Two different clones of Candida pelliculosa bloodstream infection in a tertiary neonatal intensive care unit.

INTRODUCTION: Fungemia in preterm infants results in high mortality and morbidity. The genotypes, drug susceptibilities of Candida pelliculosa strains, and clinical features of two outbreaks of neonatal candidemia caused by C. pelliculosa were analyzed, in order to provide evidence for the outbreaks and characteristics of C. pelliculosa neonatal candidemia. METHODOLOGY: The strains were genotyped by pulsed-field gel electrophoresis to investigate their genetic relatedness. The broth microdilution method was used to determine in vitro susceptibility of the isolates to antifungal drugs. Clinical features of the infected patients were collected to analyze the risks for C. pelliculosa infection. RESULTS: Fourteen neonates, hospitalized in the neonatal intensive care unit from November 2012 to October 2013, were infected by C. pelliculosa. All 14 patients were cured after treatment with fluconazole and discharged without any complications. The C. pelliculosa isolates from the 14 patients were clustered into two groups, indicating that the outbreaks were caused by two types of strains. Eight of nine strains isolated from the 2013 outbreak were clustered into the same group, while one isolate was grouped together with five isolates from the 2012 outbreak. In vitro experiments demonstrated high antifungal activity of fluconazole, voriconazole, amphotericin B, and 5-fluorocytosine to C. pelliculosa. The common symptoms of C. pelliculosa candidaemia were fever, cyanosis, polypnea, hypoactivity, and apnea. CONCLUSIONS: The current study revealed high in vitro susceptibility of C. pelliculosa to antifungals. As C. pelliculosa candidaemia cannot be characterized by clinical symptoms and routine blood testing alone, monitoring unusual strains isolated from immunodeficient hosts is very important to prevent possible outbreaks.

Balancing Positive and Negative Selection: In Vivo Evolution of Candida lusitaniae MRR1.

The evolution of pathogens in response to selective pressures present during chronic infections can influence their persistence and virulence and the outcomes of antimicrobial therapy. Because subpopulations within an infection can be spatially separated and the host environment can fluctuate, an appreciation of the pathways under selection may be most easily revealed through the analysis of numerous isolates from single infections. Here, we continued our analysis of a set of clonally derived Clavispora (Candida) lusitaniae isolates from a single chronic lung infection with a striking enrichment in the number of alleles of MRR1 Genetic and genomic analyses found evidence for repeated acquisition of gain-of-function mutations that conferred constitutive Mrr1 activity. In the same population, there were multiple alleles with both gain-of-function mutations and secondary suppressor mutations that either attenuated or abolished the constitutive activity, suggesting the presence of counteracting selective pressures. Our studies demonstrated trade-offs between high Mrr1 activity, which confers resistance to the antifungal fluconazole, host factors, and bacterial products through its regulation of MDR1, and resistance to hydrogen peroxide, a reactive oxygen species produced in the neutrophilic environment associated with this infection. This inverse correlation between high Mrr1 activity and hydrogen peroxide resistance was observed in multiple Candida species and in serially collected populations from this individual over 3 years. These data lead us to propose that dynamic or variable selective pressures can be reflected in population genomics and that these dynamics can complicate the drug resistance profile of the population.IMPORTANCE Understanding microbial evolution within patients is critical for managing chronic infections and understanding host-pathogen interactions. Here, our analysis of multiple MRR1 alleles in isolates from a single Clavispora (Candida) lusitaniae infection revealed the selection for both high and low Mrr1 activity. Our studies reveal trade-offs between high Mrr1 activity, which confers resistance to the commonly used antifungal fluconazole, host antimicrobial peptides, and bacterial products, and resistance to hydrogen peroxide. This work suggests that spatial or temporal differences within chronic infections can support a large amount of dynamic and parallel evolution and that Mrr1 activity is under both positive and negative selective pressure to balance different traits that are important for microbial survival.

Phialemoniopsis limonesiae sp. nov. causing cutaneous phaeohyphomycosis in an immunosuppressed woman.

Rare or opportunistic fungal infections are mostly described in immunosuppressed patients. We present a case of a cutaneous phaeohyphomycosis that developed on the dorsal foot in an immunosuppressed woman suffering from AIDS, caused by a novel Phialemoniopsis species. It clinically presented as an indurated violaceous plaque, surmounted by nodules exuding a sero-purulent discharge. A filamentous fungus was isolated from pus and cutaneous biopsy. ITS and LSU sequences phylogenetically resolved the fungus as an unknown species of Phialemoniopsis, which is an unresolved family within Sordariomycetes. In this study we describe the new species as Phialemoniopsis limonesiae, which clusters on a single branch clearly separated from its closest phylogenetic neighbours. This new strain showed low MIC to itraconazole, voriconazole and posaconazole.

High-performance thin-layer chromatography in combination with a yeast-based multi-effect bioassay to determine endocrine effects in environmental samples.

Effect-directed analysis (EDA) that combines effect-based methods (EBMs) with high-performance thin-layer chromatography (HPTLC) is a useful technique for spatial, temporal, and process-related effect evaluation and may provide a link between effect testing and responsible substance identification. In this study, a yeast multi endocrine-effect screen (YMEES) for the detection of endocrine effects is combined with HPTLC. Simultaneous detection of estrogenic, androgenic, and gestagenic effects on the HPTLC plate is achieved by mixing different genetically modified Arxula adeninivorans yeast strains, which contain either the human estrogen, androgen, or progesterone receptor. Depending on the yeast strain, different fluorescent proteins are formed when an appropriate substance binds to the specific hormone receptor. This allows to measure hormonal effects at different wavelengths. Two yeast cell application approaches, immersion and spraying, are compared. The sensitivity and reproducibility of the method are shown by dose-response investigations for reference compounds. The spraying approach indicated similar sensitivities and higher precisions for the tested hormones compared to immersion. The EC10s for estrone (E1), 17ß-estradiol (E2), 17α-ethinylestradiol (EE2), 5α-dihydrotestosterone (DHT), and progesterone (P4) were 95, 1.4, 10, 7.4, and 15 pg/spot, respectively. Recovery rates of E1, E2, EE2, DHT, and P4 between 88 and 120% show the usability of the general method in combination with sample enrichment by solid phase extraction (SPE). The simultaneous detection of estrogenic, androgenic, and gestagenic effects in wastewater and surface water samples demonstrates the successful application of the YMEES in such matrices. This promising method allows us to identify more than one endocrine effect on the same HPTLC plate, which saves time and material. The method could be used for comparison, evaluation, and monitoring of different river sites and wastewater treatment steps and should be tested in further studies.

Kazachstania slooffiae: An unexpected journey to a human pleural sample.

We report a case of a 50-year-old shepherd hospitalized in intensive care unit for hiatal hernia complicated by an occlusive syndrome. In post-surgery, an acute respiratory distress occurs due to mediastinitis with large pleural effusion. At the laboratory, direct examination of the pleural sample revealed the presence of pseudohyphae. Kazachstania slooffiae was identified by Mass Spectrometry and confirmed by DNA sequencing. This uncommon yeast has never been previously described in human infections. Although its pathogenicity is not well known, K. slooffiae should be considered in the case of critically ill patients.

A case-series of bloodstream infections caused by the Meyerozyma guilliermondii species complex at a reference center of oncology in Brazil.

Bloodstream infections (BSI) caused by Candida species are the fourth cause of healthcare associated infections worldwide. Non-albicans Candida species emerged in the last decades as agents of serious diseases. In this study, clinical and microbiological aspects of six patients with BSI due to the Meyerozyma (Candida) guilliermondii species complex from an oncology reference center in Brazil, were evaluated. To describe demographic and clinical characteristics, medical records of the patients were reviewed. Molecular identification of the isolates was performed by ITS1-5.8S-ITS2 region sequencing. Antifungal susceptibility was evaluated by the EUCAST method and the minimal inhibitory concentrations (MIC) assessed according to the epidemiological cutoff values. Virulence associated phenotypes of the isolates were also studied. Ten isolates from the six patients were evaluated. Five of them were identified as Meyerozyma guilliermondii and the others as Meyerozyma caribbica. One patient was infected with two M. caribbica isolates with different genetic backgrounds. High MICs were observed for fluconazole and echinocandins. Non-wild type isolates to voriconazole appeared in one patient previously treated with this azole. Additionally, two patients survived, despite infected with non-wild type strains for fluconazole and treated with this drug. All isolates produced hemolysin, which was not associated with a poor prognosis, and none produced phospholipases. Aspartic proteases, phytase, and esterase were detected in a few isolates. This study shows the reduced antifungal susceptibility and a variable production of virulence-related enzymes by Meyerozyma spp. In addition, it highlights the poor prognosis of neutropenic patients with BSI caused by this emerging species complex. LAY ABSTRACT: Our manuscript describes demographic, clinical and microbiological characteristics of patients with bloodstream infection by the Meyerozyma guilliermondii species complex at a reference center in oncology in Brazil.

Identification and antifungal susceptibility of Saprochaete clavata from invasive infections in Turkey.

Saprochaete clavata is an emerging opportunistic pathogen, that causes life-threatening infections, but there are limited evidence and information about the evaluation of in vitro antifungal susceptibility test results. The aim of this study was to determine S. clavata isolates from clinical specimens and to investigate their in vitro antifungal susceptibility. S. clavata was identified by API ID20C AUX (BioMérieux, Brussels, Belgium), MALDI TOF (Bruker Daltonik, Germany), and ITS gene region sequencing. In vitro susceptibility tests were performed using Sensititre YeastOne (TREK Diagnostic System, East Grinstead, UK). During the study period, 4,736 fungi were isolated from various clinical samples and, S. clavata was identified in eight patients with underlying diseases namely, pancreatic neoplasma, acute myeloid leukaemie, follicular lymphoma, cholelithiasis. Anidulafungin and micafungin minimum inhibitory concentration values were 1-2 and 1-4 mg/L, respectively, while those of the azole group antifungals were much lower. This is the first study in Turkey reporting isolation, identification and antifungal susceptibilities of S. clavata from clinical specimens. Higher MIC values seen in some isolates suggest that continuous monitoring of sensitivity rates and observation of regional differences will thus be useful guides in determining infection control and antifungal use policies.

Chemistry and Antioxidant Potential of Phytoconstituents from Aegle Marmelos Fruit-Shell.

BACKGROUND: Oxidative stress is among the main causes of metabolic disorders. Hence, there is a need to discover potent antioxidants for therapeutic applications. OBJECTIVE: The objective of this study has been to investigate the phytoconstituents of the methanolic extract of the hard shell of Aegle marmelos fruit and their antioxidant potential. METHODS: Methanolic extract was fractionated using different solvents by liquid-liquid extraction. Characterization of the phytoconstituents was done by using phytochemical tests and GC-MS analysis. The free radical scavenging activity, total reducing power, lipid peroxidation inhibition and cell protection assays against oxidative stress were performed with methanolic extract and its fractions. RESULTS: Therapeutically significant class of compounds, for example, polyphenols, glycosides and sterols were revealed in the hard-shell extract. Differential separation of compounds was achieved by liquid-liquid extraction using different solvents. Six compounds: 4-Hydroxybenzeneacetic acid; 5-Oxo-pyrrolidine-2-carboxylic acid methyl ester; 1-[3-Methyl-3-Butenyl] Pyrrolidine; Trans-sinapyl alcohol; 5-[Hydroxymethyl]-2-furaldehyde and 2,4- Dihydroxy-2,5-dimethyl-3[2H]-furan-3-one, identified in the fruit-shell extract, are being reported for the first time from this plant. Strong antioxidant potential of the extract was evident from efficient scavenging of free radicals. The extract also conferred protection to yeast cells against oxidative damage. CONCLUSION: Results showed that the hard shell of the Aegle marmelos fruit was a potent source for antioxidant compounds, which can be developed for therapeutic applications in the control and management of metabolic diseases.

Molecular mechanism of the response of Zygosaccharomyces rouxii to D-fructose stress by the glutathione metabolism pathway.

Zygosaccharomyces rouxii produces high levels of 4-hydroxy-2,5-dimethyl-3(2H)-furanone in YPD medium supplemented with 120 g/L D-fructose and 180 g/L NaCl after 5 d. D-fructose has a stress effect on Z. rouxii, and GSH-Px is a main enzyme involved in the defense of Z. rouxii against oxygen stress according to our previous report. In order to further explore the molecular mechanism of the glutathione metabolism pathway in Z. rouxii in response to D-fructose stress, changes in the expression of genes and proteins involved in the synthesis of glutathione precursor amino acids and enzymes were observed. In addition, changes in the intermediates related to glutathione synthesis in Z. rouxii were reported. The results indicated that some gene-encoding enzymes involved in the glutamate, cysteine and glycine biosynthesis pathways and key genes involved in glutathione synthesis were upregulated. The expression levels of other genes, except SHMT, were consistent with the qRT-PCR results. The contents of γ-glutamylcysteine and glutathione amide in the D-fructose group were higher than those in the control group. In the D-fructose stress groups, the metabolic flux towards glutathione synthesis was increased. These results might provide more in-depth and detailed theoretical support for the oxidative stress defense mechanism of Z. rouxii under D-fructose stress.

Kodamaea ohmeri infections in humans: A systematic review.

BACKGROUND: Kodamaea ohmeri, previously known as Pichia ohmeri or Yamadazyma ohmeri, belongs to the Saccharomycetaceae family and the Ascomycetae class, is the telomorphic form of C guilliermondii var. membranaefaciens and is frequently mistaken for Candida, as they belong to the same family. It has been isolated from environmental sources, such as sand, pools, seawater and fruits, while the last decades it is recognised as a rare pathogen that causes life-threatening infections in humans. The purpose of this study was to systemically review all published cases of K ohmeri infections in the literature and describe the epidemiology, microbiology, antimicrobial susceptibility, treatment and outcomes of these infections in humans. METHODS: Systematic review of PubMed (through 27th December 2019) for studies providing epidemiological, clinical, microbiological as well as treatment data and outcomes of K ohmeri infections. RESULTS: A total of 35 studies, containing data of 44 patients, were included in the analysis. The most common K ohmeri infections were those of the bloodstream, infective endocarditis and onychomycosis. Previous antibiotic use, presence of a central venous catheter, parenteral nutrition and cancer were very common among patients. Mortality was high in the case of fungemias but low for other types of infections. Amphotericin B and fluconazole are the most common agents used for treatment, even though alarming MICs for fluconazole were noted. CONCLUSIONS: This systematic review thoroughly describes infections by K ohmeri and provides information on their epidemiology, clinical presentation, microbiology, antibiotic resistance patterns, treatment and outcomes.

Targeting Cdc42 with the anticancer compound MBQ-167 inhibits cell polarity and growth in the budding yeast S. cerevisiae.

The Rho GTPase Cdc42 is highly conserved in structure and function. Mechanical or chemical cues in the microenvironment stimulate the localized activation of Cdc42 to rearrange the actin cytoskeleton and establish cell polarity. A role for Cdc42 in cell polarization was first discovered in the budding yeast Saccharomyces cerevisiae, and subsequently shown to also regulate directional motility in animal cells. Accordingly, in cancer Cdc42 promotes migration, invasion, and spread of tumor cells. Therefore, we targeted Cdc42 as a therapeutic strategy to treat metastatic breast cancer and designed the small molecule MBQ-167 as a potent inhibitor against Cdc42 and the homolog Rac. MBQ-167 inhibited cancer cell proliferation and migration in-vitro, and tumor growth and spread in-vivo in a mouse xenograft model of metastatic breast cancer. Since haploid budding yeast express a single Cdc42 gene, and do not express Rac, we used this well characterized model of polarization to define the contribution of Cdc42 inhibition to the effects of MBQ-167 in eukaryotic cells. Growth, budding pattern, and Cdc42 activity was determined in wildtype yeast or cells expressing a conditional knockdown of Cdc42 in response to vehicle or MBQ-167 treatment. As expected, growth and budding polarity were reduced by knocking-down Cdc42, with a parallel effect observed with MBQ-167. Cdc42 activity assays confirmed that MBQ-167 inhibits Cdc42 activation in yeast, and thus, bud polarity. Hence, we have validated MBQ-167 as a Cdc42 inhibitor in another biological context and present a method to screen Cdc42 inhibitors with potential as anti-metastatic cancer drugs.

Saprochaete clavata infections in patients undergoing treatment for haematological malignancies: A report of a monocentric outbreak and review of the literature.

Saprochaete clavata is a rare cause of fungaemia with deep organ involvement in patients with haematological malignancies with reported mortality rates of 60%-80%. We describe four cases of S clavata infection in a haematology unit over several months that were treated with voriconazole-based regimens. We also review the literature on factors that could contribute to earlier recognition and effective treatment of S clavata. We included all cases of culture-positive S clavata from sterile sites with associated signs of infection in patients undergoing treatment for a haematological malignancy. Isolates were identified by MALDI-TOF MS, and spectrum profiles were used to prepare clustering analysis of isolates. Susceptibility testing was performed using a commercial microtitre methods. Saprochaete clavata was isolated from the bloodstream in three cases and bronchial alveolar lavage (BAL) fluid in one case. Clustering analysis suggested strains of S clavata were clonal without evidence of divergence although a common source was not identified. Susceptibility testing yielded elevated MICs to fluconazole (8 mg/L) and echinocandins (>1-8 mg/L). All patients were treated with voriconazole-based regimens resulting in survival of 3/4 patients, who continued chemotherapy for their underlying malignancy without evidence of relapse. Saprochaete clavata is a rare but aggressive cause of breakthrough yeast infection in patients undergoing treatment for haematological malignancies, particularly patients with a prior history of echinocandin treatment. Timely initiation of appropriate treatment, aided by more rapid identification in microbiology laboratory, can reduce the risk of deep organ dissemination and patient death.

Virulence properties and sensitivity profile of Candida parapsilosis complex species and Kodamaea ohmeri isolates from onychomycosis of HIV/AIDS patients.

Cutaneous fungal infections include onychomycosis, an infection of the nail that affects both healthy and immunocompromised patients. This study investigated the in vitro hydrolytic enzymes production, adhesion and biofilm formation capacity of Candida parapsilosis complex species and Kodamaea ohmeri isolates from onychomycoses of HIV/AIDS patients and also established the antifungal sensitivity profiles of these isolates. Onychomycosis in HIV/AIDS patients showed a high prevalence of emerging yeasts, among which C. parapsilosis complex species and K. ohmeri were the most frequent. Three C. parapsilosis sensu stricto and two C. orthopsilosis isolates were resistant to amphotericin B and 83% of isolates were resistant to terbinafine. All three different species evaluated were proteinase and hemolysin producers. All isolates adhered to stainless steel and siliconized latex surfaces, and carbohydrates intensified adhesion of all isolates. Isolates adhered to keratinous nail and 50% formed biofilms with strong intensity. In multispecies or polymicrobial biofilms, C. albicans and Staphylococcus aureus regulated the biofilm formation of the analyzed species, decreasing the number of their cells in biofilms. The isolation of emerging yeast species from onychomycosis which are great producers of hydrolytic enzymes and with high adhesion and biofilm formation capacity is a result that should be considered relevant in clinical practice. In addition, half of the isolates was resistant to at least one of the tested antifungals. Taken together these data corroborate the infectious capacity and viability of these isolates under favorable conditions.

Solid-liquid Interface Screening SystemーApplication to the Screening of Antibiotic and Cytotoxic Substance-producing Fungi.

　A useful tool for the screening of fungi producing biologically active secondary metabolites such as antibiotics and cytotoxic substances has been developed. An agar plate-organic solvent interface cultivation (A/S-IFC) system, which comprised a hydrophobic organic solvent (upper phase) , a fungal mat (middle phase) and an agar plate (lower phase) , was constructed. The metabolite profiles were compared among the A/S-IFC, a traditional submerged cultivation (SmC) and an extractive liquid surface immobilization (Ext-LSI) system consisted of a hydrophobic solvent (upper phase) , a fungal cells-ballooned microspheres (middle phase) and a liquid medium (lower phase) , with high-performance liquid chromatography-photodiode array detector (HPLC-PDA) . In the A/S-IFC, many hydrophobic metabolites vastly different from those in the SmC were accumulated in the organic phase as with the Ext-LSI. For example, a valuable azaphilone, sclerotiorin, was remarkably produced into the organic phase in the A/S-IFC. The A/S-IFC was applied to the screening of antibiotic-producing fungi. As a result of paper disk method, it was found that 321 isolated among 811 strains produced antifungal metabolites (hit rate, 39.6%) . Furthermore, 8, 23, and 30 strains also produced cytotoxic metabolites against SKOV-3 (human ovary adenocarcinoma) , MESO-1 (human malignant pleural mesothelioma) , and Jurkat cells (immortalized human T lymphocyte) .

Effect of GAPDH-derived antimicrobial peptides on sensitive yeasts cells: membrane permeability, intracellular pH and H+-influx/-efflux rates.

Saccharomyces cerevisiae secretes antimicrobial peptides (AMPs) derived from glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which induce the death of several non-Saccharomyces yeasts. Previously, we demonstrated that the naturally secreted GAPDH-derived AMPs (i.e. saccharomycin) caused a loss of culturability and decreased the intracellular pH (pHi) of Hanseniaspora guilliermondii cells. In this study, we show that chemically synthesised analogues of saccharomycin also induce a pHi drop and loss of culturability in H. guilliermondii, although to a lesser extent than saccharomycin. To assess the underlying causes of the pHi drop, we evaluated the membrane permeability to H+ cations of H. guilliermondii cells, after being exposed to saccharomycin or its synthetic analogues. Results showed that the H+-efflux decreased by 75.6% and the H+-influx increased by 66.5% in cells exposed to saccharomycin at pH 3.5. Since H+-efflux via H+-ATPase is energy dependent, reduced glucose consumption would decrease ATP production and consequently H+-ATPase activity. However, glucose uptake rates were not affected, suggesting that the AMPs rather than affecting glucose transporters may affect directly the plasma membrane H+-ATPase or increase ATP leakage due to cell membrane disturbance. Thus, our study revealed that both saccharomycin and its synthetic analogues induced cell death of H. guilliermondii by increasing the proton influx and inhibiting the proton efflux.

Identification of uncommon oral yeasts from cancer patients by MALDI-TOF mass spectrometry.

BACKGROUND: Opportunistic infections due to Candida species occur frequently in cancer patients because of their inherent immunosuppression. The aim of the present study was to investigate the epidemiology of yeast species from the oral cavity of patients during treatment for oncological and haematological malignancies. METHODS: MALDI-TOF was performed to identify yeasts isolated from the oral cavity of 350 cancer patients. Moreover, antifungal susceptibility testing was performed in according to CLSI guidelines (M27-A3). RESULTS: Among 162 yeasts and yeast-like fungi isolated from the oral cavity of cancer patients, Candida albicans was the most common species (50.6%), followed by Candida glabrata (24.7%), Pichia kudriavzevii (Candida krusei (9.9%)), Candida tropicalis (4.3%), Candida dubliniensis (3.7%), Kluyveromyces marxianus (Candida kefyr (3.7%)) and Candida parapsilosis (1%). In addition, uncommon yeast species i.e., Saprochaete capitata, Saccharomyces cerevisiae, Clavispora lusitaniae (C. lusitaniae) and Pichia kluyveri (C. eremophila) were recovered from oral lesions. Oral colonization by C. albicans, non-albicans Candida species and uncommon yeasts were as follow; 55%, 44% and 1%, whereas oral infection due to C. albicans was 33.3%, non-albicans Candida species 60.6%, and uncommon yeasts 6.1%. Poor oral hygiene and xerostomia were identified as independent risk factors associated with oral yeast colonization. The overall resistance to fluconazole was 11.7% (19/162). Low MIC values were observed for anidulafungin for all Candida and uncommon yeast species. CONCLUSIONS: This current study provides insight into the prevalence and susceptibility profiles of Candida species, including emerging Candida species and uncommon yeasts, isolated from the oral cavity of Iranian cancer patients. The incidence of oral candidiasis was higher amongst patients with hematological malignancies. The majority of oral infections were caused by non-albicans Candida species which were often more resistant to anti-fungal agents. Our findings suggest that anidulafungin should be used as antifungal of choice for prophylaxis in clinically high-risk patients with documented oral colonization or infection.

Endocrine effects after ozonation of tamoxifen.

Ozonation is used as additional wastewater treatment option to remove recalcitrant micropollutants. It also removes the estrogenic activity found in wastewater but not always the anti-estrogenic activity. This can be explained by an incomplete removal of anti-estrogenic micropollutants or by formation of transformation products (TPs) which retain the activity. The present study investigates the degradation of the anti-estrogenic pharmaceutical tamoxifen in pure water, regarding TP formation and related anti-estrogenic effect using Arxula adeninivorans yeast estrogen screen (A-YES). In total, five transformation products were detected: three N-oxides and two further products (TP 270 and TP 388). For the transformation product TP 270 a correlation of the extent of formation with an increase of the anti-estrogenic activity was determined, demonstrating that transformation products from ozonation can be more active in a bioassay than the parent compounds. Our study shows also that the transformation of tamoxifen to N-oxides reduces the anti-estrogenic activity. The reactivity of amines towards ozone typically increases with pH, since only deprotonated amines react with ozone. Hence, removal of the endocrine activity by N-oxide formation may be disfavored at low pH.

Molecular Genetic Characterization of Mutagenesis Using a Highly Sensitive Single-Stranded DNA Reporter System in Budding Yeast.

Mutations are permanent alterations to the coding content of DNA. They are starting material for the Darwinian evolution of species by natural selection, which has yielded an amazing diversity of life on Earth. Mutations can also be the fundamental basis of serious human maladies, most notably cancers. In this chapter, I describe a highly sensitive reporter system for the molecular genetic analysis of mutagenesis, featuring controlled generation of long stretches of single-stranded DNA in budding yeast cells. This system is ~100- to ~1000-fold more susceptible to mutation than conventional double-stranded DNA reporters, and is well suited for generating large mutational datasets to investigate the properties of mutagens.

Magnusiomyces capitatus Peritonitis in a Child With Acute Lymphocytic Leukemia as a Breakthrough Infection During Caspofungin Therapy.

Magnusiomyces capitatus is an emerging opportunistic fungal pathogen particularly in immunocompromised patients. We report a case of a M. capitatus peritonitis in child with acute lymphocytic leukemia as a breakthrough infection during caspofungin therapy. The possibility of breakthrough infections caused by M. capitatus must be taken into consideration, particularly in immunosupressed patients being treated for systemic fungal infections by caspofungin. Although there are no defined breakpoints for susceptibility testing of M.capitatus, minimal inhibitory concentration results can be helpful for therapy. Antifungal treatment with amphotericin B lipid complex plus flucytosine can be effective against infections caused by M. capitatus.

Biotransformation of a potent anabolic steroid, mibolerone, with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina, and biological activity evaluation of its metabolites.

Seven metabolites were obtained from the microbial transformation of anabolic-androgenic steroid mibolerone (1) with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina. Their structures were determined as 10ß,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (2), 6ß,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (3), 6ß,10ß,17ß-trihydroxy-7α,17α-dimethylestr-4-en-3-one (4), 11ß,17ß-dihydroxy-(20-hydroxymethyl)-7α,17α-dimethylestr-4-en-3-one (5), 1α,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (6), 1α,11ß,17ß-trihydroxy-7α,17α-dimethylestr-4-en-3-one (7), and 11ß,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (8), on the basis of spectroscopic studies. All metabolites, except 8, were identified as new compounds. This study indicates that C. blakesleeana, and C. echinulata are able to catalyze hydroxylation at allylic positions, while M. phaseolina can catalyze hydroxylation of CH2 and CH3 groups of substrate 1. Mibolerone (1) was found to be a moderate inhibitor of ß-glucuronidase enzyme (IC50 = 42.98 ± 1.24 µM) during random biological screening, while its metabolites 2-4, and 8 were found to be inactive. Mibolerone (1) was also found to be significantly active against Leishmania major promastigotes (IC50 = 29.64 ± 0.88 µM). Its transformed products 3 (IC50 = 79.09 ± 0.06 µM), and 8 (IC50 = 70.09 ± 0.05 µM) showed a weak leishmanicidal activity, while 2 and 4 were found to be inactive. In addition, substrate 1 (IC50 = 35.7 ± 4.46 µM), and its metabolite 8 (IC50 = 34.16 ± 5.3 µM) exhibited potent cytotoxicity against HeLa cancer cell line (human cervical carcinoma). Metabolite 2 (IC50 = 46.5 ± 5.4 µM) also showed a significant cytotoxicity, while 3 (IC50 = 107.8 ± 4.0 µM) and 4 (IC50 = 152.5 ± 2.15 µM) showed weak cytotoxicity against HeLa cancer cell line. Compound 1 (IC50 = 46.3 ± 11.7 µM), and its transformed products 2 (IC50 = 43.3 ± 7.7 µM), 3 (IC50 = 65.6 ± 2.5 µM), and 4 (IC50 = 89.4 ± 2.7 µM) were also found to be moderately toxic to 3T3 cell line (mouse fibroblast). Interestingly, metabolite 8 showed no cytotoxicity against 3T3 cell line. Compounds 1-4, and 8 were also evaluated for inhibition of tyrosinase, carbonic anhydrase, and α-glucosidase enzymes, and all were found to be inactive.