Global regulation of alternative splicing during myogenic differentiation

Recent genome-wide analyses have elucidated the extent of alternative splicing (AS) in mammals, often focusing on comparisons of splice isoforms between differentiated tissues. However, regulated splicing changes are likely to be important in biological transitions such as cellular differentiation, or response to environmental stimuli. To assess the extent and significance of AS in myogenesis, we used splicing-sensitive microarray analysis of differentiating C2C12 myoblasts. We identified 95 AS events that undergo robust splicing transitions during C2C12 differentiation. More than half of the splicing transitions are conserved during differentiation of avian myoblasts, suggesting the products and timing of transitions are functionally significant. The majority of splicing transitions during C2C12 differentiation fall into four temporal patterns and were dependent on the myogenic program, suggesting that they are integral components of myogenic differentiation. Computational analyses revealed enrichment of many sequence motifs within the upstream and downstream intronic regions near the alternatively spliced regions corresponding to binding sites of splicing regulators. Western analyses demonstrated that several splicing regulators undergo dynamic changes in nuclear abundance during differentiation. These findings show that within a developmental context, AS is a highly regulated and conserved process, suggesting a major role for AS regulation in myogenic differentiation.     

Glutaredoxin-2 Is Required to Control Proton Leak through Uncoupling Protein-3

Glutathionylation has emerged as a key modification required for controlling protein function in response to changes in cell redox status. Recently, we showed that the glutathionylation state of uncoupling protein-3 (UCP3) modulates the leak of protons back into the mitochondrial matrix, thus controlling reactive oxygen species production. However, whether or not UCP3 glutathionylation is mediated enzymatically has remained unknown because previous work relied on the use of pharmacological agents, such as diamide, to alter the UCP3 glutathionylation state. Here, we demonstrate that glutaredoxin-2 (Grx2), a matrix oxidoreductase, is required to glutathionylate and inhibit UCP3. Analysis of bioenergetics in skeletal muscle mitochondria revealed that knock-out of Grx2 (Grx2^−/−) increased proton leak in a UCP3-dependent manner. These effects were reversed using diamide, a glutathionylation catalyst. Importantly, the increased leak did not compromise coupled respiration. Knockdown of Grx2 augmented proton leak-dependent respiration in primary myotubes from wild type mice, an effect that was absent in UCP3^−/− cells. These results confirm that Grx2 deactivates UCP3 by glutathionylation. To our knowledge, this is the first enzyme identified to regulate UCP3 by glutathionylation and is the first study on the role of Grx2 in the regulation of energy metabolism.     

Are mesophotic coral ecosystems distinct communities and can they serve as refugia for shallow reefs?

We analyzed an extensive dataset of over 9000 benthic and suprabenthic species found throughout the Gulf of Mexico (GoMx) to assess whether mesophotic coral ecosystems represent distinct assemblages and evaluate their potential to serve as refugia for shallow reef communities. We assessed community structure of the overall benthic community from 0 to 300 m via non-metric multidimensional scaling (NMDS) of species presence across depth bands. We used the Jaccard index of similarity to calculate the proportion of shared species between adjacent depth bands, measure species turnover with depth, and assess taxonomic overlap between shallow reefs versus progressively deeper depth bands. NMDS ordinations showed that the traditionally defined mesophotic range (30–150 m) as a whole is not a distinct community. In contrast, taxonomically distinct communities, determined by hierarchical clustering, were found at 0–70, 60–120, 110–200, and 190–300 m. Clustering highlighted an important separation in the benthic community at ~60 m, which was especially important for actinopterygian fishes. Species turnover between adjacent depths decreased with depth for all taxa combined and individual taxa, with peaks at ~60, 90–120, and 190–200 m. Fishes showed lower turnover from shallow to upper mesophotic depths (0–50 m) than all taxa combined, a substantial peak at 60 m, followed by a precipitous and continued decline in turnover thereafter. Taxonomic overlap between shallow (0–20 m) and progressively deeper zones declined steadily with depth in all taxa and individual taxa, suggesting that mid- and lower mesophotic habitats have less (but not inconsequential) potential to serve as refugia (60–150 m, 15–25% overlap with shallow habitats) than upper mesophotic zones (30–60 m, 30–45% overlap with shallow habitats) for all taxa combined. We conclude that the traditional mesophotic zone is home to three ecological communities in the GoMx, one that is confluent with shallow reefs, a distinct mesophotic assemblage spanning 60–120 m, and a third that extends onto the outer continental shelf.     

Monitoring Anaerobic Natural Attenuation of Petroleum Using a Novel In Situ Respiration Method in Low-Permeability Sediment

Assessing petroleum biodegradation rates is an important part of predicting natural attenuation in subsurface sediments. Monitoring carbon dioxide (CO₂) and methane (CH₄) produced in situ, and their radiocarbon ¹⁴C), stable carbon (¹³C) and deuterium (D). signature provide a novel method to assess anaerobic microbial processes. Our objectives were to: (1) estimate the rate of anaerobic petroleum hydrocarbon (PH) mineralization by monitoring the production of soil gas CH₄ and CO₂ in the vadose zone of low-permeability sediment, (2) evaluate the dominant microbial processes using δ¹³C and δD, and (3) determine the proportion of CH₄ and CO₂ attributable to anaerobic mineralization of PH using ¹⁴C analysis. Argon was sparged into the subsurface to dilute existing CO₂ and CH₄ concentrations. Vadose zone CO₂, CH₄, oxygen, total combustible hydrocarbons, and argon concentrations were measured for 75 days. CO₂ and CH₄ samples were collected on day 86 and analyzed for ¹⁴C, δ¹³C, and δD. Based on CH₄ soil gas production, the anaerobic biodegradation rate was estimated between 0.017 to 0.055 mg/kg soil-d. CH₄ ¹⁴C (2.6 pMC), δ¹³C (-45.64‰), and δD (-316‰) values indicated that fermentation of PH was the sale source of CH₄ in the vadose zone. CO₂ ¹⁴C (62 pMC) indicated that approximately 47% of the total CO₂ was from PH mineralization and 53% from plant root respiration. Although low-permeability sediment increases the difficulty of completely replacing in situ soil gas and assuring anaerobic conditions, this novel respiration method distinguished between anaerobic processes responsible for PH degradation.     

Break-induced loss of heterozygosity in fission yeast: dual roles for homologous recombination in promoting translocations and preventing de novo telomere addition

Loss of heterozygosity (LOH), a causal event in tumorigenesis, frequently encompasses multiple genetic loci and whole chromosome arms. However, the mechanisms leading to such extensive LOH are poorly understood. We investigated the mechanisms of DNA double-strand break (DSB)-induced extensive LOH by screening for auxotrophic marker loss approximately 25 kb distal to an HO endonuclease break site within a nonessential minichromosome in Schizosaccharomyces pombe. Extensive break-induced LOH was infrequent, resulting from large translocations through both allelic crossovers and break-induced replication. These events required the homologous recombination (HR) genes rad32(+), rad50(+), nbs1(+), rhp51(+), rad22(+), rhp55(+), rhp54(+), and mus81(+). Surprisingly, LOH was still observed in HR mutants, which resulted predominantly from de novo telomere addition at the break site. De novo telomere addition was most frequently observed in rad22Delta and rhp55Delta backgrounds, which disrupt HR following end resection. Further, levels of de novo telomere addition, while increased in ku70Delta rhp55Delta strains, were reduced in exo1Delta rhp55Delta and an rhp55Delta strain overexpressing rhp51. These findings support a model in which HR prevents de novo telomere addition at DSBs by competing for resected ends. Together, these results suggest that the mechanisms of break-induced LOH may be predicted from the functional status of the HR machinery.     

A second human antiretroviral factor, APOBEC3F, is suppressed by the HIV-1 and HIV-2 Vif proteins

The HIV-1 Vif protein suppresses the inhibition of viral replication caused by the human antiretroviral factor APOBEC3G. As a result, HIV-1 mutants that do not express the Vif protein are replication incompetent in 'nonpermissive' cells, such as primary T cells and the T-cell line CEM, that express APOBEC3G. In contrast, Vif-defective HIV-1 replicates effectively in 'permissive' cell lines, such as a derivative of CEM termed CEM-SS, that do not express APOBEC3G. Here, we show that a second human protein, APOBEC3F, is also specifically packaged into HIV-1 virions and inhibits their infectivity. APOBEC3F binds the HIV-1 Vif protein specifically and Vif suppresses both the inhibition of virus infectivity caused by APOBEC3F and virion incorporation of APOBEC3F. Surprisingly, APOBEC3F and APOBEC3G are extensively coexpressed in nonpermissive human cells, including primary lymphocytes and the cell line CEM, where they form heterodimers. In contrast, both genes are quiescent in the permissive CEM derivative CEM-SS. Together, these data argue that HIV-1 Vif has evolved to suppress at least two distinct but related human antiretroviral DNA-editing enzymes.     

Targeting the vaginal mucosa with human papillomavirus pseudovirion vaccines delivering simian immunodeficiency virus DNA

The majority of HIV infections occur via mucosal transmission. Vaccines that induce memory T and B cells in the female genital tract may prevent the establishment and systemic dissemination of HIV. We tested the immunogenicity of a vaccine that uses human papillomavirus (HPV)-based gene transfer vectors, also called pseudovirions (PsVs), to deliver SIV genes to the vaginal epithelium. Our findings demonstrate that this vaccine platform induces gene expression in the genital tract in both cynomolgus and rhesus macaques. Intravaginal vaccination with HPV16, HPV45, and HPV58 PsVs delivering SIV Gag DNA induced Gag-specific Abs in serum and the vaginal tract, and T cell responses in blood, vaginal mucosa, and draining lymph nodes that rapidly expanded following intravaginal exposure to SIV(mac251.) HPV PsV-based vehicles are immunogenic, which warrant further testing as vaccine candidates for HIV and may provide a useful model to evaluate the benefits and risks of inducing high levels of SIV-specific immune responses at mucosal sites prior to SIV infection.     

Characterization of High-Affinity Dopamine D2 Receptors and Modulation of Affinity States by Guanine Nucleotides in Cholate-Solubilized Bovine Striatal Preparations

3,4-Dihydroxyphenylethylamine (dopamine) D2 receptors, solubilized from bovine striatal membranes using a cholic acid-NaCl combination, exhibited the typical pharmacological characteristics of both agonist and antagonist binding. The rank order potency of the agonists and antagonists to displace [3H]spiroperidol binding was the same as that observed with membrane-bound receptors. Computer-assisted analysis of the [3H]spiroperidol/agonist competition curves revealed the retention of high- and low-affinity states of the D2 receptor in the solubilized preparations and the proportions of receptor subpopulations in the two affinity states were similar to those reported in membrane. Guanine nucleotide almost completely converted the high-affinity sites to low-affinity sites for the agonists. The binding of the high-affinity agonist [3H]N-n-propylnorapomorphine ([3H]NPA) was clearly demonstrated in the solubilized preparations for the first time. Addition of guanylyl-imidodiphosphate completely abolished the [3H]NPA binding. When the solubilized receptors were subjected to diethylaminoethyl-Sephacel chromatography, the dopaminergic binding sites eluted in two distinct peaks, showing six- to sevenfold purification of the receptors in the major peak. Binding studies performed on both peaks indicated that the receptor subpopulation present in the first peak may have a larger proportion of high-affinity binding sites than the second peak. The solubilized preparation also showed high-affinity binding of [35S]guanosine-5'-(gamma-thio)triphosphate, a result suggesting the presence of guanine nucleotide binding sites, which may interact with the solubilized D2 receptors. These data are consistent with the retention of the D2 receptor-guanine nucleotide regulatory protein complex in the solubilized preparations and should provide a suitable model system to study the receptor-effector interactions.