Light, Copper, Action: Visible-Light Illumination Enhances Bactericidal Activity of Copper Particles.

Bacteria are an old concern to human health, as they are responsible for nosocomial infections, and the number of antibiotic-resistant microorganisms keeps growing. Copper is known for its intrinsic biocidal properties, and therefore, it is a promising material to combat infections when added to surfaces. However, its biocidal properties in the presence of light illumination have not been fully explored, especially regarding the use of microsized particles since nanoparticles have taken over all fields of research and subjugated microparticles despite them being abundant and less expensive. Thus, the present work studied the bactericidal properties of metallic copper particles, in microscale (CuMPs) and nanoscale (CuNPs), in the absence of light and under white LED light illumination. The minimum bactericidal concentration (MBC) of CuMPs against Staphylococcus aureus that achieved a 6-log reduction was 5.0 and 2.5 mg mL-1 for assays conducted in the absence of light and under light illumination, respectively. Similar behavior was observed against Escherichia coli. The bactericidal activity under illumination provided a percentage increase in log reduction values of 65.2% for S. aureus and 166.7% for E. coli when compared to the assays under dark. This assay reproduced the testing CuNPs, which showed superior bactericidal activity since the concentration of 2.5 mg mL-1 promoted a 6-log reduction of both bacteria even under dark. Its superior bactericidal activity, which overcame the effect of illumination, was expected once the nanoscale facilitated the interaction of copper within the surface of bacteria. The results from MBC were supported by fluorescence microscopy and atomic absorption spectroscopy. Therefore, CuMPs and CuNPs proved to have size- and dose-dependent biocidal activity. However, we have shown that CuMPs photoactivity is competitive compared to that of CuNPs, allowing their application as a self-cleaning material for disinfection processes assisted by conventional light sources without additives to contain the spread of pathogens.

High nitrogen concentration causes G2/M arrest in Hanseniaspora vineae.

Yeasts have been widely used as a model to better understand cell cycle mechanisms and how nutritional and genetic factors can impact cell cycle progression. While nitrogen scarcity is well known to modulate cell cycle progression, the relevance of nitrogen excess for microorganisms has been overlooked. In our previous work, we observed an absence of proper entry into the quiescent state in Hanseniaspora vineae and identified a potential link between this behavior and nitrogen availability. Furthermore, the Hanseniaspora genus has gained attention due to a significant loss of genes associated with DNA repair and cell cycle. Thus, the aim of our study was to investigate the effects of varying nitrogen concentrations on H. vineae's cell cycle progression. Our findings demonstrated that nitrogen excess, regardless of the source, disrupts cell cycle progression and induces G2/M arrest in H. vineae after reaching the stationary phase. Additionally, we observed a viability decline in H. vineae cells in an ammonium-dependent manner, accompanied by increased production of reactive oxygen species, mitochondrial hyperpolarization, intracellular acidification, and DNA fragmentation. Overall, our study highlights the events of the cell cycle arrest in H. vineae induced by nitrogen excess and attempts to elucidate the possible mechanism triggering this absence of proper entry into the quiescent state.

Bacterial community associated with gingivitis and periodontitis in dogs.

Periodontal disease is a chronic condition characterized by bacterial adhesion, followed by biofilm formation, and subsequently by an inflammatory process that progresses to gingivitis and later to periodontitis. The variations in the oral microbiota have been associated with the progression of this disease. This study evaluated the alteration of the cultivable oral microbiota in dogs with different oral health status. Thirty dogs were selected and divided into three groups: healthy, gingivitis, and periodontitis. The collected oral samples were seeded, and colonies with distinct phenotypic characteristics were isolated and classified using sequencing of the 16S rRNA gene. The DNA sequences were aligned, and a phylogenetic tree was constructed. Simpson's diversity index was calculated, and a dissimilarity matrix based on the Jaccard similarity index was used to plot a principal coordinate analysis. A total of 119 bacteria with different colony morphologies were isolated and classified into 4 phyla, 29 genera, and 45 species based on phylogenetic analysis. The results indicated an increase in bacteria belonging to the Proteobacteria phylum and a less extended decrease in Actinobacteria, Firmicutes, and Bacteroidetes phyla in dogs with periodontal disease (gingivitis and periodontitis) compared to healthy dogs. Representatives of the genera Neisseria sp., Corynebacterium sp., Pasteurella sp., and Moraxella sp. increased through the worsening of the periodontal disease, while Staphylococcus sp. decreased. All groups exhibited moderate to high levels of biodiversity index, and the plotted PCoA show a clear separation in the oral microbiome of dogs with periodontitis compared to dogs with gingivitis and the healthy group.

Antifungal activity of essential oil from Eucalyptus staigeriana against Alternaria alternata causing of leaf spot and black rot in table grapes.

Alternaria alternata causes leaf spot and black rot diseases in leaves and grapes of grapevines, respectively, and leads to huge economic losses in table grapes production. As natural antifungal agents, essential oils (EOs), which are generally recognized as safe substances, shows strong antifungal activity against fungal phytopathogens. The aim of this study was to determine the chemical composition of Eucalyptus staigeriana EO and its in vitro and in vivo effects against A. alternata. The major compounds of E. staigeriana EO were citral (34.32%, of which 21.83% geranial and 12.49% neral), limonene (20.60%) and 1,8-cineole (12.33%). E. staigeriana EO exhibited the highest inhibitory activity on mycelial growth and conidial germination at 1 µL mL-1. Moreover, the EO was able to reduce the incidence and severity of leaf spot disease in leaves and black rot disease in table grapes caused by A. alternata. These results represent a possible alternative to reduce the use of synthetic molecules for the control of diseases in postharvest of table grapes and in vineyard.

Yeast biodiversity in honey produced by stingless bees raised in the highlands of southern Brazil.

The physicochemical characteristics and yeasts diversity in honey samples from 17 species of stingless bees of the genera Nannotrigona, Melipona, Plebeia, Scraptotrigona, and Tetragonisca cultivated in Southern Brazil were determined. The sugar content, moisture, water activity, pH, reducing sugars/total sugar ratio, and total yeast population varied significantly among the honey from the different bee species. The highest yeast population was found in the Plebeia's honey samples and correlated with their high water-activity. Sixteen yeast species were identified based on the nuclear large subunit (26S) ribosomal RNA partial sequences. The genera Starmerella and Zygosaccharomyces were found predominant, with a high prevalence of Starmerella sp., S. etchellsii, and S. apicola. Some yeast species were only identified in honey samples from specific bee species indicating a close relationship between the yeasts and the insects. For the first time, Wickerhamomyces sydowiorum in honey is being reported. In general, the yeast species isolated from stingless bee honey samples demonstrated high osmotolerance and low sugar assimilation.

Citral and geraniol induce necrotic and apoptotic cell death on Saccharomyces cerevisiae.

Essential oils and their main components, monoterpenes, have been proven to be important alternatives for the control of pathogenic and spoiling microorganisms, but the mode of action of these compounds is poorly understood. This work aimed to determine the mode of action of citral and geraniol on the model yeast Saccharomyces cerevisiae using a flow cytometry approach. Exponentially growing yeast cells were treated with different concentrations of citral and geraniol for 3 h, and evaluated for cell wall susceptibility to glucanase, membrane integrity, reactive oxygen species (ROS) accumulation, mitochondrial membrane potential, and metacaspase activity. Results provide strong evidence that citral and geraniol acute fungicidal activity against Saccharomyces cells involves the loss of membrane and cell wall integrity resulting in a dose-dependent apoptotic/necrotic cell death. However, yeast cells that escape this first cell membrane disruption, particularly evident on sub-lethal concentration, die by metacaspase-mediated apoptosis induced by the accumulation of intracellular ROS. The deleted mutant on the yca1 gene showed high tolerance to citral and geraniol.

Morphological characterization and molecular identification of Colletotrichum species associated to sweet persimmon anthracnose in Southern Brazil / Caracterização morfológica e identificação molecular de espécies de Colletotrichum associadas à antracnose de caqui doce no Sul do Brasil

The highlands of Southern Brazil contribute with 40% of Brazilian persimmon production. Although expanding, persimmon production faces major problems caused by anthracnose disease (black spot), including fruit rot and necrosis of leaves. Several Colletotrichum species (C. horii, C. gloeosporioides, among others) are implicated in persimmon anthracnose around the world. To identify Colletotrichum species associated with persimmon anthracnose in the highlands of Southern Brazil, 34 isolates were analyzed by ITS-rDNA partial region, GAPDH, and TUB2 partial gene sequences, morphological characteristics, and virulence on persimmon fruits and leaves. Data showed a high prevalence of C. horii (85.3%), that associated with its high virulence on fruits and leaves, confirm a considerable degree of host preference. Moreover, other species C. aenigma, C. asianum, C. fructicola, and C. nymphaeae, were identified, but the last three ones exhibited low virulence on fruits and were not able to produce symptoms on leaves. As far as we know this is the first reference on C. asianum in persimmon. The present data may contribute to a better understanding of the etiology of anthracnose in sweet persimmon in Southern Brazil, and it will be useful for epidemiological studies and the development of disease management measures.

As terras altas do sul do Brasil contribuem com 40% da produção brasileira de caqui. Embora em expansão, a produção de caqui enfrenta grandes problemas causados ​​pela antracnose (mancha preta), incluindo o apodrecimento dos frutos e necrose das folhas. Várias espécies de Colletotrichum (C. horii, C. gloeosporioides, entre outras) estão envolvidas com a antracnose do caqui em todo mundo. Para identificar espécies de Colletotrichum associadas à antracnose de caqui nas terras altas do sul do Brasil, 34 isolados foram analisados ​​através da região parcial de ITS-rDNA e sequências parciais dos genes GAPDH e TUB2, características morfológicas e virulência em frutos e folhas de caqui. Os dados mostraram uma alta prevalência de C. horii (85,3%), que associada à sua alta virulência em frutos e folhas, confirma um grau considerável de preferência pelo hospedeiro. Além disso, foram identificadas outras espécies C. aenigma, C. asianum, C. fructicola, e C. nymphaeae, mas as três últimas exibiram baixa virulência nos frutos e não foram capazes de produzir sintomas nas folhas. Até onde sabemos, esta é a primeira referência sobre C. asianum em caqui. Os presentes dados podem contribuir para uma melhor compreensão da etiologia da antracnose em caqui doce no sul do Brasil e isso pode ser útil para estudos epidemiológicos e para o desenvolvimento de medidas de controle da doença.

Morphological characterization and molecular identification of Colletotrichum species associated to sweet persimmon anthracnose in Southern Brazil / Caracterização morfológica e identificação molecular de espécies de Colletotrichum associadas à antracnose de caqui doce no Sul do Brasil

The highlands of Southern Brazil contribute with 40% of Brazilian persimmon production. Although expanding, persimmon production faces major problems caused by anthracnose disease (black spot), including fruit rot and necrosis of leaves. Several Colletotrichum species (C. horii, C. gloeosporioides, among others) are implicated in persimmon anthracnose around the world. To identify Colletotrichum species associated with persimmon anthracnose in the highlands of Southern Brazil, 34 isolates were analyzed by ITS-rDNA partial region, GAPDH, and TUB2 partial gene sequences, morphological characteristics, and virulence on persimmon fruits and leaves. Data showed a high prevalence of C. horii (85.3%), that associated with its high virulence on fruits and leaves, confirm a considerable degree of host preference. Moreover, other species C. aenigma, C. asianum, C. fructicola, and C. nymphaeae, were identified, but the last three ones exhibited low virulence on fruits and were not able to produce symptoms on leaves. As far as we know this is the first reference on C. asianum in persimmon. The present data may contribute to a better understanding of the etiology of anthracnose in sweet persimmon in Southern Brazil, and it will be useful for epidemiological studies and the development of disease management measures.(AU)

As terras altas do sul do Brasil contribuem com 40% da produção brasileira de caqui. Embora em expansão, a produção de caqui enfrenta grandes problemas causados ​​pela antracnose (mancha preta), incluindo o apodrecimento dos frutos e necrose das folhas. Várias espécies de Colletotrichum (C. horii, C. gloeosporioides, entre outras) estão envolvidas com a antracnose do caqui em todo mundo. Para identificar espécies de Colletotrichum associadas à antracnose de caqui nas terras altas do sul do Brasil, 34 isolados foram analisados ​​através da região parcial de ITS-rDNA e sequências parciais dos genes GAPDH e TUB2, características morfológicas e virulência em frutos e folhas de caqui. Os dados mostraram uma alta prevalência de C. horii (85,3%), que associada à sua alta virulência em frutos e folhas, confirma um grau considerável de preferência pelo hospedeiro. Além disso, foram identificadas outras espécies C. aenigma, C. asianum, C. fructicola, e C. nymphaeae, mas as três últimas exibiram baixa virulência nos frutos e não foram capazes de produzir sintomas nas folhas. Até onde sabemos, esta é a primeira referência sobre C. asianum em caqui. Os presentes dados podem contribuir para uma melhor compreensão da etiologia da antracnose em caqui doce no sul do Brasil e isso pode ser útil para estudos epidemiológicos e para o desenvolvimento de medidas de controle da doença.(AU)

Piperlongumine Induces Apoptosis in Colorectal Cancer HCT 116 Cells Independent of Bax, p21 and p53 Status.

BACKGROUND/AIM: Colorectal cancer is a common type of cancer with reported resistance to treatment, in most cases due to loss of function of apoptotic and cell-cycle proteins. Piperlongumine (PPLGM) is a natural alkaloid isolated from Piper species, with promising anti-cancer properties. This study investigated whether PPLGM is able to induce cell death in colorectal carcinoma HCT 116 cells expressing wild-type or deficient in Bax, p21 or p53. MATERIALS AND METHODS: PPLGM was extracted from roots of Piper tuberculatum. Cell viability was determined by reduction of 3-(4,5-dimethilthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic assay. Cell death was evaluated by acridine orange/ethidium bromide staining and flow cytometry. Plasmid cleavage activity and circular dichroism DNA interaction were also analyzed. RESULTS: PPLGM induced selective cell death in all cell lines (IC50 range from 10.7 to 13.9 µM) with an increase in the number of late apoptotic cells and different profiles in cell-cycle distribution. Plasmid DNA analysis showed that PPLGM does not interact directly with DNA. CONCLUSION: This paper suggests that PPLGM may be a promising candidate in colorectal cancer therapy.

Necrotic cell death induced by dithianon on Saccharomyces cerevisiae.

Dithianon is a broad-spectrum anthraquinone fungicide used to control several diseases of grapes, apples, and other fruits and vegetables. Its mode of action is described as multi-site and associated to thiol-reactivity. As other fungicides can affect non-phytopathogenic organisms as yeasts and bacteria, with impact on microbial population, diversity, and fermentation processes. In this context, we study the effect of dithianon on the model organism and fermentative yeast Saccharomyces cerevisiae in order to elucidate the mechanisms involved in yeast cell death., and explain its interference on wine fermentation kinetics. Thus for, we analyzed cellular protein and non-protein thiols, membrane and cell wall integrity, reactive oxygen species accumulation, mitochondrial membrane potential, and phosphatidylserine externalization. The results showed that when exponentially aerobic growing cells of S. cerevisiae are submitted to acute dithianon treatment they loss cell wall and membrane integrity, dying by necrosis, and this behavior is associated to a depletion of reduced proteic and non-proteic thiol groups. We also detected an important increase of cellular reactive oxygen species (ROS) associated to mitochondrial membrane potential modifications on dithianon treated cells. ROS accumulation was not associated to apoptotic cell death, but can be responsible for intracellular damages. Moreover, necrotic cell death induced by dithianon explains its effect on the kinetics of wine fermentations.

Extrinsic and Intrinsic Apoptotic Responses Induced by Shiitake Culinary-Medicinal Mushroom Lentinus edodes (Agaricomycetes) Aqueous Extract against a Larynx Carcinoma Cell Line.

Cumulative evidence from research studies has shown that the shiitake culinary-medicinal mushroom, Lentinus edodes, is an excellent source of natural antitumor agents and is capable of inhibiting cancer cell growth. However, the cell signaling pathway that leads tumor cells to apoptosis is not well understood because many chemical compounds may be acting. This study investigated the chemopreventive effects of an L. edodes aqueous extract on human HEp-2 epithelial larynx carcinoma cells and normal human MRC-5 lung fibroblasts by identifying proliferative and apoptotic pathways. The chemical characterization of the dry powder was assessed by high-performance liquid chromatography. Antiproliferative and proapoptotic effects induced by the extract were evaluated by assessing proliferative markers, cell sorting through flow cytometry, and expression levels of apoptotic proteins with Western blotting. The results suggest that inhibition of cell proliferation was more prominent in HEp-2 than in MRC-5 cells. Cell death analysis showed the appearance of cell populations in the sub-G1 phase, with late apoptotic signal increased in a dose-dependent manner. In addition, the aqueous extract induced depolarization of mitochondria, activating the generation of intracellular reactive oxygen species in HEp-2 cells. These observations suggest that L. edodes extract may exert a chemopreventive effect, regulating mitotic induction of apoptogenic signals. These findings highlight the mushroom's pharmacological potential in cancer treatment.

Necrotic and apoptotic cell death induced by Captan on Saccharomyces cerevisiae.

Captan is one of the most widely used broad-spectrum fungicide applied to control several early and late diseases of grapes, apples, and other fruits and vegetables, and as other phthalimide fungicides is defined as a multisite compound with thiol-reactivity. Captan can affect non-target organisms as yeasts, modifying microbial populations and fermentation processes. In this study, we asked whether Captan thiol-reactivity and other mechanisms are involved in acute Captan-induced cell death on aerobic growing Saccharomyces cerevisiae. Thus for, we analyze cellular protein and non-protein thiols, cell membrane integrity, reactive oxygen species accumulation, phosphatidylserine externalization, and apoptotic mutants behavior. The results showed that when submitted to acute Captan treatment most cells lost their membrane integrity and died by necrosis due to Captan reaction with thiols. However, part of the cells, even maintaining their membrane integrity, lost their culture ability. These cells showed an apoptotic behavior that may be the result of non-protein thiol depletion and consequent increase of reactive oxygen species (ROS). ROS accumulation triggers a metacaspase-dependent apoptotic cascade, as shown by the higher viability of the yca1-deleted mutant. Together, necrosis and apoptosis are responsible for the high mortality detected after acute Captan treatment of aerobically growing cells of S. cerevisiae.

Antitumor activity of Brazilian red propolis fractions against Hep-2 cancer cell line.

Continuous increases in the rates of tumor diseases have highlighted the need for identification of novel and inexpensive antitumor agents from natural sources. In this study, we investigated the effects of enriched fraction from hydroalcoholic Brazilian red propolis extract against Hep-2 cancer cell line. Initially 201 fractions were arranged in 12 groups according to their chromatographic characteristics (A-L). After an in vitro cell viability screening, J and L were further selected as promising enriched fractions for this study. The chemical characterization was performed and Biochanin A, Formononetin, and Liquiritigenin compounds were quantified. Through MTT viability assay and morphological changes observed by Giemsa and DAPI staining, the results showed that red propolis inhibited cancer cells growth. Flow cytometry results indicated effects that were partly mediated through programmed cell death as confirmed by externalization of phosphatidylserine, DNA cleaved assay, increase at SUB G1-G0 phase in cell cycle analysis and loss of mitochondrial membrane potential. In conclusion, our results demonstrated that red propolis enriched fractions promoted apoptotic effects in human cancer cells through the mechanisms involving mitochondrial perturbation. Therefore, red propolis fractions contain candidate agents for adjuvant cancer treatment, which further studies should elucidate the comprehensive mechanistic pathways.