Dynamic high-resolution computer simulation of isotachophoretic enantiomer separation and zone stability

The development of electrophoretic computer models and their use for simulation of electrophoretic processes has increased significantly during the last few years. Recently, GENTRANS and SIMUL5 were extended with algorithms that describe chemical equilibria between solutes and a buffer additive in a fast 1:1 interaction process, an approach that enables simulation of the electrophoretic separation of enantiomers. For acidic cationic systems with sodium and H3 0(+) as leading and terminating components, respectively, acetic acid as counter component, charged weak bases as samples, and a neutral CD as chiral selector, the new codes were used to investigate the dynamics of isotachophoretic adjustment of enantiomers, enantiomer separation, boundaries between enantiomers and between an enantiomer and a buffer constituent of like charge, and zone stability. The impact of leader pH, selector concentration, free mobility of the weak base, mobilities of the formed complexes and complexation constants could thereby be elucidated. For selected examples with methadone enantiomers as analytes and (2-hydroxypropyl)-β-CD as selector, simulated zone patterns were found to compare well with those monitored experimentally in capillary setups with two conductivity detectors or an absorbance and a conductivity detector. Simulation represents an elegant way to provide insight into the formation of isotachophoretic boundaries and zone stability in presence of complexation equilibria in a hitherto inaccessible way.

Electrophoretically mediated microanalysis for characterization of the enantioselective CYP3A4 catalyzed N-demethylation of ketamine

Execution of an enzymatic reaction performed in a capillary with subsequent electrophoretic analysis of the formed products is referred to as electrophoretically mediated microanalysis (EMMA). An EMMA method was developed to investigate the stereoselectivity of the CYP3A4-mediated N-demethylation of ketamine. Ketamine was incubated in a 50 μm id bare fused-silica capillary together with human CYP3A4 Supersomes using a 100 mM phosphate buffer (pH 7.4) at 37°C. A plug containing racemic ketamine and the NADPH regenerating system including all required cofactors for the enzymatic reaction was injected, followed by a plug of the metabolizing enzyme CYP3A4 (500 nM). These two plugs were bracketed by plugs of incubation buffer to ensure proper conditions for the enzymatic reaction. The rest of the capillary was filled with a pH 2.5 running buffer comprising 50 mM Tris, phosphoric acid, and 2% w/v of highly sulfated γ-cyclodextrin. Mixing of reaction plugs was enhanced via application of -10 kV for 10 s. After an incubation of 8 min at 37°C without power application (zero-potential amplification), the capillary was cooled to 25°C within 3 min followed by application of -10 kV for the separation and detection of the formed enantiomers of norketamine. Norketamine formation rates were fitted to the Michaelis-Menten model and the elucidated values for V(max) and K(m) were found to be comparable to those obtained from the off-line assay of a previous study.

Dynamic high-resolution computer simulation of electrophoretic enantiomer separations with neutral cyclodextrins as chiral selectors.

GENTRANS, a comprehensive one-dimensional dynamic simulator for electrophoretic separations and transport, was extended for handling electrokinetic chiral separations with a neutral ligand. The code can be employed to study the 1:1 interaction of monovalent weak and strong acids and bases with a single monovalent weak or strong acid or base additive, including a neutral cyclodextrin, under real experimental conditions. It is a tool to investigate the dynamics of chiral separations and to provide insight into the buffer systems used in chiral capillary zone electrophoresis (CZE) and chiral isotachophoresis. Analyte stacking across conductivity and buffer additive gradients, changes of additive concentration, buffer component concentration, pH, and conductivity across migrating sample zones and peaks, and the formation and migration of system peaks can thereby be investigated in a hitherto inaccessible way. For model systems with charged weak bases and neutral modified β-cyclodextrins at acidic pH, for which complexation constants, ionic mobilities, and mobilities of selector-analyte complexes have been determined by CZE, simulated and experimentally determined electropherograms and isotachopherograms are shown to be in good agreement. Simulation data reveal that CZE separations of cationic enantiomers performed in phosphate buffers at low pH occur behind a fast cationic migrating system peak that has a small impact on the buffer composition under which enantiomeric separation takes place.

Prognostic significance of foveal capillary drop-out and previous panretinal photocoagulation for diabetic macular oedema treated with ranibizumab

AIMS To investigate the prognostic significance of macular capillary drop-out and previous panretinal laser photocoagulation in diabetic macular oedema treated with intravitreal ranibizumab. METHODS Retrospective observational case series. Treatment-naive patients with diabetic macular oedema that had been treated with intravitreal ranibizumab as per the RESTORE study protocol for at least 12 months were included. Some patients (n=15) had previous panretinal laser photocoagulation. Best-corrected visual acuity and central retina thickness were recorded monthly. The foveal avascular zone and the perifoveal capillaries were quantitatively and qualitatively assessed on fluorescein angiography on two occasions during the observational period. RESULTS From the 46 eyes (46 patients) in this study, 13 (28%) had evidence of perifoveal capillary drop-out. Central retinal thickness was significantly thinner at baseline (p=0.02) and throughout the study period in these eyes compared with those with normal perifoveal capillaries. Both groups responded with a significant gain of best-corrected visual acuity to ranibizumab treatment (7.6±3.3 and 6.3±1.3 ETDRS letters, respectively). Eyes with previous panretinal laser photocoagulation displayed a comparable final outcome regarding function and morphology, requiring a similar intensity of intravitreal injections. CONCLUSIONS Perifoveal capillary drop-out did not limit the gain of visual acuity from intravitreal ranibizumab treatment. The reduction of central retina thickness was similar to that seen in eyes with normal perifoveal capillaries. Central retinal thickness in eyes with perifoveal capillary drop-out was generally reduced. However, this did not affect their benefit from treatment. Ranibizumab did not increase the amount of perifoveal capillary loss.

High depth-resolution laser ablation chemical analysis of additive-assisted Cu electroplating for microchip architectures

Laser ablation/ionisation mass spectrometry with a vertical resolution at a nanometre scale was applied for the quantitative characterisation of the chemical composition of additive-assisted Cu electroplated deposits used in the microchip industry. The detailed chemical analysis complements information gathered by optical techniques and allows new insights into the metal deposition process.

Capillary growth, ultrastructure remodelling and exercise training in skeletal muscle of essential hypertensive patients

AIM The aim was to elucidate whether essential hypertension is associated with altered capillary morphology and density and to what extent exercise training can normalize these parameters. METHODS To investigate angiogenesis and capillary morphology in essential hypertension, muscle biopsies were obtained from m. vastus lateralis in subjects with essential hypertension (n = 10) and normotensive controls (n = 11) before and after 8 weeks of aerobic exercise training. Morphometry was performed after transmission electron microscopy, and protein levels of several angioregulatory factors were determined. RESULTS At baseline, capillary density and capillary-to-fibre ratio were not different between the two groups. However, the hypertensive subjects had 9% lower capillary area (12.7 ± 0.4 vs. 13.9 ± 0.2 μm(2)) and tended to have thicker capillary basement membranes (399 ± 16 vs. 358 ± 13 nm; P = 0.094) than controls. Protein expression of vascular endothelial growth factor (VEGF), VEGF receptor-2 and thrombospondin-1 were similar in normotensive and hypertensive subjects, but tissue inhibitor of matrix metalloproteinase was 69% lower in the hypertensive group. After training, angiogenesis was evident by 15% increased capillary-to-fibre ratio in the hypertensive subjects only. Capillary area and capillary lumen area were increased by 7 and 15% in the hypertensive patients, whereas capillary basement membrane thickness was decreased by 17% (P < 0.05). VEGF expression after training was increased in both groups, whereas VEGF receptor-2 was decreased by 25% in the hypertensive patients(P < 0.05). CONCLUSION Essential hypertension is associated with decreased lumen area and a tendency for increased basement membrane thickening in capillaries of skeletal muscle. Exercise training may improve the diffusion conditions in essential hypertension by altering capillary structure and capillary number.

Hematology, serum chemistry, and serum protein electrophoresis ranges for free-ranging roe deer (Capreolus capreolus) in Sweden.

We present the first reference ranges for hematology (n = 35 animals), serum biochemistry (n = 62), and serum protein electrophoresis (n = 32) in physically restrained free-ranging roe deer (Capreolus capreolus). Animals were captured in box traps and physically restrained for blood sampling during the winter in Sweden, 2011-13. No clinically significant sex or age differences were found.

Impact of Taylor-Aris diffusivity on analyte and system zone dispersion in CZE assessed by computer simulation and experimental validation.

Application of pressure-driven laminar flow has an impact on zone and boundary dispersion in open tubular CE. The GENTRANS dynamic simulator for electrophoresis was extended with Taylor-Aris diffusivity which accounts for dispersion due to the parabolic flow profile associated with pressure-driven flow. Effective diffusivity of analyte and system zones as functions of the capillary diameter and the amount of flow in comparison to molecular diffusion alone were studied for configurations with concomitant action of imposed hydrodynamic flow and electroosmosis. For selected examples under realistic experimental conditions, simulation data are compared with those monitored experimentally using modular CE setups featuring both capacitively coupled contactless conductivity and UV absorbance detection along a 50 μm id fused-silica capillary of 90 cm total length. The data presented indicate that inclusion of flow profile based Taylor-Aris diffusivity provides realistic simulation data for analyte and system peaks, particularly those monitored in CE with conductivity detection.

STUDIES ON THE PATTERNS OF NUCLEOTIDE AND AMINO ACID SUBSTITUTION (ELECTROPHORESIS, MUTATION RATE, SELECTION, BASE, COMPOSITION)

Theoretical and empirical studies were conducted on the pattern of nucleotide and amino acid substitution in evolution, taking into account the effects of mutation at the nucleotide level and purifying selection at the amino acid level. A theoretical model for predicting the evolutionary change in electrophoretic mobility of a protein was also developed by using information on the pattern of amino acid substitution. The specific problems studied and the main results obtained are as follows: (1) Estimation of the pattern of nucleotide substitution in DNA nuclear genomes. The pattern of point mutations and nucleotide substitutions among the four different nucleotides are inferred from the evolutionary changes of pseudogenes and functional genes, respectively. Both patterns are non-random, the rate of change varying considerably with nucleotide pair, and that in both cases transitions occur somewhat more frequently than transversions. In protein evolution, substitution occurs more often between amino acids with similar physico-chemical properties than between dissimilar amino acids. (2) Estimation of the pattern of nucleotide substitution in RNA genomes. The majority of mutations in retroviruses accumulate at the reverse transcription stage. Selection at the amino acid level is very weak, and almost non-existent between synonymous codons. The pattern of mutation is very different from that in DNA genomes. Nevertheless, the pattern of purifying selection at the amino acid level is similar to that in DNA genomes, although selection intensity is much weaker. (3) Evaluation of the determinants of molecular evolutionary rates in protein-coding genes. Based on rates of nucleotide substitution for mammalian genes, the rate of amino acid substitution of a protein is determined by its amino acid composition. The content of glycine is shown to correlate strongly and negatively with the rate of substitution. Empirical formulae, called indices of mutability, are developed in order to predict the rate of molecular evolution of a protein from data on its amino acid sequence. (4) Studies on the evolutionary patterns of electrophoretic mobility of proteins. A theoretical model was constructed that predicts the electric charge of a protein at any given pH and its isoelectric point from data on its primary and quaternary structures. Using this model, the evolutionary change in electrophoretic mobilities of different proteins and the expected amount of electrophoretically hidden genetic variation were studied. In the absence of selection for the pI value, proteins will on the average evolve toward a mildly basic pI. (Abstract shortened with permission of author.) ^

Use of pulsed-field gel electrophoresis in surveillance of Salmonella in Houston, Texas

Background. Pulsed-field gel electrophoresis (PFGE) is a laboratory technique in which Salmonella DNA banding patterns are used as molecular fingerprints for epidemiologic study for "PFGE clusters". State and national health departments (CDC) use PFGE to detect clusters of related cases and to discover common sources of bacteria in outbreaks. ^ Objectives. Using Houston Department of Health and Human Services (HDHHS) data, the study sought: (1) to describe the epidemiology of Salmonella in Houston, with PFGE subtype as a variable; and (2) to determine whether PFGE patterns and clusters detected in Houston were local appearances of PFGE patterns or clusters that occurred statewide. ^ Methods. During the years 2002 to 2005, the HDHHS collected and analyzed data from routine surveillance of Salmonella. We implemented a protocol, between May 1, 2007 and December 31, 2007, in which PFGE patterns from local cases were sent via e-mail to the Texas Department of State Health Services, to verify whether the local PFGE patterns were also part of statewide clusters. PFGE was performed from 106 patients providing a sample from which Salmonella was isolated in that time period. Local PFGE clusters were investigated, with the enhanced picture obtained by linking local PFGE patterns to PFGE patterns at the state and national level. ^ Results. We found that, during the years 2002 to 2005, there were 66 PFGE clusters, ranging in size from 2 to 22 patients within each cluster. Between different serotypes, there were marked differences in the sizes of PFGE clusters. A common source or risk factor was found in fewer than 5 of the 66 PFGE clusters. With the revised protocol, we found that 19 of 66 local PFGE patterns were indistinguishable from PFGE patterns at Texas DSHS. During the eight months, we identified ten local PFGE clusters with a total of 42 patients. The PFGE pattern for eight of the ten clusters matched the PFGE patterns for cases reported to Texas DSHS from other geographic areas. Five of the ten PFGE patterns matched PFGE patterns for clusters under investigation at PulseNet at the national level. HDHHS epidemiologists identified a mode of transmission in two of the ten local clusters and a common risk factor in a third local cluster. ^ Conclusion. In the extended-study protocol, Houston PFGE patterns were linked to patterns seen at the state and national level. The investigation of PFGE clusters was more efficacious in detecting a common transmission when local data were linked to state and national data. ^

Entorno histórico y social de la aparición del microchip

El siguiente proyecto es un desarrollo histórico-científico acerca de la notoria importancia que supuso la aparición del microchip o circuito integrado1. El desarrollo de este trabajo ha sido una investigación bibliográfica en contenidos webs, enciclopedias y libros. El trabajo contiene un estudio sobre los transistores que fue el componente que dio paso al circuito integrado además de ser uno de los mayores inventos del siglo XX, además, se propone una pequeña inmersión a la época histórica del momento de la aparición del transistor. Al igual que con el transistor, se hace un estudio acerca del circuito integrado, pero en este caso siendo más extenso ya que es el objeto de estudio de este PFC. Para este componente sí que podemos encontrar una explicación más exhaustiva acerca de su fabricación, materiales. Además también podemos encontrar el momento históricosocial de la época bajo estudio. Para finalizar con el proyecto, se hace un breve repaso de los ejemplos de aplicación del circuito integrado y así poder hacer hincapié de la revolución tecnológica que supuso el descubrimiento del microchip. ABSTRACT. The following work is a historical and scientific development regarding the fundamental importance the emergence of the microchip. The development of this work has consisted of a bibliographic research of web contents, encyclopedias and books. The paper contains a study about the transistors, component that propitiated the integrated circuit and was one of the most important inventions of the XXth century. Also is proposed a short historical immersion in the time that preceded the coming of the transistor. As well as with the transistor, a study of the integrated circuit is carried out, yet with deeper insight, for that is the central aim of this Final Project report. For this component a more exhaustive explanation of its manufacture process, materials and theories can be provided. Also, the historical and social of that time is described. To complete the report, a brief review is done about examples of applications of the integrated circuit and thus highlight the technological revolution that the microchip development brought.

Technical note: Comparison of automated ribosomal intergenic spacer analysis (ARISA) and denaturing gradient gel electrophoresis (DGGE) to assess bacterial diversity in the rumen of sheep

The aim of this study was to compare automated ribosomal intergenic spacer analysis (ARISA) and denaturing gradient gel electrophoresis (DGGE) techniques to assess bacterial diversity in the rumen of sheep. Sheep were fed 2 diets with 70% of either alfalfa hay or grass hay, and the solid (SOL) and liquid (LIQ) phases of the rumen were sampled immediately before feeding (0 h) and at 4 and 8 h postfeeding. Both techniques detected similar differences between forages, with alfalfa hay promoting greater (P < 0.05) bacterial diversity than grass hay. In contrast, whereas ARISA analysis showed a decrease (P < 0.05) of bacterial diversity in SOL at 4 h postfeeding compared with 0 and 8 h samplings, no variations (P > 0.05) over the postfeeding period were detected by DGGE. The ARISA technique showed lower (P < 0.05) bacterial diversity in SOL than in LIQ samples at 4 h postfeeding, but no differences (P > 0.05) in bacterial diversity between both rumen phases were detected by DGGE. Under the conditions of this study, the DGGE was not sensitive enough to detect some changes in ruminal bacterial communities, and therefore ARISA was considered more accurate for assessing bacterial diversity of ruminal samples. The results highlight the influence of the fingerprinting technique used to draw conclusions on factors affecting ruminal bacterial diversity.

Disjoining pressure and the film-height-dependent surface tension of thin liquid films: New insight from capillary wave fluctuations

In this paper we review simulation and experimental studies of thermal capillary wave fluctuations as an ideal means for probing the underlying disjoining pressure and surface tensions, and more generally, fine details of the Interfacial Hamiltonian Model. We discuss recent simulation results that reveal a film-height-dependent surface tension not accounted for in the classical Interfacial Hamiltonian Model. We show how this observation may be explained bottom-up from sound principles of statistical thermodynamics and discuss some of its implications