Diversity and abundance of freshwater Actinobacteria along environmental gradients in the brackish northern Baltic Sea

Actinobacteria are highly abundant in pelagic freshwater habitats and also occur in estuarine environments such as the Baltic Sea. Because of gradients in salinity and other environmental variables estuaries offer natural systems for examining factors that determine Actinobacteria distribution. We studied abundance and community structure of Bacteria and Actinobacteria along two transects in the northern Baltic Sea. Quantitative (CARD-FISH) and qualitative (DGGE and clone libraries) analyses of community composition were compared with environmental parameters. Actinobacteria accounted for 22–27% of all bacteria and the abundance changed with temperature. Analysis of 549 actinobacterial 16S rRNA sequences from four clone libraries revealed a dominance of the freshwater clusters ac I and ac IV, and two new subclusters ( ac I-B scB-5 and ac IV-E) were assigned. Whereas ac I was present at all stations, occurrence of ac II and ac IV differed between stations and was related to dissolved organic carbon (DOC) and chlorophyll a (Chl a ) respectively. The prevalence of the ac I-A and ac I-B subclusters changed in relation to total phosphorus (Tot-P) and Chl a respectively. Community structure of Bacteria and Actinobacteria differed between the river station and all other stations, responding to differences in DOC, Chl a and bacterial production. In contrast, the composition of active Actinobacteria (analysis based on reversely transcribed RNA) changed in relation to salinity and Tot-P. Our study suggests an important ecological role of Actinobacteria in the brackish northern Baltic Sea. It highlights the need to address dynamics at the cluster or subcluster phylogenetic levels to gain insights into the factors regulating distribution and composition of Actinobacteria in aquatic environments.     

Community stability under different correlation structures of species’ environmental responses

The outcome of species interactions in a variable environment is expected to depend on how similarly different species react to variation in environmental conditions. We study community stability (evenness and species diversity) in competitive communities that are either closed or subjected to random migration, under different regimes of environmental forcing. Community members respond to environmental variation: (i) independently (IR), (ii) in a positively correlated way (CR), or (iii) hierarchically, according to niche differences (HR). Increasing the amplitude of environmental variation and environmental reddening both reduce species evenness in closed communities through a reduction in species richness and increased skew in species abundances, under all three environmental response scenarios, although autocorrelation only has a minor effect with HR. Open communities show important qualitative differences, according to changes in the correlation structure of species’ environmental responses. There is an intermediate minimum in evenness for HR communities with increasing environmental amplitude, explained by the interaction of changes in species richness and changes in the variance of within-species environmental responses across the community. Changes in autocorrelation also lead to qualitative differences between IR, CR and HR communities. Our results highlight the importance of considering mechanistically derived, hierarchical environmental correlations between species when addressing the influence of environmental variation on ecological communities, not only uniform environmental correlation across all species within a community.     

Inhibition of human Chk1 causes increased initiation of DNA replication, phosphorylation of ATR targets, and DNA breakage

Human checkpoint kinase 1 (Chk1) is an essential kinase required to preserve genome stability. Here, we show that Chk1 inhibition by two distinct drugs, UCN-01 and CEP-3891, or by Chk1 small interfering RNA (siRNA) leads to phosphorylation of ATR targets. Chk1-inhibition triggered rapid, pan-nuclear phosphorylation of histone H2AX, p53, Smc1, replication protein A, and Chk1 itself in human S-phase cells. These phosphorylations were inhibited by ATR siRNA and caffeine, but they occurred independently of ATM. Chk1 inhibition also caused an increased initiation of DNA replication, which was accompanied by increased amounts of nonextractable RPA protein, formation of single-stranded DNA, and induction of DNA strand breaks. Moreover, these responses were prevented by siRNA-mediated downregulation of Cdk2 or the replication initiation protein Cdc45, or by addition of the CDK inhibitor roscovitine. We propose that Chk1 is required during normal S phase to avoid aberrantly increased initiation of DNA replication, thereby protecting against DNA breakage. These results may help explain why Chk1 is an essential kinase and should be taken into account when drugs to inhibit this kinase are considered for use in cancer treatment.     

Escapement success of silver eels from a German river system is low compared to management‐based estimates

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Regulation and Gene Expression Profiling of NKG2D Positive Human Cytomegalovirus-Primed CD4(+) T-Cells

NKG2D is a stimulatory receptor expressed by natural killer (NK) cells, CD8(+) T-cells, and γδ T-cells. NKG2D expression is normally absent from CD4(+) T-cells, however recently a subset of NKG2D(+) CD4(+) T-cells has been found, which is specific for human cytomegalovirus (HCMV). This particular subset of HCMV-specific NKG2D(+) CD4(+) T-cells possesses effector-like functions, thus resembling the subsets of NKG2D(+) CD4(+) T-cells found in other chronic inflammations. However, the precise mechanism leading to NKG2D expression on HCMV-specific CD4(+) T-cells is currently not known. In this study we used genome-wide analysis of individual genes and gene set enrichment analysis (GSEA) to investigate the gene expression profile of NKG2D(+) CD4(+) T-cells, generated from HCMV-primed CD4(+) T-cells. We show that the HCMV-primed NKG2D(+) CD4(+) T-cells possess a higher differentiated phenotype than the NKG2D(-) CD4(+) T-cells, both at the gene expression profile and cytokine profile. The ability to express NKG2D at the cell surface was primarily determined by the activation or differentiation status of the CD4(+) T-cells and not by the antigen presenting cells. We observed a correlation between CD94 and NKG2D expression in the CD4(+) T-cells following HCMV stimulation. However, knock-down of CD94 did not affect NKG2D cell surface expression or signaling. In addition, we show that NKG2D is recycled at the cell surface of activated CD4(+) T-cells, whereas it is produced de novo in resting CD4(+) T-cells. These findings provide novel information about the gene expression profile of HCMV-primed NKG2D(+) CD4(+) T-cells, as well as the mechanisms regulating NKG2D cell surface expression.     

The metabolic role of isoleucine in detoxification of ammonia in cultured mouse neurons and astrocytes

Cerebral hyperammonemia is a hallmark of hepatic encephalopathy, a debilitating condition arising secondary to liver disease. Pyruvate oxidation including tricarboxylic acid (TCA) cycle metabolism has been suggested to be inhibited by hyperammonemia at the pyruvate and -ketoglutarate dehydrogenase steps. Catabolism of the branched-chain amino acid isoleucine provides both acetyl-CoA and succinyl-CoA, thus by-passing both the pyruvate dehydrogenase and the -ketoglutarate dehydrogenase steps. Potentially, this will enable the TCA cycle to work in the face of ammonium-induced inhibition. In addition, this will provide the -ketoglutarate carbon skeleton for glutamate and glutamine synthesis by glutamate dehydrogenase and glutamine synthetase (astrocytes only), respectively, both reactions fixing ammonium. Cultured cerebellar neurons (primarily glutamatergic) or astrocytes were incubated in the presence of either [U-¹³C]glucose (2.5 mM) and isoleucine (1 mM) or [U-¹³C]isoleucine and glucose. Cell cultures were treated with an acute ammonium chloride load of 2 (astrocytes) or 5 mM (neurons and astrocytes) and incorporation of ¹³C-label into glutamate, aspartate, glutamine and alanine was determined employing mass spectrometry. Labeling from [U-¹³C]glucose in glutamate and aspartate increased as a result of ammonium-treatment in both neurons and astrocytes, suggesting that the TCA cycle was not inhibited. Labeling in alanine increased in neurons but not in astrocytes, indicating elevated glycolysis in neurons. For both neurons and astrocytes, labeling from [U-¹³C]isoleucine entered glutamate and aspartate albeit to a lower extent than from [U-¹³C]glucose. Labeling in glutamate and aspartate from [U-¹³C]isoleucine was decreased by ammonium treatment in neurons but not in astrocytes, the former probably reflecting increased metabolism of unlabeled glucose. In astrocytes, ammonia treatment resulted in glutamine production and release to the medium, partially supported by catabolism of [U-¹³C]isoleucine. In conclusion, i) neuronal and astrocytic TCA cycle metabolism was not inhibited by ammonium and ii) isoleucine may provide the carbon skeleton for synthesis of glutamate/glutamine in the detoxification of ammonium.     

Rapid genotyping of the osteoporosis-associated polymorphic transcription factor Sp1 binding site in the COL1A1 gene by pyrosequencing

We describe a novel polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) sequencingbased assay for rapid genotyping of the polymorphic Sp1 binding site in the COL1A1 gene (1). A single nucleotide G-->T substitution polymorphism at this GC-rich site has recently been reported to be a predictive genetic marker for low bone mineral density (BMD). To simplify screening for this marker, we optimized PCR conditions and subjected the amplicons to pyrosequencing, which is a convenient high-throughput sequence analysis technique, readily amenable to automation. The analysis of 200 deidentified convenience DNA samples extracted from blood revealed genotype frequences in Hardy-Weinberg equilibrium (SS 68.0%, Ss 28.5%, and ss 3.5%) in agreement with other studies of European populations. This study demonstrates for the first time that pyrosequencing can be used for rapid identification of the osteoporosis-associated single nucleotide polymorphism (SNP) in the COL1A1 gene.     

Environmental Variation Generates Environmental Opportunist Pathogen Outbreaks

Many socio-economically important pathogens persist and grow in the outside host environment and opportunistically invade host individuals. The environmental growth and opportunistic nature of these pathogens has received only little attention in epidemiology. Environmental reservoirs are, however, an important source of novel diseases. Thus, attempts to control these diseases require different approaches than in traditional epidemiology focusing on obligatory parasites. Conditions in the outside-host environment are prone to fluctuate over time. This variation is a potentially important driver of epidemiological dynamics and affect the evolution of novel diseases. Using a modelling approach combining the traditional SIRS models to environmental opportunist pathogens and environmental variability, we show that epidemiological dynamics of opportunist diseases are profoundly driven by the quality of environmental variability, such as the long-term predictability and magnitude of fluctuations. When comparing periodic and stochastic environmental factors, for a given variance, stochastic variation is more likely to cause outbreaks than periodic variation. This is due to the extreme values being further away from the mean. Moreover, the effects of variability depend on the underlying biology of the epidemiological system, and which part of the system is being affected. Variation in host susceptibility leads to more severe pathogen outbreaks than variation in pathogen growth rate in the environment. Positive correlation in variation on both targets can cancel the effect of variation altogether. Moreover, the severity of outbreaks is significantly reduced by increase in the duration of immunity. Uncovering these issues helps in understanding and controlling diseases caused by environmental pathogens.