Usage of primary cells to delineate IFN-gamma-responsive DNA elements in the HLA-DRA promoter and to identify a novel IFN-gamma-enhanced nuclear factor.

The IFN-gamma regulation of the HLA-DRA gene was examined in a primary cell type, the astrocyte. Site-specific mutagenesis of the DRA promoter reveals that three known sequences, S, X, and Y, are required for an optimal IFN-gamma response. Specifically for the X sequence, the X1, but not the X2, site is involved in IFN-gamma regulation of HLA-DRA in the astrocyte. Most interesting, a novel IFN-gamma-enhanced protein (IFNEX) with specificity for the X element has consistently been observed in nuclear extracts made from primary astrocytes. The correlation of the functional importance of X1 in IFN-gamma-regulated DRA expression and the enhancement of IFNEX by IFN-gamma strongly suggests that IFNEX may play a crucial role in IFN-gamma-regulated class II MHC gene expression.     

Trehalase activity and its regulation during growth of Saccharomyces cerevisiae.

Trehalase activity decreased in 95% at the onset of the transition phase of growth of S. cerevisiae. The question which we raised was whether this phenomenon was due to proteolysis or to conversion of the enzyme to a less active form (dephosphorylation). Immunological methods allowed to identify the presence of the trehalase protein during cell growth. At the same stage of growth, an increase in the non-phosphorylated enzyme was detected "in vitro". Results utilizing mutant strains also indicated that regulation occurred by interconversion of forms. The same mechanism also seems to control trehalase activity in non proliferating conditions.     

Permeabilization of yeast for in situ determination of α-glucosidase

A quantitative in situ assay of yeast α-glucosidase involving permeabilization of the cells by freezing and thawing is described. The assay was applied to different strains in different physiological states and was shown to give results comparable to those obtained with total cell homogenates. The primary advantage of the in situ assay was the possibility of analyzing a large number of samples from the same culture during a growth curve using a very reduced cell mass.     

Using variation partitioning techniques to quantify the effects of invasive alien species on native urban bird assemblages

Quantifying the impacts that invasive alien species (IAS) cause on affected systems is not an easy task. Here, we explore the application of variation partitioning techniques to measure and control for the effects of possible confounding factors when studying the impact that feral pigeons, European starlings, and house sparrows cause on native urban bird communities in Mexico. We argue that these IAS are provoking a severe impact on whole assemblages of native passerines only if (a) their marginal effect is statistically significant, (b) it remains so after partialling out other explanatory variables, and (c) is larger than (or similar to) the conditional effect of these covariates. We censused passerine bird assemblages and measured habitat variables in a number of greenspaces in three replicate study areas. Then, by means of partial redundancy analyses, we decomposed the total variability in bird data as a function of IAS, physical variables and vegetation data. In one of the study areas the marginal effect of IAS on native assemblages was significant, but the conditional effect was not. We conclude that this IAS effect was confounded with other explanatory variables. In the other study areas, no (marginal or partial) significant effect was found. Without invoking interspecific competition, our results support the opportunistic hypothesis, according to which IAS can exploit ecological conditions in modern cities that native species cannot even tolerate. Finally, apart from the Precautionary Principle, we found no scientific justification to control the abundance of the three IAS in our study areas.     

The development and characterization of an E. coli O25B bioconjugate vaccine

Extraintestinal pathogenic Escherichia coli (ExPEC) cause a wide range of clinical diseases such as bacteremia and urinary tract infections. The increase of multidrug resistant ExPEC strains is becoming a major concern for the treatment of these infections and E. coli has been identified as a critical priority pathogen by the WHO. Therefore, the development of vaccines has become increasingly important, with the surface lipopolysaccharide constituting a promising vaccine target. This study presents genetic and structural analysis of clinical urine isolates from Switzerland belonging to the serotype O25. Approximately 75% of these isolates were shown to correspond to the substructure O25B only recently described in an emerging clone of E. coli sequence type 131. To address the high occurrence of O25B in clinical isolates, an O25B glycoconjugate vaccine was prepared using an E. coli glycosylation system. The O antigen cluster was integrated into the genome of E. coli W3110, thereby generating an E. coli strain able to synthesize the O25B polysaccharide on a carrier lipid. The polysaccharide was enzymatically conjugated to specific asparagine side chains of the carrier protein exotoxin A (EPA) of Pseudomonas aeruginosa by the PglB oligosaccharyltransferase from Campylobacter jejuni. Detailed characterization of the O25B-EPA conjugate by use of physicochemical methods including NMR and GC-MS confirmed the O25B polysaccharide structure in the conjugate, opening up the possibility to develop a multivalent E. coli conjugate vaccine containing O25B-EPA.

     

3′-End processing of histone pre-mRNAs in Drosophila: U7 snRNP is associated with FLASH and polyadenylation factors

3′-End cleavage of animal replication-dependent histone pre-mRNAs is controlled by the U7 snRNP. Lsm11, the largest component of the U7-specific Sm ring, interacts with FLASH, and in mammalian nuclear extracts these two proteins form a platform that recruits the CPSF73 endonuclease and other polyadenylation factors to the U7 snRNP. FLASH is limiting, and the majority of the U7 snRNP in mammalian extracts exists as a core particle consisting of the U7 snRNA and the Sm ring. Here, we purified the U7 snRNP from Drosophila nuclear extracts and characterized its composition by mass spectrometry. In contrast to the mammalian U7 snRNP, a significant fraction of the Drosophila U7 snRNP contains endogenous FLASH and at least six subunits of the polyadenylation machinery: symplekin, CPSF73, CPSF100, CPSF160, WDR33, and CstF64. The same composite U7 snRNP is recruited to histone pre-mRNA for 3′-end processing. We identified a motif in Drosophila FLASH that is essential for the recruitment of the polyadenylation complex to the U7 snRNP and analyzed the role of other factors, including SLBP and Ars2, in 3′-end processing of Drosophila histone pre-mRNAs. SLBP that binds the upstream stem–loop structure likely recruits a yet-unidentified essential component(s) to the processing machinery. In contrast, Ars2, a protein previously shown to interact with FLASH in mammalian cells, is dispensable for processing in Drosophila. Our studies also demonstrate that Drosophila symplekin and three factors involved in cleavage and polyadenylation—CPSF, CstF, and CF I_m—are present in Drosophila nuclear extracts in a stable supercomplex.     

Never ending story: a lesson in using sampling efficiency methods with ground beetles

Biological inventory is a crucial activity in life sciences field research. However, it is sometimes time-consuming and laborious to take representative samplings of communities, especially in the case of invertebrates. In this paper, we address the issue of sampling efficiency and its influence on obtained results. As a study system, we used data on epigeic carabid beetles (Carabidae) collected in 1999–2001 in the Warta River valley of western Poland. We trapped a total of 17,722 individuals belonging to 108 species. However, due to rarefaction methods, the expected number of species was estimated at 134–140, suggesting that from 26 to 32 species are missing from the material, even expressed as a huge number of collected specimens. The estimated probability that another captured individual will represent a new species (i.e. a species that was not already recorded) is 0.0010. In order to record all the species present in the study area, another 193,338 individuals need to be sampled (abundance-based approach) or another 1,871 samples need to be collected (incidence-based approach). This means that the collected material should be 10.9 times greater (or 7.9 times greater for incidence-based data) than what was actually collected in order to record all the species present in the study area. The results show that, in practice, full inventory is simply nearly impossible to achieve, and this knowledge should be included in inventory planning. Therefore, we argue that species accumulation curves and unseen species estimators need to be carefully examined and threshold probability of detecting a new species should be built into the design of inventory science. The ratio between recorded and estimated species richness and the estimated efficiency of further sampling can be easily computed with available freeware software and should be incorporated when performing biological inventories.     

Molecular diagnosis of cystic echinococcosis in humans from central Poland

The identity of the causative agent of cystic echinococcosis (CE) in humans from central Poland receiving treatment between 2000 and 2010 was determined. A total of 47 samples obtained after hepatectomy were examined and protoscoleces were identified in wet preparations in 27 cases. Using DNA extracted from the samples, two mitochondrial regions (nad1 and cox1 genes) were amplified and the nad1 fragment was sequenced. This PCR analysis confirmed the presence of Echinococcus species in 30 cases and nad1 sequence alignments showed identity with the G7 (pig) strain, Echinococcus canadensis. These data demonstrate that the pig strain of this parasite is the most frequent causative agent of human cystic echinococcosis in central Poland.     

Can Body Condition and Somatic Indices be Used to Evaluate Metal-Induced Stress in Wild Small Mammals?

Wildlife is exposed to natural (e.g., food availability and quality, parasitism) and anthropogenic stressors (e.g., habitat fragmentation, toxicants). Individual variables (e.g., age, gender) affect behaviour and physiology of animals. Together, these parameters can create both great inter-individual variations in health indicators and interpretation difficulties. We investigated the relevance of body condition and somatic indices (liver, kidneys) as indicators of health status in wood mice (Apodemus sylvaticus, n = 560) captured along a metal pollution gradient in four landscape types (30 sampling squares 500-m sided). The indices were calculated using a recently proposed standard major axis regression instead of an ordinary least square regression. After considering age and gender for the body condition index, no landscape type influence was detected in the indices. However, important index variability was observed between sampling squares; this effect was included as a random effect in linear models. After integrating all individual and environmental variables that may affect the indices, cadmium (Cd) concentrations in both the liver and kidneys were negatively related to body condition and liver indices only for individuals from highly contaminated sites. Lead in the liver was negatively related to the liver index, and Cd in kidneys was positively linked to the kidney index, potentially suggesting metal-induced stress. However, interpretation of these indices as a wildlife ecotoxicology tool should be performed with caution due to the sensitivity of potentially confounding variables (e.g., individual factors and environmental parameters).