A simple device for multiplex ELISA made from melt-extruded plastic microcapillary film

We present a simple device for multiplex quantitative enzyme-linked immunosorbant assays (ELISA) made from a novel melt-extruded microcapillary film (MCF) containing a parallel array of 200µm capillaries along its length. To make ELISA devices different protein antigens or antibodies were immobilised inside individual microcapillaries within long reels of MCF extruded from fluorinated ethylene propylene (FEP). Short pieces of coated film were cut and interfaced with a pipette, allowing sequential uptake of samples and detection solutions into all capillaries from a reagent well. As well as being simple to produce, these FEP MCF devices have excellent light transmittance allowing direct optical interrogation of the capillaries for simple signal quantification. Proof of concept experiments demonstrate both quantitative and multiplex assays in FEP MCF devices using a standard direct ELISA procedure and read using a flatbed scanner. This new multiplex immunoassay platform should find applications ranging from lab detection to point-of-care and field diagnostics.

Using HIT to deliver integrated care for the frail elderly in the UK: current barriers and future challenges

In this paper we briefly describe the results of a 3 year project examining the use of Health Information Technologies (e.g., electronic patient record systems) to deliver integrated care. In particular, we focus on one group of patient (the frail elderly) and efforts to design an e-health supported healthcare pathway (the frail elderly pathway – FEP). The aim of FEP is to bring together clinicians and staff from health and social care and allow them to share patient information. Our findings show that progress in delivering a fully-supported and working FEP has been slow, not least because of the difficulties experienced by healthcare staff in using current IT systems. In addition, there are many strategic and technical issues which remain unresolved (e.g., systems interoperability).

Exposure of Escherichia coli and Salmonella enterica serovar Typhimurium to triclosan induces a species-specific response, including drug detoxification

Objectives: The use of triclosan within various environments has been linked to the development of multiple drug resistance (MDR) through the increased expression of efflux pumps such as AcrAB-ToIC. In this work, we investigate the effect of triclosan exposure in order to ascertain the response of two species to the presence of this widely used biocide. Methods: The transcriptomes of Salmonella enterica serovar Typhimurium SL1344 and Escherichia coli K-12 MG1655 after exposure to the MIC of triclosan (0.12 mg/L) were determined in microarray experiments. Phenotypic validation of the transcriptomic data included RT-PCR, ability to form a biofilm and motility assays. Results: Despite important differences in the triclosan-dependent transcriptomes of the two species, increased expression of efflux pump component genes was seen in both. Increased expression of soxS was observed in Salmonella Typhimurium, however, within E. coli, decreased expression was seen. Expression of fabBAGI in Salmonella Typhimurium was decreased, whereas in E. coli expression of fabABFH was increased. Increased expression of ompR and genes within this regulon (e.g. ompC, csgD and ssrA) was seen in the transcriptome of Salmonella Typhimurium. An unexpected response of E. coli was the differential expression of genes within operons involved in iron homeostasis; these included fhu, fep and ent. Conclusions: These data indicate that whilst a core response to triclosan exposure exists, the differential transcriptome of each species was different. This suggests that E. coli K-12 should not be considered the paradigm for the Enterobacteriaceae when exploring the effects of antimicrobial agents.

In-situ comparison of the NOy instruments flown in MOZAIC and SPURT

Two aircraft instruments for the measurement of total odd nitrogen (NOy) were compared side by side aboard a Learjet A35 in April 2003 during a campaign of the AFO2000 project SPURT (Spurengastransport in der Tropopausenregion). The instruments albeit employing the same measurement principle (gold converter and chemiluminescence) had different inlet configurations. The ECO-Physics instrument operated by ETH-Zürich in SPURT had the gold converter mounted outside the aircraft, whereas the instrument operated by FZ-Jülich in the European project MOZAIC III (Measurements of ozone, water vapour, carbon monoxide and nitrogen oxides aboard Airbus A340 in-service aircraft) employed a Rosemount probe with 80 cm of FEP-tubing connecting the inlet to the gold converter. The NOy concentrations during the flight ranged between 0.3 and 3 ppb. The two data sets were compared in a blind fashion and each team followed its normal operating procedures. On average, the measurements agreed within 7%, i.e. within the combined uncertainty of the two instruments. This puts an upper limit on potential losses of HNO3 in the Rosemount inlet of the MOZAIC instrument. Larger transient deviations were observed during periods after calibrations and when the aircraft entered the stratosphere. The time lag of the MOZAIC instrument observed in these instances is in accordance with the time constant of the MOZAIC inlet line determined in the laboratory for HNO3.

Culture bag systems for clinical applications of adult human neural crest-derived stem cells

Introduction Facing the challenging treatment of neurodegenerative diseases as well as complex craniofacial injuries such as those common after cancer therapy, the field of regenerative medicine increasingly relies on stem cell transplantation strategies. Here, neural crest-derived stem cells (NCSCs) offer many promising applications, although scale up of clinical-grade processes prior to potential transplantations is currently limiting. In this study, we aimed to establish a clinical-grade, cost-reducing cultivation system for NCSCs isolated from the adult human nose using cGMP-grade Afc-FEP bags. Methods We cultivated human neural crest-derived stem cells from inferior turbinate (ITSCs) in a cell culture bag system using Afc-FEP bags in human blood plasma-supplemented medium. Investigations of viability, proliferation and expression profile of bag-cultured ITSCs were followed by DNA-content and telomerase activity determination. Cultivated ITSCs were introduced to directed in vitro differentiation assays to assess their potential for mesodermal and ectodermal differentiation. Mesodermal differentiation was determined using an enzyme activity assay (alkaline phosphatase, ALP), respective stainings (Alizarin Red S, Von Kossa and Oil Red O), and RT-PCR, while immunocytochemistry and synaptic vesicle recycling were applied to assay neuroectodermal differentiation of ITSCs. Results When cultivated within Afc-FEP bags, ITSCs grew three-dimensionally in a human blood plasma-derived matrix, thereby showing unchanged morphology, proliferation capability, viability and expression profile in comparison to three dimensionally-cultured ITSCs growing in standard cell culture plastics. Genetic stability of bag-cultured ITSCs was further accompanied by unchanged telomerase activity. Importantly, ITSCs retained their potential to differentiate into mesodermal cell types, particularly including ALP-active, Alizarin Red S-, and Von Kossa-positive osteogenic cell types, as well as adipocytes positive in Oil Red O assays. Bag culture further did not affect the potential of ITSCs to undergo differentiation into neuroectodermal cell types coexpressing β-III-tubulin and MAP2 and exhibiting the capability for synaptic vesicle recycling. Conclusions Here, we report for the first time the successful cultivation of human NCSCs within cGMP-grade Afc-FEP bags using a human blood plasma-supplemented medium. Our findings particularly demonstrate the unchanged differentiation capability and genetic stability of the cultivated NCSCs, suggesting the great potential of this culture system for future medical applications in the field of regenerative medicine.

Estudo de resistência da bactéria Escherichia Coli a antibióticos em infecções urinárias na população da Região Autónoma da Madeira

As infecções do tracto urinário (ITU) são doenças infecciosas comuns e distinguem-se em dois grandes grupos: o das infecções do tracto urinário superior e o das infecções do tracto urinário inferior. O principal agente etiológico envolvido nas ITU é a bactéria Escherichia coli (E. coli), representando 80-95% dos invasores do tracto urinário na população em geral. É mais comum entre os adultos do sexo feminino, principalmente com actividade sexual, embora as crianças, mulheres grávidas e idosos também apresentam grande susceptibilidade. Paralelamente, a problemática da resistência aos antimicrobianos é um tema actual e muito debatido internacionalmente. Como tal, o presente estudo teve como principal objectivo contribuir para a identificação fiável da bactéria E. coli em infecções urinárias bem como a determinação do seu perfil de resistência a antibióticos. Os dados apresentados referem-se a um estudo efectuado entre Outubro de 2008 e Junho de 2009 no Laboratório de Análises Clínicas Castro Fernandes – MMC. Foram analisadas 6522 amostras de urina, das quais 390 corresponderam a uroculturas positivas. Verificou-se que o sexo feminino tem uma maior propensão para infecções urinárias, havendo uma tendência para o aumento das ITU com a idade. Na interpretação da cultura revelou-se importante a informação dada pelo exame cultural acerca do número de leucócitos. Concluiu-se que nas uroculturas cujo número de colónias foi superior a 105 com menos de três espécies de colónias diferentes na cultura, o número de leucócitos foi superior a 30 milhares de células por μL, o que sugere a existência de infecção urinária. No estudo do perfil de sensibilidade da bactéria E. coli isolada, para a família dos betalactâmicos (Ampicilina (AMP), Amoxicilina/Ácido Clavulânico (AMC), Piperacilina/Tazobactam (PIP/TZP)), família das cefalosporinas (Cefalotina (KF), Cefuroxima (CXM), Cefuroxima Acetil (CA), Cefotaxima (CTX), Ceftazidima (CAZ), Cefepima (FEP)), família dos aminoglicosídeos (Amicacina (AM), Gentamicina (GM), Tobramicina (TOB)) e para as nitrofurantoínas (F), verificou-se uma grande sensibilidade e pouca resistência em termos quantitativos, mas para a família das quinolonas (Ciprofloxacina (CIP), Levofloxacina (LEV), Norfloxacina (NOR)) e para os Trimetoprim/Sulfametoxazol (TMP/SMX) verificou-se resistências quantitativamente superiores aos das famílias anteriormente referidas, sugerindo valores convergentes aos da tendência europeia. Foram identificados famílias de fenótipos tais como: os beta-lactâmicos, os aminoglicosídeos, quinolonas, tetraciclinas, furanos e trimetoprim/sulfamidas.E. coli é um microganismo cujo fenótipo selvagem não tem resistências intrínsecas. Todas as resistências são adquiridas. Actualmente, a realização dos antibiogramas para E. coli tornou-se fundamental tendo em vista os factores ecológicos, genéticos, ambientais e pelo facto de que mais isolados são resistentes à maioria dos antibióticos. Como conclusão, este estudo permitirá alertar para um problema sério que é uma grande ameaça à Saúde Pública.

Stability of atomic condensed systems with attractive two-body interactions

In the present report, we review recent investigations that we have conducted on the stability of atomic condensed systems, when the two-body interaction is attractive. In particular, the dynamics that occurs in the condensate due to nonconservative terms is considered in the context of an extension of the mean-field Gross-Pitaevskii approximation. Considering the relative intensity of the nonconservative parameters, chaotic and solitonic solutions are verified. Also discussed is the possibility of a liquid-gas phase transition in the presence of positive three-body elastic collisions.

Uso de esterco associado à adubação mineral na produção de mudas de mamoeiro (Carica papaya L.)

Na composição do substrato para produção de mudas de mamoeiro, recomenda-se o uso de adubação orgânica, que traz como vantagens a melhoria das características físicas, químicas e biológicas do solo, criando um ambiente favorável ao desenvolvimento inicial das mudas, bem como boa resposta do mamoeiro. Os materiais orgânicos desempenham ainda um importante papel na nutrição das plantas como fornecedores de nutrientes. Contudo, observa-se que existe uma grande variação nas recomendações e quase sempre a adubação orgânica está associada à adubação mineral. Neste contexto, o objetivo deste trabalho foi o de verificar os efeitos do uso de esterco de curral associado ou não à adubação mineral no substrato sobre a formação de mudas de mamoeiro. O Experimento foi conduzido na FEP/UNESP/Câmpus de Ilha Solteira-SP. O delineamento utilizado foi em blocos, em esquema de parcelas subdivididas, sendo parcelas as cultivares e subparcelas os substratos. Cada parcela com 5 repetições e 10 plantas úteis por subparcela. Para a comparação das médias, utilizou-se o teste de Tukey. Com base nos resultados obtidos, pode-se concluir que: a) Na formação das mudas de mamoeiro, o esterco de curral pode ser utilizado sem a necessidade de adubação mineral com o superfosfato simples e o cloreto de potássio; b) O esterco de curral foi capaz de fornecer às mudas de mamoeiro os nutrientes N, P, K, Ca, Mg e Cu, necessários para seu desenvolvimento até o transplantio para o campo.

Manejo químico das plantas daninhas Euphorbia heterophylla e Bidens pilosa em sistema de plantio direto da cultura de soja

Objetivou-se neste trabalho avaliar a eficácia agronômica e os efeitos fitotóxicos de herbicidas aplicados em pré-plantio no manejo de Euphorbia heterophylla e Bidens pilosa, em sistema de plantio direto da cultura de soja. O experimento foi desenvolvido na safra 2000/2001, na FEP-UNESP, no município de Selvíria-MS. A variedade de soja utilizada foi a 'Conquista', e o delineamento experimental adotado foi o de blocos ao acaso, com sete tratamentos e quatro repetições, com oito linhas da cultura, totalizando 20 m² cada parcela. Os tratamentos foram os seguintes: glyphosate (720 e 960 g ha-1), chlorimuron-ethyl + glyphosate (10 g + 720 e 960 g ha-1), glyphosate + 2,4-D (960 + 868 g ha-1) e testemunhas no mato e no limpo. As aplicações foram realizadas com pulverizador de pressão constante (CO2) de 45 lb pol-2, com barra equipada com quatro bicos do tipo leque Teejet 110.03 XR, espaçados de 0,5 m, e volume de calda de 250 L ha-1. Concluiu-se que os tratamentos testados são eficientes na dessecação de E. heterophylla e B. pilosa e seletivos às plantas de soja. O herbicida chlorimuron-ethyl (10 g ha-1) aplicado juntamente com o glyphosate, no manejo, proporciona ao tratamento um efeito residual significativo para E. heterophylla e B. pilosa, reduzindo a infestação das plantas daninhas durante o ciclo da cultura da soja.

Characterization and catalytic activity of iron(III) mono(4-N-methyl pyridyl)-tris(halophenyl) porphyrins in homogeneous and heterogeneous systems

The synthesis, characterization and catalytic activity of the cationic iron porphyrins Fe[M(4-N-MePy)TDCPP]Cl-2 and Fe[M(4-N-MePy)TFPP]Cl-2 in the epoxidation of (Z)-cyclooctene by PhIO in homogeneous solution and supported on silica gel (SG), imidazole propyl gel (IPG) or SG modified with 2-(4-sulfonatophenyl)ethyl groups (SiSO3) have been accomplished. When supported on IPG, both cationic FeP bind to the support via Fe-imidazole coordination. Fe[M(4-N-MePy)TDCPP]IPG contains a mixture of low-spin bis-coordinated (FeP)-P-III and high-spin mono-coordinated (FeP)-P-III species, whereas Fe[M(4-N-MePy)TFPP]IPG only contains high-spin mono-coordinated (FeP)-P-III. These FePIPG catalysts also contain (FeP)-P-II species, whose presence was confirmed by EPR spectroscopy using NO as a paramagnetic probe. Both cationic FePs coordinate to SG through Fe-O ligation and they are present as high-spin (FeP)-P-III species. The cationic FePs supported on SiSO3- are also high-spin (FeP)-P-III species and they bind to the support via electrostatic interaction between the 4-N-methylpyridyl groups and the SO3- groups present on the matrix. In homogeneous solution, both Fe[M(4-N-MePy)TDCPP]Cl-2 and Fe[M(4-N-MePy)TFPP]Cl-2 have similar catalytic activity to Fe(TDCPP)Cl and Fe(TFPP)Cl, leading to cis-epoxycyclooctane yields of 92%. When supported on inorganic matrices,both FePs lead to epoxide yields comparable to their homogeneous analogues and their anchoring enables catalyst recovery and re-use. Recycling of Fe[M(4-N-MePy)TDCPP]SiSO3- shows that this FeP maintains its activity in a second reaction. (C) 1999 Elsevier B.V. B.V. All rights reserved.

Factors which affect the catalytic activity of iron(III) meso tetrakis(2,6-dichlorophenyl)porphyrin chloride in homogeneous system

An optimization study of the reaction conditions of Fe(TDCPP)Cl when it is used as catalyst in the hydroxylation of cyclohexane by iodosylbenzene (PhIO) has been carried out, It was found that Fe(TDCPP)Cl follows the classical PhIO mechanism described for Fe(TPP)Cl, which involves the monomeric active species Fe-IV(O)P-+. (I). In the optimized condition ([Fe(TDCPP) = 3.0 X 10(-4) mol l(-1) in 1,2-dichloroethane (DCE); ultrasound stirring at 0 degrees C; PhIO/FeP molar ratio = 100), this FeP led to a yield of cyclohexanol (C-ol) of 96% and a turnover number of 96, Therefore, Fe(TDCPP)Cl may be considered a good biomimetic model and a very stable, resistant and selective catalyst, which yields C-ol as the sole product. DCE showed to be a better solvent than dichloromethane (DCM), 1 DCE:1 MeOH mixture or acetonitrile (ACN). Since the Fe-IV(O)P-+. is capable of abstracting hydrogen atom from DCM, MeOH or ACN, the solvent competes with the substrate. Presence of O-2 lowers the yield of C-ol, as it can further oxidize this alcohol to carboxylic acid in the presence of radicals, Presence of H2O also causes a decrease in the yield, since it converts the active species I into Fe-IV(OH)P, which cannot oxidize cyclohexane. Addition of excess imidazole or OH- to the system results in a decrease in the yield of C-ol, due to the formation of the hexacoordinated complexes Fe(TDCPP)Im(2)(+) (low-spin, beta(2) = 2.5 X 10(8) mol(-2) l(2)) and Fe(TDCPP)(OH)(2)(-) (high-spin, beta(2) = 6.3 X 10(7) mol(-2) l(2)), the formation of both Fe(TDCPP)Im(2)(+) and Fe(TDCPP)(OH)(2)(-) complexes were confirmed by EPR studies. The catalytic activities of Fe(TDCPP)C and Fe(TFPP)Cl were compared, the unusually high yields of C-ol with Fe(TFPP)Cl obtained when ultrasound, DCM and O-2 atmosphere were used, suggest that a parallel mechanism involving the mu-oxo dimer form, O-2 and radicals may also be occurring with this FeP, besides the PhIO mechanism.

Characterization and catalytic activity of 2,6-dichlorophenyl substituted iron(III) porphyrin supported on silica gel and imidazole propyl gel

In this work we have made use of the study of the interaction between Fe(TDCPP)(+) and the axial ligands OH- and imidazole in order to help characterize the heterogenized catalysts Fe(TDCPP)SG and Fe(TDCPP)IPG through UV-VIS and EPR spectroscopies and thus, better understand their different catalytic activity in the oxidation of cyclohexane by PhIO. We have found out that in Fe(TDCPP)SG (containing 1.2 X 10(-6) mol Fe(TDCPP)(+)/g of support), the FeP bis-coordinates to silica gel through Fe-O coordination and it is high-spin (FeP)-P-III species. In Fe(TDCPP)IPG 1 (containing 1.1 X 10(-6) mol Fe(TDCPP)(+) and 2.2 X 10(-4) mol imidazole/g of support), the FeP is bis-ligated to imidazole propyl gel through Fe-imidazole coordination and using NO as a paramagnetic probe, we present evidence that Fe(TDCPP)(+) is present as a mixture of low-spin (FeP)-P-III and (FeP)-P-II species. This catalyst led to a relative low yield of cyclohexanol (25%) because the bis-coordination of the (FeP)-P-III to the support partially blocks the reaction between Fe(TDCPP)(+) and PhIO, thus leading to the formation of only a small amount of the active species Fe-IV(OP+, while the (FeP)-P-II species do not react with the oxygen donor. Increasing the amount of Fe(TDCPP)(+) and decreasing the amount of imidazole in the support led to the obtention of high-spin (FeP)-P-III EPR signals in the spectra of Fe(TDCPP)IPG 5 (containing 4.4 X 10(-6) mol Fe(TDCPP)(+) and 2.2 X 10(-5) mol imidazole/g of IPG), together with low-spin (FeP)-P-III species. This latter catalyst led to better cyclohexanol yields (67%) than Fe(TDCPP)IPG 1. Fe(TDCPP)IPG 5 was further used in a study of the optimization of its catalytic activity and in recycling experiments in the optimized conditions. Recycling oxidation reactions of Fe(TDCPP)IPG 5 led to a total turnover number of 201 and total cyclohexanol yield of 201%, which could not be attained with Fe(TDCPP)Cl in homogeneous solution (turnover = 96) due to the difficulty in recovering and reusing it.

Study of the catalytical intermediates of metalloporphyrins supported on imidazole propyl gel

In this work, the catalytic intermediates for Fe(TPP)(+), Fe(TDCPP)(+), Fe(TFPP)(+), Mn(TPP)(+) and Mn(TDCPP)(+) supported on imidazole propyl gel with PhIO were studied by UV-Vis spectrophotometry. For Fe(TPP)+ and Fe(TFPP)+ the study was also monitored by EPR spectroscopy. The active catalytic intermediate observed for FeP-IPG is the ore-iron (IV) porphyrin pi cation radical Fe-IV(O)P.+, which is evidenced by a decrease in the intensity of the Sorer band. The total re-establishment of the initial Soret band intensity for Fe(TDCPP)IPG and Fe(TFPP)IPG at the end of the reaction shows that they were completely recovered, There are advantages in following the reactions of PNO with unsubstituted Fe(TPP)(+) and Mn(TPP)(+) on IPG by UV-Vis, since they were slower and allowed to 'see' the intermediate species without spectral interference from the recovered catalyst, since they are only partially recovered. With Fe(TPP)IPG, a band at 580 nm was detected at the beginning of the reaction, indicating the possible formation of a Fe-OIPh intermediate. Supporting Mn(TPP)(+) on IPG leads to a shift of band V from 478 nm to 488 nm. In the reaction of MnP-IPG with PhIO, we observed the disappearance of the band in 488 nm and the appearance of a band in 412 nm, which corresponds to the active catalytic intermediate Mn-V(O)P as the main component, as is expected for a more efficient system. The recovery of supported catalysts observed in these experiments was further proved with the possibility of their successive recyclings in cyclohexane oxidation reactions by PhIO.

Characterization of iron(III)porphyrin-hydroxo complexes in organic media through UV-Vis and EPR spectroscopies

The interaction of OH- with Fe(TPP)(+), Fe(TDCPP)(+), Fe(TMP)(+) and Fe(TFPP)(+) in 1,2-dichloroethane was studied by titrating FeP solutions with aliquots of a solution of tetrabutylammonium hydroxide in acetonitrile. The number of OH- ions (n) coordinated to the FeP and the stability constants (beta(n)) for the FeP-OH- complexes were calculated from UV-Vis absorbance data and iron spin states were determined through EPR spectroscopy, Fe(TMP) (+) forms a high-spin mono-hydroxo complex, while Fe(TPP)I and Fe(TDCPP)(+) form high-spin bis-hydroxo complexes. To our knowledge, this is the first time that the formation of bis-hydroxo complexes from Fe(TPP) (+) has been reported, and this was possible because the studies were carried out in basic organic media, In this same medium, Fe-III-Fe-II reduction upon OH- addition to Fe(TFPP) (+) was observed, without concomitant formation of the mu-oxo dimeric species [Fe(TFPP)](2)O. (C) 1999 Elsevier B.V., All rights reserved.

Using shifts in the electronic emission curve to evaluate polymer surface degradation

The polymer surface degradation and/or modification evolution of Teflon FEP and Mylar C films caused by a low energy electron beam were analyzed using a new method that consists in measuring the second crossover energy shift in the electronic emission curve. Upon prolonged irradiation, the second crossover energy shifts irreversibly to lower values in Teflon FEP but to higher values in Mylar C, indicating distinct mechanisms of surface degradation for the two polymers. The method represents a relatively inexpensive way to monitor early stages of surface degradation since the secondary electron emission comes from a maximum depth below the geometric surface of 100 mn in insulators. (C) 2001 Elsevier B.V. Ltd. All rights reserved.