ALBERT

Description: Background: The diploid (2n = 2x = 14) strawberry model plant Fragaria vesca ssp. vesca 'Hawaii 4' was employed for functional analysis of expressed DNA sequences initially identified as being unique to Fragaria and of unknown or poorly understood function. 'Hawaii 4' is prominent in strawberry research due to its ease of Agrobacterium-mediated transformation and regenerability, and its status as the source of the first complete strawberry genomic sequence. Our studies of a set of transformants have documented intriguing, construct-associated effects on leaf morphology, and provide important and unexpected insights into the performance of the 'Hawaii 4' transformation and regeneration system. Results: Following Agrobacterium-mediated transformation of leaf explants with gene constructs carried by Gateway(R) vectors, plants were regenerated using a modified version of an established 'Hawaii 4' protocol. Expanding upon the findings of prior studies, we documented that plantlet regeneration was occurring via a somatic embryogenic rather than an organogenic developmental pathway. Among transformants, several variations in leaf morphology were observed. Unexpectedly, a particular leaf variant type, occurring in ~17% of all regenerants independent of construct type, was found to be attributable to tetraploidy. The tetraploidy-associated alteration in leaf morphology could be differentiated from the leaf morphology of diploid regenerants on the basis of a quantitative ratio of leaf dimensions: B/A, where B is the width of the central leaflet and A is the overall width of the trifoliate leaf. Variant effects on leaf morphology of four different transgenic constructs were also documented, and were in all cases distinguishable from the effects of tetraploidy. Conclusions: These results define opportunities to optimize the existing 'Hawaii 4' protocol by focusing on treatments that specifically promote somatic embryogenesis. The reported morphological metric and descriptions will guide future transgenic studies using the 'Hawaii 4' model system by alerting researchers to the potential occurrence of polyploid regenerants, and to differentiating the effects on leaf morphology due to polyploidy versus transgenic manipulations. Finally, an intriguing spectrum of leaf morphology alterations resulting from manipulation of expressed sequences of uncertain function is documented, providing a foundation for detailed studies of the respective genes and their functional roles.

Description: Background: The basidiomycete Moniliophthora roreri is the causal agent of Frosty pod rot (FPR) disease of cacao (Theobroma cacao), the source of chocolate, and FPR is one of the most destructive diseases of this important perennial crop in the Americas. This hemibiotroph infects only cacao pods and has an extended biotrophic phase lasting up to sixty days, culminating in plant necrosis and sporulation of the fungus without the formation of a basidiocarp. Results: We sequenced and assembled 52.3 Mb into 3,298 contigs that represent the M. roreri genome. Of the 17,920 predicted open reading frames (OFRs), 13,760 were validated by RNA-Seq. Using read count data from RNA sequencing of cacao pods at 30 and 60 days post infection, differential gene expression was estimated for the biotrophic and necrotrophic phases of this plant-pathogen interaction. The sequencing data were used to develop a genome based secretome for the infected pods. Of the 1,535 genes encoding putative secreted proteins, 1,355 were expressed in the biotrophic and necrotrophic phases. Analysis of the data revealed secretome gene expression that correlated with infection and intercellular growth in the biotrophic phase and invasive growth and plant cellular death in the necrotrophic phase. Conclusion: Genome sequencing and RNA-Seq was used to determine and validate the Moniliophthora roreri genome and secretome. High sequence identity between Moniliophthora roreri genes and Moniliophthora perniciosa genes supports the taxonomic relationship with Moniliophthora perniciosa and the relatedness of this fungus to other basidiomycetes. Analysis of RNA-Seq data from infected plant tissues revealed differentially expressed genes in the biotrophic and necrotrophic phases. The secreted protein genes that were upregulated in the biotrophic phase are primarily associated with breakdown of the intercellular matrix and modification of the fungal mycelia, possibly to mask the fungus from plant defenses. Based on the transcriptome data, the upregulated secreted proteins in the necrotrophic phase are hypothesized to be actively attacking the plant cell walls and plant cellular components resulting in necrosis. These genes are being used to develop a new understanding of how this disease interaction progresses and to identify potential targets to reduce the impact of this devastating disease.

Description: Background: Transcription factors (TFs) and histone modifications (HMs) play critical roles in gene expression by regulating mRNA transcription. Modeling frameworks have been developed to integrate high-throughput -omics data, with the aim of elucidating the regulatory logic that results from the interactions of DNA, TFs and HMs. These models have yielded an unexpected and poorly understood result: that TFs and HMs are statistically redundant in explaining mRNA transcript abundance at a genome-wide level. Results: We constructed predictive models of gene expression by integrating RNA-seq, TF and HM ChIP-seq and DNase I hypersensitivity data for two mammalian cell types. All models identified genome-wide statistical redundancy both within and between TFs and HMs, as previously reported. To investigate potential explanations, groups of genes were constructed for ontology-classified biological processes. Predictive models were constructed for each process to explore the distribution of statistical redundancy. We find significant variation in the predictive capacity of TFs and HMs across these processes and demonstrate the predictive power of HMs to be inversely proportional to process enrichment for housekeeping genes. Conclusion: It is well-established that the roles played by TFs and HMs are not functionally redundant. Instead, we attribute the statistical redundancy reported in this and previous genome-wide modeling studies to the heterogeneous distribution of HMs across chromatin domains. Furthermore, we conclude that statistical redundancy between individual TFs can be readily explained nucleosome-mediated cooperative binding. This could possibly help the cell confer regulatory robustness by rejecting signaling noise and allowing control via multiple pathways.

Description: Background: Bovine respiratory disease complex (BRDC) is an infectious disease of cattle that is caused by a combination of viral and/or bacterial pathogens. Selection for cattle with reduced susceptibility to respiratory disease would provide a permanent tool for reducing the prevalence of BRDC. The objective of this study was to identify BRDC susceptibility loci in pre-weaned Holstein calves as a prerequisite to using genetic improvement as a tool for decreasing the prevalence of BRDC. High density SNP genotyping with the Illumina BovineHD BeadChip was conducted on 1257 male and 757 female Holstein calves from California (CA), and 767 calves identified as female from New Mexico (NM). Of these, 1382 were classified as BRDC cases, and 1396 were classified as controls, with all phenotypes assigned using the McGuirk health scoring system. During the acquisition of blood for DNA isolation, two deep pharyngeal and one mid-nasal diagnostic swab were obtained from each calf for the identification of bacterial and viral pathogens. Genome-wide association analyses were conducted using four analytical approaches (EIGENSTRAT, EMMAX-GRM, GBLUP and FvR). The most strongly associated SNPs from each individual analysis were ranked and evaluated for concordance. The heritability of susceptibility to BRDC in pre-weaned Holstein calves was estimated. Results: The four statistical approaches produced highly concordant results for 373 top ranked SNPs that defined 126 chromosomal regions for the CA population. Similarly, in NM, 370 SNPs defined 138 genomic regions that were identified by all four approaches. When the two populations were combined (i.e., CA + NM) and analyzed, 324 SNPs defined 116 genomic regions that were associated with BRDC across all analytical methods. Heritability estimates for BRDC were 21% for both CA and NM as individual populations, but declined to 13% when the populations were combined. Conclusions: Four analytical approaches utilizing both single and multi-marker association methods revealed common genomic regions associated with BRDC susceptibility that can be further characterized and used for genomic selection. Moderate heritability estimates were observed for BRDC susceptibility in pre-weaned Holstein calves, thereby supporting the application of genomic selection to reduce the prevalence of BRDC in U.S. Holsteins.

Description: Background: Retinal ganglion cell (RGC) loss is one of the earliest and most important cellular changes in glaucoma. The DARC (Detection of Apoptosing Retinal Cells) technology enables in vivo real-time non-invasive imaging of single apoptosing retinal cells in animal models of glaucoma and Alzheimer's disease. To date, apoptosing RGCs imaged using DARC have been counted manually. This is time-consuming, labour-intensive, vulnerable to bias, and has considerable inter- and intra-operator variability. Results: A semi-automated algorithm was developed which enabled automated identification of apoptosing RGCs labeled with fluorescent Annexin-5 on DARC images. Automated analysis included a pre-processing stage involving local-luminance and local-contrast "gain control", a "blob analysis" step to differentiate between cells, vessels and noise, and a method to exclude non-cell structures using specific combined 'size' and 'aspect' ratio criteria. Apoptosing retinal cells were counted by 3 masked operators, generating 'Gold-standard' mean manual cell counts, and were also counted using the newly developed automated algorithm. Comparison between automated cell counts and the mean manual cell counts on 66 DARC images showed significant correlation between the two methods (Pearson's correlation coefficient 0.978 (p 〈 0.001), R Squared = 0.956. The Intraclass correlation coefficient was 0.986 (95% CI 0.977-0.991, p 〈 0.001), and Cronbach's alpha measure of consistency = 0.986, confirming excellent correlation and consistency. No significant difference (p = 0.922, 95% CI: -5.53 to 6.10) was detected between the cell counts of the two methods. Conclusions: The novel automated algorithm enabled accurate quantification of apoptosing RGCs that is highly comparable to manual counting, and appears to minimise operator-bias, whilst being both fast and reproducible. This may prove to be a valuable method of quantifying apoptosing retinal cells, with particular relevance to translation in the clinic, where a Phase I clinical trial of DARC in glaucoma patients is due to start shortly.

Description: A study in BMC Evolutionary Biology represents the most comprehensive effort to clarify the phylogeny of green plants using sequences from the plastid genome. This study highlights the strengths and limitations of plastome data for resolving the green plant phylogeny, and points toward an exciting future for plant phylogenetics, during which the vast and largely untapped territory of nuclear genomes will be explored.

Description: Background: Copper is essential for the survival of aerobic organisms. If copper is not properly regulated in the body however, it can be extremely cytotoxic and genetic mutations that compromise copper homeostasis result in severe clinical phenotypes. Understanding how cells maintain optimal copper levels is therefore highly relevant to human health. Results: We found that addition of copper (Cu) to culture medium leads to increased respiratory growth of yeast, a phenotype which we then systematically and quantitatively measured in 5050 homozygous diploid deletion strains. Cu's positive effect on respiratory growth was quantitatively reduced in deletion strains representing 73 different genes, the function of which identify increased iron uptake as a cause of the increase in growth rate. Conversely, these effects were enhanced in strains representing 93 genes. Many of these strains exhibited respiratory defects that were specifically rescued by supplementing the growth medium with Cu. Among the genes identified are known and direct regulators of copper homeostasis, genes required to maintain low vacuolar pH, and genes where evidence supporting a functional link with Cu has been heretofore lacking. Roughly half of the genes are conserved in man, and several of these are associated with Mendelian disorders, including the Cu-imbalance syndromes Menkes and Wilson's disease. We additionally demonstrate that pharmacological agents, including the approved drug disulfiram, can rescue Cu-deficiencies of both environmental and genetic origin. Conclusions: A functional screen in yeast has expanded the list of genes required for Cu-dependent fitness, revealing a complex cellular system with implications for human health. Respiratory fitness defects arising from perturbations in this system can be corrected with pharmacological agents that increase intracellular copper concentrations.

Description: Background: The current inability to culture the entirety of observed bacteria is well known and with the advent of ever more powerful molecular tools, that can survey bacterial communities at previously unattainable depth, the gap in our capacity to culture and define all of these species increases exponentially. This gap has essentially become the rate limiting step in determining how the knowledge of which species are present in a sample can be applied to understand the role of these species in an ecosystem or disease process. A case in point is periodontal disease, which is the most widespread oral disease in dogs. If untreated the disease results in significant pain, eventual loss of the dentition and potentially an increased risk of systemic diseases. Previous molecular based studies have identified the bacterial species associated with periodontal disease in dogs; however without cultured strains from many of these species it has not been possible to study whether they play a role in the disease process. Results: Using a quantitative polymerase chain reaction (qPCR) directed approach a range of microbiological media were screened and optimized to enrich for previously uncultivated target species. A systematic screening methodology was then employed to isolate the species of interest. In cases where the target species were not cultivable in isolation, helper strains grown underneath a nitrocellulose membrane were used to provide the necessary growth factors. This guided media optimization approach enabled the purification of 14 species, 8 of which we had previously been unable to cultivate in isolation. It is also applicable to the targeted isolation of isolates from species that have previously been cultured (for example to study intra-species variation) as demonstrated by the successful isolation of 6 targeted isolates of already cultured species. Conclusions: To our knowledge this is the first time this combination of qPCR guided media optimization, strategic screening and helper strain support has been used successfully to isolate previously uncultured bacteria. This approach can be applied to any uncultured bacterial species where knowledge of their nutritional requirements or low relative abundance impedes their isolation.

Description: Background: Differential distribution of DNA methylation on the parental alleles of imprinted genes distinguishes the alleles from each other and dictates their parent of origin-specific expression patterns. While differential DNA methylation at primary imprinting control regions is inherited via the gametes, additional allele-specific DNA methylation is acquired at secondary sites during embryonic development and plays a role in the maintenance of genomic imprinting. The precise mechanisms by which this somatic DNA methylation is established at secondary sites are not well defined and may vary as methylation acquisition at these sites occurs at different times for genes in different imprinting clusters. Results: In this study, we show that there is also variability in the timing of somatic DNA methylation acquisition at multiple sites within a single imprinting cluster. Paternal allele-specific DNA methylation is initially acquired at similar stages of post-implantation development at the linked Dlk1 and Gtl2 differentially methylated regions (DMRs). In contrast, unlike the Gtl2-DMR, the maternal Dlk1-DMR acquires DNA methylation in adult tissues. Conclusions: These data suggest that the acquisition of DNA methylation across the Dlk1/Gtl2 imprinting cluster is variable. We further found that the Dlk1 differentially methylated region displays low DNA methylation fidelity, as evidenced by the presence of hemimethylation at approximately one-third of the methylated CpG dyads. We hypothesize that the maintenance of DNA methylation may be less efficient at secondary differentially methylated sites than at primary imprinting control regions.

Description: Background: As high throughput sequencing continues to grow more commonplace, the need to disseminate the resulting data via web applications continues to grow. Particularly, there is a need to disseminate multiple versions of related gene and protein sequences simultaneously--whether they represent alleles present in a single species, variations of the same gene among different strains, or homologs among separate species. Often this is accomplished by displaying all versions of the sequence at once in a manner that is not intuitive or space-efficient and does not facilitate human understanding of the data. Web-based applications needing to disseminate multiple versions of sequences would benefit from a drop-in module designed to effectively disseminate these data.FindingsSnipViz is a client-side software tool designed to disseminate multiple versions of related gene and protein sequences on web sites. SnipViz has a space-efficient, interactive, and dynamic interface for navigating, analyzing and visualizing sequence data. It is written using standard World Wide Web technologies (HTML, Javascript, and CSS) and is compatible with most web browsers. SnipViz is designed as a modular client-side web component and may be incorporated into virtually any web site and be implemented without any programming. Conclusions: SnipViz is a drop-in client-side module for web sites designed to efficiently visualize and disseminate gene and protein sequences. SnipViz is open source and is freely available at https://github.com/yeastrc/snipviz.