Viability and sunlight sensitivity of oak pollen and its implications for pollen-mediated gene flow

Pollen-mediated gene flow and the male reproductive success of wind-pollinated trees depend on the initial viability of the pollen and the changes that occur in its viability during transport in the atmosphere. The viability of Quercus robur pollen was determined before and during exposure to sunlight by in vitro germination and the fluorescein diacetate reaction (FCR) in 2002 and 2003, respectively. These experiments allowed us to calculate initial pollen viability and pollen sensitivity to sunlight. The germination test revealed a lower initial pollen viability (25–65%) than the FCR (53–92%). Following 9.5 h of irradiation the viability was reduced to 75–100% as determined by the in vitro germination test or to 40–70% as determined by the FCR. The actual values of initial pollen viability and pollen sensitivity to sunlight were used to define a range of values for modelling pollen dispersal using the mesoscale meteorological model METRAS. The deposition patterns of viable pollen varied by as much as a factor of 14 by changing the viability parameters in the range of the observed values. This suggests significant differences in male reproductive success. Variations in initial pollen viability have stronger effects on the gene-flow pattern than do variations in pollen sensitivity to sunlight. In particular, pollen distribution throughout the local environment is shaped by the initial pollen viability, while pollen sensitivity to sunlight mainly influences long-distance pollen dispersal.This revised version was published online in February 2005 with corrections to an incorrect sentence in the Results section. Under Pollen Sensitivity it should read: decreased faster when assessed by the germination test than by the FCR.     

Thesaurus-based disambiguation of gene symbols

Background  

Massive text mining of the biological literature holds great promise of relating disparate information and discovering new knowledge. However, disambiguation of gene symbols is a major bottleneck.     

Functional studies of signaling pathways in peri-implantation development of the mouse embryo by RNAi

Background

Studies of gene function in the mouse have relied mainly on gene targeting via homologous recombination. However, this approach is difficult to apply in specific windows of time, and to simultaneously knock-down multiple genes. Here we report an efficient method for dsRNA-mediated gene silencing in late cleavage-stage mouse embryos that permits examination of phenotypes at post-implantation stages.

Results

We show that introduction of Bmp4 dsRNA into intact blastocysts by electroporation recapitulates the genetic Bmp4 null phenotype at gastrulation. It also reveals a novel role for Bmp4 in the regulation the anterior visceral endoderm specific gene expression and its positioning. We also show that RNAi can be used to simultaneously target several genes. When applied to the three murine isoforms of Dishevelled, it leads to earlier defects than previously observed in double knock-outs. These include severe delays in post-implantation development and defects in the anterior midline and neural folds at headfold stages.

Conclusion

Our results indicate that the BMP4 signalling pathway contributes to the development of the anterior visceral endoderm, and reveal an early functional redundancy between the products of the murine Dishevelled genes. The proposed approach constitutes a powerful tool to screen the functions of genes that govern the development of the mouse embryo.     

Behavioural and physiological consequences of acute social defeat in growing gilts: effects of the social environment

Endocrine, behavioural and immunologic processes, together with body growth, were evaluated in gilts that were defeated at 10 weeks of age in resident-intruder tests. Immediately after defeat, gilts were either separated from or reunited with a familiar conspecific (litter-mate; always a barrow). Gilts were assigned to one of four treatments: (a) DI: defeat, followed by isolation (separation from original litter-mate; n=8); (b) I: no defeat, isolation (control group; n=9); (c) DP; defeat, followed by pair-housing (reunion with original litter-mate; n=8); and (d) P: no defeat, pair-housing (control group; n=8). The following general conclusions were derived: (1) social defeat caused pronounced short-term elevations in hypothalamic-pituitary-adrenal (HPA) and sympathetic-adrenal medullary activities, and of prolactin levels. Moreover, as soon as 1h after defeat, percentages of blood lymphocytes and neutrophilic granulocytes were, respectively, decreased and increased; (2) social defeat had some long-lasting influence on behaviour and physiology, but isolation predominantly determined responses in the longer term. Defeat, as well as isolation, resulted in increased cardiovascular activities compared to P controls, as observed in a novel object test (NOT: +7 days) and an aversion test (AVT: +14 days). Moreover, defeated as well as isolated gilts did not habituate to a repeated novel environment test (NET: -7, +2 and +7 days) in terms of frequencies of vocalising, whereas P controls did. Isolation, through the separation from any other pig, was responsible for the other observed long-term characteristics, which developed progressively. Isolated gilts showed high mobilities and high cortisol responses in the repeated NET (+7 days), not being habituated. This contrasted the reactions of pair-housed gilts, which were much reduced. In addition to their high cardiovascular activities in the NOT and the AVT, isolated gilts also displayed higher heart rates in the repeated NET and during human presence following the NOT, compared to pair-housed gilts. Finally, isolated gilts were more inhibited to approach a novel object (in the NOT) than pair-housed pigs; and (3) stress responses of defeated gilts were modulated by the subsequent social environment. Stimulation of the HPA-axis (plasma- and salivary cortisol) was prolonged in those defeated gilts which were isolated (observed in the first hour). Changes in leucocyte subsets were still observed after 3 days in DI, but were 'normalised' within 1 day in DP gilts. Two days after defeat, habituation to the repeated NET in terms of mobility and salivary cortisol responses occurred in control and DP gilts, but not in DI gilts. We argue that these effects of the social environment shortly after defeat were related to a stress-reducing effect of a stable social relationship, i.e. social support.     

Selecting Populations for Non-Analogous Climate Conditions Using Universal Response Functions: The Case of Douglas-Fir in Central Europe

Identifying populations within tree species potentially adapted to future climatic conditions is an important requirement for reforestation and assisted migration programmes. Such populations can be identified either by empirical response functions based on correlations of quantitative traits with climate variables or by climate envelope models that compare the climate of seed sources and potential growing areas. In the present study, we analyzed the intraspecific variation in climate growth response of Douglas-fir planted within the non-analogous climate conditions of Central and continental Europe. With data from 50 common garden trials, we developed Universal Response Functions (URF) for tree height and mean basal area and compared the growth performance of the selected best performing populations with that of populations identified through a climate envelope approach. Climate variables of the trial location were found to be stronger predictors of growth performance than climate variables of the population origin. Although the precipitation regime of the population sources varied strongly none of the precipitation related climate variables of population origin was found to be significant within the models. Overall, the URFs explained more than 88% of variation in growth performance. Populations identified by the URF models originate from western Cascades and coastal areas of Washington and Oregon and show significantly higher growth performance than populations identified by the climate envelope approach under both current and climate change scenarios. The URFs predict decreasing growth performance at low and middle elevations of the case study area, but increasing growth performance on high elevation sites. Our analysis suggests that population recommendations based on empirical approaches should be preferred and population selections by climate envelope models without considering climatic constrains of growth performance should be carefully appraised before transferring populations to planting locations with novel or dissimilar climate.     

Genetic polymorphims of estrogen receptor alpha −397 PvuII (T>C) and −351 XbaI (A>G) in a portuguese population: prevalence and relation with breast cancer susceptibility

Estrogen receptor alpha (ERα), that mediates the biologic effects of estrogen in estrogen-sensitive tissues like breast, is genetically polymorphic. To evaluate the association between ?397 PvuII (T>C) and ?351 XbaI (A>G) restriction fragment length polymorphisms (RFLPs) in intron 1 of ERα gene and susceptibility of breast cancer, we undertook a case–control study in BRCA1 185delAG and 5382insC/BRCA2 6174delT negative Portuguese women. The study population consisted of 107 patients with histological diagnosis of breast cancer and 121 women with no history of breast cancer. Genomic DNA was extracted from blood samples and genotyping analyses were performed by PCR–RFLP. XbaI polymorphism was associated with a significant reduced risk of breast cancer for carriers of the x allele in homozygozity (OR 0.178; 95 % CI 0.070–0.456; P < 0.001) or heterozigozity (OR 0.223; 95 % CI 0.089–0.561; P = 0.001). The PvuII polymorphism was associated with a non-significantly reduced risk. The combined analysis of PvuII and XbaI polymorphisms revealed none synergistic effect of the two genotypes, except for simultaneous carriers of pp and xx genotypes, that have a reduced risk of breast cancer (OR 0.226; 95 % CI 0.049–1.035; P = 0.044). The combination of PvuII and XbaI genotypes into haplotypes showed that carriers of two copies of the px (ppxx) haplotype had a reduced risk of breast cancer (OR 0.405; 95 % CI 0.194–0.843; P = 0.014), compared with PX (PPXX + PPXx + PpXX + PpXx) haplotypes. PvuII and XbaI polymorphisms were in linkage disequilibrium both in cases (D = 0.044, r² = 0.049, X² = 5.216, P = 0.022) and controls (D = 0.090, r² = 0.139, X² = 16.819, P < 0.001), but not in the entire sample population analyzed as a whole (D = 0.087, r² = 0.0076, X² = 1.733, P = 0.188). In conclusion, in this case–control study we found that ERα gene XbaI polymorphism may modify individual susceptibility for breast cancer in this population.     

THE COMBINED USE OF WHOLE CUPHEA SEEDS CONTAINING MEDIUM CHAIN FATTY ACIDS AND AN EXOGENOUS LIPASE IN PIGLET NUTRITION

In search for an alternative for nutritional antimicrobials in piglet feeding, the effects of adding whole Cuphea seeds, as a natural source of medium chain fatty acids (MCFA), with known antimicrobial effects, and an exogenous lipase to a weaner diet were studied. The foregut flora, the gut morphology, some digestive parameters and the zootechnical performance of weaned piglets were investigated. Thirty newly weaned piglets, initial weight 7.0 ± 0.4 kg, were divided according to litter, sex and weight in two groups (control diet; Cuphea+lipase diet). The Cuphea seeds (lanceolata and ignea) (50 g kg^?1) were substituted for soybean oil (15 g kg^?1), Alphacell (25 g kg^?1) and soy protein isolate (10 g kg^?1) in the control diet. Also 500 mg kg^?1 microbial lipase was added to the Cuphea diet. The piglets were weighted individually on days 0, 3, 7, 14 and 16. Feed intake was recorded per pen during days 0 to 3, 3 to 7, 7 to 14 and 14 to 16. On day 7 five piglets of each experimental group were euthanized for counting the gastric and small intestinal gut flora and for gut morphology at two sites of the small intestine (proximal, distal). The results indicate a trend towards improved performances parameters by feeding Cuphea + lipase. The enzymic released MCFA (1.7 g kg^?1 fresh gastric contents) tended to decrease the number of Coliforms in the proximal small intestine, but increased the number in the stomach and distal small intestine. With Cuphea, the number of Streptococci was significantly lower in small intestine, but not in the stomach, while the number of Lactobacilli was significantly lower in the distal small intestine and tended to be lower in the stomach and proximal small intestine. No differences between the diets were noted for the total anaerobic microbial load in the stomach or in the gut. Feeding Cuphea+lipase resulted in a significantly greater villus height (distal small intestine) and a lesser crypt depth (proximal and distal small intestine) and greater villus/crypt ratio depth (proximal and distal small intestine). The intra-epithelial lymphocyte (IEL) counts per 100 enterocytes were significantly decreased in the proximal small intestine and tended to decrease in the distal small intestine by feeding the Cuphea+lipase diet. Both phenomena are indicative for a more healthy and better functional state of the mucosa. Present results are in line with foregoing research, showing that manipulation of the gut ecosystem by the enzymic in situ released MCFA in the stomach and foregut can result in improved performances of the piglets, which makes the concept a potential alternative for in-feed nutritional antibiotics.     

Impact of Untreated Obstructive Sleep Apnea on Left and Right Ventricular Myocardial Function and Effects of CPAP Therapy

Background

Obstructive sleep apnea (OSA) has deteriorating effect on LV function, whereas its impact on RV function is controversial. We aimed to determine the effect of OSA and continuous positive airway pressure (CPAP) treatment on left and right ventricular (LV, RV) function using transthoracic echocardiography (TTE) and 2 dimensional speckle tracking (2D ST) analysis of RV deformation capability.

Methods and Results

82 patients with OSA and need for CPAP therapy were prospectively enrolled and underwent TTE at study inclusion and after 6 months of follow up (FU). Multivariate regression analysis revealed an independent association between baseline apical right ventricular longitudinal strain (RV-Sl), BMI and the severity of OSA (apical RV-Sl: P = 0.0002, BMI: P = 0.02). After CPAP therapy, LV functional parameters (LVEF: P<0.0001, LV performance index: P = 0.03, stroke volume: P = 0.042), and apical RV-Sl (P = 0.001) improved significantly. The effect of CPAP therapy was related to severity of OSA (LVEF: AHI 5–14, 66.4±8.8%, 68.5±10.6% [P = ns]; AHI 15–30∶59.8±7.7%, 68.6±9.3% [P = 0.002]; AHI>30∶54.1±12.4%, 68.2±13.6%[P<0.0001]; apical RV-Sl: AHI 5–14: −17.3±8.7%, −16.0±10.8% [P = ns], AHI 15–30: −9.8±6.0%, −15.4±10.9% [P = 0.028], AHI>30: −6.3±5.7%, −17.9±11.2% [P<0.0001]).

Conclusions

OSA seems to have deteriorating effect on LV and RV function. We found a beneficial effect of CPAP on LV and RV functional parameters predominately in patients with severe OSA. 2D speckle tracking might be of value to determine early changes in global and regional right ventricular function.     

Intratibial Injection of Human Multiple Myeloma Cells in NOD/SCID IL-2Rγ(Null) Mice Mimics Human Myeloma and Serves as a Valuable Tool for the Development of Anticancer Strategies

Background

We systematically analyzed multiple myeloma (MM) cell lines and patient bone marrow cells for their engraftment capacity in immunodeficient mice and validated the response of the resulting xenografts to antimyeloma agents.

Design and Methods

Using flow cytometry and near infrared fluorescence in-vivo-imaging, growth kinetics of MM cell lines L363 and RPMI8226 and patient bone marrow cells were investigated with use of a murine subcutaneous bone implant, intratibial and intravenous approach in NOD/SCID, NOD/SCID treated with CD122 antibody and NOD/SCID IL-2Rγ(null) mice (NSG).

Results

Myeloma growth was significantly increased in the absence of natural killer cell activity (NSG or αCD122-treated NOD/SCID). Comparison of NSG and αCD122-treated NOD/SCID revealed enhanced growth kinetics in the former, especially with respect to metastatic tumor sites which were exclusively observed therein. In NSG, MM cells were more tumorigenic when injected intratibially than intravenously. In NOD/SCID in contrast, the use of juvenile long bone implants was superior to intratibial or intravenous cancer cell injection. Using the intratibial NSG model, mice developed typical disease symptoms exclusively when implanted with human MM cell lines or patient-derived bone marrow cells, but not with healthy bone marrow cells nor in mock-injected animals. Bortezomib and dexamethasone delayed myeloma progression in L363- as well as patient-derived MM cell bearing NSG. Antitumor activity could be quantified via flow cytometry and in vivo imaging analyses.

Conclusions

Our results suggest that the intratibial NSG MM model mimics the clinical situation of the disseminated disease and serves as a valuable tool in the development of novel anticancer strategies.     

Magneto immuno-PCR: a novel immunoassay based on biogenic magnetosome nanoparticles

We describe an innovative modification of the Immuno-PCR technology for automatable high sensitive antigen detection. The Magneto Immuno-PCR (M-IPCR) is based on antibody-functionalized biogenic magnetosome nanoparticles revealing major advantages over synthetic magnetic particles. The general principle of the M-IPCR is similar to that of a two-sided (sandwich) immunoassay. However, antibody-functionalized magnetosome conjugates were employed for the immobilization and magnetic enrichment of the signal generating detection complex enabling the establishment of a surface independent immunoassay. To this end, the M-IPCR was carried out by simultaneously tagging the antigen with the reagent for read-out, i.e., a conjugate comprising the specific antibody and DNA fragments, in the presence of the antibody-functionalized magnetosomes. To demonstrate the general functionality of the M-IPCR, the detection of recombinant Hepatitis B surface Antigen (HBsAg) in human serum was established. We observed a detection limit of 320pg/ml of HBsAg using the M-IPCR, which was about 100-fold more sensitive than the analogous Magneto-ELISA, established in parallel for comparison purposes.