RESUMEN
Three-dimensional (3-D) cell cultures are beneficial models for mimicking the complexities of in vivo tissues, especially in tumour studies where transport limitations can complicate response to cancer drugs. 3-D optical microscopy techniques are less involved than traditional embedding and sectioning, but are impeded by optical scattering properties of the tissues. Confocal and even two-photon microscopy limit sample imaging to approximately 100-200 µm depth, which is insufficient to image hypoxic spheroid cores. Optical clearing methods have permitted high-depth imaging of tissues without physical sectioning, but they are difficult to implement for smaller 3-D cultures due to sample loss in solution exchange. In this work, we demonstrate a microfluidic platform for high-throughput on-chip optical clearing of breast cancer spheroids using the SeeDB, Clear(T2), and ScaleSQ clearing methods. Although all three methods are able to effectively clear the spheroids, we find that SeeDB and ScaleSQ more effectively clear the sample than Clear(T2); however, SeeDB induces green autofluorescence while ScaleS causes sample expansion. Our unique on-chip implementation permits clearing arrays of 3-D cultures using perfusion while monitoring the 3-D cultures throughout the process, enabling visualization of the clearing endpoint as well as monitoring of transient changes that could induce image artefacts. Our microfluidic device is compatible with on-chip 3-D cell culture, permitting the use of on-chip clearing at the endpoint after monitoring the same spheroids during their culture. This on-chip method has the potential to improve readout from 3-D cultures, facilitating their use in cell-based assays for high-content drug screening and other applications.
RESUMEN
Creating multicellular tumor spheroids is critical for characterizing anticancer treatments since they may provide a better model of the tumor than conventional monolayer culture. Moreover, tumor cell interaction with the extracellular matrix can determine cell organization and behavior. In this work, a microfluidic system was used to form cell-laden core-shell beads which incorporate elements of the extracellular matrix and support the formation of multicellular spheroids. The bead core (comprising a mixture of alginate, collagen, and reconstituted basement membrane, with gelation by temperature control) and shell (comprising alginate hydrogel, with gelation by ionic crosslinking) were simultaneously formed through flow focusing using a cooled flow path into the microfluidic chip. During droplet gelation, the alginate acts as a fast-gelling shell which aids in preventing droplet coalescence and in maintaining spherical droplet geometry during the slower gelation of the collagen and reconstituted basement membrane components as the beads warm up. After droplet gelation, the encapsulated MCF-7 cells proliferated to form uniform spheroids when the beads contained all three components: alginate, collagen, and reconstituted basement membrane. The dose-dependent response of the MCF-7 cell tumor spheroids to two anticancer drugs, docetaxel and tamoxifen, was compared to conventional monolayer culture.
RESUMEN
We present a novel and simple method for patterning oxygen-sensitive polystyrene thin films and demonstrate its potential for integration with microfluidic lab-on-a-chip devices. Optical oxygen sensing films composed of polystyrene with an embedded luminescent oxygen-sensitive dye present a convenient option for the measurement of oxygen levels in microfluidic and lab-on-a-chip devices; however, patterning and integrating the films with poly(dimethylsiloxane) (PDMS) microfluidic devices has proven difficult due to a residue after dry etch patterning that inhibits subsequent PDMS bonding. Our new method uses mask-less laser ablation by a commercial laser ablation system to define the outline of the structures and subsequent bulk film removal by aqueous lift-off. Because the bulk film is peeled or lifted off of the substrate rather than etched, the process is compatible with standard PDMS plasma bonding. We used ToF-SIMS analysis to investigate how laser ablation facilitates this fabrication process as well as why dry etching polystyrene inhibits PDMS plasma bonding. The results of this analysis showed evidence of chemical species formed during the laser ablation and dry etching processes that can produce these effects. Our new method's mask-less nature, simplicity, speed, and compatibility with PDMS bonding make it ideally suited for single-use lab-on-a-chip applications. To demonstrate the method's compatibility with PDMS microfluidics, we also present a demonstration of the sensors' integration into a microfluidic oxygen gradient generator device.
Asunto(s)
Dispositivos Laboratorio en un Chip , Oxígeno/química , Poliestirenos/química , Calibración , Espectrometría de Masas , MicrofluídicaRESUMEN
We present for the first time the integration of nanohole arrays for surface plasmon resonance (SPR) sensing together with SU-8 polymer microfluidic channels containing special packaging structures for chip-to-chip and world-to-chip interconnect. Primary steps towards an optical biospecies detection device are presented including observing the effect of period on transmission peak location, examining new materials for the enclosed microchannels, and demonstrating nanohole array SPR transmission data through water contained in the microchannels. Additionally, the enclosed microchannels are integrated with interconnect structures that facilitate interfacing with macro-scale test equipment and microfluidic components and systems such as lab-on-a-chip. Results demonstrate the potential of the polymer microchannels with integrated nanohole arrays and interconnect as a packaged optical SPR detection device.
Asunto(s)
Técnicas Biosensibles/instrumentación , Compuestos Epoxi/química , Técnicas Analíticas Microfluídicas/instrumentación , Nanotecnología/instrumentación , Dispositivos Ópticos , Polímeros/química , Resonancia por Plasmón de Superficie/instrumentación , Transductores , Técnicas Biosensibles/métodos , Diseño de Equipo , Análisis de Falla de Equipo , Nanotecnología/métodos , Porosidad , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Resonancia por Plasmón de Superficie/métodosRESUMEN
S100A12 is a member of the S100 family of EF-hand calcium-modulated proteins. Together with S100A8 and S100A9, it belongs to the calgranulin subfamily, i.e. it is mainly expressed in granulocytes, although there is an increasing body of evidence of expression in keratinocytes and psoriatic lesions. As well as being linked to inflammation, allergy and neuritogenesis, S100A12 is involved in host-parasite response, as are the other two calgranulins. Recent data suggest that the function of the S100-family proteins is modulated not only by calcium, but also by other metals such as zinc and copper. Previously, the structure of human S100A12 in low-calcium and high-calcium structural forms, crystallized in space groups R3 and P2(1), respectively, has been reported. Here, the structure of S100A12 in complex with copper (space group P2(1)2(1)2; unit-cell parameters a = 70.6, b = 119.0, c = 90.2 A) refined at 2.19 A resolution is reported. Comparison of anomalous difference electron-density maps calculated with data collected with radiation of wavelengths 1.37 and 1.65 A shows that each monomer binds a single copper ion. The copper binds at an equivalent site to that at which another S100 protein, S100A7, binds zinc. The results suggest that copper binding may be essential for the functional role of S100A12 and probably the other calgranulins in the early immune response.
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Cobre/química , Proteínas S100/química , Secuencia de Aminoácidos , Sitios de Unión , Calcio/química , Calcio/metabolismo , Cobre/metabolismo , Cristalización , Cristalografía por Rayos X , Motivos EF Hand , Interacciones Huésped-Parásitos , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas S100/metabolismo , Proteína S100A12 , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Electricidad Estática , Zinc/química , Zinc/metabolismoRESUMEN
S100A12 is a member of the S100 subfamily of EF-hand calcium-binding proteins; it has been shown to be one of the ligands of the 'receptor for advanced glycation end products' (RAGE) that belongs to the immunoglobulin superfamily and is involved in diabetes, Alzheimer's disease, inflammation and tumour invasion. The structure of the dimeric form of native S100A12 from human granulocytes in the presence of calcium in space group R3 has previously been reported. Here, the structure of a second crystal form in space group P2(1) (unit-cell parameters a = 53.9, b = 100.5, c = 112.7A, beta = 94.6 degrees) solved at 2.7A resolution by molecular replacement using the R3 structure as a search model is reported. Like most S100 proteins, S100A12 is a dimer. However, in the P2(1) crystal form dimers of S100A12 are arranged in a spherical hexameric assembly with an external diameter of about 55 A stabilized by calcium ions bound between adjacent dimers. The putative target-binding sites of S100A12 are located at the outer surface of the hexamer, making it possible for the hexamer to bind several targets. It is proposed that the S100A12 hexameric assembly might interact with three extracellular domains of the receptor, bringing them together into large trimeric assemblies.
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Proteínas de Unión al Calcio/química , Proteínas S100 , Transducción de Señal/fisiología , Sitios de Unión , Biopolímeros/química , Western Blotting , Proteínas de Unión al Calcio/fisiología , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Conformación Proteica , Proteína S100A12RESUMEN
Spatio-temporal data on cytotaxonomic identifications of larvae of different members of the Simulium damnosum complex collected from rivers in southern Ghana and south-western Togo from 1975 until 1997 were analysed. When the data were combined, the percentages of savannah blackflies (S. damnosum sensu stricto and S. sirbanum) in the samples were shown to have been progressively increasing since 1975. The increases were statistically significant (P < 0.001), but the rates of increase were not linear. Further analyses were conducted according to the collection seasons and locations of the samples, to account for possible biases such as savannah flies occurring further south in the dry season or a preponderance of later samples from northern rivers having more savannah flies. These analyses showed that the increasing trend was statistically significant (P < 0.0001) only during the periods April to June and October to December. The presence of adult savannah flies carrying infective larvae (L3) indistinguishable from those of Onchocerca volvulus in the study zone was confirmed by examinations of captured flies. The percentages of savannah flies amongst the human-biting populations and the percentages with L3s in the head were higher during dry seasons than wet seasons and the savannah species were found furthest south (5 degrees 25'N) in the dry season. Comparisons of satellite images taken in 1973 and 1990 over a study area in south-western Ghana encompassing stretches of the Tano and Bia rivers demonstrated that there have been substantial increases in urban and savannah areas, at the expense of forest. This was so not only for the whole images but also for subsamples of the images taken at 1, 2, 4, 8 and 16 km distant from sites alongside the River Tano. At every distance from the river, the percentages of pixels classified as urban or savannah have increased in 1990 compared with 1973, while those classified as degraded or dense forest have decreased. The possibility that the proportionate increases in savannah forms of the vectors of onchocerciasis, and hence in the likelihood of the transmission of savannah strains of the disease in formerly forested areas, were related to the decreases in forest cover is discussed.
Asunto(s)
Conservación de los Recursos Naturales , Oncocercosis/transmisión , Simuliidae/aislamiento & purificación , Animales , Ecosistema , Monitoreo del Ambiente , Ghana , Insectos Vectores , Larva , Estaciones del Año , TogoRESUMEN
During cardiac surgery for transposition of the great arteries at age 7 weeks, a female infant received blood, fresh frozen plasma and platelet transfusions. Eleven days postoperatively, she developed bloody diarrhoea, fever, an erythematous macular rash, hepatomegaly, seizures and pancytopaenia. A clinical diagnosis of transfusion related graft-versus-host disease (GVHD) was supported by skin histopathology. DNA polymorphism studies confirmed that circulating lymphocytes in peripheral blood and infiltrating cells in the skin were foreign in origin and were derived from transfused blood cells. No underlying immunodeficiency was identified. Treatment with steroids cyclosporin and antithymocyte globulin was unsuccessful and death occurred 2 months after surgery. The features of fever, rash, diarrhoea, liver dysfunction and pancytopaenia which characterize GVHD may mimic drug reactions or viral infection. In addition to histological features on skin biopsy. DNA polymorphism studies on skin and blood samples provide a unique and sensitive method to confirm GVHD. Irradiation of blood products should be considered for acutely compromised infants requiring urgent cardiac surgery.
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ADN Satélite/genética , Enfermedad Injerto contra Huésped/diagnóstico , Enfermedad Injerto contra Huésped/genética , Complicaciones Intraoperatorias/diagnóstico , Complicaciones Intraoperatorias/etiología , Polimorfismo Genético/genética , Reacción a la Transfusión , Resultado Fatal , Femenino , Enfermedad Injerto contra Huésped/sangre , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Humanos , Inmunofenotipificación , Lactante , Complicaciones Intraoperatorias/sangre , Complicaciones Intraoperatorias/tratamiento farmacológico , Repeticiones de Microsatélite/genética , Transposición de los Grandes Vasos/cirugíaRESUMEN
Immunoselection has been used to identify human lymphocytes that have undergone spontaneous mutation resulting in the loss of expression of one of the codominant HLA-A alleles. Approximately 35% of such mutations are the consequence of mitotic recombination events. Mitotic recombination is the result of nonsister chromatid exchange that leaves the mutated cell homozygous for all loci distal to the crossover point. The location of the crossover has been regionalized for 99 independently derived mutant lymphocyte clones by identification of their loss or retention of heterozygosity at seven reference polymorphic loci on chromosome 6. If a polymorphic locus of unknown map position is studied in clones from this ordered set of mitotic recombinants, and clones that display loss of heterozygosity and retention of heterozygosity of the locus are observed, then the map position of the locus is between the appropriate reference loci of the ordered set. The newly mapped locus becomes a reference locus in turn. In this way the mitotic recombinant mutant clones can be used to generate an ordered set of crossover points with a theoretical resolution limited only by the number of mutants generated. In this paper such a set of mutants is used to refine or confirm the map position of eight polymorphic loci on chromosome 6.
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Mapeo Cromosómico/métodos , Cromosomas Humanos Par 6 , Mitosis/genética , Polimorfismo Genético , Intercambio Genético , Estudios de Evaluación como Asunto , Antígenos HLA-A/genética , Humanos , Linfocitos/ultraestructura , Mutación , Recombinación GenéticaRESUMEN
DNA sequences containing tandem dinucleotide repeats represent an abundant source of DNA polymorphism in human and other eukaryotic genomes. Here we describe a novel technique for the identification and characterization of regions of DNA containing these repetitive elements. Using primers designed to recognize tandem dinucleotide repeat sequences and limiting dilution of a target genomic library enable amplification by polymerase chain reaction (PCR) of single-target molecules containing dinucleotide repeats. Amplified material was sequenced by the PCR direct method and by the resultant sequences used to design locus-specific primers. This study identified and characterized four anonymous dinucleotide repeat sequences, three of which exhibited polymorphism. Although developed for dinucleotide repeats, the technique is universally applicable to repeat DNA elements of a size usually analyzed by PCR. The technique is comparatively rapid, eliminates library screening and its associated manipulations, and compares favorably with existing methods for the recovery of repetitive DNAs.
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Oligonucleótidos/química , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Secuencias Repetitivas de Ácidos Nucleicos , Secuencia de Bases , Humanos , Datos de Secuencia MolecularRESUMEN
We investigated whether the polymerase chain reaction (PCR) could be used to determine the mechanism of mutation in lymphocyte clones mutated at the HLA-A locus. Three polymorphisms, at Factor XIIIA, D6S109, and intron 3 of the HLA-A gene, were used to study a series of clones previously characterised by Southern blotting (SB) at multiple loci on chromosome 6. For detection of loss of heterozygosity, the results of PCR and SB were concordant in 140 of 141 clones when polymorphism in the Factor XIIIA region was studied and in 144 of 145 clones when polymorphism in the HLA-A gene was studied. For classification of the mechanism of mutation, PCR and SB gave the same result in 88 of 92 clones (96%) when a combination of the HLA-A and Factor XIIIA polymorphisms was used and in 46 of 47 clones (98%) when a combination of the HLA-A and D6S109 polymorphisms was used. The results indicate that PCR provides a simple and reliable method for categorising mutations at the HLA-A locus as arising from mitotic recombination, deletion, or from presumptive minor changes within the gene. Rare events such as gene conversion, nondisjunction, or large deletions extending to the telomere will be misclassified. However, such events are rare for mutations at this locus.
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Análisis Mutacional de ADN/métodos , Genes MHC Clase I , Antígenos HLA-A/genética , Mutación , Reacción en Cadena de la Polimerasa , Secuencia de Bases , Southern Blotting , Cromosomas Humanos Par 6 , Clonación Molecular , Cartilla de ADN , Conversión Génica , Eliminación de Gen , Heterocigoto , Humanos , Linfocitos/ultraestructura , Datos de Secuencia Molecular , Mutagénesis , Polimorfismo de Longitud del Fragmento de Restricción , Recombinación Genética , Reproducibilidad de los Resultados , Transglutaminasas/genéticaRESUMEN
The number and molecular nature of in vivo mutations in relation to age was studied at the autosomal HLA-A locus in human lymphocytes. Mutant lymphocytes were isolated by immunoselection, cloned at limiting dilution and enumerated, and the HLA-A gene and other polymorphic gene loci on chromosome 6 were studied by Southern blotting to determine gene dosage and loss of heterozygosity. Results of 167 assays in 73 individuals showed that the total number of mutant lymphocytes increased significantly with age from a geometric mean frequency of 0.71 x 10(-5) in neonates to 6.53 x 10(-5) in elderly individuals. Analysis of rearrangement of T lymphocyte receptor beta or gamma chain genes gave a best estimate of 3.3% for the proportion of mutant lymphocytes detected which are clonally related. Molecular study of 434 mutants from 31 individuals showed no change on Southern blotting in 64.7%, gene deletion in 2.8% and mitotic recombination in 32.5%. Two mutants due to gene conversion but no mutants due to non-disjunction were detected. The number of 'no change' and recombination mutants increased significantly with age. There was a significant difference between individuals in the proportion of mutants which resulted from mitotic recombination and the data suggested that the proportion was bimodally distributed. The point of crossing-over in recombination mutants was predominantly randomly distributed between the HLA-A locus and the centromere.
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Envejecimiento/genética , Mutación , Adolescente , Adulto , Anciano , Antígenos HLA/genética , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Linfocitos/ultraestructura , Persona de Mediana Edad , Recombinación GenéticaRESUMEN
Gene rearrangement and monoclonality have been detected in normal cells and in lymphoproliferative disease by using the polymerase chain reaction and primers for the V and J regions of the Ig heavy chain gene or T-cell receptor gamma-chain gene. Using the Ig primers monoclonality was detected in 20 of 20 normal B-lymphocyte clones and in 39 of 52 cases of various types of B-lymphoproliferative disease, but not in 11 cases of T-lymphoproliferative disease. Using the T-cell receptor primers, monoclonality was detected in 186 of 192 normal T-lymphocyte clones, in 11 of 11 cases of T-lymphoproliferative disease, in 9 of 12 cases of B-acute lymphocytic leukemia, and in 1 of 21 cases of B-non-Hodgkin's lymphoma, but not in nine cases of B-chronic lymphocytic leukemia nor in 10 cases of myeloma. Monoclonality was detected in material obtained by lymph node aspiration in four of six additional cases of non-Hodgkin's lymphoma. It was not detected in 10 cases of acute myeloid leukemia nor in four cases of reactive lymphadenopathy. Detection of gene rearrangement by the polymerase chain reaction has a number of advantages over Southern blotting and is likely to become the initial diagnostic technique of choice to detect monoclonality.
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Reordenamiento Génico de la Cadena gamma de los Receptores de Antígenos de los Linfocitos T/genética , Trastornos Linfoproliferativos/genética , Anticuerpos Monoclonales/inmunología , Linfocitos B/patología , Secuencia de Bases , Southern Blotting , Humanos , Linfocitos/citología , Linfocitos/patología , Linfocitos/ultraestructura , Trastornos Linfoproliferativos/patología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/patologíaRESUMEN
The mutations present in vivo in normal human cells were studied at the HLA-A locus by isolating mutant lymphocytes using antibody-complement immunoselection and cloning at limiting dilution. The molecular basis for mutation in 127 mutant lymphocytes from 10 individuals was determined by studying a variety of polymorphic gene loci on both arms of chromosome 6. No change was detected in 78 mutants (61.4%), gene deletion was detected in 11 (8.7%), and mitotic recombination was detected in 38 (29.9%). Neither gene conversion nor chromosome loss was detected. These observations document the mechanisms responsible for gene loss in normal human cells in vivo, emphasize the importance of mitotic recombination, and indicate the similarity between mutational mechanisms in normal cells and in cancer cells.
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Cromosomas Humanos Par 6 , Antígenos HLA-A/genética , Mutación , Células Cultivadas , Deleción Cromosómica , Mapeo Cromosómico , Tamización de Portadores Genéticos , Marcadores Genéticos/análisis , Glutatión Transferasa/genética , Humanos , Linfocitos/inmunología , Mitosis , Recombinación GenéticaRESUMEN
Three anonymous DNA probes ACH207, ACH224, and ACH202, isolated from a flow-purified chromosome 16 library and mapped to defined intervals of human chromosome 16, detected restriction fragment length polymorphisms (RFLPs). The RFLPs were of simple two allele types. The ACH207 (D16S4) probe detected a TaqI and an MspI RFLP with polymorphism information content (PIC) values of 0.30 and 0.27; the ACH224 (D16S5) probe detected an RsaI RFLP, PIC value of 0.34; and the ACH202 (D16S4) probe detected an XbaI RFLP, PIC value of 0.22.
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Cromosomas Humanos Par 16 , ADN/aislamiento & purificación , Polimorfismo Genético , Polimorfismo de Longitud del Fragmento de Restricción , Alelos , Mapeo Cromosómico , ADN/genética , Enzimas de Restricción del ADN , Desoxirribonucleasa HindIII , Frecuencia de los Genes , HumanosRESUMEN
Mutations in human lymphocytes are commonly due to gene deletion. To investigate the mechanism of deletion for autosomal genes, we immunoselected lymphocytes mutated at the HLA-A locus and cloned them for molecular analysis. Of 36 mutant clones that showed deletion of the selected HLA-A allele, 8 had resulted from a simple gene deletion, whereas 28 had resulted from a more complex mutational event involving reduplication of the nonselected HLA-A allele as indicated by hybridization intensity on Southern blots. In 3 of the 28 clones, retention of heterozygosity at the HLA-B locus indicated that the reduplication was due to recombination between the two chromosomes 6; but in the remaining 25 clones, distinction could not be made between recombination and chromosome reduplication. The results indicate that mutations in normal somatic cells frequently result in hemizygosity or homozygosity at gene loci and, thereby, resemble the mutations thought to be important in the etiology of various forms of cancer.
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Linfocitos/análisis , Familia de Multigenes , Mutación , Neoplasias/genética , Alelos , Deleción Cromosómica , Cromosomas Humanos Par 6 , Clonación Molecular , Antígenos HLA/genética , Antígenos HLA-A , Antígeno HLA-A2 , Antígenos HLA-B , Humanos , Hibridación de Ácido NucleicoRESUMEN
Anonymous DNA probes specific for human chromosome 16 were isolated from a flow-purified human chromosome 16 library. The library was constructed at the Lawrence Livermore National Laboratory. Twenty-nine clones containing a unique or low-copy DNA insert were isolated. Of these, six were assigned to chromosome 16 and regionally mapped and 12 were shown not to map to chromosome 16. One clone mapped to 16pter----16p13.1, one clone to 16p11.1----16q13, one clone to 16q13----16q22.1, and three clones to 16q22.1----16q24. An additional clone from the same library was mapped to 16q13----16q22.1.