Kisspeptin-10 Improves Testicular Redox Status but Does Not Alter the Unfolded Protein Response (UPR) That Is Downregulated by Hypothyroidism in a Rat Model.

Hypothyroidism compromises the testicular redox status and is associated with reduced sperm quality and infertility in men. In this regard, studies have demonstrated the antioxidant potential of kisspeptin in reproductive and metabolic diseases. In this study, we evaluate the effects of kisspeptin-10 (Kp10) on the testicular redox, as well as mediators of the unfolded protein response (UPR) in adult rats with hypothyroidism. Adult male Wistar rats were randomly separated into the Control (n = 15), Hypo (n = 13) and Hypo + Kp10 (n = 14) groups, and hypothyroidism was induced with 6-propyl-2-thiouracil (PTU) for three months. In the last month, half of the hypothyroid animals received Kp10. Testis samples were collected for enzymatic, immunohistochemical and/or gene evaluation of mediators of oxidative stress (TBARs, lipid hydroperoxides (LOOH), ROS, peroxynitrite, SOD, CAT and GPX), endoplasmic reticulum stress (GRP78, ATF6, PERK, CHOP, HO-1 and sXBP1) and antiapoptocytes (BCL-2). Hypothyroidism increased apoptosis index, TBARS and LOOH concentrations, and reduced testicular gene expression of Sod1, Sod2 and Gpx1, as well as the expression of Grp78, Atf6, Ho1 and Chop. Treatment with Kp10, in turn, reduced testicular apoptosis and the production of peroxynitrite, while increased SOD1 and GPX ½ expression, and enzymatic activity of CAT, but did not affect the lower expression of UPR mediators caused by hypothyroidism. This study demonstrated that hypothyroidism causes oxidative stress and dysregulated the UPR pathway in rat testes and that, although Kp10 does not influence the low expression of UPR mediators, it improves the testicular redox status, configuring it as an important antioxidant factor in situations of thyroid dysfunction.

Genotyping Analysis of Cryptococcus deuterogattii and Correlation with Virulence Factors and Antifungal Susceptibility by the Clinical and Laboratory Standards Institute and the European Committee on Antifungal Susceptibility Testing Methods.

Data about the relationship between their molecular types, virulence factors, clinical presentation, antifungal susceptibility profile, and outcome are still limited for Cryptococcus deuterogattii. This study aimed to evaluate the molecular and phenotypic characteristics of 24 C. deuterogattii isolates from the southeast region of Brazil. The molecular characterization was performed by multilocus sequence typing (MLST). The antifungal susceptibility profile was obtained according to CLSI-M27-A3 and EUCAST-EDef 7.1 methods. The virulence factors were evaluated using classic techniques. The isolates were divided into four populations. The molecular analysis suggests recombinant events in most of the groups evaluated. Resistance and susceptibility dose-dependent to fluconazole were evidenced in four isolates (16%) by EUCAST and in four isolates (16%) by CLSI methods. The agreement at ±two dilutions for both methods was 100% for itraconazole, ketoconazole, and voriconazole, 96% for amphotericin B, and 92% for fluconazole. Significant differences in virulence factor expression and antifungal susceptibility to itraconazole and amphotericin B were found. The mixed infection could be suggested by the presence of variable sequence types, differences in virulence factor production, and decreased antifungal susceptibility in two isolates from the same patient. The data presented herein corroborate previous reports about the molecular diversity of C. deuterogattii around the world.

Redox profile and mediators of the unfolded protein response (UPR) in the placenta of rats during pregnancy.

CONTEXT: Proliferation, differentiation, migration and apoptosis of trophoblastic cells are influenced by hypoxia, as well as adequate modulation of oxidative stress and the unfolded protein response (UPR) pathway. AIMS: We aimed to evaluate the expression profile of redox and UPR mediators in the placenta of rats throughout pregnancy. METHODS: Placental expression of hypoxia-inducible factor 1α (HIF1α), 8-Hydroxy-2'-deoxyguanosine (8-OHdG), superoxide dismutase 1 (SOD1), glutathione peroxidase (GPX), catalase (Cat), activating transcription factor 6 (ATF6), protein kinase RNA-like endoplasmic reticulum kinase (PERK), 78 kD glucose-regulated protein (GRP78) and C/EBP-homologous protein (CHOP), as well as reactive oxygen species (ROS) and peroxynitrite production, were evaluated in Wistar rats on the 10th, 12th, 14th, 16th and 18th day of pregnancy (DP). KEY RESULTS: Increased immunostaining of HIF1α was observed on the 16th and 18th DP, while 8-OHdG and ROS production were greater on the 14th DP. SOD1 and Cat had increased immunostaining on the 14th and 18th DP, while staining of GPX1/2, GRP78 and CHOP was greater on the 18th DP. With regard to gene expression, Hif1α and Sod1 showed increased mRNA expression on the 12th and 16th DP, while Gpx1 had increased expression on the 10th and 16th DP. Cat , Perk and Grp78 gene expression was greater on the 14th DP, unlike Atf6 , which showed greater expression on the 12th DP. In contrast, Chop maintained increased expression from the 12th to the 18th DP. CONCLUSIONS: The placental expression of redox and UPR mediators in rats is influenced by gestational age, with greater expression in periods of greater HIF1α and 8-OHdG expression and at the end of the pregnancy. IMPLICATIONS: This study provides data on the physiological modulation of redox and UPR mediators during placental development in rats.

Organoselenium Has a Potent Fungicidal Effect on Cryptococcus neoformans and Inhibits the Virulence Factors.

Cryptococcosis therapy is often limited by toxicity problems, antifungal tolerance, and high costs. Studies approaching chalcogen compounds, especially those containing selenium, have shown promising antifungal activity against pathogenic species. This work aimed to evaluate the in vitro and in vivo antifungal potential of organoselenium compounds against Cryptococcus neoformans. The lead compound LQA_78 had an inhibitory effect on C. neoformans planktonic cells and dispersed cells from mature biofilms at similar concentrations. The fungal growth inhibition led to an increase in budding cells arrested in the G2/M phase, but the compound did not significantly affect structural cell wall components or chitinase activity, an enzyme that regulates the dynamics of the cell wall. The compound also inhibited titan cell (Tc) and enlarged capsule yeast (NcC) growth and reduced the body diameter and capsule thickness associated with increased capsular permeability of both virulent morphotypes. LQA_78 also reduced fungal melanization through laccase activity inhibition. The fungicidal activity was observed at higher concentrations (16 to 64 µg/mL) and may be associated with augmented plasma membrane permeability, ROS production, and loss of mitochondrial membrane potential. While LQA_78 is a nonhemolytic compound, its cytotoxic effects were cell type dependent, exhibiting no toxicity on Galleria mellonella larvae at a dose ≤46.5 mg/kg. LQA_78 treatment of larvae infected with C. neoformans effectively reduced the fungal burden and inhibited virulent morphotype formation. To conclude, LQA_78 displays fungicidal action and inhibits virulence factors of C. neoformans. Our results highlight the potential use of LQA_78 as a lead molecule for developing novel pharmaceuticals for treating cryptococcosis.

Maternal hypothyroidism causes oxidative stress and endoplasmic reticulum stress in the maternal-fetal interface of rats.

Maternal hypothyroidism is associated with pre-eclampsia and intrauterine growth restriction, gestational diseases involving oxidative stress (OS) and endoplasmic reticulum stress (ERS) in the placenta. However, it is not known whether hypothyroidism also causes OS and ERS at the maternal-fetal interface. The aim was to evaluate the fetal-placental development and the expression of mediators of OS and of the unfolded protein response (UPR) in the maternal-fetal interface of hypothyroid rats. Hypothyroidism was induced in Wistar rats with propylthiouracil and the fetal-placental development and placental and decidual expression of antioxidant, hypoxia, and UPR mediators were analyzed at 14 and 18 days of gestation (DG), as well the expression of 8-OHdG and MDA, and reactive oxygen species (ROS) and peroxynitrite levels. Hypothyroidism reduced fetal weight at 14 and 18 DG, in addition to increasing the percentage of fetal death and reducing the weight of the uteroplacental unit at 18 DG. At 14 DG, there was greater decidual and/or placental immunostaining of Hif1α, 8-OHdG, MDA, SOD1, GPx1/2, Grp78 and CHOP in hypothyroid rats, while there was a reduction in placental and/or decidual gene expression of Sod1, Gpx1, Atf6, Perk, Ho1, Xbp1, Grp78 and Chop in the same gestational period. At 18 DG, hypothyroidism increased the placental ROS levels and the decidual and/or placental immunostaining of HIF1α, 8-OHdG, MDA, ATF4, GRP78 and CHOP, while it reduced the immunostaining and enzymatic activity of SOD1, CAT, GST. Hypothyroidism increased the placental mRNA expression of Hifα, Nrf2, Sod2, Gpx1, Cat, Perk, Atf6 and Chop at 18 DG, while decreasing the decidual expression of Sod2, Cat and Atf6. These findings demonstrated that fetal-placental restriction in female rats with hypothyroidism is associated with hypoxia and dysregulation in placental and decidual expression of UPR mediators and antioxidant enzymes, and activation of oxidative stress and endoplasmic reticulum stress at the maternal-fetal interface.

Antimicrobial and Immunomodulatory Activities of Dried Extracts of Echinacea Purpurea

Abstract The use of Echinacea purpurea (EP), a plant native from North America, is widely diffused throughout the world, presenting many pharmacological applications, mainly for the treatment of infections of respiratory and urinary tracts. Due to the widespread commercialization of EP-based products, an effective evaluation of their pharmacological properties is essential to assure efficacy during clinical use. In this study, in vitro tests were performed to evaluate the antimicrobial activity of dried extracts of EP by the microdilution method. In addition, a phagocytosis model was employed to assess the immunomodulatory potential of the extracts. The increase in reactive oxygen species production, as well as the intracellular proliferation rate of Cryptococcus gatti after phagocytosis by macrophages in the presence of EP dried extracts were also evaluated. The analyzed samples showed no significant antibacterial activity; however, a slight antifungal activity was verified. Positive effects of EP extracts on the modulation of cellular immune response were observed in different experiments, indicating that this mechanism may contribute to the control and treatment of infections.

High genetic variability of clinical and environmental Cryptococcus gattii isolates from Brazil.

Among Cryptococcus gattii genotypes, VGII has gained pivotal relevance in epidemiological, clinical and genetic contexts due to its association with several outbreaks in temperate regions and due to the high variability of this genotype. The aim of this study was to compare 25 isolates of C. gattii from the Southeast region of Brazil with previously described isolates from other regions of the country and around the world. Among the 25 isolates, 24 were VGII and one was VGI. All of them were newly identified. Three new allele types (AT) (AT47 for the URA5 locus, AT56 for the LAC1 locus, and AT96 for the IGS1 region) were also described. Compared with other Brazilian isolates, those from the Southeast region presented the greatest haplotype diversity. In general, the regions presented different sequence types (STs), and only nine STs were found in more than one location. GoeBURST analysis showed two large groups among the Brazilian isolates. The largest group consists of 59 STs predominantly from the North and Northeast regions; the other large group includes 57 STs from the Southeast and Midwest regions. In a global context the South American isolates presented the highest genetic diversity (STs = 145, haplotype diversity (Hd) = 0.999 and π = 0.00464), while the African populations showed the lowest genetic diversity (STs = 3, Hd = 0.667 and π = 0.00225). These results confirm that the Brazilian C. gattii VGII population is highly diverse and reinforce the hypothesis of dispersion of this genotype from South America.

Corrigendum: Characterization of Aspergillus fumigatus Extracellular Vesicles and Their Effects on Macrophages and Neutrophils Functions.

[This corrects the article DOI: 10.3389/fmicb.2019.02008.].

Characterization of Aspergillus fumigatus Extracellular Vesicles and Their Effects on Macrophages and Neutrophils Functions.

Extracellular vesicles (EVs) has been considered an alternative process for intercellular communication. EVs release by filamentous fungi and the role of vesicular secretion during fungus-host cells interaction remain unknown. Here, we identified the secretion of EVs from the pathogenic filamentous fungus, Aspergillus fumigatus. Analysis of the structure of EVs demonstrated that A. fumigatus produces round shaped bilayer structures ranging from 100 to 200 nm size, containing ergosterol and a myriad of proteins involved in REDOX, cell wall remodeling and metabolic functions of the fungus. We demonstrated that macrophages can phagocytose A. fumigatus EVs. Phagocytic cells, stimulated with EVs, increased fungal clearance after A. fumigatus conidia challenge. EVs were also able to induce the production of TNF-α and CCL2 by macrophages and a synergistic effect was observed in the production of these mediators when the cells were challenged with the conidia. In bone marrow-derived neutrophils (BMDN) treated with EVs, there was enhancement of the production of TNF-α and IL-1ß in response to conidia. Together, our results demonstrate, for the first time, that A. fumigatus produces EVs containing a diverse set of proteins involved in fungal physiology and virulence. Moreover, EVs are biologically active and stimulate production of inflammatory mediators and fungal clearance.

Acanthamoeba polyphaga mimivirus prevents amoebal encystment-mediating serine proteinase expression and circumvents cell encystment.

Acanthamoeba is a genus of free-living amoebas distributed worldwide. Few studies have explored the interactions between these protozoa and their infecting giant virus, Acanthamoeba polyphaga mimivirus (APMV). Here we show that, once the amoebal encystment is triggered, trophozoites become significantly resistant to APMV. Otherwise, upon infection, APMV is able to interfere with the expression of a serine proteinase related to amoebal encystment and the encystment can no longer be triggered.

Antifungal activity of extracts from Piper aduncum leaves prepared by different solvents and extraction techniques against dermatophytes Trichophyton rubrum and Trichophyton interdigitale

The effects of different solvents and extraction techniques upon the phytochemical profile and anti-Trichophyton activity of extracts from Piper aduncum leaves were evaluated. Extract done by maceration method with ethanol has higher content of sesquiterpenes and antifungal activity. This extract may be useful as an alternative treatment for dermatophytosis.

Photodynamic inhibition of Trichophyton rubrum: in vitro activity and the role of oxidative and nitrosative bursts in fungal death.

OBJECTIVES: Antimicrobial photodynamic inhibition (aPI) is based on the use of a light source and a photosensitizer to kill pathogens. Little is known about aPI of dermatophytic fungi and its mechanism of action. We aimed to evaluate aPI of Trichophyton rubrum. METHODS: We performed tests using toluidine blue (TBO) as a photosensitizer and a 630 nm light-emitting diode (LED) as a source of light to target 12 T. rubrum isolates. Susceptibility testing with cyclopiroxolamine, time-kill curves and quantification of reactive oxygen species (ROS), peroxynitrite (ONOO·) and nitric oxide (NO·) were performed. RESULTS: The optimal conditions for in vitro aPI were 10 mg/L for TBO and 48 J/cm(2) for LED; these conditions were fungicidal or inhibited >98% of fungal growth depending on the strain tested. LED or TBO treatment alone did not inhibit growth. The MICs of cyclopiroxolamine were 2.0 mg/L for 90% of the strains. Analysis of time-kill curves revealed that pathogen death occurred 24 h post-treatment. Quantification of ROS, ONOO· and NO· revealed improvement after aPI. CONCLUSIONS: Photodynamic inhibition was more efficient in promoting cell death than the antifungal cyclopiroxolamine against T. rubrum. ROS, ONOO· and NO· were important in the fungicidal activity of aPI. A suggested mechanism for this activity is that TBO is excited by LED light (630 nm), reacts with biomolecules and increases the availability of transition electrons and substrates for nitric oxide synthase, thereby increasing the oxidative and nitrosative bursts in the fungal cell.

Avaliação do crescimento de Staphylococcus aureus em músculo bovino sob diferentes formas de descongelamento e tempos de congelamento / Evaluation of growth of Staphylococcus aureus in bovine muscle in different forms of thawing and freezing time

Uma amostra de músculo bovino moído foi selecionada em dividida em dez porções de 25 g cada. Uma delas foi utilizada para a determinação do teor basal de S. aureus e as outras nove foram propositalmente contaminadas com 10² UFC de S. aureus (ATCC 29213) e submetidas ao congelamento por 24, 72 ou 96 horas. Após o período de congelamento, as amostras foram submetidas ao descongelamento em geladeira, temperatura ambiente ou micro-ondas. Posteriormente, foi quantificado o teor de S. aureus em cada porção. Os teores obtidos após 24 h de congelamento foram superiores aos outros tempos estudados (p<0,05). Foi verificada diferença significativa (p<0,05) quando foram comparados os três modos de descongelamentos estudados. O descongelamento no micro-ondas foi aquele que propiciou melhor redução dos níveis de S. aureus no produto, seguido da geladeira e temperatura ambiente. Pela análise dos resultados, percebe-se que o descongelamento à temperatura ambiente, freqüentemente executado em domicílios, exibe sérios riscos de multiplicação microbiana, tornando o produto inadequado ao consumo. Espera-se que os resultados aqui obtidos possam contribuir para o melhor conhecimento sobre o crescimento de S. aureus em produtos cárneos, em diferentes tempos de congelamento e formas de descongelamento.

Evaluation of susceptibility of Trichophyton mentagrophytes and Trichophyton rubrum clinical isolates to antifungal drugs using a modified CLSI microdilution method (M38-A).

Onychomycosis is a common adult human mycosis, and dermatophytes of the Trichophyton genera are the most common causative agent. Many antimycotic agents are safe and highly effective for the treatment of dermatophytosis, and are available for clinical practice. Successful treatment depends on the ability of antifungal drugs to eradicate the fungal isolates. The aim of this work was to determine the MICs of four antifungal drugs (fluconazole, itraconazole, terbinafine and griseofulvin) recognized for ungual dermatophytosis treatment caused by Trichophyton species, especially Trichophyton mentagrophytes and Trichophyton rubrum. MICs were determined using a broth microdilution method in accordance with Clinical and Laboratory Standards Institute approved standard M38-A with some modifications, such as an incubation temperature of 28 degrees C, an incubation time of 7 days and inocula constituted of only microconidia. The results showed that the activities of terbinafine and itraconazole were significantly higher (MICs of <0.007-0.031 and 0.015-0.25 microg ml(-1), respectively) than other tested agents. All isolates had reduced susceptibility to fluconazole (1-64 microg ml(-1)). The MIC of griseofulvin varied among strains (MICs of 0.062-1 microg ml(-1)). The parameters adopted to perform susceptibility testing of T. rubrum and T. mentagrophytes to antifungal agents appeared to be suitable and reliable, and could contribute to the possible development of a standard protocol.

In vitro methods for antifungal susceptibility testing of Trichophyton spp.

In general, methods to test the susceptibility of fungi to antifungal drugs require standardized techniques, but so far there is no methodology that is widely applicable to dermatophytes. Here we introduced modifications to the protocols from documents of the National Committee for Clinical Laboratory Standards (CLSI) M38-A and the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) that are usually applied to moulds and fermentative yeasts, in order to adjust the conditions for the growth of dermatophytes. The modifications included: growth on potato dextrose agar supplemented with 2% in-house rice flour to encourage sporulation, the addition of 2% glucose to the culture media (RPMI-1640), and an incubation temperature of 28 degrees C. In addition, the incubation period was 7d, the minimum inhibitory concentration (MIC) was defined as 80% growth inhibition endpoints for azole agents, and the inocula only contained microconidia. Results obtained by both tested methodologies were very similar to the ones reported by other researchers. MIC90 (MIC at which 90% of isolates tested were inhibited) values were identical for four out of five antifungal drugs tested and there was only a difference of one or two dilutions when MIC50 values were compared. Although the modifications introduced did not interfere with the results, more studies are necessary to establish a standard technique to test susceptibility of dermatophytes to antifungal drugs.

Ocorrência de Staphylococcus aureus em fisioterapeutas que atuam em clínicas e hospitais de Governador Valadares – Minas Gerais / Staphylococcus aureus occurrence in physical therapists of Governador Valadares – Minas Gerais

Trinta profissionais fisioterapeutas foram selecionados para verificar o estado de portador de Staphylococcus aureus na mucosa nasal. A coleta foi realizada por meio de um swab esterilizado que, posteriormente foi inserido em um tubo contendo caldo de enriquecimento. Para o isolamento e identificação utilizaram-se as provas bioquímicas manita, Dnase, catalase e coagulase. Verificou-se que todos os indivíduos pesquisados eram portadores de uma espécie de estafi lococo, sendo que 52% eram portadores de S. aureus, 43% de S. epidermidis e 5% possuíam S. saprophyticcus. Fatores como idade, sexo, área de atuação profissional, internações prévias e uso de medicamentos não influenciaram de forma significativa os resultados obtidos (p<0,05). O presente estudo demonstrou que a maioria dos profissionais pesquisados possuía S. aureus na mucosa nasal. O estado de portador assintomático desse microrganismo pelos fisioterapeutas pode apresentar risco potencial de transmissão aos pacientes por eles atendidos.

Thirty physical therapists from Governador Valadares (Southeast of Brazil) were selected to investigate the nasal carrier occurrence of Staphylococcus aureus. Specimens for culture were collected from the nares with cotton swabs. The nasal swabs were cultured on enrichment broth. After that, culture was transferred to manitol salt agar, Dnase agar and citrated serum to identify the isolates. Our data revealed that all investigated professionals were nasal carrier of one Staphylococcus specie (52% - S. aureus; 43% - S. epidermidis; 5% - S. saprophyticcus). Conditions as age, sex, place of work, medicine use and previous diseases didn’t significantly influence obtained results (p<0.05). This work demonstrated that the majority of investigated professionals was nasal carriers of S. aureus, and there is a transmission risk of the microorganism to their patients.