Multilocus sequence types of Finnish bovine <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> isolates and their attribution to human infections

Background  

Campylobacter jejuni is the most common bacterial cause of human gastroenteritis worldwide. Due to the sporadic nature of infection, sources often remain unknown. Multilocus sequence typing (MLST) has been successfully applied to population genetics of Campylobacter jejuni and mathematical modelling can be applied to the sequence data. Here, we analysed the population structure of a total of 250 Finnish C. jejuni isolates from bovines, poultry meat and humans collected in 2003 using a combination of Bayesian clustering (BAPS software) and phylogenetic analysis.     

Fracture prevention in COPD patients; a clinical 5-step approach

Although osteoporosis and its related fractures are common in patients with COPD, patients at high risk of fracture are poorly identified, and consequently, undertreated. Since there are no fracture prevention guidelines available that focus on COPD patients, we developed a clinical approach to improve the identification and treatment of COPD patients at high risk of fracture. We organised a round-table discussion with 8 clinical experts in the field of COPD and fracture prevention in the Netherlands in December 2013. The clinical experts presented a review of the literature on COPD, osteoporosis and fracture prevention. Based on the Dutch fracture prevention guideline, they developed a 5-step clinical approach for fracture prevention in COPD. Thereby, they took into account both classical risk factors for fracture (low body mass index, older age, personal and family history of fracture, immobility, smoking, alcohol intake, use of glucocorticoids and increased fall risk) and COPD-specific risk factors for fracture (severe airflow obstruction, pulmonary exacerbations and oxygen therapy). Severe COPD (defined as postbronchodilator FEV₁ < 50% predicted) was added as COPD-specific risk factor to the list of classical risk factors for fracture. The 5-step clinical approach starts with case finding using clinical risk factors, followed by risk evaluation (dual energy X-ray absorptiometry and imaging of the spine), differential diagnosis, treatment and follow-up. This systematic clinical approach, which is evidence-based and easy-to-use in daily practice by pulmonologists, should contribute to optimise fracture prevention in COPD patients at high risk of fracture.     

Genetic diversity,genetic drift and mixed mating system in small subpopulations of <Emphasis Type="Italic">Dyckia ibiramensis</Emphasis>, a rare endemic bromeliad from Southern Brazil

Dyckia ibiramensis is a naturally rare, endemic and threatened bromeliad which occurs naturally on 4 km of rocky river outcroppings in Southern Brazil. For this study, subpopulations of the species were characterized based on size and genetics, to compile information for in situ and ex situ conservation strategies. A census of the rosettes was undertaken for each subpopulation and seven allozyme polymorphic loci were used to estimate genetic diversity and structure of adults and offspring and assess the mating system. In general, the subpopulations were small and most of the rosettes were aggregated into clumps. The species showed a high genetic diversity ([^(H)]_e = 0.219 \hat{H}_{e} = 0.219 ) and significant fixation index ([^(f)] = 0.642, \hat{f} = 0.642, P ≤ 0.05). The estimate of differentiation among all adult subpopulations indicate pronounced genetic structure ([^(G)]^￠_ST = 0.674 \hat{G}^{\prime}_{ST} = 0.674 ). D. ibiramensis has a mixed mating system and multilocus outcrossing rates [^(t)]_m \hat{t}_{m} were variable between subpopulations. This study demonstrates the importance of in situ preservation of all subpopulations for the maintenance of species diversity. For effective ex situ conservation, it would be necessary to collect seeds from 52 to 99 seed-rosettes, depending on the target population.     

Pachybrachis sassii, a new species from the Mediterranean Giglio Island (Italy) (Coleoptera, Chrysomelidae, Cryptocephalinae)

Pachybrachis sassii, new species is described from Giglio Island, of the Tuscan Archipelago (Italy). The new species belongs to the nominotypical subgenus and is closely related to Pachybrachis salfii Burlini, 1957, from which it differs in the shape of the median lobe of the aedeagus and in the pattern of the yellow raised spots on the elytra and pronotum. Ecological observations are made. The neotype of Pachybrachis salfii from Colloreto, Monte Pollino (Italy) is designated.     

Single-cell analysis of ploidy and centrosomes underscores the peculiarity of normal hepatocytes

Polyploidization is the most well recognized feature of the liver. Yet, a quantitative and behavioral analysis of centrosomes and DNA content in normal hepatocytes has been limited by the technical challenges of methods available. By using a novel approach employing FISH for chromosomes 18, X and Y we provide, for the first time, a detailed analysis of DNA copies during physiological development in the liver at single cell level. We demonstrate that aneuploidy and unbalanced DNA content in binucleated hepatocytes are common features in normal adult liver. Despite the common belief that hepatocytes contain 1, 2 or no more than 4 centrosomes, our double staining for centrosome associated proteins reveals extranumerary centrosomes in a high percentage of cells as early as 15 days of age. We show that in murine liver the period between 15 days and 1.5 months marks the transition from a prevalence of mononucleated cells to up to 75% of binucleated cells. Our data demonstrate that this timing correlates with a switch in centrosomes number. At 15 days the expected 1 or 2 centrosomes converge with several hepatocytes that contain 3 centrosomes; at 1.5 months the percentage of cells with 3 centrosomes decreases concomitantly with the increase of cells with more than 4 centrosomes. Our analysis shows that the extranumerary centrosomes emerge in concomitance with the process of binucleation and polyploidization and maintain α-tubulin nucleation activity. Finally, by integrating interphase FISH and immunofluorescent approaches, we detected an imbalance between centrosome number and DNA content in liver cells that deviates from the equilibrium expected in normal cells. We speculate that these unique features are relevant to the peculiar biological function of liver cells which are continuously challenged by stress, a condition that could predispose to genomic instability.     

Sex hormone modulation of cell growth and apoptosis of the human monocytic/macrophage cell line

Sex hormones seem to modulate the immune/inflammatory responses by different mechanisms in female and male rheumatoid arthritis patients. The effects of 17β-oestradiol and of testosterone were tested on the cultured human monocytic/macrophage cell line (THP-1) activated with IFN-γ in order to investigate their role in cell proliferation and apoptosis. Activated human THP-1 cells were cultured in the presence of 17β-oestradiol and testosterone (final concentration, 10 nM). The evaluation of markers of cell proliferation included the NF-κB DNA-binding assay, the NF-κB inhibition complex, the proliferating cell nuclear antigen expression and the methyl-tetrazolium salt test. Apoptosis was detected by the annexin V-propidium assay and by the cleaved poly-ADP ribose polymerase expression. Specific methods included flow analysis cytometry scatter analysis, immunocytochemistry and western blot analysis. Cell growth inhibition and increased apoptosis were observed in testosterone-treated THP-1 cells. Increased poly-ADP ribose polymerase-cleaved expression and decreased proliferating cell nuclear antigen expression, as well as an increase of IκB-α and a decrease of the IκB-α phosphorylated form (ser 32), were found in testosterone-treated THP-1 cells. However, the NF-κB DNA binding was found increased in 17β-oestradiol-treated THP-1 cells. The treatment with staurosporine (enhancer of apoptosis) induced decreased NF-κB DNA binding in all conditions, but particularly in testosterone-treated THP-1 cells. Treatment of THP-1 by sex hormones was found to influence cell proliferation and apoptosis. Androgens were found to increase the apoptosis, and oestrogens showed a protective trend on cell death – both acting as modulators of the NF-κB complex.     

Spatial and bathymetric trends in Harpacticoida (Copepoda) community structure in the Northern Gulf of Mexico deep-sea

Harpacticoid copepod community structure was analyzed at 43 stations in the northern Gulf of Mexico deep-sea to test regional and bathymetric patterns of diversity in relation to environmental variables and topographic complexity of the continental slope. Depth, longitude, and proximity to the Florida Escarpment significantly affect average phylogenetic diversity, but basins and canyons do not. Multivariate analysis reveals a significant inverse relationship between diversity and POM flux, which is confirmed by significant region-scale depth and longitude differences. Although species richness declines linearly with increasing depth, the expected number of species (rarefraction) is maximized at approximately 1200 m, and average taxonomic and phylogenetic diversity continue to increase with depth, suggesting greater morphological or functional harpacticoid diversity with increasing depth. Most stations have unique species compositions, suggesting high regional (2200 species) and global (10⁵-10⁶ species) diversity by extrapolation. Therefore, processes maintaining harpacticoid diversity in the northern Gulf of Mexico deep-sea seem to rely on both small-scale dispersal and large-scale food supply mechanisms.     

A G-rich element forms a G-quadruplex and regulates BACE1 mRNA alternative splicing

β-Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is the transmembrane aspartyl protease that catalyzes the first cleavage step in the proteolysis of the APP to the amyloid β-protein (Aβ), a process involved in the pathogenesis of Alzheimer disease. BACE1 pre-mRNA undergoes complex alternative splicing, the regulation of which is not well understood. We identified a G-rich sequence within exon 3 of BACE1 involved in controlling splice site selection. Mutation of the G-rich sequence decreased use of the normal 5' splice site of exon 3, which leads to full-length and proteolytically active BACE1, and increased use of an alternative splice site, which leads to a shorter, essentially inactive isoform. Nuclease protection assays, nuclear magnetic resonance, and circular dichroism spectroscopy revealed that this sequence folds into a G-quadruplex structure. Several proteins were identified as capable of binding to the G-rich sequence, and one of these, heterogeneous nuclear ribonucleoprotein H, was found to regulate BACE1 exon 3 alternative splicing and in a manner dependent on the G-rich sequence. Knockdown of heterogeneous nuclear ribonucleoprotein H led to a decrease in the full-length BACE1 mRNA isoform as well as a decrease in Aβ production from APP, suggesting new possibilities for therapeutic approaches to Alzheimer's disease.     

RAD51 Mutants Cause Replication Defects and Chromosomal Instability

RAD51 is important for restarting stalled replication forks and for repairing DNA double-strand breaks (DSBs) through a pathway called homology-directed repair (HDR). However, analysis of the consequences of specific RAD51 mutants has been difficult since they are toxic. Here we report on the dominant effects of two human RAD51 mutants defective for ATP binding (K133A) or ATP hydrolysis (K133R) expressed in mouse embryonic stem (ES) cells that also expressed normal mouse RAD51 from the other chromosome. These cells were defective for restarting stalled replication forks and repairing breaks. They were also hypersensitive to camptothecin, a genotoxin that generates breaks specifically at the replication fork. In addition, these cells exhibited a wide range of structural chromosomal changes that included multiple breakpoints within the same chromosome. Thus, ATP binding and hydrolysis are essential for chromosomal maintenance. Fusion of RAD51 to a fluorescent tag (enhanced green fluorescent protein [eGFP]) allowed visualization of these proteins at sites of replication and repair. We found very low levels of mutant protein present at these sites compared to normal protein, suggesting that low levels of mutant protein were sufficient for disruption of RAD51 activity and generation of chromosomal rearrangements.