Grid flexibility and patching techniques

The numerical determination of combustor flowfields is of great value to the combustor designer. An a priori knowledge of the flow behavior can speed the combustor design process and reduce the number of experimental test rigs required to arrive at an optimal design. Even 2-D steady incompressible isothermal flow predictions are of use; many codes of this kind are available, each employing different techniques to surmount the difficulties arising from the nonlinearity of the governing equations and from typically irregular combustor geometries. Mapping techniques (algebraic and elliptic PDE), and adaptive grid methods (both multi-grid and grid embedding) as applied to axisymmetric combustors are discussed

Transcriptome analysis indicates dominant effects on ribosome and mitochondrial function of a premature termination codon mutation in the zebrafish gene psen2

Published: July 13, 2020PRESENILIN 2 (PSEN2) is one of the genes mutated in early onset familial Alzheimer's disease (EOfAD). PSEN2 shares significant amino acid sequence identity with another EOfAD-related gene PRESENILIN 1 (PSEN1), and partial functional redundancy is seen between these two genes. However, the complete range of functions of PSEN1 and PSEN2 is not yet understood. In this study, we performed targeted mutagenesis of the zebrafish psen2 gene to generate a premature termination codon close downstream of the translation start with the intention of creating a null mutation. Homozygotes for this mutation, psen2S4Ter, are viable and fertile, and adults do not show any gross psen2-dependent pigmentation defects, arguing against significant loss of γ-secretase activity. Also, assessment of the numbers of Dorsal Longitudinal Ascending (DoLA) interneurons that are responsive to psen2 but not psen1 activity during embryogenesis did not reveal decreased psen2 function. Transcripts containing the S4Ter mutation show no evidence of destabilization by nonsense-mediated decay. Forced expression in zebrafish embryos of fusions of psen2S4Ter 5' mRNA sequences with sequence encoding enhanced green fluorescent protein (EGFP) indicated that the psen2S4Ter mutation permits utilization of cryptic, novel downstream translation start codons. These likely initiate translation of N-terminally truncated Psen2 proteins lacking late endosomal/lysosomal localization sequences and that obey the "reading frame preservation rule" of PRESENILIN EOfAD mutations. Transcriptome analysis of entire brains from a 6-month-old family of wild type, heterozygous and homozygous psen2S4Ter female siblings revealed profoundly dominant effects on gene expression likely indicating changes in ribosomal, mitochondrial, and anion transport functions.Haowei Jiang, Stephen Martin Pederson, Morgan Newman, Yang Dong, Karissa Barthelson, Michael Lardell

BgaA acts as an adhesin to mediate attachment of some pneumococcal strains to human epithelial cells

Streptococcus pneumoniae colonization of the respiratory tract is an essential precursor for pneumococcal disease. To colonize efficiently, bacteria must adhere to the epithelial-cell surface. S. pneumoniae possesses surface-associated exoglycosidases that are capable of sequentially deglycosylating human glycans. Two exoglycosidases, neuraminidase (NanA) and β-galactosidase (BgaA), have previously been shown to contribute to S. pneumoniae adherence to human epithelial cells, as deletion of either of these genes results in reduced adherence. It has been suggested that these enzymes may modulate adherence by cleaving sugars to reveal a receptor on host cells. Pretreatment of epithelial cells with exogenous neuraminidase restores the adherence of a nanA mutant, whereas pretreatment with β-galactosidase does not restore the adherence of a bgaA mutant. These data suggest that BgaA may not function to reveal a receptor, and implicate an alternative role for BgaA in adherence. Here we demonstrate that β-galactosidase activity is not required for BgaA-mediated adherence. Addition of recombinant BgaA (rBgaA) to adherence assays and pretreatment of epithelial cells with rBgaA both significantly reduced the level of adherence of the parental strain, but not the BgaA mutant. One possible explanation of these data is that BgaA is acting as an adhesin and that rBgaA is binding to the receptor, preventing bacterial binding. A bead-binding assay demonstrated that BgaA can bind directly to human epithelial cells, supporting the hypothesis that BgaA is an adhesin. Preliminary characterization of the epithelial-cell receptor suggests that it is a glycan in the context of a glycosphingolipid. To further establish the relevance of this adherence mechanism, we demonstrated that BgaA-mediated adherence contributed to adherence of a recent clinical isolate to primary human epithelial cells. Together, these data suggest a novel role for BgaA as an adhesin and suggest that this mechanism could contribute to adherence of at least some pneumococcal strains in vivo

Brain transcriptomes of zebrafish and mouse Alzheimer's disease knock-in models imply early disrupted energy metabolism

Accepted manuscriptEnergy production is the most fundamentally important cellular activity supporting all other functions, particularly in highly active organs such as brains, Here we summarise transcriptome analyses of young adult (pre-disease) brains from a collection of eleven early-onset familial Alzheimer’s disease (EOfAD)-like and non-EOfAD-like mutations in three zebrafish genes. The one cellular activity consistently predicted as affected by only the EOfAD-like mutations is oxidative phosphorylation that produces most of the brain’s energy. All the mutations were predicted to affect protein synthesis. We extended our analysis to knock-in mouse models of APOE alleles and found the same effect for the late onset Alzheimer’s disease risk allele 4. Our results support a common molecular basis for initiation of the pathological processes leading to both early and late onset forms of Alzheimer’s disease and illustrate the utility of zebrafish and of knock-in, single EOfAD mutation models for understanding the causes of this disease.Karissa Barthelson, Morgan Newman, Michael Lardell

No observed effect on brain vasculature of Alzheimer's disease-related mutations in the zebrafish presenilin 1 gene

Previously, we found that brains of adult zebrafish heterozygous for Alzheimer's disease-related mutations in their presenilin 1 gene (psen1, orthologous to human PSEN1) show greater basal expression levels of hypoxia responsive genes relative to their wild type siblings under normoxia, suggesting hypoxic stress. In this study, we investigated whether this might be due to changes in brain vasculature. We generated and compared 3D reconstructions of GFP-labelled blood vessels of the zebrafish forebrain from heterozygous psen1 mutant zebrafish and their wild type siblings. We observed no statistically significant differences in vessel density, surface area, overall mean diameter, overall straightness, or total vessel length normalised to the volume of the telencephalon. Our findings do not support that changes in vascular morphology are responsible for the increased basal expression of hypoxia responsive genes in psen1 heterozygous mutant brains.Karissa Barthelson, Morgan Newman, Cameron J. Nowell and Michael Lardell

Brain transcriptome analysis of a protein-truncating mutation in sortilin-related receptor 1 associated with early-onset familial alzheimer's disease indicates early effects on mitochondrial and ribosome function

BACKGROUND: The early cellular stresses leading to Alzheimer's disease (AD) remain poorly understood because we cannot access living, asymptomatic human AD brains for detailed molecular analyses. Sortilin-related receptor 1 (SORL1) encodes a multi-domain receptor protein genetically associated with both rare, early-onset familial AD (EOfAD) and common, sporadic, late-onset AD (LOAD). SORL1 protein has been shown to act in the trafficking of the amyloid β A4 precursor protein (AβPP) that is proteolysed to form one of the pathological hallmarks of AD, amyloid-β (Aβ) peptide. However, other functions of SORL1 in AD are less well understood. OBJECTIVE: To investigate the effects of heterozygosity for an EOfAD-like mutation in SORL1 on the brain transcriptome of young-adult mutation carriers using zebrafish as a model organism. METHODS: We performed targeted mutagenesis to generate an EOfAD-like mutation in the zebrafish orthologue of SORL1 and performed RNA-sequencing on mRNA isolated from the young adult brains of siblings in a family of fish either wild type (non-mutant) or heterozygous for the EOfAD-like mutation. RESULTS: We identified subtle differences in gene expression indicating changes in mitochondrial and ribosomal function in the mutant fish. These changes appear to be independent of changes in mitochondrial content or the expression of AβPP-related proteins in zebrafish. CONCLUSION: These findings provided evidence supporting that EOfAD mutations in SORL1 affect mitochondrial and ribosomal function and provide the basis for future investigation elucidating the nature of these effects.Karissa Barthelson, Stephen Martin Pederson, Morgana Newman, Michael Lardell

PRESENILIN 1 mutations causing early-onset familial alzheimer's disease or familial acne inversa differ in their effects on genes facilitating energy metabolism and signal transduction

BACKGROUND: The most common cause of early-onset familial Alzheimer's disease (EOfAD) is mutations in PRESENILIN 1 (PSEN1) allowing production of mRNAs encoding full-length, but mutant, proteins. In contrast, a single known frameshift mutation in PSEN1 causes familial acne inversa (fAI) without EOfAD. The molecular consequences of heterozygosity for these mutation types, and how they cause completely different diseases, remains largely unexplored. OBJECTIVE: To analyze brain transcriptomes of young adult zebrafish to identify similarities and differences in the effects of heterozygosity for psen1 mutations causing EOfAD or fAI. METHODS: RNA sequencing was performed on mRNA isolated from the brains of a single family of 6-month-old zebrafish siblings either wild type or possessing a single, heterozygous EOfAD-like or fAI-like mutation in their endogenous psen1 gene. RESULTS: Both mutations downregulate genes encoding ribosomal subunits, and upregulate genes involved in inflammation. Genes involved in energy metabolism appeared significantly affected only by the EOfAD-like mutation, while genes involved in Notch, Wnt and neurotrophin signaling pathways appeared significantly affected only by the fAI-like mutation. However, investigation of direct transcriptional targets of Notch signaling revealed possible increases in γ-secretase activity due to heterozygosity for either psen1 mutation. Transcriptional adaptation due to the fAI-like frameshift mutation was evident. CONCLUSION: We observed both similar and contrasting effects on brain transcriptomes of the heterozygous EOfAD-like and fAI-like mutations. The contrasting effects may illuminate how these mutation types cause distinct diseases.Karissa Barthelson, Yang Dong, Morgan Newman and Michael Lardell

Transcriptome analyses of 7-day-old zebrafish larvae possessing a familial Alzheimer’s disease-like mutation in psen1 indicate effects on oxidative phosphorylation, ECM and MCM functions, and iron homeostasis

Background: Early-onset familial Alzheimer’s disease (EOfAD) is promoted by dominant mutations, enabling the study of Alzheimer’s disease (AD) pathogenic mechanisms through generation of EOfAD-like mutations in animal models. In a previous study, we generated an EOfAD-like mutation, psen1(Q96_K97del), in zebrafish and performed transcriptome analysis comparing entire brains from 6-month-old wild type and heterozygous mutant fish. We identified predicted effects on mitochondrial function and endolysosomal acidification. Here we aimed to determine whether similar effects occur in 7 day post fertilization (dpf) zebrafish larvae that might be exploited in screening of chemical libraries to find ameliorative drugs. Results: We generated clutches of wild type and heterozygous psen1(Q96_K97del) 7 dpf larvae using a paired-mating strategy to reduce extraneous genetic variation before performing a comparative transcriptome analysis. We identified 228 differentially expressed genes and performed various bioinformatics analyses to predict cellular functions. Conclusions: Our analyses predicted a significant effect on oxidative phosphorylation, consistent with our earlier observations of predicted effects on ATP synthesis in adult heterozygous psen1(Q96_K97del) brains. The dysregulation of minichromosome maintenance protein complex (MCM) genes strongly contributed to predicted effects on DNA replication and the cell cycle and may explain earlier observations of genome instability due to PSEN1 mutation. The upregulation of crystallin gene expression may be a response to defective activity of mutant Psen1 protein in endolysosomal acidification. Genes related to extracellular matrix (ECM) were downregulated, consistent with previous studies of EOfAD mutant iPSC neurons and postmortem late onset AD brains. Also, changes in expression of genes controlling iron ion transport were observed without identifiable changes in the prevalence of transcripts containing iron responsive elements (IREs) in their 3′ untranslated regions (UTRs). These changes may, therefore, predispose to the apparent iron dyshomeostasis previously observed in 6-month-old heterozygous psen1(Q96_K97del) EOfAD-like mutant brains.Yang Dong, Morgan Newman, Stephen M. Pederson, Karissa Barthelson, Nhi Hin and Michael Lardell

Pneumococcal 6-phosphogluconate-dehydrogenase, a putative adhesin, induces protective immune response in mice

For most bacteria, adherence to human cells is achieved by bacterial lectins binding to mammalian surface glyconjugates. 6-Phosphogluconate dehydrogenase (6PGD) was identified by us as one of Streptococcus pneumoniae cell wall lectin proteins, which elicits an age-dependent immune response in humans. This study assesses the role of 6PGD in S. pneumoniae pathogenesis as an adhesin and its ability to elicit a protective immune response in mice. Recombinant 6PGD (r6PGD) was cloned from S. pneumoniae serotype 3 (strain WU2). r6PGD interference in adhesion of three genetically unrelated unencapsulated pneumococcal strains (3·8, 14·8 and R6) and two genetically unrelated encapsulated pneumococcal strains (WU2 and D39) to A549 type II lung carcinoma cell was tested. BALB/c mice were immunized with r6PGD and boosted after 3 weeks. Immunized mice were challenged intranasally with a lethal dose of S. pneumoniae. r6PGD inhibited 90% and 80% of pneumococcal adhesion to the A549 cells of three unencapsulated S. pneumoniae strains and two encapsulated S. pneumoniae strains, respectively, in a concentration-dependent manner (P< 0·05). Antibodies to r6PGD produced in mice significantly inhibited bacterial adhesion to A549 cell (P< 0·05). Immunization of mice with r6PGD protected 60% (P< 0·001) of mice for 5 days and 40% (P< 0·05) of the mice for 21 days following intranasal lethal challenge. We have identified 6PGD as a surface-located immunogenic lectin protein capable of acting as an adhesin. 6PGD importance to bacterial pathogenesis was demonstrated by the ability of r6PGD to elicit a protective immune response in mice