Ubiquitination of CD86 is a key mechanism in regulating antigen presentation by dendritic cells

Dendritic cells (DCs) require costimulatory molecules such as CD86 to efficiently activate T cells for the induction of adaptive immunity. DCs maintain minimal levels of CD86 expression at rest, but upregulate levels upon LPS stimulation. LPS-stimulated DCs produce the immune suppressive cytokine IL-10 that acts in an autocrine manner to regulate CD86 levels. Interestingly, the underlying molecular mechanism behind the tight control of CD86 is not completely understood. In this study, we report that CD86 is ubiquitinated in DCs via MARCH1 E3 ubiquitin ligase and that this ubiquitination plays a key role in CD86 regulation. Ubiquitination at lysine 267 played the most critical role for this regulation. CD86 is ubiquitinated in MARCH1-deficient DCs to a much lesser degree than in wild-type DCs, which also correlated with a significant increase in CD86 expression. Importantly, CD86 is continuously ubiquitinated in DCs following activation by LPS, and this was due to the autocrine IL-10 inhibition of MARCH1 downregulation. Accordingly, DCs lacking MARCH1 and DCs expressing ubiquitination-resistant mutant CD86 both failed to regulate CD86 in response to autocrine IL-10. DCs expressing ubiquitination-resistant mutant CD86 failed to control their T cell-activating abilities at rest as well as in response to autocrine IL-10. These studies suggest that ubiquitination serves as an important mechanism by which DCs control CD86 expression and regulate their Ag-presenting functions.     

Nuclear localization of the Escherichia coli cytolethal distending toxin CdtB subunit

Cytolethal distending toxin (CDT) is a heterotrimeric protein toxin produced by several bacterial pathogens. Cells exposed to CDT die from either activation of the mitotic checkpoint cascade or apoptosis. Introduction of the purified CdtB subunit, a homologue of mammalian type I DNase, into cells mimics the action of the CDT holotoxin. Mutant CdtBs lacking DNase activity are devoid of biological activity. Chromosomal DNA appears to be the CDT target; thus, nuclear translocation of CdtB must precede cytolethal activity. Examination of the CdtB sequence indicates the presence of putative candidate bipartite nuclear localization signals (NLS). Here, we examine the functionality of the two potential NLS sequences found in the Escherichia coli CdtB-II. Nuclear translocation of EcCdtB-II was examined by monitoring the localization of an EcCdtB-II-EGFP fusion in Cos-7 cells. Our results indicated that EGFP-EcCdtB-II localized to the nucleus. The candidate EcCdtB-II-II NLS sequences were modified by site-directed mutagenesis such that tandem arginine residues were changed to threonine and serine respectively. Mutation of both putative NLS sequences had no effect on EcCdtB-II-associated DNase activity; however, cell cycle arrest and nuclear localization were significantly impaired in cells that received CDT reconstituted from the EcCdtB-II-DeltaNLS mutants. When HeLa cells were electroporated with the EcCdtB-II-DeltaNLS1 and the EcCdtB-II-NLS double mutants, toxicity was not observed, whereas the activity of EcCdtB-II-DeltaNLS2 was similar to that of wild-type EcCdtB-II. These data indicate that the putative NLS sequences are important for CDT-mediated action arrest and that they are likely to function in the nuclear translocation of EcCdtB-II.     

Comparative evaluation of vacuum-based surface sampling methods for collection of Bacillus spores

In this study, four commonly-used sampling devices (vacuum socks, 37 mm 0.8 μm mixed cellulose ester (MCE) filter cassettes, 37 mm 0.3 μm polytetrafluoroethylene (PTFE) filter cassettes, and 3M™ forensic filters) were comparatively evaluated for their ability to recover surface-associated spores. Aerosolized spores (~ 10⁵ CFU cm^− 2) of a Bacillus anthracis surrogate were allowed to settle onto three material types (concrete, carpet, and upholstery). Ten replicate samples were collected using each vacuum method, from each material type. Stainless steel surfaces, inoculated simultaneously with test materials, were sampled with pre-moistened wipes. Wipe recoveries were utilized to normalize vacuum-based recoveries across trials. Recovery (CFU cm^− 2) and relative recovery (vacuum recovery/wipe recovery) were determined for each method and material type. Recoveries and relative recoveries ranged from 3.8 × 10³ to 7.4 × 10⁴ CFU cm^− 2 and 0.035 to 1.242, respectively. ANOVA results indicated that the 37 mm MCE method exhibited higher relative recoveries than the other methods when used for sampling concrete or upholstery. While the vacuum sock resulted in the highest relative recoveries on carpet, no statistically significant difference was detected. The results of this study may be used to guide selection of sampling approaches following biological contamination incidents.     

Development of an oligonucleotide probe targeting 16S rRNA and its application for detection and quantitation of the ruminal bacterium Synergistes jonesii in a mixed-population chemostat.

Radiolabelled and fluorescent-dye-conjugated oligonucleotide probes which targeted rRNA sequences were developed for the enumeration of the ruminal bacterium Synergistes jonesii 78-1 in mixed culture. Two probes were tested, and both were highly specific for the respective complementary sequences of the target organism. Individual cells of S. jonesii in pure and mixed cultures were clearly visualized in situ by hybridization with the fluorescent-dye-conjugated probe but could not be detected in natural samples. Therefore the radiolabelled probe was used to monitor the population of S. jonesii introduced into a chemostat which simulated the rumen ecosystem. The S. jonesii probe did not hybridize to RNA extracted from the culture prior to inoculation with the target organism. After inoculation, S. jonesii rRNA represented 4.5% of the total bacterial rRNA and then rapidly declined to < 0.2% before increasing to about 1% of the total bacterial rRNA during the following 3 weeks. This study demonstrates that rRNA-targeted probes could be used for tracking organisms introduced into the rumen ecosystem.     

Expression of a modified Neocallimastix patriciarum xylanase in Butyrivibrio fibrisolvens digests more fibre but cannot effectively compete with highly fibrolytic bacteria in the rumen

AIMS: This study investigated the competitive abilities of two Neocallimastix patriciarum-derived xylanases constructs in Butyrivibrio fibrisolvens H17c (xynA and pUMSX) and their ability to compete in vivo. METHODS AND RESULTS: The digestibility of neutral detergent fibre (NDF) increased during co-culture of xynA or pUMSX and weakly cellulolytic, but not with highly cellulolytic, ruminococci. Competition studies among xynA, pUMSX and cellulolytic consortia demonstrated that xynA was the fittest. XynA did not persist at high levels in the rumen and was undetectable after 22 days. CONCLUSION: The construction of recombinant xylanolytic B. fibrisolvens does improve the digestibility of fibre above that of the native, but digestibility is still less than that of the most potent fibre digesters such as ruminococci. SIGNIFICANCE AND IMPACT OF THE STUDY: Fibre digestion may be improved by genetic manipulation of ruminal bacteria but ecological parameters, such as persistence in vivo and the niche of the organism, must be taken into account.     

Population structure of island-associated dolphins: Evidence from photo-identification of common bottlenose dolphins (Tursiops truncatus) in the main Hawaiian Islands

Management agencies often use geopolitical boundaries as proxies for biological boundaries. In Hawaiian waters a single stock is recognized of common bottlenose dolphins, Tursiops truncatus , a species that is found both in open water and near-shore among the main Hawaiian Islands. To assess population structure, we photo-identified 336 distinctive individuals from the main Hawaiian Islands, from 2000 to 2006. Their generally shallow-water distribution, and numerous within-year and between-year resightings within island areas suggest that individuals are resident to the islands, rather than part of an offshore population moving through the area. Comparisons of identifications obtained from Kaua'i/Ni'ihau, O'ahu, the 4-island area, and the island of Hawai'i showed no evidence of movements among these island groups, although movements from Kaua'i to Ni'ihau and among the 4-islands were documented. A Bayesian analysis examining the probability of missing movements among island groups, given our sample sizes for different areas, indicates that interisland movement rates are less than 1% per year with 95% probability. Our results suggest the existence of multiple demographically independent populations of island-associated common bottlenose dolphins around the main Hawaiian islands.     

Effect of tea saponin on methanogenesis,microbial community structure and expression of mcrA gene,in cultures of rumen micro‐organisms

Aims: To determine the in‐vitro effect and mode of action of tea saponin on the rumen microbial community and methane production. Methods and Results: Saponin extracted from tea seeds was added to (1) an in‐vitro fermentation inoculated with rumen fluid and (2) a pure culture of Methanobrevibacter ruminantium. Methane production and expression of the methyl coenzyme‐M reductase subunit A (mcrA) were monitored in both cultures. Abundance of methanogens, protozoa, rumen fungi and cellulolytic bacteria were quantified using real‐time PCR, and bacterial diversity was observed using denaturing gradient gel electrophoresis. Addition of tea saponin significantly reduced methane production and mcrA gene expression in the ruminal fermentation but not with the pure culture of M. ruminantium. The abundance of protozoa and fungi were significantly decreased 50% and 79% respectively but methanogen numbers were not affected, and Fibrobacter succinogenes increased by 41%. Bacterial diversity was similar in cultures with or without tea saponin. Conclusions: Tea saponin appeared to reduce methane production by inhibiting protozoa and presumably lowering methanogenic activity of protozoal‐associated methanogens. Significance and Impact of the Study: Tea saponin may be useful as a supplement to indirectly inhibit methane production in ruminants without a deleterious effect on rumen function.