Prevalence and Phylogenetic Analysis of Hepatitis B in Captive and Wild-Living Pileated Gibbons (Hylobates pileatus) in Cambodia

International Journal of Primatology - The risk of transmitting novel pathogens from released animals to wild conspecifics is an important consideration for reintroduction initiatives. Apart from...     

Gamma radiation increases endonuclease-dependent L1 retrotransposition in a cultured cell assay

Long Interspersed Elements (LINE-1s, L1s) are the most active mobile elements in the human genome and account for a significant fraction of its mass. The propagation of L1 in the human genome requires disruption and repair of DNA at the site of integration. As Barbara McClintock first hypothesized, genotoxic stress may contribute to the mobilization of transposable elements, and conversely, element mobility may contribute to genotoxic stress. We tested the ability of genotoxic agents to increase L1 retrotransposition in a cultured cell assay. We observed that cells exposed to gamma radiation exhibited increased levels of L1 retrotransposition. The L1 retrotransposition frequency was proportional to the number of phosphorylated H2AX foci, an indicator of genotoxic stress. To explore the role of the L1 endonuclease in this context, endonuclease-deficient tagged L1 constructs were produced and tested for their activity in irradiated cells. The activity of the endonuclease-deficient L1 was very low in irradiated cells, suggesting that most L1 insertions in irradiated cells still use the L1 endonuclease. Consistent with this interpretation, DNA sequences that flank L1 insertions in irradiated cells harbored target site duplications. These results suggest that increased L1 retrotransposition in irradiated cells is endonuclease dependent. The mobilization of L1 in irradiated cells potentially contributes to genomic instability and could be a driving force for secondary mutations in patients undergoing radiation therapy.     

Exogenous and endogenous TLR ligands activate anti-chromatin and polyreactive B cells

Autoreactive B cells may become activated in a T-independent manner via synergistic engagement of the BCR and TLRs. Using the VH3H9 Ig H chain transgene to track anti-chromatin B cells, we demonstrate that VH3H9/Vlambda1 anti-chromatin B cells proliferate in response to stimulatory oligodeoxynucleotides containing CpG motifs, suggesting that these autoreactive B cells are responsive to TLR9 signaling. Strikingly, some VH3H9 B cells, but not the well-characterized VH3H9/Vlambda1 B cells, proliferate spontaneously in culture medium. This proliferation is blocked by inhibitory CpG oligodeoxynucleotides, implicating the TLR9 (or possibly TLR7) pathway. Most hybridomas generated from the proliferating cells are polyreactive, and one exhibits binding to nuclear Ags but not to the other Ags tested. Thus, B cells carrying autoreactive and/or polyreactive specificities may be susceptible to T cell-independent activation via dual engagement of the BCR and TLRs.     

Degradation of polycyclic aromatic hydrocarbons dissolved in Tween 80 surfactant solutions by Sphingomonas paucimobilis EPA 505

The degradation rates of mixtures of pyrene (PYR), fluoranthene (FLA), and phenanthrene (PHE) by Sphingomonas paucimobilis EPA 505 were measured in the presence of the nonionic surfactant Tween 80. For strain EPA 505, FLA and PHE are growth substrates, while PYR is not. Linear degradation rates ranging from 0.05 to 2.2 mg x L(-1) x h(-1) were observed for FLA, PYR, and PHE at approximately 10(7) colony-forming units (CFU)/mL. At lower biomass, PYR degradation exhibited lognormal degradation. The degradation rates of PYR, FLA, and PHE increased with increasing biomass and substrate concentration. At high FLA concentrations, FLA degradation rates were faster in the presence of surfactant than in the absence of surfactant, suggesting that some of the FLA was transported directly into the cell from the micellar phase. In mixtures, PHE was the preferred substrate and was utilized first, followed by FLA and then PYR. Once the competing substrates were degraded, the remaining substrate was degraded at the same rate or faster than the rate found in the single-substrate system. Based on the results with Tween 80, it appears that PHE, PYR, and FLA are competing for the same enzymatic sites.     

Determination of L1 retrotransposition kinetics in cultured cells

L1 retrotransposons are autonomous retroelements that are active in the human and mouse genomes. Previously, we developed a cultured cell assay that uses a neomycin phosphotransferase (neo) retrotransposition cassette to determine relative retrotransposition frequencies among various L1 elements. Here, we describe a new retrotransposition assay that uses an enhanced green fluorescent protein (EGFP) retrotransposition cassette to determine retrotransposition kinetics in cultured cells. We show that retrotransposition is not detected in cultured cells during the first 48 h post-transfection, but then proceeds at a continuous high rate for at least 16 days. We also determine the relative retrotransposition rates of two similar human L1 retrotransposons, L1_RP and L1.3. L1_RP retrotransposed in the EGFP assay at a rate of ~0.5% of transfected cells/day, ~3-fold higher than the rate measured for L1.3. We conclude that the new assay detects near real time retrotransposition in a single cell and is sufficiently sensitive to differentiate retrotransposition rates among similar L1 elements. The EGFP assay exhibits improved speed and accuracy compared to the previous assay when used to determine relative retrotransposition frequencies. Furthermore, the EGFP cassette has an expanded range of experimental applications.     

Mobile elements and the human genome

Genomic DNA is often thought of as the stable template of heredity, largely dormant and unchanging, apart from perhaps the occasional point mutation. But it has become increasingly clear that DNA is dynamic rather than static, being subjected to rearrangements, insertions and deletions. Much of this plasticity can be attributed to transposable elements and their genomic relatives.     

Persistence and selection of an expanded B-cell clone in the setting of rituximab therapy for Sj?gren’s syndrome

Introduction

Subjects with primary Sjögren’s syndrome (SjS) have an increased risk of developing B-cell lymphoma and may harbor monoclonal B-cell expansions in the peripheral blood. Expanded B-cell clones could be pathogenic, and their persistence could exacerbate disease or predispose toward the development of lymphoma. Therapy with anti-CD20 (rituximab) has the potential to eliminate expanded B-cell clones and thereby potentially ameliorate disease. This study was undertaken to identify and track expanded B-cell clones in the blood of subjects with primary SjS who were treated with rituximab.

Methods

To determine whether circulating B-cell clones in subjects with primary SjS emerge or remain after B cell-depleting therapy with rituximab, we studied the antibody heavy-chain repertoire. We performed single-memory B-cell and plasmablast sorting and antibody heavy-chain sequencing in six rituximab-treated SjS subjects over the course of a 1-year follow-up period.

Results

Expanded B-cell clones were identified in four out of the six rituximab-treated SjS subjects, based upon the independent amplification of sequences with identical or highly similar VH, DH, and JH gene segments. We identified one SjS subject with a large expanded B-cell clone that was present prior to therapy and persisted after therapy. Somatic mutations in the clone were numerous but did not increase in frequency over the course of the 1-year follow-up, suggesting that the clone had been present for a long period of time. Intriguingly, a majority of the somatic mutations in the clone were silent, suggesting that the clone was under chronic negative selection.

Conclusions

For some subjects with primary SjS, these data show that (a) expanded B-cell clones are readily identified in the peripheral blood, (b) some clones are not eliminated by rituximab, and (c) persistent clones may be under chronic negative selection or may not be antigen-driven. The analysis of sequence variation among members of an expanded clone may provide a novel means of measuring the chronicity and selection of expanded B-cell populations in humans.     

Homozygous mutations in PXDN cause congenital cataract, corneal opacity, and developmental glaucoma

Anterior segment dysgenesis describes a group of heterogeneous developmental disorders that affect the anterior chamber of the eye and are associated with an increased risk of glaucoma. Here, we report homozygous mutations in peroxidasin (PXDN) in two consanguineous Pakistani families with congenital cataract-microcornea with mild to moderate corneal opacity and in a consanguineous Cambodian family with developmental glaucoma and severe corneal opacification. These results highlight the diverse ocular phenotypes caused by PXDN mutations, which are likely due to differences in genetic background and environmental factors. Peroxidasin is an extracellular matrix-associated protein with peroxidase catalytic activity, and we confirmed localization of the protein to the cornea and lens epithelial layers. Our findings imply that peroxidasin is essential for normal development of the anterior chamber of the eye, where it may have a structural role in supporting cornea and lens architecture as well as an enzymatic role as an antioxidant enzyme in protecting the lens, trabecular meshwork, and cornea against oxidative damage.