The pgip family in soybean and three other legume species: evidence for a birth-and-death model of evolution

Background Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) plant cell wall glycoproteins involved in plant immunity. They are typically encoded by gene families with a small number of gene copies whose evolutionary origin has been poorly investigated. Here we report the complete characterization of the full complement of the pgip family in soybean (Glycine max [L.] Merr.) and the characterization of the genomic region surrounding the pgip family in four legume species. Results BAC clone and genome sequence analyses showed that the soybean genome contains two pgip loci. Each locus is composed of three clustered genes that are induced following infection with the fungal pathogen Sclerotinia sclerotiorum (Lib.) de Bary, and remnant sequences of pgip genes. The analyzed homeologous soybean genomic regions (about 126 Kb) that include the pgip loci are strongly conserved and this conservation extends also to the genomes of the legume species Phaseolus vulgaris L., Medicago truncatula Gaertn. and Cicer arietinum L., each containing a single pgip locus. Maximum likelihood-based gene trees suggest that the genes within the pgip clusters have independently undergone tandem duplication in each species. Conclusions The paleopolyploid soybean genome contains two pgip loci comprised in large and highly conserved duplicated regions, which are also conserved in bean, M. truncatula and C. arietinum. The genomic features of these legume pgip families suggest that the forces driving the evolution of pgip genes follow the birth-and-death model, similar to that proposed for the evolution of resistance (R) genes of NBS-LRR-type.

Chromogenic in situ hybridization compared with other approaches to evaluate HER2/neu status in breast carcinomas

Human epidermal growth factor receptor 2 (HER2) has been evaluated in breast cancer patients to identify those most likely to benefit from herceptin-targeted therapy. HER2 amplification, detected in 20-30% of invasive breast tumors, is associated with reduced survival and metastasis. The most frequently used technique for evaluating HER2 protein status as a routine procedure is immunohistochemistry (IHC). HER2 copy number alterations have also been evaluated by fluorescence in situ hybridization (FISH) in moderate immunoexpression (IHC 2+) cases. An alternative procedure to evaluate gene amplification is chromogenic in situ hybridization (CISH), which has some advantages over FISH, including the correlation between HER2 status and morphological features. Other methodologies have also been used, such as silver-enhanced in situ hybridization (SISH) and quantitative real-time RT-PCR, to determine the number of HER2 gene copies and expression, respectively. Here we will present a short and comprehensive review of the current advances concerning HER2 evaluation in human breast cancer.

The 5S rDNA family evolves through concerted and birth-and-death evolution in fish genomes: an example from freshwater stingrays

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Alterações no número de cópias genômicas em mastocitomas cutâneos caninos

Pós-graduação em Medicina Veterinária - FCAV

Validation of absolute quantitative real-time PCR for the diagnosis of streptococcus agalactiae in fish

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Chromatin Structure of Ribosomal RNA Genes in Dipterans and Its Relationship to the Location of Nucleolar Organizers

Nucleoli, nuclear organelles in which ribosomal RNA is synthesized and processed, emerge from nucleolar organizers (NORs) located in distinct chromosomal regions. In polytene nuclei of dipterans, nucleoli of some species can be observed under light microscopy exhibiting distinctive morphology: Drosophila and chironomid species display well-formed nucleoli in contrast to the fragmented and dispersed nucleoli seen in sciarid flies. The available data show no apparent relationship between nucleolar morphology and location of NORs in Diptera. The regulation of rRNA transcription involves controlling both the transcription rate per gene as well as the proportion of rRNA genes adopting a proper chromatin structure for transcription, since active and inactive rRNA gene copies coexist in NORs. Transcription units organized in nucleosomes and those lacking canonical nucleosomes can be analyzed by the method termed psoralen gel retarding assay (PGRA), allowing inferences on the ratio of active to inactive rRNA gene copies. In this work, possible connections between chromosomal location of NORs and proportion of active rRNA genes were studied in Drosophila melanogaster, and in chironomid and sciarid species. The data suggested a link between location of NORs and proportion of active rRNA genes since the copy number showing nucleosomal organization predominates when NORs are located in the pericentric heterochromatin. The results presented in this work are in agreement with previous data on the chromatin structure of rRNA genes from distantly related eukaryotes, as assessed by the PGRA.

Redundancy and recombination in the Echinococcus AgB multigene family: is there any similarity with protozoan contingency genes?

Numerous genetic variants of the Echinococcus antigen B (AgB) are encountered within a single metacestode. This could be a reflection of gene redundancy or the result of a somatic hypermutation process. We evaluate the complexity of the AgB multigene family by characterizing the upstream promoter regions of the 4 already known genes (EgAgB1-EgAgB4) and evaluating their redundancy in the genome of 3 Echinococcus species (E. granulosus, E. ortleppi and E. multilocularis) using PCR-based approaches. We have ascertained that the number of AgB gene copies is quite variable, both within and between species. The most repetitive gene seems to be AgB3, of which there are more than 110 copies in E. ortleppi. For E. granulosus, we have cloned and characterized 10 distinct upstream promoter regions of AgB3 from a single metacestode. Our sequences suggest that AgB1 and AgB3 are involved in gene conversion. These results are discussed in light of the role of gene redundancy and recombination in parasite evasion mechanisms of host immunity, which at present are known for protozoan organisms, but virtually unknown for multicellular parasites.

Questing Dermacentor reticulatus harbouring Babesia canis DNA associated with outbreaks of canine babesiosis in the Swiss Midlands

In 2011 and 2012, outbreaks of clinical canine babesiosis were observed in 2 areas of the Swiss Midlands that had no history of this disease so far. In one area, cases of canine babesiosis occurred over 2 consecutive tick seasons. The outbreaks involved 29 dogs, 4 of which died. All dogs were infected with large Babesia sp. as diagnosed in Giemsa-stained blood smears and/or PCR. These were identified as B. canis (formerly known as B. canis canis) by subsequent partial sequencing of the 18S rRNA gene of Babesia sp. Interestingly, the sequence indicated either a genotype with heterogeneity in the ssrRNA gene copies or double infection with different B. canis isolates. None of the dogs had a recent travel history, but one had frequently travelled to Hungary and had suffered twice from clinical babesiosis 18 and 24 months prior to the outbreak in autumn 2011. Retrospective sequencing of a stored blood DNA sample of this dog revealed B. canis, with an identical sequence to the Babesia involved in the outbreaks. For the first time in Switzerland, the partial 18S rRNA gene of B. canis could be amplified from DNA isolated from 19 out of 23 adult Dermacentor reticulatus ticks flagged in the same area. The sequence was identical to that found in the dogs. Furthermore, one affected dog carried a female D. reticulatus tick harbouring B. canis DNA. Our findings illustrate that, under favourable biogeographic and climatic conditions, the life-cycle of B. canis can relatively rapidly establish itself in previously non-endemic areas. Canine babesiosis should therefore always be a differential diagnosis when dogs with typical clinical signs are presented, regardless of known endemic areas.

Detection and quantification of Flavobacterium psychrophilum in water and fish tissue samples by quantitative real time PCR.

BACKGROUND Flavobacterium psychrophilum is the agent of Bacterial Cold Water Disease and Rainbow Trout Fry Syndrome, two diseases leading to high mortality. Pathogen detection is mainly carried out using cultures and more rapid and sensitive methods are needed. RESULTS We describe a qPCR technique based on the single copy gene β' DNA-dependent RNA polymerase (rpoC). Its detection limit was 20 gene copies and the quantification limit 103 gene copies per reaction. Tests on spiked spleens with known concentrations of F. psychrophilum (106 to 101 cells per reaction) showed no cross-reactions between the spleen tissue and the primers and probe. Screening of water samples and spleens from symptomless and infected fishes indicated that the pathogen was already present before the outbreaks, but F. psychrophilum was only quantifiable in spleens from diseased fishes. CONCLUSIONS This qPCR can be used as a highly sensitive and specific method to detect F. psychrophilum in different sample types without the need for culturing. qPCR allows a reliable detection and quantification of F. psychrophilum in samples with low pathogen densities. Quantitative data on F. psychrophilum abundance could be useful to investigate risk factors linked to infections and also as early warning system prior to potential devastating outbreak.

Biogeochemical investigation of the benthic nitrogen cycle in the Arabian Sea off Pakistan

A pronounced deficit of nitrogen (N) in the oxygen minimum zone (OMZ) of the Arabian Sea suggests the occurrence of heavy N-loss that is commonly attributed to pelagic processes. However, the OMZ water is in direct contact with sediments on three sides of the basin. Contribution from benthic N-loss to the total N-loss in the Arabian Sea remains largely unassessed. In October 2007, we sampled the water column and surface sediments along a transect cross-cutting the Arabian Sea OMZ at the Pakistan continental margin, covering a range of station depths from 360 to 1430 m. Benthic denitrification and anammox rates were determined by using 15N-stable isotope pairing experiments. Intact core incubations showed declining rates of total benthic N-loss with water depth from 0.55 to 0.18 mmol N m**-2 day**-1. While denitrification rates measured in slurry incubations decreased from 2.73 to 1.46 mmol N m**-2 day**-1 with water depth, anammox rates increased from 0.21 to 0.89 mmol N m**-2 day**-1. Hence, the contribution from anammox to total benthic N-loss increased from 7% at 360 m to 40% at 1430 m. This trend is further supported by the quantification of cd1-containing nitrite reductase (nirS), the biomarker functional gene encoding for cytochrome cd1-Nir of microorganisms involved in both N-loss processes. Anammox-like nirS genes within the sediments increased in proportion to total nirS gene copies with water depth. Moreover, phylogenetic analyses of NirS revealed different communities of both denitrifying and anammox bacteria between shallow and deep stations. Together, rate measurement and nirS analyses showed that anammox, determined for the first time in the Arabian Sea sediments, is an important benthic N-loss process at the continental margin off Pakistan, especially in the sediments at deeper water depths. Extrapolation from the measured benthic N-loss to all shelf sediments within the basin suggests that benthic N-loss may be responsible for about half of the overall N-loss in the Arabian Sea.

Spatial and seasonal distribution of Thaumarchaeota in the water column and sediment of the southern North Sea

We have examined the spatial and seasonal distribution of Thaumarchaeota in the water column and sediment of the southern North Sea using the specific intact polar lipid (IPL) hexose, phosphohexose (HPH) crenarchaeol, as well as thaumarchaeotal 16S rRNA gene abundances and expression. In the water column, a higher abundance of Thaumarchaeota was observed in the winter season than in the summer, which is in agreement with previous studies, but this was not the case in the sediment where Thaumarchaeota were most abundant in spring and summer. This observation corresponds well with the idea that ammonia availability is a key factor in thaumarchaeotal niche determination. In the surface waters of the southern North Sea, we observed a spatial variability in HPH crenarchaeol, thaumarchaeotal 16S rRNA gene abundance and transcriptional activity that corresponded well with the different water masses present. In bottom waters, a clear differentiation based on water masses was not observed; instead, we suggest that observed differences in thaumarchaeotal abundance with depth may be related to resuspension from the sediment. This could be due to suspension of benthic Thaumarchaeota to the water column or due to delivery of e.g. resuspended sediment or ammonium to the water column, which could be utilized by pelagic Thaumarchaeota. This study has shown that the seasonality of Thaumarchaeota in water and sediment is different and highlights the importance of water masses, currents and sedimentary processes in determining the spatial abundance of Thaumarchaeota in the southern North Sea.

Ladderane lipid analysis and pore water and sediment chemistry of sediment cores from the continental shelf and slope off northwest Africa

The presence and abundance of anaerobic ammonium-oxidizing (anammox) bacteria was investigated in continental shelf and slope sediments (300-3000 m water depth) off northwest Africa in a combined approach applying quantitative polymerase chain reaction (q-PCR) analysis of anammox-specific 16S rRNA genes and anammox-specific ladderane biomarker lipids. We used the presence of an intact ladderane monoether lipid with a phosphocholine (PC) headgroup as a direct indicator for living anammox bacteria and compared it with the abundance of ladderane core lipids derived from both living and dead bacterial biomass. All investigated sediments contained ladderane lipids, both intact and core lipids, in agreement with the presence of anammoxspecific 16S rRNA gene copies, indicating that anammox occurs at all sites. Concentrations of ladderane core lipids in core top sediments varied between 0.3 and 97 ng g**-1 sediment, with the highest concentrations detected at the sites located on the shelf at shallower water depths between 300 and 500 m. In contrast, the C20 [3]-ladderane monoether-PC lipid was most abundant in a core top sediment from 1500 m water depth. Both anammox-specific 16S rRNA gene copy numbers and the concentration of the C20 [3]-ladderane monoether-PC lipid increased downcore in sediments located at greater water depths, showing highest concentrations of 1.2 x 10**8 copies g**-1 sediment and 30 pg g**-1 sediment, respectively, at the deepest station of 3000 m water depth. This suggests that the relative abundance of anammox bacteria is higher in sediments at intermediate to deep water depths where carbon mineralization rates are lower but where anammox is probably more important than denitrification.

Comparison of fecal indicators with pathogenic bacteria and rotavirus in rural Bangladesh groundwater

Groundwater is routinely analyzed for fecal indicators but direct comparisons of fecal indicators to the presence of bacterial and viral pathogens are rare. This study was conducted in rural Bangladesh where the human population density is high, sanitation is poor, and groundwater pumped from shallow tubewells is often contaminated with fecal bacteria. Five indicator microorganisms (E. coli, total coliform, F+RNA coliphage, Bacteroides and human-associated Bacteroides (HuBacteroides)) and various environmental parameters were compared to the direct detection of waterborne pathogens by quantitative PCR in groundwater pumped from 50 tubewells. Rotavirus was detected in groundwater filtrate from the largest proportion of tubewells (40%), followed by Shigella (10%), Vibrio (10%), and pathogenic E. coli (8%). Spearman rank correlations and sensitivity-specificity calculations indicate that some, but not all, combinations of indicators and environmental parameters can predict the presence of pathogens. Culture-dependent fecal indicator bacteria measured on a single date did not predict bacterial pathogens, but annually averaged monthly measurements of culturable E. coli did improve prediction for total bacterial pathogens. F+RNA coliphage were neither correlated nor sufficiently sensitive towards rotavirus, but were predictive of bacterial pathogens. A qPCR-based E. coli assay was the best indicator for the bacterial pathogens, rotavirus and all pathogens combined. Since groundwater cannot be excluded as a significant source of diarrheal disease in Bangladesh and neighboring countries with similar characteristics, the need to develop more effective methods for screening tubewells with respect to microbial contamination is necessary.