The anti-inflammatory effects of cytokine suppressive anti-inflammatory drugs Tenilsetam and Longvida curcumin on microglia numbers in the GFAP-IL6 mouse model

Chronic neuroinflammation has been observed to be a key factor in the progress of neurodegenerative diseases including Alzheimer’s disease (AD). Microglia serve as resident macrophages within the central nervous system (CNS) and become activated upon contact with entities that are released from dying neurons during neurodegenerative processes, these include neuro-filaments, Aβ aggregates and tau containing neurofibrillary tangles, exposed naked DNA and oligodendrocytic myelin fragments. This prompts microglial and astrocyte activation which ultimately causes further neuronal damage including synaptic depredation. Neuronal tissue within the central nervous system can become particularly vulnerable to chronic neuroinflammation as a vicious self-perpetuating cycle of inflammation causes chronic neuronal decay. It is thought that preventing and or reducing chronic neuroinflammation by anti-inflammatory drugs will reduce chronic neuroinflammation, thus disrupting the cycle of microglial and astrocyte activation and resulting neurodegeneration. This thesis compares the short and long-term anti-inflammatory properties of two drugs: The nootropic drug Tenilsetam (±)-3-(2-thienyl)-2-piperazinone (CAS 997, Tenilsetam) and the herbal spice curcumin (1E,6E)-1,7-bis (4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) (by Verdure Sciences) in the GFAP-IL6 mouse model. This research project found that Tenilsetam decreased the total number of TSPO+ microglia in the hippocampus of 8-month-old GFAP-IL6 mice along with Iba1+ microglia in the cerebellum and hippocampus of GFAP-IL6 mice at 8 months of age and in the cerebellum at 18 months of age. Microglial density in the cerebellum of GFAP-IL6 mice decreased to a similar level after both 5 and 15 months of feeding, with volume loss prevented in by Tenilsetam at 8 months. Longvida curcumin was found to decrease the total number of Iba-l+ microglia in the cerebellum of GFAP-IL6 mice at 8 months of age. When compared to Tenilsetam, Longvida curcumin had statistically similar Iba-1+ microglial numbers in the cerebellum of GFAP-IL6 mice at 8 months of age. Comparing these results and considering the efficacy of Tenilsetam, it can be inferred that due to the similarity in microglial population within the same genotype, Longvida curcumin is as effective as Tenilsetam at preventing neuroinflammation in the cerebellum of GFAP-IL6 mice at 8 months of age

Phenotypic characterization and candidate gene analysis of a short kernel and brassinosteroid insensitive mutant from hexaploid oat (Avena sativa)

In an ethyl methanesulfonate oat (Avena sativa) mutant population we have found a mutant with striking differences to the wild-type (WT) cv. Belinda. We phenotyped the mutant and compared it to the WT. The mutant was crossed to the WT and mapping-by-sequencing was performed on a pool of F2 individuals sharing the mutant phenotype, and variants were called. The impacts of the variants on genes present in the reference genome annotation were estimated. The mutant allele frequency distribution was combined with expression data to identify which among the affected genes was likely to cause the observed phenotype. A brassinosteroid sensitivity assay was performed to validate one of the identified candidates. A literature search was performed to identify homologs of genes known to be involved in seed shape from other species. The mutant had short kernels, compact spikelets, altered plant architecture, and was found to be insensitive to brassinosteroids when compared to the WT. The segregation of WT and mutant phenotypes in the F2 population was indicative of a recessive mutation of a single locus. The causal mutation was found to be one of 123 single-nucleotide polymorphisms (SNPs) spanning the entire chromosome 3A, with further filtering narrowing this down to six candidate genes. In-depth analysis of these candidate genes and the brassinosteroid sensitivity assay suggest that a Pro303Leu substitution in AVESA.00010b.r2.3AG0419820.1 could be the causal mutation of the short kernel mutant phenotype. We identified 298 oat proteins belonging to orthogroups of previously published seed shape genes, with AVESA.00010b.r2.3AG0419820.1 being the only of these affected by a SNP in the mutant. The AVESA.00010b.r2.3AG0419820.1 candidate is functionally annotated as a GSK3/SHAGGY-like kinase with homologs in Arabidopsis, wheat, barley, rice, and maize, with several of these proteins having known mutants giving rise to brassinosteroid insensitivity and shorter seeds. The substitution in AVESA.00010b.r2.3AG0419820.1 affects a residue with a known gain-of function substitution in Arabidopsis BRASSINOSTEROID-INSENSITIVE2. We propose a gain-of-function mutation in AVESA.00010b.r2.3AG0419820.1 as the most likely cause of the observed phenotype, and name the gene AsGSK2.1. The findings presented here provide potential targets for oat breeders, and a step on the way towards understanding brassinosteroid signaling, seed shape and nutrition in oats

Structural and functional consequences of removing the N-terminal domain from the magnesium chelatase ChlH subunit of Thermosynechococcus elongatus

Magnesium chelatase (MgCH) initiates chlorophyll biosynthesis by catalysing the ATP-dependent insertion of Mg2+ into protoporphyrin. This large enzyme complex comprises ChlH, I and D subunits, with I and D involved in ATP hydrolysis, and H the protein that handles the substrate and product. The 148 kDa ChlH subunit has a globular N-terminal domain attached by a narrow linker to a hollow cage-like structure. Following deletion of this ~18 kDa domain from the Thermosynechoccus elongatus ChlH, we used single particle reconstruction to show that the apo- and porphyrin-bound forms of the mutant subunit consist of a hollow globular protein with three connected lobes; superposition of the mutant and native ChlH structures shows that, despite the clear absence of the N-terminal ‘head’ region, the rest of the protein appears to be correctly folded. Analyses of dissociation constants shows that the ΔN159ChlH mutant retains the ability to bind protoporphyrin and the Gun4 enhancer protein, although the addition of I and D subunits yields an extremely impaired active enzyme complex. Addition of the Gun4 enhancer protein, which stimulates MgCH activity significantly especially at low Mg2+ concentrations, partially reactivates the ΔN159ChlH–I–D mutant enzyme complex, suggesting that the binding site or sites for Gun4 on H do not wholly depend on the N-terminal domain

The mosaic oat genome gives insights into a uniquely healthy cereal crop

Cultivated oat (Avena sativa L.) is an allohexaploid (AACCDD, 2n = 6x = 42) thought to have been domesticated more than 3,000 years ago while growing as a weed in wheat, emmer and barley fields in Anatolia1,2. Oat has a low carbon footprint, substantial health benefits and the potential to replace animal-based food products. However, the lack of a fully annotated reference genome has hampered efforts to deconvolute its complex evolutionary history and functional gene dynamics. Here we present a high-quality reference genome of A. sativa and close relatives of its diploid (Avena longiglumis, AA, 2n = 14) and tetraploid (Avena insularis, CCDD, 2n = 4x = 28) progenitors. We reveal the mosaic structure of the oat genome, trace large-scale genomic reorganizations in the polyploidization history of oat and illustrate a breeding barrier associated with the genome architecture of oat. We showcase detailed analyses of gene families implicated in human health and nutrition, which adds to the evidence supporting oat safety in gluten-free diets, and we perform mapping-by-sequencing of an agronomic trait related to water-use efficiency. This resource for the Avena genus will help to leverage knowledge from other cereal genomes, improve understanding of basic oat biology and accelerate genomics-assisted breeding and reanalysis of quantitative trait studies

Magnesium Chelatase: Insights into the first Step of Chlorophyll Biosynthesis

The enzyme magnesium chelatase inserts magnesium into protoporphyrin IX (Proto) to produce to magnesium protoporphyrin IX, the first unique intermediate of the chlorophyll biosynthetic pathway. Magnesium chelatase is composed of three distinct proteins termed I (molecular weight ~40 kDa), D (~70 kDa) and H (~140 kDa). Defining the individual properties and structure of the magnesium chelatase components and their role in the reaction mechanism is important for a full understanding of the first step in magnesium tetrapyrrole biosynthesis. The three components of magnesium chelatase show significant conservation at the protein sequence level, which extends from bacteriochlorophyll synthesising purple non-sulfur bacteria and green sulfur bacteria to chlorophyll synthesising eukaryotes and cyanobacteria. In Paper I, eight mutants of the H gene (Xantha-f) from barley were characterised at the molecular level and provide explanations for the yellow phenotypes of germinating mutant seedlings. In this thesis magnesium chelatase from the photosynthetic bacterium Rhodobacter capsulatus has been used as a model system since much of the pioneering work has been conducted on this organism. Magnesium chelatase requires ATP to insert magnesium into Proto. There has been conflicting results as to the ATPase activity of the H subunit. In Paper II it was demonstrated that ATP hydrolysis can be attributed the I subunit and not the H. The unprecedented discovery of an iron-sulfur cluster in the H subunit of R. capsulatus is described in Paper III. The cysteine motif that coordinates this iron-sulfur cluster is only present in five other facultative proteobacteria and absent in all oxygenic or anaerobic species. The function of this cluster is yet to be established. In Paper IV the first insights into the structure of an H subunit is presented. Electron microscopy and single-particle reconstruction was used to solve the structure in the apo and substrate bound conformations at a resolution of 25 Å, and revealed a conformational change upon Proto binding. Limited proteolysis and construction of truncated H polypeptides provided supporting information to propose a cooperative substrate binding model. The binding of porphyrin to the H subunit was further investigated in Paper V using tryptophan fluorescence quenching to detect a high affinity porphyrin binding site in the nanomolar range. Alanine mutagenesis of the H subunit implicated key residues involved in porphyrin binding and catalysis. The work presented in this thesis has expanded our understanding of the magnesium chelatase, particularly in respect to the H subunit. With three different proteins and three substrates, it is clear that magnesium chelatase is an elaborate molecular machine that is proving to be far more complicated than ever expected

NfCR1, the first non-LTR retrotransposon characterized in the Australian lungfish genome, Neoceratodus forsteri, shows similarities to CR1-like elements

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Characterization of eight barley xantha-f mutants deficient in magnesium chelatase

Magnesium chelatase (EC 6.6.1.1) catalyses the insertion of magnesium into protoporphyrin IX, the first unique step of the chlorophyll biosynthetic pathway. The enzyme is composed of three different subunits of approximately 40, 70 and 140 kDa. In barley (Hordeum vulgare L.) the subunits are encoded by the genes Xantha-h, Xantha-g and Xantha-f. In the 1950s, eight induced xantha-f mutants were isolated. In this work we characterized these mutations at the DNA level and provided explanations for their phenotypes. The xantha-f10 mutation is a 3 bp deletion, resulting in a polypeptide lacking the glutamate residue at position 424. The leaky mutation xantha-f26 has a missense mutation leading to a M632R exchange. The xantha-f27 and -f40 are deletions of 14 and 2 bp, respectively, resulting in truncated polypeptides of 1104 and 899 amino acid residues, respectively. Mutation xantha-f41 is an in-frame deletion that removes A439, L440, Q441 and V442 from the resulting protein. Mutation xantha-f58 is most likely a deletion of the whole Xantha-f gene, as no DNA fragments could be detected by PCR or southern blot experiments. The slightly leaky xantha-f60 and non-leaky -f68 mutations each have a missense mutation causing a P393L and G794E exchange in the polypeptide, respectively

Exophiala macquariensis sp. nov., a cold adapted black yeast species recovered from a hydrocarbon contaminated sub-Antarctic soil

A new black yeast species, Exophiala macquariensis is described that is a member of the ascomycete family Herpotrichiellaceae, order Chaetothyriales. The genus Exophiala is comprised of opportunistic pathogens isolated from clinical specimens as well as species recovered from hydrocarbon contaminated environments. Several species have been reported to be able to degrade benzene, toluene, ethylbenzene and xylenes. Here, a novel species of Exophiala (CZ06) previously isolated from a Sub-Antarctic, Macquarie Island soil that was spiked with Special Antarctic Blend diesel fuel (SAB) is described. This isolate has the capacity of toluene biodegradation at cold temperatures. Multilocus sequence typing showed that this fungus was closely related to the pathogenic species Exophiala salmonis and Exophiala equina. With the capacity to utilise hydrocarbons as a sole carbon source at 10 °C, this fungus has great potential for future bioremediation applications

PCR-mediated deletion of plasmid DNA

The PCR-mediated plasmid DNA deletion method is a simple approach to delete DNA sequences from plasmids using only one round of PCR, with two primers, and without ligation or purification prior to in vivo recombination. By using only PCR, the method is sequence independent and, as shown in this study, is applicable to various sizes of plasmids and deletions using minimal primer design