Differential Effects of Reactive Oxygen Species on IgG versus IgM Levels in TLR-Stimulated B Cells.

It is becoming increasingly evident that reactive oxygen species (ROS) have critical roles as "second messengers" in cell signaling. In B cells, ROS can be generated either as a byproduct of mitochondrial respiration, as a result of the endoplasmic reticulum stress response induced by high production of Igs, or by the activation of NADPH oxidase (NOX) complexes. Having previously shown that costimulation of B cells via TLR 9 and the TLR-related receptor RP105 drives maturation of human peripheral blood B cells into Ig-producing cells, we aimed to study the role of ROS generated during this vital process. To this end, the ROS levels were either reduced by the NOX inhibitor VAS2870 or by the ROS scavenger N-acetyl cysteine (NAC). We revealed that TLR9/RP105-mediated stimulation of human B cells involved a rapid activation of NOX. Moreover, VAS2870 blocked the TLR9/RP105-induced B cell activation and thereby all Ig production. Importantly, we showed that ROS targeted by NAC was selectively required for IgG but not for IgM production. The endoplasmic reticulum stress response in the TLR9/RP105-stimulated cells was higher in IgG+ than in IgG- cells and was reduced by NAC in IgG+ cells only. Of note, we revealed that substantially higher levels of IgG than IgM were produced per cell and that IgG+ cells produced significantly higher ROS levels than IgG- cells. Taken together, our results imply that NAC-targeted ROS may be particularly important for sustaining the high Ig production in IgG+ B cells.

Development of an All-Marine 3D Printed Bioactive Hydrogel Dressing for Treatment of Hard-to-Heal Wounds.

Current standard wound care involves dressings that provide moisture and protection; however, dressings providing active healing are still scarce and expensive. We aimed to develop an ecologically sustainable 3D printed bioactive hydrogel-based topical wound dressing targeting healing of hard-to-heal wounds, such as chronic or burn wounds, which are low on exudate. To this end, we developed a formulation composed of renewable marine components; purified extract from unfertilized salmon roe (heat-treated X, HTX), alginate from brown seaweed, and nanocellulose from tunicates. HTX is believed to facilitate the wound healing process. The components were successfully formulated into a 3D printable ink that was used to create a hydrogel lattice structure. The 3D printed hydrogel showed a HTX release profile enhancing pro-collagen I alpha 1 production in cell culture with potential of promoting wound closure rates. The dressing has recently been tested on burn wounds in Göttingen minipigs and shows accelerated wound closure and reduced inflammation. This paper describes the dressings development, mechanical properties, bioactivity, and safety.

Uracil-DNA glycosylases SMUG1 and UNG2 coordinate the initial steps of base excision repair by distinct mechanisms.

DNA glycosylases UNG and SMUG1 excise uracil from DNA and belong to the same protein superfamily. Vertebrates contain both SMUG1 and UNG, but their distinct roles in base excision repair (BER) of deaminated cytosine (U:G) are still not fully defined. Here we have examined the ability of human SMUG1 and UNG2 (nuclear UNG) to initiate and coordinate repair of U:G mismatches. When expressed in Escherichia coli cells, human UNG2 initiates complete repair of deaminated cytosine, while SMUG1 inhibits cell proliferation. In vitro, we show that SMUG1 binds tightly to AP-sites and inhibits AP-site cleavage by AP-endonucleases. Furthermore, a specific motif important for the AP-site product binding has been identified. Mutations in this motif increase catalytic turnover due to reduced product binding. In contrast, the highly efficient UNG2 lacks product-binding capacity and stimulates AP-site cleavage by APE1, facilitating the two first steps in BER. In summary, this work reveals that SMUG1 and UNG2 coordinate the initial steps of BER by distinct mechanisms. UNG2 is apparently adapted to rapid and highly coordinated repair of uracil (U:G and U:A) in replicating DNA, while the less efficient SMUG1 may be more important in repair of deaminated cytosine (U:G) in non-replicating chromatin.

The region of XRCC1 which harbours the three most common nonsynonymous polymorphic variants, is essential for the scaffolding function of XRCC1.

XRCC1 functions as a non-enzymatic, scaffold protein in single strand break repair (SSBR) and base excision repair (BER). Here, we examine different regions of XRCC1 for their contribution to the scaffolding functions of the protein. We found that the central BRCT1 domain is essential for recruitment of XRCC1 to sites of DNA damage and DNA replication. Also, we found that ectopic expression of the region from residue 166-436 partially rescued the methyl methanesulfonate (MMS) hypersensitivity of XRCC1-deficient EM9 cells, suggesting a key role for this region in mediating DNA repair. The three most common amino acid variants of XRCC1, Arg194Trp, Arg280His and Arg399Gln, are located within the region comprising the NLS and BRCT1 domains, and these variants may be associated with increased incidence of specific types of cancer. While we could not detect differences in the intra-nuclear localization or the ability to support recruitment of POLß or PNKP to micro-irradiated sites for these variants relative to the conservative protein, we did observe lower foci intensity after micro-irradiation and a reduced stability of the foci with the Arg280His and Arg399Gln variants, respectively. Furthermore, when challenged with MMS or hydrogen peroxide, we detected small but consistent differences in the repair profiles of cells expressing these two variants in comparison to the conservative protein.