Sec22b and Stx4 Depletion Has No Major Effect on Cross-Presentation of PLGA Microsphere-Encapsulated Antigen and a Synthetic Long Peptide In Vitro.

The induction of CTL responses by vaccines is important to combat infectious diseases and cancer. Biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres and synthetic long peptides are efficiently internalized by professional APCs and prime CTL responses after cross-presentation of Ags on MHC class I molecules. Specifically, they mainly use the cytosolic pathway of cross-presentation that requires endosomal escape, proteasomal processing, and subsequent MHC class I loading of Ags in the endoplasmic reticulum (ER) and/or the endosome. The vesicle SNARE protein Sec22b has been described as important for this pathway by mediating vesical trafficking for the delivery of ER-derived proteins to the endosome. As this function has also been challenged, we investigated the role of Sec22b in cross-presentation of the PLGA microsphere-encapsulated model Ag OVA and a related synthetic long peptide. Using CRISPR/Cas9-mediated genome editing, we generated Sec22b knockouts in two murine C57BL/6-derived APC lines and found no evidence for an essential role of Sec22b. Although pending experimental evidence, the target SNARE protein syntaxin 4 (Stx4) has been suggested to promote cross-presentation by interacting with Sec22b for the fusion of ER-derived vesicles with the endosome. In the current study, we show that, similar to Sec22b, Stx4 knockout in murine APCs had very limited effects on cross-presentation under the conditions tested. This study contributes to characterizing cross-presentation of two promising Ag delivery systems and adds to the discussion about the role of Sec22b/Stx4 in related pathways. Our data point toward SNARE protein redundancy in the cytosolic pathway of cross-presentation.

Effects of the residual ammonium concentration on NOB repression during partial nitritation with granular sludge.

Partial nitritation was stably achieved in a bench-scale airlift reactor (1.5L) containing granular sludge. Continuous operation at 20 °C treating low-strength synthetic wastewater (50 mg N-NH4+/L and no COD) achieved nitrogen loading rates of 0.8 g N-NH4+/(L·d) during partial nitritation. The switch between nitrite-oxidizing bacteria (NOB) repression and NOB proliferation was observed when ammonium concentrations in the reactor were below 2-5 mg N-NH4+/L for DO concentrations lower than 4 mg O2/L at 20 °C. Nitrospira spp. were detected to be the dominant NOB population during the entire reactor operation, whereas Nitrobacter spp. were found to be increasing in numbers over time. Stratification of the granule structure, with ammonia-oxidizing bacteria (AOB) occupying the outer shell, was found to be highly important in the repression of NOB in the long term. The pH gradient in the granule, containing a pH difference of ca. 0.4 between the granule surface and the granule centre, creates a decreasing gradient of ammonia towards the centre of the granule. Higher residual ammonium concentration enhances the ammonium oxidation rate of those cells located further away from the granule surface, where the competition for oxygen between AOB and NOB is more important, and it contributes to the stratification of both populations in the biofilm.

Syntrophic associations from hypersaline soda lakes converting organic acids and alcohols to methane at extremely haloalkaline conditions.

Until now anaerobic oxidation of VFA at high salt-pH has been demonstrated only at sulfate-reducing conditions. Here, we present results of a microbiological investigation of anaerobic conversion of organic acids and alcohols at methanogenic conditions by syntrophic associations enriched from hypersaline soda lakes in Central Asia. Sediment incubation experiments showed active, albeit very slow, methane formation from acetate, propionate, butyrate and C2 C4 alcohols at pH 10 and various levels of salinity. Enrichments of syntrophic associations using hydrogenotrophic members of the genus Methanocalculus from soda lakes as partners resulted in several highly enriched cultures converting acetate, propionate, butyrate, benzoate and EtOH to methane. Most syntrophs belonged to Firmicutes, while the propionate-oxidizer formed a novel lineage within the family Syntrophobacteraceae in the Deltaproteobacteria. The acetate-oxidizing syntroph was identified as 'Ca. Syntrophonatronum acetioxidans' previously found to oxidize acetate at sulfate-reducing conditions up to salt-saturating concentrations. Butyrate and a benzoate-degrading syntrophs represent novel genus-level lineages in Syntrophomonadales which are proposed as Candidatus taxons 'Syntrophobaca', 'Syntrophocurvum' and 'Syntropholuna'. Overall, despite very slow growth, the results indicated the presence of a functionally competent syntrophic community in hypersaline soda lakes, capable of efficient oxidation of fermentation products to methane at extremely haloalkaline conditions.

Methanogenesis at extremely haloalkaline conditions in the soda lakes of Kulunda Steppe (Altai, Russia).

Microbial methanogenesis at extreme conditions of saline alkaline soda lakes has, so far, been poorly investigated. Despite the obvious domination of sulfidogenesis as the therminal anaerobic process in the hypersaline soda lakes of Kulunda Steppe (Altai, southwestern Siberia), high concentrations of methane were detected in the anaerobic sediments. Potential activity measurements with different substrates gave results significantly deviating from what is commonly found in hypersaline habitats with neutral pH. In particular, not only a non-competitive methylotrophic pathway was active, but also lithotrophic and, in some cases, even acetate-dependent methanogenesis was found to be present in hypersaline soda lake sediments. All three pathways were functioning exclusively within the alkaline pH range between 8 and 10.5, while the salt concentration was the key factor influencing the activity. Methylotrophic and, to a lesser extent, lithotrophic methanogenesis were active up to soda-saturating conditions (4 M total Na(+)). Acetate-dependent methanogenesis was observed at salinities below 3 M total Na(+). Detection of methanogens in sediments using the mcrA gene as a functional marker demonstrated domination of methylotrophic genera Methanolobus and Methanosalsum and lithotrophic Methanocalculus. In a few cases, acetoclastic Methanosaeta was detected, as well as two deep lineage methanogens. Cultivation results corresponded well to the mcrA-based observations. Enrichments for natronophilic methylotrophic methanogens resulted in isolation of Methanolobus strains at moderate salinity, while at salt concentrations above 2 M Na(+) a novel member of the genus Methanosalsum was dominating. Enrichments with H2 or formate invariably resulted in domination of close relatives of Methanocalculus natronophilus. Enrichments with acetate at low salt concentration yielded two acetoclastic alkaliphilic Methanosaeta cultures, while at salinity above 1 M Na(+) syntrophic associations were apparently responsible for the observed acetate conversion to methane. Overall, the results indicated the presence of functionally structured and active methanogenic populations in Siberian hypersaline soda lakes.