well as benign lesions. It is possible that SIS/PDGFB expression within the epithelial cell components of nonmalignant as well as malignant breast lesions may be in part responsible for the stromal reaction.
Asunto(s)
Neoplasias de la Mama/genética , Carcinoma/genética , Factor de Crecimiento Derivado de Plaquetas/genética , Proteínas Proto-Oncogénicas/genética , Northern Blotting , Neoplasias de la Mama/patología , Carcinoma/patología , Epitelio/fisiología , Regulación de la Expresión Génica , Humanos , Hibridación de Ácido Nucleico , Proteínas Proto-Oncogénicas c-sis , ARN Mensajero/genéticaRESUMEN
Several oncogenes have been reported to be expressed in normal and malignant hematopoietic cells. Since these studies have almost exclusively been done by Northern and dot blot hybridization techniques using mixed populations of cells, any conclusions concerning quantitative changes in gene expression are difficult to document. We have developed a rapid and sensitive RNA-in situ hybridization technique permitting detection of as few as five copies of mRNA per cell. Using this technique we have studied the expression of two genes, c-myc and c-sis, in acute leukemia patients as well as hematologically normal individuals. We have found that expression levels of myc and often sis are higher (greater than 5-fold) in hematopoietic cells obtained from leukemia patients than in normal hematopoietic cells. In regenerating marrow, there is a dramatic increase in the frequency of cells expressing myc at the level of five to 10 copies without the presence of any cells expressing myc at the high levels found in acute leukemia. This is completely different from leukemic remission marrow in which we find a subpopulation of cells which express myc at very high levels. At this time, the leukemic origin of this abnormal cell population is likely because of the close correlation we find between gene overexpression and leukemic phenotype as identified by double-labeling experiments. It appears that gene overexpression may be a more sensitive or an earlier marker for leukemic cells and that such an assay could be used in the detection of residual disease.
Asunto(s)
Regulación de la Expresión Génica , Leucemia Mieloide Aguda/genética , Oncogenes , Médula Ósea/fisiología , Regeneración Ósea , Humanos , Microscopía Fluorescente , Hibridación de Ácido Nucleico , ARNRESUMEN
A series of 60 acute nonlymphocytic leukemias (ANLL) was analyzed for the expression of terminal deoxynucleotidyl transferase (TdT). The detected TdT+ cells were studied in detail by use of double marker analyses for TdT and differentiation markers, such as myeloid markers (CD13 and CD33), B cell markers, T cell markers, and the precursor antigen CD34. In 15 (25%) of these leukemic cell samples, we found no TdT+ cells or low percentages of CD10+TdT+ cells; the latter probably represent precursor B cells. In the other 45 (75%) ANLL myeloid marker+TdT+ CD10- cells were detected, ranging from 0.1-10% (n = 24) or over 10% (n = 21) of mononuclear cells. Interestingly, a higher frequency of CD34 positivity was found on the TdT+ cells as compared to the TdT- cells, suggesting that the TdT+ cells represent an immature leukemic subpopulation. Therefore, it may be speculated that the TdT+ subpopulation contains the clonogenic ANLL cells. In two patients, in whom immunologic marker analysis was performed at initial diagnosis as well as at relapse, an expansion of the TdT+ subpopulation was documented at relapse, which may reflect a reduced differentiation capacity of the leukemic cells. Previous studies on a series of nonleukemic bone marrow and blood samples revealed that normal counterparts of myeloid marker+TdT+ cells are rare in bone marrow (less than 0.03%, if they occur at all) and that such cells are not detectable in blood. Therefore myeloid marker TdT double stainings may be useful to monitor the TdT+ leukemic subpopulation in patients with a TdT+ ANLL during and after chemotherapy. Our preliminary results on the follow-up of two such patients support this hypothesis.
Asunto(s)
Biomarcadores de Tumor/análisis , Pruebas Enzimáticas Clínicas , ADN Nucleotidilexotransferasa/análisis , Leucemia Mieloide Aguda/diagnóstico , Adolescente , Adulto , Anciano , Antígenos de Diferenciación Mielomonocítica/análisis , Biomarcadores de Tumor/sangre , Médula Ósea/enzimología , Médula Ósea/inmunología , Niño , Preescolar , ADN Nucleotidilexotransferasa/sangre , Femenino , Estudios de Seguimiento , Humanos , Lactante , Leucemia Mieloide Aguda/inmunología , Masculino , Persona de Mediana Edad , RecurrenciaRESUMEN
Three human brain-derived cell lines (including two of astrocytic origin) were exposed in vitro to the human immunodeficiency virus (HIV), the etiologic agent of immunodeficiency in AIDS. In all three lines, HIV transcripts were detected by in situ hybridisation in 20-30% of cells 48 h after infection. Synthesis of virus gag gene products p24 and p55 was demonstrated by immunoblotting. No cytopathic effects typical of HIV-infected human T lymphocytes were observed. Our data indicate that HIV is neurotropic, and support the hypothesis that this virus may infect astrocytes in the brain.
Asunto(s)
VIH/patogenicidad , Neuroglía/microbiología , Anticuerpos Monoclonales , Línea Celular , Efecto Citopatogénico Viral , ADN/análisis , ADN Viral/análisis , Proteína Ácida Fibrilar de la Glía/genética , VIH/genética , Humanos , Técnicas de Inmunoadsorción , Hibridación de Ácido Nucleico , Transcripción GenéticaRESUMEN
Studies on mechanisms for allograft rejection are focused on recognition of major histocompatibility complex (MHC) antigens. In addition, there is evidence for non-MHC-mediated alloreactivity, possibly evoked by tissue-specific antigens. To measure cellular immune responses toward tissue-specific alloantigens, we isolated endothelial cells and smooth muscle cells from small pieces of human atrium at the time of transplantation. Endothelial cells were scraped off the endocardium after trypsin digestion and cultured in fibronectin-coated dishes. Smooth muscle cells were obtained by outgrowth of small pieces of atrium in a culture flask. Morphologic and immunologic characterization showed only minor differences between endothelial and smooth muscle cells cultured from atrium and cells cultured from umbilical vein (endothelial cells) and artery (smooth muscle cells). Furthermore, we studied the proliferative immune responses with endothelial and smooth muscle cells as stimulator cells, with and without induction of MHC class II antigens on these cells by addition of interferon-gamma to the culture. Peripheral blood mononuclear cells showed a proliferative response to donor human atrium endothelial cells, even without pre-incubation with interferon-gamma. Human atrium smooth muscle cells caused only a weak triggering of the mononuclear cells, irrespective of interferon-gamma pre-incubation. Immunofluorescence studies demonstrated HLA-DR expression on these endothelial and smooth muscle cells. These observations may indicate a role for non-MHC, probably tissue-specific, alloantigens expressed by endothelial cells in human cardiac allograft rejection.
Asunto(s)
Rechazo de Injerto/inmunología , Trasplante de Corazón/inmunología , Miocardio/inmunología , Endotelio/inmunología , Técnica del Anticuerpo Fluorescente , Antígenos HLA-DR/inmunología , Atrios Cardíacos , Antígenos de Histocompatibilidad Clase II/análisis , Humanos , Isoantígenos/inmunología , Prueba de Cultivo Mixto de Linfocitos , Músculo Liso/inmunología , Especificidad de Órganos/inmunologíaRESUMEN
SUMMARY: MR imaging at 7T has a high sensitivity for cerebral microbleed detection. We identified mIP processing conditions with an optimal balance between the number of visually detected microbleeds and the number of sections on 7T MR imaging. Even with optimal mIP processing, the limited size of some of the microbleeds and the susceptibility effects of other adjacent structures were a challenge for visual detection, which led to a modest inter-rater agreement, mainly due to missed microbleeds. Automated lesion-detection techniques may be required to optimally benefit from the increased spatial resolution offered by 7T MR imaging.
Asunto(s)
Hemorragia Cerebral/patología , Procesamiento de Imagen Asistido por Computador/métodos , Procesamiento de Imagen Asistido por Computador/normas , Imagen por Resonancia Magnética/métodos , Imagen por Resonancia Magnética/normas , Anciano , Anciano de 80 o más Años , Circulación Cerebrovascular , Humanos , Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Imagen por Resonancia Magnética/estadística & datos numéricos , Microcirculación , Persona de Mediana Edad , Variaciones Dependientes del Observador , Estudios Prospectivos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Axillary metastatic lymphadenopathy with no primary tumour identified in the breast on physical examination, mammography or ultrasound is referred to as occult breast cancer. The goal of this systematic review is to give an overview of the value and additional considerations of using breast MRI in occult breast cancer. METHODS: The databases of Pubmed, Embase, CINAHL and the Cochrane library were searched for studies addressing the use of breast MRI in occult breast cancer. Cross-referencing was used to find additional articles. RESULTS: 8 retrospective studies were included. Breast MRI can detect an otherwise occult breast cancer in more than two thirds of patients with a high sensitivity but lower specificity. In 80% of patients MRI detected lesions could be localized again by using ultrasound. Furthermore the size and localization of the lesions found on MRI most often correlated closely with findings at pathology. Breast MRI also provided the possibility of breast conserving surgery in one thirds of patients. CONCLUSION: Breast MRI can result in additional detection of otherwise occult lesions in occult breast cancer. Because of low specificity of malignant lesion detection by breast MRI, lesions should be histologically confirmed. This can be achieved either by MRI or ultrasound guided biopsy, as long as all MRI detected lesions are histologically checked. Routine application of breast MRI in occult breast cancer may also alter locoregional treatment by offering the possibility of breast conserving surgery in one thirds of patients.
Asunto(s)
Neoplasias de la Mama/diagnóstico , Mama/patología , Imagen por Resonancia Magnética , Mamografía , Axila , Biopsia con Aguja , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/patología , Neoplasias de la Mama/cirugía , Femenino , Humanos , Metástasis Linfática , Imagen por Resonancia Magnética Intervencional , Mastectomía Segmentaria , Sensibilidad y EspecificidadAsunto(s)
Médula Ósea/metabolismo , Leucemia/metabolismo , Leucocitos Mononucleares/metabolismo , Linfocitos/metabolismo , Oncogenes , Proteínas Proto-Oncogénicas/biosíntesis , Proto-Oncogenes , Regulación de la Expresión Génica , Humanos , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Eritroblástica Aguda/patología , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , ARN Neoplásico/análisis , Células Tumorales Cultivadas/metabolismoRESUMEN
In this work, we investigated processing methods to obtain subgrain sizes from electron backscattered diffraction data using samples of experimentally deformed calcite (CaCO(3)) polycrystals. The domain boundary hierarchy method, based on area measurements of domains enclosed by boundaries larger than a given misorientation angle, was applied to these calcite samples and was found to be limited by: (i) topological problems; (ii) undersampling of large grains; and (iii) artefacts caused by nonindexing. We tested two alternative methods that may reduce the problems: (i) the measured linear intercept hierarchy method, based on measurements of linear intercept between boundaries having larger misorientations than a given minimum angle; and (ii) the calculated linear intercept hierarchy method, based on the total length of boundaries having misorientations larger than a given minimum angle. The measured linear intercept hierarchy method was found to produce results more representative for the microstructure than the calculated linear intercept hierarchy method, because the calculated linear intercept hierarchy method has a significant uncertainty related to the grid-based nature of the measurements. Preliminary results on calcite suggest that the measured linear intercept hierarchy method is related, in a complex way, to deformation conditions such as stress, strain and temperature as well as to the characteristics of subgrain rotation and grain boundary migration processes.
RESUMEN
A procedure for quantitatively binding covalently closed circular DNA to nitrocellulose is detailed. The procedure involves making DNA saturated with respect to Nat at 25 degrees C, denaturing the DNA by heating to 80-100 degrees C, then passing the DNA-NaI solution through a nitrocellulose membrane at 50-60 degrees C. Form I DNA remains denatured throughout the loading procedure and is quantitatively deposited on the membrane as denatured DNA. The mechanism of binding DNA to nitrocellulose in NaI appears to be different from the binding promoted by NaCl.
Asunto(s)
Colodión , ADN Circular , Yoduros , Yoduro de Sodio , Membranas Artificiales , Desnaturalización de Ácido Nucleico , Hibridación de Ácido NucleicoRESUMEN
This paper describes methods that are commonly used for performing mRNA in situ hybridizations. Each stage of the procedure has been analyzed to identify the parameters that most significantly affect the final cell morphology and sensitivity of the system. We have identified key elements of the procedure as the fixation employed, the type of polynucleotide probe and label chosen, and the detection system used. By optimizing these critical components, we have developed a procedure for performing mRNA in situ hybridizations that takes 2-4 hours and has a sensitivity of 1-10 molecules of mRNA per cell. This system has been used to detect levels of oncogene expression in normal bone marrow and peripheral blood. It is possible to detect the expression of three oncogenes (c-myc, c-sis, and c-abl) simultaneously in a small population of cells from the peripheral blood of leukemic patients.
Asunto(s)
Hibridación de Ácido Nucleico , ARN Mensajero/análisis , Animales , Línea Celular , Células Cultivadas , Sondas de ADN , Técnica del Anticuerpo Fluorescente , Humanos , ARN Mensajero/genéticaRESUMEN
Quick-blot, a method for selectively immobilizing either mRNA or DNA on nitrocellulose, is described in detail. Essential elements of the procedure for immobilizing DNA include tissue lysis, proteinase K treatment, solubilization of nucleic acids in hot 12.2 molal NaI, passage through a nitrocellulose filter, and acetylation of residual protein with acetic anhydride. Advantages include speed, quantitative recovery, low background, and elimination of the usual baking step. Essential elements of the procedure for selectively immobilizing mRNA include dissolving cells in Brij-35 and desoxycholate, proteinase K treatment, solubilizing nucleic acids in room temperature 12.2 molal NaI, filtration through nitrocellulose, and acetylation of residual protein. Advantages include selective immobilization of mRNA but not tRNA, rRNA, or DNA, and the maintenance of biological activity of the immobilized mRNA. Control experiments to optimize the procedures and examples of their application are shown.
Asunto(s)
ADN/aislamiento & purificación , Hibridación de Ácido Nucleico , ARN Mensajero/aislamiento & purificación , Células/análisis , Colodión , ADN Bacteriano/aislamiento & purificación , ADN Viral/aislamiento & purificación , Células HeLa , Humanos , Métodos , Yoduro de SodioRESUMEN
In 12.2 molal NaI and at 25 degrees C or below, mRNA bound to nitrocellulose while DNA and rRNA did not. Neither the poly(A) tract nor the cap were required for binding. The immobilized RNA could be translated, reverse transcribed, hybridized with radioactive probes, or released for further manipulation. mRNA was efficiently transferred from polyacrylamide to nitrocellulose in NaI. Baking was not required to fix NaI-immobilized mRNA to nitrocellulose. When cells dissolved in 12.2 molal NaI were filtered through nitrocellulose, mRNA became selectively bound (quickblot). The quick-blot system utilizing protease and detergents to prepare cells for NaI solubilization was especially suitable in quantitative, rapid screening of cells for expression of specific genes. Expression of highly repeated DNA sequences was detected in human leukemia cells.
Asunto(s)
Colodión , Yoduros , ARN Mensajero , Yoduro de Sodio , Regulación de la Expresión Génica , Humanos , Leucemia/genética , Hibridación de Ácido Nucleico , Poli A , Biosíntesis de Proteínas , ARN Mensajero/análisis , ARN Neoplásico/análisis , SolubilidadRESUMEN
A rapid, sensitive, inexpensive in situ hybridization technique, using 30-mer 16S rRNA probes, can specifically differentiate two closely related Bacillus spp., B. polymyxa and B. macerans. The 16S rRNA probes were labeled with a rhodamine derivative (Texas Red), and quantitative fluorescence measurements were made on individual bacterial cells. The microscopic fields analyzed were selected by phase-contrast microscopy, and the fluorescence imaging analyses were performed on 16 to 67 individual cells. The labeled 16S rRNA probe, POL, whose sequence was a 100% match with B. polymyxa 16S rRNA but only a 60% match with B. macerans 16S rRNA, gave quantitative fluorescence ratio measurements that were 34.8-fold higher for B. polymyxa cells than for B. macerans cells. Conversely, the labeled probe, MAC, which matched B. polymyxa 16S rRNA in 86.6% of its positions and B. macerans 16S rRNA in 100% of its positions, gave quantitative fluorescence measurements that were 59.3-fold higher in B. macerans cells than in B. polymyxa cells. Control probes, whose 16S rRNA sequence segment (P-M) was present in both B. polymyxa and B. macerans as well as a panprokaryotic probe (16S), having a 100% match with all known bacteria, hybridized equally well with both organisms. These latter hybridizations generated very high fluorescence signals, but their comparative fluorescence ratios (the differences between two organisms) were low. The control paneukaryotic probe (28S), which had less than 30% identity for both B. macerans and B. polymyxa, did not hybridize with either organism.