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Introduction: We assessed a genetic risk score for Alzheimer's disease (AD-GRS) and apolipoprotein E (APOE4) in an exploratory neuroimaging substudy of the FINGER trial., Methods: 1260 at-risk older individuals without dementia were randomized to multidomain lifestyle intervention or health advice. N = 126 participants underwent magnetic resonance imaging (MRI), and N = 47 positron emission tomography (PET) scans (Pittsburgh Compund B [PiB], Fluorodeoxyglucose) at baseline; N = 107 and N = 38 had repeated 2-year scans., Results: The APOE4 allele, but not AD-GRS, was associated with baseline lower hippocampus volume (β = -0.27, p = 0.001), greater amyloid deposition (β = 0.48, p = 0.001), 2-year decline in hippocampus (β = -0.27, p = 0.01), total gray matter volume (β = -0.25, p = 0.01), and cortical thickness (β = -0.28, p = 0.003). In analyses stratified by AD-GRS (below vs above median), the PiB composite score increased less in intervention versus control in the higher AD-GRS group (β = -0.60, p = 0.03)., Discussion: AD-GRS and APOE4 may have different impacts on potential intervention effects on amyloid, that is, less accumulation in the higher-risk group (AD-GRS) versus lower-risk group (APOE)., Highlights: First study of neuroimaging and AD genetics in a multidomain lifestyle intervention. Possible intervention effect on brain amyloid deposition may rely on genetic risk. AD-GRS and APOE4 allele may have different impacts on amyloid during intervention., (© 2024 The Authors. Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

Introduction: Genome-wide association studies (GWAS) are fundamental for identifying loci associated with diseases. However, they require replication in other ethnicities., Methods: We performed GWAS on sporadic Alzheimer's disease (AD) including 539 patients and 854 controls from Argentina and Chile. We combined our results with those from the European Alzheimer and Dementia Biobank (EADB) in a meta-analysis and tested their genetic risk score (GRS) performance in this admixed population., Results: We detected apolipoprotein E ε4 as the single genome-wide significant signal (odds ratio  = 2.93 [2.37-3.63], P = 2.6 × 10 -23 ). The meta-analysis with EADB summary statistics revealed four new loci reaching GWAS significance. Functional annotations of these loci implicated endosome/lysosomal function. Finally, the AD-GRS presented a similar performance in these populations, despite the score diminished when the Native American ancestry rose., Discussion: We report the first GWAS on AD in a population from South America. It shows shared genetics modulating AD risk between the European and these admixed populations., Highlights: This is the first genome-wide association study on Alzheimer's disease (AD) in a population sample from Argentina and Chile. Trans-ethnic meta-analysis reveals four new loci involving lysosomal function in AD. This is the first independent replication for TREM2L, IGH-gene-cluster, and ADAM17 loci. A genetic risk score (GRS) developed in Europeans performed well in this population. The higher the Native American ancestry the lower the GRS values., (© 2023 The Authors. Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

Objective: The objective of this study was to aggregate data for the first genomewide association study meta-analysis of cluster headache, to identify genetic risk variants, and gain biological insights., Methods: A total of 4,777 cases (3,348 men and 1,429 women) with clinically diagnosed cluster headache were recruited from 10 European and 1 East Asian cohorts. We first performed an inverse-variance genomewide association meta-analysis of 4,043 cases and 21,729 controls of European ancestry. In a secondary trans-ancestry meta-analysis, we included 734 cases and 9,846 controls of East Asian ancestry. Candidate causal genes were prioritized by 5 complementary methods: expression quantitative trait loci, transcriptome-wide association, fine-mapping of causal gene sets, genetically driven DNA methylation, and effects on protein structure. Gene set and tissue enrichment analyses, genetic correlation, genetic risk score analysis, and Mendelian randomization were part of the downstream analyses., Results: The estimated single nucleotide polymorphism (SNP)-based heritability of cluster headache was 14.5%. We identified 9 independent signals in 7 genomewide significant loci in the primary meta-analysis, and one additional locus in the trans-ethnic meta-analysis. Five of the loci were previously known. The 20 genes prioritized as potentially causal for cluster headache showed enrichment to artery and brain tissue. Cluster headache was genetically correlated with cigarette smoking, risk-taking behavior, attention deficit hyperactivity disorder (ADHD), depression, and musculoskeletal pain. Mendelian randomization analysis indicated a causal effect of cigarette smoking intensity on cluster headache. Three of the identified loci were shared with migraine., Interpretation: This first genomewide association study meta-analysis gives clues to the biological basis of cluster headache and indicates that smoking is a causal risk factor. ANN NEUROL 2023;94:713-726., (© 2023 The Authors. Annals of Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

Alzheimer disease (AD) is the most common form of senile dementia, with high incidence late in life in many populations including Caribbean Hispanic (CH) populations. Such admixed populations, descended from more than one ancestral population, can present challenges for genetic studies, including limited sample sizes and unique analytical constraints. Therefore, CH populations and other admixed populations have not been well represented in studies of AD, and much of the genetic variation contributing to AD risk in these populations remains unknown. Here, we conduct genome-wide analysis of AD in multiplex CH families from the Alzheimer Disease Sequencing Project (ADSP). We developed, validated, and applied an implementation of a logistic mixed model for admixture mapping with binary traits that leverages genetic ancestry to identify ancestry-of-origin loci contributing to AD. We identified three loci on chromosome 13q33.3 associated with reduced risk of AD, where associations were driven by Native American (NAM) ancestry. This AD admixture mapping signal spans the FAM155A , ABHD13 , TNFSF13B , LIG4, and MYO16 genes and was supported by evidence for association in an independent sample from the Alzheimer's Genetics in Argentina-Alzheimer Argentina consortium (AGA-ALZAR) study with considerable NAM ancestry. We also provide evidence of NAM haplotypes and key variants within 13q33.3 that segregate with AD in the ADSP whole-genome sequencing data. Interestingly, the widely used genome-wide association study approach failed to identify associations in this region. Our findings underscore the potential of leveraging genetic ancestry diversity in recently admixed populations to improve genetic mapping, in this case for AD-relevant loci., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Introduction: The metabolic routes altered in Alzheimer's disease (AD) brain are poorly understood. As the metabolic pathways are evolutionarily conserved, the metabolic profiles carried out in animal models of AD could be directly translated into human studies. Methods: We performed untargeted Nuclear Magnetic Resonance metabolomics in hippocampus of McGill-R-Thy1-APP transgenic (Tg) rats, a model of AD-like cerebral amyloidosis and the translational potential of these findings was assessed by targeted Gas Chromatography-Electron Impact-Mass Spectrometry in plasma of participants in the German longitudinal cohort AgeCoDe. Results: In rat hippocampus 26 metabolites were identified. Of these 26 metabolites, nine showed differences between rat genotypes that were nominally significant. Two of them presented partial least square-discriminant analysis (PLS-DA) loadings with the larger absolute weights and the highest Variable Importance in Projection (VIP) scores and were specifically assigned to nicotinamide adenine dinucleotide (NAD) and nicotinamide (Nam). NAD levels were significantly decreased in Tg rat brains as compared to controls. In agreement with these results, plasma of AD patients showed significantly reduced levels of Nam in respect to cognitively normal participants. In addition, high plasma levels of Nam showed a 27% risk reduction of progressing to AD dementia within the following 2.5 years, this hazard ratio is lost afterwards. Discussion: To our knowledge, this is the first report showing that a decrease of Nam plasma levels is observed couple of years before conversion to AD, thereby suggesting its potential use as biomarker for AD progression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Dalmasso, Arán, Galeano, Perin, Giavalisco, Martino Adami, Novack, Castaño, Cuello, Scherer, Maier, Wagner, Riedel-Heller, Ramirez and Morelli.)

Amyloid-beta 42 (Aβ42) and phosphorylated tau (pTau) levels in cerebrospinal fluid (CSF) reflect core features of the pathogenesis of Alzheimer's disease (AD) more directly than clinical diagnosis. Initiated by the European Alzheimer & Dementia Biobank (EADB), the largest collaborative effort on genetics underlying CSF biomarkers was established, including 31 cohorts with a total of 13,116 individuals (discovery n = 8074; replication n = 5042 individuals). Besides the APOE locus, novel associations with two other well-established AD risk loci were observed; CR1 was shown a locus for Aβ42 and BIN1 for pTau. GMNC and C16orf95 were further identified as loci for pTau, of which the latter is novel. Clustering methods exploring the influence of all known AD risk loci on the CSF protein levels, revealed 4 biological categories suggesting multiple Aβ42 and pTau related biological pathways involved in the etiology of AD. In functional follow-up analyses, GMNC and C16orf95 both associated with lateral ventricular volume, implying an overlap in genetic etiology for tau levels and brain ventricular volume., (© 2022. The Author(s).)

Alzheimer's disease has a long asymptomatic phase that offers a substantial time window for intervention. Using this window of opportunity will require early diagnostic and prognostic biomarkers to detect Alzheimer's disease pathology at predementia stages, thus allowing identification of patients who will most probably progress to dementia of the Alzheimer's type and benefit from specific disease-modifying therapies. Consequently, we searched for CSF proteins associated with disease progression along with the clinical disease staging. We measured the levels of 184 proteins in CSF samples from 556 subjective cognitive decline and mild cognitive impairment patients from three independent memory clinic longitudinal studies (Spanish ACE, n = 410; German DCN, n = 93; German Mannheim, n = 53). We evaluated the association between protein levels and clinical stage, and the effect of protein levels on the progression from mild cognitive impairment to dementia of the Alzheimer's type. Mild cognitive impairment subjects with increased CSF level of matrix metalloproteinase 10 (MMP-10) showed a higher probability of progressing to dementia of the Alzheimer's type and a faster cognitive decline. CSF MMP-10 increased the prediction accuracy of CSF amyloid-β 42 (Aβ42), phospho-tau 181 (P-tau181) and total tau (T-tau) for conversion to dementia of the Alzheimer's type. Including MMP-10 to the [A/T/(N)] scheme improved considerably the prognostic value in mild cognitive impairment patients with abnormal Aβ42, but normal P-tau181 and T-tau, and in mild cognitive impairment patients with abnormal Aβ42, P-tau181 and T-tau. MMP-10 was correlated with age in subjects with normal Aβ42, P-tau181 and T-tau levels. Our findings support the use of CSF MMP-10 as a prognostic marker for dementia of the Alzheimer's type and its inclusion in the [A/T/(N)] scheme to incorporate pathologic aspects beyond amyloid and tau. CSF level of MMP-10 may reflect ageing and neuroinflammation., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Characterization of the genetic landscape of Alzheimer's disease (AD) and related dementias (ADD) provides a unique opportunity for a better understanding of the associated pathophysiological processes. We performed a two-stage genome-wide association study totaling 111,326 clinically diagnosed/'proxy' AD cases and 677,663 controls. We found 75 risk loci, of which 42 were new at the time of analysis. Pathway enrichment analyses confirmed the involvement of amyloid/tau pathways and highlighted microglia implication. Gene prioritization in the new loci identified 31 genes that were suggestive of new genetically associated processes, including the tumor necrosis factor alpha pathway through the linear ubiquitin chain assembly complex. We also built a new genetic risk score associated with the risk of future AD/dementia or progression from mild cognitive impairment to AD/dementia. The improvement in prediction led to a 1.6- to 1.9-fold increase in AD risk from the lowest to the highest decile, in addition to effects of age and the APOE ε4 allele., (© 2022. The Author(s).)

Genetic discoveries of Alzheimer's disease are the drivers of our understanding, and together with polygenetic risk stratification can contribute towards planning of feasible and efficient preventive and curative clinical trials. We first perform a large genetic association study by merging all available case-control datasets and by-proxy study results (discovery n = 409,435 and validation size n = 58,190). Here, we add six variants associated with Alzheimer's disease risk (near APP, CHRNE, PRKD3/NDUFAF7, PLCG2 and two exonic variants in the SHARPIN gene). Assessment of the polygenic risk score and stratifying by APOE reveal a 4 to 5.5 years difference in median age at onset of Alzheimer's disease patients in APOE ɛ4 carriers. Because of this study, the underlying mechanisms of APP can be studied to refine the amyloid cascade and the polygenic risk score provides a tool to select individuals at high risk of Alzheimer's disease.

The IPDGC (The International Parkinson Disease Genomics Consortium) and EADB (Alzheimer Disease European DNA biobank) are listed correctly as an author to the article, however, they were incorrectly listed more than once.

The genetic variant rs72824905-G (minor allele) in the PLCG2 gene was previously associated with a reduced Alzheimer's disease risk (AD). The role of PLCG2 in immune system signaling suggests it may also protect against other neurodegenerative diseases and possibly associates with longevity. We studied the effect of the rs72824905-G on seven neurodegenerative diseases and longevity, using 53,627 patients, 3,516 long-lived individuals and 149,290 study-matched controls. We replicated the association of rs72824905-G with reduced AD risk and we found an association with reduced risk of dementia with Lewy bodies (DLB) and frontotemporal dementia (FTD). We did not find evidence for an effect on Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS) risks, despite adequate sample sizes. Conversely, the rs72824905-G allele was associated with increased likelihood of longevity. By-proxy analyses in the UK Biobank supported the associations with both dementia and longevity. Concluding, rs72824905-G has a protective effect against multiple neurodegenerative diseases indicating shared aspects of disease etiology. Our findings merit studying the PLCγ2 pathway as drug-target.

Rare coding variants in TREM2, PLCG2, and ABI3 were recently associated with the susceptibility to Alzheimer's disease (AD) in Caucasians. Frequencies and AD-associated effects of variants differ across ethnicities. To start filling the gap on AD genetics in South America and assess the impact of these variants across ethnicity, we studied these variants in Argentinian population in association with ancestry. TREM2 (rs143332484 and rs75932628), PLCG2 (rs72824905), and ABI3 (rs616338) were genotyped in 419 AD cases and 486 controls. Meta-analysis with European population was performed. Ancestry was estimated from genome-wide genotyping results. All variants show similar frequencies and odds ratios to those previously reported. Their association with AD reach statistical significance by meta-analysis. Although the Argentinian population is an admixture, variant carriers presented mainly Caucasian ancestry. Rare coding variants in TREM2, PLCG2, and ABI3 also modulate susceptibility to AD in populations from Argentina, and they may have a European heritage.

The specific roles of Notch in progressive adulthood neurodegenerative disorders have begun to be unraveled in recent years. A number of independent studies have shown significant increases of Notch expression in brains from patients at later stages of sporadic Alzheimer's disease (AD). However, the impact of Notch canonical signaling activation in the pathophysiology of AD is still elusive. To further investigate this issue, 2-month-old wild-type (WT) and hemizygous McGill-R-Thy1-APP rats (Tg(+/-)) were injected in CA1 with lentiviral particles (LVP) expressing the transcriptionally active fragment of Notch, known as Notch Intracellular Domain (NICD), (LVP-NICD), or control lentivirus particles (LVP-C). The Tg(+/-) rat model captures presymptomatic aspects of the AD pathology, including intraneuronal amyloid beta (Aβ) accumulation and early cognitive deficits. Seven months after LVP administration, Morris water maze test was performed, and brains isolated for biochemical and histological analysis. Our results showed a learning impairment and a worsening of spatial memory in LVP-NICD- as compared to LVP-C-injected Tg(+/-) rats. In addition, immuno histochemistry, ELISA multiplex, Western blot, RT-qPCR, and 1 H-NMR spectrometry of cerebrospinal fluid (CSF) indicated that chronic expression of NICD promoted hippocampal vessel thickening with accumulation of Aβ in brain microvasculature, alteration of blood-brain barrier (BBB) permeability, and a decrease of CSF glucose levels. These findings suggest that, in the presence of early Aβ pathology, expression of NICD may contribute to the development of microvascular abnormalities, altering glucose transport at the BBB with impact on early decline of spatial learning and memory.

Presenilin 2 gene (PSEN2) mutations account for <5% of all early-onset familial Alzheimer's disease (EOFAD) cases and only 13 have strong evidence for pathogenicity. We aimed to investigate the presence of PSEN2 mutation p.N141I and characterize the clinical phenotypes in 2 Argentine pedigrees (AR2 and AR3) with clinical symptoms of EOFAD. Detailed clinical assessments and genetic screening for PSEN2 and APOE genes were carried out in 19 individuals of AR2 and AR3 families. The p.N141I mutation was identified in all affected subjects and was associated with prominent early onset, rapidly progressive dementia, neurologic, and behavioral symptoms. AR2 and AR3 families share the same Volga German ancestry as all the families reported presenting this mutation. To our knowledge, this is the first report of PSEN2 mutation p.N141I in Argentina and even more, in South America. Our contribution increases the total number of described families carrying this mutation and help to improve the characterization of clinical phenotype in EOFAD associated to PSEN2 mutations., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Histone H2Ba of Toxoplasma gondii was expressed as recombinant protein (rH2Ba) and used to generate antibody in mouse that is highly specific. Antibody recognizing rH2Ba detects a single band in tachyzoite lysate of the expected molecular weight (12kDa). By indirect immunofluorescence (IFA) in in vitro grown tachyzoites and bradyzoites, the signal was detected only in the parasite nucleus. The nucleosome composition of H2Ba was determined through co-immunoprecipitation assays. H2Ba was detected in the same immunocomplex as H2A.X, but not with H2A.Z. Through chromatin immunoprecipitation (ChIP) assays and qPCR, it was observed that H2Ba is preferentially located at promoters of inactive genes and silent regions, accompanying H2A.X and opposed to H2A.Z/H2B.Z dimers., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Chromosome ends are composed of telomeric repeats and subtelomeric regions, which are patchworks of genes interspersed with repeated elements. Although chromosome ends display similar arrangements in different species, their sequences are highly divergent. In addition, these regions display a particular nucleosomal composition and bind specific factors, therefore producing a special kind of heterochromatin. Using data from currently available draft genomes we have characterized these putative Telomeric Associated Sequences in Toxoplasma gondii., Results: An all-vs-all pairwise comparison of T. gondii assembled chromosomes revealed the presence of conserved regions of ∼ 30 Kb located near the ends of 9 of the 14 chromosomes of the genome of the ME49 strain. Sequence similarity among these regions is ∼ 70%, and they are also highly conserved in the GT1 and VEG strains. However, they are unique to Toxoplasma with no detectable similarity in other Apicomplexan parasites. The internal structure of these sequences consists of 3 repetitive regions separated by high-complexity sequences without annotated genes, except for a gene from the Toxoplasma Specific Family. ChIP-qPCR experiments showed that nucleosomes associated to these sequences are enriched in histone H4 monomethylated at K20 (H4K20me1), and the histone variant H2A.X, suggesting that they are silenced sequences (heterochromatin). A detailed characterization of the base composition of these sequences, led us to identify a strong long-range compositional bias, which was similar to that observed in other genomic silenced fragments such as those containing centromeric sequences, and was negatively correlated to gene density., Conclusions: We identified and characterized a region present in most Toxoplasma assembled chromosomes. Based on their location, sequence features, and nucleosomal markers we propose that these might be part of subtelomeric regions of T. gondii. The identified regions display a unique trinucleotide compositional bias, which is shared (despite the lack of any detectable sequence similarity) with other silenced sequences, such as those making up the chromosome centromeres. We also identified other genomic regions with this compositional bias (but no detectable sequence similarity) that might be functionally similar.

Toxoplasma gondii is an apicomplexan intracellular protozoan parasite responsible for toxoplasmosis, a disease with considerable medical and economic impact worldwide. Toxoplasma gondii cells never lose the nuclear envelope and their chromosomes do not condense. Here, we tested the murine monoclonal antibody PL2-6, which labels epichromatin (a conformational chromatin epitope based on histones H2A and H2B complexed with DNA), in T. gondii cultured in human fibroblasts. This epitope is present at the exterior chromatin surface of interphase nuclei and on the periphery of mitotic chromosomes in higher eukaryotes. PL2-6 reacted with T. gondii H2A and H2B histones in Western blot (WB) assays. In addition, the antibody reacted with the nuclear fraction of tachyzoites, as a single band coincident with H2B histone. In the T. gondii tachyzoite stage, PL2-6 also had peripheral nuclear localization, as observed by epifluorescence/confocal microscopy and immunoelectron microscopy. Confocal analysis showed that epichromatin is slightly polarized to one face of the parasite exterior chromatin surface. In replicating tachyzoites, PL2-6 also labels the exterior chromatin surface, covering the face of both segregating nuclei, facing the plasma membrane of the mother cell. The possible role of epichromatin in T. gondii is discussed.

Toxoplasma gondii is a leading cause of neurological birth defects and a serious opportunistic pathogen. The authors and others have found that Toxoplasma uses a unique nucleosome composition supporting a fine gene regulation together with other factors. Post-translational modifications in histones facilitate the establishment of a global chromatin environment and orchestrate DNA-related biological processes. Histone acetylation is one of the most prominent post-translational modifications influencing gene expression. Histone acetyltransferases and histone deacetylases have been intensively studied as potential drug targets. In particular, histone deacetylase inhibitors have activity against apicomplexan parasites, underscoring their potential as a new class of antiparasitic compounds. In this review, we summarize what is known about Toxoplasma histone acetyltransferases and histone deacetylases, and discuss the inhibitors studied to date. Finally, the authors discuss the distinct possibility that the unique nucleosome composition of Toxoplasma, which harbors a nonconserved H2Bv variant histone, might be targeted in novel therapeutics directed against this parasite.

Protozoan parasites have tremendously diverse lifestyles that require adaptation to a remarkable assortment of different environmental conditions. In order to complete their life cycles, protozoan parasites rely on fine-tuning gene expression. In general, protozoa use novel regulatory elements, transcription factors, and epigenetic mechanisms to regulate their transcriptomes. One of the most surprising findings includes the nature of their histones--these primitive eukaryotes lack some histones yet harbor novel histone variants of unknown function. In this review, we describe the histone components of different protozoan parasites based on literature and database searching. We summarize the key discoveries regarding histones and histone variants and their impact on chromatin regulation in protozoan parasites. In addition, we list histone genes IDs, sequences, and genomic localization of several protozoan parasites and Microsporidia histones, obtained from a thorough search of genome databases. We then compare these findings with those observed in higher eukaryotes, allowing us to highlight some novel aspects of epigenetic regulation in protists and to propose questions to be addressed in the upcoming years.

Toxoplasma gondii is among the most successful parasites, with nearly half of the human population chronically infected. Recently a link between the T. gondii Hsp90 chaperone machinery and parasite development was observed. Here, the T. gondii Hsp90 co-chaperones p23 and Hip were identified mining the Toxoplasma- database (www.toxodb.org). Their identity was confirmed by domain structure and blast analysis. Additionally, analysis of the secondary structure and studies on the chaperone function of the purified protein verified the p23 identity. Studies of co-immunoprecipitation (co-IP) identified two different types of complexes, one comprising at least Hip-Hsp70-Hsp90 and another containing at least p23-Hsp90. Indirect immunofluorescence assays showed that Hip is localized in the cytoplasm in tachyzoites and as well in bradyzoites. For p23 in contrast, a solely cytoplasmic localization was only observed in the tachyzoite stage whereas nuclear and cytosolic distribution and co-localization with Hsp90 was observed in bradyzoites. These results indicate that the T. gondii Hsp90-heterocomplex cycle is similar to the one proposed for higher eukaryotes, further highlighting the implication of the Hsp90/p23 in parasite development. Furthermore, co-IP experiments of tachyzoite/bradyzoite lysates with anti-p23 antiserum and identification of the complexed proteins together with the use of the curated interaction data available from different source (orthologs and Plasmodium databases) allowed us to construct an interaction network (interactome) covering the dynamics of the Hsp90 chaperone machinery.

Toxoplasma gondii is an obligate intracellular parasite. Toxoplasmosis is incurable because of its ability to differentiate from the rapidly replicating tachyzoite stage into a latent cyst form (bradyzoite stage). Gene regulation pertinent to Toxoplasma differentiation involves histone modification, but very little is known about the histone proteins in this early branching eukaryote. Here, we report the characterization of three H2A histones, variants H2AX and H2AZ, and a canonical H2A1. H2AZ is the minor parasite H2A member. H2A1 and H2AX both have an SQ motif, but only H2AX has a complete SQ(E/D)varphi (where varphi denotes a hydrophobic residue) known to be phosphorylated in response to DNA damage. We show that a novel H2B variant interacts with H2AZ and H2A1 but not with H2AX. Chromatin immunoprecipitation (ChIP) revealed that H2AZ and H2Bv are enriched at active genes while H2AX is enriched at repressed genes as well as the silent TgIRE repeat element. During DNA damage, we detected an increase in H2AX phosphorylation as well as increases in h2a1 and h2ax transcription. We found that expression of h2ax, but not h2a1 or h2az, increases in bradyzoites generated in vitro. Similar analysis performed on mature bradyzoites generated in vivo, which are arrested in G0, showed that h2az and h2ax are expressed but h2a1 is not, consistent with the idea that h2a1 is the canonical histone orthologue in the parasite. The increase of H2AX, which localizes to silenced areas during bradyzoite differentiation, is consistent with the quiescent nature of this stage of the life cycle. Our results indicate that the early-branching eukaryotic parasite Toxoplasma contains nucleosomes of novel composition, which is likely to impact multiple facets of parasite biology, including the clinically important process of bradyzoite differentiation.

• Polyamine biosynthesis inhibitors were used to study polyamine metabolism during the germination of Sclerotinia sclerotiorum ascospores, and to evaluate the potential of polyamine biosynthesis inhibition for the control of ascospore-borne diseases in plants. • The effects of inhibitors on ascospore germination, free polyamine levels, ornithine decarboxylase activity and development of disease symptoms on tobacco (Nicotiana tabacum) leaf discs inoculated with ascospores were determined. • α-Difluoromethylornithine inhibited ornithine decarboxylase and decreased free spermidine levels, but had no effect on ascospore germination. Both, the spermidine synthase inhibitor cyclohexylamine and the S-adenosyl-methionine decarboxylase inhibitor methylglyoxal bis-[guanyl hydrazone] decreased free spermidine levels, but only the latter inhibited ascospore germination, at concentrations of 5 mm or higher. Lesion development on leaf discs was reduced by cyclohexylamine and methylglyoxal bis-[guanyl hydrazone], but not by α-difluoromethylornithine. In the absence of inhibitors, dormant ascospores contained higher polyamine levels than mycelium. • Ascospore germination did not depend on ornithine decarboxylase activity and inhibitors of this enzyme will probably have a limited potential for the control of ascospore-borne plant diseases. On the contrary, spermidine synthase and S-adenosyl-methionine decarboxylase could be more suitable targets for fungicidal action. The relative insensitivity of ascospore germination to polyamine biosynthesis inhibitors may be caused by their high polyamine content.

The effects of the putrescine analogue 1-aminooxy-3-aminopropane on fungal polyamine metabolism were evaluated using Sclerotinia sclerotiorum as an experimental model. The compound inhibited ornithine decarboxylase, spermidine synthase, and S -adenosyl-methionine decarboxylase in mycelial extracts. Addition of 1-aminooxy-3-aminopropane at 1 mM to the culture medium did not reduce mycelial growth and caused a 29% decrease in free spermidine and a two-fold increase in free spermine. When added 4.5 h before the determination of ornithine decarboxylase, 1-aminooxy-3-aminopropane reduced in vivo activity of this enzyme by 40-50%. When added 48 h before the determination, 1-aminooxy-3-aminopropane at 0.01 and 0.1 mM caused a slight increase of in vivo ornithine decarboxylase activity, while it had no effect at 1 mM. Comparison of the action of 1-aminooxy-3-aminopropane with that of other inhibitors of polyamine biosynthesis suggested that its effects on in vivo ornithine decarboxylase activity resulted from a balance between direct inhibition of enzyme activity and indirect stimulation of enzyme synthesis and/or activity mediated by the decrease in spermidine levels, which in turn was due to inhibition of spermidine synthase and S -adenosyl-methionine decarboxylase. The potential of 1-aminooxy-3-aminopropane as a tool for studies on fungal polyamine metabolism and for the control of plant diseases of fungal origin is discussed.