RESUMO
The two-photon photopolymerization of resins by focused laser light in principle enables the fabrication of structures with details below the diffraction limit. However, the method can be highly susceptible to aberrations, which hinders the fabrication of structures that are larger than, e.g., the working distance of the microscope objective. Here, two-photon polymerization is extended to the fabrication of macroscopic structures by making use of medium numerical-aperture microscope objectives. By introducing a substrate holder movable in the axial direction it is possible to keep the focusing conditions constant and to fabricate very large structures with heights that are not limited by the working distance of the objective. Moreover, the constant focusing conditions enable us to quantify spherical aberrations by experimental mapping of the optical point-spread function, which manifests itself in the shape of singe photo-polymerized voxels. By monitoring such shapes it is possible to minimize aberrations. Effective aberration control enables us to fabricate large but detailed biomedical scaffolds with interconnected pores, e.g., in the shape of a human stirrup bone.
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Chromosome 14 breakpoints in malignant human lymphocytes cluster on the long (q) arm near bands q11 and q32. An inversion of chromosome 14 due to breaks in q11.2 and q32.3 has now been found in a newly established childhood T-cell lymphoma cell line and confirmed in T-cell chronic lymphocytic leukemia. A translocation was also found between chromosomes 10 and 14 with a breakpoint at 14q11.2 in another T-cell lymphoma cell line. It is proposed that a proximal region on chromosome 14 in or near sub-band q11.2 is related to T-cell function. Rearrangements in this region may affect the growth of T lymphocytes and be involved in the development of T-cell malignancies.
Assuntos
Aberrações Cromossômicas , Leucemia Linfoide/genética , Linfoma/genética , Linhagem Celular , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Masculino , Pessoa de Meia-Idade , Linfócitos T , Translocação GenéticaRESUMO
We describe a simple and reliable procedure for obtaining a flat plateau on top of standard silicon nitride atomic force microscopy tips by scanning them over the focus of a high-numerical-aperture objective illuminated by near-infrared ultrashort laser pulses. Flattened tips produced this way exhibit a plateau that is parallel to the substrate when the cantilever is mounted. They represent a valid and cost-effective alternative to commercially available plateau tips.
Assuntos
Desenho de Equipamento/instrumentação , Desenho de Equipamento/métodos , Microscopia de Força Atômica/instrumentação , Nanotecnologia/instrumentação , Transdutores , Análise de Falha de Equipamento , Microscopia de Força Atômica/métodos , Nanotecnologia/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
We investigate the dependence of the spot size in single-emitter confocal imaging on the degree of saturation. We show that single-emitter spots are broadened and flattened significantly already at excitation intensities well below saturation. The resulting single-emitter spot shapes thus deviate significantly from the excitation point spread function.We show and support by Monte Carlo simulations that fitting of a single spot is sufficient to extract the saturation intensity and the maximum emission rate of a single emitter with high accuracy. Our results will be of interest in all areas of single-emitter studies.
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Attempts were made to establish lymphoid cell lines from the cultured peripheral blood lymphocytes of six patients with chronic lymphocytic leukemia. In only one case was cell growth obtained following the addition of exogenous transforming Epstein-Barr virus, and those cell cultures proved not to have acquired the ability to proliferate permanently. In the same case, cell lines were established spontaneously from the peripheral blood without addition of Epstein-Barr virus. The cells which grew spontaneously were large, were occasionally weakly surface adherent, and grew in suspension as loose clumps or as single cells. They were negative for surface immunoglobulins and spontaneous rosette formation with sheep erythrocytes and positive for intracytoplasmic immunoglobulins (Fc and C3 receptors). At an early passage, the spontaneous lines had an aneuploid karyotype with some triploid and some tetraploid cells. Structural chromosomal aberrations include a 14q+. Electron microscopy of the chronic lymphocytic leukemia lines revealed relatively smooth surfaces with numerous mitochondria, widespread vacuolization, and numerous unusual "myelin" figures. Five to 10% of the cells were phagocytic as detected by internalization of latex particles; however, they were Epstein-Barr nuclear antigen positive. The nature of these cells and their possible relationship to the etiology of chronic lymphocytic leukemia are discussed.
Assuntos
Transformação Celular Viral , Herpesvirus Humano 4 , Leucemia Linfoide/patologia , Linhagem Celular , Imunofluorescência , Humanos , Leucemia Linfoide/microbiologia , Microscopia Eletrônica de Varredura , Fagocitose , Formação de RosetaRESUMO
A method to identify single molecules rapidly and with high efficiency based on simple probability considerations is proposed. In principle, any property of a detected photon in a single-molecule fluorescence experiment, e.g., emission wavelength, arrival time after pulsed excitation, and polarization, can be analyzed within the framework of the outlined methodology. Monte Carlo simulations show that less than 500 photons are needed to assign an observed single molecule to one out of four species with a confidence level higher than 99.9%. We show that single dye molecules of four different dyes embedded in a polymer film can be identified with time-correlated single-photon counting spectrally resolved in two channels.
RESUMO
For two decades, cytogenetic studies have been used to rule in (or out) the Philadelphia (Ph1) chromosome associated with chronic myeloid leukemia. Beyond this single purpose, chromosome studies have generally not been utilized in or applied to the practice of hematology-oncology. This report presents male and female patients, teens to 70s in age, with representative hematologic disorders, in whom the cytogenetic findings were useful clinically. These cases illustrate the following principles: (1) hematologic disorders can be characterized by chromosome analysis; (2) chromosome findings help in the diagnosis, prognosis, and treatment of blood diseases; (3) blood and bone marrow samples can be processed routinely for cytogenetic analysis; (4) these samples can be transported long distances from clinic to laboratory; and (5) the contemporary practice of hematology and oncology requires chromosome analysis for fuller evaluation and understanding of hematologic conditions.
Assuntos
Cromossomos Humanos 21-22 e Y , Cromossomos Humanos 6-12 e X , Leucemia/genética , Translocação Genética , Adulto , Idoso , Medula Óssea/ultraestrutura , Aberrações Cromossômicas/genética , Transtornos Cromossômicos , Feminino , Hematócrito , Humanos , Cariotipagem , Leucemia Mieloide/genética , Leucemia Mieloide Aguda/genética , Linfoma/genética , Masculino , Pessoa de Meia-Idade , Pancitopenia/genéticaRESUMO
Specific chromosome changes occur in the initiation and progression of cancer. A translocation between chromosomes 14 and 18 arises as a primary cytogenetic event in the formation of non-Hodgkin, non-Burkitt lymphomas (BL), while a translocation between chromosomes 2 and 8 is seen in BL and BL-type acute lymphocytic leukemia (ALL-L3) with expression of kappa (kappa) light immunoglobulin chains. These two translocations were detected in a lymphocytic malignancy expressing not kappa, but lambda (lambda) light chains. The anomalous light chain expression, it appears, provides the key clue indicating that the translocation between chromosomes 14 and 18 arose first during lymphoma formation in a cell committed to lambda chain synthesis and the translocation between chromosomes 2 and 8 occurred in the transformation to ALL. This sequence of cytogenetic events is consistent with the clinical course from lymphocytic lymphoma to ALL, the immunologic phenotype of the malignancy, and the concept of a cascade of chromosome changes eventuating in aggressive cancer.
Assuntos
Neoplasias da Mama/genética , Cadeias Leves de Imunoglobulina/biossíntese , Cadeias lambda de Imunoglobulina/biossíntese , Linfoma Folicular/genética , Cromossomos Humanos 1-3 , Cromossomos Humanos 13-15 , Cromossomos Humanos 16-18 , Cromossomos Humanos 6-12 e X , Feminino , Humanos , Leucemia Linfoide/genética , Pessoa de Meia-Idade , Translocação GenéticaRESUMO
One gauge of creativity in medical genetics and dysmorphology is the eponym. Conditions named for people reflect their powers of observation and analysis. We examined the fields and ages of 210 eponymous physicians and scientists whose biographies were published by Peter and Greta Beighton [1986] in The Man Behind the Syndrome. Twenty fields were represented with the dominant fields being, in order, pediatrics, neurology, general and internal medicine, pathology, radiology, and orthopedics. The ages of the eponymous workers averaged 43 years at the time of their relevant publications. Fully a fifth of the contributions were made by persons aged 30 years or less, or 60 years or more, suggesting that eponymous fame may come to workers of talent independent of age.
Assuntos
Criatividade , Genética Médica , Adulto , Fatores Etários , Idoso , Humanos , Pessoa de Meia-Idade , Síndrome , Terminologia como AssuntoRESUMO
GUSB, the gene for beta-glucuronidase, has been localized to the proximal long arm of chromosome 7 between 7q11.2 and 7q22. Deficiency of beta-glucuronidase results in mucopolysaccharidosis type VII (MPS VII, Sly syndrome). The enzymatic defect has been demonstrated in cultured skin fibroblasts, leukocytes and serum of affected patients. An 8-yr-old boy presented with manifestations similar to MPS VII (mental retardation, short stature, "coarse" facial appearance, mild skeletal involvement and recurrent lower respiratory tract infection) but other, discrepant abnormalities, e.g., bilateral iris colobomata and cleft palate. Normal activity of beta-glucuronidase was found in the patient's leukocytes. Chromosome analysis disclosed an interstitial deletion of 7q with one breakpoint at the interface between bands 11.22 and 11.23 and the other breakpoint within band 21.1. DNA from this patient's leukocytes was analyzed for dosage of GUSB sequences. This locus appeared to be present at the normal diploid level. These findings suggest that GUSB is not in the portion of chromosome 7 deleted in our case, narrowing the smallest region of overlap to 7q21.1----7q22. We therefore assign the beta-glucuronidase gene to 7q21.1----7q22.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 7 , Anormalidades Congênitas/enzimologia , Genes , Glucuronidase/genética , Criança , Mapeamento Cromossômico , Clonagem Molecular , Humanos , Linfócitos/citologia , Linfócitos/enzimologia , MasculinoRESUMO
We present prenatal diagnosis and follow-up examination of an individual with a de novo paracentric inversion of the long are of chromosome 14. A literature search documented 19 other cases of paracentric inversion of 14q. The outcome of each of these cases is specified together with that of this current case. Four of the 20 cases, all XY, manifested significant abnormalities with mental retardation and microcephaly present in 3 of the 4 cases; 15% (2/13) of familial cases had abnormalities and 40% (2/5) of de novo cases had abnormalities.
Assuntos
Inversão Cromossômica , Cromossomos Humanos Par 14 , Diagnóstico Pré-Natal , Cromossomo X , Adulto , Amniocentese , Bandeamento Cromossômico , Mapeamento Cromossômico , Feminino , Humanos , Recém-Nascido , Cariotipagem , Masculino , Gravidez , Cromossomo YRESUMO
The absorption dipole orientation of single fluorescent molecules is determined by mapping the spatial distribution of the squared electric field components in a high-numerical-aperture laser focus. Annular illumination geometry and the vicinity of a plane dielectric/air interface strongly enhance the longitudinal field component and the transverse fields perpendicular to the polarization direction. As a result, all three excitation field components in the focus are of comparable magnitude. The scheme holds promise to monitor rotational diffusion of single molecules in complex environments.
RESUMO
The fluorescence of single terrylene molecules in a crystalline host is investigated at room temperature by scanning confocal optical microscopy. Photon arrival times are analyzed in terms of interphoton time distributions, second order correlation functions, and the variance of the photon number probability distribution. Antibunching at short times and bunching behavior for longer times is observed, associated with sub- and super-Poissonian statistics, respectively. A rate-equation analysis of the molecular level populations indicates an accelerated reverse intersystem crossing.
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In an interesting experiment of nature, we observed the discordant onset of fetal cardiac activity between the two gestations of a heterotopic pregnancy. After initially detecting cardiac activity only in the tubal gestational sac, we later observed cardiac activity in the intrauterine sac 6 days after laparoscopic salpingectomy. This case illustrates the variable onset of cardiac activity. We advise expectant management of the intrauterine pregnancy in a heterotopic gestation when cardiac activity is not detected initially in the intrauterine sac.
Assuntos
Coração Fetal/fisiologia , Gravidez Tubária , Gravidez , Ultrassonografia Pré-Natal , Adulto , Feminino , Coração Fetal/diagnóstico por imagem , HumanosRESUMO
BACKGROUND: Febrile morbidity following in vitro fertilization and embryo transfer (IVF-ET) is a rare but possibly serious complication. This report describes a case of salpingitis after IVF-ET and discusses the possible reasons for febrile morbidity following this common procedure. CASE: A 37-year-old woman undergoing IVF-ET for tubal factor infertility developed sudden, severe pelvic pain, fever, and leukocytosis 24 hours after ET. Laparoscopy revealed bilateral suppurative pyosalpinges with cystic, hemorrhagic ovaries. Pain, fever, and leukocytosis resolved with conservative surgery and intravenous antibiotic therapy. CONCLUSIONS: This case presents laparoscopic documentation of a rare complication of oocyte aspiration and/or ET, namely, salpingitis. Possibilities for the development of salpingitis following IVF-ET include activation of quiescent bacteria within the fallopian tubes from a previous pelvic infection, puncture of the bowel during oocyte aspiration, inoculation of the pelvis with cervicovaginal flora during oocyte aspiration, and introduction of bacteria-laden secretions or air into the fallopian tubes during ET. Although rare, the possibility of severe pelvic infection following IVF-ET warrants consideration of prophylactic antibiotic coverage.
Assuntos
Transferência Embrionária , Infecções por Escherichia coli/complicações , Fertilização in vitro , Febre de Causa Desconhecida/etiologia , Ooforite/diagnóstico , Salpingite/complicações , Adulto , Infecções por Escherichia coli/diagnóstico , Feminino , Humanos , Salpingite/microbiologiaRESUMO
A gene locus for ataxia-telangiectasia (A-T) is in chromosome region 11q22 to 11q23 and predisposes to cancer. Ataxia-telangiectasia patients appear to have two separate clinical patterns of malignancy. One pattern involves solid tumors, which have not been stressed and which include malignancies in the oral cavity, breast, stomach, pancreas, ovary, and bladder. Detection of a solid tumor in an A-T patient should serve as a warning. It heralds a markedly elevated risk of another malignancy in that patient. The second pattern of neoplasia in A-T is well recognized and consists of lymphocytic leukemia and non-Hodgkin's lymphoma. These malignancies may relate to immunodeficiency in A-T and to chromosome breakage and rearrangement, which are a feature of A-T. These two patterns of malignancy may be truly separate and reflect different mechanisms of malignancy in A-T, or they may not really be separate but instead reflect a single mechanism of malignancy. The situation in A-T is reminiscent of that in the acquired immunodeficiency syndrome (AIDS), in which Kaposi's sarcoma occurs with mild immunodeficiency and pneumocystis carinii pneumonia occurs with more profound immunodeficiency owing to the human immunodeficiency virus. Next to pulmonary disease, cancer is the leading cause of death in A-T.
Assuntos
Ataxia Telangiectasia/genética , Cromossomos Humanos Par 11 , Neoplasias/genética , Ataxia Telangiectasia/complicações , Mapeamento Cromossômico , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Neoplasias/complicaçõesRESUMO
The dysplastic nevus syndrome (DNS) is an autosomal dominant trait characterized by multiple atypical skin moles and a propensity to malignant melanoma. We studied chromosomes from a three-generation DNS family in 1977. Cells cultured from normal skin and dysplastic nevi showed an elevation in chromosome rearrangements with nonrandom breakpoints and clonal proliferation marked by chromosome change. The DNS qualifies as a chromosome instability disorder, the first known to manifest dominant inheritance and a clear discernible premalignant state. The DNS road to malignancy may logically proceed by genomic alterations including translocations, duplications, and deletions.
Assuntos
Aberrações Cromossômicas , Síndrome do Nevo Displásico/genética , Melanoma/genética , Lesões Pré-Cancerosas/genética , Neoplasias Cutâneas/genética , Humanos , MasculinoRESUMO
Translocations of chromosomes occurring in human cancer cells appear specific to the type of cell from which the cancer arises. To explain the action of these translocations, we propose dual position effect: Position effect of the cis type transforms the cell to malignancy, while position effect of the trans type permits the normal homologous chromosome to express a normal gene product. Thus, the translocation itself has to do with malignant transformation, while the normal homologous chromosome performs its normal function. This dual concept is illustrated by t(8;14) in Burkitt's lymphoma. The concept can be tested with t(2;18) and t(8;22) in lymphoid malignancies and with other translocations and chromosome rearrangements marking human cancer cells.
Assuntos
Neoplasias/genética , Ataxia Telangiectasia/genética , Linfoma de Burkitt/genética , Regulação da Expressão Gênica , Humanos , Imunoglobulinas/genética , Leucemia Linfoide/genética , Leucemia Mieloide/genética , Translocação GenéticaRESUMO
Whole-arm Robertsonian rearrangements are common constitutional changes of chromosomes that possess unusual properties. They occur spontaneously, are not inducible by ionizing radiation or clastogenic chemicals, show an exceptionally high meiotic mutation rate and nonrandom chromosome composition. A sample of 30 Robertsonian rearrangements in cancer patients revealed only three to have been acquired, none as a primary or significant secondary cancer chromosome change. The frequency of Robertsonian rearrangements is in the range of one per 1,100 at birth versus one acquired per 10,000 in cancer. Consistent with the prediction that Robertsonian rearrangements are recombinants, a subfamily of alpha satellite DNA displays selective homology between the centromeric regions of acrocentric autosomes proportional to their preferential entry into Robertsonian chromosomes. Recombination leading to Robertsonian rearrangements is common in meiosis, but is rare in mitosis. Hence, Robertsonian rearrangements are rare in cancer.
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Neoplasias/genética , Recombinação Genética , Translocação Genética , DNA Satélite/genética , HumanosRESUMO
Fragile sites on chromosomes are points at which rearrangements tend to occur nonrandomly. Because translocations between chromosomes #7 and #14 occur nonrandomly in normal cultured lymphocytes, we analyzed chromosomes #7 and #14 in 53,580 cultured lymphocytes and 109,300 other human cells. We found one rearrangement per 1,218 lymphocytes. These rearrangements were not restricted to translocations but included inversions and hitherto undetected duplications and deletions. In lymphocytes cultured for only 48 hours, rearrangements were seen indicating their presence in vivo. The breakpoints were exclusively in chromosome bands 7p13, 7q35, 14q11, and 14q32. The predisposition to form these rearrangements appeared nonrandom and inherited. These four bands act as if they contain fragile sites limited to lymphocytes. Fragility was not observed in these bands in cells from amniotic fluid, bone marrow, skin, or chorionic villi. Bands 7p13, 7q35, and 14q11 contain T-cell receptor (TCR) genes, whereas, band 14q32 contains the immunoglobulin heavy (IgH) chain locus. Rearrangements of these bands may result from molecular recombination between TCR or between TCR and IgH genes forming TCR/TCR and TCR/IgH chimeric genes important to understanding lymphocyte development and neoplasia. TCR/IgH chimeric genes have been found in T- and B-cell malignancy.