Microfluidic devices for DNA sequencing: sample preparation and electrophoretic analysis

Modern DNA sequencing 'factories' have revolutionized biology by completing the human genome sequence, but in the race to completion we are left with inefficient, cumbersome, and costly macroscale processes and supporting facilities. During the same period, microfabricated DNA sequencing, sample processing and analysis devices have advanced rapidly toward the goal of a 'sequencing lab-on-a-chip'. Integrated microfluidic processing dramatically reduces analysis time and reagent consumption, and eliminates costly and unreliable macroscale robotics and laboratory apparatus. A microfabricated device for high-throughput DNA sequencing that couples clone isolation, template amplification, Sanger extension, purification, and electrophoretic analysis in a single microfluidic circuit is now attainable.     

Myeloid cell function in MRP-14 (S100A9) null mice

Myeloid-related protein 14 (MRP-14) and its heterodimeric partner, MRP-8, are cytosolic calcium-binding proteins, highly expressed in neutrophils and monocytes. To understand the function of MRP-14, we performed targeted disruption of the MRP-14 gene in mice. MRP-14(-/-) mice showed no obvious phenotype and were fertile. MRP-8 mRNA but not protein is present in the myeloid cells of these mice, suggesting that the stability of MRP-8 protein is dependent on MRP-14 expression. A compensatory increase in other proteins was not detected in cells lacking MRP-8 and MRP-14. Although the morphology of MRP-14(-/-) myeloid cells was not altered, they were significantly less dense. When Ca(2+) responses were investigated, there was no change in the maximal response to the chemokine MIP-2. At lower concentrations, however, there was reduced responsiveness in MRP-14(-/-) compared with MRP-14(+/+) neutrophils. This alteration in the ability to flux Ca(2+) did not impair the ability of the MRP-14(-/-) neutrophils to respond chemotactically to MIP-2. In addition, the myeloid cell functions of phagocytosis, superoxide burst, and apoptosis were unaffected in MRP-14(-/-) cells. In an in vivo model of peritonitis, MRP-14(-/-) mice showed no difference from wild-type mice in induced inflammatory response. The data indicate that MRP-14 and MRP-8 are dispensable for many myeloid cell functions.     

Baseline Levels of Potentially Toxic Elements in Pampas Soils

The soils of the Pampas are thought to be generally non-contaminated but there is growing evidence of trace element accumulation at some specific sites. The goal of this study was to measure the current levels of the main Potentially Toxic Elements (PTE) in the top horizon and in specific soil profiles so that we would establish the baseline concentrations of these elements. Eighty-eight top soils and three soil profiles were sampled. The samples were acid digested. Arsenic, boron, barium, cadmium, cobalt, chromium, copper, lead, manganese, mercury, molybdenum, nickel, silver, selenium and zinc were determined with inductively coupled argon plasma emission spectrometry (ICPES).

All of the values found are within the normal range for uncontaminated soils as reported from several continents. Elements with high environmental risk potential are lower than the admissible range of the European Union and some of them are orders of magnitude lower than those of the United States Environmental Protection Agency (US-EPA) 501 levels. Potentially Toxic Elements contents increased with depth or showed a maximum concentration at the B2 horizon. This is related to the parent material and the pedogenetic processes but not to recent contamination. Soil profiles showed higher concentrations of PTE in clayey horizons. However, these relationships did not appear in top soil samples in any soil Great Group studied. The shown data establishes a baseline for PTE concentrations for Pampas soils.     

Conversion of capillary electrophoresis microchip genotyping data for analysis with Genetic Profiler software

The collection and conversion of 4-color fluorescent genotyping data from capillary array electrophoresis microchip devices and its conversion to a format easily and rapidly analyzed by Genetic Profiler genotyping software is presented. Microchip fluorescence intensity data are acquired and stored as 4-color tab-delimited text. These files are converted to electrophoretic signal data (ESD) files using a utility program (TEXT-to-ESD) written in C. TEXT-to-ESD generates an ESD file by converting text data to binary data and then appending a 632-byte ESD-file trailer. Up to 96 ESD files are then assembled into a run folder and imported into Genetic Profiler, where data are reduced to 4-color electropherograms and analyzed. In this manner, DNA fragment sizing data acquired with our high-speed electrophoretic microchip devices can be rapidly analyzed using robust commercial software. Additionally, the conversion program allows sizing of data with Genetic Profiler that have been preprocessed using other third-party software, such as BaseFinder.     

Probing the photoreaction mechanism of phytochrome through analysis of resonance Raman vibrational spectra of recombinant analogues

Resonance Raman spectra of native and recombinant analogues of oat phytochrome have been obtained and analyzed in conjunction with normal mode calculations. On the basis of frequency shifts observed upon methine bridge deuteration and vinyl and C(15)-methine bridge saturation of the chromophore, intense Raman lines at 805 and 814 cm(-)(1) in P(r) and P(fr), respectively, are assigned as C(15)-hydrogen out-of-plane (HOOP) wags, lines at 665 cm(-)(1) in P(r) and at 672 and 654 cm(-)(1) in P(fr) are assigned as coupled C=C and C-C torsions and in-plane ring twisting modes, and modes at approximately 1300 cm(-)(1) in P(r) are coupled N-H and C-H rocking modes. The empirical assignments and normal mode calculations support proposals that the chromophore structures in P(r) and P(fr) are C(15)-Z,syn and C(15)-E,anti, respectively. The intensities of the C(15)-hydrogen out-of-plane, C=C and C-C torsional, and in-plane ring modes in both P(r) and P(fr) suggest that the initial photochemistry involves simultaneous bond rotations at the C(15)-methine bridge coupled to C(15)-H wagging and D-ring rotation. The strong nonbonded interactions of the C- and D-ring methyl groups in the C(15)-E,anti P(fr) chromophore structure indicated by the intense 814 cm(-1) C(15) HOOP mode suggest that the excited state of P(fr) and its photoproduct states are strongly coupled.     

Population differences in density and resource allocation of ornamental tail feathers in the barn swallow

Many organisms show well‐defined latitudinal clines in morphology, which appear to be caused by spatially varying natural selection, resulting in different optimal phenotypes in each location. Such spatial variability raises an interesting question, with different prospects for the action of sexual selection on characters that have a dual purpose, such as locomotion and sexual attraction. The outermost tail feathers of barn swallows (Hirundo rustica) represent one such character, and their evolution has been a classic model subject to intense debate. In the present study, we examined individuals from four European populations to analyze geographical variation in the length and mass of tail feathers in relation to body size and wing size. Tail feather length differed between sexes and populations, and such variation was a result of the effects of natural selection, acting through differences in body size and wing size, as well as the effects of sexual selection that favours longer tails. The extra enlargement of the tail promoted by sexual selection (i.e. beyond the natural selection optimum) could be achieved by increasing investment in ornaments, and by modifying feather structure to produce longer feathers of lower density. These two separate processes accounting for the production of longer and more costly tail feathers and less dense feathers, respectively, are consistent with the hypothesis that both Zahavian and Fisherian mechanisms may be involved in the evolution of the long tails of male barn swallows. We hypothesize that the strength of sexual selection increases with latitude because of the need for rapid mating as a result of the short duration of the breeding season at high latitudes. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 925–936.     

The core genome of the anaerobic oral pathogenic bacterium <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis>

Background  

The Gram negative anaerobic bacterium Porphyromonas gingivalis has long been recognized as a causative agent of periodontitis. Periodontitis is a chronic infectious disease of the tooth supporting tissues eventually leading to tooth-loss. Capsular polysaccharide (CPS) of P. gingivalis has been shown to be an important virulence determinant. Seven capsular serotypes have been described. Here, we used micro-array based comparative genomic hybridization analysis (CGH) to analyze a representative of each of the capsular serotypes and a non-encapsulated strain against the highly virulent and sequenced W83 strain. We defined absent calls using Arabidopsis thaliana negative control probes, with the aim to distinguish between aberrations due to mutations and gene gain/loss.     

The Occurrence and Ape-to-Ape Transmission of the Entodiniomorphid Ciliate Troglodytella abrassarti in Captive Gorillas

ABSTRACT. Entodiniomorphid ciliates are often present in the colons of wild apes. In captive apes the infection tends to gradually disappear, with the exception of Troglodytella abrassarti . We used fecal examinations to screen the gorillas ( Gorilla gorilla gorilla ) in European (Czech Republic, UK) and Australian Zoos to explore the ape-to-ape transmission pattern of T. abrassarti . Gorillas from two out of three European Zoos were positive for T. abrassarti , while gorillas from the Australian Zoo were negative. We documented a horizontal transmission of T. abrassarti to a non-infected adult gorilla introduced into a Troglodytella -positive group in the Prague Zoo and traced the origin of the ciliate infection to the Paignton Zoo (UK) using serial fecal examinations. During this study, two infant gorillas born in the Prague Zoo (CZ) first became positive for T. abrassarti at the age of 9 mo. Ciliate morphology and the sequencing of the small subunit rRNA gene and the internal transcribed spacer rDNA spacer region revealed that T. abrassarti affects both captive gorillas and chimpanzees. We conclude that zoo transport plays a major role in the distribution of T. abrassarti among captive gorillas.     

An empirical demonstration of the ideal free distribution: Little Grebes Tachybaptus ruficollis breeding in intensive agricultural landscapes

The ideal free distribution (IFD) model predicts that a density‐dependent mechanism operates to regulate habitat selection and reproductive performance. We studied a Little Grebe Tachybaptus ruficollis population, which breeds on irrigation ponds in the Vega Baja Valley (southeastern Spain) to test the premises of the IFD model. These ponds are highly dynamic because they are managed according to agricultural requirements, and are subject to different levels of disturbance, which can change the quality of individual ponds across the landscape. Surveys were carried out during the breeding season from 2002 to 2006, with reproduction performance estimated during two consecutive breeding seasons, 2003 and 2004. Occupation frequency differed from random, indicating preference for some ponds over others. Habitat features such as pond construction and design, the presence of submerged vegetation, vegetation along the shore and reed beds, and pond area correlated with occupation frequency and might be considered to be indicators of pond quality. Ponds were occupied sequentially from best to worst. Thus, when the population size increased, the number of low‐quality ponds occupied also increased. High‐quality ponds held more breeding pairs than low‐quality ones, resulting in the mean reproductive success per breeding pair being independent of pond quality. Little Grebes therefore occupy ponds in a manner consistent with the expectations of the IFD model.     

Vibrational communication and mating behaviour of Dichelops melacanthus (Hemiptera: Pentatomidae) recorded from loudspeaker membranes and plants

Vibrational communication is important for successful mating in various stink bugs species. The vibrational signals from males and females of Dichelops melacanthus Dallas (Hemiptera: Pentatomidae) are recorded from a nonresonant substrate (i.e. a loudspeaker membrane) to characterize the temporal and spectral properties of these vibrational signals, as well as on a resonant substrate (i.e. bean plants) to obtain information about how these signals are altered when they are transmitted through the plants. On the loudspeaker membrane, D. melacanthus males and females emit only one male or one female song, respectively. However, when the insects are placed on bean leaves, a more complex repertoire is recorded, with three different songs for each sex. The first female and male songs appear to have calling functions and the third male and female songs are emitted during courtship. The second female and male songs are emitted after the first song, although their functions in mating behaviour are not clear. The identified repertoire is similar to those of other Neotropical stink bugs, starting with songs 1 and 2 and developing into song 3. Frequency modulation is observed in the female songs recorded from the loudspeaker membrane and the plants. The signals recorded from plants present higher harmonic peaks compared with the signals recorded from the loudspeaker membrane. The presence of species and sex‐specific songs during mating confirms the important role of vibrational communication in mate location and recognition. The temporal and spectral characteristic signals are influenced by the substrate used to record the songs emitted by D. melacanthus.