The pleiotropic phenotype of Apc mutations in the mouse: allele specificity and effects of the genetic background

Familial adenomatous polyposis (FAP) is a human cancer syndrome characterized by the development of hundreds to thousands of colonic polyps and extracolonic lesions including desmoid fibromas, osteomas, epidermoid cysts, and congenital hypertrophy of the pigmented retinal epithelium. Afflicted individuals are heterozygous for mutations in the APC gene. Detailed investigations of mice heterozygous for mutations in the ortholog Apc have shown that other genetic factors strongly influence the phenotype. Here we report qualitative and quantitative modifications of the phenotype of Apc mutants as a function of three genetic variables: Apc allele, p53 allele, and genetic background. We have found major differences between the Apc alleles Min and 1638N in multiplicity and regionality of intestinal tumors, as well as in incidence of extracolonic lesions. By contrast, Min mice homozygous for either of two different knockout alleles of p53 show similar phenotypic effects. These studies illustrate the classic principle that functional genetics is enriched by assessing penetrance and expressivity with allelic series. The mouse permits study of an allelic gene series on multiple genetic backgrounds, thereby leading to a better understanding of gene action in a range of biological processes.     

Detection of Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial (e.g., viruses, bacteria and parasite) infections, they also play an important role in the pathophysiology of infectious diseases, inflammatory diseases, and possibly in autoimmune diseases. Thus, the intensity and duration of TLR responses against infectious diseases must be tightly controlled. It follows that understanding the structural integrity of sensor receptors, their ligand interactions and signaling components is essential for subsequent immunological protection. It would also provide important opportunities for disease modification through sensor manipulation. Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between the sensors and their ligands still remain poorly defined. We have recently identified a novel mechanism of TLR activation by its natural ligand, which has not been previously observed ^1,2. It suggests that ligand-induced TLR activation is tightly controlled by Neu1 sialidase activation. We have also reported that Neu1 tightly regulates neurotrophin receptors like TrkA and TrkB ³, which involve Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in complex with the receptors ⁴. The sialidase assay has been initially use to find a novel ligand, thymoquinone, in the activation of Neu4 sialidase on the cell surface of macrophages, dendritic cells and fibroblast cells via GPCR Gαi proteins and MMP-9 ⁵. For TLR receptors, our data indicate that Neu1 sialidase is already in complex with TLR-2, -3 and -4 receptors, and is induced upon ligand binding to either receptor. Activated Neu1 sialidase hydrolyzes sialyl α-2,3-linked β-galactosyl residues distant from ligand binding to remove steric hinderance to TLR-4 dimerization, MyD88/TLR4 complex recruitment, NFkB activation and pro-inflammatory cell responses. In a collaborative report, Neu1 sialidase has been shown to regulate phagocytosis in macrophage cells ⁶. Taken together, the sialidase assay has provided us with powerful insights to the molecular mechanisms of ligand-induced receptor activation. Although the precise relationship between Neu1 sialidase and the activation of TLR, Trk receptors has yet to be fully elucidated, it would represent a new or pioneering approach to cell regulation pathways.Download video file.^{(57M, mov)}     

The Vi Capsular Polysaccharide Enables Salmonella enterica Serovar Typhi to Evade Microbe-Guided Neutrophil Chemotaxis

Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever, a disseminated infection, while the closely related pathogen S. enterica serovar Typhimurium (S. Typhimurium) is associated with a localized gastroenteritis in humans. Here we investigated whether both pathogens differ in the chemotactic response they induce in neutrophils using a single-cell experimental approach. Surprisingly, neutrophils extended chemotactic pseudopodia toward Escherichia coli and S. Typhimurium, but not toward S. Typhi. Bacterial-guided chemotaxis was dependent on the presence of complement component 5a (C5a) and C5a receptor (C5aR). Deletion of S. Typhi capsule biosynthesis genes markedly enhanced the chemotactic response of neutrophils in vitro. Furthermore, deletion of capsule biosynthesis genes heightened the association of S. Typhi with neutrophils in vivo through a C5aR-dependent mechanism. Collectively, these data suggest that expression of the virulence-associated (Vi) capsular polysaccharide of S. Typhi obstructs bacterial-guided neutrophil chemotaxis.     

Gear modifications reduced humpback whale entanglements in a commercial rock lobster fishery

Entanglement of whales in fishing gear occurs globally and where populations are recovering from past exploitation, entanglement frequency is likely to increase. The Western Australian population of humpback whales (Megaptera novaeangliae) is growing rapidly, yet from 1990 to 2010 the number of whales reported entangled in gear from the pot-based western rock lobster fishery was relatively stable at around one per year. However, from 2010, reported entanglements increased, reaching a maximum of 17 in 2013. This increase occurred immediately after a shift to a year-round quota-based fishery that eliminated the annual 4½-month closure that coincided with the whale migration. Gear modifications that eliminated surface rope, shortened rope lengths, and reduced float numbers were implemented in June 2014 to reduce whale entanglements. The effectiveness of these modifications was evaluated using a Bayesian model that incorporated changes in humpback whale population size, entanglement reporting probability, fishing effort, and whale migration timing. Our analyses indicate that gear modifications reduced entanglement in fishing gear from the rock lobster fishery by at least 25% (with 95% probability), with a median reduction of 64%. The model also showed that the greatest entanglement risk occurs on the northward migration and in water depths of 55–73 m.     

Differential Selection of Cells with Proviral c-myc and c-erbB Integrations after Avian Leukosis Virus Infection

Avian leukosis virus (ALV) infection induces bursal lymphomas in chickens after proviral integration within the c-myc proto-oncogene and induces erythroblastosis after integration within the c-erbB proto-oncogene. A nested PCR assay was used to analyze the appearance of these integrations at an early stage of tumor induction after infection of embryos. Five to eight distinct proviral c-myc integration events were amplified from bursas of infected 35-day-old birds, in good agreement with the number of transformed bursal follicles arising with these integrations. Cells containing these integrations are remarkably common, with an estimated 1 in 350 bursal cells having proviral c-myc integrations. These integrations were clustered within the 3′ half of c-myc intron 1, in a pattern similar to that observed in bursal lymphomas. Bone marrow and spleen showed a similar number and pattern of integrations clustered within 3′ c-myc intron 1, indicating that this region is a common integration target whether or not that tissue undergoes tumor induction. While all tissues showed equivalent levels of viral infection, cells with c-myc integrations were much more abundant in the bursa than in other tissues, indicating that cells with proviral c-myc integrations are preferentially expanded within the bursal environment. Proviral integration within the c-erbB gene was also analyzed, to detect clustered c-erbB intron 14 integrations associated with erythroblastosis. Proviral c-erbB integrations were equally abundant in the bone marrow, spleen, and bursa. These integrations were randomly situated upstream of c-erbB exon 15, indicating that cells carrying 3′ intron 14 integrations must be selected during induction of erythroblastosis.     

Extensive diversity of ionizing-radiation-resistant bacteria recovered from Sonoran Desert soil and description of nine new species of the genus Deinococcus obtained from a single soil sample

The ionizing-radiation-resistant fractions of two soil bacterial communities were investigated by exposing an arid soil from the Sonoran Desert and a nonarid soil from a Louisiana forest to various doses of ionizing radiation using a (60)Co source. The numbers of surviving bacteria decreased as the dose of gamma radiation to which the soils were exposed increased. Bacterial isolates surviving doses of 30 kGy were recovered from the Sonoran Desert soil, while no isolates were recovered from the nonarid forest soil after exposure to doses greater than 13 kGy. The phylogenetic diversities of the surviving culturable bacteria were compared for the two soils using 16S rRNA gene sequence analysis. In addition to a bacterial population that was more resistant to higher doses of ionizing radiation, the diversity of the isolates was greater in the arid soil. The taxonomic diversity of the isolates recovered was found to decrease as the level of ionizing-radiation exposure increased. Bacterial isolates of the genera Deinococcus, Geodermatophilus, and Hymenobacter were still recovered from the arid soil after exposure to doses of 17 to 30 kGy. The recovery of large numbers of extremely ionizing-radiation-resistant bacteria from an arid soil and not from a nonarid soil provides further ecological support for the hypothesis that the ionizing-radiation resistance phenotype is a consequence of the evolution of other DNA repair systems that protect cells against commonly encountered environmental stressors, such as desiccation. The diverse group of bacterial strains isolated from the arid soil sample included 60 Deinococcus strains, the characterization of which revealed nine novel species of this genus.     

Screening random peptide libraries with subacute sclerosing panencephalitis brain-derived recombinant antibodies identifies multiple epitopes in the C-terminal region of the measles virus nucleocapsid protein

Infectious and inflammatory diseases of the CNS are often characterized by a robust B-cell response that manifests as increased intrathecal immunoglobulin G (IgG) synthesis and the presence of oligoclonal bands. We previously used laser capture microdissection and single-cell PCR to analyze the IgG variable regions of plasma cells from the brain of a patient with subacute sclerosing panencephalitis (SSPE). Five of eight human IgG1 recombinant antibodies (rAbs) derived from SSPE brain plasma cell clones recognized the measles virus (MV) nucleocapsid protein, confirming that the antibody response in SSPE targets primarily the agent causing disease. In this study, as part of our work on antigen identification, we used four rAbs to probe a random phage-displayed peptide library to determine if epitopes within the MV nucleocapsid protein could be identified with SSPE brain rAbs. All four of the SSPE rAbs enriched phage-displayed peptide sequences that reacted specifically to their panning rAb by enzyme-linked immunosorbent assay. BLASTP searches of the NCBI protein database revealed clear homologies in three peptides and different amino acid stretches within the 65 C-terminal amino acids of the MV nucleocapsid protein. The specificities of SSPE rAbs to these regions of the MV nucleocapsid protein were confirmed by binding to synthetic peptides or to short cDNA expression products. These results indicate the feasibility of using peptide screening for antigen discovery in central nervous system inflammatory diseases of unknown etiology, such as multiple sclerosis, neurosarcoidosis, or Behcet's syndrome.