RESUMO
Thrombotic thrombocytopenic purpura (TTP) remains a serious illness with potentially life-threatening complications. The following case of a TTP patient describes a serious relapse with exacerbation in spite of adequately initiated therapy and highlights the necessity of interdisciplinary expertise in the treatment of the disease.
Assuntos
Púrpura Trombocitopênica Trombótica , Humanos , Púrpura Trombocitopênica Trombótica/diagnóstico , RecidivaRESUMO
Using scattering scanning nearfield infrared microscopy (s-SNIM), we have imaged the nanoscale phase separation of mixed polystyrene-poly(methyl methacrylate) (PS-PMMA) brushes and investigated changes in the top layer as a function of solvent exposure. We deduce that the top-layer of the mixed brushes is composed primarily of PMMA after exposure to acetone, while after exposure to toluene this changes to PS. Access to simultaneously measured topographic and chemical information allows direct correlation of the chemical morphology of the sample with topographic information. Our results demonstrate the potential of s-SNIM for chemical mapping based on distinct infrared absorption properties of polymers with a high spatial resolution of 80 nm × 80 nm.
RESUMO
To obtain insight into the potential role of the cytoskeleton on lipid mixing behavior in plasma membranes, the current study explores the influence of physisorbed actin filaments (F-actin) on lipid-lipid phase separations in planar model membrane systems containing raft-mimicking lipid mixtures of well-defined compositions using a complementary experimental approach of epifluorescence microscopy, fluorescence anisotropy, wide-field single molecule fluorescence microscopy, and interfacial rheometry. In particular, we have explored the impact of F-actin on cholesterol (CHOL)-phospholipid interactions, which are considered important for the formation of CHOL-enriched lipid raft domains. By using epifluorescence microscopy, we show that physisorbed filamentous actin (F-actin) alters the domain size of lipid-lipid phase separations in the presence of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS) and cholesterol (CHOL). In contrast, no actin-induced modification in lipid-lipid phase separations is observed in the absence of POPS or when POPS is replaced by another anionic lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG). Wide-field single molecule fluorescence microscopy on binary lipid mixtures indicate that PS and PG lipids show similar electrostatic interactions with physisorbed actin filaments. Complementary fluorescence anisotropy experiments on binary PS lipid-containing lipid mixtures are provided to illustrate the actin-induced segregation of anionic lipids. The similarity of electrostatic interactions between actin and both anionic lipids suggests that the observed differences in actin-mediated perturbations of lipid phase separations are caused by distinct PS lipid-CHOL versus PG lipid-CHOL interactions. We hypothesize that the actin cytoskeleton and some peripheral membrane proteins may alter lipid-lipid phase separations in plasma membranes in a similar way by interacting with PS lipids.
Assuntos
Actinas/química , Colesterol/química , Citoesqueleto/metabolismo , Bicamadas Lipídicas/química , Membranas Artificiais , Fosfatidilserinas/química , Polarização de Fluorescência , Microscopia de FluorescênciaRESUMO
Human papillomavirus (HPV) plays a key role in the development of cervical and laryngeal cancers. The aim of our study was to compare the performance of a new hydrogel-based HPV genotyping biochip assay (Biochip) to a commercially available and CE-marked conventional PCR followed by reverse hybridization (GenID-PCR). One hundred twenty-three samples were available for the study. Of these samples, 101/123 were gynecological swabs, 8/123 were swabs or biopsy samples of genital warts, 7/123 were biopsy samples of otorhinolaryngeal lesions, 5/123 were samples of skin warts, and 2/123 were samples of orolabial abnormalities. These molecular methods for HPV genotyping showed comparable sensitivity and specificity. However, 19/123 of the results were discrepant. Specifically, Biochip showed better performance in the detection of multiple infections, especially when more than one high-risk genotype was present. Due to the different probe configurations used in the two assays, GenID-PCR achieves only group-specific detection of many HPV genotypes, whereas Biochip allows for specific identification. Overall, the newly developed HPV chip system (Biochip) proved to be a suitable tool for HPV detection and genotyping; it also proved to be superior for establishing HPV genotyping methods.
Assuntos
Análise de Sequência com Séries de Oligonucleotídeos/métodos , Papillomaviridae/classificação , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/virologia , Colo do Útero/virologia , Feminino , Humanos , Laringe/virologia , Boca/virologia , Papillomaviridae/genética , Sensibilidade e Especificidade , Pele/virologiaRESUMO
Peptide-polymer hybrid molecules are being introduced, where one part of the molecule (i.e., the peptide) promotes the adhesion of living cells, whereas the other part of the molecule (i.e., the synthetic polymer) is known to prevent cell adhesion. The hybrid copolymer, poly(dimethylacrylamide) (PDMAA)-glycine-arginine-glycine-aspartic acid-serine-proline (GRGDSP) was synthesized by first preparing an initiator-modified peptide and in a second step growing the PDMAA block directly off the peptide through atom transfer radical polymerization (ATRP). The PDMAA block length can be varied by adjusting appropriate polymerization conditions, thereby changing progressively the amount of the cell-repelling part of the molecule. The hybrid copolymer was further used to prepare surface-attached peptide-polymer monolayers at planar solid glass substrates through a photochemical immobilization process. By blending of the hybrid copolymer with PDMAA homopolymer (i.e., without peptide), the apparent peptide film concentration can be varied in a very simple manner. The adhesion of human skin fibroblast cells in serum-free medium was investigated as a function of the amount of peptide-polymer in the solution used for film preparation. Cells do not adhere to a pure PDMAA monolayer; however, already 0.02 wt % of peptide in the film is enough to induce cell adhesion, and 0.1 wt % promotes stress-fiber formation within adherent cells. Using lithographical means, chemically micropatterned peptide-polymer films were prepared that allow for a spatial control of the adhesion of living cells and thus they constitute a simple platform for the design of live-cell biochips.
Assuntos
Acrilamidas/química , Fibroblastos/citologia , Fibroblastos/fisiologia , Oligopeptídeos/química , Polímeros/química , Acrilamidas/farmacologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Humanos , Oligopeptídeos/farmacologia , Polímeros/farmacologia , Propriedades de SuperfícieRESUMO
In photolithographic processes, the light inducing the photochemical reactions is confined to a small volume, which enables direct writing of micro- and nanoscale features onto solid surfaces without the need of a predefined photomask. The direct writing process can be used to generate topographic patterns through photopolymerization or photo-cross-linking or can be employed to use light to generate chemical patterns on the surface with high spatial control, which would make such processes attractive for bioapplications. The prospects of maskless photolithography technologies with a focus on two-photon lithography and scanning-probe-based photochemical processes based on scanning near-field optical microscopy or beam pen lithography are discussed.
RESUMO
This retrospective cohort study examined the clinical significance of isolated Staphylococcus aureus central venous catheter (CVC) tip cultures (i.e., positive tip cultures without concomitant positive blood cultures). Subsequent S. aureus bacteraemia was found in nine (12%) of 77 patients at a median time of 4 days after CVC removal. A high co-morbidity score and no effective antibiotic treatment within 48 h of CVC removal were independent risk-factors for septic complications following multivariate analysis. A matched case-control study that compared the above cohort with patients with CVC tip cultures negative for S. aureus supported the significance of these findings.
Assuntos
Cateterismo Venoso Central/efeitos adversos , Cateteres de Demora/microbiologia , Infecções Estafilocócicas/fisiopatologia , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/patogenicidade , Idoso , Bacteriemia/microbiologia , Estudos de Casos e Controles , Meios de Cultura , Feminino , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Choque Séptico/microbiologia , Infecções Estafilocócicas/microbiologiaRESUMO
BACKGROUND: Calcific degeneration is the major drawback of bioprostheses. None of the numerous preventive approaches omitted calcification. Previous studies showed that cellular surface seeding decreases calcium uptake in vitro but achievement of coverage remains problematic. A new approach is presented masking glutaraldehyde residues with a polymer layer allowing cell seeding. The aim of this study was to evaluate different polymers for suitability. METHODS: Ten polymers--covalently bound to glass--were tested for their ability to seed animal and human cells. Quality of coverage was evaluated by light and scanning electron microscopy, and polymers were characterized physicochemically. RESULTS: Quality of cellular growth was similar for canine and human cells. Five polymers allowed excellent surface coverage, two led to a decrease of cell adherence, and four to poor cellular growth. No correlation between molecular weight, thickness, hydrophilicity, or charge of the polymer and cell growth was found. CONCLUSIONS: Polymer monolayers can promote cellular growth but without correlation to physicochemical characteristics. Polymers covalently bound to biologic tissue appear to be a promising approach for achieving cellular coverage of biomaterials.
Assuntos
Bioprótese , Adesão Celular/fisiologia , Materiais Revestidos Biocompatíveis , Análise de Falha de Equipamento , Polímeros , Animais , Divisão Celular/fisiologia , Endotélio/citologia , Fibroblastos/citologia , Humanos , Microscopia Eletrônica de Varredura , CoelhosRESUMO
Our strategy to provide the structural basis for the build-up of functional tethered membranes focuses on three approaches: the first one is based on the pre-organization of a monomolecular layer of a lipopolymer at the water/air interface which is then transferred to a solid support. Prior to deposition, the substrate is coated with a layer of benzophenone-derivatized silane molecules that allow for a stable covalent attachment by photo-cross-linking of some of the monomer units of the lipopolymer to the support. An alternative concept realizes a layer-by-layer deposition of the various structural elements: (1) the attachment layer with the reactive sites for the chemical stabilization; (2) a polymer 'cushion' prepared by adsorption and simultaneous or subsequent partial covalent binding to the reactive sites; and (3) a lipid monolayer transferred from the water/air interface, that contains a certain amount of lipids with reactive headgroups which, upon binding to the polymer tether, act as anchor lipids stabilizing the whole monolayer/cushion-composite. And finally, we build peptide-supported monolayers by first (self-) assembling amino acid sequences of various lengths via a SH-group near their N-terminus onto Au substances and use then their COO(-)-terminus to chemically attach phosphatidyl-ethanolamine lipids to form a stable monolayer of lipid-peptide conjugates. All the individual preparation steps and the various resulting (multi-) layers are characterized by surface plasmon spectroscopy, X-ray and neutron-reflectometry, contact angle measurements, IR spectroscopy, fluorescence microscopy, scanning probe microscopies, as well as, electrochemical techniques. For all tethering systems, the final membranes' architecture is obtained by fusing lipid vesicles onto the lipid monolayer. Proteins can be incorporated by either fusing vesicles that are loaded with the respective receptors, pores, or ion pumps via a reconstitution procedure, or via a transfer directly from a micellar solution to the pre-formed lipid bilayer at the solid support by a dialysis step. Two structural/dynamical features of tethered membranes which are considered to be of particular functional relevance, i.e. the degree of water uptake and, hence, the degree of swelling of the polymer support, as well as the lateral mobility of the lipid molecules in the membrane, are tested by surface plasmon optics and by measurements of the fluorescence recovery after photobleaching (FRAP), respectively. The results confirm that the presented preparation protocols yield fluid bilayers that mimic certain relevant properties of biological membranes. The functional characterization of tethered membranes, which is briefly summarized, is based on various electrochemical techniques, in particular, impedance spectroscopy, cyclic voltammetry, and chronoamperometric studies. The results obtained for reconstituted H(+)-ATPase from chloroplasts and E. coli and for cytochrome oxidase (with and without cytochrome c) confirm the incorporation of the proteins in an active form, thus, opening opportunities for novel sensor formats or offering a completely new model membrane system.
Assuntos
Bicamadas Lipídicas , Membranas Artificiais , Biopolímeros , Proteínas de Membrana/química , Modelos Biológicos , Bombas de Próton/químicaRESUMO
The fluid transport produced by rectangular shaped, magnetically actuated artificial cilia of 70 µm length and 20 µm width was determined by means of phase-locked Micro Particle Image Velocimetry (µPIV) measurements in a closed microfluidic chamber. The phase-averaged flow produced by the artificial cilia reached up to 130 µm s(-1) with an actuation cycle frequency of 10 Hz. Analysis of the measured flow data indicate that the present system is capable of achieving volume flow rates of V[combining dot above](cilia) = 14 ± 4 µl min(-1) in a micro channel of 0.5 × 5 mm(2) cross-sectional area when no back pressure is built up. This corresponds to an effective pressure gradient of 6 ± 1 Pa m(-1), which equals a pressure difference of 0.6 ± 0.1 mPa over a distance of 100 µm between two rows of cilia. These results were derived analytically from the measured velocity profile by treating the cilia as a thin boundary layer. While the cilia produce phase-averaged velocities of the order of O(10(2)µm s(-1)), time-resolved measurements showed that the flow field reverses two times during one actuation cycle inducing instantaneous velocities of up to approximately 2 mm s(-1). This shows that the flow field is dominated by fluid oscillations and flow rates are expected to increase if the beating motion of the cilia is further improved.
Assuntos
Cílios/química , Magnetismo , Microfluídica/instrumentação , Cílios/fisiologia , Microfluídica/métodos , ViscosidadeRESUMO
In this paper we quantitatively analyse the performance of magnetically-driven artificial cilia for lab-on-a-chip applications. The artificial cilia are fabricated using thin polymer films with embedded magnetic nano-particles and their deformation is studied under different external magnetic fields and flows. A coupled magneto-mechanical solid-fluid model that accurately captures the interaction between the magnetic field, cilia and fluid is used to simulate the cilia motion. The elastic and magnetic properties of the cilia are obtained by fitting the results of the computational model to the experimental data. The performance of the artificial cilia with a non-uniform cross-section is characterised using the numerical model for two channel configurations that are of practical importance: an open-loop and a closed-loop channel. We predict that the flow and pressure head generated by the artificial cilia can be as high as 18 microlitres per minute and 3 mm of water, respectively. We also study the effect of metachronal waves on the flow generated and show that the fluid propelled increases drastically compared to synchronously beating cilia, and is unidirectional. This increase is significant even when the phase difference between adjacent cilia is small. The obtained results provide guidelines for the optimal design of magnetically-driven artificial cilia for microfluidic propulsion.
Assuntos
Cílios/química , Magnetismo , Técnicas Analíticas Microfluídicas/instrumentação , Simulação por Computador , ViscosidadeRESUMO
Mutational analysis of oncogenes is critical for our understanding of cancer development. Oncogenome screening has identified a fibroblast growth factor receptor 4 (FGFR4) Y367C mutation in the human breast cancer cell line MDA-MB453. Here, we investigate the consequence of this missense mutation in cancer cells. We show that MDA-MB453 cells harbouring the mutation are insensitive to FGFR4-specific ligand stimulation or inhibition with an antagonistic antibody. Furthermore, the FGFR4 mutant elicits constitutive phosphorylation leading to an activation of the mitogen-activated protein kinase cascade as shown by an enhanced Erk1/2 phosphorylation. Cloning and ectopic expression of the FGFR4 Y367C mutant in HEK293 cells revealed high pErk levels and enhanced cell proliferation. Based on these findings, we propose that FGFR4 may be a driver of tumour growth, particularly when highly expressed or stabilized and constitutively activated through genetic alterations. As such, FGFR4 presents an option for further mutational screening in tumours and is an attractive cancer target with the therapeutic potential.
Assuntos
Regulação Neoplásica da Expressão Gênica , Mutação de Sentido Incorreto , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Genes Dominantes , Humanos , Immunoblotting , Camundongos , Células NIH 3T3 , Fosforilação/efeitos dos fármacos , Interferência de RNA , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaAssuntos
Negro ou Afro-Americano , Processos Grupais , Liderança , Comportamento Verbal , Adulto , Humanos , Masculino , Meio SocialRESUMO
The diffusion of dilute colloids in contact with swollen polymer brushes has been studied by evanescent wave dynamic light scattering. Two polystyrene nanogels with 16 nm and 42 nm radius were put into contact with three polystyrene brushes with varying grafting densities. Partial penetration of the nanogels within the brushes was revealed by the evanescent wave penetration depth-dependent scattering intensities. The experimental short-time diffusion coefficients of the penetrating particles were measured and found to strongly slow down as the nanoparticles get deeper into the brushes. The slow down is much more marked for the smaller (16 nm) nanogels, suggesting a size exclusion type of mechanism and the existence of a characteristic length scale present in the outer part of the brush.
RESUMO
In an effort to control particle diffusion near surfaces, we have studied the dynamics of colloidal hard spheres and soft compliant star copolymers on surfaces coated with polymer brushes using evanescent wave dynamic light scattering. The same experiments provide information on the brush structure and confined particle motion. The penetration into dense polydisperse brushes is size- and solvent-dependent.
Assuntos
Poliestirenos/química , Difusão , Tamanho da Partícula , Propriedades de Superfície , Fatores de TempoRESUMO
We present a novel concept to process human blood on a spinning polymer disk for the determination of the hematocrit level by simple visual inspection. The microfluidic disk which is spun by a macroscopic drive unit features an upstream metering structure and a downstream blind channel where the centrifugally enforced sedimentation of the blood is performed. The bubble-free priming of the blind channel is governed by centrifugally assisted capillary filling along the sloped hydrophilic side-wall and the lid as well as the special shape of the dead end of the two-layer channel. The hematocrit is indicated at the sharp phase boundary between the plasma and the segregated cellular pellet on a disk-imprinted calibrated scale. This way, we conduct the hematocrit determination of human blood within 5 min at a high degree of linearity (R(2) = 0.999) and at a high accuracy (CV = 4.7%) spanning over the physiological to pathological working range. As all processing steps including the priming, the metering to a defined volume as well as the centrifugation are executed automatically during rotation, the concept is successfully demonstrated in a conventional PC-CDROM drive while delivering the same performance (R(2) = 0.999, CV = 4.3%).
Assuntos
Centrifugação/instrumentação , Hematócrito/instrumentação , Microfluídica/instrumentação , Centrifugação/métodos , Análise Custo-Benefício , Desenho de Equipamento , Análise de Falha de Equipamento , Alemanha , Hematócrito/métodos , Microfluídica/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A case of a total knee arthroplasty infection with Actinomyces naeslundii is described. The difficulties of therapeutic decision-making are emphasized.
Assuntos
Actinomicose/diagnóstico , Artrite Infecciosa/microbiologia , Artroplastia do Joelho , Actinomyces/isolamento & purificação , Actinomicose/tratamento farmacológico , Actinomicose/cirurgia , Artrite Infecciosa/diagnóstico , Artrite Infecciosa/tratamento farmacológico , Artrite Infecciosa/cirurgia , Feminino , Humanos , Pessoa de Meia-Idade , ReoperaçãoRESUMO
We have investigated the electrolyte-induced collapse of a polyelectrolyte brush covalently attached to a planar solid surface. Positively charged poly-4-vinyl [N-methyl-pyridinium] (MePVP) brushes were prepared in situ at the surface by free radical chain polymerization using a surface-immobilized initiator monolayer ("grafting from" technique) and 4-vinylpyridine as the monomer, followed by a polymer-analogous quaternization reaction. The height of the brushes was measured as a function of the external salt concentration via multiple-angle null ellipsometry. As predicted by mean-field theory, the height of the MePVP brushes remains unaffected by the addition of low amounts of external salt. At higher salt concentrations the brush height decreases. The extent to which the brush shrinks strongly depends on the nature of the salt present in the environment. MePVP brushes collapse to almost the dry layer thickness upon the addition of potassium iodide to a contacting aqueous medium. In contrast, the collapse of MePVP brushes having bromide or chloride counterions is much less pronounced. These brushes remain in a highly swollen state even after large amounts of salt have been added to the solution.