Comparative proteomics reveals evidence for evolutionary diversification of rodent seminal fluid and its functional significance in sperm competition

During insemination, males of internally fertilizing speciestransfer a complex array of seminal fluid proteins to the femalereproductive tract. These proteins can have profound effectson female reproductive physiology and behavior and are thoughtto mediate postcopulatory sexual selection and intersexual conflict.Such selection may cause seminal fluid to evolve rapidly, withpotentially important consequences for speciation. Here we investigatethe evolution of seminal fluid proteins in a major mammalianradiation, the muroid rodents, by quantifying diversity in seminalfluid proteome composition for the first time across a broadrange of closely related species. Using comparative proteomicstechniques to identify and cross-match proteins, we demonstratethat rodent seminal fluid is highly diverse at the level ofboth proteomes and individual proteins. The striking interspecificheterogeneity in seminal fluid composition revealed by our surveyfar exceeds that seen in a second proteome of comparable complexity,skeletal muscle, indicating that the complement of proteinsexpressed in seminal fluid may be subject to rapid diversification.We further show that orthologous seminal fluid proteins exhibitsubstantial interspecific variation in molecular mass. Becausethis variation cannot be attributed to differential glycosylationor radical differences in termination sites, it is stronglysuggestive of rapid amino acid divergence. Sperm competitionis implicated in generating such divergence for at least onemajor seminal fluid protein in our study, SVS II, which is responsiblefor copulatory plug formation via transglutaminase-catalyzedcross-linking after insemination. We show that the molecularmass of SVS II is positively correlated with relative testissize across species, which could be explained by selection foran increased number of cross-linking sites involved in the formationof the copulatory plug under sperm competition.     

Safety and Tolerability of Conserved Region Vaccines Vectored by Plasmid DNA,Simian Adenovirus and Modified Vaccinia Virus Ankara Administered to Human Immunodeficiency Virus Type 1-Uninfected Adults in a Randomized,Single-Blind Phase I Trial

Trial Design

HIV-1 vaccine development has advanced slowly due to viral antigenic diversity, poor immunogenicity and recently, safety concerns associated with human adenovirus serotype-5 vectors. To tackle HIV-1 variation, we designed a unique T-cell immunogen HIVconsv from functionally conserved regions of the HIV-1 proteome, which were presented to the immune system using a heterologous prime-boost combination of plasmid DNA, a non-replicating simian (chimpanzee) adenovirus ChAdV-63 and a non-replicating poxvirus, modified vaccinia virus Ankara. A block-randomized, single-blind, placebo-controlled phase I trial HIV-CORE 002 administered for the first time candidate HIV-1- vaccines or placebo to 32 healthy HIV-1/2-uninfected adults in Oxford, UK and elicited high frequencies of HIV-1-specific T cells capable of inhibiting HIV-1 replication in vitro. Here, detail safety and tolerability of these vaccines are reported.

Methods

Local and systemic reactogenicity data were collected using structured interviews and study-specific diary cards. Data on all other adverse events were collected using open questions. Serum neutralizing antibody titres to ChAdV-63 were determined before and after vaccination.

Results

Two volunteers withdrew for vaccine-unrelated reasons. No vaccine-related serious adverse events or reactions occurred during 190 person-months of follow-up. Local and systemic events after vaccination occurred in 27/32 individuals and most were mild (severity grade 1) and predominantly transient (<48 hours). Myalgia and flu-like symptoms were more strongly associated with MVA than ChAdV63 or DNA vectors and more common in vaccine recipients than in placebo. There were no intercurrent HIV-1 infections during follow-up. 2/24 volunteers had low ChAdV-63-neutralizing titres at baseline and 7 increased their titres to over 200 with a median (range) of 633 (231-1533) post-vaccination, which is of no safety concern.

Conclusions

These data demonstrate safety and good tolerability of the pSG2.HIVconsv DNA, ChAdV63.HIVconsv and MVA.HIVconsv vaccines and together with their high immunogenicity support their further development towards efficacy studies.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01151319","term_id":"NCT01151319"}}NCT01151319     

Indene bioconversion by a toluene inducible dioxygenase of Rhodococcus sp. I24

Rhodococcus sp. I24 can oxygenate indene via at least three independent enzyme activities: (i) a naphthalene inducible monooxygenase (ii) a naphthalene inducible dioxygenase, and (iii) a toluene inducible dioxygenase (TID). Pulsed field gel analysis revealed that the I24 strain harbors two megaplasmids of 340 and 50 kb. Rhodococcus sp. KY1, a derivative of the I24 strain, lacks the 340 kb element as well as the TID activity. Southern blotting and sequence analysis of an indigogenic, I24-derived cosmid suggested that an operon encoding a TID resides on the 340 kb element. Expression of the tid operon was induced by toluene but not by naphthalene. In contrast, naphthalene did induce expression of the nid operon, encoding the naphthalene dioxygenase in I24. Cell free protein extracts of Escherichia coli cells expressing tidABCD were used in HPLC-based enzyme assays to characterize the indene bioconversion of TID in vitro. In addition to 1-indenol, indene was transformed to cis-indandiol with an enantiomeric excess of 45.2% of cis-(1S,2R)-indandiol over cis-(1R,2S)-indandiol, as revealed by chiral HPLC analysis. The K_m of TID for indene was 380 M. The enzyme also dioxygenated naphthalene to cis-dihydronaphthalenediol with an activity of 78% compared to the formation of cis-indandiol from indene. The K_m of TID for naphthalene was 28 M. TID converted only trace amounts of toluene to 1,2-dihydro-3-methylcatechol after prolonged incubation time. The results indicate the role of the tid operon in the bioconversion of indene to 1-indenol and cis-(1S,2R)-indandiol by Rhodococcus sp. I24.     

Symmetrical Dose-Dependent DNA-Methylation Profiles in Children with Deletion or Duplication of 7q11.23

Epigenetic dysfunction has been implicated in a growing list of disorders that include cancer, neurodevelopmental disorders, and neurodegeneration. Williams syndrome (WS) and 7q11.23 duplication syndrome (Dup7) are rare neurodevelopmental disorders with broad phenotypic spectra caused by deletion and duplication, respectively, of a 1.5-Mb region that includes several genes with a role in epigenetic regulation. We have identified striking differences in DNA methylation across the genome between blood cells from children with WS or Dup7 and blood cells from typically developing (TD) children. Notably, regions that were differentially methylated in both WS and Dup7 displayed a significant and symmetrical gene-dose-dependent effect, such that WS typically showed increased and Dup7 showed decreased DNA methylation. Differentially methylated genes were significantly enriched with genes in pathways involved in neurodevelopment, autism spectrum disorder (ASD) candidate genes, and imprinted genes. Using alignment with ENCODE data, we also found the differentially methylated regions to be enriched with CCCTC-binding factor (CTCF) binding sites. These findings suggest that gene(s) within 7q11.23 alter DNA methylation at specific sites across the genome and result in dose-dependent DNA-methylation profiles in WS and Dup7. Given the extent of DNA-methylation changes and the potential impact on CTCF binding and chromatin regulation, epigenetic mechanisms most likely contribute to the complex neurological phenotypes of WS and Dup7. Our findings highlight the importance of DNA methylation in the pathogenesis of WS and Dup7 and provide molecular mechanisms that are potentially shared by WS, Dup7, and ASD.     

The Relationship between Imputation Error and Statistical Power in Genetic Association Studies in Diverse Populations

Genotype-imputation methods provide an essential technique for high-resolution genome-wide association (GWA) studies with millions of single-nucleotide polymorphisms. For optimal design and interpretation of imputation-based GWA studies, it is important to understand the connection between imputation error and power to detect associations at imputed markers. Here, using a 2 × 3 chi-square test, we describe a relationship between genotype-imputation error rates and the sample-size inflation required for achieving statistical power at an imputed marker equal to that obtained if genotypes at the marker were known with certainty. Surprisingly, typical imputation error rates (∼2%–6%) lead to a large increase in the required sample size (∼10%–60%), and in some African populations whose genotypes are particularly difficult to impute, the required sample-size increase is as high as ∼30%–150%. In most populations, each 1% increase in imputation error leads to an increase of ∼5%–13% in the sample size required for maintaining power. These results imply that in GWA sample-size calculations investigators will need to account for a potentially considerable loss of power from even low levels of imputation error and that development of additional genomic resources that decrease imputation error will translate into substantial reduction in the sample sizes needed for imputation-based detection of the variants that underlie complex human diseases.     

Mutation discovery in mice by whole exome sequencing

We report the development and optimization of reagents for in-solution, hybridization-based capture of the mouse exome. By validating this approach in a multiple inbred strains and in novel mutant strains, we show that whole exome sequencing is a robust approach for discovery of putative mutations, irrespective of strain background. We found strong candidate mutations for the majority of mutant exomes sequenced, including new models of orofacial clefting, urogenital dysmorphology, kyphosis and autoimmune hepatitis.     

Transcription profiling of acute temperature stress in the Antarctic plunderfish Harpagifer antarcticus

Harpagifer antarcticus (the Antarctic plunderfish), a shallow-water benthic fish distributed around the Antarctic Peninsula, is a member of the notothenioid family, one of whose adaptations to the cold waters of Antarctica has been the loss of the classic heat shock response. In order to gain a more comprehensive understanding of the effects of temperature stress on H. antarcticus, we constructed a liver cDNA library and a 10,371 feature microarray. This was hybridized with material from a time course series of animals held at 6°C for 48h. The resulting expression profiles show that this fish displays the classical vertebrate acute inflammatory response. There was also a pronounced signal for increased energy requirements via up-regulation of genes involved in the β oxidation of fatty acids and also a strong signature of response to oxidative stress. Genes in the latter category did not include the "classic" antioxidants such as glutathione S-transferase, but genes involved in the production of reducing potential in the form of NADPH, peroxisome proliferation via peroxisomal acyl co-enzyme A oxidase 1 and genes known to be up-regulated by hypoxia-inducible factor 1 (HIF1). These identifications provide clear support for oxygen being the whole animal limiting factor at least in acute short-term temperature challenges. The classical heat shock proteins were not up-regulated during this trial, although numerous clones for each were present on the gene chip, confirming the lack of this response in this species. These data significantly increase our knowledge of the cellular stress response from animals in this unique environment.     

Bacterial and fungal communities in bulk soil and rhizospheres of aluminum-tolerant and aluminum-sensitive maize (Zea mays L.) lines cultivated in unlimed and limed Cerrado soil

Liming of acidic soils can prevent aluminum toxicity and improve crop production. Some maize lines show aluminum (Al) tolerance, and exudation of organic acids by roots has been considered to represent an important mechanism involved in the tolerance. However, there is no information about the impact of liming on the structures of bacterial and fungal communities in Cerrado soil, nor if there are differences between the microbial communities from the rhizospheres of Al-tolerant and Al-sensitive maize lines. This study evaluated the effects of liming on the structure of bacterial and fungal communities in bulk soil and rhizospheres of Al-sensitive and Al-tolerant maize (Zea mays L.) lines cultivated in Cerrado soil by PCR-DGGE, 30 and 90 days after sowing. Bacterial fingerprints revealed that the bacterial communities from rhizospheres were more affected by aluminum stress in soil than by the maize line (Al-sensitive or Al-tolerant). Differences in bacterial communities were also observed over time (30 and 90 days after sowing), and these occurred mainly in the Actinobacteria. Conversely, fungal communities from the rhizosphere were weakly affected either by liming or by the rhizosphere, as observed from the DGGE profiles. Furthermore, only a few differences were observed in the DGGE profiles of the fungal populations during plant development when compared with bacterial communities. Cloning and sequencing of 16S rRNA gene fragments obtained from dominant DGGE bands detected in the bacterial profiles of the Cerrado bulk soil revealed that Actinomycetales and Rhizobiales were among the dominant ribotypes.