Multiplex genotype determination at a large number of gene loci.

To facilitate large-scale genotype analysis, an efficient PCR-based multiplex approach has been developed. For simultaneously amplifying the target sequences at a large number of genetic loci, locus-specific primers containing 5' universal tails are used. Attaching the universal tails to the target sequences in the initial PCR steps allows replacement of all specific primers with a pair of primers identical to the universal tails and converts the multiplex amplification into "uniplex." Simultaneous amplification of 26 genetic loci with this approach is described. The multiplex amplification can be coupled with genotype determination. By incorporating a single-base mismatch between a primer and the template into the target sequences, a polymorphic site can be converted into a desirable restriction fragment length polymorphism when it is necessary. In this way, the allelic PCR products for the polymorphic loci can be discriminated by gel electrophoresis after restriction enzyme digestion. In this study, 32 loci were typed in such a multiplex way.

Biodiversity of Archaea and floral of two inland saltern ecosystems in the Alto Vinalopó Valley, Spain

Background. The extraction of salt from seawater by means of coastal solar salterns is a very well-described process. Moreover, the characterization of these environments from ecological, biochemical and microbiological perspectives has become a key focus for many research groups all over the world over the last 20 years. In countries such as Spain, there are several examples of coastal solar salterns (mainly on the Mediterranean coast) and inland solar salterns, from which sodium chloride is obtained for human consumption. However, studies focused on the characterization of inland solar salterns are scarce and both the archaeal diversity and the plant communities inhabiting these environments remain poorly described. Results. Two of the inland solar salterns (termed Redonda and Penalva), located in the Alto Vinalopó Valley (Alicante, Spain), were characterized regarding their geological and physico-chemical characteristics and their archaeal and botanical biodiversity. A preliminary eukaryotic diversity survey was also performed using saline water. The chemical characterization of the brine has revealed that the salted groundwater extracted to fill these inland solar salterns is thalassohaline. The plant communities living in this environment are dominated by Sarcocornia fruticosa (L.) A.J. Scott, Arthrocnemum macrostachyum (Moris) K. Koch, Suaeda vera Forsk. ex Gmelin (Amaranthaceae) and several species of Limonium (Mill) and Tamarix (L). Archaeal diversity was analyzed and compared by polymerase chain reaction (PCR)-based molecular phylogenetic techniques. Most of the sequences recovered from environmental DNA samples are affiliated with haloarchaeal genera such as Haloarcula, Halorubrum, Haloquadratum and Halobacterium, and with an unclassified member of the Halobacteriaceae. The eukaryote Dunaliella was also present in the samples. Conclusions. To our knowledge, this study constitutes the first analysis centered on inland solar salterns located in the southeastern region of Spain. The results obtained revealed that the salt deposits of this region have marine origins. Plant communities typical of salt marshes are present in this ecosystem and members of the Halobacteriaceae family can be easily detected in the microbial populations of these habitats. Possible origins of the haloarchaea detected in this study are discussed.

Maximal Number of Successors in a Nginar(1) Process

2000 Mathematics Subject Classification: 60J80, 60J20, 60J10, 60G10, 60G70, 60F99.

Isotopes content, mineralogical composition, Uranium and Thorium contents and determined activity ratios from METEOR cruise M74/3 in 2007

Authigenic carbonate deposits have been sampled with the remotely operated vehicle 'MARUM-QUEST 4000 m' from five methane seeps between 731 and 1823 m water depth along the convergent Makran continental margin, offshore Pakistan (northern Arabian Sea). Two seeps on the upper slope are located within the oxygen minimum zone (OMZ; ca. 100 to 1100 m water depth), the other sites are situated in oxygenated water below the OMZ (below 1100 m water depth). The carbonate deposits vary with regard to their spatial extent, sedimentary fabrics, and associated seep fauna: Within the OMZ, carbonates are spatially restricted and associated with microbial mats, whereas in the oxygenated zone below the OMZ extensive carbonate crusts are exposed on the seafloor with abundant metazoans (bathymodiolin mussels, tube worms, galatheid crabs). Aragonite and Mg-calcite are the dominant carbonate minerals, forming common early diagenetic microcrystalline cement and clotted to radial-fibrous cement. The delta18O carbonate values range from 1.3 to 4.2 per mil V-PDB, indicating carbonate precipitation at ambient bottom-water temperature in shallow sediment depth. Extremely low delta13Ccarbonate values (as low - 54.6per mil V-PDB) point to anaerobic oxidation of methane (AOM) as trigger for carbonate precipitation, with biogenic methane as dominant carbon source. Prevalence of biogenic methane in the seepage gas is corroborated by delta13C methane values ranging from - 70.3 to - 66.7per mil V-PDB, and also by back-calculations considering delta 13C methane values of carbonate and incorporated lipid biomarkers.

Relative abundance of prokaryotes in sediments of the Håkon Mosby mud volcano (Z1-Z3, all habitats)

CARD-FISH was performed as previously described in Ruff et al., (2013; doi:10.1371/journal.pone.0072627) with the following modifications. 4-6 µl of 25-fold diluted sediment were used for filtration. Archaeal cell walls were permeabilized with 0.1M HCl for 2 min to detect ANME-3 cells, or Proteinase K solution (15 µg ml-1 (Merck, Darmstadt, Germany) in 0.05 M EDTA (pH 8), 0.1 M Tris-HCl (pH 8), 0.5 M NaCl) for 2-4 min at room temperature for all other archaea. Bacterial cell walls were permeabilized with lysozyme solution (1000kU/ml) for 60 min at 37°. Cells were stained with DAPI (1µg/ml), embedded in mounting medium and counted in 40-60 independent microscopic fields using an Axiophot II epifluorescence microscope (Carl Zeiss, Jena, Germany).

Microbial community composition in sediments of the Hydrate Ridge (Cascadian Margin) collected during RV SONNE cruises SO143 (1999) and SO148-1 (2000)

Cold seep environments such as sediments above outcropping hydrate at Hydrate Ridge (Cascadia margin off Oregon) are characterized by methane venting, high sulfide fluxes caused by the anaerobic oxidation of methane, and the presence of chemosynthetic communities. This investigation deals with the diversity and distribution of sulfate-reducing bacteria, some of which are directly involved in the anaerobic oxidation of methane as syntrophic partners of the methanotrophic archaea. The composition and activity of the microbial communities at methane vented and nonvented sediments are compared by quantitative methods including total cell counts, fluorescence in situ hybridization (FISH). Bacteria involved in the degradation of particulate organic carbon (POC) are as active and diverse as at other productive margin sites of similar water depths. The availability of methane supports a two orders of magnitude higher microbial biomass (up to 9.6×10**10cells/cm**3). Sediment samples were obtained during RV SONNE cruises SO143-2 and SO148-1 at the crest of southern Hydrate Ridge at the Cascadia convergent margin off the coast of Oregon. Sediment cores of 20 - 40 cm length were obtained using a video-guided multiple corer from gas hydrate bearing sediments and from reference sites not enriched in methane in the surface sediments. Samples for total cell counts were obtained from 1 cm core slices, fixed with 2% formaldehyde and stored cold (4°C) and the quantification of aggregates was done via epifluorescence microscopy after staining the sediments with Acridine Orange Direct Counts (AODC) according to the method of Meyer- Reil (1983, doi:10.1007/BF00395813). Total cell counts were defined as the sum of single cells plus the aggregated cells in the syntrophic consortia. DAPI staining was used to measure ANME2/DSS aggregate sizes via epifluorescence microscopy of FISH-treated samples. For FISH, subsamples of sediment cores were sliced into 1 cm intervals and fixed for 2-3 h with 3% formaldehyde (final concentration), washed twice with 1×PBS (10 mM sodium phosphate; 130 mM NaCl), and finally stored in 1×PBS/EtOH (1:1) at -20°C.

Petroleomics By Electrospray Ionization Ft-icr Mass Spectrometry Coupled To Partial Least Squares With Variable Selection Methods: Prediction Of The Total Acid Number Of Crude Oils.

Negative-ion mode electrospray ionization, ESI(-), with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) was coupled to a Partial Least Squares (PLS) regression and variable selection methods to estimate the total acid number (TAN) of Brazilian crude oil samples. Generally, ESI(-)-FT-ICR mass spectra present a power of resolution of ca. 500,000 and a mass accuracy less than 1 ppm, producing a data matrix containing over 5700 variables per sample. These variables correspond to heteroatom-containing species detected as deprotonated molecules, [M - H](-) ions, which are identified primarily as naphthenic acids, phenols and carbazole analog species. The TAN values for all samples ranged from 0.06 to 3.61 mg of KOH g(-1). To facilitate the spectral interpretation, three methods of variable selection were studied: variable importance in the projection (VIP), interval partial least squares (iPLS) and elimination of uninformative variables (UVE). The UVE method seems to be more appropriate for selecting important variables, reducing the dimension of the variables to 183 and producing a root mean square error of prediction of 0.32 mg of KOH g(-1). By reducing the size of the data, it was possible to relate the selected variables with their corresponding molecular formulas, thus identifying the main chemical species responsible for the TAN values.

Influence of English language in the number of citations of articles published in Brazilian journals of Ophthalmology

PURPOSE: To determine the association between language and number of citations of ophthalmology articles published in Brazilian journals. METHODS: This study was a systematic review. Original articles were identified by review of documents published at the two Brazilian ophthalmology journals indexed at Science Citation Index Expanded - SCIE [Arquivos Brasileiros de Oftalmologia (ABO) and Revista Brasileira de Oftalmologia (RBO)]. All document types (articles and reviews) listed at SCIE in English (English Group) or in Portuguese (Portuguese Group) from January 1, 2008 to December 31, 2009 were included, except: editorial materials; corrections; letters; and biographical items. The primary outcome was the number of citations through the end of second year after publication date. Subgroup analysis included likelihood of citation (cited at least once versus no citation), journal, and year of publication. RESULTS: The search at the web of science revealed 382 articles [107 (28%) in the English Group and 275 (72%) in the Portuguese Group]. Of those, 297 (77.7%) were published at the ABO and 85 (23.3%) at the RBO. The citation counts were statistically significantly higher (P<0.001) in the English Group (1.51 - SD 1.98 - range 0 to 11) compared with the Portuguese Group (0.57 - SD 1.06 - range 0 to 7). The likelihood citation was statistically significant higher (P<0.001) in the English Group (70/107 - 65.4%) compared with the Portuguese Group (89/275 - 32.7%). There were more articles published in English at the ABO (98/297 - 32.9%) than at the RBO (9/85 - 10.6%) [P<0.001]. There were no significant difference (P=0.967) at the proportion of articles published in English at the years 2008 (48/172 - 27.9%) and 2009 (59/210 - 28.1%). CONCLUSION: The number of citations of articles published in Portuguese at Brazilian ophthalmology journals is lower than the published in English. The results of this study suggest that the editorial boards should strongly encourage the authors to adopt English as the main language in their future articles.

Relationship among malocclusion, number of occlusal pairs and mastication

This study evaluated the relationship among malocclusion, number of occlusal pairs, masticatory performance, masticatory time and masticatory ability in completely dentate subjects. Eighty healthy subjects (mean age = 19.40 ± 4.14 years) were grouped according to malocclusion diagnosis (n = 16): Class I, Class Class II-2, Class III and Normocclusion (control). Number of occlusal pairs was determined clinically. Masticatory performance was evaluated by the sieving method, and the time used for the comminute test food was registered as the masticatory time. Masticatory ability was measured by a dichotomic self-perception questionnaire. Statistical analysis was done by one-way ANOVA, ANOVA on ranks, Chi-Square and Spearman tests. Class II-1 and III malocclusion groups presented a smaller number of occlusal pairs than Normocclusion (p < 0.0001), Class I (p < 0.001) and II-2 (p < 0.0001) malocclusion groups. Class I, and III malocclusion groups showed lower masticatory performance values compared to Normocclusion (p < 0.05) and Class II-2 (p < 0.05) malocclusion groups. There were no differences in masticatory time (p = 0.156) and ability (χ2 = 3.58/p= 0.465) among groups. Occlusal pairs were associated with malocclusion (rho = 0.444/p < 0.0001) and masticatory performance (rho = 0.393/p < 0.0001), but malocclusion was not correlated with masticatory performance (rho = 0.116/p= 0.306). In conclusion, masticatory performance and ability were not related to malocclusion, and subjects with Class I, II-1 and III malocclusions presented lower masticatory performance because of their smaller number of occlusal pairs.