The UBC Domain Is Required for BRUCE to Promote BRIT1/MCPH1 Function in DSB Signaling and Repair Post Formation of BRUCE-USP8-BRIT1 Complex

BRUCE is implicated in the regulation of DNA double-strand break response to preserve genome stability. It acts as a scaffold to tether USP8 and BRIT1, together they form a nuclear BRUCE-USP8-BRIT1 complex, where BRUCE holds K63-ubiquitinated BRIT1 from access to DSB in unstressed cells. Following DSB induction, BRUCE promotes USP8 mediated deubiquitination of BRIT1, a prerequisite for BRIT1 to be released from the complex and recruited to DSB by binding to γ-H2AX. BRUCE contains UBC and BIR domains, but neither is required for the scaffolding function of BRUCE mentioned above. Therefore, it remains to be determined whether they are required for BRUCE in DSB response. Here we show that the UBC domain, not the BIR domain, is required for BRUCE to promote DNA repair at a step post the formation of BRUCE-USP8-BRIT1 complex. Mutation or deletion of the BRUCE UBC domain did not disrupt the BRUCE-USP8-BRIT1 complex, but impaired deubiquitination and consequent recruitment of BRIT1 to DSB. This leads to impaired chromatin relaxation, decreased accumulation of MDC1, NBS1, pATM and RAD51 at DSB, and compromised homologous recombination repair of DNA DSB. These results demonstrate that in addition to the scaffolding function in complex formation, BRUCE has an E3 ligase function to promote BRIT1 deubiquitination by USP8 leading to accumulation of BRIT1 at DNA double-strand break. These data support a crucial role for BRUCE UBC activity in the early stage of DSB response.     

Snap-25 is polarized to axons and abundant along the axolemma: an immunogold study of intact neurons

SNAP-25, synaptosomal associated protein of 25 kDa, is reported to be a t-SNARE (target receptor associated with the presynaptic plasma membrane) involved in the docking and fusion of synaptic vesicles. We present here the first ultrastructural localization of SNAP-25 in intact neurons by pre-embedding EM immunocytochemistry in rat brains, hippocampal slice cultures, and PC12 cells. In differentiated neurons, SNAP-25 labeling was clearly membrane-associated. The labeling was most prominent in the plasma membrane of axons and excluded from the plasma membranes of soma and dendrites. Furthermore, SNAP-25 did not appear to be restricted to the synaptic junctions. SNAP-25 labeling was seen in the cytoplasm of the soma and large dendrites, mostly associated with the Golgi complexes. There were also some SNAP-25 labeled tubulo-vesicular structures in the cytoplasm of the soma and the axons, but rarely in the smaller dendrites. In PC12 cells, after 5–10 minutes of high potassium (75 mM) stimulation in the presence of HRP, SNAP-25 labeling appeared, additionally, on HRP-filled early endosomes. After a longer (20–30 minutes) HRP incubation, most of the later stage endosomes and lysosomes were loaded with HRP but they were negative for SNAP-25. These results suggest that SNAP-25 is sorted out of these late endosomal compartments, and that the bulk of the SNAP-25 protein is probably recycled back to the axolemma from the early endosomes. In contrast, in those samples which were incubated with HRP for longer periods, there were still some SNAP-25–positive vesicular structures which were HRP-negative. These structures most likely represent anterograde vesicles that carry newly synthesized SNAP-25 from the soma to the axolemma by axonal transport. SNAP-25 appears to be sorted at the Golgi complex to reach the axolemma specifically. Its widespread distribution all along the axolemma does not support the view of SNAP-25 as a t-SNARE limited for synaptic exocytosis.     

Splenic gene expression profiling in White Leghorn layer inoculated with the Salmonella enterica serovar Enteritidis

Salmonella enterica serovar Enteritidis (SE) is a foodborne pathogen that can threaten human health through contaminated poultry products. Live poultry, chicken eggs and meat are primary sources of human salmonellosis. To understand the genetic resistance of egg‐type chickens in response to SE inoculation, global gene expression in the spleen of 20‐week‐old White Leghorn was measured using the Agilent 4 × 44 K chicken microarray at 7 and 14 days following SE inoculation (dpi). Results showed that there were 1363 genes significantly differentially expressed between inoculated and non‐inoculated groups at 7 dpi (I7/N7), of which 682 were up‐regulated and 681 were down‐regulated genes. By contrast, 688 differentially expressed genes were observed at 14 dpi (I14/N14), of which 371 were up‐regulated genes and 317 were down‐regulated genes. There were 33 and 28 immune‐related genes significantly differentially expressed in the comparisons of I7/N7 and I14/N14 respectively. Functional annotation revealed that several Gene Ontology (GO) terms related to immunity were significantly enriched between the inoculated and non‐inoculated groups at 14 dpi but not at 7 dpi, despite a similar number of immune‐related genes identified between I7/N7 and I14/N14. The immune response to SE inoculation changes with different time points following SE inoculation. The complicated interaction between the immune system and metabolism contributes to the immune responses to SE inoculation of egg‐type chickens at 14 dpi at the onset of lay. GC, TNFSF8, CD86, CD274, BLB1 and BLB2 play important roles in response to SE inoculation. The results from this study will deepen the current understanding of the genetic response of the egg‐type chicken to SE inoculation at the onset of egg laying.     

Marine incursions,cryptic species and ecological diversification in Amazonia: the biogeographic history of the croaker genus Plagioscion (Sciaenidae)

Aim We propose a phylogenetic hypothesis for the marine‐derived sciaenid genus Plagioscion in the context of geomorphology and adaptation to freshwaters of South America, and assess the extent to which contemporary freshwater hydrochemical gradients influence diversification within a widely distributed Plagioscion species, Plagioscion squamosissimus. Location Amazon Basin and South America. Methods Using nuclear and mitochondrial DNA sequence data, phylogenetic analyses were conducted on the five nominal Plagioscion species, together with representatives from Pachyurus and Pachypops, using character and model‐based methods. Genealogical relationships and population genetic structure of 152 P. squamosissimus specimens sampled from the five major rivers and three hydrochemical settings/‘colours’ (i.e. white, black and clear water) of the Amazon Basin were assessed. Results Phylogenetic analyses support the monophyly of Plagioscion in South America and identify two putative cryptic species of Plagioscion. Divergence estimates suggest that the Plagioscion ancestor invaded South America via a northern route during the late Oligocene to early Miocene. Within P. squamosissimus a strong association of haplotype and water colour was observed, together with significant population structure detected between water colours. Main conclusions Our analyses of Plagioscion are consistent with a biogeographic scenario of early Miocene marine incursions into South America. Based on our phylogenetic results, the fossil record, geomorphological history and distributional data of extant Plagioscion species, we propose that marine incursions into western Venezuela between the late Oligocene and early Miocene were responsible for the adaptation to freshwaters in Plagioscion species. Following the termination of the marine incursions during the late Miocene and the establishment of the modern Amazon River, Plagioscion experienced a rapid diversification. Plagioscion squamosissimus arose during that time. The formation of the Amazon River probably facilitated population and range expansions for this species. Further, the large‐scale hydrochemical gradients within the Amazon Basin appear to be acting as ecological barriers maintaining population discontinuities in P. squamosissimus even in the face of gene flow. Our results highlight the importance of divergent natural selection through time in the generation and maintenance of sciaenid diversity in Amazonia.     

A doubly auxotrophic CHO-K1 cell line for the production of recombinant monoclonal antibodies

Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for recombinant protein production due to their hardiness, ease of transfection, and production of glycan structures similar to those in natural human monoclonal antibodies. To enhance the usefulness of CHO-K1 cells we developed a new selection system based on double auxotrophy. We used CRISPR-Cas9 to knockout the genes that encode the bifunctional enzymes catalyzing the last two steps in the de novo synthesis of pyrimidines and purines (uridine monophosphate synthase and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase [ATIC], respectively). Survival of these doubly auxotrophic cells depends on the provision of sources of purines and pyrimidines or on the transfection and integration of open reading frames encoding these two enzymes. We successfully used one such double auxotroph (UA10) to select for stable transfectants carrying (a) the recombinant tumor necrosis factor-α receptor fusion protein etanercept and (b) the heavy and light chains of the anti-Her2 monoclonal antibody trastuzumab. Transfectant clones produced these recombinant proteins in a stable manner and in substantial amounts. The availability of this double auxotroph provides a rapid and efficient selection method for the serial or simultaneous transfer of genes for multiple polypeptides of choice into CHO cells using readily available purine- and pyrimidine-free commercial media.     

Systematic Differences in Signal Emitting and Receiving Revealed by PageRank Analysis of a Human Protein Interactome

Most protein PageRank studies do not use signal flow direction information in protein interactions because this information was not readily available in large protein databases until recently. Therefore, four questions have yet to be answered: A) What is the general difference between signal emitting and receiving in a protein interactome? B) Which proteins are among the top ranked in directional ranking? C) Are high ranked proteins more evolutionarily conserved than low ranked ones? D) Do proteins with similar ranking tend to have similar subcellular locations? In this study, we address these questions using the forward, reverse, and non-directional PageRank approaches to rank an information-directional network of human proteins and study their evolutionary conservation. The forward ranking gives credit to information receivers, reverse ranking to information emitters, and non-directional ranking mainly to the number of interactions. The protein lists generated by the forward and non-directional rankings are highly correlated, but those by the reverse and non-directional rankings are not. The results suggest that the signal emitting/receiving system is characterized by key-emittings and relatively even receivings in the human protein interactome. Signaling pathway proteins are frequent in top ranked ones. Eight proteins are both informational top emitters and top receivers. Top ranked proteins, except a few species-related novel-function ones, are evolutionarily well conserved. Protein-subunit ranking position reflects subunit function. These results demonstrate the usefulness of different PageRank approaches in characterizing protein networks and provide insights to protein interaction in the cell.     

Serum alpha-tocopherol, all-trans retinol, total lipids and cholesterol in the black rhinoceros (Diceros bicornis)

1. Mean concentration of serum alpha-tocopherol (Vitamin E) in 28 free-living black rhinoceroses sedated during translocation in Zimbabwe was 1.92 (SD, 0.43) mg/l. 2. Alpha-tocopherol was not detectable (less than 0.15 mg/l) in five captive black rhinoceroses held at London Zoo. 3. Circulating levels of all-trans retinol (Vitamin A) were not different between the two groups. 4. The low level of alpha-tocopherol in captive rhinoceroses suggests a risk of acute haemolytic anaemia.     

Cables1 protects p63 from proteasomal degradation to ensure deletion of cells after genotoxic stress

The p63 gene product regulates epithelial morphogenesis and female germline integrity. In this study, we show that cyclin‐dependent kinase 5 and Abl enzyme substrate 1 (Cables1) interacts with the trans‐activating (TA) p63α isoform to protect it from proteasomal degradation. Using the female germline of Cables1‐null mice as an in vivo model, we demonstrate further that oocytes lacking Cables1 exhibit lower basal levels of TAp63α and reduced accumulation of phosphorylated TAp63α in response to genotoxic stress. This in turn enhances the survival of these cells after ionizing radiation exposure. Thus, Cables1 modulates p63 protein stability and function during genotoxic stress.