Mirage: A Novel Multiple Protein Sequence Alignment Tool

A fundamental problem in computational biology is the organization of many related sequences into a multiple sequence alignment (MSA) [2]. MSAs have a range of research applications, such as inferring phylogeny [22] and identifying regions of conserved sequence that indicate functional similarity [18]. In the case of protein isoforms, MSAs are valuable tools for transitively annotating post-translational modifications (PTMs) by enabling information transfer between known PTM sites and the sites that they align to [11]. For protein MSA tools, one challenging biological phenomenon is alternative splicing, wherein identical genomic sequence will differentially select from a subset of available coding regions (exons), depending on the biochemical environment [21]. Traditional methods struggle to align the islands of non-homologous sequence produced by alternative splicing, and frequently compensate for the penalties incurred from aligning non-identical characters by aligning small pieces of relatively similar sequence from alternative exons in a way that avoids extreme gap penalties but falsely indicates sequence homology. Presented here is Mirage, a novel protein MSA tool capable of accurately aligning alternatively spliced proteins by first mapping proteins to the genomic sequence that encoded them and then aligning proteins to one another based on the relative positions of their coding DNA. This method of transitive alignment demonstrates an awareness of intron splice site locations and resolves the problems associated with alternative splicing in traditional MSA tools

Common CHD8 Genomic Targets Contrast With Model-Specific Transcriptional Impacts of CHD8 Haploinsufficiency.

The packaging of DNA into chromatin determines the transcriptional potential of cells and is central to eukaryotic gene regulation. Case sequencing studies have revealed mutations to proteins that regulate chromatin state, known as chromatin remodeling factors, with causal roles in neurodevelopmental disorders. Chromodomain helicase DNA binding protein 8 (CHD8) encodes a chromatin remodeling factor with among the highest de novo loss-of-function mutation rates in patients with autism spectrum disorder (ASD). However, mechanisms associated with CHD8 pathology have yet to be elucidated. We analyzed published transcriptomic data across CHD8 in vitro and in vivo knockdown and knockout models and CHD8 binding across published ChIP-seq datasets to identify convergent mechanisms of gene regulation by CHD8. Differentially expressed genes (DEGs) across models varied, but overlap was observed between downregulated genes involved in neuronal development and function, cell cycle, chromatin dynamics, and RNA processing, and between upregulated genes involved in metabolism and immune response. Considering the variability in transcriptional changes and the cells and tissues represented across ChIP-seq analysis, we found a surprisingly consistent set of high-affinity CHD8 genomic interactions. CHD8 was enriched near promoters of genes involved in basic cell functions and gene regulation. Overlap between high-affinity CHD8 targets and DEGs shows that reduced dosage of CHD8 directly relates to decreased expression of cell cycle, chromatin organization, and RNA processing genes, but only in a subset of studies. This meta-analysis verifies CHD8 as a master regulator of gene expression and reveals a consistent set of high-affinity CHD8 targets across human, mouse, and rat in vivo and in vitro studies. These conserved regulatory targets include many genes that are also implicated in ASD. Our findings suggest a model where perturbation to dosage-sensitive CHD8 genomic interactions with a highly-conserved set of regulatory targets leads to model-specific downstream transcriptional impacts

Efficacy Of Heated Hydrous Ethanol Injection For Improving Emissions From Dual Fuel Diesel Engines

University of Minnesota M.S.M.E. thesis. May 2017. Major: Mechanical Engineering. Advisor: William Northrop. 1 computer file (PDF); xii, 76 pages.With emissions standards becoming ever more stringent, aftermarket dual-fuel solutions are being developed to allow legacy diesel engines to reach higher regulatory emissions tiers. Manufacturers are reluctant to adopt dual-fuel systems due to perceived lack of consumer interest. However, the use of aftermarket dual-fuel systems with partially renewable fuels has sparked interest in limited markets. Previous research has shown slight emissions reduction benefits from fumigation with 120 proof hydrous ethanol in a diesel engine using a commercially available dual-fuel system. However, the findings do not match manufacturer claims of emissions reductions. The work presented here examines the design, development, performance, and emissions from an engine equipped with a novel aftermarket port fuel injection (PFI) dual fuel system with a fuel heating system integrated into the fuel injector rail. Finite element modeling techniques in ANSYS were used to optimize the heat exchanger and analyze its performance. In addition, cross-sections and flow path lines were created in ANSYS to examine thermal profiles and flow turbulence at varying ethanol flow rates. A John Deere 4045HF475 Tier 2 diesel engine was retrofitted with a custom PFI rail designed to inject hydrous ethanol with the ability to preheat the ethanol using circulated hot engine coolant to improve the vaporization and mixing of the secondary fuel and air in the intake port. Port-injected fuel flow was controlled by varying injector pulse width and throttle position was adjusted manually to maintain testing mode parameters. Heated ethanol, unheated ethanol, and diesel only operating modes were run over a modified ISO 8178 eight-point test plan. Fumigant energy fraction (FEF), defined as the amount of energy provided by the fumigant based on the lower heating value (LHV) divided by the total fuel energy, up to 37% was achieved in the experiments. Ethanol fuel rail heat exchanger effectiveness decreased with increasing FEF and log-mean temperature difference (LMTD) increased. These opposite effects were likely due to dimensional design constraints of the heat exchanger limiting the heat transfer. Experiments found that with increasing FEF, engine NO emissions decreased, whereas NO2, CO, THC, and ethanol emissions increased. NO emissions reductions were countered by increasing NO2, resulting in constant NOX emissions. Soot concentrations produced varying trends, but with a tendency to decrease overall at high FEF. Preheating the ethanol with circulated engine coolant yielded few benefits to engine out-emissions. This study showed that the dual-fuel heated PFI rail system provided modest emissions benefits over diesel-only operation. Preheating the liquid ethanol was not as successful as anticipated because ethanol’s high latent heat of vaporization dominated over the sensible heat required to heat the liquid prior to the injectors

Common CHD8 Genomic Targets Contrast With Model-Specific Transcriptional Impacts of CHD8 Haploinsufficiency

The packaging of DNA into chromatin determines the transcriptional potential of cells and is central to eukaryotic gene regulation. Case sequencing studies have revealed mutations to proteins that regulate chromatin state, known as chromatin remodeling factors, with causal roles in neurodevelopmental disorders. Chromodomain helicase DNA binding protein 8 (CHD8) encodes a chromatin remodeling factor with among the highest de novo loss-of-function mutation rates in patients with autism spectrum disorder (ASD). However, mechanisms associated with CHD8 pathology have yet to be elucidated. We analyzed published transcriptomic data across CHD8 in vitro and in vivo knockdown and knockout models and CHD8 binding across published ChIP-seq datasets to identify convergent mechanisms of gene regulation by CHD8. Differentially expressed genes (DEGs) across models varied, but overlap was observed between downregulated genes involved in neuronal development and function, cell cycle, chromatin dynamics, and RNA processing, and between upregulated genes involved in metabolism and immune response. Considering the variability in transcriptional changes and the cells and tissues represented across ChIP-seq analysis, we found a surprisingly consistent set of high-affinity CHD8 genomic interactions. CHD8 was enriched near promoters of genes involved in basic cell functions and gene regulation. Overlap between high-affinity CHD8 targets and DEGs shows that reduced dosage of CHD8 directly relates to decreased expression of cell cycle, chromatin organization, and RNA processing genes, but only in a subset of studies. This meta-analysis verifies CHD8 as a master regulator of gene expression and reveals a consistent set of high-affinity CHD8 targets across human, mouse, and rat in vivo and in vitro studies. These conserved regulatory targets include many genes that are also implicated in ASD. Our findings suggest a model where perturbation to dosage-sensitive CHD8 genomic interactions with a highly-conserved set of regulatory targets leads to model-specific downstream transcriptional impacts

In vivo targeted DamID identifies CHD8 genomic targets in fetal mouse brain.

Funder: Royal SocietyFunder: Agouron InstituteGenetic studies of autism have revealed causal roles for chromatin remodeling gene mutations. Chromodomain helicase DNA binding protein 8 (CHD8) encodes a chromatin remodeler with significant de novo mutation rates in sporadic autism. However, relationships between CHD8 genomic function and autism-relevant biology remain poorly elucidated. Published studies utilizing ChIP-seq to map CHD8 protein-DNA interactions have high variability, consistent with technical challenges and limitations associated with this method. Thus, complementary approaches are needed to establish CHD8 genomic targets and regulatory functions in developing brain. We used in utero CHD8 Targeted DamID followed by sequencing (TaDa-seq) to characterize CHD8 binding in embryonic mouse cortex. CHD8 TaDa-seq reproduced interaction patterns observed from ChIP-seq and further highlighted CHD8 distal interactions associated with neuronal loci. This study establishes TaDa-seq as a useful alternative for mapping protein-DNA interactions in vivo and provides insights into the regulatory targets of CHD8 and autism-relevant pathophysiology associated with CHD8 mutations

Pbx Regulates Patterning of the Cerebral Cortex in Progenitors and Postmitotic Neurons

SummaryWe demonstrate using conditional mutagenesis that Pbx1, with and without Pbx2+/− sensitization, regulates regional identity and laminar patterning of the developing mouse neocortex in cortical progenitors (Emx1-Cre) and in newly generated neurons (Nex1-Cre). Pbx1/2 mutants have three salient molecular phenotypes of cortical regional and laminar organization: hypoplasia of the frontal cortex, ventral expansion of the dorsomedial cortex, and ventral expansion of Reelin expression in the cortical plate of the frontal cortex, concomitant with an inversion of cortical layering in the rostral cortex. Molecular analyses, including PBX ChIP-seq, provide evidence that PBX promotes frontal cortex identity by repressing genes that promote dorsocaudal fate

Comparison of tagging single-nucleotide polymorphism methods in association analyses

Several methods to identify tagging single-nucleotide polymorphisms (SNPs) are in common use for genetic epidemiologic studies; however, there may be loss of information when using only a subset of SNPs. We sought to compare the ability of commonly used pairwise, multimarker, and haplotype-based tagging SNP selection methods to detect known associations with quantitative expression phenotypes. Using data from HapMap release 21 on unrelated Utah residents with ancestors from northern and western Europe (CEPH-Utah, CEU), we selected tagging SNPs in five chromosomal regions using ldSelect, Tagger, and TagSNPs. We found that SNP subsets did not substantially overlap, and that the use of trio data did not greatly impact SNP selection. We then tested associations between HapMap genotypes and expression phenotypes on 28 CEU individuals as part of Genetic Analysis Workshop 15. Relative to the use of all SNPs (n = 210 SNPs across all regions), most subset methods were able to detect single-SNP and haplotype associations. Generally, pairwise selection approaches worked extremely well, relative to use of all SNPs, with marked reductions in the number of SNPs required. Haplotype-based approaches, which had identified smaller SNP subsets, missed associations in some regions. We conclude that the optimal tagging SNP method depends on the true model of the genetic association (i.e., whether a SNP or haplotype is responsible); unfortunately, this is often unknown at the time of SNP selection. Additional evaluations using empirical and simulated data are needed

Transcriptional Regulation of Enhancers Active in Protodomains of the Developing Cerebral Cortex

SummaryElucidating the genetic control of cerebral cortical (pallial) development is essential for understanding function, evolution, and disorders of the brain. Transcription factors (TFs) that embryonically regulate pallial regionalization are expressed in gradients, raising the question of how discrete domains are generated. We provide evidence that small enhancer elements active in protodomains integrate broad transcriptional information. CreERT2 and GFP expression from 14 different enhancer elements in stable transgenic mice allowed us to define a comprehensive regional fate map of the pallium. We explored transcriptional mechanisms that control the activity of the enhancers using informatics, in vivo occupancy by TFs that regulate cortical patterning (CoupTFI, Pax6, and Pbx1), and analysis of enhancer activity in Pax6 mutants. Overall, the results provide insights into how broadly expressed patterning TFs regulate the activity of small enhancer elements that drive gene expression in pallial protodomains that fate map to distinct cortical regions