Engineered Fluorescent Strains of Cryptococcus neoformans: a Versatile Toolbox for Studies of Host-Pathogen Interactions and Fungal Biology, Including the Viable but Nonculturable State.

Cryptococcus neoformans is an opportunistic fungal pathogen known for its remarkable ability to infect and subvert phagocytes. This ability provides survival and persistence within the host and relies on phenotypic plasticity. The viable but nonculturable (VBNC) phenotype was recently described in C. neoformans, whose study is promising in understanding the pathophysiology of cryptococcosis. The use of fluorescent strains is improving host interaction research, but it is still underexploited. Here, we fused histone H3 or the poly(A) binding protein (Pab) to enhanced green fluorescent protein (eGFP) or mCherry, obtaining a set of C. neoformans transformants with different colors, patterns of fluorescence, and selective markers (hygromycin B resistance [Hygr] or neomycin resistance [Neor]). We validated their similarity to the parental strain in the stress response, the expression of virulence-related phenotypes, mating, virulence in Galleria mellonella, and survival within murine macrophages. PAB-GFP, the brightest transformant, was successfully applied for the analysis of phagocytosis by flow cytometry and fluorescence microscopy. Moreover, we demonstrated that an engineered fluorescent strain of C. neoformans was able to generate VBNC cells. GFP-tagged Pab1, a key regulator of the stress response, evidenced nuclear retention of Pab1 and the assembly of cytoplasmic stress granules, unveiling posttranscriptional mechanisms associated with dormant C. neoformans cells. Our results support that the PAB-GFP strain is a useful tool for research on C. neoformans. IMPORTANCE Cryptococcus neoformans is a human-pathogenic yeast that can undergo a dormant state and is responsible for over 180,000 deaths annually worldwide. We engineered a set of fluorescent transformants to aid in research on C. neoformans. A mutant with GFP-tagged Pab1 improved fluorescence-based techniques used in host interaction studies. Moreover, this mutant induced a viable but nonculturable phenotype and uncovered posttranscriptional mechanisms associated with dormant C. neoformans. The experimental use of fluorescent mutants may shed light on C. neoformans-host interactions and fungal biology, including dormant phenotypes.

Pilocarpine-induced seizures associate with modifications of LSD1/CoREST/HDAC1/2 epigenetic complex and repressive chromatin in mice hippocampus.

Epilepsy is a neurological disorder of genetic or environmental origin characterized by recurrent spontaneous seizures. A rodent model of temporal lobe epilepsy is induced by a single administration of pilocarpine, a non-selective cholinergic muscarinic receptor agonist. The molecular changes associated with pilocarpine-induced seizures are still poorly described. Epigenetic multiprotein complexes that regulate gene expression by changing the structure of chromatin impose transcriptional memories. Among the epigenetic enzymes relevant to the epileptogenic process is lysine-specific demethylase 1 (LSD1, KDM1A), which regulates the expression of genes that control neuronal excitability. LSD1 forms complexes with the CoREST family of transcriptional corepressors, which are molecular bridges that bring HDAC1/2 and LSD1 enzymes to deacetylate and demethylate the tail of nucleosomal histone H3. To test the hypothesis that LSD1-complexes are involved in initial modifications associated with pilocarpine-induced epilepsy, we studied the expression of main components of LSD1-complexes and the associated epigenetic marks on isolated neurons and the hippocampus of pilocarpine-treated mice. Using a single injection of 300 mg/kg of pilocarpine and after 24 h, we found that protein levels of LSD1, CoREST2, and HDAC1/2 increased, while CoREST1 decreased in the hippocampus. In addition, we observed increased histone H3 lysine 9 di- and trimethylation (H3K9me2/3) and decreased histone H3 lysine 4 di and trimethylation (H3K4me2/3). Similar findings were observed in cultured hippocampal neurons and HT-22 hippocampal cell line treated with pilocarpine. In conclusion, our data show that muscarinic receptor activation by pilocarpine induces a global repressive state of chromatin and prevalence of LSD1-CoREST2 epigenetic complexes, modifications that could underlie the pathophysiological processes leading to epilepsy.

Evolutionary Dynamics of the Repetitive DNA in the Karyotypes of Pipa carvalhoi and Xenopus tropicalis (Anura, Pipidae).

The large amphibian genomes contain numerous repetitive DNA components that have played an important role in the karyotypic diversification of this vertebrate group. Hypotheses based on the presumable primitive karyotype (2n = 20) of the anurans of the family Pipidae suggest that they have evolved principally through intrachromosomal rearrangements. Pipa is the only South American pipid, while all the other genera are found in Africa. The divergence of the South American lineages from the African ones occurred at least 136 million years ago and is thought to have had a strong biogeographic component. Here, we tested the potential of the repetitive DNA to enable a better understanding of the differentiation of the karyotype among the family Pipidae and to expand our capacity to interpret the chromosomal evolution in this frog family. Our results indicate a long history of conservation in the chromosome bearing the H3 histone locus, corroborating inferences on the chromosomal homologies between the species in pairs 6, 8, and 9. The chromosomal distribution of the microsatellite motifs also provides useful markers for comparative genomics at the chromosome level between Pipa carvalhoi and Xenopus tropicalis, contributing new insights into the evolution of the karyotypes of these species. We detected similar patterns in the distribution and abundance of the microsatellite arrangements, which reflect the shared organization in the terminal/subterminal region of the chromosomes between these two species. By contrast, the microsatellite probes detected a differential arrangement of the repetitive DNA among the chromosomes of the two species, allowing longitudinal differentiation of pairs that are identical in size and morphology, such as pairs 1, 2, 4, and 5. We also found evidence of the distinctive composition of the repetitive motifs of the centromeric region between the species analyzed in the present study, with a clear enrichment of the (CA) and (GA) microsatellite motifs in P. carvalhoi. Finally, microsatellite enrichment in the pericentromeric region of chromosome pairs 6, 8, and 9 in the P. carvalhoi karyotype, together with interstitial telomeric sequences (ITS), validate the hypothesis that pericentromeric inversions occurred during the chromosomal evolution of P. carvalhoi and reinforce the role of the repetitive DNA in the remodeling of the karyotype architecture of the Pipidae.

JMJD1B, a novel player in histone H3 and H4 processing to ensure genome stability.

BACKGROUND: Maintaining a proper supply of soluble histones throughout the cell cycle is important to ensure chromatin and genome stability. Following their synthesis, histones undergo a series of maturation steps to prepare them for deposition onto chromatin. RESULTS: Here, we identify the lysine demethylase JMJD1B as a novel player in the maturation cascade that contributes to regulate histone provision. We find that depletion of JMJD1B increases the protein levels of the histone chaperone tNASP leading to an accumulation of newly synthesized histones H3 and H4 at early steps of the histone maturation cascade, which perturbs chromatin assembly. Furthermore, we find a high rate of JMJD1B mutations in cancer patients, and a correlation with genomic instability. CONCLUSIONS: Our data support a role for JMJD1B in fine-tuning histone supply to maintain genome integrity, opening novel avenues for cancer therapeutics.

Perinatal exposure to bisphenol A impacts in the mammary gland morphology of adult Mongolian gerbils.

The endocrine disruptive effects caused by bisphenol A (BPA) are well known. Despite this, to date, evaluation of its long term effects is limited, meaning that there is still much to be unveiled in terms of alterations caused by perinatal exposure to BPA. Our aim was to determine if perinatal exposure to two different doses of BPA causes long term morphological and molecular alteration effects in the mammary gland (MG). We evaluated MG from Mongolian gerbil offspring exposed perinatally (during gestation and lactation) to 50 or 5000 µg/kg/day BPA. At 90 days of age the animals were subjected to a single dose of N-nitroso-N-methylurea in order to mimic a carcinogenic environment. At 6 months of age, animals in estrous were euthanized for morphological evaluation of the MGs. The MG architecture presented considerable changes in terms of detached epithelial cells, inflammation, glandular hyperplasia, and collagen fiber deposition. Furthermore, a higher index of epithelial cell proliferation was detected in comparison to the intact control group. In addition, we verified a higher molecular expression of EZH2 in the vehicle treated group, indicating that corn oil applied alone can alter the expression of this epigenetic biomarker. In conclusion, BPA perinatal exposure promotes significant changes in glandular cytoarchitecture and increases glandular epithelium proliferation rate, leading to the retention of stem-like properties. This event could compromise the fate and differentiation potential of mammary epithelium.

Isoflavonoids from Brazilian red propolis down-regulate the expression of cancer-related target proteins: A pharmacogenomic analysis.

Vestitol and neovestitol are bioactive isoflavonoids isolated from Brazilian red propolis, a unique Apis melifera type of propolis botanically originated from Dalbergia ecastophyllum. Although these molecules have relevant biological effects, including anticancer and immunomodulatory activities, their mechanism(s) of action and the affected pathways remain largely unknown. Here, we carried out a pharmacogenomic analysis to investigate the effects of vestitol and neovestitol on the whole-genome expression in human tumor cells, particularly cancer-related target proteins. HeLa cells were exposed to the compounds at IC20 and genomic information of treated cells was analyzed using the Illumina transcriptome system and GeneGo MetaCore software. Our results showed that vestitol (IC20  = 214.7 µM) reduced the expression of genes enrolled with the alpha tubulin (fold -3.7), tubulin in microtubules (fold -3.7), and histone h3 (fold = -3.03), and that treatment with neovestitol (IC20  = 102.91 µM) downregulated prostaglandin E synthase gene (fold = -3.12), which are considered ideal targets for anticancer therapy. These data open avenues for the study of vestitol and neovestitol as potential promising candidates for anticancer therapy. Toxicological, non-clinical, and clinical validation of the findings presented herein is needed.

Extraction of DNA from micro-tissue for bat species identification.

Bat populations are declining worldwide. Accurate identification is essential to promote species' conservation. However, minimal morphological differences and a high rate of cryptic species make identification difficult, unless voucher specimens are kept, a controversial issue today. The objective of this work was to standardize a method of extracting non-lethal DNA using bats' uropatagium micro-tissue, aiming the molecular identification of species that occur in the region of Maringá PR. The method standardized was efficient, and does not cause serious damage to bats. For future field studies, collection of micro-tissue and morphometry of the specimens will be sufficient for accurate identification.

Supplementation with Brazil nuts and green tea extract regulates targeted biomarkers related to colorectal cancer risk in humans.

Se and green tea have been shown in epidemiological, observational and preclinical studies to be inversely related to the risk of developing colorectal cancer (CRC). However, there are limited studies to evaluate their regulatory effects on genes/proteins that relate to CRC oncogenesis in human subjects, such as selenoproteins, WNT signalling pathway, inflammation and methylation. This study examined the effects of supplementation of Se using Brazil nuts and green tea extract (GTE) capsules, alone and in combination, on targeted biomarkers. In total, thirty-two volunteers (>50 years of age) with plasma Se≤1·36 µmol/l were randomised to one of three treatment groups: nine to Se (approximately 48 µg/d) as six Brazil nuts, eleven to four GTE capsules (800 mg (-)-epigallocatechin-3-gallate) and twelve to a combination of Brazil nuts and GTE. Blood and rectal biopsies were obtained before and after each intervention. Plasma Se levels, rectal selenoprotein P (SePP) and ß-catenin mRNA increased significantly in subjects consuming Brazil nuts alone or in combination, whereas rectal DNA methyltransferase (DNMT1) and NF-κB mRNA were reduced significantly in subjects consuming GTE alone or in combination. None of the interventions significantly affected rectal acetylated histone H3 or Ki-67 expression at the protein level or plasma C-reactive protein. Effects of the combination of Brazil nuts and GTE did not differ from what would be expected from either agent alone. In conclusion, supplementation of Brazil nuts and/or GTE regulates targeted biomarkers related to CRC oncogenesis, specifically genes associated with selenoproteins (SePP), WNT signalling (ß-catenin), inflammation (NF-κB) and methylation (DNMT1). Their combination does not appear to provide additional effects compared with either agent alone.

cAMP-Induced Histones H3 Dephosphorylation Is Independent of PKA and MAP Kinase Activations and Correlates With mTOR Inactivation.

cAMP is a second messenger well documented to be involved in the phosphorylation of PKA, MAP kinase, and histone H3 (H3). Early, we reported that cAMP also induced H3 dephosphorylation in a variety of proliferating cell lines. Herein, it is shown that cAMP elicits a biphasic H3 dephosphorylation independent of PKA activation in cycling cells. H89, a potent inhibitor of PKA catalytic sub-unite, could not abolish this effect. Additionally, H89 induces a rapid and biphasic H3 serine 10 dephosphorylation, while a decline in the basal phosphorylation of CREB/ATF-1 is observed. Rp-cAMPS, an analog of cAMP and specific inhibitor of PKA, is unable to suppress cAMP-mediated H3 dephosphorylation, whereas Rp-cAMPS effectively blocks CREB/ATF-1 hyper-phosphorylation by cAMP and its inducers. Interestingly, cAMP exerts a rapid and profound H3 dephosphorylation at much lower concentration (50-fold lower, 0.125 mM) than the concentration required for maximal CREB/ATF-1 phosphorylation (5 mM). Much higher cAMP concentration is required to fully induce CREB/ATF-1 gain in phosphate (5 mM), which correlates with the inhibition of H3 dephosphorylation. Also, the dephosphorylation of H3 does not overlap at onset of MAP kinase phosphorylation pathways, p38 and ERK. Surprisingly, rapamycin (an mTOR inhibitor), cAMP, and its natural inducer isoproterenol, elicit identical dephosphorylation kinetics on both S6K1 ribosomal kinase (a downstream mTOR target) and H3. Finally, cAMP-induced H3 dephosphorylation is PP1/2-dependent. The results suggest that a pathway, requiring much lower cAMP concentration to that required for CREB/ATF-1 hyper-phosphorylation, is responsible for histone H3 dephosphorylation and may be linked to mTOR down regulation.

Spreading of heterochromatin and karyotype differentiation in two Tropidacris Scudder, 1869 species (Orthoptera, Romaleidae).

Tropidacris Scudder, 1869 is a genus widely distributed throughout the Neotropical region where speciation was probably promoted by forest reduction during the glacial and interglacial periods. There are no cytogenetic studies of Tropidacris, and information allowing inference or confirmation of the evolutionary events involved in speciation within the group is insufficient. In this paper, we used cytogenetic markers in two species, Tropidacriscollaris (Stoll, 1813) and Tropidacriscristatagrandis (Thunberg, 1824), collected in different Brazilian biomes. Both species exhibited 2n=24,XX for females and 2n=23,X0 for males. All chromosomes were acrocentric. There were some differences in the karyotype macrostructure, e.g. in the chromosome size. A wide interspecific variation in the chromosome banding (C-banding and CMA3/DAPI staining) indicated strong differences in the distribution of repetitive DNA sequences. Specifically, Tropidacriscristatagrandis had a higher number of bands in relation to Tropidacriscollaris. FISH with 18S rDNA revealed two markings coinciding with the NORs in both species. However, two analyzed samples of Tropidacriscollaris revealed a heterozygous condition for the rDNA site of S10 pair. In Tropidacriscollaris, the histone H3 genes were distributed on three chromosome pairs, whereas in Tropidacriscristatagrandis, these genes were observed on 14 autosomes and on the X chromosome, always in terminal regions. Our results demonstrate that, although the chromosome number and morphology are conserved in the genus, Tropidacriscristatagrandis substantially differs from Tropidacriscollaris in terms of the distribution of repetitive sequences. The devastation and fragmentation of the Brazilian rainforest may have led to isolation between these species, and the spreading of these repetitive sequences could contribute to speciation within the genus.