Reconstitution of the Kaposi's sarcoma-associated herpesvirus nuclear egress complex and formation of nuclear membrane vesicles by coexpression of ORF67 and ORF69 gene products

The Kaposi's sarcoma-associated herpesvirus nuclear egress complex is composed of two proteins, ORF67 and ORF69. In this study, we have recapitulated the KSHV complex by coexpression of these two proteins in insect cells using expression from recombinant baculoviruses. The proteins form a complex at the nuclear membrane as judged by live-cell analysis of protein fusions tagged with green fluorescent protein (GFP) and mCherry. Ultrastructural analysis of infected cells showed that ORF67 expression results in reduplication of the nuclear membrane. When the two proteins are expressed together, numerous virion-size nuclear membrane-derived vesicles were evident at the nuclear margins.     

Microbial iron acquisition is influenced by spatial and temporal conditions in a glacial influenced river and estuary system

In Arctic regions, glaciers are major sources of iron to rivers and streams; however, estuaries are considered iron sinks due to the coagulation and flocculation processes that occur at higher salinities. It is unknown how iron dynamics in a glacial influenced river and estuary environment affect microbial mechanisms for iron acquisition. Microbial taxonomic and functional sequencing was performed on samples taken throughout the year from the Kenai River and the estuary, Alaska. Despite distinct iron, sodium, and other nutrient concentrations, the river and estuary did not have statistically different microbial communities nor was time of sampling significant. However, ferrous iron transport (Feo) system genes were more abundant in river environments, while siderophore genes were more abundant and diverse in estuary environments. Siderophore transport and iron storage genes were found in all samples, but gene abundance and distribution were potentially influenced by physical drivers such as discharge rates and nutrient distributions. Differences in iron metabolism between river and estuary ecosystems indicate environmental conditions drive microbial mechanisms to sequester iron. This could have implications for iron transport as the Arctic continues to warm.     

LBNP treadmill exercise maintains spine function and muscle strength in identical twins during 28-day simulated microgravity.

The purpose of this study was to determine whether lower body negative pressure (LBNP) treadmill exercise maintains lumbar spinal compressive properties, curvature, and back muscle strength after 28 days of 6 degrees head-down tilt (HDT) bed rest (BR). We hypothesize that LBNP treadmill exercise will maintain lumbar spine compressibility, lumbar lordosis and back muscle strength after 28 days of 6 degrees HDT bed rest. Fifteen healthy identical twin pairs (14 women and 16 men) participated in this study. One identical twin was randomly assigned to the nonexercise control (Con) group, and their sibling was assigned to the exercise (Ex) group. The lumbar spine was significantly more compressible Post-BR compared with Pre-BR in the Con (P=0.01). Lumbar spine compressibility Post-BR was not significantly different compared with Pre-BR in the Ex group (P=0.89). In both the Con and Ex groups, there were no significant changes Post-BR in lumbar lordosis compared with Pre-BR. Back muscle strength significantly decreased in the Con group Post-BR (P=0.002), whereas in the Ex group back muscle strength was not significantly different from Pre-BR values. A significant increase in lumbar spine compressibility in the Con group suggests that spinal deconditioning to gravity occurs during 28-day bed rest. Changes in the mechanical properties of the lumbar spine may be an early indicator of lumbar intervertebral disk degeneration. Supine LBNP treadmill exercise provides axial loads to the lumbar spine and may prevent lumbar spine deconditioning associated with HDT bed rest.     

Evoked Potential Audiograms of the Nurse Shark (Ginglymostoma cirratum) and the Yellow Stingray (Urobatis jamaicensis)

The hearing thresholds of the nurse shark, Ginglymostoma cirratum, and the yellow stingray, Urobatis jamaicensis, were measured using auditory evoked potentials (AEP). Stimuli were calibrated using a pressure-velocity probe so that the acoustic field could be completely characterized. The results show similar hearing thresholds for both species and similar hearing thresholds to previously measured audiograms for the lemon shark, Negaprion brevirostris, and the horn shark, Heterodontis francisi. All of these audiograms suggest poor hearing abilities, raising questions about field studies showing attraction of sharks to acoustic signals. By extrapolating the particle acceleration thresholds into estimates of their equivalent far-field sound pressure levels, it appears that these sharks cannot likely detect most of the sounds that have attracted sharks in the field.     

TRIM5α does not affect simian immunodeficiency virus SIV(mac251) replication in vaccinated or unvaccinated Indian rhesus macaques following intrarectal challenge exposure

TRIM5α is a natural resistance factor that binds retroviral capsid proteins and restricts virus replication. The B30.2/SPRY domain of TRIM5α is polymorphic in rhesus macaques, and some alleles are associated with reduced simian immunodeficiency virus (SIV) SIV(mac251) and SIV(smE543) replication in vivo. We determined the distribution of TRIM5α alleles by PCR and sequence analysis of the B30.2/SPRY domain in a cohort of 82 macaques. Thirty-nine of these macaques were mock vaccinated, 43 were vaccinated with either DNA-SIV/ALVAC-SIV/gp120, ALVAC-SIV/gp120, or gp120 alone, and all were exposed intrarectally to SIV(mac251) at one of three doses. We assessed whether the TRIM5α genotype of the macaques affected the replication of challenge virus by studying the number of SIV variants transmitted, the number of exposures required, the SIV(mac251) viral level in plasma and tissue, and the CD4(+) T-cell counts. Our results demonstrated that TRIM5α alleles, previously identified as restrictive for SIV(mac251) replication in vivo following intravenous exposure, did not affect SIV(mac251) replication following mucosal exposure, regardless of prior vaccination, challenge dose, or the presence of the protective major histocompatibility complex alleles (MamuA01(+), MamuB08(+), or MamuB017(+)). The TRIM5α genotype had no apparent effect on the number of transmitted variants or the number of challenge exposures necessary to infect the animals. DNA sequencing of the SIV(mac251) Gag gene of the two stocks used in our study revealed SIV(mac239)-like sequences that are predicted to be resistant to TRIM5α restriction. Thus, the TRIM5α genotype does not confound results of mucosal infection of rhesus macaques with SIV(mac251).     

Use of damaged DNA and dNTP substrates by the error-prone DNA polymerase X from African swine fever virus

The structural specificity that translesion DNA polymerases often show for a particular class of lesions suggests that the predominant criterion of selection during their evolution has been the capacity for lesion tolerance and that the error-proneness they display when copying undamaged templates may simply be a byproduct of this adaptation. Regardless of selection criteria/evolutionary history, at present both of these properties coexist in these enzymes, and both properties confer a fitness advantage. The repair polymerase, Pol X, encoded by the African swine fever virus (ASFV) is one of the most error-prone polymerases known, leading us to previously hypothesize that it may work in tandem with the exceptionally error-tolerant ASFV DNA ligase to effect viral mutagenesis. Here, for the first time, we test whether the error-proneness of Pol X is coupled with a capacity for lesion tolerance by examining its ability to utilize the types of damaged DNA and dNTP substrates that are expected to be relevant to ASFV. We (i) test Pol X's ability to both incorporate opposite to and extend from ubiquitous oxidative purine (7,8-dihydro-8-oxoguanine), oxidative pyrimidine (5,6-dihydroxy-5,6-dihydrothymine), and noncoding (AP site) lesions, in addition to 5,6-dihydrothymine, (ii) determine the catalytic efficiency and dNTP specificity of Pol X when catalyzing incorporation opposite to, and when extending from, 7,8-dihydro-8-oxoguanine in a template/primer context, and (iii) quantitate Pol X-catalyzed incorporation of the damaged nucleotide 8-oxo-dGTP opposite to undamaged templates in the context of both template/primer and a single-nucleotide gap. Our findings are discussed in light of ASFV biology and the mutagenic DNA repair hypothesis described above.     

Significant Variation for Bio-oil Compounds After Pyrolysis/Gas Chromatography–Mass Spectrometry of Cobs and Stover Among Five Near-Isogenic Brown Midrib Hybrids in Maize

We analyzed five near-isogenic brown midrib hybrids in maize via pyrolysis/gas chromatography–mass spectrometry (Py/GC-MS) in order to determine how differing lignin composition and structure impacts individual bio-oil compounds. Twenty-six compounds were analyzed for differences among the five hybrids and between cob and stover materials. We found statistically significant differences for 9 compounds, when comparing the 5 hybrids, and 17 significant differences when comparing maize cobs with stover. Our data indicate that it may be possible to predict phenolic compounds within bio-oil based on cell wall lignin composition. The genetic variation observed in this study suggests that bio-oil quality can be improved by plant breeding.     

siRNA-induced liver ApoB knockdown lowers serum LDL-cholesterol in a mouse model with human-like serum lipids

Increased serum apolipoprotein (apo)B and associated LDL levels are well-correlated with an increased risk of coronary disease. ApoE^–/– and low density lipoprotein receptor (LDLr)^–/– mice have been extensively used for studies of coronary atherosclerosis. These animals show atherosclerotic lesions similar to those in humans, but their serum lipids are low in apoB-containing LDL particles. We describe the development of a new mouse model with a human-like lipid profile. Ldlr CETP^+/– hemizygous mice carry a single copy of the human CETP transgene and a single copy of a LDL receptor mutation. To evaluate the apoB pathways in this mouse model, we used novel short-interfering RNAs (siRNA) formulated in lipid nanoparticles (LNP). ApoB siRNAs induced up to 95% reduction of liver ApoB mRNA and serum apoB protein, and a significant lowering of serum LDL in Ldlr CETP^+/– mice. ApoB targeting is specific and dose-dependent, and it shows lipid-lowering effects for over three weeks. Although specific triglycerides (TG) were affected by ApoB mRNA knockdown (KD) and the total plasma lipid levels were decreased by 70%, the overall lipid distribution did not change. Results presented here demonstrate a new mouse model for investigating additional targets within the ApoB pathways using the siRNA modality.     

Role of ßarrestins in bradykinin B2 receptor-mediated signalling

G protein-coupled receptors (GPCRs) can engage multiple pathways to activate ERK1/2 via both G proteins and/or ßarrestin. Receptor recruitment of ßarrestin is also important for GPCR desensitization, internalization and resensitization. Modulation of the receptor/ßarrestin interaction through modification of either component would presumably alter the output generated by receptor activation. Here we examined how ßarrestins regulate bradykinin (BK) B2 receptor (B2R) signalling and desensitization by either truncating ßarrestin1 or ßarrestin2 or by alanine substitution of a serine/threonine cluster in the C-terminal tail of B2R (B2R-4A), conditions which all affect the avidity of the B2R/ßarrestin complex. We first demonstrate that BK-mediated ERK1/2 activation is biphasic containing an early peak (between 2-5 min) followed by sustained activation for at least 60 min. The early but not the sustained phase was predictably affected by inhibition of either Gαq/11 or Gαi/o, whereas loss of ßarrestin2 but not ßarrestin1 resulted in diminished prolonged ERK1/2 activation. ßarrestin2's role was further examined using a truncation mutant with augmented avidity for the agonist-occupied receptor, revealing an increase in both immediate and extended ERK1/2 signalling. We also show that ERK1/2 is recruited to the B2R/ßarrestin complex on endosomes as well as the plasma membrane. Moreover, we investigated ßarrestin's role using the B2R-4A, which is deficient in ßarrestin binding and does not internalize. We show that ERK1/2 signalling downstream of the receptor is entirely G protein-dependent and receptor-mediated intracellular calcium mobilization studies revealed a lack of desensitization. Functionally, the lack of desensitization resulted in increased cell growth and migration compared to the wild-type receptor, which was sensitive to MEK inhibition. These results highlight ßarrestin's crucial role in the maintenance of proper B2R signalling.