The Leber Congenital Amaurosis Protein AIPL1 and EB Proteins Co-Localize at the Photoreceptor Cilium

Purpose

The aim of this study was to investigate the interaction and co-localization of novel interacting proteins with the Leber congenital amaurosis (LCA) associated protein aryl hydrocarbon receptor interacting protein-like 1 (AIPL1).

Methods

The CytoTrapXR yeast two-hybrid system was used to screen a bovine retinal cDNA library. A novel interaction between AIPL1 and members of the family of EB proteins was confirmed by directed yeast two-hybrid analysis and co-immunoprecipitation assays. The localization of AIPL1 and the EB proteins in cultured cells and in retinal cryosections was examined by immunofluorescence microscopy and cryo-immunogold electron microscopy.

Results

Yeast two-hybrid (Y2H) analysis identified the interaction between AIPL1 and the EB proteins, EB1 and EB3. EB1 and EB3 were specifically co-immunoprecipitated with AIPL1 from SK-N-SH neuroblastoma cells. In directed 1:1 Y2H analysis, the interaction of EB1 with AIPL1 harbouring the LCA-causing mutations A197P, C239R and W278X was severely compromised. Immunofluorescent confocal microscopy revealed that AIPL1 did not co-localize with endogenous EB1 at the tips of microtubules, endogenous EB1 at the microtubule organising centre following disruption of the microtubule network, or with endogenous β-tubulin. Moreover, AIPL1 did not localize to primary cilia in ARPE-19 cells, whereas EB1 co-localized with the centrosomal marker pericentrin at the base of primary cilia. However, both AIPL1 and the EB proteins, EB1 and EB3, co-localized with centrin-3 in the connecting cilium of photoreceptor cells. Cryo-immunogold electron microscopy confirmed the co-localization of AIPL1 and EB1 in the connecting cilia in human retinal photoreceptors.

Conclusions

AIPL1 and the EB proteins, EB1 and EB3, localize at the connecting cilia of retinal photoreceptor cells, but do not co-localize in the cellular microtubule network or in primary cilia in non-retinal cells. These findings suggest that AIPL1 function in these cells is not related to the role of EB proteins in microtubule dynamics or primary ciliogenesis, but that their association may be related to a specific role in the specialized cilia apparatus of retinal photoreceptors.     

Vitamin D deficiency exacerbates COPD-like characteristics in the lungs of cigarette smoke-exposed mice

Background

Chronic obstructive pulmonary disease (COPD) is characterized by excessive inflammation and disturbed bacterial clearance in the airways. Although cigarette smoke (CS) exposure poses a major risk, vitamin D deficiency could potentially contribute to COPD progression. Many in vitro studies demonstrate important anti-inflammatory and antibacterial effects of vitamin D, but a direct contribution of vitamin D deficiency to COPD onset and disease progression has not been explored.

Methods

In the current study, we used a murine experimental model to investigate the combined effect of vitamin D deficiency and CS exposure on the development of COPD-like characteristics. Therefore, vitamin D deficient or control mice were exposed to CS or ambient air for a period of 6 (subacute) or 12 weeks (chronic). Besides lung function and structure measurements, we performed an in depth analysis of the size and composition of the cellular infiltrate in the airways and lung parenchyma and tested the ex vivo phagocytic and oxidative burst capacity of alveolar macrophages.

Results

Vitamin D deficient mice exhibited an accelerated lung function decline following CS exposure compared to control mice. Furthermore, early signs of emphysema were only observed in CS-exposed vitamin D deficient mice, which was accompanied by elevated levels of MMP-12 in the lung. Vitamin D deficient mice showed exacerbated infiltration of inflammatory cells in the airways and lung parenchyma after both subacute and chronic CS exposure compared to control mice. Furthermore, elevated levels of typical proinflammatory cytokines and chemokines could be detected in the bronchoalveolar lavage fluid (KC and TNF-α) and lung tissue (IP-10, MCP-1, IL-12) of CS-exposed vitamin D deficient mice compared to control mice. Finally, although CS greatly impaired the ex vivo phagocytic and oxidative burst function of alveolar macrophages, vitamin D deficient mice did not feature an additional defect.

Conclusions

Our data demonstrate that vitamin D deficiency both accelerates and aggravates the development of characteristic disease features of COPD. As vitamin D deficiency is highly prevalent, large randomized trials exploring effects of vitamin D supplementation on lung function decline and COPD onset are needed.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0271-x) contains supplementary material, which is available to authorized users.     

Characterization of the cultivable bacterial populations associated with field grown Brassica napus L.: an evaluation of sampling and isolation protocols

Plant‐associated bacteria are intensively investigated concerning their characteristics for plant growth promotion, biocontrol mechanisms and enhanced phytoremediation efficiency. To obtain endophytes, different sampling and isolation protocols are used although their representativeness is not always clearly demonstrated. The objective of this study was to acquire representative pictures of the cultivable bacterial root, stem and leaf communities for all Brassica napus L. individuals growing on the same field. For each plant organ, genotypic identifications of the endophytic communities were performed using three replicates. Root replicates were composed of three total root systems, whereas stem and leaf replicates needed to consist of six independent plant parts in order to be representative. Greater variations between replicates were found when considering phenotypic characteristics. Correspondence analysis revealed reliable phenotypic results for roots and even shoots, but less reliable ones for leaves. Additionally, realistic Shannon–Wiener biodiversity indices were calculated for all three organs and showed similar Evenness factors. Furthermore, it was striking that all replicates and thus the whole plant contained Pseudomonas and Bacillus strains although aboveground and belowground plant tissues differed in most dominant bacterial genera and characteristics.     

Identification and characterization of novel factors that act in the nonsense‐mediated mRNA decay pathway in nematodes,flies and mammals

Nonsense‐mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNAs harboring premature termination codons (PTCs). We have conducted a genome‐wide RNAi screen in Caenorhabditis elegans that resulted in the identification of five novel NMD genes that are conserved throughout evolution. Two of their human homologs, GNL2 (ngp‐1) and SEC13 (npp‐20), are also required for NMD in human cells. We also show that the C. elegans gene noah‐2, which is present in Drosophila melanogaster but absent in humans, is an NMD factor in fruit flies. Altogether, these data identify novel NMD factors that are conserved throughout evolution, highlighting the complexity of the NMD pathway and suggesting that yet uncovered novel factors may act to regulate this process.     

The Potential of the Ni-Resistant TCE-Degrading Pseudomonas putida W619-TCE to Reduce Phytotoxicity and Improve Phytoremediation Efficiency of Poplar Cuttings on A Ni-TCE Co-Contamination

To examine the potential of Pseudomonas putida W619-TCE to improve phytoremediation of Ni-TCE co-contamination, the effects of inoculation of a Ni-resistant, TCE-degrading root endophyte on Ni-TCE phytotoxicity, Ni uptake and trichloroethylene (TCE) degradation of Ni-TCE-exposed poplar cuttings are evaluated.

After inoculation with P. putida W619-TCE, root weight of non-exposed poplar cuttings significantly increased. Further, inoculation induced a mitigation of the Ni-TCE phytotoxicity, which was illustrated by a diminished exposure-induced increase in activity of antioxidative enzymes. Considering phytoremediation efficiency, inoculation with P. putida W619-TCE resulted in a 45% increased Ni uptake in roots as well as a slightly significant reduction in TCE concentration in leaves and TCE evapotranspiration to the atmosphere.

These results indicate that endophytes equipped with the appropriate characteristics can assist their host plant to deal with co-contamination of toxic metals and organic contaminants during phytoremediation. Furthermore, as poplar is an excellent plant for biomass production as well as for phytoremediation, the obtained results can be exploited to produce biomass for energy and industrial feedstock applications in a highly productive manner on contaminated land that is not suited for normal agriculture. Exploiting this land for biomass production could contribute to diminish the conflict between food and bioenergy production.     

Actual and Preferred Place of Death of Home-Dwelling Patients in Four European Countries: Making Sense of Quality Indicators

Background

Dying at home and dying at the preferred place of death are advocated to be desirable outcomes of palliative care. More insight is needed in their usefulness as quality indicators. Our objective is to describe whether “the percentage of patients dying at home” and “the percentage of patients who died in their place of preference” are feasible and informative quality indicators.

Methods and Findings

A mortality follow-back study was conducted, based on data recorded by representative GP networks regarding home-dwelling patients who died non-suddenly in Belgium (n = 1036), the Netherlands (n = 512), Italy (n = 1639) or Spain (n = 565). “The percentage of patients dying at home” ranged between 35.3% (Belgium) and 50.6% (the Netherlands) in the four countries, while “the percentage of patients dying at their preferred place of death” ranged between 67.8% (Italy) and 86.0% (Spain). Both indicators were strongly associated with palliative care provision by the GP (odds ratios of 1.55–13.23 and 2.30–6.63, respectively). The quality indicator concerning the preferred place of death offers a broader view than the indicator concerning home deaths, as it takes into account all preferences met in all locations. However, GPs did not know the preferences for place of death in 39.6% (the Netherlands) to 70.3% (Italy), whereas the actual place of death was known in almost all cases.

Conclusion

GPs know their patients’ actual place of death, making the percentage of home deaths a feasible indicator for collection by GPs. However, patients’ preferred place of death was often unknown to the GP. We therefore recommend using information from relatives as long as information from GPs on the preferred place of death is lacking. Timely communication about the place where patients want to be cared for at the end of life remains a challenge for GPs.     

Bilateral field advantage in visual enumeration

A number of recent studies have demonstrated superior visual processing when the information is distributed across the left and right visual fields than if the information is presented in a single hemifield (the bilateral field advantage). This effect is thought to reflect independent attentional resources in the two hemifields and the capacity of the neural responses to the left and right hemifields to process visual information in parallel. Here, we examined whether a bilateral field advantage can also be observed in a high-level visual task that requires the information from both hemifields to be combined. To this end, we used a visual enumeration task--a task that requires the assimilation of separate visual items into a single quantity--where the to-be-enumerated items were either presented in one hemifield or distributed between the two visual fields. We found that enumerating large number (>4 items), but not small number (<4 items), exhibited the bilateral field advantage: enumeration was more accurate when the visual items were split between the left and right hemifields than when they were all presented within the same hemifield. Control experiments further showed that this effect could not be attributed to a horizontal alignment advantage of the items in the visual field, or to a retinal stimulation difference between the unilateral and bilateral displays. These results suggest that a bilateral field advantage can arise when the visual task involves inter-hemispheric integration. This is in line with previous research and theory indicating that, when the visual task is attentionally demanding, parallel processing by the neural responses to the left and right hemifields can expand the capacity of visual information processing.     

Neisseria gonorrhoeae infection induces altered amphiregulin processing and release

Adhesion of the human pathogen Neisseria gonorrhoeae has established effects on the host cell and evokes a variety of cellular events including growth factor activation. In the present study we report that infection with N. gonorrhoeae causes altered amphiregulin processing and release in human epithelial cells. Amphiregulin is a well-studied growth factor with functions in various cell processes and is upregulated in different forms cancer and proliferative diseases. The protein is prototypically cleaved on the cell surface in response to external stimuli. We demonstrate that upon infection, a massive upregulation of amphiregulin mRNA is seen. The protein changes its subcellular distribution and is also alternatively cleaved at the plasma membrane, which results in augmented release of an infection-specific 36 kDa amphiregulin product from the surface of human cervical epithelial cells. Further, using antibodies directed against different domains of the protein we could determine the impact of infection on pro-peptide processing. In summary, we present data showing that the infection of N. gonorrhoeae causes an alternative amphiregulin processing, subcellular distribution and release in human epithelial cervical cells that likely contribute to the predisposition cellular abnormalities and anti-apoptotic features of N. gonorrhoeae infections.     

Collagen fibre orientation in human bridging veins

Biomechanics and Modeling in Mechanobiology - Bridging veins (BVs) drain the blood from the cerebral cortex into dural sinuses. BVs have one end attached to the brain and the other to the superior...     

Expressed 2-5A synthetase genes and pseudogenes in the marine sponge Geodia barretti

In cattle, bovine leukocyte antigens (BoLAs) have been extensively used as markers for bovine diseases and immunological traits. In this study, we sequenced alleles of the BoLA class II loci, BoLA-DRB3 and BoLA-DQA1, from 650 Japanese cattle from six herds [three herds (507 animals) of Japanese Black cattle and three herds (143 animals) of Holstein cattle] using polymerase chain reaction-sequence-based typing (PCR-SBT) methods. We identified 26 previously reported distinct DRB3 alleles in the two populations: 22 in Japanese Black and 17 in Holstein. The number of DRB3 alleles detected in each herd ranged from 9 to 20. Next, we identified 15 previously reported distinct DQA1 alleles: 13 in Japanese Black and 10 in Holstein. The number of alleles in each herd ranged from 6 to 10. Thus, allelic divergence is significantly greater for DRB3 than for DQA1. A population tree on the basis of the frequencies of the DRB3 and DQA1 alleles showed that, although the genetic distance differed significantly between the two cattle breeds, it was closely related within the three herds of each breed. In addition, Wu-Kabat variability analysis indicated that the DRB3 gene was more polymorphic than the DQA1 gene in both breeds and in all herds, and that the majority of the hypervariable positions within both loci corresponded to pocket-forming residues. The DRB3 and DQA1 heterozygosity for both breeds within each herd were calculated based on the Hardy-Weinberg equilibrium. Only one Japanese Black herd showed a significant difference between the expected and observed heterozygosity at both loci. This is the first report presenting a detailed study of the allelic distribution of BoLA-DRB3 and -DQA1 genes in Japanese Black and Holstein cattle from different farms in Japan. These results may help to develop improved livestock breeding strategies in the future.