Chemical and cytotoxicity profiles of 11 pink pepper (Schinus spp.) samples via non-targeted hyphenated high-performance thin-layer chromatography.

INTRODUCTION: Pink pepper is a worldwide used spice that corresponds to the berries of two species, Schinus terebinthifolia Raddi or S. molle L. (Anacardiaceae). Toxic and allergic reactions by ingestion or contact with these plants were reported, and classical in vitro studies have highlighted the cytotoxic properties of apolar extracts from the fruits. OBJECTIVES: Perform a non-targeted screening of 11 pink pepper samples for the detection and identification of individual cytotoxic substances. METHODS: After reversed-phase high-performance thin-layer chromatography (RP-HPTLC) separation of the extracts and multi-imaging (UV/Vis/FLD), cytotoxic compounds were detected by bioluminescence reduction from luciferase reporter cells (HEK 293 T-CMV-ELuc) applied directly on the adsorbent surface, followed by elution of detected cytotoxic substance into atmospheric-pressure chemical ionization high-resolution mass spectrometry (APCI-HRMS). RESULTS: Separations for mid-polar and non-polar fruit extracts demonstrated the selectivity of the method to different substance classes. One cytotoxic substance zone was tentatively assigned as moronic acid, a pentacyclic triterpenoid acid. CONCLUSION: The developed non-targeted hyphenated RP-HPTLC-UV/Vis/FLD-bioluminescent cytotoxicity bioassay-FIA-APCI-HRMS method was successfully demonstrated for cytotoxicity screening (bioprofiling) and respective cytotoxin assignment.

Bioactivity Profiling and Quantification of Gastrodin in Gastrodia elata Cultivated in the Field versus Facility via Hyphenated High-Performance Thin-Layer Chromatography.

Gastrodia elata (Orchidaceae) is native to mountainous areas of Asia and is a plant species used in traditional medicine for more than two thousand years. The species was reported to have many biological activities, such as neuroprotective, antioxidant, and anti-inflammatory activity. After many years of extensive exploitation from the wild, the plant was added to lists of endangered species. Since its desired cultivation is considered difficult, innovative cultivation methods that can reduce the costs of using new soil in each cycle and at the same time avoid contamination with pathogens and chemicals are urgently needed on large scale. In this work, five G. elata samples cultivated in a facility utilizing electron beam-treated soil were compared to two samples grown in the field concerning their chemical composition and bioactivity. Using hyphenated high-performance thin-layer chromatography (HPTLC) and multi-imaging (UV/Vis/FLD, also after derivatization), the chemical marker compound gastrodin was quantified in the seven G. elata rhizome/tuber samples, which showed differences in their contents between facility and field samples and between samples collected during different seasons. Parishin E was also found to be present. Combining HPTLC with on-surface (bio)assays, the antioxidant activity and inhibition of acetylcholinesterase as well as the absence of cytotoxicity against human cells were demonstrated and compared between samples.

PAF signaling plays a role in obesity-induced adipose tissue remodeling.

BACKGROUND/OBJECTIVES: Platelet-activating factor receptor (PAFR) activation controls adipose tissue (AT) expansion in animal models. Our objective was twofold: (i) to check whether PAFR signaling is involved in human obesity and (ii) investigate the PAF pathway role in hematopoietic or non-hematopoietic cells to control adipocyte size. MATERIALS/SUBJECTS AND METHODS: Clinical parameters and adipose tissue gene expression were evaluated in subjects with obesity. Bone marrow (BM) transplantation from wild-type (WT) or PAFR-/- mice was performed to obtain chimeric PAFR-deficient mice predominantly in hematopoietic or non-hematopoietic-derived cells. A high carbohydrate diet (HC) was used to induce AT remodeling and evaluate in which cell compartment PAFR signaling modulates it. Also, 3T3-L1 cells were treated with PAF to evaluate fat accumulation and the expression of genes related to it. RESULTS: PAFR expression in omental AT from humans with obesity was negatively correlated to different corpulence parameters and more expressed in the stromal vascular fraction than adipocytes. Total PAFR-/- increased adiposity compared with WT independent of diet-induced obesity. Differently, WT mice receiving PAFR-/--BM exhibited similar adiposity gain as WT chimeras. PAFR-/- mice receiving WT-BM showed comparable augmentation in adiposity as total PAFR-/- mice, demonstrating that PAFR signaling modulates adipose tissue expansion through non-hematopoietic cells. Indeed, the PAF treatment in 3T3-L1 adipocytes reduced fat accumulation and expression of adipogenic genes. CONCLUSIONS: Therefore, decreased PAFR signaling may favor an AT accumulation in humans and animal models. Importantly, PAFR signaling, mainly in non-hematopoietic cells, especially in adipocytes, appears to play a significant role in regulating diet-induced AT expansion.

A Trypanosoma cruzi zinc finger protein that is implicated in the control of epimastigote-specific gene expression and metacyclogenesis.

Trypanosoma cruzi has three biochemically and morphologically distinct developmental stages that are programmed to rapidly respond to environmental changes the parasite faces during its life cycle. Unlike other eukaryotes, Trypanosomatid genomes contain protein coding genes that are transcribed into polycistronic pre-mRNAs and have their expression controlled by post-transcriptional mechanisms. Transcriptome analyses comparing three stages of the T. cruzi life cycle revealed changes in gene expression that reflect the parasite adaptation to distinct environments. Several genes encoding RNA binding proteins (RBPs), known to act as key post-transcriptional regulatory factors, were also differentially expressed. We characterized one T. cruzi RBP, named TcZH3H12, which contains a zinc finger domain and is up-regulated in epimastigotes compared to trypomastigotes and amastigotes. TcZC3H12 knockout (KO) epimastigotes showed decreased growth rates and increased capacity to differentiate into metacyclic trypomastigotes. Transcriptome analyses comparing wild type and TcZC3H12 KOs revealed a TcZC3H12-dependent expression of epimastigote-specific genes such as genes encoding amino acid transporters and proteins associated with differentiation (PADs). RNA immunoprecipitation assays showed that transcripts from the PAD family interact with TcZC3H12. Taken together, these findings suggest that TcZC3H12 positively regulates the expression of genes involved in epimastigote proliferation and also acts as a negative regulator of metacyclogenesis.

Vaccinia virus dissemination requires p21-activated kinase 1.

The orthopoxvirus vaccinia virus (VACV) interacts with both actin and microtubule cytoskeletons in order to generate and spread progeny virions. Here, we present evidence demonstrating the involvement of PAK1 (p21-activated kinase 1) in the dissemination of VACV. Although PAK1 activation has previously been associated with optimal VACV entry via macropinocytosis, its absence does not affect the production of intracellular mature virions (IMVs) and extracellular enveloped virions (EEVs). Our data demonstrate that low-multiplicity infection of PAK1(-/-) MEFs leads to a reduction in plaque size followed by decreased production of both IMVs and EEVs, strongly suggesting that virus spread was impaired in the absence of PAK1. Confocal and scanning electron microscopy showed a substantial reduction in the amount of VACV-induced actin tails in PAK1(-/-) MEFs, but no significant alteration in the total amount of cell-associated enveloped virions (CEVs). Furthermore, the decreased VACV dissemination in PAK1(-/-) cells was correlated with the absence of phosphorylated ARPC1 (Thr21), a downstream target of PAK1 and a key regulatory subunit of the ARP2/3 complex, which is necessary for the formation of actin tails and viral spread. We conclude that PAK1, besides its role in virus entry, also plays a relevant role in VACV dissemination.

Cytotoxicity Screening of Sterculia striata A.St.-Hil. & Naudin (Chichá) and Arachis hypogaea L. (Peanut) and Comparative Chemical Profiles Before and After in Vitro Digestion.

This study traced the cytotoxicity, antioxidant activity, and phytochemical profile before and after in vitro digestion of nuts from Sterculia striata A. St.-Hil. & Naudin (Malvaceae) (chichá or monkey's peanut), a native plant from Brazil, in comparison with Arachis hypogaea L. (peanut). The antioxidant activity in the 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and Ferric Reducing Antioxidant Power Assay (FRAP) assays was lower in chichá when compared with peanuts, corroborating the lower concentration of polyphenols. None of the samples studied showed significant cytotoxicity in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromideDAD: diode-array detection (MTT) assays. In vitro digestion altered the phytochemical profile in both plants, increasing the concentration of rutin in fresh and roasted chichá but only in raw peanuts. In roasted peanuts, rutin was converted into quercetin. Chichá nuts have been used by the local population for centuries, and the identification of their bioactive components can be useful to promote their benefits as a functional food.

Planar bioluminescent cytotoxicity assay via genetically modified adherent human reporter cell lines, applied to authenticity screening of Saussurea costus root.

The hyphenation of high-performance thin-layer chromatography to effect-directed assays is a very straightforward way to detect individual bioactive zones, and at the same time, to investigate several samples simultaneously. The combination of the separation technique with adherent human cells applied on the same surface was recently shown to be possible. Since on-surface adherent cell assays are in their infancy, a planar bioluminescent cytotoxicity assay was developed to expand the possibilities. Human embryonic kidney (HEK) 293 or HeLa (cervical carcinoma) cells were chosen because of their fast growth rates and high rates of successful transfection, being suitable for the generation of genetically modified reporter cells. For the first time, HeLa cells were visualized on the wettable reversed phase plate surface using digital microscopy. For the generation of bioluminescent reporter cells, vectors for the expression of three luciferase enzymes of various origins were tested. The genetically modified HEK 293T-CMV-ELuc cells were the best suitable for the new planar cytotoxicity assay due to the faster growth rate, robustness, and stronger bioluminescence signal. The stable expression of luciferase under the control of a strong constitutive promoter allowed the cells to be used for the determination of the cytotoxicity of Saussurea costus root samples obtained from the market and to assess the authenticity of these samples. Any cytotoxic zone was detected as a dark zone inhibiting the cell bioluminescence. Five replicates of the dose-response curve confirmed the good assay performance and the cytotoxicity of a zone, which was assigned to costunolide and dehydrocostus lactone. By this, the proof-of-principle of the new planar bioluminescent cytotoxicity assay, which does not require expensive licensing, was successful.

Disruption of Active Trans-Sialidase Genes Impairs Egress from Mammalian Host Cells and Generates Highly Attenuated Trypanosoma cruzi Parasites.

Trans-sialidases (TS) are unusual enzymes present on the surface of Trypanosoma cruzi, the causative agent of Chagas disease. Encoded by the largest gene family in the T. cruzi genome, only few members of the TS family have catalytic activity. Active trans-sialidases (aTS) are responsible for transferring sialic acid from host glycoconjugates to mucins, also present on the parasite surface. The existence of several copies of TS genes has impaired the use of reverse genetics to study this highly polymorphic gene family. Using CRISPR-Cas9, we generated aTS knockout cell lines displaying undetectable levels of TS activity, as shown by sialylation assays and labeling with antibodies that recognize sialic acid-containing mucins. In vitro infection assays showed that disruption of aTS genes does not affect the parasite's capacity to invade cells or to escape from the parasitophorous vacuole but resulted in impaired differentiation of amastigotes into trypomastigotes and parasite egress from the cell. When inoculated into mice, aTS mutants were unable to establish infection even in the highly susceptible gamma interferon (IFN-γ) knockout mice. Mice immunized with aTS mutants were fully protected against a challenge infection with the virulent T. cruzi Y strain. Altogether, our results confirmed the role of aTS as a T. cruzi virulence factor and indicated that aTS play a major role during the late stages of intracellular development and parasite egress. Notably, mutants lacking TS activity are completely avirulent in animal models of infection and may be used as a live attenuated vaccine against Chagas disease. IMPORTANCE Trypanosoma cruzi is the causative agent of Chagas disease, a neglected tropical disease that affects approximately 6 to 8 million people and for which there is no effective treatment or vaccine. The parasite expresses a family of surface proteins, named trans-sialidases, responsible for transferring sialic acid from host glycoconjugates to parasite mucins. Although recognized as a main virulence factor, the multiple roles of these proteins during infection have not yet been fully characterized, mainly because the presence of several copies of aTS genes has impaired their study using reverse genetics. By applying CRISPR-Cas9, we generated aTS knockout parasites and showed that, although aTS parasite mutants were able to infect cells in vitro, they have an impaired capacity to egress from the infected cell. Importantly, aTS mutants lost the ability to cause infection in vivo but provided full protection against a challenge infection with a virulent strain.

The interplay between Araçatuba virus and host signaling pathways: role of PI3K/Akt in viral replication.

In this study, we describe the interaction between Araçatuba virus (ARAV), a naturally occurring Brazilian vaccinia virus isolated from an outbreak at a dairy farm, and the host cell's signal transduction pathways. Even though ARAV infection led to phosphorylation of MAPKs MEK/ERK, JNK, and p38MAPK, genetic or pharmacological inhibition of these pathways had no impact on viral replication. We also provide evidence that ARAV stimulated the phosphorylation of Akt (PKB) at serine 473 (S473-P), a signaling event that is required for full activation of Akt during the infectious cycle. Furthermore, pharmacological inhibition of PI3K (LY294002) abrogated ARAV-induced Akt activation (S473-P) and affected early and late viral gene expression, which was followed by a decrease in virus yield (~1 log). Taken together, our data shed some light onto the biological differences between ARAV and vaccinia virus strain WR (VACV-WR), which could contribute, at least in part, to the low-virulence phenotype displayed by ARAV. Thus, while the requirement for the PI3K/Akt pathway for successful ARAV replication is also shared with VACV-WR and cowpox virus strain BR (CPXV-BR), ARAV showed a lower replicative capacity, as well as a smaller plaque-size phenotype after infection of A31 cells when compared to VACV-WR.

Proliferative Effect of Food Plant Species from Brazil's Cerrado on Cultured Fibroblasts.

Brazilian Cerrado is the second largest biome in South America and contains many unstudied valuable plant species rich in bioactive substances. In this study we investigated the phenolic content and proliferative effects on cultured fibroblasts of 32 extracts of different polarities prepared from 11 plants found in Cerrado regions. Eight extracts from six species increased cell proliferation and significantly induced ATP production by the cells. Four of these extracts were obtained from plants used as food, specifically from its fruits or seeds. A high phenolic content for these eight extracts, which directly correlated with the induction of cell proliferation, was corroborated by mass spectrometry analysis. We suggest that the bioactive substance content of these species shows an interesting potential use in cosmetic and food industry, which can contribute to the conservation and sustainable development of this region.

Publisher Correction: Aspirin metabolite sodium salicylate selectively inhibits transcriptional activity of ATF6α and downstream target genes.

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Evidence of traditionality of Brazilian medicinal plants: The case studies of Stryphnodendron adstringens (Mart.) Coville (barbatimão) barks and Copaifera spp. (copaíba) oleoresin in wound healing.

ETHNOPHARMACOLOGICAL RELEVANCE: The World Health Organization (WHO) recognizes the potential of plants used in secular traditional medicine and considers this an important source of evidence to assess their effectiveness and safety. Brazil is rich in biodiversity and traditional uses based on the Amerindian culture. However, many processes started with the arrival of the Portuguese in the year 1500. The successive economic cycles, for example, led to destruction of native vegetation and an intense cultural erosion. As a consequence, the information about the use of plants in the past centuries are dispersed and without interpretation. In this study a methodology to evidence the traditionality of Brazilian plants was demonstrated using data about barbatimão barks (Stryphnodendron adstringens (Mart.) Coville - Fabaceae) and Copaiba oleoresin (Copaifera spp. - Fabaceae) in wound healing, was established. MATERIAL AND METHODS: Data about use of the plants were recovered from bibliography published between 1576 and 2011. The books (101) were classified using weights, considering the date of publication and the source of Information. Older books that describe primary information received weight 10, while books written more recently and with secondary information received weight 0.4. A score for each category of medicinal use was calculated based on the books weights and the frequency of citation. A review about the current use of both plants was also performed from ethnobotanical studies published in journals. RESULTS AND DISCUSSION: The traditional secular use of barks of barbatimão and oleoresin of copaiba to treat wounds was confirmed based on the historic bibliographic research. The most frequent use of barbatimão in a timeline of 500 years of Brazil's history, was as astringent, whereas for copaíba was as healing of skin and mucosal lesions. The continuous and current use of these plants to treat wounds, confirmed by recent ethnobotanical studies, is an indicative of the resilience of these remedies and their effectiveness. CONCLUSION: The use of preparations containing barbatimão barks and copaiba oleoresin can be considered effective in the treatment of wounds. Nonetheless, it is necessary to improve the quality of the formulas as established by WHO.

Aspirin metabolite sodium salicylate selectively inhibits transcriptional activity of ATF6α and downstream target genes.

In response to ER stress, activating transcription factor 6 (ATF6) traffics from ER to Golgi apparatus where it is activated by cleavage before being translocated as transcription factor to the cell nucleus. In this work we describe ATF6α as a newly target of the aspirin metabolite sodium salicylate (NaSal). NaSal treatment of cells induces increases in ATF6α mRNA and protein levels, but these events are not accompanied by ATF6 activation. Conversely, NaSal inhibited ATF6 transactivating activity elicited by various ER stress-inducing stimuli in different cell types. This resulted in reduced expression of a subset of ATF6α target genes. Mechanistically, exposure of cells to NaSal results in ATF6α trapping at the Golgi apparatus, thus preventing nuclear translocation. This study provides evidence that NaSal compound restrains the activity of ATF6α, thereby preventing activation of a specific subset of ER-stress responsive genes implicated in different cellular responses.