Utilization of newly developed immobilized enzyme reactors for preparation and study of immunoglobulin G fragments

The newly developed immobilized enzyme reactors (IMERs) with proteolytic enzymes chymotrypsin, trypsin or papain were used for specific fragmentation of high molecular-mass and heterogeneous glycoproteins immunoglobulin G (IgG) and crystallizable fragment of IgG (Fc). The efficiency of splitting or digestion were controlled by RP-HPLC. The specificity of digestion by trypsin reactor was controlled by MS. IMERs (trypsin immobilized on magnetic microparticles focused in a channel of magnetically active microfluidic device) was used for digestion of the whole IgG molecule. The sufficient conditions for IgG digestion in microfluidic device (flow rate, ratio S:E, pH, temperature) were optimized. It was confirmed that the combination of IMERs with microfluidic device enables efficient digestion of highly heterogeneous glycoproteins such as IgG in extremely short time and minimal reaction volume.     

A pyrosequencing-tailored nucleotide barcode design unveils opportunities for large-scale sample multiplexing

Multiplexed high-throughput pyrosequencing is currently limited in complexity (number of samples sequenced in parallel), and in capacity (number of sequences obtained per sample). Physical-space segregation of the sequencing platform into a fixed number of channels allows limited multiplexing, but obscures available sequencing space. To overcome these limitations, we have devised a novel barcoding approach to allow for pooling and sequencing of DNA from independent samples, and to facilitate subsequent segregation of sequencing capacity. Forty-eight forward–reverse barcode pairs are described: each forward and each reverse barcode unique with respect to at least 4 nt positions. With improved read lengths of pyrosequencers, combinations of forward and reverse barcodes may be used to sequence from as many as n² independent libraries for each set of ‘n’ forward and ‘n’ reverse barcodes, for each defined set of cloning-linkers. In two pilot series of barcoded sequencing using the GS20 Sequencer (454/Roche), we found that over 99.8% of obtained sequences could be assigned to 25 independent, uniquely barcoded libraries based on the presence of either a perfect forward or a perfect reverse barcode. The false-discovery rate, as measured by the percentage of sequences with unexpected perfect pairings of unmatched forward and reverse barcodes, was estimated to be <0.005%.     

Foraging in the ant Camponotus mus: nectar-intake rate and crop filling depend on colony starvation

The effects of colony starvation on the dynamics of nectar collection were studied in individual workers of the ant Camponotus mus. A laboratory colony was first deprived of carbohydrates for 15days, and thereafter fed daily ad libitum with diluted honey until satiation. During these two successive experimental phases, the probability of feeding, crop filling and fluid-intake rates were recorded daily for individual foragers collecting a 10% (w/w) sucrose solution. The feeding responses of individuals varied with the nutritional state of the colony. When the colony was deprived of sugar, acceptance of the sucrose solution was higher than under satiation. Feeding time increased with increasing starvation. During deprivation workers fed nearly continuously on the solution, whereas a number of feeding interruptions occurred under satiation. Crop filling also increased with increasing starvation, and showed a marked decrease when the colony was satiated. Fluid-intake rate during the deprivation phase was roughly twice that during the satiation phase. This matched well with the difference in sucking frequency recorded during ingestion in satiated and starved workers, which was also higher during starvation. Results indicate that the responsiveness of foragers, determined by the nutritional state of the colony, influenced both foraging decisions and the dynamics of fluid intake.     

Class discovery and classification of tumor samples using mixture modeling of gene expression data--a unified approach

MOTIVATION: The DNA microarray technology has been increasingly used in cancer research. In the literature, discovery of putative classes and classification to known classes based on gene expression data have been largely treated as separate problems. This paper offers a unified approach to class discovery and classification, which we believe is more appropriate, and has greater applicability, in practical situations. RESULTS: We model the gene expression profile of a tumor sample as from a finite mixture distribution, with each component characterizing the gene expression levels in a class. The proposed method was applied to a leukemia dataset, and good results are obtained. With appropriate choices of genes and preprocessing method, the number of leukemia types and subtypes is correctly inferred, and all the tumor samples are correctly classified into their respective type/subtype. Further evaluation of the method was carried out on other variants of the leukemia data and a colon dataset.     

Use of olfaction for sexual recognition in the subterranean rodent<Emphasis Type="Italic">Ctenomys talarum</Emphasis>

The ability of the tuco-tucoCtenomys talarum Thomas, 1898 to recognize sex by olfactory cues contained in urine, faeces and soiled shavings was tested by using preference tests. Nonbreeding tuco-tucos selected odours from opposite-sex rather than same-sex conspecifics. This pattern differed between sexes: females spent more time sniffing male than female odours for all scent sources whereas males did not show any difference in the time they spent investigating odours of each sex for each tested odour sources. Dissimilarities in odour selections between sexes may be attributed to a different combination of factors involved in olfactory interest for each sex. The function of gender cues recognition is discussed.     

Strategies to Avoid Wrongly Labelled Genomes Using as Example the Detected Wrong Taxonomic Affiliation for Aeromonas Genomes in the GenBank Database

Around 27,000 prokaryote genomes are presently deposited in the Genome database of GenBank at the National Center for Biotechnology Information (NCBI) and this number is exponentially growing. However, it is not known how many of these genomes correspond correctly to their designated taxon. The taxonomic affiliation of 44 Aeromonas genomes (only five of these are type strains) deposited at the NCBI was determined by a multilocus phylogenetic analysis (MLPA) and by pairwise average nucleotide identity (ANI). Discordant results in relation to taxa assignation were found for 14 (35.9%) of the 39 non-type strain genomes on the basis of both the MLPA and ANI results. Data presented in this study also demonstrated that if the genome of the type strain is not available, a genome of the same species correctly identified can be used as a reference for ANI calculations. Of the three ANI calculating tools compared (ANI calculator, EzGenome and JSpecies), EzGenome and JSpecies provided very similar results. However, the ANI calculator provided higher intra- and inter-species values than the other two tools (differences within the ranges 0.06–0.82% and 0.92–3.38%, respectively). Nevertheless each of these tools produced the same species classification for the studied Aeromonas genomes. To avoid possible misinterpretations with the ANI calculator, particularly when values are at the borderline of the 95% cutoff, one of the other calculation tools (EzGenome or JSpecies) should be used in combination. It is recommended that once a genome sequence is obtained the correct taxonomic affiliation is verified using ANI or a MLPA before it is submitted to the NCBI and that researchers should amend the existing taxonomic errors present in databases.     

Paralarval development,abundance, and distribution of Pterygioteuthis hoylei (Cephalopoda: Oegopsida: Pyroteuthidae) in the Gulf of California,México

The morphology and ecology of paralarvae (PL) through to the juvenile stages are unknown in most cephalopod species in spite of their importance as indicators of biodiversity, of adult spawning grounds, and as key basis for fisheries management programs. We describe, for the first time, the morphological development of Pterygioteuthis hoylei (Pfeffer 1912 Pfeffer, G. 1912. Die Cephalopoden der Plankton-Expedition. Zugleich eine monographische übersicht der Oegopsiden Cephalopoden. Ergebnisse der Plankton-Expedition der Humboldt-stiftung, 2: 1–815.  [Google Scholar]) and used cytochrome C oxidase for genetic identification of specimens, linking PL with both juveniles and adults. Pterygioteuthis hoylei represents the most abundant species of squid in oblique plankton collections in the Gulf of California, México. PL were collected in plankton tows made in the Gulf of California from 2004 to 2007. Almost all morphological features followed an increasing size/count trend. Most specimens were collected during March–May, accounting for 71.8% of total cephalopod paralarval abundance, and in November (13.5%). Size, abundance and distribution analyses indicate a high abundance of recently hatched PL (1.1–2.0 mm mantle length) off Bahía Concepción to Bahía de La Paz in these months along with the highest abundance of juveniles and adults in the same area. These results indicate that reproductive activity of P. hoylei takes place in the southern Gulf of California in late winter to early spring, and in November, representing an important spawning and hatching area for this species.