Immunization with herpes simplex virus 2 (HSV-2) genes plus inactivated HSV-2 is highly protective against acute and recurrent HSV-2 disease

To date, no vaccine that is safe and effective against herpes simplex virus 2 (HSV-2) disease has been licensed. In this study, we evaluated a DNA prime-formalin-inactivated-HSV-2 (FI-HSV2) boost vaccine approach in the guinea pig model of acute and recurrent HSV-2 genital disease. Five groups of guinea pigs were immunized and intravaginally challenged with HSV-2. Two groups were primed with plasmid DNAs encoding the secreted form of glycoprotein D2 (gD2t) together with two genes required for viral replication, either the helicase (UL5) and DNA polymerase (UL30) genes or the single-stranded DNA binding protein (UL29) and primase (UL52) genes. Both DNA-primed groups were boosted with FI-HSV2 formulated with monophosphoryl lipid A (MPL) and alum adjuvants. Two additional groups were primed with the empty backbone plasmid DNA (pVAX). These two groups were boosted with MPL and alum (MPL-alum) together with either formalin-inactivated mock HSV-2 (FI-Mock) or with FI-HSV2. The final group was immunized with gD2t protein in MPL-alum. After challenge, 0/9 animals in the group primed with UL5, UL30, and gD2t DNAs and all 10 animals in the mock-immunized control group (pVAX-FI-Mock) developed primary lesions. All mock controls developed recurrent lesions through day 100 postchallenge. Only 1 guinea pig in the group primed with pVAX DNA and boosted with FI-HSV2 (pVAX-FI-HSV2 group) and 2 guinea pigs in the group primed with UL5, UL30, and gD2t DNAs and boosted with FI-HSV2 (UL5, UL30, gD2t DNA-FI-HSV2 group) developed recurrent lesions. Strikingly, the UL5, UL30, gD2t DNA-FI-HSV2 group showed a 97% reduction in recurrent lesion days compared with the mock controls, had the highest reduction in days with recurrent disease, and contained the lowest mean HSV-2 DNA load in the dorsal root ganglia.     

Activation of NAD(P)H oxidase by lipid hydroperoxides: mechanism of oxidant-mediated smooth muscle cytotoxicity

Oxidized lipids, such as 13-hydroperoxyoctadecadienoic acid (13-HPODE), have been implicated in the pathogenesis of atherosclerosis. 13-HPODE, a constituent of oxidized low-density lipoproteins, can induce cytotoxicity of vascular smooth muscle cells (SMC), which may facilitate plaque destabilization and/or rupture. 13-HPODE-induced cytotoxicity has been linked to oxidative stress, although the mechanisms by which this occurs are unknown. In the present study, we show that 13-HPODE and 9-HPODE (10-30 microM) increased superoxide (O2*-) production and induced cytotoxicity in SMC. The 13-HPODE-induced increase in O2*- was blocked by transfecting the cells with antisense oligonucleotides against p22phox, suggesting that the O2*- was produced by NAD(P)H oxidase. Similar concentrations of the corresponding HPODE reduction products, 13-hydroxyoctadecadienoic acid (13-HODE) and 9-HODE, neither increased O2*- production nor induced cytotoxicity, while 4-hydroxy nonenal (4-HNE), an unsaturated aldehyde lipid peroxidation product, induced cytotoxicity without increasing O2*- production. Treatment with superoxide dismutase or Tiron to scavenge O2*-, or transfection with p22phox antisense oligonucleotides to inhibit O2*- production, attenuated 13-HPODE-induced cytotoxicity, but not that induced by 4-HNE. These findings suggest that activation of NAD(P)H oxidase, and production of O2*-, play an important role in lipid hydroperoxide-induced smooth muscle cytotoxicity.     

Whole Exome Re-Sequencing Implicates CCDC38 and Cilia Structure and Function in Resistance to Smoking Related Airflow Obstruction

Chronic obstructive pulmonary disease (COPD) is a leading cause of global morbidity and mortality and, whilst smoking remains the single most important risk factor, COPD risk is heritable. Of 26 independent genomic regions showing association with lung function in genome-wide association studies, eleven have been reported to show association with airflow obstruction. Although the main risk factor for COPD is smoking, some individuals are observed to have a high forced expired volume in 1 second (FEV₁) despite many years of heavy smoking. We hypothesised that these “resistant smokers” may harbour variants which protect against lung function decline caused by smoking and provide insight into the genetic determinants of lung health. We undertook whole exome re-sequencing of 100 heavy smokers who had healthy lung function given their age, sex, height and smoking history and applied three complementary approaches to explore the genetic architecture of smoking resistance. Firstly, we identified novel functional variants in the “resistant smokers” and looked for enrichment of these novel variants within biological pathways. Secondly, we undertook association testing of all exonic variants individually with two independent control sets. Thirdly, we undertook gene-based association testing of all exonic variants. Our strongest signal of association with smoking resistance for a non-synonymous SNP was for rs10859974 (P = 2.34×10⁻⁴) in CCDC38, a gene which has previously been reported to show association with FEV₁/FVC, and we demonstrate moderate expression of CCDC38 in bronchial epithelial cells. We identified an enrichment of novel putatively functional variants in genes related to cilia structure and function in resistant smokers. Ciliary function abnormalities are known to be associated with both smoking and reduced mucociliary clearance in patients with COPD. We suggest that genetic influences on the development or function of cilia in the bronchial epithelium may affect growth of cilia or the extent of damage caused by tobacco smoke.     

Determining the quality and complexity of next-generation sequencing data without a reference genome

We describe an open-source kPAL package that facilitates an alignment-free assessment of the quality and comparability of sequencing datasets by analyzing k-mer frequencies. We show that kPAL can detect technical artefacts such as high duplication rates, library chimeras, contamination and differences in library preparation protocols. kPAL also successfully captures the complexity and diversity of microbiomes and provides a powerful means to study changes in microbial communities. Together, these features make kPAL an attractive and broadly applicable tool to determine the quality and comparability of sequence libraries even in the absence of a reference sequence. kPAL is freely available at https://github.com/LUMC/kPAL.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0555-3) contains supplementary material, which is available to authorized users.     

Epigenome-Wide DNA Methylation in Hearing Ability: New Mechanisms for an Old Problem

Epigenetic regulation of gene expression has been shown to change over time and may be associated with environmental exposures in common complex traits. Age-related hearing impairment is a complex disorder, known to be heritable, with heritability estimates of 57–70%. Epigenetic regulation might explain the observed difference in age of onset and magnitude of hearing impairment with age. Epigenetic epidemiology studies using unrelated samples can be limited in their ability to detect small effects, and recent epigenetic findings in twins underscore the power of this well matched study design. We investigated the association between venous blood DNA methylation epigenome-wide and hearing ability. Pure-tone audiometry (PTA) and Illumina HumanMethylation array data were obtained from female twin volunteers enrolled in the TwinsUK register. Two study groups were explored: first, an epigenome-wide association scan (EWAS) was performed in a discovery sample (n = 115 subjects, age range: 47–83 years, Illumina 27 k array), then replication of the top ten associated probes from the discovery EWAS was attempted in a second unrelated sample (n = 203, age range: 41–86 years, Illumina 450 k array). Finally, a set of monozygotic (MZ) twin pairs (n = 21 pairs) within the discovery sample (Illumina 27 k array) was investigated in more detail in an MZ discordance analysis. Hearing ability was strongly associated with DNA methylation levels in the promoter regions of several genes, including TCF25 (cg01161216, p = 6.6×10⁻⁶), FGFR1 (cg15791248, p = 5.7×10⁻⁵) and POLE (cg18877514, p = 6.3×10⁻⁵). Replication of these results in a second sample confirmed the presence of differential methylation at TCF25 (p(replication) = 6×10⁻⁵) and POLE (p(replication) = 0.016). In the MZ discordance analysis, twins'' intrapair difference in hearing ability correlated with DNA methylation differences at ACP6 (cg01377755, r = −0.75, p = 1.2×10⁻⁴) and MEF2D (cg08156349, r = −0.75, p = 1.4×10⁻⁴). Examination of gene expression in skin, suggests an influence of differential methylation on expression, which may account for the variation in hearing ability with age.     

Docosatetraenoic acid in endothelial cells: formation, retroconversion to arachidonic acid, and effect on prostacyclin production

Cultured bovine aortic endothelial cells convert arachidonic acid to docosatetraenoic acid and also take up docosatetraenoic acid from the extracellular fluid. After a 24-h incubation with biosynthetically prepared [3H]docosatetraenoic acid, about 20% of the cellular fatty acid radioactivity was converted to arachidonic acid. Furthermore, in pulse-chase experiments, the decrease in phospholipid docosatetraenoic acid content was accompanied by an increase in arachidonic acid, providing additional evidence for retroconversion. These findings suggest that one possible function of docosatetraenoic acid in endothelial cells is to serve as a source of arachidonic acid. The endothelial cells can release docosatetraenoic acid when they are stimulated with ionophore A23187, but they do not form appreciable amounts of eicosanoids from docosatetraenoic acid. Enrichment of the endothelial cells with docosatetraenoic acid reduced their capacity to produce prostacyclin (PGI2) in response to ionophore A23187. This may be related to the fact that docosatetraenoic acid enrichment caused a 40% reduction in the arachidonic acid content of the inositol phosphoglycerides. In addition, less prostacyclin was formed when the enriched cells were incubated with arachidonic acid, suggesting that docosatetraenoic acid also may act as an inhibitor of prostaglandin synthesis in endothelial cells.     

THE STABILITY OF VITAMIN B6 ACCUMULATION AND PYRIDOXAL KINASE ACTIVITY IN RABBIT BRAIN AND CHOROID PLEXUS

The effects of changes in the concentrations of pyridoxal phosphate and blogenic amines in brain on: (I) pyridoxal kinase (EC 2.7.1.35) activity in brain and choroid plexus; and (2) vitamin B₆ accumulation by brain slices and isolated, intact choroid plexuses were studied. New Zealand white rabbits were treated parenterally with 200 mg/kg pyridoxine-HCl for 3 days or 120 mg/kg 4-deoxypyridoxine HCI or 5 mg/kg reserpine I day before death. After these treatments the mean concentration of pyridoxal phosphate in brain was elevated by 39% by pyridoxine and decreased by 57% by 4-deoxypyridoxine. Reserpine had no effect. However, the ability of brain slices and isolated, intact choroid plexuses from the treated rabbits to accumulate [³H] vitamin B₆ (with [³H]pyridoxine in the medium) was not different from untreated controls. Also, the specific activity of pyridoxal kinase in brain and choroid plexus of treated rabbits was not different from controls. These results show that vitamin B₆ accumulation and pyridoxal kinase activity in brain and choroid plexus are independent of both pyridoxal phosphate and reserpine-sensitive biogenic amine concentrations in brain. In vitro studies with pyridoxal kinase showed that. in both choroid plexus and brain. pyridoxal kinase was a single enzyme with a molecular weight of 43.000 and a K_m , for pyridoxine of 2.0 μM Crude and partially-purified pyridoxal kinase from brain was not inhibited by biogenic amines (1 mM) or pyridoxal phosphate (5 μM). These in vitro data are consistent with the lack of effect of changes in pyridoxal phosphate and biogenic amine concentrations (in brain) on pyridoxal kinase activity in brain in vivo.     

Nuclear choreography: interpretations from living cells

The advent of green fluorescent protein technology, its use in photobleaching experiments and the development of methods to rapidly acquire images and analyze complex datasets have opened the door to unraveling the mechanisms of nuclear functions in living cells. Studies over the past few years have characterized the movement of chromatin, nuclear proteins and nuclear bodies and, in some cases, correlated their dynamics with energy dependence, cell cycle progression, developmental changes, factor targeting and nuclear position. The mechanisms by which nuclear components move or are restrained have important implications for understanding not only the efficacy of nuclear functions but also the regulation of developmental programs and cellular growth.