The ISG12 family constitute small hydrophic proteins involved in apoptosis. This is the first comparison of the apoptotic potentials of the full-length ISG12A protein and the three ISG12A variants. The differential apoptotic potentials of these proteins might have an impact on the strategies to monitor and interpret their dysregulation associated with many disorders.

The classical pathway of complement is important for protection against pathogens and in maintaining tissue homeostasis, but excessive or aberrant activation is directly linked to numerous pathologies. We describe the development and in vitro characterization of C1qNb75, a single domain antibody (nanobody) specific for C1q, the pattern recognition molecule of the classical pathway. C1qNb75 binds to the globular head modules of human C1q with sub-nanomolar affinity and impedes classical pathway mediated hemolysis by IgG and IgM. Crystal structure analysis revealed that C1qNb75 recognizes an epitope primarily located in the C1q B-chain that overlaps with the binding sites of IgG and IgM. Thus, C1qNb75 competitively prevents C1q from binding to IgG and IgM causing blockade of complement activation by the classical pathway. Overall, C1qNb75 represents a high-affinity nanobody-based inhibitor of IgG-and IgM-mediated activation of the classical pathway and may serve as a valuable reagent in mechanistic and functional studies of complement, and as an efficient inhibitor of complement under conditions of excessive CP activation.

Upon infection of Escherichia coli by bacteriophage Qβ, the virus-encoded β-subunit recruits host translation elongation factors EF-Tu and EF-Ts and ribosomal protein S1 to form the Qβ replicase holoenzyme complex, which is responsible for amplifying the Qβ (+)-RNA genome. Here, we use X-ray crystallography, NMR spectroscopy, as well as sequence conservation, surface electrostatic potential and mutational analyses to decipher the roles of the β-subunit and the first two oligonucleotide-oligosaccharide-binding domains of S1 (OB1–2) in the recognition of Qβ (+)-RNA by the Qβ replicase complex. We show how three basic residues of the β subunit form a patch located adjacent to the OB2 domain, and use NMR spectroscopy to demonstrate for the first time that OB2 is able to interact with RNA. Neutralization of the basic residues by mutagenesis results in a loss of both the phage infectivity in vivo and the ability of Qβ replicase to amplify the genomic RNA in vitro. In contrast, replication of smaller replicable RNAs is not affected. Taken together, our data suggest that the β-subunit and protein S1 cooperatively bind the (+)-stranded Qβ genome during replication initiation and provide a foundation for understanding template discrimination during replication initiation.

β‐Mannosidase is a lysosomal enzyme from the glycosyl hydrolase family 2 that cleaves the single β(1–4)‐linked mannose at the nonreducing end of N‐glycosylated proteins, and plays an important role in the polysaccharide degradation pathway. Mutations in the MANBA gene, which encodes the β‐mannosidase, can lead to the lysosomal storage disease β‐mannosidosis, as well as nystagmus, an eye condition characterized by involuntary eye movements. Here, we present the first structures of a mammalian β‐mannosidase in both the apo‐ and mannose‐bound forms. The structure is similar to previously determined β‐mannosidase structures with regard to domain organization and fold, however, there are important differences that underlie substrate specificity between species. Additionally, in contrast to most other ligand‐bound β‐mannosidases from bacterial and fungal sources where bound sugars were in a boat‐like conformation, we find the mannose in the chair conformation. Evaluation of known disease mutations in the MANBA gene provides insight into their impact on disease phenotypes. Together, these results will be important for the design of therapeutics for treating diseases caused by β‐mannosidase deficiency. Database Structural data are available in the Protein Data Bank under the accession numbers http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6DDT and http://www.rcsb.org/pdb/search/structidSearch.do?structureId=6DDU.

Precision gene editing occurs at a low percentage in mammalian cells using Cas9. Colocalization of donor with Cas9MAV and PEI delivery raises HDR occurrence..