Use of freeze-cracking in ontogenetic research in <Emphasis Type="Italic">Macrostomum lignano</Emphasis> (Macrostomida,Rhabditophora)

A method for studying whole mount flatworm embryos based on freeze-cracking of the eggs is described. This method allows successful immunohistological and immunocytological studies of whole mount embryos. It does not require the use of sharpened needles or a microinjection system to puncture the eggshell. Moreover, this method is more practical and less time-consuming than classical puncturing and much cheaper than the use of a microinjection system. The advantages of this method are illustrated by results of several immunolocalisation experiments in the macrostomid flatworm Macrostomum lignano. The optimal procedure and crucial steps for this method are discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Survival and colonization of nematodes in a composting process

Nematodes are omnipresent in composts and are active in virtually all stages of the composting process. Major shifts in species composition, life strategies, and feeding behavior occur during the composting process. Due to the heat peak, nematodes can be virtually absent, but several taxa appear immediately when the temperatures drop. These comprise both taxa present before the heat peak and new taxa. However, it is not known how nematodes populate the compost. In this study, we aimed to assess the survival and colonization capacity of nematodes in compost. Our results showed that composting processes inaccessible to insects or not in contact with soil did not significantly influence nematode succession during composting. However, differences between treatments were found for some specific taxa (i.e., for Acrostichus sp., Neodiplogasteridae sp., Nygolaimoides sp., and Rhabditidae sp. 1), illustrating the importance of insects for the dispersal of nematodes to compost. Experiments in the lab with the blue bottle fly as a possible carrier demonstrated actual transport of nematodes isolated from compost by the fly (i.e., Halicephalobus cfr. gingivalis, Diploscapter coronatus, Diplogasteritus sp., Acrostichus sp., and Mesorhabditis sp.). Juveniles and dauer stages of Aphelenchoides sp., Panagrolaimus sp., and rhabditids survived an experimentally induced temperature peak, while members of Tylenchidae did not. In conclusion, our results indicate that the rapidly changing nematode community in compost is the result of both differential survival and colonization capacities.     

Linear Ubiquitination of NEMO Negatively Regulates the Interferon Antiviral Response through Disruption of the MAVS-TRAF3 Complex

The RIG-I/Mda5 sensors recognize viral intracellular RNA and trigger host antiviral responses. RIG-I signals through the adaptor protein MAVS, which engages various TRAF family members and results in type I interferon (IFNs) and proinflammatory cytokine production via activation of IRFs and NF-κB, respectively. Both the IRF and NF-κB pathways also require the adaptor protein NEMO. We determined that the RIG-I pathway is differentially regulated by the linear ubiquitin assembly complex (LUBAC), which consists of the E3 ligases HOIL-1L, HOIP, and the accessory protein SHARPIN. LUBAC downregulated virus-mediated IFN induction by targeting NEMO for linear ubiquitination. Linear ubiquitinated NEMO associated with TRAF3 and disrupted the MAVS-TRAF3 complex, which inhibited IFN activation while stimulating NF-κB-dependent signaling. In SHARPIN-deficient MEFs, vesicular stomatitis virus replication was decreased due to increased IFN production. Linear ubiquitination thus switches NEMO from a positive to a negative regulator of RIG-I signaling, resulting in an attenuated IFN response.     

Rap1 Can Bypass the FAK-Src-Paxillin Cascade to Induce Cell Spreading and Focal Adhesion Formation

We developed new image analysis tools to analyse quantitatively the extracellular-matrix-dependent cell spreading process imaged by live-cell epifluorescence microscopy. Using these tools, we investigated cell spreading induced by activation of the small GTPase, Rap1. After replating and initial adhesion, unstimulated cells exhibited extensive protrusion and retraction as their spread area increased, and displayed an angular shape that was remodelled over time. In contrast, activation of endogenous Rap1, via 007-mediated stimulation of Epac1, induced protrusion along the entire cell periphery, resulting in a rounder spread surface, an accelerated spreading rate and an increased spread area compared to control cells. Whereas basal, anisotropic, spreading was completely dependent on Src activity, Rap1-induced spreading was refractory to Src inhibition. Under Src inhibited conditions, the characteristic Src-induced tyrosine phosphorylations of FAK and paxillin did not occur, but Rap1 could induce the formation of actomyosin-connected adhesions, which contained vinculin at levels comparable to that found in unperturbed focal adhesions. From these results, we conclude that Rap1 can induce cell adhesion and stimulate an accelerated rate of cell spreading through mechanisms that bypass the canonical FAK-Src-Paxillin signalling cascade.     

Association of FcRn Heavy Chain Encoding Gene (FCGRT) Polymorphisms with IgG Content in Bovine Colostrum

In this study, we evaluated haplotypes of the bovine FCGRT (encoding the FcRn heavy chain) and their relationship to the IgG concentration in bovine colostrum. Four single nucleotide polymorphism (SNP), classified into five haplotypes, were identified in a total of 49 Holstein-Frisians cows. Haplotype 5 was found to be significantly associated with a high IgG level ([OR] = 9.90, 95%CI = 1.11–88.34, p = 0.016) and haplotype 2 exhibited a similar trend ([OR] = 2.89, 95%CI = 1.17–7.11, p = 0.019).     

Genome-Wide Gene Expression Analysis of Bordetella pertussis Isolates Associated with a Resurgence in Pertussis: Elucidation of Factors Involved in the Increased Fitness of Epidemic Strains

Bordetella pertussis (B. pertussis) is the causative agent of whooping cough, which is a highly contagious disease in the human respiratory tract. Despite vaccination since the 1950s, pertussis remains the most prevalent vaccine-preventable disease in developed countries. A recent resurgence pertussis is associated with the expansion of B. pertussis strains with a novel allele for the pertussis toxin (ptx) promoter ptxP3 in place of resident ptxP1 strains. The recent expansion of ptxP3 strains suggests that these strains carry mutations that have increased their fitness. Compared to the ptxP1 strains, ptxP3 strains produce more Ptx, which results in increased virulence and immune suppression. In this study, we investigated the contribution of gene expression changes of various genes on the increased fitness of the ptxP3 strains. Using genome-wide gene expression profiling, we show that several virulence genes had higher expression levels in the ptxP3 strains compared to the ptxP1 strains. We provide the first evidence that wildtype ptxP3 strains are better colonizers in an intranasal mouse infection model. This study shows that the ptxP3 mutation and the genetic background of ptxP3 strains affect fitness by contributing to the ability to colonize in a mouse infection model. These results show that the genetic background of ptxP3 strains with a higher expression of virulence genes contribute to increased fitness.     

Smelling the Diagnosis: The Electronic Nose as Diagnostic Tool in Inflammatory Arthritis. A Case-Reference Study

Objective

To investigate whether exhaled breath analysis using an electronic nose can identify differences between inflammatory joint diseases and healthy controls.

Methods

In a cross-sectional study, the exhaled breath of 21 rheumatoid arthritis (RA) and 18 psoriatic arthritis (PsA) patients with active disease was compared to 21 healthy controls using an electronic nose (Cyranose 320; Smiths Detection, Pasadena, CA, USA). Breathprints were analyzed with principal component analysis, discriminant analysis, and area under curve (AUC) of receiver operating characteristics (ROC) curves. Volatile organic compounds (VOCs) were identified by gas chromatography and mass spectrometry (GC-MS), and relationships between breathprints and markers of disease activity were explored.

Results

Breathprints of RA patients could be distinguished from controls with an accuracy of 71% (AUC 0.75, 95% CI 0.60–0.90, sensitivity 76%, specificity 67%). Breathprints from PsA patients were separated from controls with 69% accuracy (AUC 0.77, 95% CI 0.61–0.92, sensitivity 72%, specificity 71%). Distinction between exhaled breath of RA and PsA patients exhibited an accuracy of 69% (AUC 0.72, 95% CI 0.55–0.89, sensitivity 71%, specificity 72%). There was a positive correlation in RA patients of exhaled breathprints with disease activity score (DAS28) and number of painful joints. GC-MS identified seven key VOCs that significantly differed between the groups.

Conclusions

Exhaled breath analysis by an electronic nose may play a role in differential diagnosis of inflammatory joint diseases. Data from this study warrant external validation.