Natural compulsive-like behaviour in the deer mouse (Peromyscus maniculatus bairdii) is associated with altered gut microbiota composition

Obsessive–compulsive disorder (OCD) is a psychiatric illness that significantly impacts affected patients and available treatments yield suboptimal therapeutic response. Recently, the role of the gut–brain axis (GBA) in psychiatric illness has emerged as a potential target for therapeutic exploration. However, studies concerning the role of the GBA in OCD are limited. To investigate whether a naturally occurring obsessive–compulsive‐like phenotype in a rodent model, that is large nest building in deer mice, is associated with perturbations in the gut microbiome, we investigated and characterised the gut microbiota in specific‐pathogen‐free bred and housed large (LNB) and normal (NNB) nest‐building deer mice of both sexes (n = 11 per group, including three males and eight females). Following baseline characterisation of nest‐building behaviour, a single faecal sample was collected from each animal and the gut microbiota analysed. Our results reveal the overall microbial composition of LNB animals to be distinctly different compared to controls (PERMANOVA p < .05). While no genera were found to be significantly differentially abundant after correcting for multiple comparisons, the normal phenotype showed a higher loading of Prevotella and Anaeroplasma, while the OC phenotype demonstrated a higher loading of Desulfovermiculus, Aestuariispira, Peptococcus and Holdemanella (cut‐off threshold for loading at 0.2 in either the first or second component of the PCA). These findings not only provide proof‐of‐concept for continued investigation of the GBA in OCD, but also highlight a potential underlying aetiological association between alterations in the gut microbiota and the natural development of obsessive–compulsive‐like behaviours

Comparison of functional domains of the cytotoxic protein NS3 of different orbiviruses

African horsesickness (AHS), caused by the African horsesickness virus (AHSV) (Coetzer&Erasmus, 1994), has 10 segments of double stranded RNA (Verwoerd et al., 1970). These encode seven structural proteins, namely VP1, VP2, VP3, VP4, VP5, VP6 and VP7 (Verwoerd et al., 1972; Huismans&Van Dijk, 1990) as well as four nonstructural proteins (NS1, NS2, NS3 and NS3A) (Van Dijk&Huismans, 1988). NS3 is cytotoxic when expressed in insect cells causing membrane permeabilisation and cell death (Van Staden et al., 1995). This characteristic was postulated to be associated with the two hydrophobic domains (Van Staden et al., 1998; Van Niekerk et al., 2001(a)). Although NS3 has the same basic structure and function in all orbiviruses it differs greatly on nucleic acid sequence level as well as in the degree of variation that is found within the protein (Sailleau et al., 1997; Huismans et al., 2004). Therefore the main aim of this study was to identify which regions or amino acids remain conserved and might therefore play a role in the structure or function of NS3. The focus was mainly on the two hydrophobic domains as well as the spacer region between the hydrophobic domains. The sequence variation analyses between AHSV, BTV and EHDV NS3 revealed that there is a conserved threonine-serine motif in HD1, a conserved aspartic acid at the beginning of the spacer region as well as a conserved asparagine in HD2. The conservation of these residues between the different serogroups indicates that they might have some functional or structural role in the NS3 protein. NS3 protein sequences in the AHSV ã phylogenetic cluster has three extra amino acids in the spacer region at aa 149-151. There is also a significant difference in the spacer region length between EEV NS3 and AHSV NS3, which might have an effect on the topology of the respective proteins. In the spacer region of AHSV NS3, three regions were targeted for mutation analyses: substitution of the conserved isoleucine (aa 141) with a neutral amino acid, deletion of three amino acids (aa 149-151), and insertion of 15 additional amino acids. Three mutants were generated and assayed: the IÄS mutant, the KGDdel mutant and the 15 aa insert mutant. The cytotoxicity of the IÄS and KGD deletion mutants was exactly the same as the native NS3 protein, while the 15 aa insertion resulted in a slightly decreased cytotoxicity. This was the same as the EEV NS3 cytotoxicity level in insect cells. The fluorescent localisation and subcellular fractionation studies showed that both the IÄS mutation and 15 aa insertion affected the localisation of NS3 within the cell and also increased the solubility of the protein. This indicated that even though the membrane localisation of NS3 was disrupted it still had a cytotoxic role in the cell. It could be that wild type NS3 has both a membrane associated and a soluble fraction within cells, and that the soluble fraction is indeed responsible for the observed cytotoxicity and not the membrane fraction as assumed previously. It is therefore postulated that NS3 might have different domains that have various roles within the cell.Dissertation (MSc)--University of Pretoria, 2009.Geneticsunrestricte

Investigation and characterisation of the genetic variation in the coding region of the glycine N-acyltransferase gene

PhD (Biochemistry), North-West University, Potchefstroom Campus, 2015Thorough investigation of the glycine conjugation pathway has been neglected over the last 30 years. Environmental factors, nutrition, and the chronic use of medications are increasing the exposure of humans to benzoate and drugs that are metabolized to acyl-CoA intermediates. Glycine conjugation of mitochondrial acyl-CoAs, catalysed by glycine N-acyltransferase (GLYAT, E.C. 2.3.1.13), is an important metabolic pathway responsible for maintaining adequate levels of free coenzyme A (CoASH). However, because of the small number of pharmaceutical drugs that are conjugated to glycine, the pathway has not yet been characterised in detail. Therefore, one of the objectives of this thesis was to develop a better understanding of glycine conjugation and its role in metabolism. In humans and animals a number of endogenous and xenobiotic organic acids are conjugated to glycine. Glycine conjugation has generally been assumed to be a detoxification mechanism, increasing the water solubility of organic acids in order to facilitate urinary excretion. However, recently it was proposed that the role of the amino acid conjugations, including glycine conjugation, is to regulate systemic levels of amino acids that are also utilised as neurotransmitters in the central nervous systems of animals. The glycine deportation hypothesis was based on the observation that, compared to glucuronidation, glycine conjugation does not significantly increase the water solubility of aromatic acids. A thorough review of the literature for this thesis showed that the major role of glycine conjugation, however, is to dispose of the end products of phenylpropionate metabolism. The review also introduced the new perspective that mitochondrial glycine conjugation prevents the accumulation of benzoate in the mitochondrial matrix by forming hippuric acid a less lipophilic conjugate that can be more readily transported out of the mitochondria. Although organic anion transporters can export benzoate from the matrix, this process would likely be futile because benzoic acid can simply diffuse back into the matrix. Hippurate, however, is significantly less lipophilic and therefore less capable of diffusing into the matrix. It is therefore not the transport out of the mitochondrial matrix that is facilitated by glycine conjugation, but rather the ability of the glycine conjugates to re-enter the matrix that is decreased. Lastly, glycine conjugation of benzoate also exacerbates the dietary deficiency of glycine in humans. Because the resulting shortage of glycine can negatively influence brain neurochemistry and the synthesis of collagen, nucleic acids, porphyrins, and other important metabolites, the risks of using benzoate as a preservative should not be underestimated. To date, no defect of the glycine conjugation pathway has been reported and this, together with the fact that GLYAT plays an important role in hepatic metabolism, suggests that this pathway is essential for survival. GLYAT activity affects mitochondrial ATP production, glycine availability, CoASH availability and the toxicity of various organic acids. Therefore, variation in the glycine conjugation pathway could influence liver cancer, musculoskeletal development and mitochondrial energy metabolism. Significant interindividual variation exists in glycine conjugation capacity. The molecular basis for this variability is not known. The main aim of this thesis was to investigate and characterise the genetic variation in the coding region of the GLYAT gene. This was accomplished by firstly, investigating the influence of non-synonymous single nucleotide polymorphisms (SNPs) on the enzyme activity of a recombinant human GLYAT and secondly, by analysing the level of genetic variation in the coding region of the GLYAT gene using existing worldwide population data. To investigate the influence of non-synonymous SNPs in the GLYAT gene on the enzyme activity, a recombinant human GLYAT was prepared, and characterised. Site-directed mutagenesis was used to generate six variants of the enzyme (K16N; S17T; R131H; N156S; F168L; R199C). The variants were expressed, purified, and enzymatically characterised. The enzyme activities of the K16N, S17T and R131H variants were similar to that of the wild-type, whereas the N156S variant was more active, the F168L variant less active, and the R199C variant was inactive. The results showed that SNP variations in the human GLYAT gene can influence the kinetic properties of the enzyme. The genetic variation data of the human GLYAT open reading frame (ORF) available on public databases was investigated by formulating the hypothesis that due to the essential nature of the glycine conjugation pathway, the genetic variation in the ORF of the GLYAT gene should be low and that deleterious alleles will be found at low frequencies. Data from the i) 1000 Genome Project, ii) the HapMap Project, and iii) the Khoi-San/Bantu Sequencing Project was downloaded from available databases. Sequence data of the coding region of a small cohort of South African Afrikaner Caucasian individuals was also generated and included in the analyses. In the GLYAT ORF of the 1537 individuals analysed, only two haplotypes (S156 and T17S156) out of 14 haplotypes were identified in all populations as having the highest haplotype frequencies (70% and 20% respectively). The S156C199 and S156H131 haplotypes, which have a deleterious effect on the enzyme activity of a recombinant human GLYAT, were detected at very low frequencies. The results of this study indicated that the GLYAT ORF is remarkably conserved, which supports the hypothesis that the glycine conjugation pathway is an essential detoxification pathway. The findings presented in this thesis highlight the importance that future investigations should determine the in vivo capacity of the glycine conjugation pathway for the detoxification of benzoate and other xenobiotics.Doctora

Variations de couleur du plumage dans le genre Agapornis : une critique

The genus Agapornis consists of nine small African parrot species that are globally well known as pets, but are also found in their native habitat. Illegal trapping, poaching and habitat destruction are the main threats these birds face in the wild. In aviculture, Agapornis breeding is highly popular all across the globe. Birds are mainly selected based on their plumage colour variations but very little molecular research has been conducted on this topic. There are 30 known colour variations amongst the nine species and most of these are inherited as Mendelian traits. However, to date none of the genes or polymorphisms linked to these variations have been identified or verified. Due to unethical breeding practices, the need for the development of molecular tests such as identification verification tests or species identity tests is growing. Future research is paramount to ensure the conservation of wild populations as well as aiding breeders in improving breeding strategies.Le genre Agapornis se compose de neuf petites espèces de perroquets africains qui sont mondialement connus comme animaux de compagnie, mais qui se trouvent également dans leur habitat d’origine. Le piégeage illégal, le braconnage et la destruction de l’habitat sont les principales menaces auxquelles ces oiseaux sont confrontés dans la nature. En aviculture, l’élevage Agapornis est très populaire dans le monde entier. Les oiseaux sont principalement sélectionnés en fonction des variations des couleurs de leur plumage, mais très peu de recherches moléculaires ont été menées sur ce sujet. Il existe 30 variations de couleur connues parmi les neuf espèces et la plupart d’entre elles sont héritées des caractères Mendéliens. Cependant, à ce jour, aucun des gènes ou polymorphismes liés à ces variations n’a été identifié ou vérifié. En raison des pratiques de sélection contraires à l’éthique, la nécessité de développer des tests moléculaires tels que des tests de vérification de l’identification ou des tests d’identité d’espèce est de plus en plus importante. Les recherches futures sont primordiales pour assurer la conservation des populations sauvages et aider les éleveurs à améliorer leurs stratégies de sélection.The Technology and Human Resources for Industry Programme (THRIP), which forms part of the Department of Trade and Industry of the South African government project ID number TP13082831254, Lumegen Laboratories as well as the Technology Innovation Agency, SABDI grant (407/01 SABDI 16/1016).http://www.tandfonline.com/loi/tost202020-03-01hj2019Animal and Wildlife Science

Plumage colour variations in the Agapornis genus: a review

The genus Agapornis consists of nine small African parrot species that are globally well known as pets, but are also found in their native habitat. Illegal trapping, poaching and habitat destruction are the main threats these birds face in the wild. In aviculture, Agapornis breeding is highly popular all across the globe. Birds are mainly selected based on their plumage colour variations but very little molecular research has been conducted on this topic. There are 30 known colour variations amongst the nine species and most of these are inherited as Mendelian traits. However, to date none of the genes or polymorphisms linked to these variations have been identified or verified. Due to unethical breeding practices, the need for the development of molecular tests such as identification verification tests or species identity tests is growing. Future research is paramount to ensure the conservation of wild populations as well as aiding breeders in improving breeding strategies.Keywords: avi culture, love birds, molecular breeding tools, Psittacida

Functional Characterisation of Three Glycine N-Acyltransferase Variants and the Effect on Glycine Conjugation to Benzoyl–CoA

The glycine conjugation pathway in humans is involved in the metabolism of natural substrates and the detoxification of xenobiotics. The interactions between the various substrates in this pathway and their competition for the pathway enzymes are currently unknown. The pathway consists of a mitochondrial xenobiotic/medium-chain fatty acid: coenzyme A (CoA) ligase (ACSM2B) and glycine N-acyltransferase (GLYAT). The catalytic mechanism and substrate specificity of both of these enzymes have not been thoroughly characterised. In this study, the level of evolutionary conservation of GLYAT missense variants and haplotypes were analysed. From these data, haplotype variants were selected (156Asn &gt; Ser, [17Ser &gt; Thr,156Asn &gt; Ser] and [156Asn &gt; Ser,199Arg &gt; Cys]) in order to characterise the kinetic mechanism of the enzyme over a wide range of substrate concentrations. The 156Asn &gt; Ser haplotype has the highest frequency and the highest relative enzyme activity in all populations studied, and hence was used as the reference in this study. Cooperative substrate binding was observed, and the kinetic data were fitted to a two-substrate Hill equation. The coding region of the GLYAT gene was found to be highly conserved and the rare 156Asn &gt; Ser,199Arg &gt; Cys variant negatively affected the relative enzyme activity. Even though the 156Asn &gt; Ser,199Arg &gt; Cys variant had a higher affinity for benzoyl-CoA (s0.5,benz = 61.2 µM), kcat was reduced to 9.8% of the most abundant haplotype 156Asn &gt; Ser (s0.5,benz = 96.6 µM), while the activity of 17Ser &gt; Thr,156Asn &gt; Ser (s0.5,benz = 118 µM) was 73% of 156Asn &gt; Ser. The in vitro kinetic analyses of the effect of the 156Asn &gt; Ser,199Arg &gt; Cys variant on human GLYAT enzyme activity indicated that individuals with this haplotype might have a decreased ability to metabolise benzoate when compared to individuals with the 156Asn &gt; Ser variant. Furthermore, the accumulation of acyl-CoA intermediates can inhibit ACSM2B leading to a reduction in mitochondrial energy production

Development of an SNP-based parentage verification panel for lovebirds

Please read abstract in the article.File S1 NGS sequencing, SNP discovery initial SNP panel selection, samples genotyped and construction of SNP‐based parentage verification panel.File S2 MAF and HO values of each SNP.File S3 Suggested relationships and parentage verification results.The Technology Innovation Agency, SABDI grant (407/01 SABDI 16/1016).https://onlinelibrary.wiley.com/journal/136520522020-12-01hj2019Animal and Wildlife Science

Development of an SNP

File S1 NGS sequencing, SNP discovery initial SNP panel selection, samples genotyped and construction of SNP‐based parentage verification panel.File S2 MAF and HO values of each SNP.File S3 Suggested relationships and parentage verification results.Please read abstract in the article.The Technology Innovation Agency, SABDI grant (407/01 SABDI 16/1016).https://onlinelibrary.wiley.com/journal/136520522020-12-01hj2019Animal and Wildlife Science

Draft de novo genome sequence of Agapornis roseicollis for application in avian breeding

In aviculture, lovebirds are considered one of the most popular birds to keep. This African parakeet is known for its range of plumage colors and ease to tame. Plumage variation is the most important price-determining trait of these birds, and also the main selection criterion for breeders. Currently, no genetic screening tests for traits of economic importance or to confirm pedigree data are available for any of the nine lovebird species. As a starting point to develop these tests, the de novo genome of Agapornis roseicollis (rosy-faced lovebird) was sequenced, assembled, and annotated. Sequencing was done on the Illumina HiSeq 2000 platform and the assembly was performed using SOAPdenovo v2.04. The genome was found to be 1.1 Gb in size and 16,044 genes were identified and annotated. This compared well with other previously sequenced avian genomes, such as the chicken, zebra finch, and budgerigar. To assess genome completeness, the number of benchmarking universal single-copy orthologs were identified in the genome. This was compared to other previously assembled avian genomes and the results indicated that the genome will be useful in the development of genetic screening tests to aid lovebird breeders in selecting breeding pairs.http://www.tandfonline.com/loi/labt202018-10-16hj2017Animal and Wildlife Science

The glycine N-acyltransferases, GLYAT and GLYATL1, contribute to the detoxification of isovaleryl-CoA - an in-silico and in vitro validation

Isovaleric acidemia (IVA), due to isovaleryl-CoA dehydrogenase (IVD) deficiency, results in the accumulation of isovaleryl-CoA, isovaleric acid and secondary metabolites. The increase in these metabolites decreases mitochondrial energy production and increases oxidative stress. This contributes to the neuropathological features of IVA. A general assumption in the literature exists that glycine N-acyltransferase (GLYAT) plays a role in alleviating the symptoms experienced by IVA patients through the formation of N-isovalerylglycine. GLYAT forms part of the phase II glycine conjugation pathway in the liver and detoxifies excess acyl-CoA’s namely benzoyl-CoA. However, very few studies support GLYAT as the enzyme that conjugates isovaleryl-CoA to glycine. Furthermore, GLYATL1, a paralogue of GLYAT, conjugates phenylacetyl-CoA to glutamine. Therefore, GLYATL1 might also be a candidate for the formation of N-isovalerylglycine. Based on the findings from the literature review, we proposed that GLYAT or GLYATL1 can form N-isovalerylglycine in IVA patients. To test this hypothesis, we performed an in-silico analysis to determine which enzyme is more likely to conjugate isovaleryl-CoA with glycine using AutoDock Vina. Thereafter, we performed in vitro validation using purified enzyme preparations. The in-silico and in vitro findings suggested that both enzymes could form N-isovaleryglycine albeit at lower affinities than their preferred substrates. Furthermore, an increase in glycine concentration does not result in an increase in N-isovalerylglycine formation. The results from the critical literature appraisal, in-silico, and in vitro validation, suggest the importance of further investigating the reaction kinetics and binding behaviors between these substrates and enzymes in understanding the pathophysiology of IVA