New cryptic karyotypic differences between cattle (Bos taurus) and goat (Capra hircus).

Cattle (Bos taurus) and goat (Capra hircus) belong to the Bovidae family, and they share a common ancestor 19.7-21.5 Ma ago (MYA). The Bovidae family apparently experienced a rapid species radiation in the middle Miocene. The present day cattle and goat possess the same diploid chromosome number (2n = 60) and structurally similar autosomes, except that a small subcentromeric portion of cattle chromosome nine has been translocated to goat chromosome 14. In this study, we adopted a new strategy that involves the use of bioinformatics approach to detect unknown cryptic chromosome divergences between cattle and goat using and subsequent validation using the fluorescence in situ hybridization (FISH) of bacterial artificial chromosome clones. We identified two hypothetical discrepancies between the cattle and goat genome assemblies: an inversion in the goat chromosome 13 and a transposition in the goat chromosome 6. The FISH technique allowed clear validation of the existence of a new 7.4 Mb chromosomal inversion in the goat chromosome 13. Regarding the transposition in the goat chromosome six, FISH analyses revealed that the cattle and goat genomes shared the same organization, with the assembly of the goat genome being the correct one. Moreover, we defined, for the first time, the size and orientation of the translocated fragment involved in the evolutionary translocation between cattle chromosomes 9 and goat chromosome 14. Our results suggest that bioinformatics represents an efficient method for detecting cryptic chromosome divergences among species.

Analysis of the structural integrity of SU-8-based optofluidic systems for small-molecule crystallization studies.

The use of SU-8-based optofluidic systems (OFS) is validated as an affordable and easy alternative to expensive glass device manufacturing for small-molecule crystallization studies and, in comparison with other polymers, able to withstand most organic solvents. A comparison between two identical OFS (using SU-8 and poly(dimethylsiloxane), PDMS) against the 36 most commonly used organic solvents for small-molecule crystallization studies have confirmed both the structural and optical stability of the SU-8, whereas PDMS suffered from unsealing or tearing in most cases. In order to test its compatibility, measurements before and after 24 h of continued exposure against solvents have been pursued. Here, three aspects have been considered: in the macroscale, swelling has been determined by analyzing the variations in the optical path in the OFS. For determining compatibility at microscale, fabricated SU-8 micropatterns were solvent-etched and subsequently characterized by scanning electron microscopy (SEM). Roughness of the polymer has also been studied through atomic force microscopy (AFM) measurements at the nanoscale. Experimental measurements of PDMS swelling were in accordance with previously reported observations, while SU-8 displayed a great stability against all the tested solvents. Through this experimental procedure we also show that the OFS are suitable for real-time, on-chip, UV-vis spectroscopy. Micro- and nanoscale observations did not show apparent corrosion on SU-8 surface. Also, two commonly used carrier fluids for microdroplet generation (FC-70 Fluorinert oil and silicone oil) were also tested against the different solvents with the aim of providing useful information for later microbatch experiments.

Classification and characterization of different white grape juices by using a hybrid electronic tongue.

A multisensor system combined with multivariate analysis is applied for the characterization and classification of white grape juices. The proposed system, known as hybrid electronic tongue, consists of an array of electrochemical microsensors and a colorimetric optofluidic system. A total of 25 white grape juices representing the large variability of vines grown in the Northwest Iberian Peninsula were studied. The data obtained were treated with Principal Component Analysis (PCA) and Soft Independent Modeling Class Analogy (SIMCA). The first tool was used to train the system with the reference genotypes -Albariño, Muscat à Petit Grains Blanc and Palomino- and the second to study the feasibility of the hybrid electronic tongue to distinguish between different grape juice varieties. The results show that the three reference genotypes are well differentiated in the PCA model and this can be used to interpolate the rest of varieties and predict their basic characteristics. Besides, using the SIMCA, the system demonstrates high potential for classifying and discriminating grape varieties.

Application of an e-tongue to the analysis of monovarietal and blends of white wines.

This work presents a multiparametric system capable of characterizing and classifying white wines according to the grape variety and geographical origin. Besides, it quantifies specific parameters of interest for quality control in wine. The system, known as a hybrid electronic tongue, consists of an array of electrochemical microsensors-six ISFET based sensors, a conductivity sensor, a redox potential sensor and two amperometric electrodes, a gold microelectrode and a microelectrode for sensing electrochemical oxygen demand--and a miniaturized optofluidic system. The test sample set comprised eighteen Catalan monovarietal white wines from four different grape varieties, two Croatian monovarietal white wines and seven bi- and trivarietal mixtures prepared from the Catalan varieties. Different chemometric tools were used to characterize (i.e., Principal Component Analysis), classify (i.e., Soft Independent Modeling Class Analogy) and quantify (i.e., Partial-Least Squares) some parameters of interest. The results demonstrate the usefulness of the multisensor system for analysis of wine.

Cell analysis using a multiple internal reflection photonic lab-on-a-chip.

Here we present a protocol for analyzing cell cultures using a photonic lab-on-a-chip (PhLoC). By using a broadband light source and a spectrometer, the spectrum of a given cell culture with an arbitrary population is acquired. The PhLoC can work in three different regimes: light scattering (using label-free cells), light scattering plus absorption (using stained cells) and, by subtraction of the two former regimes, absorption (without the scattering band). The acquisition time of the PhLoC is ∼30 ms. Hence, it can be used for rapid cell counting, dead/live ratio estimation or multiparametric measurements through the use of different dyes. The PhLoC, including microlenses, micromirrors and microfluidics, is simply fabricated in a single-mask process (by soft lithographic methods) using low-cost materials. Because of its low cost it can easily be implemented for point-of-care applications. From raw substrates to final results, this protocol can be completed in 29 h.

Ecuaciones de transformación de valores de HbA 1c obtenidos por un método trazable al método de referencia de la IFCC a valores trazables a los esquemas de estandarización japonés y norteamericano / Equation for transforming HbA 1c values obtained by a method traceable to the IFCC reference method to values traceable to the Japanese and the United States standardization schemes

Introducción. Las mediciones de HbA1c se consideran la piedra angular en el control del paciente diabético. Se han publicado ecuaciones para la transformación de valores trazables desde el método de referencia hasta los sistemas de estandarización nacionales, sin embargo, existen diversos inconvenientes relacionados con su obtención como el bajo número de muestras empleado. El objetivo de este estudio ha sido desarrollar dos ecuaciones que permitan la transformación de resultados trazables al sistema de referencia a valores trazables a los sistemas de estandarización japonés y estadounidense y compararlas con las ecuaciones previamente publicadas. Material y métodos. Se midió la HbA1c en 6.002 muestras de sangre mediante dos analizadores, uno alineado al sistema de estandarización japonés y posteriormente al sistema de estandarización estadounidense y otro al método de referencia. Se obtuvieron las ecuaciones de transformación mediante regresión lineal simple y se efectuó una comparación estadística de los resultados obtenidos mediante las ecuaciones aquí desarrolladas y las anteriormente publicadas. Resultados. La ecuaciones obtenidas fueron JDS/JSCC (%) = 1,0545*IFCC (%) + 1,129% (R2=0,9972) y NGSP (%) = 0,9293*IFCC (%) + 2,1054 (R2=0,9979). Se observaron diferencias estadísticamente significativas entre los resultados obtenidos con el uso de las ecuaciones previamente publicadas, las desarrolladas en el presente estudio y los resultados obtenidos directamente del analizador. Conclusiones. Estas nuevas ecuaciones son útiles aunque sólo para ser empleadas en nuestro laboratorio con objeto de transformar resultados alineados con el método de referencia a resultados equivalentes a los del sistema de estandarización japonés y estadounidense y viceversa. Pese a que existen diferencias estadísticamente significativas respecto a los valores obtenidos directamente del analizador son unas ecuaciones considerablemente robustas debido al elevado número de muestras empleado para obtenerlas. Las pequeñas diferencias observadas no serían clínicamente significativas, ya que la diferencia entre las medianas de los resultados obtenidos directamente del analizador o obtenidos mediante ecuaciones es inferior a 0,1% (AU)

Introduction. HbA1c measurements are the cornerstone in monitoring the diabetic patient. Master equations have been published to transform values from the reference method to values traceable to national standardization systems. There are many disadvantages in these equations, such as the low sample number used and heterogeneity of methods used to develop equations. The aim of this study was to obtain two equations for reference method values transformation to values traceable to the Japanese and United States standardization schemes and compare them with the already published equations. Material and methods. HbA1c was measured in 3002 blood samples on two analyzers calibrated with traceably to the Japanese and the United States schemes and to the reference method. The equations for transforming the values were developed by simple linear regression methods and compared with the already published master equation. Results. The equations obtained were JDS/JSCC (%) = 1.0545*IFCC (%) + 1.129% (R2=0.9972) and NGSP (%) = 0.9293*IFCC (%) + 2.1054 (R2=0.9979). Statistically significant differences were found between results obtained with the previously published equations and the equations developed in this study and those obtained directly from the analyzers. Conclusions. These new developed master equations are useful tools, although only for use in our laboratory, to transform reference method values to the Japanese and United States schemes values. Despite them show statistically significant differences with real values directly obtained from the analyzer, they are considerably robust due to the high number of samples used to obtain them. The small differences in the values obtained with the equations developed in this study compared to the direct values from the analyzers will not affect clinical management of diabetes as the difference in medians between the values obtained from the analyzer and values obtained using the equations is less than 0.1% (AU)

Poly(dimethylsiloxane) photonic microbioreactors based on segmented waveguides for local absorbance measurement.

We present the development of microbioreactors (MBRs) based on poly(dimethylsiloxane) (PDMS) segmented waveguides (SWG) for local absorbance measurements. Two different MBRs were studied, either using symmetric or asymmetric SWG (being defined as MBR-S and MBR-A, respectively). Their optical and fluidic performances were numerically analyzed, showing robustness from an optical point of view and distinct fluid flow profile. The optical characterization was done in two steps. Initially, the experimental limit of detection (LOD) and the sensitivity were determined for two different analytes (fluorescein and methylorange). With both systems, a similar limit of detection for both analytes was obtained, being in the micromolar level. Their sensitivities were 20.2±0.3 (×10⁻³) A.U./µM and 5.5±0.2 (×10⁻³) A.U./µM for fluorescein and methylorange, respectively. Once validated its applicability for local absorbance measurements, a continuous cultivation of Saccharomyces cerevisiae was done to test the viability of the proposed systems for photonic MBRs. Concretely, the cell growth was locally monitored inside the MBR during 33 h. Spectral analysis showed that the determination of the culture parameters were wavelength dependant, with a growth rate of 0.39±0.02 h⁻¹ and a doubling time of 1.65±0.09 h at an optimal wavelength of 469.9±0.3 nm. Besides the easy and monolithic integration of the SWG into poly(dimethylsiloxane) microfluidic systems, the results presented here are very promising for the application in any disposable photonic lab-on-a-chip systems used for online analysis or photonic MBRs.

Gene promoter evolution targets the center of the human protein interaction network.

Assessing the contribution of promoters and coding sequences to gene evolution is an important step toward discovering the major genetic determinants of human evolution. Many specific examples have revealed the evolutionary importance of cis-regulatory regions. However, the relative contribution of regulatory and coding regions to the evolutionary process and whether systemic factors differentially influence their evolution remains unclear. To address these questions, we carried out an analysis at the genome scale to identify signatures of positive selection in human proximal promoters. Next, we examined whether genes with positively selected promoters (Prom+ genes) show systemic differences with respect to a set of genes with positively selected protein-coding regions (Cod+ genes). We found that the number of genes in each set was not significantly different (8.1% and 8.5%, respectively). Furthermore, a functional analysis showed that, in both cases, positive selection affects almost all biological processes and only a few genes of each group are located in enriched categories, indicating that promoters and coding regions are not evolutionarily specialized with respect to gene function. On the other hand, we show that the topology of the human protein network has a different influence on the molecular evolution of proximal promoters and coding regions. Notably, Prom+ genes have an unexpectedly high centrality when compared with a reference distribution (P=0.008, for Eigenvalue centrality). Moreover, the frequency of Prom+ genes increases from the periphery to the center of the protein network (P=0.02, for the logistic regression coefficient). This means that gene centrality does not constrain the evolution of proximal promoters, unlike the case with coding regions, and further indicates that the evolution of proximal promoters is more efficient in the center of the protein network than in the periphery. These results show that proximal promoters have had a systemic contribution to human evolution by increasing the participation of central genes in the evolutionary process.

Hybrid electronic tongue based on optical and electrochemical microsensors for quality control of wine.

A multiparametric system able to classify red and white wines according to the grape varieties and for analysing some specific parameters is presented. The system, known as hybrid electronic tongue, consists of an array of electrochemical microsensors and a colorimetric optofluidic system. The array of electrochemical sensors is composed of six ISFETs based sensors, a conductivity sensor, a redox potential sensor and two amperometric electrodes, an Au microelectrode and a microelectrode for sensing electrochemical oxygen demand. The optofluidic system is entirely fabricated in polymer technology and comprises a hollow structure, air mirrors, microlenses and self-alignment structures. The data obtained from these sensors has been treated with multivariate advanced tools; Principal Component Analysis (PCA), for the patterning recognition and classification of wine samples, and Partial-Least Squares (PLS) regression, for quantification of several chemical and optical parameters of interest in wine quality. The results have demonstrated the utility of this system for distinguishing the samples according to the grape variety and year vintage and for quantifying several sample parameters of interest in wine quality control.

Cell screening using disposable photonic lab on a chip systems.

A low-cost photonic lab on a chip with three different working regimes for cell screening is presented. The proposed system is able to perform scattering, scattering + absorption, and absorption measurements without any modification. Opposite to the standard flow cytometers, in this proposed configuration, a single 30 ms scan allows to obtain information regarding the cell optical properties. An additional novelty is that the whole spectrum is obtained and analyzed, being then possible to determine for each regime which is the optimal working wavelength that would provide the best performance in terms of sensitivity and limit of detection (LOD). Experimental results have provided with an LOD of 54.9 +/- 0.7 cells (in the scattering regime using unlabeled cells), 53 +/- 1 cells (in the scattering + absorption regime using labeled cells), and 105 +/- 4 cells (in the absorption regime using labeled cells). Finally, the system has also been used for measuring the dead/live cell ratio, obtaining LODs between 7.6 +/- 0.4% and 6.7 +/- 0.3%, depending on the working regime used.

Comparison of three expression systems for heterologous xylanase production by S. cerevisiae in defined medium.

The influence of the auxotrophic deficiencies of the host strain and expression vector selection on the production of a heterologous protein was investigated. Heterologous xylanase production by two prototrophic S. cerevisiae transformants, containing either a plasmid-based, YEp-type expression system or an integrative, YIp-type expression system, were compared with production by an auxotrophic transformant, containing an identical YEp-type expression system, in batch and continuous cultivation, using a chemically defined medium. Heterologous xylanase production by the auxotrophic strains in defined medium was critically dependent on the availability of amino acids, as extracellular xylanase production increased dramatically when amino acids were over-consumed from the medium to the point of saturating the cell. Saturation with amino acids, indicated by an increased leakage of amino acids from the cell, was thus a prerequisite for high level of heterologous protein production by the auxotrophic strain. Maximal xylanase production levels by the auxotrophic strain corresponded to the levels obtained with a similar prototrophic strain during cultivation in defined medium without amino acids. Superfluous auxotrophic markers thus had a strong deleterious effect on heterologous protein production by recombinant yeasts, and the use of such strains should be limited to initial exploratory investigations. The increased copy number and foreign gene dosage of the YEp-based expression vector, stabilized by the ura3 fur1 autoselection system, significantly improved production levels of heterologous xylanase, compared to the YIp system, which is based on a single integration into the yeast genome. No evidence was found of the possible saturation of the host secretory capacity by multicopy overexpression. Stable production of heterologous xylanase at high levels by the prototrophic YEp-based recombinant strain, compared to the YIp system, was demonstrated.