Changes in microbiota of rainbow trout caused by sediments contamination

The abundance, composition and hydrocarbon-degrading bacteria, as possible biomarkers of contamination with oil hydrocarbons, of autochthonous and alochtonous microbiota of the digestive tract of rainbow trout have been estimated. The samples of the bottom sediments for microbiological tests have been collected and a response of natural bacterial communities in the digestive tract of rainbow trout and nutritional changes has been investigated. Experimental fish have been fed with a mixture of three substances with the aim to assess the influence of hydrocarbon-degrading bacteria contained in the sediments on the microbiota of rainbow trout’s digestive tracts. The abundance values of rainbow trout intestinal heterotrophic bacteria were found to change depending on alochtonous microbiota of different bottom sediments given to the experimental fish with food in vitro. According to the results of our research, it is likely that the changes in the abundance values of the microbiota of the digestive tract of fish and in the proportions of functional groups of the bacteria allow us to determine changes in the functional activity of bacteria depending on food composition. Any relative increase or decrease of abundance or activity of alochtonous microbiota allows the prediction of toxic effects of the contaminants on animals and the environment.     

Development and Application of Genomic Control Methods for Genome-Wide Association Studies Using Non-Additive Models

Genome-wide association studies (GWAS) comprise a powerful tool for mapping genes of complex traits. However, an inflation of the test statistic can occur because of population substructure or cryptic relatedness, which could cause spurious associations. If information on a large number of genetic markers is available, adjusting the analysis results by using the method of genomic control (GC) is possible. GC was originally proposed to correct the Cochran-Armitage additive trend test. For non-additive models, correction has been shown to depend on allele frequencies. Therefore, usage of GC is limited to situations where allele frequencies of null markers and candidate markers are matched. In this work, we extended the capabilities of the GC method for non-additive models, which allows us to use null markers with arbitrary allele frequencies for GC. Analytical expressions for the inflation of a test statistic describing its dependency on allele frequency and several population parameters were obtained for recessive, dominant, and over-dominant models of inheritance. We proposed a method to estimate these required population parameters. Furthermore, we suggested a GC method based on approximation of the correction coefficient by a polynomial of allele frequency and described procedures to correct the genotypic (two degrees of freedom) test for cases when the model of inheritance is unknown. Statistical properties of the described methods were investigated using simulated and real data. We demonstrated that all considered methods were effective in controlling type 1 error in the presence of genetic substructure. The proposed GC methods can be applied to statistical tests for GWAS with various models of inheritance. All methods developed and tested in this work were implemented using R language as a part of the GenABEL package.     

Overwinter survival depends on immune defence and body length in male Aquarius najas water striders

Winter survival of organisms has a crucial effect on their fitness in a seasonal environment. We tested whether overwinter survival of male water striders, Aquarius najas (De Geer) (Heteroptera: Gerridae), is associated with the time of season they leave the water for winter diapause. This was performed by comparing parameters of males leaving the water at the beginning of autumn and 1 month later. The results show significantly higher overwinter survival in males of the early diapausing group. They were found to be larger than late diapausing individuals. Overall, overwinter survival was found to be associated with body length. The strength of immune defence measured as encapsulation response against nylon monofilament appeared to be stronger in the early than in the late dia‐pausing group. Body length had an effect on the encapsulation rates of water striders, with larger males having a stronger encapsulation response. The amount of lipids was larger in males of the early diapausing group, and the relationship between body length and absolute lipid reserves was positive in both groups. However, a correlation between body reserves and body length was not found when lipid content was expressed as a proportion of total body weight. The results suggest that body length is the main variable linking overwinter survival and resistance against pathogens and parasitoids. This study shows that differences in resistance can explain differential winter survival of larger and smaller A. najas water striders.     

Body size of newborns in relation to mother's ethnicity and education: a pilot study from Vilnius City (Lithuania), 2005-2010

The purpose of this study was to analyse body size indices of newborns in Vilnius city (Lithuania) during 2005-2010 in relation with mother's education and ethnicity, and in parallel with the changes of socio-economic situation during the recent years. The present results were based on data (N = 18,084) from the Vilnius University Clinic of Obstetrics and Gynaecology. Comparison of the present Lithuanian data with other newborn studies was made. The analysis of socio-economic and demographic indicators of Lithuania, and the comparison of Gross Domestic Product of various countries was performed. The comparison of body size of newborns' data from the different countries showed that Lithuanian newborns were among the biggest babies. Some statistically significant differences in body size of newborns from different ethnic groups were established. Body length of Lithuanian newborns (M = 52.6 cm, SD = 2.5) was higher than length of Russian, Ukrainian and Romanian newborns. Body weight of Lithuanian newborns (M = 3511 g, SD = 485) was bigger than birth weight of Russian, Polish, Ukrainian and Romanian newborns. The analysis of newborns size by mother's education showed that body weight of neonates from mothers with the university education and from each other education group was bigger in comparison with the babies from respectively lower education group. The comparison of newborns weight by mother's ethnicity in relation to education level revealed nearly no discrepancies between size of newborns from mothers with the same education level at different ethnic group. The analysis of birth parameters by year has not established a statistically significant difference between the mean values for the body weight and body length of the whole investigated contingent of the full-term, single-birth newborns from Vilnius city during the 2005-2010. However, the tendency has been revealed that newborns from mothers with lower education were the most susceptible to negative economic changes after the 2008.     

CNN3 Regulates Trophoblast Invasion and Is Upregulated by Hypoxia in BeWo Cells

CNN3 is an ubiquitously expressed F-actin binding protein, shown to regulate trophoblast fusion and hence seems to play a role in the placentation process. In this study we demonstrate that CNN3 levels are upregulated under low oxygen conditions in the trophoblast cell line BeWo. Since hypoxia is discussed to be a pro-migratory stimulus for placental cells, we examined if CNN3 is involved in trophoblast invasion. Indeed, when performing a matrigel invasion assay we were able to show that CNN3 promotes BeWo cell invasion. Moreover, CNN3 activates the MAPKs ERK1/2 and p38 in trophoblast cells and interestingly, both kinases are involved in BeWo invasion. However, when we repeated the experiments under hypoxic conditions, CNN3 did neither promote cell invasion nor MAPK activation. These results indicate that CNN3 promotes invasive processes by the stimulation of ERK1/2 and/or p38 under normoxic conditions in BeWo cells, but seems to have different functions at low oxygen levels. We further speculated that CNN3 expression might be altered in human placentas derived from pregnancies complicated by IUGR and preeclampsia, since these placental disorders have been described to go along with impaired trophoblast invasion. Our studies show that, at least in our set of placenta samples, CNN3 expression is neither deregulated in IUGR nor in preeclampsia. In summary, we identified CNN3 as a new pro-invasive protein in trophoblast cells that is induced under low oxygen conditions.     

Ligand‐Induced Compaction of the PEX5 Receptor‐Binding Cavity Impacts Protein Import Efficiency into Peroxisomes

Peroxisomes entirely rely on the import of their proteome across the peroxisomal membrane. Recognition efficiencies of peroxisomal proteins vary by more than 1000‐fold, but the molecular rationale behind their subsequent differential import and sorting has remained enigmatic. Using the protein cargo alanine‐glyoxylate aminotransferase as a model, an unexpected increase from 34 to 80% in peroxisomal import efficiency of a single‐residue mutant has been discovered. By high‐resolution structural analysis, we found that it is the recognition receptor PEX5 that adapts its conformation for high‐affinity binding rather than the cargo protein signal motif as previously thought. During receptor recognition, the binding cavity of the receptor shrinks to one third of its original volume. This process is impeded in the wild‐type protein cargo because of a bulky side chain within the recognition motif, which blocks contraction of the PEX5 binding cavity. Our data provide a new insight into direct protein import efficiency by removal rather than by addition of an apparent specific sequence signature that is generally applicable to peroxisomal matrix proteins and to other receptor recognition processes.      

Integration of conventional quantitative and phospho‐proteomics reveals new elements in activated Jurkat T‐cell receptor pathway maintenance

Recent years have seen a constant development of tools for the global assessment of phosphoproteins. Here, we outline a concept for integrating approaches for quantitative proteomics and phosphoproteomics. The strategy was applied to the analysis of changes in signalling and protein synthesis occurring after activation of the T‐cell receptor (TCR) pathway in a T‐cell line (Jurkat cells). For this purpose, peptides were obtained from four biological replicates of activated and control Jurkat T‐cells and phosphopeptides enriched via a TiO₂‐based chromatographic step. Both phosphopeptide‐enriched and flow‐through fractions were analyzed by LC–MS. We observed 1314 phosphopeptides in the enriched fraction whereas 19 were detected in the flow‐through, enabling the quantification of 414 and eight phosphoproteins in the respective fractions. Pathway analysis revealed the differential regulation of many metabolic pathways. Among the quantified proteins, 11 kinases with known TCR‐related function were detected. A kinase‐substrate database search for the phosphosites identified also confirmed the activity of a further ten kinases. In total, these two approaches provided evidence of 19 unique TCR‐related kinases. The combination of phosphoproteomics and conventional quantitative shotgun analysis leads to a more comprehensive assessment of the signalling networks needed for the maintenance of the activated status of Jurkat T‐cells.     

Evolutionary Trade-Offs Underlie the Multi-faceted Virulence of Staphylococcus aureus

Bacterial virulence is a multifaceted trait where the interactions between pathogen and host factors affect the severity and outcome of the infection. Toxin secretion is central to the biology of many bacterial pathogens and is widely accepted as playing a crucial role in disease pathology. To understand the relationship between toxicity and bacterial virulence in greater depth, we studied two sequenced collections of the major human pathogen Staphylococcus aureus and found an unexpected inverse correlation between bacterial toxicity and disease severity. By applying a functional genomics approach, we identified several novel toxicity-affecting loci responsible for the wide range in toxic phenotypes observed within these collections. To understand the apparent higher propensity of low toxicity isolates to cause bacteraemia, we performed several functional assays, and our findings suggest that within-host fitness differences between high- and low-toxicity isolates in human serum is a contributing factor. As invasive infections, such as bacteraemia, limit the opportunities for onward transmission, highly toxic strains could gain an additional between-host fitness advantage, potentially contributing to the maintenance of toxicity at the population level. Our results clearly demonstrate how evolutionary trade-offs between toxicity, relative fitness, and transmissibility are critical for understanding the multifaceted nature of bacterial virulence.     

Synchronized mammalian cell culture: Part I—A physical strategy for synchronized cultivation under physiological conditions

Conventional analysis and optimization procedures of mammalian cell culture processes mostly treat the culture as a homogeneous population. Hence, the focus is on cell physiology and metabolism, cell line development, and process control strategy. Impact on cultivations caused by potential variations in cellular properties between different subpopulations, however, has not yet been evaluated systematically. One main cause for the formation of such subpopulations is the progress of all cells through the cell cycle. The interaction of potential cell cycle specific variations in the cell behavior with large‐scale process conditions can be optimally determined by means of (partially) synchronized cultivations, with subsequent population resolved model analysis. Therefore, it is desirable to synchronize a culture with minimal perturbation, which is possible with different yield and quality using physical selection methods, but not with frequently used chemical or whole‐culture methods. Conventional nonsynchronizing methods with subsequent cell‐specific, for example, flow cytometric analysis, can only resolve cell‐limited effects of the cell cycle. In this work, we demonstrate countercurrent‐flow centrifugal elutriation as a useful physical method to enrich mammalian cell populations within different phases of a cell cycle, which can be further cultivated for synchronized growth in bioreactors under physiological conditions. The presented combined approach contrasts with other physical selection methods especially with respect to the achievable yield, which makes it suitable for bioreactor scale cultivations. As shown with two industrial cell lines (CHO‐K1 and human AGE1.HN), synchronous inocula can be obtained with overall synchrony degrees of up to 82% in the G1 phase, 53% in the S phase and 60% in the phase, with enrichment factors ( ) of 1.71, 1.79, and 4.24 respectively. Cells are able to grow with synchrony in bioreactors over several cell cycles. This strategy, combined with population‐resolved model analysis and parameter extraction as described in the accompanying paper, offers new possibilities for studies of cell lines and processes at levels of cell cycle and population under physiological conditions. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:165–174, 2015