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.