RESUMO
Full exploitation of fibre Raman probes has been limited by the obstruction of weak Raman signals by background fluorescence of the sample and the intrinsic Raman signal of the delivery fibre. Here we utilised functionalised gold nanoshells (NS) to take advantage of the surface-enhanced Raman spectroscopy (SERS) effect to enhance the pH responsive spectrum of 4-mercaptobenzoic acid (MBA). However, the fibre background is still dominant. Using the photon arrival time-resolving capability of a CMOS single-photon avalanche diode (SPAD) based line sensor, we recover the SERS spectrum without a fibre background in a 10 s measurement. In this manner, pH sensing through a multimode fibre at a low excitation power that is safe for future in vivo applications, with short acquisition times (10 or 60 s), is demonstrated. A measurement precision of ± 0.07 pH units is thus achieved.
RESUMO
Conventional two dimensional (2D) monolayer cell culture has been considered the 'gold standard' technique for in vitro cellular experiments. However, the need for a model that better mimics the three dimensional (3D) architecture of tissue in vivo has led to the development of Multicellular Tumour Spheroids (MTS) as a 3D tissue culture model. To some extent MTS mimic the environment of in vivo tumours where, for example, oxygen and nutrient gradients develop, protein expression changes and cells form a spherical structure with regions of proliferation, senescence and necrosis. This review focuses on the development of techniques for chemical analysis of MTS as a tool for understanding in vivo tumours and a platform for more effective drug and therapy discovery. While traditional monolayer techniques can be translated to 3D models, these often fail to provide the desired spatial resolution and z-penetration for live cell imaging. More recently developed techniques for overcoming these problems will be discussed with particular reference to advances in instrument technology for achieving the increased spatial resolution and imaging depth required.
Assuntos
Neoplasias/patologia , Esferoides Celulares/química , Animais , Humanos , Neoplasias/tratamento farmacológico , Esferoides Celulares/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Intracellular redox potential is a highly regulated cellular characteristic and is critically involved in maintaining cellular health and function. The dysregulation of redox potential can result in the initiation and progression of numerous diseases. Redox potential is determined by the balance of oxidants and reductants in the cell and also by pH. For this reason a technique for quantitative measurement of intracellular redox potential and pH is highly desirable. In this paper we demonstrate how surface enhanced Raman scattering (SERS) nanosensors can be used for multiplexed measurement of both pH and redox potential in live single cells.
Assuntos
Espaço Intracelular/química , Espaço Intracelular/metabolismo , Nanotecnologia , Análise Espectral Raman , Linhagem Celular , Sobrevivência Celular , Humanos , Concentração de Íons de Hidrogênio , OxirreduçãoRESUMO
Bats subject to high rates of fatalities at wind-energy facilities are of high conservation concern due to the long-term, cumulative effects they have, but the impact on broader bat populations can be difficult to assess. One reason is the poor understanding of the geographic source of individual fatalities and whether they constitute migrants or more local individuals. Here, we used stable hydrogen isotopes, trace elements and species distribution models to determine the most likely summer geographic origins of three different bat species (Lasiurus borealis, L. cinereus, and Lasionycteris noctivagans) killed at wind-energy facilities in Ohio and Maryland in the eastern United States. In Ohio, 41.6%, 21.3%, 2.2% of all individuals of L. borealis, L. cinereus, and L. noctivagans, respectively, had evidence of movement. In contrast, in Maryland 77.3%, 37.1%, and 27.3% of these same species were classified as migrants. Our results suggest bats killed at a given wind facility are likely derived from migratory as well as resident populations. Finally, there is variation in the proportion of migrants killed between seasons for some species and evidence of philopatry to summer roosts. Overall, these results indicate that the impact of wind-energy facilities on bat populations occurs across a large geographic extent, with the proportion of migrants impacted likely to vary across species and sites. Similar studies should be conducted across a broader geographic scale to understand the impacts on bat populations from wind-energy facilities.
Assuntos
Quirópteros , Vento , Humanos , Animais , Estados Unidos , Maryland , Estações do Ano , OhioRESUMO
An MRSA assay requiring neither labeling nor amplification of target DNA has been developed. Sequence specific binding of fragments of bacterial genomic DNA is detected at femtomolar concentrations using electrochemical impedance spectroscopy (EIS). This has been achieved using systematic optimisation of probe chemistry (PNA self-assembled monolayer film on gold electrode), electrode film structure (the size and nature of the chemical spacer) and DNA fragmentation, as these are found to play an important role in assay performance. These sensitivity improvements allow the elimination of the PCR step and DNA labeling and facilitate the development of a simple and rapid point of care test for MRSA. Assay performance is then evaluated and specific direct detection of the MRSA diagnostic mecA gene from genomic DNA, extracted directly from bacteria without further treatment is demonstrated for bacteria spiked into saline (10(6) cells per mL) on gold macrodisc electrodes and into human wound fluid (10(4) cells per mL) on screen printed gold electrodes. The latter detection level is particularly relevant to clinical requirements and point of care testing where the general threshold for considering a wound to be infected is 10(5) cells per mL. By eliminating the PCR step typically employed in nucleic acid assays, using screen printed electrodes and achieving sequence specific discrimination under ambient conditions, the test is extremely simple to design and engineer. In combination with a time to result of a few minutes this means the assay is well placed for use in point of care testing.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Técnicas Eletroquímicas , Staphylococcus aureus Resistente à Meticilina , Sistemas Automatizados de Assistência Junto ao Leito/normas , Infecções Estafilocócicas/diagnóstico , Humanos , Reação em Cadeia da PolimeraseRESUMO
The 7.6(5) eV nuclear magnetic-dipole transition in a single 229Th3+ ion may provide the foundation for an optical clock of superb accuracy. A virtual clock transition composed of stretched states within the 5F(5/2) electronic ground level of both nuclear ground and isomeric manifolds is proposed. It is shown to offer unprecedented systematic shift suppression, allowing for clock performance with a total fractional inaccuracy approaching 1×10(-19).
RESUMO
We have produced laser-cooled Wigner crystals of 229Th3+ in a linear Paul trap. The magnetic dipole (A) and electric quadrupole (B) hyperfine constants for four low-lying electronic levels and the relative isotope shifts with respect to 232Th3+ for three low-lying optical transitions are measured. Using the hyperfine B constants in conjunction with prior atomic structure calculations, a new value of the spectroscopic nuclear electric quadrupole moment Q=3.11(16) eb is deduced. These results are a step towards optical excitation of the low-lying isomer level in the 229Th nucleus.
RESUMO
This paper investigates the properties of a simple DNA-based nanodevice capable of detecting single base mutations in unlabeled nucleic acid target sequences. Detection is achieved by a two-stage process combining first complementary-base hybridization of a target and then a conformational change as molecular recognition criteria. A probe molecule is constructed from a single DNA strand designed to adopt a partial cruciform structure with a pair of exposed (unhybridized) strands. Upon target binding, a switchable cruciform construct (similar to a Holliday junction) is formed which can adopt open and closed junction conformations. Switching between these forms occurs by junction folding in the presence of divalent ions. It has been shown from the steady-state fluorescence of judiciously labeled constructs that there are differences between the fluorescence resonance energy transfer (FRET) efficiencies of closed forms, dependent on the target sequence near the branch point, where the arms of the cruciform cross. This difference in FRET efficiency is attributed to structural variations between these folded junctions with their different branch point sequences arising from the single base mutations. This provides a robust means for the discrimination of single nucleotide mismatches in a specific region of the target. In this paper, these structural differences are analyzed by fitting observed time-resolved donor fluorescence decay data to a Gaussian distribution of donor-acceptor separations. This shows the closest mean separation (approximately 40 A) for the perfectly matched case, whereas larger separations (up to 50 A) are found for the single point mutations. These differences therefore indicate a structural basis for the observed FRET differences in the closed configuration which underpins the operation of these devices as biosensors capable of resolving single base mutations.
Assuntos
Pareamento Incorreto de Bases , DNA/química , DNA/genética , Nanotecnologia , Conformação de Ácido Nucleico , Sequência de Bases , Transferência Ressonante de Energia de Fluorescência , Coloração e RotulagemRESUMO
Previously unobtainable measurements of alveolar pH were obtained using an endoscope-deployable optrode. The pH sensing was achieved using functionalized gold nanoshell sensors and surface enhanced Raman spectroscopy (SERS). The optrode consisted of an asymmetric dual-core optical fiber designed for spatially separating the optical pump delivery and signal collection, in order to circumvent the unwanted Raman signal generated within the fiber. Using this approach, we demonstrate a ~100-fold increase in SERS signal-to-fiber background ratio, and demonstrate multiple site pH sensing with a measurement accuracy of ± 0.07 pH units in the respiratory acini of an ex vivo ovine lung model. We also demonstrate that alveolar pH changes in response to ventilation.
RESUMO
A Holliday junction (HJ) consists of four DNA double helices, with a branch point discontinuity at the intersection of the component strands. At low ionic strength, the HJ adopts an open conformation, with four widely spaced arms, primarily due to strong electrostatic repulsion between the phosphate groups on the backbones. At high ionic strength, screening of this repulsion induces a switch to a more compact (closed) junction conformation. Fluorescent labelling with dyes placed on the HJ arms allows this conformational switch to be detected optically using fluorescence resonance energy transfer (FRET), producing a sensitive fluorescent output of the switch state. This paper presents a systematic and quantitative survey of the switch characteristics of such a labelled HJ. A short HJ (arm length 8 bp) is shown to be prone to dissociation at low switching ion concentration, whereas an HJ of arm length 12 bp is shown to be stable over all switching ion concentrations studied. The switching characteristics of this HJ have been systematically and quantitatively studied for a variety of switching ions, by measuring the required ion concentration, the sharpness of the switching transition and the fluorescent output intensity of the open and closed states. This stable HJ is shown to have favourable switch characteristics for a number of inorganic switching ions, making it a promising candidate for use in nanoscale biomolecular switch devices.
Assuntos
DNA Cruciforme/química , DNA Cruciforme/efeitos dos fármacos , Corantes Fluorescentes/química , Íons/farmacologia , Conformação de Ácido Nucleico/efeitos dos fármacos , Espectrometria de Fluorescência , Espermidina/farmacologiaRESUMO
Using a radioisotope labeling technic, the ability of bovine and porcine insulin antigens to induce lymphocyte transformation was tested with cells from the peripheral blood of thirty nondiabetic controls, fifty established insulin-dependent diabetics with no evidence of insulin allergy, and ten newly diagnosed diabetics (five untreated, five insulin-treated for less than three weeks). Lymphocytes from twenty-six (42 per cent) of the diabetics showed significant blastogenesis to bovine or porcine insulin, as compared with two (7 per cent) of controls; the phenomenon was shown by both established and newly diagnosed patients including four who had never recieved insulin. The results indicate that cellular hypersensitivity to insulin, as judged by an in vitro test, is relatively common in insulin-treated diabetics without in vivo evidence of allergy, and suggest that hypersensitivity may also be present in untreated diabetics. Lymphocytes from twenty-one of the twenty-six diabetics who responded to intact insulin were further tested using bovine and porcine insulin A chain bovine B chain as antigens. The A chain of either insulin induced significant blastogenesis in only one diabetic but bovine B chain induced significant blastogenesis in fourteen (67 per cent) of the patients tested. These results suggest that B chain is the major antigenic site determining cellular hypersensitivity to insulin. Diabetes 24:36-43, January, 1975.
Assuntos
Diabetes Mellitus/imunologia , Insulina/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Adolescente , Adulto , Idoso , Animais , Complexo Antígeno-Anticorpo , Bovinos , Diabetes Mellitus/diagnóstico , Diabetes Mellitus/tratamento farmacológico , Hipersensibilidade a Drogas , Feminino , Humanos , Insulina/uso terapêutico , Masculino , Pessoa de Meia-Idade , SuínosRESUMO
In a study of 972 patients with diabetes mellitus, humoral pancreatic islet-cell antibodies (I.C.Ab.) were detected in highest prevalence in insulin-treated diabetics with (38 per cent) and without (22 per cent) associated overt organ-specific autoimmune disease (A.I.D.) where consideration was not given to the duration of diabetes. They were also detected in 8 per cent of diabetics treated with oral hypoglycemic agents (O.H.A.), but not in diabetics requiring diet alone and in only 0.5 per cent of 434 control subjects. Six per cent of 522 patients with overt organ-specific A.I.D. but not diagnosed to be diabetic had I.C.Ab.s. I.C.Ab.s were present in the sera of 2 per cent of 157 first-degree relatives of I.C.Ab.-positive subjects. In insulin-treated diabetics and, to a lesser extent, in diabetics not requiring insulin, the prevalence of humoral I.C.Ab. was strongly dependent of the duration of the diabetes, being 60 per cent during the first year from diagnosis in the insulin-treated group and falling to 20 per cent at two to five years and to 5 per cent at 10-20 years. The prevalence of I.C.Ab. in insulin-treated diabetics showed no correlation with the patient's age at the time of testing when the duration of diabetes was taken into account. Diabetics who did not require insulin for treatment but who were I.C.Ab.-positive showed a significant tendency to subsequently require insulin and to have a higher prevalence of other autoantibodies than insulin-independent diabetics who were I.C.Ab.-negative. Persistence of I.C.Ab. for more than five years from diagnosis of diabetes was associated with coexistent overt organ-specific A.I.D. and with HLA-B8, A1, and A1 + B8.
Assuntos
Anticorpos , Doenças Autoimunes/imunologia , Diabetes Mellitus/imunologia , Antígenos HLA , Antígenos de Histocompatibilidade , Ilhotas Pancreáticas/imunologia , Adulto , Doenças Autoimunes/complicações , Complicações do Diabetes , Diabetes Mellitus/tratamento farmacológico , Feminino , Humanos , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêutico , Masculino , Fatores de TempoRESUMO
Neutrophils can attach to E-selectin under flow. Proposed ligands for E-selectin carry SLe(x)-type glycans. The leukocyte beta2 integrins are glycosylated with SLe(x). Thus, we speculated that beta2 integrins could support attachment to E-selectin. To test this hypothesis, we coated 10-microm-diameter microspheres with purified CD11b/CD18 (alphaMbeta2) and investigated the adhesion of the resulting alphaMbeta2 microspheres to E-selectin. Under in vitro flow conditions, the alphaMbeta2 microspheres attached to Chinese hamster ovary cells expressing E-selectin (CHO-E) and 4-h interleukin-1beta-activated human umbilical vein endothelial cells (HUVEC). At a shear stress of 1.8 dynes/cm2, the attachment events were eliminated by pretreatment of the cellular monolayers with a mAb to E-selectin. alphaMbeta2 microspheres did not attach to untransfected CHO cells or unactivated HUVEC at 1.8 dynes/cm2. Taken together, the results strongly suggest that the CD11b/CD18-E-selectin bond has sufficient biophysical properties to mediate attachment of neutrophil-sized particles to E-selectin under flow.
Assuntos
Antígenos CD18/metabolismo , Selectina E/metabolismo , Antígeno de Macrófago 1/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Antígenos CD18/imunologia , Células CHO/citologia , Células CHO/metabolismo , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Cricetinae , Cricetulus , Selectina E/genética , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Fibrinogênio/metabolismo , Gangliosídeos/metabolismo , Glicosilação , Humanos , Antígeno de Macrófago 1/imunologia , Microesferas , Neuraminidase/farmacologia , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/metabolismo , Reologia , Antígeno Sialil Lewis X , Transfecção , Veias UmbilicaisRESUMO
Cobalt(iii) tetrahedral capsules have been prepared using an assembly-followed-by-oxidation protocol from a cobalt(ii) precursor and a readily derivatizable pyridyl-triazole ligand system. Experiments designed to probe the constitutional dynamics show that these architectures are in a non-equilibrium state. A preliminary investigation into the host-guest chemistry of a water-soluble derivative shows it can bind and differentiate a range of different neutral organic molecules. The stability of this ensemble also permits the study of guest-binding at high salt concentrations.
RESUMO
In atherosclerosis, endothelial cells at sites of stenosis experience elevated levels of shear stress. We have constructed a series of shear stress-inducible transcription units (SITUs) expressing the luciferase reporter gene and determined their activation by fluid shear stress in transfected endothelial cells. Chimeric promoters were constructed that comprised basal transcription factor-binding sites coupled to a shear stress response element (SSRE). We have used consensus binding sites for transcription factors NF-kappaB, Ap1, Sp1, Oct1, and Egr-1/Sp1 in either the presence or absence of the previously defined "GAGACC" SSRE. The response of the promoters to shear stress was determined after transfection into human umbilical vein endothelial cells (HUVECs). After transient transfection into HUVECs, fluid shear stress activated the promoters by between two- and eightfold. The most responsive SITUs comprised an overlapping Sp1/Egr-1-binding site linked to a TATA box with (SP5) or without (SP7) the GAGACC SSRE. Instillation of SP5 DNA in vivo into the left carotid artery of rabbit and subsequent generation of a stenosis using a mechanical wire occluder caused a 10-fold upregulation of luciferase reporter gene expression at the site of vessel occlusion. These vectors show promise for therapeutic gene expression at sites of occlusive vascular disease.
Assuntos
Doenças Cardiovasculares/terapia , Endotélio Vascular/metabolismo , Terapia Genética , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Células Cultivadas , Primers do DNA , Humanos , Coelhos , Transcrição Gênica , TransfecçãoRESUMO
The healing of tissue involves a wide range of molecular, cellular, and physiological events that are coordinated in a temporally specific manner. The cellular transcription factor early growth response factor 1 (Egr-1) is expressed minutes after acute injury and serves to stimulate the production of a class of growth factors whose role is to promote tissue repair. We have studied the effects of Egr-1 expression at the site of dermal wounding in rodents. We find that Egr-1 promotes angiogenesis in vitro and in vivo, increases collagen production, and accelerates wound closure. These results show that Egr-1 gene therapy accelerates the normal healing process and raises the potential use of this therapeutic transcription factor for any aspect of tissue repair.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas Imediatamente Precoces , Fatores de Transcrição/genética , Cicatrização , Animais , Biolística , Colágeno/biossíntese , DNA Complementar/metabolismo , Proteína 1 de Resposta de Crescimento Precoce , Fatores de Crescimento Endotelial/biossíntese , Imunofluorescência , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Linfocinas/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Neovascularização Fisiológica/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Ratos , Ratos Sprague-Dawley , Pele/metabolismo , Fatores de Tempo , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta1 , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , beta-Galactosidase/metabolismoRESUMO
A gene encoding bovine prochymosin (PC) was fused to the coding sequence (phoA) for the Escherichia coli alkaline phosphatase (AP) signal peptide and expressed in E. coli under the control of the phoA promoter. Upon induction, an AP-PC fusion protein was produced which was neither processed nor exported into the periplasm. We investigated this lack of secretion by constructing a series of gene fusions in which different regions of the PC gene were inserted between the coding regions of the AP leader and mature protein. Analysis of the cellular location of the proteins encoded by these fusions revealed that a region of PC (between amino acids 6 and 29) prevented processing and secretion of an AP-PC fusion when inserted near to the AP signal peptide. In contrast, when this 'blocking sequence' was inserted elsewhere in AP the hybrid proteins were efficiently processed and translocation was initiated.
Assuntos
Fosfatase Alcalina/genética , Quimosina/genética , Clonagem Molecular , Precursores Enzimáticos/genética , Escherichia coli/genética , Genes Bacterianos , Genes , Regiões Promotoras Genéticas , Sinais Direcionadores de Proteínas/genética , Animais , Bovinos , Quimosina/biossíntese , Precursores Enzimáticos/biossíntese , Escherichia coli/enzimologia , Plasmídeos , Sinais Direcionadores de Proteínas/biossíntese , Proteínas Recombinantes de Fusão/biossínteseRESUMO
Endothelial dysfunction plays a major role in the pathogenesis of atherosclerosis. Pro-inflammatory cytokines such as interleukin-1 beta and tumour necrosis factor alpha activate endothelial cells changing their resting phenotype to become pro-adhesive, pro-thrombotic and pro-atherogenic. Phage display in vivo biopanning has been used to identify peptide sequences that home to diseased regions of the vessel wall in low density lipoprotein receptor (LDLr) knockout mice. In LDLr knockout mice, peptide sequence determinants exhibiting organ specificity have been isolated. These sequences have applications for gene delivery, drug delivery and for improving contrast agents for vascular imaging.
Assuntos
Arteriosclerose/metabolismo , Sistemas de Liberação de Medicamentos , Receptores de LDL/metabolismo , Animais , Artérias/metabolismo , Arteriosclerose/patologia , Arteriosclerose/terapia , Bacteriófagos/genética , Sítios de Ligação , Ligação Competitiva , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Rim/metabolismo , Camundongos , Camundongos Knockout , Especificidade de Órgãos , Biblioteca de Peptídeos , Peptídeos/metabolismo , Receptores de LDL/deficiência , Receptores de LDL/genética , Análise de Sequência de ProteínaRESUMO
The measurement process in fluorophotometry inherently involves a loss of information due to the finite sampling volume of the instrument. Mathematically, the effect is expressed as a convolution of the actual fluorescein distribution with the spread function of the instrument. Scattering by the ocular media can increase the spread function over that due to the instrument alone. A method is proposed for deconvolution of vitreous fluorophotometry data. Simulation studies and analyses of patient data demonstrate recovery of information with this method, including enhancement of retinal peaks and resolution of detail in the posterior vitreous which was not apparent from the original scans.
Assuntos
Microscopia de Fluorescência/métodos , Fotometria/métodos , Corpo Vítreo/fisiologia , Apresentação de Dados , Humanos , MatemáticaRESUMO
An esterase which hydrolyses the cephalosporin antibiotic, cefuroxime axetil has been isolated from rat intestinal washings and purified. Closely related cefuroxime esters were extremely poor substrates, but p-nitrophenyl acetate and alpha-naphthyl acetate were slowly hydrolysed by the purified enzyme. Analysis by gel filtration gave an Mr = 51,000 and on SDS-polyacrylamide gel electrophoresis the esterase resolved into two main bands of Mr = 31,500 and 26,800. Analytical isoelectric focusing resolved purified esterase into multiple forms active toward alpha-naphthyl acetate, the isoelectric points of which ranged from pH 4.5 to 6.3. The esterase bound specifically to Con A-Sepharose suggesting it could be a glycoprotein. Esterase activity was unaffected by the presence of dihydroxy bile salts (1-8 mM) and inhibition studies using organophosphates and eserine salicylate have classified the enzyme as a carboxylesterase.