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Momordica charantia L. (Cucurbitaceae), popularly known as "bitter melon" or "bitter gourd," is a climbing plant well-adapted to tropical countries. This plant is used traditionally to treat several conditions including diabetes mellitus, inflammation, liver dysfunctions, and cancer. Given the widespread ethnopharmacological use, this study aimed to examine the cytogenetic, maternal, and developmental toxicity attributed to exposure to dry extract of M. charantia leaves using Allium cepa and Wistar rats as test models. First, phytochemical characterization of the dry extract by high performance liquid chromatography (HPLC) analyses was performed. Then, Allium cepa roots were exposed to three different concentrations of the dry extract (0.25, 0.5, or 1 mg/ml) to determine the mitotic index, frequency of chromosomal aberrations, and nuclear abnormalities. In addition, pregnant Wistar rats were administered either 500; 1,000 or 2,000 mg/kg dry extract during the gestational period (GD) days 6-15, and subsequently possible toxic effect on the dams and fetuses were recorded. HPLC analyses confirmed rutin as the main secondary metabolite present in the dry extract. In the Allium cepa test, the dry extract was cytotoxic. In Wistar rats, dry extract administration reduced water and feed intake and mean body mass gain, indicating maternal toxicity during the organogenesis period. However, the dry extract did not markedly affect reproductive outcome parameters evaluated. Regarding developmental toxicity assessment, the dry extract treatment did not significantly alter number of skeletal malformations in the offspring. Data demonstrated that the dry extract of M. charantia leaves presents cytotoxicity and low maternal toxicity, indicating indiscriminate use needs to be avoided.

Coumarins and chalcones are compounds widely found in plants or obtained by synthetic methods which possess several biological properties including antioxidant, anti-inflammatory, and antitumor effects. A series of coumarin-chalcone hybrids were synthesized to improve their biological actions and reduce potential adverse effects. Considering the applications of these molecules, a coumarin-chalcone hybrid [7-methoxy-3-(E)-3-(3,4,5-trimethoxyphenyl) acryloyl-2 H-chromen-2-one] (4-MET) was synthesized and the genotoxic, cytotoxic, and protective effects assessed against damage induced by different mutagens. First, in silico tools were used to predict biological activity of 4-MET which indicated a chemopreventive potential. Subsequently, the genotoxic/antigenotoxic activities of 4-MET were determined both in vitro (Ames test) and in vivo (micronucleus (MN) test and comet assay). In addition, molecular docking simulations were performed between 4-MET and glutathione reductase, an important cellular detoxifying enzyme. Our results indicated that 4-MET was not mutagenic in the Ames test; however, when co-treated with sodium azide or 4-nitroquinoline 1-oxide (4-NQO), 4-MET significantly reduced the harmful actions of these mutagens. Except for a cytotoxic effect after 120 hr treatment, 4-MET alone did not produce cytotoxicity or genotoxicity in the MN test and comet assay. Nonetheless, all treatments of 4-MET with cyclophosphamide (CPA) showed a chemoprotective effect against DNA damage induced by CPA. Further, molecular docking analysis indicated a strong interaction between 4-MET and the catalytic site of glutathione reductase. These effects may be related to (1) damage prevention, (2) interaction with detoxifying enzymes, and (3) DNA-repair induction. Therefore, data demonstrated that 4-MET presents a favorable profile to be used in chemopreventive therapies.

Pedunculagin (PD), an ellagitannin found in different plant species, possesses several pharmaceutical properties, including antitumor, antioxidant, gastroprotective, hepatoprotective, and anti-inflammatory properties. However, the effects of PD alone on DNA remain to be determined. The aim of this study was to investigate the potential cytotoxic, genotoxic, and antigenotoxic activities of PD isolated from Plinia cauliflora seeds using in silico and in vitro assays. To elucidate the biological activities of PD, in silico tools indicative of antioxidant, antineoplastic, and chemopreventive activities of PD were used. Subsequently, the mutagenic/antimutagenic effects of PD were later assessed using bacteria with the Ames test, and the cytotoxic, genotoxic, and antigenotoxic effects utilizing human lymphocytes as evidenced by trypan blue exclusion test and CometChip assay. In silico analysis indicated potential antioxidant, chemopreventive, free radical scavenger, and cytostatic activities of PD. In the Ames test, PD was found to be not mutagenic; however, this plant component protected DNA against damage-mediated by mutagens 4-nitroquinoline-1-oxide and sodium azide. Regarding human lymphocytes, PD alone was cytotoxic and genotoxic; however, it also reduced DNA damage induced by doxorubicin at co- and post-treatment. In conclusion, PD showed genotoxic, antigenotoxic and cytotoxic effects in human lymphocytes and antimutagenic effects in bacteria.

Vernonanthura polyanthes , popularly known as 'assa-peixe', is widely used in Brazil for therapeutic purpose mainly to treat respiratory tract problems. However, few studies investigated its chemical safety. In this way, we first obtained the V. polyanthes leaf aqueous extract ( Vp LAE) and three fractions (aqueous; n-butanol, n-BF; and ethyl acetate), and we chemically characterized this material. Then, the cytogenotoxic potential of the Vp LAE and its fractions was investigated against human erythrocytes and lymphocytes using Trypan blue exclusion test of cell viability and CometChip. The phytochemical screening of V. polyanthes leaf revealed the presence of total phenolic compounds, flavonoids, tannins, coumarins, terpenic compounds, and cardioactive heterosides. n-BF presented the highest total phenolic, flavonoids, and tannins contents and, consequently, the highest antioxidant activity, according to the DPPH free radical scavenging method. Although the Vp LAE and its fractions did not cause death of erythrocytes, the cells acquired an echinocytic form. Regarding lymphocytes, Vp LAE and its fractions presented cytotoxicity and genotoxicity. When Vp LAE or its fractions were co-treated with doxorubicin (DXR), a recognized cytotoxic drug, we observed an enhancement of DXR cytotoxicity against lymphocytes, but the DXR genotoxicity decreased around 15%. Since the Vp LAE and its fractions increased the DXR cytotoxicity and decreased its genotoxicity, further studies should be conducted for the development of an adjuvant drug from this extract to reduce the side effects of chemotherapy. Moreover, the indiscriminate use of 'assa-peixe' by local people should be discouraged.

BP-3 is one of the most used organic UV filters. However, its widespread use and release into aquatic environment can induce ecotoxicological impact on aquatic organisms. Thus, the aim of the current study is to evaluate the gills and liver of freshwater fish Poecilia reticulata subjected to acute exposure (96 h) to BP-3 at environmentally relevant concentrations (10-1000 ng L -1 ). The study was based on adopting qualitative and semi-quantitative approach to assess histopathological changes and integrated the biomarker response in order to investigate organ-specific responses to BP-3 exposure. BP-3 has induced high histopathological index associated with circulatory disturbances, as well as with regressive and immunological changes in gills, whereas the hepatic histopathological index was associated with circulatory disturbances. Moreover, lower BP-3 concentrations were mostly associated with changes in gills, whereas higher BP-3 concentration was mostly linked to hepatic changes. In conclusion, acute exposure to BP-3 at environmentally relevant concentrations had stronger impact on gills than on the liver of P. reticulata, which confirmed organ-specific responses to UV filters., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Silymarin (SM), a standardized extract derived from Silybum marianum (L.) Gaertn, is primarily composed of flavonolignans, with silibinin (SB) as its major active constituent. The present study aimed to evaluate the antigenotoxic activities of SM and SB using the alkaline comet assay in whole blood cells and to assess their effects on the expression of genes associated with carcinogenesis and chemopreventive processes. Different concentrations of SM or SB (1.0, 2.5, 5.0, and 7.5 mg/ml) were used in combination with the DNA damage-inducing agent methyl methanesulfonate (MMS, 800 μ M) to evaluate their genoprotective potential. To investigate the role of SM and SB in modulating gene expression, we performed quantitative real-time PCR (qRT-PCR) analysis of five genes that are known to be involved in DNA damage, carcinogenesis, and/or chemopreventive mechanisms. Treatment with SM or SB was found to significantly reduce the genotoxicity of MMS, upregulate the expression of PTEN and BCL2 , and downregulate the expression of BAX and ABL1 . We observed no significant changes in ETV6 expression levels following treatment with SM or SB. In conclusion, both SM and SB exerted antigenotoxic activities and modulated the expression of genes related to cell protection against DNA damage.

Vernonanthura polyanthes (Spreng.) A.J. Vega & Dematt. (Asteraceae), known as "assa-peixe", has been used in ethnomedicine for the treatment of various diseases such as bronchitis, pneumonia, hemoptysis, persistent cough, internal abscesses, gastric and kidney stone pain. Moreover, some studies demonstrated that species of Genus Vernonia present antifungal activity. Due to the biological relevance of this species, the aim of this study was to investigate the toxic, genotoxic, antigenotoxic and antifungal potential of V. polyanthes leaves aqueous extract in somatic cells of Drosophila melanogaster or against Candida spp. The aqueous extract of the plant showed no toxic, genotoxic and antigenotoxic activity in the experimental conditions tested using the wing somatic mutation and recombination test (SMART/wing). However, when the extract was associated with doxorubicin, used in this work as a positive control, the mutagenic potential of doxorubicin was enhanced, increasing the number of mutations in D. melanogaster somatic cells. In the other hand, no inhibitory activity against Candida spp. was observed for V. polyanthes leaves aqueous extract using agar-well diffusion assay. More studies are necessary to reveal the components present in the V. polyanthes leaves aqueous extract that could contribute to potentiate the doxorubicin genotoxicity.

Carbonic anhydrases (CA) belong to the family of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate. In the present work, we characterized the cDNAs of four Paracoccidioides CAs (CA1, CA2, CA3, and CA4). In the presence of CO2, there was not a significant increase in fungal ca1, ca2 and ca4 gene expression. The ca1 transcript was induced during the mycelium-to-yeast transition, while ca2 and ca4 gene expression was much higher in yeast cells, when compared to mycelium and mycelium-to-yeast transition. The ca1 transcript was induced in yeast cells recovered directly from liver and spleen of infected mice, while transcripts for ca2 and ca4 were down-regulated. Recombinant CA1 (rCA1) and CA4 (rCA4), with 33 kDa and 32 kDa respectively, were obtained from bacteria. The enzymes rCA1 (β-class) and rCA4 (α-class) were characterized regarding pH, temperature, ions and amino acids addition influence. Both enzymes were stable at pHs 7.5-8.5 and temperatures of 30-35 °C. The enzymes were dramatically inhibited by Hg+2 and activated by Zn+2, while only rCA4 was stimulated by Fe2+. Among the amino acids tested (all in L configuration), arginine, lysine, tryptophan and histidine enhanced residual activity of rCA1 and rCA4.

Adaptive changes of carbohydrate and lipid metabolism induced by 7, 15, 30, 60, 90, 150 and 200 days of fasting were investigated in red tilapia (Oreochromis sp.). Plasma glucose, lactate and free fatty acids (FFA) levels, liver and muscle glycogen and total lipid contents and rates of FFA release from mesenteric adipose tissue (MAT) were measured. Plasma glucose levels showed significant differences only after 90 days of fasting, when glycemia was 34% lower (50±5mg.dL-1) than fed fish values (74±1mg.dL-1), remaining relatively constant until 200 days of fasting. The content of liver glycogen ("15%) in fed tilapia fell 40% in 7 days of food deprivation. In 60, 90 and 150 days of fasting, plasma FFA levels increased 49%, 64% and 90%, respectively, compared to fed fish values. In agreement with the increase in plasma FFA, fasting induced a clear increase in lipolytic activity of MAT incubated in vitro. Addition of isobutylmethylxanthine (cAMP-phosphodiesterase inhibitor) and isoproterenol (non selective beta adrenergic agonist) to the incubation medium induced a reduction of lipolysis in fasted fish, differently to what was observed in mammal adipose tissue. This study allowed a physiological assessment of red tilapia response to starvation.

Functional foods include any natural product that presents health-promoting effects, thereby reducing the risk of chronic diseases. Cerrado fruits are considered a source of bioactive substances, mainly phenolic compounds, making them important functional foods. Despite this, the losses of natural vegetation in the Cerrado are progressive. Hence, the knowledge propagation about the importance of the species found in Cerrado could contribute to the preservation of this biome. This review provides information about Cerrado fruits and highlights the structures and pharmacologic potential of functional compounds found in these fruits. Compounds detected in Caryocar brasiliense Camb. (pequi), Dipteryx alata Vog. (baru), Eugenia dysenterica DC. (cagaita), Eugenia uniflora L. (pitanga), Genipa americana L. (jenipapo), Hancornia speciosa Gomes (mangaba), Mauritia flexuosa L.f. (buriti), Myrciaria cauliflora (DC) Berg (jabuticaba), Psidium guajava L. (goiaba), Psidium spp. (araçá), Solanum lycocarpum St. Hill (lobeira), Spondias mombin L. (cajá), Annona crassiflora Mart. (araticum), among others are reported here.

Iron is an essential micronutrient for almost all organisms, including fungi. Usually, fungi can uptake iron through receptor-mediated internalization of a siderophore or heme, and/or reductive iron assimilation (RIA). Traditionally, the RIA pathway consists of ferric reductases (Fres), ferroxidase (Fet3) and a high-affinity iron permease (Ftr1). Paracoccidioides spp. genomes do not present an Ftr1 homolog. However, this fungus expresses zinc regulated transporter homologs (Zrts), members of the ZIP family of membrane transporters that are able in some organisms to transport zinc and iron. A 2,3,5-triphenyltetrazolium chloride (TTC)-overlay assay indicates that both Pb01 and Pb18 express a ferric reductase activity; however, (59)Fe uptake assays indicate that only in Pb18 is this activity coupled to a reductase-dependent iron uptake pathway. In addition, Zrts are up-regulated in iron deprivation, as indicated by RNAseq and qRT-PCR using Pb01 transcripts. RNAseq strategy also demonstrated that transcripts related to siderophore uptake and biosynthesis are up-regulated in iron-deprived condition. The data suggest that the fungus could use both a non-classical RIA, comprising ferric reductases and Fe/Zn permeases (Zrts), and siderophore uptake pathways under iron-limited conditions. The study of iron metabolism reveals novel surface molecules that could function as accessible targets for drugs to block iron uptake and, consequently, inhibit pathogen's proliferation.

Background: The Fungal Genome Initiative of the Broad Institute, in partnership with the Paracoccidioides research community, has recently sequenced the genome of representative isolates of this human-pathogen dimorphic fungus: Pb18 (S1), Pb03 (PS2) and Pb01. The accomplishment of future high-throughput, genome-wide, functional genomics will rely upon appropriate molecular tools and straightforward techniques to streamline the generation of stable loss-of-function phenotypes. In the past decades, RNAi has emerged as the most robust genetic technique to modulate or to suppress gene expression in diverse eukaryotes, including fungi. These molecular tools and techniques, adapted for RNAi, were up until now unavailable for P. brasiliensis., Methodology/principal Findings: In this paper, we report Agrobacterium tumefaciens mediated transformation of yeast cells for high-throughput applications with which higher transformation frequencies of 150±24 yeast cell transformants per 1×106 viable yeast cells were obtained. Our approach is based on a bifunctional selective marker fusion protein consisted of the Streptoalloteichus hindustanus bleomycin-resistance gene (Shble) and the intrinsically fluorescent monomeric protein mCherry which was codon-optimized for heterologous expression in P. brasiliensis. We also report successful GP43 gene knock-down through the expression of intron-containing hairpin RNA (ihpRNA) from a Gateway-adapted cassette (cALf) which was purpose-built for gene silencing in a high-throughput manner. Gp43 transcript levels were reduced by 73.1±22.9% with this approach., Conclusions/significance: We have a firm conviction that the genetic transformation technique and the molecular tools herein described will have a relevant contribution in future Paracoccidioides spp. functional genomics research.

Iron is a micronutrient required by almost all living organisms, including fungi. Although this metal is abundant, its bioavailability is low either in aerobic environments or within mammalian hosts. As a consequence, pathogenic microorganisms evolved high affinity iron acquisition mechanisms which include the production and uptake of siderophores. Here we investigated the utilization of these molecules by species of the Paracoccidioides genus, the causative agents of a systemic mycosis. It was demonstrated that iron starvation induces the expression of Paracoccidioides ortholog genes for siderophore biosynthesis and transport. Reversed-phase HPLC analysis revealed that the fungus produces and secretes coprogen B, which generates dimerumic acid as a breakdown product. Ferricrocin and ferrichrome C were detected in Paracoccidioides as the intracellular produced siderophores. Moreover, the fungus is also able to grow in presence of siderophores as the only iron sources, demonstrating that beyond producing, Paracoccidioides is also able to utilize siderophores for growth, including the xenosiderophore ferrioxamine. Exposure to exogenous ferrioxamine and dimerumic acid increased fungus survival during co-cultivation with macrophages indicating that these molecules play a role during host-pathogen interaction. Furthermore, cross-feeding experiments revealed that Paracoccidioides siderophores promotes growth of Aspergillus nidulans strain unable to produce these iron chelators. Together, these data denote that synthesis and utilization of siderophores is a mechanism used by Paracoccidioides to surpass iron limitation. As iron paucity is found within the host, siderophore production may be related to fungus pathogenicity.

Iron is essential for the proliferation of fungal pathogens during infection. The availability of iron is limited due to its association with host proteins. Fungal pathogens have evolved different mechanisms to acquire iron from host; however, little is known regarding how Paracoccidioides species incorporate and metabolize this ion. In this work, host iron sources that are used by Paracoccidioides spp. were investigated. Robust fungal growth in the presence of the iron-containing molecules hemin and hemoglobin was observed. Paracoccidioides spp. present hemolytic activity and have the ability to internalize a protoporphyrin ring. Using real-time PCR and nanoUPLC-MSE proteomic approaches, fungal growth in the presence of hemoglobin was shown to result in the positive regulation of transcripts that encode putative hemoglobin receptors, in addition to the induction of proteins that are required for amino acid metabolism and vacuolar protein degradation. In fact, one hemoglobin receptor ortholog, Rbt5, was identified as a surface GPI-anchored protein that recognized hemin, protoporphyrin and hemoglobin in vitro. Antisense RNA technology and Agrobacterium tumefaciens-mediated transformation were used to generate mitotically stable Pbrbt5 mutants. The knockdown strain had a lower survival inside macrophages and in mouse spleen when compared with the parental strain, which suggested that Rbt5 could act as a virulence factor. In summary, our data indicate that Paracoccidioides spp. can use hemoglobin as an iron source most likely through receptor-mediated pathways that might be relevant for pathogenic mechanisms.

The cell wall of pathogenic microbes acts as an initial barrier that is in contact with hostile environments. Several proteins are associated to the cell wall, including the glucanosyltransferases, which are attached through glycosylphosphatidylinositol anchors to the wall. Here, we characterized the Paracoccidioides beta-1,3-glucanosyltranferase ( Gel ) family of proteins that showed significant homology to proteins belonging to the GH72 family. Immunoassays demonstrated Gel1p associated with the cell wall and with the nucleus. For Gel2p, immune labeling was associated with the cell wall and cytoplasm. Genetic complementation studies in Saccharomyces cerevisiae demonstrated that Gel2p is able to participate in the maintenance of fungal cell wall integrity, as it was able to restore the lack of Gas1p activity in a gas1Δ mutant; Gel1p was not able to do the same. On the other hand, Gel1p restores telomeric silencing in a gas1Δ mutant, providing strong support that Gel1p can be involved in transcriptional silencing in Paracoccidioides. Use of the in vivo yeast two-hybrid system revealed proteins that interact with Paracoccidioides Gel proteins, supporting new insights into the function of Gel family members and suggesting that they could play other roles than those established at the fungal cell wall., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Iron, copper, and zinc are essential for all living organisms. Moreover, the homeostasis of these metals is vital to microorganisms during pathogenic interactions with a host. Most pathogens have developed specific mechanisms for the uptake of micronutrients from their hosts in order to counteract the low availability of essential ions in infected tissues. We report here an analysis of genes potentially involved in iron, copper, and zinc uptake and homeostasis in the fungal pathogens Paracoccidioides brasiliensis, Cryptococcus neoformans var. grubii, and Cryptococcus gattii. Although prior studies have identified certain aspects of metal regulation in Cryptococcus species, little is known regarding the regulation of these elements in P. brasiliensis. We also present amino acid sequences analyses of deduced proteins in order to examine possible conserved domains. The genomic data reveals, for the first time, genes associated to iron, copper, and zinc assimilation and homeostasis in P. brasiliensis. Furthermore, analyses of the three fungal species identified homologs to genes associated with high-affinity uptake systems, vacuolar and mitochondrial iron storage, copper uptake and reduction, and zinc assimilation. However, homologs to genes involved in siderophore production were only found in P. brasiliensis. Interestingly, in silico analysis of the genomes of P. brasiliensisPb01, Pb03, and Pb18 revealed significant differences in the presence and/or number of genes involved in metal homeostasis, such as in genes related to iron reduction and oxidation. The broad analyses of the genomes of P. brasiliensis, C. neoformans var. grubii, and C. gattii for genes involved in metal homeostasis provide important groundwork for numerous interesting future areas of investigation that are required in order to validate and explore the function of the identified genes and gene pathways.