SUMMARY We report that in the presence of signal 1 (NF-κB), the NLRP3 inflammasome was activated by mitochondrial apoptotic signaling that licensed production of interleukin-1β (IL-1β). NLRP3 secondary signal activators such as ATP induced mitochondrial dysfunction and apoptosis, resulting in release of oxidized mitochondrial DNA (mtDNA) into the cytosol, where it bound to and activated the NLRP3 inflammasome. The anti-apoptotic protein Bcl-2 inversely regulated mitochondrial dysfunction and NLRP3 inflammasome activation. Mitochondrial DNA directly induced NLRP3 inflammasome activation, because macrophages lacking mtDNA had severely attenuated IL-1β production, yet still underwent apoptosis. Both binding of oxidized mtDNA to the NLRP3 inflammasome and IL-1β secretion could be competitively inhibited by the oxidized nucleoside, 8-OH-dG. Thus, our data reveal that oxidized mtDNA released during programmed cell death causes activation of the NLRP3 inflammasome. These results provide a missing link between apoptosis and inflammasome activation, via binding of cytosolic oxidized mtDNA to the NLRP3 inflammasome.

The prevention of aggregation in therapeutic antibodies is of great importance to the biopharmaceutical industry. In our investigation, acid-induced aggregation of monoclonal IgG1 and IgG2 antibodies was studied at pH 3.5 as a function of salt concentration and buffer type. The extent of aggregation was estimated using a native cation-exchange chromatography (CEX) method based on the loss of soluble monomer. This approach allowed quantitative analysis of antibody aggregation kinetics for individual and mixed protein solutions. Information regarding the aggregation mechanism was gained by assessing stabilities of intact antibodies relative to their Fc and Fab fragments. The role of protein thermodynamic stability in aggregation was deduced from differential scanning calorimetry (DSC). The rate of aggregation under conditions mimicking the viral inactivation step during monoclonal antibody (mAb) processing was found to be strongly dependent on the antibody subclass (IgG1 vs IgG2). At 25 °C, IgG1s were resistant to low pH aggregation, but IgG2s aggregated readily in the presence of salt. The observed distinction between IgG1 and IgG2 aggregation resulted from differential stability of the corresponding C(H)2 domains. This was further confirmed by experimenting with an IgG1 molecule containing an aglycosylated C(H)2 domain. Interestingly, comparative analysis of two buffer systems (based on acetic acid vs citric acid) revealed differences in mAb aggregation under identical pH conditions. Evidence is provided for the importance of the total acid concentration for antibody aggregation at low pH. The effects of C(H)2 instability and solution composition on aggregation are significant and deserve careful consideration during the development of mAb- or Fc-based therapeutics.

SummaryMicroRNAs are a group of endogenously expressed, singlestranded, 18-24 nt RNAs that regulate diverse cellular pathways. Although documented evidence indicates that some microRNAs can function as oncogenes or tumor-suppressors, the role of miR-214 in regulating human cervical cancer cells remains unexplored. We determined the expression level of miR-214 and found it is downregulated in cervical cancer compared with normal tissue. Overexpression of miR-214 in HeLa cells, a human cervical cancer cell line, significantly inhibited cell proliferation according to the MTT and colony forming assays. HeLa cells that stably overexpress miR-214 downregulate the expression of MEK3 and JNK1 at both mRNA and protein levels. Further investigation revealed that miR-214 regulates the expression of MEK3 and JNK1 by targeting the 3 0 UTRs of these genes. Collectively, these results suggest that miR-214 negatively regulates HeLa cell proliferation by targeting the noncoding regions of MEK3 and JNK1 mRNAs.

SUMMARY Notch2, but not Notch1, plays indispensable roles in kidney organogenesis and Notch2 haploinsufficiency is associated with Alagille syndrome. We proposed that proximal nephron fates are regulated by a threshold that requires nearly all available free Notch intracellular domains (ICDs), but we could not identify the mechanism explaining why Notch2 (N2) is more important than Notch1 (N1). By generating mice that swap their ICDs, we establish that overall protein concentration, expression domain, or ICD amino acid composition does not account for the differential requirement for these receptors. Instead, we find that the N2 extracellular domain (ECD) increases Notch protein localized to the cell surface during kidney development and is cleaved more efficiently upon ligand binding. This context-specific asymmetry in NICD release efficiency is further enhanced by Fringe. Our results indicate that elevating N1 surface level could compensate for the loss of N2 signal in specific cell contexts.