Simple and versatile heterodyne whole-field interferometer for phase optics characterization

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Copy number variation of individual cattle genomes using next-generation sequencing

Copy number variations (CNVs) affect a wide range of phenotypic traits; however, CNVs in or near segmental duplication regions are often intractable. Using a read depth approach based on next-generation sequencing, we examined genome-wide copy number differences among five taurine (three Angus, one Holstein, and one Hereford) and one indicine (Nelore) cattle. Within mapped chromosomal sequence, we identified 1265 CNV regions comprising similar to 55.6-Mbp sequence-476 of which (similar to 38%) have not previously been reported. We validated this sequence-based CNV call set with array comparative genomic hybridization (aCGH), quantitative PCR (qPCR), and fluorescent in situ hybridization (FISH), achieving a validation rate of 82% and a false positive rate of 8%. We further estimated absolute copy numbers for genomic segments and annotated genes in each individual. Surveys of the top 25 most variable genes revealed that the Nelore individual had the lowest copy numbers in 13 cases (similar to 52%, chi(2) test; P-value <0.05). In contrast, genes related to pathogen- and parasite-resistance, such as CATHL4 and ULBP17, were highly duplicated in the Nelore individual relative to the taurine cattle, while genes involved in lipid transport and metabolism, including APOL3 and FABP2, were highly duplicated in the beef breeds. These CNV regions also harbor genes like BPIFA2A (BSP30A) and WC1, suggesting that some CNVs may be associated with breed-specific differences in adaptation, health, and production traits. By providing the first individualized cattle CNV and segmental duplication maps and genome-wide gene copy number estimates, we enable future CNV studies into highly duplicated regions in the cattle genome.

Chemistry of Biotites and Whole Rocks from the Rapakivi Itu Complex (Late Precambrian), State of São Paulo, SE Brazil

Chemical analyses for biotites and their host rocks from the Cabreúva (three facies) and Salto (five facies) intrusions from the multiple-centered rapakivi Itu Complex, State of São Paulo, Brazil, are presented and compared. The Cabreúva intrusion comprises different kinds of mainly even-grained biotite and hornblende-bearing syenogranites, monzogranites and quartz syenites and the Salto intrusion several types of mainly porphyritic biotite syenogranites, some of them hornblende-bearing. The biotites from the Salto intrusion (S-micas) show a more restricted composition than those from the Cabreúva intrusion (C-micas). This reflects the chemical variability of the two bodies which is smaller in the Salto intrusion and larger in the Cabreúva pluton. In the AlIV x Fet/(Fet+Mg) diagram the S- and C-micas show similar AlIV contents, around 2.2-2.3, but C-micas have higher Fet/(Fet+Mg) ratios (0.7-0.9) compared to those of S-micas (0.5-0.6). In the Mg:(Al+Fe+3+Ti):(Fe+2+Mn) diagram the S-micas are defined as Fe+2-biotites and the C-micas occupy the area between the Fe+2-biotites and the siderophyllite/lepidomelane fields, slightly overlapping the latter. In the Al2O3 × FeOt, MgO × FeOt, Al2O3 x MgO and Alt x Mg diagrams, the S-micas always lie on the calc-alkaline/alkaline boundary (or in the subalkaline field) whereas the C-micas systematically plot in the alkaline field, reflecting the higher alkalis content of the Cabreúva intrusion. In the Fet/(Fet+Mg) x SiO2 diagram, the S-micas lie on a smooth line whereas the C-micas from the different facies are separated by distinct chemical gaps reflecting the major or minor chemical overlapping of the facies from the Salto and Cabreúva intrusions.

Identification and complete sequencing of novel human transcripts through the use of mouse orthologs and testis cDNA sequences

The correct identification of all human genes, and their derived transcripts, has not yet been achieved, and it remains one of the major aims of the worldwide genomics community. Computational programs suggest the existence of 30,000 to 40,000 human genes. However, definitive gene identification can only be achieved by experimental approaches. We used two distinct methodologies, one based on the alignment of mouse orthologous sequences to the human genome, and another based on the construction of a high-quality human testis cDNA library, in an attempt to identify new human transcripts within the human genome sequence. We generated 47 complete human transcript sequences, comprising 27 unannotated and 20 annotated sequences. Eight of these transcripts are variants of previously known genes. These transcripts were characterized according to size, number of exons, and chromosomal localization, and a search for protein domains was undertaken based on their putative open reading frames. In silico expression analysis suggests that some of these transcripts are expressed at low levels and in a restricted set of tissues.

Development and validation of a Xanthomonas axonopodis pv. citri DNA microarray platform (XACarray) generated from the shotgun libraries previously used in the sequencing of this bacterial genome

Background. From shotgun libraries used for the genomic sequencing of the phytopathogenic bacterium Xanthomonas axonopodis pv. citri (XAC), clones that were representative of the largest possible number of coding sequences (CDSs) were selected to create a DNA microarray platform on glass slides (XACarray). The creation of the XACarray allowed for the establishment of a tool that is capable of providing data for the analysis of global genome expression in this organism. Findings. The inserts from the selected clones were amplified by PCR with the universal oligonucleotide primers M13R and M13F. The obtained products were purified and fixed in duplicate on glass slides specific for use in DNA microarrays. The number of spots on the microarray totaled 6,144 and included 768 positive controls and 624 negative controls per slide. Validation of the platform was performed through hybridization of total DNA probes from XAC labeled with different fluorophores, Cy3 and Cy5. In this validation assay, 86% of all PCR products fixed on the glass slides were confirmed to present a hybridization signal greater than twice the standard deviation of the deviation of the global median signal-to-noise ration. Conclusions. Our validation of the XACArray platform using DNA-DNA hybridization revealed that it can be used to evaluate the expression of 2,365 individual CDSs from all major functional categories, which corresponds to 52.7% of the annotated CDSs of the XAC genome. As a proof of concept, we used this platform in a previously work to verify the absence of genomic regions that could not be detected by sequencing in related strains of Xanthomonas. © 2010 Moreira et al; licensee BioMed Central Ltd.

Two-wavelength whole-field interferometry setup for thermal lens study

In this paper we present a new approach for thermal lens analysis using a two-wavelength DSPI (Digital Speckle Pattern Interferometry) setup for wavefront sensing. The employed geometry enables the sensor to detect wavefronts with small phase differences and inherent aberrations found in induced lenses. The wavefronts was reconstructed by four-stepping fringe evaluation and branch-cut unwrapping from fringes formed onto a diffusive glass. Real-time single-exposure contour interferograms could be obtained in order to get discernible and low-spacial frequency contour fringes and obtain low-noise measurements. In our experiments we studied the thermal lens effect in a 4% Er-doped CaO-Al2O3 glass sample. The diode lasers were tuned to have a contour interval of around 120 μm. The incident pump power was longitudinally and collinearly oriented with the probe beams. Each interferogram described a spherical-like wavefront. Using the ABCD matrix formalism we obtained the induced lens dioptric power from the thermal effect for different values of absorbed pump power. © 2012 Copyright SPIE.

Investigation of Partamona helleri (Apidae, Meliponini) B chromosome origin. An approach by microdissection and whole chromosome painting

The stingless bee Partamona helleri in southeast Brazil shows the regular chromosome number 2n = 34 and a variable number of up to four minute B1 or B2 chromosomes. Previous cytogenetic analyses have indicated morphological similarities between the B1 chromosome and chromosome segments in the regular karyotype. In this study, microdissection and chromosome painting were employed along with C banding, NOR banding, and base-specific fluorochrome staining to investigate the origin of the B1 chromosome in P. helleri. B1-generated probe hybridized exclusively to B1 chromosomes. This result suggests an independent origin from the regular karyotype or, alternatively, that the B chromosome may have suffered substantial genetic alterations along its independent evolution. The absence of higher dosages of these small B chromosomes in this population of P. helleri may be related to the existence of either a genetic or cytogenetic constraint in the establishment of such high numbered karyotypes. © 2012 INRA, DIB and Springer-Verlag, France.

Are NORs Always Located on Homeologous Chromosomes? A FISH Investigation with rDNA and Whole Chromosome Probes in Gymnotus Fishes (Gymnotiformes)

Gymnotus (Gymnotiformes, Gymnotidae) is the most diverse known Neotropical electric knife fish genus. Cytogenetic studies in Gymnotus demonstrate a huge karyotypic diversity for this genus, with diploid numbers ranging from 34 to 54. The NOR are also variable in this genus, with both single and multiple NORs described. A common interpretation is that the single NOR pair is a primitive trait while multiple NORs are derivative. However this hypothesis has never been fully tested. In this report we checked if the NOR-bearing chromosome and the rDNA site are homeologous in different species of the genus Gymnotus: G. carapo (2n = 40, 42, 54), G. mamiraua (2n = 54), G. arapaima (2n = 44), G. sylvius (2n = 40), G. inaequilabiatus (2n = 54) and G. capanema (2n = 34), from the monophyletic group G. carapo (Gymnotidae-Gymnotiformes), as well as G. jonasi (2n = 52), belonging to the G1 group. They were analyzed with Fluorescence in situ hybridization (FISH) using 18S rDNA and whole chromosome probes of the NOR-bearing chromosome 20 (GCA20) of G. carapo (cytotype 2n = 42), obtained by Fluorescence Activated Cell Sorting. All species of the monophyletic G. carapo group show the NOR in the same single pair, confirmed by hybridization with CGA20 whole chromosome probe. In G. jonasi the NORs are multiple, and located on pairs 9, 10 and 11. In G. jonasi the GCA20 chromosome probe paints the distal half of the long arm of pair 7, which is not a NOR-bearing chromosome. Thus these rDNA sequences are not always in the homeologous chromosomes in different species thus giving no support to the hypothesis that single NOR pairs are primitive traits while multiple NORs are derived. The separation of groups of species in the genus Gymnotus proposed by phylogenies with morphologic and molecular data is supported by our cytogenetic data. © 2013 Milhomem et al.

Photodynamic and Antibiotic Therapy Impair the Pathogenesis of Enterococcus faecium in a Whole Animal Insect Model

Enterococcus faecium has emerged as one of the most important pathogens in healthcare-associated infections worldwide due to its intrinsic and acquired resistance to many antibiotics, including vancomycin. Antimicrobial photodynamic therapy (aPDT) is an alternative therapeutic platform that is currently under investigation for the control and treatment of infections. PDT is based on the use of photoactive dye molecules, widely known as photosensitizer (PS). PS, upon irradiation with visible light, produces reactive oxygen species that can destroy lipids and proteins causing cell death. We employed Galleria mellonella (the greater wax moth) caterpillar fatally infected with E. faecium to develop an invertebrate host model system that can be used to study the antimicrobial PDT (alone or combined with antibiotics). In the establishment of infection by E. faecium in G. mellonella, we found that the G. mellonella death rate was dependent on the number of bacterial cells injected into the insect hemocoel and all E. faecium strains tested were capable of infecting and killing G. mellonella. Antibiotic treatment with ampicillin, gentamicin or the combination of ampicillin and gentamicin prolonged caterpillar survival infected by E. faecium (P = 0.0003, P = 0.0001 and P = 0.0001, respectively). In the study of antimicrobial PDT, we verified that methylene blue (MB) injected into the insect followed by whole body illumination prolonged the caterpillar survival (P = 0.0192). Interestingly, combination therapy of larvae infected with vancomycin-resistant E. faecium, with antimicrobial PDT followed by vancomycin, significantly prolonged the survival of the caterpillars when compared to either antimicrobial PDT (P = 0.0095) or vancomycin treatment alone (P = 0.0025), suggesting that the aPDT made the vancomycin resistant E. faecium strain more susceptible to vancomycin action. In summary, G. mellonella provides an invertebrate model host to study the antimicrobial PDT and to explore combinatorial aPDT-based treatments.

A simple, rapid method for the extraction of whole fire ant venom (Insecta: Formicidae: Solenopsis)

The invasive fire ant Solenopsis invicta is medically important because its venom is highly potent. However, almost nothing is known about fire ant venom proteins because obtaining even milligram-amounts of these proteins has been prohibitively challenging. We present a simple and fast method of obtaining whole venom compounds from large quantities of fire ants. For this, we separate the ants are from the nest soil, immerse them in dual-phase mixture of apolar organic solvent and water, and evaporate each solvent phase in separate. The remaining extract from the aqueous phase is largely made up of ant venom proteins. We confirmed this by using 2D gel electrophoresis while also demonstrating that our new approach yields the same proteins obtained by other authors using less efficient traditional methods. © 2013 Elsevier Ltd.

Study of whole genome linkage disequilibrium in Nellore cattle

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Changes during chilled storage of whole tilapia and short-term frozen storage of tilapia fillets

This study evaluated the physicochemical changes in Nile tilapia (n = 82, 373.71 ± 61.91 g) refrigerated for up to 92 h and in the frozen fillets. The tilapias were captured with nets, slaughtered by ice and water shock (1:1) in a temperature of approximately 2°C for 30 min, and stored refrigerated at 4°C in polystyrene boxes containing ice. The fish were filleted, and filets were weighed and frozen. The drip loss and protein were determined after 23 days of frozen storage. After 4 h of storage, all fish were in full rigor mortis. The pH of the muscles decreased for up to 45 h of the storage period. The fillets obtained from tilapia stored for more than 72 h lost more weight and protein. Thus, the filleting or processing of tilapia should be done before 72 h of cold storage, since deterioration of the fish starts to occur after this period. Copyright © Taylor & Francis Group, LLC.