Genetic evidence supporting the association of protease and protease inhibitor genes with inflammatory bowel disease: a systematic review

As part of the European research consortium IBDase, we addressed the role of proteases and protease inhibitors (P/PIs) in inflammatory bowel disease (IBD), characterized by chronic mucosal inflammation of the gastrointestinal tract, which affects 2.2 million people in Europe and 1.4 million people in North America. We systematically reviewed all published genetic studies on populations of European ancestry (67 studies on Crohn's disease [CD] and 37 studies on ulcerative colitis [UC]) to identify critical genomic regions associated with IBD. We developed a computer algorithm to map the 807 P/PI genes with exact genomic locations listed in the MEROPS database of peptidases onto these critical regions and to rank P/PI genes according to the accumulated evidence for their association with CD and UC. 82 P/PI genes (75 coding for proteases and 7 coding for protease inhibitors) were retained for CD based on the accumulated evidence. The cylindromatosis/turban tumor syndrome gene (CYLD) on chromosome 16 ranked highest, followed by acylaminoacyl-peptidase (APEH), dystroglycan (DAG1), macrophage-stimulating protein (MST1) and ubiquitin-specific peptidase 4 (USP4), all located on chromosome 3. For UC, 18 P/PI genes were retained (14 proteases and 4 protease inhibitors), with a considerably lower amount of accumulated evidence. The ranking of P/PI genes as established in this systematic review is currently used to guide validation studies of candidate P/PI genes, and their functional characterization in interdisciplinary mechanistic studies in vitro and in vivo as part of IBDase. The approach used here overcomes some of the problems encountered when subjectively selecting genes for further evaluation and could be applied to any complex disease and gene family.

Hostile takeover by Plasmodium: reorganization of parasite and host cell membranes during liver stage egress

The protozoan parasite Plasmodium is transmitted by female Anopheles mosquitoes and undergoes obligatory development within a parasitophorous vacuole in hepatocytes before it is released into the bloodstream. The transition to the blood stage was previously shown to involve the packaging of exoerythrocytic merozoites into membrane-surrounded vesicles, called merosomes, which are delivered directly into liver sinusoids. However, it was unclear whether the membrane of these merosomes was derived from the parasite membrane, the parasitophorous vacuole membrane or the host cell membrane. This knowledge is required to determine how phagocytes will be directed against merosomes. Here, we fluorescently label the candidate membranes and use live cell imaging to show that the merosome membrane derives from the host cell membrane. We also demonstrate that proteins in the host cell membrane are lost during merozoite liberation from the parasitophorous vacuole. Immediately after the breakdown of the parasitophorous vacuole membrane, the host cell mitochondria begin to degenerate and protein biosynthesis arrests. The intact host cell plasma membrane surrounding merosomes allows Plasmodium to mask itself from the host immune system and bypass the numerous Kupffer cells on its way into the bloodstream. This represents an effective strategy for evading host defenses before establishing a blood stage infection.

Causes and consequences of past and projected Scandinavian summer temperatures, 500-2100 AD

Tree rings dominate millennium-long temperature reconstructions and many records originate from Scandinavia, an area for which the relative roles of external forcing and internal variation on climatic changes are, however, not yet fully understood. Here we compile 1,179 series of maximum latewood density measurements from 25 conifer sites in northern Scandinavia, establish a suite of 36 subset chronologies, and analyse their climate signal. A new reconstruction for the 1483–2006 period correlates at 0.80 with June–August temperatures back to 1860. Summer cooling during the early 17th century and peak warming in the 1930s translate into a decadal amplitude of 2.9°C, which agrees with existing Scandinavian tree-ring proxies. Climate model simulations reveal similar amounts of mid to low frequency variability, suggesting that internal ocean-atmosphere feedbacks likely influenced Scandinavian temperatures more than external forcing. Projected 21st century warming under the SRES A2 scenario would, however, exceed the reconstructed temperature envelope of the past 1,500 years.

How reliable is Ki-67 immunohistochemistry in grade 2 breast carcinomas? A QA study of the Swiss Working Group of Breast- and Gynecopathologists

Adjuvant chemotherapy decisions in breast cancer are increasingly based on the pathologist's assessment of tumor proliferation. The Swiss Working Group of Gyneco- and Breast Pathologists has surveyed inter- and intraobserver consistency of Ki-67-based proliferative fraction in breast carcinomas. Methods Five pathologists evaluated MIB-1-labeling index (LI) in ten breast carcinomas (G1, G2, G3) by counting and eyeballing. In the same way, 15 pathologists all over Switzerland then assessed MIB-1-LI on three G2 carcinomas, in self-selected or pre-defined areas of the tumors, comparing centrally immunostained slides with slides immunostained in the different laboratoires. To study intra-observer variability, the same tumors were re-examined 4 months later. Results The Kappa values for the first series of ten carcinomas of various degrees of differentiation showed good to very good agreement for MIB-1-LI (Kappa 0.56–0.72). However, we found very high inter-observer variabilities (Kappa 0.04–0.14) in the read-outs of the G2 carcinomas. It was not possible to explain the inconsistencies exclusively by any of the following factors: (i) pathologists' divergent definitions of what counts as a positive nucleus (ii) the mode of assessment (counting vs. eyeballing), (iii) immunostaining technique, and (iv) the selection of the tumor area in which to count. Despite intensive confrontation of all participating pathologists with the problem, inter-observer agreement did not improve when the same slides were re-examined 4 months later (Kappa 0.01–0.04) and intra-observer agreement was likewise poor (Kappa 0.00–0.35). Conclusion Assessment of mid-range Ki-67-LI suffers from high inter- and intra-observer variability. Oncologists should be aware of this caveat when using Ki-67-LI as a basis for treatment decisions in moderately differentiated breast carcinomas.

Novel agents targeting the IGF-1R/PI3K pathway impair cell proliferation and survival in subsets of medulloblastoma and neuroblastoma

The receptor tyrosine kinase (RTK)/phosphoinositide 3-kinase (PI3K) pathway is fundamental for cancer cell proliferation and is known to be frequently altered and activated in neoplasia, including embryonal tumors. Based on the high frequency of alterations, targeting components of the PI3K signaling pathway is considered to be a promising therapeutic approach for cancer treatment. Here, we have investigated the potential of targeting the axis of the insulin-like growth factor-1 receptor (IGF-1R) and PI3K signaling in two common cancers of childhood: neuroblastoma, the most common extracranial tumor in children and medulloblastoma, the most frequent malignant childhood brain tumor. By treating neuroblastoma and medulloblastoma cells with R1507, a specific humanized monoclonal antibody against the IGF-1R, we could observe cell line-specific responses and in some cases a strong decrease in cell proliferation. In contrast, targeting the PI3K p110α with the specific inhibitor PIK75 resulted in broad anti-proliferative effects in a panel of neuro- and medulloblastoma cell lines. Additionally, sensitization to commonly used chemotherapeutic agents occurred in neuroblastoma cells upon treatment with R1507 or PIK75. Furthermore, by studying the expression and phosphorylation state of IGF-1R/PI3K downstream signaling targets we found down-regulated signaling pathway activation. In addition, apoptosis occurred in embryonal tumor cells after treatment with PIK75 or R1507. Together, our studies demonstrate the potential of targeting the IGF-1R/PI3K signaling axis in embryonal tumors. Hopefully, this knowledge will contribute to the development of urgently required new targeted therapies for embryonal tumors.

eIF4E is an important determinant of adhesion and pseudohyphal growth of the yeast S. cerevisiae

eIF4E, the cytoplasmatic cap-binding protein, is required for efficient cap-dependent translation. We have studied the influence of mutations that alter the activity and/or expression level of eIF4E on haploid and diploid cells in the yeast S. cerevisiae. Temperature-sensitive eIF4E mutants with reduced levels of expression and reduced cap-binding affinity clearly show a loss in haploid adhesion and diploid pseudohyphenation upon starvation for nitrogen. Some of these mutations affect the interaction of the cap-structure of mRNAs with the cap-binding groove of eIF4E. The observed reduction in adhesive and pseudohyphenating properties is less evident for an eIF4E mutant that shows reduced interaction with p20 (an eIF4E-binding protein) or for a p20-knockout mutant. Loss of adhesive and pseudohyphenating properties was not only observed for eIF4E mutants but also for knockout mutants of components of eIF4F such as eIF4B and eIF4G1. We conclude from these experiments that mutations that affect components of the eIF4F-complex loose properties such as adhesion and pseudohyphal differentiation, most likely due to less effective translation of required mRNAs for such processes.

Effect of Methodological and Ecological Approaches on Heterogeneity of Nest-Site Selection of a Long-Lived Vulture

The application of scientific-based conservation measures requires that sampling methodologies in studies modelling similar ecological aspects produce comparable results making easier their interpretation. We aimed to show how the choice of different methodological and ecological approaches can affect conclusions in nest-site selection studies along different Palearctic meta-populations of an indicator species. First, a multivariate analysis of the variables affecting nest-site selection in a breeding colony of cinereous vulture (Aegypius monachus) in central Spain was performed. Then, a meta-analysis was applied to establish how methodological and habitat-type factors determine differences and similarities in the results obtained by previous studies that have modelled the forest breeding habitat of the species. Our results revealed patterns in nesting-habitat modelling by the cinereous vulture throughout its whole range: steep and south-facing slopes, great cover of large trees and distance to human activities were generally selected. The ratio and situation of the studied plots (nests/random), the use of plots vs. polygons as sampling units and the number of years of data set determined the variability explained by the model. Moreover, a greater size of the breeding colony implied that ecological and geomorphological variables at landscape level were more influential. Additionally, human activities affected in greater proportion to colonies situated in Mediterranean forests. For the first time, a meta-analysis regarding the factors determining nest-site selection heterogeneity for a single species at broad scale was achieved. It is essential to homogenize and coordinate experimental design in modelling the selection of species' ecological requirements in order to avoid that differences in results among studies would be due to methodological heterogeneity. This would optimize best conservation and management practices for habitats and species in a global context.

Occurrence of vasospasm and infarction in relation to a focal monitoring sensor in patients after SAH: placing a bet when placing a probe?

INTRODUCTION Vasospastic brain infarction is a devastating complication of aneurysmal subarachnoid hemorrhage (SAH). Using a probe for invasive monitoring of brain tissue oxygenation or blood flow is highly focal and may miss the site of cerebral vasospasm (CVS). Probe placement is based on the assumption that the spasm will occur either at the dependent vessel territory of the parent artery of the ruptured aneurysm or at the artery exposed to the focal thick blood clot. We investigated the likelihood of a focal monitoring sensor being placed in vasospasm or infarction territory on a hypothetical basis. METHODS From our database we retrospectively selected consecutive SAH patients with angiographically proven (day 7-14) severe CVS (narrowing of vessel lumen >50%). Depending on the aneurysm location we applied a standard protocol of probe placement to detect the most probable site of severe CVS or infarction. We analyzed whether the placement was congruent with existing CVS/infarction. RESULTS We analyzed 100 patients after SAH caused by aneurysms located in the following locations: MCA (n = 14), ICA (n = 30), A1CA (n = 4), AcoA or A2CA (n = 33), and VBA (n = 19). Sensor location corresponded with CVS territory in 93% of MCA, 87% of ICA, 76% of AcoA or A2CA, but only 50% of A1CA and 42% of VBA aneurysms. The focal probe was located inside the infarction territory in 95% of ICA, 89% of MCA, 78% of ACoA or A2CA, 50% of A1CA and 23% of VBA aneurysms. CONCLUSION The probability that a single focal probe will be situated in the territory of severe CVS and infarction varies. It seems to be reasonably accurate for MCA and ICA aneurysms, but not for ACA or VBA aneurysms.

Up in the Tree - The Overlooked Richness of Bryophytes and Lichens in Tree Crowns

Assessing diversity is among the major tasks in ecology and conservation science. In ecological and conservation studies, epiphytic cryptogams are usually sampled up to accessible heights in forests. Thus, their diversity, especially of canopy specialists, likely is underestimated. If the proportion of those species differs among forest types, plot-based diversity assessments are biased and may result in misleading conservation recommendations. We sampled bryophytes and lichens in 30 forest plots of 20 m x 20 m in three German regions, considering all substrates, and including epiphytic litter fall. First, the sampling of epiphytic species was restricted to the lower 2 m of trees and shrubs. Then, on one representative tree per plot, we additionally recorded epiphytic species in the crown, using tree climbing techniques. Per tree, on average 54% of lichen and 20% of bryophyte species were overlooked if the crown was not been included. After sampling all substrates per plot, including the bark of all shrubs and trees, still 38% of the lichen and 4% of the bryophyte species were overlooked if the tree crown of the sampled tree was not included. The number of overlooked lichen species varied strongly among regions. Furthermore, the number of overlooked bryophyte and lichen species per plot was higher in European beech than in coniferous stands and increased with increasing diameter at breast height of the sampled tree. Thus, our results indicate a bias of comparative studies which might have led to misleading conservation recommendations of plot-based diversity assessments.

Organic vs. Conventional Grassland Management: Do N-15 and C-13 Isotopic Signatures of Hay and Soil Samples Differ?

Distinguishing organic and conventional products is a major issue of food security and authenticity. Previous studies successfully used stable isotopes to separate organic and conventional products, but up to now, this approach was not tested for organic grassland hay and soil. Moreover, isotopic abundances could be a powerful tool to elucidate differences in ecosystem functioning and driving mechanisms of element cycling in organic and conventional management systems. Here, we studied the delta N-15 and delta C-13 isotopic composition of soil and hay samples of 21 organic and 34 conventional grasslands in two German regions. We also used Delta delta N-15 (delta N-15 plant - delta N-15 soil) to characterize nitrogen dynamics. In order to detect temporal trends, isotopic abundances in organic grasslands were related to the time since certification. Furthermore, discriminant analysis was used to test whether the respective management type can be deduced from observed isotopic abundances. Isotopic analyses revealed no significant differences in delta C-13 in hay and delta C-13 in both soil and hay between management types, but showed that delta C-13 abundances were significantly lower in soil of organic compared to conventional grasslands. delta C-15 values implied that management types did not substantially differ in nitrogen cycling. Only delta C-13 in soil and hay showed significant negative relationships with the time since certification. Thus, our result suggest that organic grasslands suffered less from drought stress compared to conventional grasslands most likely due to a benefit of higher plant species richness, as previously shown by manipulative biodiversity experiments. Finally, it was possible to correctly classify about two third of the samples according to their management using isotopic abundances in soil and hay. However, as more than half of the organic samples were incorrectly classified, we infer that more research is needed to improve this approach before it can be efficiently used in practice.

Epidemiology of Brucellosis and Q Fever in Linked Human and Animal Populations in Northern Togo

BACKGROUND: Although brucellosis (Brucella spp.) and Q Fever (Coxiella burnetii) are zoonoses of global importance, very little high quality data are available from West Africa. METHODS/PRINCIPAL FINDINGS: A serosurvey was conducted in Togo's main livestock-raising zone in 2011 in 25 randomly selected villages, including 683 people, 596 cattle, 465 sheep and 221 goats. Additionally, 464 transhumant cattle from Burkina Faso were sampled in 2012. The serological analyses performed were the Rose Bengal Test and ELISA for brucellosis and ELISA and the immunofluorescence assay (IFA) for Q Fever Brucellosis did not appear to pose a major human health problem in the study zone, with only 7 seropositive participants. B. abortus was isolated from 3 bovine hygroma samples, and is likely to be the predominant circulating strain. This may explain the observed seropositivity amongst village cattle (9.2%, 95%CI:4.3-18.6%) and transhumant cattle (7.3%, 95%CI:3.5-14.7%), with an absence of seropositive small ruminants. Exposure of livestock and people to C. burnetii was common, potentially influenced by cultural factors. People of Fulani ethnicity had greater livestock contact and a significantly higher seroprevalence than other ethnic groups (Fulani: 45.5%, 95%CI:37.7-53.6%; non-Fulani: 27.1%, 95%CI:20.6-34.7%). Appropriate diagnostic test cut-off values in endemic settings requires further investigation. Both brucellosis and Q Fever appeared to impact on livestock production. Seropositive cows were more likely to have aborted a foetus during the previous year than seronegative cows, when adjusted for age. This odds was 3.8 times higher (95%CI: 1.2-12.1) for brucellosis and 6.7 times higher (95%CI: 1.3-34.8) for Q Fever. CONCLUSIONS: This is the first epidemiological study of zoonoses in Togo in linked human and animal populations, providing much needed data for West Africa. Exposure to Brucella and C. burnetii is common but further research is needed into the clinical and economic impact.

Toxoplasma gondii Infection in Kyrgyzstan: Seroprevalence, Risk Factor Analysis, and Estimate of Congenital and AIDS-Related Toxoplasmosis

Background HIV-prevalence, as well as incidence of zoonotic parasitic diseases like cystic echinococcosis, has increased in the Kyrgyz Republic due to fundamental socio-economic changes after the breakdown of the Soviet Union. The possible impact on morbidity and mortality caused by Toxoplasma gondii infection in congenital toxoplasmosis or as an opportunistic infection in the emerging AIDS pandemic has not been reported from Kyrgyzstan. Methodology/Principal Findings We screened 1,061 rural and 899 urban people to determine the seroprevalence of T. gondii infection in 2 representative but epidemiologically distinct populations in Kyrgyzstan. The rural population was from a typical agricultural district where sheep husbandry is a major occupation. The urban population was selected in collaboration with several diagnostic laboratories in Bishkek, the largest city in Kyrgyzstan. We designed a questionnaire that was used on all rural subjects so a risk-factor analysis could be undertaken. The samples from the urban population were anonymous and only data with regard to age and gender was available. Estimates of putative cases of congenital and AIDS-related toxoplasmosis in the whole country were made from the results of the serology. Specific antibodies (IgG) against Triton X-100 extracted antigens of T. gondii tachyzoites from in vitro cultures were determined by ELISA. Overall seroprevalence of infection with T. gondii in people living in rural vs. urban areas was 6.2% (95%CI: 4.8–7.8) (adjusted seroprevalence based on census figures 5.1%, 95% CI 3.9–6.5), and 19.0% (95%CI: 16.5–21.7) (adjusted 16.4%, 95% CI 14.1–19.3), respectively, without significant gender-specific differences. The seroprevalence increased with age. Independently low social status increased the risk of Toxoplasma seropositivity while increasing numbers of sheep owned decreased the risk of seropositivity. Water supply, consumption of unpasteurized milk products or undercooked meat, as well as cat ownership, had no significant influence on the risk for seropositivity. Conclusions We present a first seroprevalence analysis for human T. gondii infection in the Kyrgyz Republic. Based on these data we estimate that 173 (95% CI 136–216) Kyrgyz children will be born annually to mothers who seroconverted to toxoplasmosis during pregnancy. In addition, between 350 and 1,000 HIV-infected persons are currently estimated to be seropositive for toxoplasmosis. Taken together, this suggests a substantial impact of congenital and AIDS-related symptomatic toxoplasmosis on morbidity and mortality in Kyrgyzstan.

RP1 is a phosphorylation target of CK2 and is involved in cell adhesion

RP1 (synonym: MAPRE2, EB2) is a member of the microtubule binding EB1 protein family, which interacts with APC, a key regulatory molecule in the Wnt signalling pathway. While the other EB1 proteins are well characterized the cellular function and regulation of RP1 remain speculative to date. However, recently RP1 has been implicated in pancreatic cancerogenesis. CK2 is a pleiotropic kinase involved in adhesion, proliferation and anti-apoptosis. Overexpression of protein kinase CK2 is a hallmark of many cancers and supports the malignant phenotype of tumor cells. In this study we investigate the interaction of protein kinase CK2 with RP1 and demonstrate that CK2 phosphorylates RP1 at Ser(236) in vitro. Stable RP1 expression in cell lines leads to a significant cleavage and down-regulation of N-cadherin and impaired adhesion. Cells expressing a Phospho-mimicking point mutant RP1-ASP(236) show a marked decrease of adhesion to endothelial cells under shear stress. Inversely, we found that the cells under shear stress downregulate endogenous RP1, most likely to improve cellular adhesion. Accordingly, when RP1 expression is suppressed by shRNA, cells lacking RP1 display significantly increased cell adherence to surfaces. In summary, RP1 phosphorylation at Ser(236) by CK2 seems to play a significant role in cell adhesion and might initiate new insights in the CK2 and EB1 family protein association.

Epidemiology of masked and white-coat hypertension: the family-based SKIPOGH study

Objective We investigated factors associated with masked and white-coat hypertension in a Swiss population-based sample. Methods The Swiss Kidney Project on Genes in Hypertension is a family-based cross-sectional study. Office and 24-hour ambulatory blood pressure were measured using validated devices. Masked hypertension was defined as office blood pressure<140/90 mmHg and daytime ambulatory blood pressure≥135/85 mmHg. White-coat hypertension was defined as office blood pressure≥140/90 mmHg and daytime ambulatory blood pressure<135/85 mmHg. Mixed-effect logistic regression was used to examine the relationship of masked and white-coat hypertension with associated factors, while taking familial correlations into account. High-normal office blood pressure was defined as systolic/diastolic blood pressure within the 130–139/85–89 mmHg range. Results Among the 652 participants included in this analysis, 51% were female. Mean age (±SD) was 48 (±18) years. The proportion of participants with masked and white coat hypertension was respectively 15.8% and 2.6%. Masked hypertension was associated with age (odds ratio (OR) = 1.02, p = 0.012), high-normal office blood pressure (OR = 6.68, p<0.001), and obesity (OR = 3.63, p = 0.001). White-coat hypertension was significantly associated with age (OR = 1.07, p<0.001) but not with education, family history of hypertension, or physical activity. Conclusions Our findings suggest that physicians should consider ambulatory blood pressure monitoring for older individuals with high-normal office blood pressure and/or who are obese.

Fixed and Flexible: Coexistence of Obligate and Facultative Migratory Strategies in a Freshwater Fish

Migration is an important event in many animal life histories, but the degree to which individual animals participate in seasonal migrations often varies within populations. The powerful ecological and evolutionary consequences of such partial migration are now well documented, but the underlying mechanisms are still heavily debated. One potential mechanism of partial migration is between-individual variation in body condition, where animals in poor condition cannot pay the costs of migration and hence adopt a resident strategy. However, underlying intrinsic traits may overrule such environmental influence, dictating individual consistency in migratory patterns. Unfortunately, field tests of individual consistency compared to the importance of individual condition on migratory propensity are rare. Here we analyse 6 years of field data on roach migration, gathered by tagging almost 3000 individual fish and monitoring their seasonal migrations over extended periods of time. Our aims were to provide a field test of the role of condition in wild fish for migratory decisions, and also to assess individual consistency in migratory tendency. Our analyses reveal that (1) migratory strategy, in terms of migration/residency, is highly consistent within individuals over time and (2) there is a positive relationship between condition and the probability of migration, but only in individuals that adopt a migratory strategy at some point during their lives. However, life-long residents do not differ in condition to migrants, hence body condition is only a good predictor of migratory tendency in fish with migratory phenotypes and not a more general determinant of migratory tendency for the population. As resident individuals can achieve very high body condition and still remain resident, we suggest that our data provides some of the first field evidence to show that both facultative and obligate strategies can co-exist within populations of migratory animals.