Novel target genes and a valid biomarker panel identified for cholangiocarcinoma

Cholangiocarcinoma is notoriously difficult to diagnose, and the mortality rate is high due to late clinical presentation. CpG island promoter methylation is frequently seen in cancer development. In the present study, we aimed at identifying novel epigenetic biomarkers with the potential to improve the diagnostic accuracy of cholangiocarcinoma. Microarray data analyses of cholangiocarcinoma cell lines treated with epigenetic drugs and their untreated counterparts were compared with previously published gene expression profiles of primary tumors and with non-malignant controls. Genes responding to the epigenetic treatment that were simultaneously downregulated in primary cholangiocarcinoma compared with controls (n = 43) were investigated for their promoter methylation status in cancer cell lines from the gastrointestinal tract. Genes commonly methylated in cholangiocarcinoma cell lines were subjected to quantitative methylation-specific polymerase chain reaction in a total of 93 clinical samples (cholangiocarcinomas and non-malignant controls). CDO1, DCLK1, SFRP1 and ZSCAN18, displayed high methylation frequencies in primary tumors and were unmethylated in controls. At least one of these four biomarkers was positive in 87% of the tumor samples, with a specificity of 100%. In conclusion, the novel methylation-based biomarker panel showed high sensitivity and specificity for cholangiocarcinoma. The potential of these markers in early diagnosis of this cancer type should be further explored.     

Community dynamics of intertidal soft-bottom mussel beds over two decades

Macrozoobenthos communities in the North Sea showed pronounced changes over the past decade in relation to an increasing number of invasive species and climate change. We analysed data sets spanning 22 years on abundance, biomass and species composition of intertidal soft bottom mussel beds near the island of Sylt (German Bight) in the Northern Wadden Sea, based on surveys from 1983/1984, 1990, 1993 and from 1999 to 2005. Mussel bed area and blue mussel biomass decreased, and a change in the dominance structure in the associated community comparing 1984 to mid-1990s with the period from 1999 to 2005 was observed. Coverage of the mussel beds with the algae Fucus vesiculosus decreased since the end of the 1990s. Within the study period biomass and densities of the associated community increased significantly. Dominance structure changed mainly because of increasing abundances of associated epibenthic taxa. Apart from the Pacific oyster Crassostrea gigas all other alien species were already present in the area during the study period. Community changes already started before Pacific oysters became abundant. An attempt is made to evaluate effects on the observed changes of decreasing mussel biomass, ageing of mussel beds, decreasing fucoid coverage and increasing abundances of invader. All four factors are assumed to contribute to changing community structure of intertidal mussel beds.     

First observations of temperature tolerances of adult male snow crab (Chionoecetes opilio) from the Barents Sea population and the effects on the fisheries strategy

This study investigates the effects of temperature on the survival, food intake, oxygen consumption and growth during long-term live holding of captive male snow crab (Chionoecetes opilio) (average?=?0.7?kg). The crabs were held at three different temperatures, 3, 6 and 9°C. The trials were done using groups of snow crabs held in nine land-based holding tanks (three replicates per temperature treatment). The results showed that temperature had a significant effect on survival. The survival rate at 3°C (61%) was significantly higher than at 6°C (33%) and at 9°C (28%). Specific oxygen consumption rates of unfed crab at 6°C were significantly higher than at 9°C and 3°C. In summary, the current study shows that the Barents Sea snow crab have a narrow temperature range in which they thrive compared with the Barents Sea red king crab (Paralithodes camtschaticus). Barents Sea snow crab has similar metabolic and physiological attributes to other major snow crab populations. In conditions when ambient temperatures are approximately 6°C, it may prove beneficial for animal welfare and also be financially advantageous to reduce ambient water temperatures in live snow crab holding facilities on boat or on land.     

Hemispheric Lateralization of Motor Thresholds in Relation to Stuttering

Stuttering is a complex speech disorder. Previous studies indicate a tendency towards elevated motor threshold for the left hemisphere, as measured using transcranial magnetic stimulation (TMS). This may reflect a monohemispheric motor system impairment. The purpose of the study was to investigate the relative side-to-side difference (asymmetry) and the absolute levels of motor threshold for the hand area, using TMS in adults who stutter (n = 15) and in controls (n = 15). In accordance with the hypothesis, the groups differed significantly regarding the relative side-to-side difference of finger motor threshold (p = 0.0026), with the stuttering group showing higher motor threshold of the left hemisphere in relation to the right. Also the absolute level of the finger motor threshold for the left hemisphere differed between the groups (p = 0.049). The obtained results, together with previous investigations, provide support for the hypothesis that stuttering tends to be related to left hemisphere motor impairment, and possibly to a dysfunctional state of bilateral speech motor control.     

Chaperone-usher fimbriae in a diverse selection of Gallibacterium genomes

Background

Fimbriae are bacterial cell surface organelles involved in the pathogenesis of many bacterial species, including Gallibacterium anatis, in which a F17-like fimbriae of the chaperone-usher (CU) family was recently shown to be an important virulence factor and vaccine candidate. To reveal the distribution and variability of CU fimbriae 22 genomes of the avian host-restricted bacteria Gallibacterium spp. were investigated. Fimbrial clusters were classified using phylogeny-based and conserved domain (CD) distribution-based approaches. To characterize the fimbriae in depth evolutionary analysis and in vitro expression of the most prevalent fimbrial clusters was performed.

Results

Overall 48 CU fimbriae were identified in the genomes of the examined Gallibacterium isolates. All fimbriae were assigned to γ4 clade of the CU fimbriae of Gram-negative bacteria and were organized in four-gene clusters encoding a putative major fimbrial subunit, a chaperone, an usher and a fimbrial adhesin. Five fimbrial clusters (Flf-Flf4) and eight conserved domain groups were defined to accommodate the identified fimbriae. Although, the number of different fimbrial clusters in individual Gallibacterium genomes was low, there was substantial amino acid sequence variability in the major fimbrial subunit and the adhesin proteins. The distribution of CDs among fimbrial clusters, analysis of their flanking regions, and evolutionary comparison of the strains revealed that Gallibacterium fimbrial clusters likely underwent evolutionary divergence resulting in highly host adapted and antigenically variable fimbriae. In vitro, only the fimbrial subunit FlfA was expressed in most Gallibacterium strains encoding this protein. The absence or scarce expression of the two other common fimbrial subunits (Flf1A and Flf3A) indicates that their expression may require other in vitro or in vivo conditions.

Conclusions

This is the first approach establishing a systematic fimbria classification system within Gallibacterium spp., which indicates a species-wide distribution of γ₄ CU fimbriae among a diverse collection of Gallibacterium isolates. The expression of only one out of up to three fimbriae present in the individual genomes in vitro suggests that fimbriae expression in Gallibacterium is highly regulated. This information is important for future attempts to understand the role of Gallibacterium fimbriae in pathogenesis, and may prove useful for improved control of Gallibacterium infections in chickens.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1093) contains supplementary material, which is available to authorized users.     

Apoptosis inducer NGFI-B is degraded by the proteasome and stabilized by treatment with EGF

NGFI-B is a nuclear receptor and immediate early gene that is upregulated in many different tumour cell lines. As it is involved in cell death and survival, it has been suggested as a target for anti-cancer drugs. The protein level of NGFI-B is important for its functions and may be regulated through induction or stabilization. NGFI-B protein stability was studied using the protein synthesis inhibitor cycloheximide in CV1 cells transiently transfected with NGFI-B. Inhibiting the proteasome with MG132 stabilized NGFI-B, indicating that the proteasome is responsible for break-down of NGFI-B, as it is for many nuclear receptors. In order to determine regions responsible for the break-down of NGFI-B two N-terminal regions with high PEST-scores were deleted. Deletion of amino acids 122-195 containing a PEST-sequence which includes an ERK2 phosphorylation target, gave a more stable protein. In addition, treatment of the cells with the ERK2 activator EGF increased the stability of wild type NGFI-B. We then tested whether a mutation at threonine 142 influenced the stability of NGFI-B. We found that the phosphorylation-mimicking mutant NGFI-B T142E had an increased stability, while the non-phosphorylable mutant (T142A) showed similar stability to the wild type. Thus, EGF-stimulation of cells may be a mechanism for priming the cells for effects of NGFI-B by increasing its stability.     

Assessment of Fusion Gene Status in Sarcomas Using a Custom Made Fusion Gene Microarray

Sarcomas are relatively rare malignancies and include a large number of histological subgroups. Based on morphology alone, the differential diagnoses of sarcoma subtypes can be challenging, but the identification of specific fusion genes aids correct diagnostication. The presence of individual fusion products are routinely investigated in Pathology labs. However, the methods used are time-consuming and based on prior knowledge about the expected fusion gene and often the most likely break-point. In this study, 16 sarcoma samples, representing seven different sarcoma subtypes with known fusion gene status from a diagnostic setting, were investigated using a fusion gene microarray. The microarray was designed to detect all possible exon-exon breakpoints between all known fusion genes in a single analysis. An automated scoring of the microarray data from the 38 known sarcoma-related fusion genes identified the correct fusion gene among the top-three hits in 11 of the samples. The analytical sensitivity may be further optimised, but we conclude that a sarcoma-fusion gene microarray is suitable as a time-saving screening tool to identify the majority of the correct fusion genes.