Unexpected photobleaching of Alexa 488 in a fixed bacterial sample during 2-photon excitation

A sample of fixed bacterial cells was examined by immunofluorescence microscopy using an Alexa 488 conjugated secondary antibody for visualization. Excitation using visible light confirmed the expected photostability of this fluorophore; however, when using 2-photon excitation, Alexa 488 was rapidly and substantially photobleached. The unexpected instability of Alexa 488 under certain conditions may have deleterious consequences if not anticipated and accommodated in experimental protocols.     

The canine isolate Lactobacillus acidophilus LAB20 adheres to intestinal epithelium and attenuates LPS-induced IL-8 secretion of enterocytes in vitro

Background

For a good probiotic candidate, the abilities to adhere to intestinal epithelium and to fortify barrier function are considered to be crucial for colonization and functionality of the strain. The strain Lactobacillus acidophilus LAB20 was isolated from the jejunum of a healthy dog, where it was found to be the most pre-dominant lactobacilli. In this study, the adhesion ability of LAB20 to intestinal epithelial cell (IECs) lines, IECs isolated from canine intestinal biopsies, and to canine, porcine and human intestinal mucus was investigated. Further, we studied the ability of LAB20 to fortify the epithelial cell monolayer and to reduce LPS-induced interleukin (IL-8) release from enterocytes.

Results

We found that LAB20 presented higher adhesion to canine colonic mucus as compared to mucus isolated from porcine colon. LAB20 showed adhesion to HT-29 and Caco-2 cell lines, and importantly also to canine IECs isolated from canine intestinal biopsies. In addition, LAB20 increased the transepithelial electrical resistance (TER) of enterocyte monolayers and thus strengthened the intestinal barrier function. The strain showed also anti-inflammatory capacity in being able to attenuate the LPS-induced IL-8 production of HT-29 cells.

Conclusion

In conclusion, canine indigenous strain LAB20 is a potential probiotic candidate for dogs adhering to the host epithelium and showing intestinal barrier fortifying and anti-inflammatory effects.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-014-0337-9) contains supplementary material, which is available to authorized users.     

Recycling of peroxiredoxin IV provides a novel pathway for disulphide formation in the endoplasmic reticulum

Disulphide formation in the endoplasmic reticulum (ER) is catalysed by members of the protein disulphide isomerase (PDI) family. These enzymes can be oxidized by the flavoprotein ER oxidoreductin 1 (Ero1), which couples disulphide formation with reduction of oxygen to form hydrogen peroxide (H(2)O(2)). The H(2)O(2) produced can be metabolized by ER-localized peroxiredoxin IV (PrxIV). Continuous catalytic activity of PrxIV depends on reduction of a disulphide within the active site to form a free thiol, which can then react with H(2)O(2). Here, we demonstrate that several members of the PDI family are able to directly reduce this PrxIV disulphide and in the process become oxidized. Furthermore, we show that altering cellular expression of these proteins within the ER influences the efficiency with which PrxIV can be recycled. The oxidation of PDI family members by PrxIV is a highly efficient process and demonstrates how oxidation by H(2)O(2) can be coupled to disulphide formation. Oxidation of PDI by PrxIV may therefore increase efficiency of disulphide formation by Ero1 and also allows disulphide formation via alternative sources of H(2)O(2).     

Molecular evidence places the swallow bug genus Oeciacus Stål within the bat and bed bug genus Cimex Linnaeus (Heteroptera: Cimicidae)

The genera Cimex Linnaeus and Oeciacus Stål (Heteroptera: Cimicidae) are common haematophagous ectoparasites of bats or birds in the Holarctic region. Both their phylogenetic relationship and the systematics of the entire family previously were based on data from morphology and host relationships. Relationships among nine species of the genus Cimex and three species of the genus Oeciacus were analysed here using two mitochondrial and three nuclear genes. Cimex was shown to be paraphyletic with respect to Oeciacus. Oeciacus is thus proposed as a synonym of Cimex. The characteristic phenotype of Oeciacus results from the specific host association with different species of swallows (Hirundinidae). The morphological characters that have been used as diagnostic for the genera were shown to be valid and can be further used for determination at species level. The present analyses recovered the four traditional morphologically defined species groups of the genus Cimex. However, their relationships were poorly resolved – only the C. hemipterus group showed a well‐supported relationship to the C. pipistrelli group. The molecular differentiation within the Palaearctic C. pipistrelli and the Nearctic C. pilosellus species groups correlates with their karyotype differentiation. Furthermore, the analyses suggest poly‐ or paraphyly of the former genus Oeciacus. Either way this indicates there is a large amount of host‐associated phenotypic convergence in either bat‐ or bird‐associated groups of species. The probability of host choice and subsequent switch in Cimicidae are discussed and possible scenarios of the evolution of host association in species of Cimex are suggested.