Approaches to analyse dynamic microbial communities such as those seen in cystic fibrosis lung

Microbial communities play vital roles in many aspects of our lives, although our understanding of microbial biogeography and community profiles remains unclear. The number of microbes or the diversity of the microbes, even in small environmental niches, is staggering. Current microbiological methods used to analyse these communities are limited, in that many microorganisms cannot be cultured. Even for the isolates that can be cultured, the expense of identifying them definitively is much too high to be practical. Many recent molecular technologies, combined with bioinformatic tools, are raising the bar by improving the sensitivity and reliability of microbial community analysis. These tools and techniques range from those that attempt to understand a microbial community from their length heterogeneity profiles to those that help to identify the strains and species of a random sampling of the microbes in a given sample. These technologies are reviewed here, using the microbial communities present in the lungs of cystic fibrosis patients as a paradigm

Bacterial regulon modeling and prediction based on systematic \u3ci\u3ecis\u3c/i\u3e regulatory motif analyses

Regulons are the basic units of the response system in a bacterial cell, and each consists of a set of transcriptionally co-regulated operons. Regulon elucidation is the basis for studying the bacterial global transcriptional regulation network. In this study, we designed a novel co-regulation score between a pair of operons based on accurate operon identification and cis regulatory motif analyses, which can capture their co-regulation relationship much better than other scores. Taking full advantage of this discovery, we developed a new computational framework and built a novel graph model for regulon prediction. This model integrates the motif comparison and clustering and makes the regulon prediction problem substantially more solvable and accurate. To evaluate our prediction, a regulon coverage score was designed based on the documented regulons and their overlap with our prediction; and a modified Fisher Exact test was implemented to measure how well our predictions match the co-expressed modules derived from E. coli microarray gene-expression datasets collected under 466 conditions. The results indicate that our program consistently performed better than others in terms of the prediction accuracy. This suggests that our algorithms substantially improve the state-of-the-art, leading to a computational capability to reliably predict regulons for any bacteria

Comparison of Three Assembly Strategies for a Heterozygous Seedless Grapevine Genome Assembly

BackgroundDe novo heterozygous assembly is an ongoing challenge requiring improved assembly approaches. In this study, three strategies were used to develop de novo Vitis vinifera ‘Sultanina’ genome assemblies for comparison with the inbred V. vinifera (PN40024 12X.v2) reference genome and a published Sultanina ALLPATHS-LG assembly (AP). The strategies were: 1) a default PLATANUS assembly (PLAT_d) for direct comparison with AP assembly, 2) an iterative merging strategy using METASSEMBLER to combine PLAT_d and AP assemblies (MERGE) and 3) PLATANUS parameter modifications plus GapCloser (PLAT*_GC).ResultsThe three new assemblies were greater in size than the AP assembly. PLAT*_GC had the greatest number of scaffolds aligning with a minimum of 95% identity and ≥1000 bp alignment length to V. vinifera (PN40024 12X.v2) reference genome. SNP analysis also identified additional high quality SNPs. A greater number of sequence reads mapped back with zero-mismatch to the PLAT_d, MERGE, and PLAT*_GC (\u3e94%) than was found in the AP assembly (87%) indicating a greater fidelity to the original sequence data in the new assemblies than in AP assembly. A de novo gene prediction conducted using seedless RNA-seq data predicted \u3e 30,000 coding sequences for the three new de novo assemblies, with the greatest number (30,544) in PLAT*_GC and only 26,515 for the AP assembly. Transcription factor analysis indicated good family coverage, but some genes found in the VCOST.v3 annotation were not identified in any of the de novo assemblies, particularly some from the MYB and ERF families.ConclusionsThe PLAT_d and PLAT*_GC had a greater number of synteny blocks with the V. vinifera (PN40024 12X.v2) reference genome than AP or MERGE. PLAT*_GC provided the most contiguous assembly with only 1.2% scaffold N, in contrast to AP (10.7% N), PLAT_d (6.6% N) and Merge (6.4% N). A PLAT*_GC pseudo-chromosome assembly with chromosome alignment to the reference genome V. vinifera, (PN40024 12X.v2) provides new information for use in seedless grape genetic mapping studies. An annotated de novo gene prediction for the PLAT*_GC assembly, aligned with VitisNet pathways provides new seedless grapevine specific transcriptomic resource that has excellent fidelity with the seedless short read sequence data

Metastatic model of HPV+ oropharyngeal squamous cell carcinoma demonstrates heterogeneity in tumor metastasis

Human papillomavirus induced (HPV+) cancer incidence is rapidly rising, comprising 60–80% of oropharyngeal squamous cell carcinomas (OPSCCs); while rare, recurrent/metastatic disease accounts for nearly all related deaths. An in vivo pre-clinical model for these invasive cancers is necessary for testing new therapies. We characterize an immune competent recurrent/metastatic HPV+ murine model of OPSSC which consists of four lung metastatic (MLM) cell lines isolated from an animal with HPV+ OPSCC that failed cisplatin/radiation treatment. These individual metastatic clonal cell lines were tested to verify their origin (parental transgene expression and define their physiological properties: proliferation, metastatic potential, heterogeneity and sensitivity/resistance to cisplatin and radiation. All MLMs retain expression of parental HPV16 E6 and E7 and degrade P53 yet are heterogeneous from one another and from the parental cell line as defined by Illumina expression microarray. Consistent with this, reverse phase protein array defines differences in protein expression/activation between MLMs as well as the parental line. While in vitro growth rates of MLMs are slower than the parental line, in vivo growth of MLM clones is greatly enhanced. Moreover, in vivo resistance to standard therapies is dramatically increased in 3 of the 4 MLMs. Lymphatic and/or lung metastasis occurs 100% of the time in one MLM line. This recurrent/metastatic model of HPV+ OPSCC retains the characteristics evident in refractory human disease (heterogeneity, resistance to therapy, metastasis in lymph nodes/lungs) thus serving as an ideal translational system to test novel therapeutics. Moreover, this system may provide insights into the molecular mechanisms of metastasis

Functional Genomics of Acclimation and Adaptation in Response to Thermal Stress in Daphnia

Background: Gene expression regulation is one of the fundamental mechanisms of phenotypic plasticity and is expected to respond to selection in conditions favoring phenotypic response. The observation that many organisms increase their stress tolerance after acclimation to moderate levels of stress is an example of plasticity which has been long hypothesized to be based on adaptive changes in gene expression. We report genome-wide patterns of gene expression in two heat-tolerant and two heat-sensitive parthenogenetic clones of the zooplankton crustacean Daphnia pulex exposed for three generations to either optimal (18°C) or substressful (28°C) temperature. Results: A large number of genes responded to temperature and many demonstrated a significant genotype-byenvironment (GxE) interaction. Among genes with a significant GxE there were approximately equally frequent instances of canalization, i.e. stronger plasticity in heat-sensitive than in heat-tolerant clones, and of enhancement of plasticity along the evolutionary vector toward heat tolerance. The strongest response observed is the across-the-board down-regulation of a variety of genes occurring in heat-tolerant, but not in heat-sensitive clones. This response is particularly obvious among genes involved in core metabolic pathways and those responsible for transcription, translation and DNA repair. Conclusions: The observed down-regulation of metabolism, consistent with previous findings in yeast and Drosophila, may reflect a general compensatory stress response. The associated down-regulation of DNA repair pathways potentially creates a trade-off between short-term benefits of survival at high temperature and long-term costs of accelerated mutation accumulation

Requirement for commissureless2 function during dipteran insect nerve cord development

BACKGROUND: In Drosophila melanogaster, commissureless (comm) function is required for proper nerve cord development. Although comm orthologs have not been identified outside of Drosophila species, some insects possess orthologs of Drosophila comm2, which may also regulate embryonic nerve cord development. Here, this hypothesis is explored through characterization of comm2 genes in two disease vector mosquitoes. RESULTS: Culex quinquefasciatus (West Nile and lymphatic filiariasis vector) has three comm2 genes that are expressed in the developing nerve cord. Aedes aegypti (dengue and yellow fever vector) has a single comm2 gene that is expressed in commissural neurons projecting axons toward the midline. Loss of comm2 function in both A. aegypti and D. melanogaster was found to result in loss of commissure defects that phenocopy the frazzled (fra) loss of function phenotypes observed in both species. Loss of fra function in either insect was found to result in decreased comm2 transcript levels during nerve cord development. CONCLUSIONS: The results of this investigation suggest that Fra down-regulates repulsion in precrossing commissural axons by regulating comm2 levels in both A. aegypti and D. melanogaster, both of which require Comm2 function for proper nerve cord development

Functional genomics of acclimation and adaptation in response to thermal stress in Daphnia

BACKGROUND: Gene expression regulation is one of the fundamental mechanisms of phenotypic plasticity and is expected to respond to selection in conditions favoring phenotypic response. The observation that many organisms increase their stress tolerance after acclimation to moderate levels of stress is an example of plasticity which has been long hypothesized to be based on adaptive changes in gene expression. We report genome-wide patterns of gene expression in two heat-tolerant and two heat-sensitive parthenogenetic clones of the zooplankton crustacean Daphnia pulex exposed for three generations to either optimal (18°C) or substressful (28°C) temperature. RESULTS: A large number of genes responded to temperature and many demonstrated a significant genotype-by-environment (GxE) interaction. Among genes with a significant GxE there were approximately equally frequent instances of canalization, i.e. stronger plasticity in heat-sensitive than in heat-tolerant clones, and of enhancement of plasticity along the evolutionary vector toward heat tolerance. The strongest response observed is the across-the-board down-regulation of a variety of genes occurring in heat-tolerant, but not in heat-sensitive clones. This response is particularly obvious among genes involved in core metabolic pathways and those responsible for transcription, translation and DNA repair. CONCLUSIONS: The observed down-regulation of metabolism, consistent with previous findings in yeast and Drosophila, may reflect a general compensatory stress response. The associated down-regulation of DNA repair pathways potentially creates a trade-off between short-term benefits of survival at high temperature and long-term costs of accelerated mutation accumulation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2164-15-859) contains supplementary material, which is available to authorized users

A deficiency in SUMOylation activity disrupts multiple pathways leading to neural tube and heart defects in Xenopus embryos

Adenovirus protein, Gam1, triggers the proteolytic destruction of the E1 SUMO-activating enzyme. Microinjection of an empirically determined amount of Gam1 mRNA into one-cell Xenopus embryos can reduce SUMOylation activity to undetectable, but nonlethal, levels, enabling an examination of the role of this post-translational modification during early vertebrate development.https://doi.org/10.1186/s12864-019-5773-

Clinically actionable secondary findings in 130 triads from sub-Saharan African families with non-syndromic orofacial clefts

Abstract Introduction The frequency and implications of secondary findings (SFs) from genomic testing data have been extensively researched. However, little is known about the frequency or reporting of SFs in Africans, who are underrepresented in large‐scale population genomic studies. The availability of data from the first whole‐genome sequencing for orofacial clefts in an African population motivated this investigation. Methods In total, 130 case‐parent trios were analyzed for SFs within the ACMG SFv.3.0 list genes. Additionally, we filtered for four more genes (HBB, HSD32B, G6PD and ACADM). Results We identified 246 unique variants in 55 genes; five variants in four genes were classified as pathogenic or likely pathogenic (P/LP). The P/LP variants were seen in 2.3% (9/390) of the subjects, a frequency higher than ~1% reported for diverse ethnicities. On the ACMG list, pathogenic variants were observed in PRKAG (p. Glu183Lys). Variants in the PALB2 (p. Glu159Ter), RYR1 (p. Arg2163Leu) and LDLR (p. Asn564Ser) genes were predicted to be LP. Conclusion This study provides information on the frequency and pathogenicity of SFs in an African cohort. Early risk detection will help reduce disease burden and contribute to efforts to increase knowledge of the distribution and impact of actionable genomic variants in diverse populations