Production of tannase from a newly isolated yeast, Geotrichum cucujoidarum using agro-residues.

With an aim of producing commercially important tannase enzyme using cheap and readily available agro-residues, leaves of Indian Gooseberry (Phyllanthus emblica) and Jamun (Syzygium cumini), peels of Lemon (Citrus limon), and Pomegranate (Punica granatum) were screened. Newly isolated Geotrichum cucujoidarum was utilized for the study. Preliminary studies indicated that tannase titer obtained is not proportional to the tannin content of the agro-residues and solid state fermentation superior compared to submerged fermentation. Jamun mixed with lemon peel in equal proportion supplemented with minerals under solid-state fermentation gave a tannase titer of 15.46 U/g dry solids. Through successful implantation of Plackett-Burman design, yeast extract concentration, inoculum volume, and amount of substrate were found to be the most significant factors. Further optimization of these three factors through Response Surface Methodology resulted in the 1.7-fold increase in tannase titer. Validation experiments using 3.97 g of Jamun leaves + lemon peel powder mixed with a nutrient solution having (w/v) yeast extract - 1.1%, dextrose - 3%, Urea - 1.125%, potassium chloride - 0.1%, magnesium sulfate heptahydrate - 0.1% with the initial pH of 5, inoculated with 2.48 ml of inoculum gave a tannase titer of 26.43 U/g dry solids after 6 days of solid-state fermentation.

New Data on Effects of SkQ1 and SkQT1 on Rat Liver Mitochondria and Yeast Cells.

Mitochondria are involved in many processes in eukaryotic cells. They play a central role in energy conservation and participate in cell metabolism and signaling pathways. Mitochondria are the main source of reactive oxygen species, excessive generation of which provokes numerous pathologies and cell death. One of the most promising approaches to the attenuation of oxidative stress in mitochondria is the use of targeted (i.e., transported exclusively into mitochondria) lipophilic cationic antioxidants. These compounds offer advantages over conventional water-soluble antioxidants because they induce the so-called "mild uncoupling" and can prevent collapse of the membrane potential in low, nontoxic concentrations. A novel mitochondria-targeted antioxidant, SkQT1, was synthesized and tested within the framework of the research project guided by V. P. Skulachev. The results of these experiments were initially reported in 2013; however, one publication was not able to accommodate all the data on the SkQT1 interactions with isolated mitochondria and cells. Here, we examined comparative effects of SkQT1 and SkQ1 on rat liver mitochondria (with broader spectrum of energy parameters being studied) and yeast cells. SkQT1 was found to be less effective uncoupler, depolarizing agent, inhibitor of respiration and ATP synthesis, and "opener" of a nonspecific pore compared to SkQ1. At the same time SkQ1 exhibited higher antioxidant activity. Both SkQT1 and SkQ1 prevented oxidative stress and mitochondria fragmentation in yeast cells exposed to t-butyl hydroperoxide and promoted cell survival, with SkQT1 being more efficient than SkQ1. Together with the results presented in 2013, our data suggest that SkQT1 is the most promising mitochondria-targeted antioxidant that can be used for preventing various pathologies associated with the oxidative stress in mitochondria.

Genotyping and Antifungal Susceptibility of Dipodascus capitatus Isolated in a Neonatal Intensive Care Unit of a Sicilian Hospital.

In August 2015, Dipodascus capitatus was isolated from two patients admitted to the neonatal intensive care unit. Nosocomial acquisition of the fungus was suspected and epidemiological studies were undertaken. The patients were simultaneously hospitalized, and the comparison of the two isolates by two independent molecular typing methods have confirmed clonal dissemination of a single strain of D. capitatus. Antimicrobial susceptibility testing was useful for identifying the appropriated antifungal therapy in micafungin. To our knowledge these are the first described cases of neonatal D. capitatus infection and also the first report of successful treatment by micafungin.

[Role of surgery in Saprochaete capitata (S. capitata) sepsis in a patient with acute myeloid leukemia]. / Rol de la cirugía en el manejo de la infección invasora por Saprochaete capitata.

Saprochaete capitata (S. capitata) fungal sepsis is a severe condition with a clinical presentation that is similar to other yeast originated fungal sepsis. It is observed in patients with hematological malignancies such as acute myeloid leukemia and neutropenia. We report a 23 year old male presenting with cough, fever and malaise. A bone marrow biopsy led to the diagnosis of acute myeloid leukemia. During the first cycle of chemotherapy the patient presented fever: blood cultures were positive for Klebsiella pneumoniae. Despite antimicrobial treatment, fever persisted; a computed tomography showed a focal splenic lesion; a left exudative pleural effusion appeared. A Matrix Assisted Laser Desorption Ionization-Time of Flight mass spectrometry identified the presence of S. capitata. After multiple antifungal treatments and pleural cavity cleansing by means of videothoracoscopy and laparoscopic splenectomy, the infection resolved and the patient completed his chemotherapy.

Subcutaneous infection caused by Blastoschizomyces capitatus post allogeneic hematopoietic transplant and its successful treatment with voriconazole.

We describe a 33-year-old man with relapsed acute myelogenous leukemia who developed subcutaneous nodules >6 months after allogeneic hematopoietic stem cell transplant. These nodules were caused by Blastoschizomyces capitatus. The lesions progressed after treatment with a posaconazole suspension. The lesions resolved after switching to voriconazole, which was given for 21 weeks. B. capitatus is a rare infection affecting immunocompromised patients, which responds to azoles.

Optimizing biocatalytic potential of Dipodascus australiensis M-2 for degrading lignin under laboratory conditions.

In present research, a potent fungal strain was isolated from paper mill effluent (black liquor) in order to investigate its potential for the biodegradation of lignin. Two step strategy was used to screen most efficient fungal strain having ability to growin MSM-black liquor medium and to degrade alkali lignin.The results of initial screening indicated that the strain M-2 produced comparatively higher ligninolytic zone on MSN agar plates supplemented with black liquor (BL) and alkali ligninase compared to the other isolates.The results of 18S rRNA gene sequencing revealed that strain M-2 showed ≥ 99% sequence homology with Dipodasceus australiansis.The process for the biodegradation of lignin was optimized using Taguchi Orthogonal Array design. Under optimized conditions of pH 9, 40 °C and 4% inoculum, a maximum of 89% lignin was degraded with 41% color reduction after 8 days of incubation period by Dipodasceus australiansis M-2. The pH and temperature were found to be significant terms with the p-values of 0.002 and 0.001 respectively. The laccase activity of the Dipodascus australiensis was found to be maximum of 1.511 U/mL. The HPLC analysis of lignin biodegradation indicated sharp transformation of peaks as compared to the control. Our results suggested that the strain Dipodascus australiensis M-2 possess excellent lignin degradation and color reduction capability and can be applied in waste treatment systems for pulp and paper mill effluent. In present work we are reporting first hand information regarding biodegradation of lignin by a potent strain of Dipodascus australiensis and statistical optimization of the bioprocess.

Mitochondria from Dipodascus (Endomyces) magnusii and Yarrowia lipolytica yeasts did not undergo a Ca²âº-dependent permeability transition even under anaerobic conditions.

In this study we used tightly-coupled mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts. The two yeast strains are good alternatives to Saccharomyces cerevisiae, being aerobes containing well structured mitochondria (thus ensuring less structural limitation to observe their appreciable swelling) and fully competent respiratory chain with three invariantly functioning energy conservation points, including Complex I, that can be involved in induction of the canonical Ca²âº/P(i)-dependent mitochondrial permeability transition (mPTP pore) with an increased open probability when electron flux increases(Fontaine et al. J Biol Chem 273: 25734­25740, 1998; Bernardi et al. FEBS J 273:2077­2099, 2006). High amplitude swelling and collapse of the membrane potential were used as parameters for demonstrating pore opening. Previously (Kovaleva et al. J Bioenerg Biomembr 41:239­249, 2009; Kovaleva et al. Biochemistry (Moscow) 75: 297­303, 2010) we have shown that mitochondria from Y.lipolytica and D. magnusii were very resistant to the Ca²âºoverload combined with varying concentrations of P(i),palmitic acid, SH-reagents, carboxyatractyloside (an inhibitor of ADP/ATP translocator), as well as depletion of intramitochondrial adenine nucleotide pools, deenergization of mitochondria, and shifting to acidic pH values in the presence of high [P(i)]. Here we subjected yeast mitochondria to other conditions known to induce an mPTP in animal and plant mitochondria, namely to Ca²âº overload under hypoxic conditions (anaerobiosis). We were unable to observe Ca²âº-induced high permeability of the inner membrane of D. magnusii and Y. lipolytica yeast mitochondria under anaerobic conditions, thus suggesting that an mPTP-like pore, if it ever occurs in yeast mitochondria, is not coupled with the Ca²âº uptake. The results provide the first demonstration of ATP-dependent energization of yeast mitochondria under conditions of anaerobiosis.

Effect of prooxidants on yeast mitochondria.

Tightly coupled mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts were used in this study. The two yeasts are aerobes containing the fully competent respiratory chain with three energy conservation sites. Interaction of the yeast mitochondria with prooxidants (diamide, menadione, oxaloacetate, phenylarsine oxide, hydrogen peroxide, t-butyl peroxide, and ascorbate plus Fe(2+)) was studied. The prooxidants, depending on their chemical nature, either caused uncoupling (e.g., activated state 4 respiration) or inhibited oxidation of respiratory substrates. All of the agents dissipated the membrane potential without megachannel formation (no large-scale swelling of mitochondria was observed). Except for combined application of ascorbate and Fe(2+), the prooxidant-induced decrease in the membrane potential was specifically prevented by ATP, even in the cases when classic antioxidants, e.g., N-acetylcysteine, were ineffective. No permeabilization of yeast mitochondria was observed under concerted action of prooxidants and Ca(2+), suggesting that an mPTP-like pore, if it ever occurs in yeast mitochondria, is not coupled with Ca(2+) uptake.

In vitro antifungal activities of isavuconazole and comparators against rare yeast pathogens.

We compared the in vitro activities of isavuconazole, posaconazole, voriconazole, and fluconazole against Dipodascus capitatus (n = 21), Saccharomyces cerevisiae (n = 20), Rhodotorula mucilaginosa (n = 18), and Trichosporon spp. (n = 15). The MIC(50)s, MIC(90)s, and MIC ranges (in microg/ml) obtained using the CLSI M27-A3 procedure were as follows: isavuconazole, 0.125, 0.5, and < or = 0.015 to 2; posaconazole, 0.5, 2, and < or = 0.015 to > 16; voriconazole, 0.125, 2, and < or = 0.015 to 8; and fluconazole, 4, > 128, and < or = 0.125 to > 128. Isavuconazole showed potent activity against the isolates studied.

Interaction of yeast mitochondria with fatty acids and mitochondria-targeted lipophilic cations.

The effect of fatty acids and mitochondria-targeted lipophilic cations (SkQ1, SkQ3, MitoQ, and C(12)TPP) on tightly-coupled mitochondria from yeasts Dipodascus (Endomyces) magnusii and Yarrowia lipolytica was investigated. Micromolar concentrations of saturated and unsaturated fatty acids were found to decrease the membrane potential, which was recovered almost totally by ATP and BSA. At low, micromolar concentrations, mitochondria-targeted lipophilic cations are "relatively weak, mild uncouplers", at higher concentrations they inhibit respiration in state 3, and at much higher concentrations they induce swelling of mitochondria, possibly due to their prooxidant and detergent action. At very low, not uncoupling concentrations, mitochondria-targeted lipophilic cations profoundly promote (potentiate) the uncoupling effect of fatty acids. It is conceivable that the observed uncoupling effect of lipophilic cations can be, at least partially, due to their interactions with the endogenous pool of fatty acids.

Induction of a non-specific permeability transition in mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts.

In this study we used tightly-coupled mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts, possessing a respiratory chain with the usual three points of energy conservation. High-amplitude swelling and collapse of the membrane potential were used as parameters for demonstrating induction of the mitochondrial permeability transition due to opening of a pore (mPTP). Mitochondria from Y. lipolytica, lacking a natural mitochondrial Ca(2+) uptake pathway, and from D. magnusii, harboring a high-capacitive, regulated mitochondrial Ca(2+) transport system (Bazhenova et al. J Biol Chem 273:4372-4377, 1998a; Bazhenova et al. Biochim Biophys Acta 1371:96-100, 1998b; Deryabina and Zvyagilskaya Biochemistry (Moscow) 65:1352-1356, 2000; Deryabina et al. J Biol Chem 276:47801-47806, 2001) were very resistant to Ca(2+) overload. However, exposure of yeast mitochondria to 50-100 microM Ca(2+) in the presence of the Ca(2+) ionophore ETH129 induced collapse of the membrane potential, possibly due to activation of the fatty acid-dependent Ca(2+)/nH(+)-antiporter, with no classical mPTP induction. The absence of response in yeast mitochondria was not simply due to structural limitations, since large-amplitude swelling occurred in the presence of alamethicin, a hydrophobic, helical peptide, forming voltage-sensitive ion channels in lipid membranes. Ca(2+)- ETH129-induced activation of the Ca(2+)/H(+)-antiport system was inhibited and prevented by bovine serum albumin, and partially by inorganic phosphate and ATP. We subjected yeast mitochondria to other conditions known to induce the permeability transition in animal mitochondria, i.e., Ca(2+) overload (in the presence of ETH129) combined with palmitic acid (Mironova et al. J Bioenerg Biomembr 33:319-331, 2001; Sultan and Sokolove Arch Biochem Biophys 386:37-51, 2001), SH-reagents, carboxyatractyloside (an inhibitor of the ADP/ATP translocator), depletion of intramitochondrial adenine nucleotide pools, deenergization of mitochondria, and shifting to acidic pH values in the presence of high phosphate concentrations. None of the above-mentioned substances or conditions induced a mPTP-like pore. It is thus evident that the permeability transition in yeast mitochondria is not coupled with Ca(2+) uptake and is differently regulated compared to the mPTP of animal mitochondria.

Cervical spondylodiscitis caused by Blastoschizomyces capitatus.

A 37-year-old woman, during her second remission of acute myeloid leukemia, presented with severe neck pain and cervico-brachial neuralgia. Investigation revealed a C5-C6 spondylodiscitis. A CT-guided anterior biopsy decompressed the mass, immediately alleviated the symptoms, and isolated a rare yeast: Blastoschizomyces capitatus. To our knowledge, only three cases of spondylodiscitis with this yeast have been described. Six months of voriconazole and liposomal amphotericin B treatment produced a complete resolution on CT and MRI imaging. However, the ongoing severe yeast infection prevented the planned bone marrow allograft.

Spondylodiscitis due to an emergent fungal pathogen: Blastoschizomyces capitatus, a case report and review of the literature.

The study includes a case report and a literature review. The main objective of this study is to present a case of spondylodiscitis due to a fungal pathogen, Blastoschizomyces capitatus and to review the published literature on this emergent fungus in etiology of spondylodiscitis, and osteomyelitis. Osteoarticular involvement due to B. capitatus has been reported in six cases, and vertebral involvement has been seen in five of them. All of these cases had underlying malignancy. Infection is usually advanced at presentation. Case notes and online databases were reviewed. Organism was isolated from bone material in all of the cases and antibiotic treatment by antifungal agents cured the infection. We present another case of infectious spondylodiscitis due to B. capitaus, which is reported first in Turkey and tried to attract attendance to this emergent fungal pathogen as an etiologic agent of spine infections in cancer patients.

[Investigation of in vitro activities of fluconazole, itraconazole and voriconazole against clinical isolates of Blastoschizomyces capitatus (Geotrichum capitatum) by two different methods]. / Flukonazol, itrakonazol ve vorikonazolün klinik Blastoschizomyces capitatus (Geotrichum capitatum) izolatlarina karsi in vitro aktivitesinin iki farkli yöntemle arastirilmasi.

Blastoschizomyces capitatus is an emerging opportunistic pathogen that may lead to invasive infections particularly in neutropenic patients. In this study, the in vitro activities of fluconazole, itraconazole and voriconazole against 15 clinical B. copitatus isolates were determined by microdilution method (MD) and Etest. The isolation and identification of the isolates were done by standard mycological methods. MD tests were done in accordance with CLSI microdilution method (M27A-2). Etest was performed according to the instructions of the manufacturer (AB Biodisk, Sweden) by using RPMI-2% glucose. Since susceptibility breakpoints were not yet established for B. copitatus, only the distribution of the MIC values obtained for the tested antifungals were given. MIC values were determined after 48 h incubation and by using MIC-2 value for all the drugs tested. At 48 h, MIC90 values obtained by MD and Etest were 16 and 32 microg/ml, 0.5 and 1 microg/ml, 0.5 and 1 microg/ml for fluconazole, itraconazole and voriconazole, respectively. These results suggested that voriconazole and itraconazole had favorable activity against B. capitatus isolates. However, the activity of fluconazole remained poor and limited at least for a significant number of isolates. Percent agreement of Etest with MD method within +/-1 dilution range and at 48 hour for fluconazole, itraconazole and voriconazole were 86.7%, 80% and 73.3%, respectively, suggesting a higher agreement of the two methods for fluconazole as compared to itraconazole and voriconazole. Etest tended to generate 1-2 fold higher MICs as compared to MD MICs for most of the isolates of this particular fungus. In conclusion, further studies are required for determination of the optimal susceptibility testing method and the MIC breakpoints for B. capitatus.

Magnusiomyces capitatus: a new and emerging pathogen linked to keratomycosis.

We describe our experience managing a case of fungal keratitis in a 71-year-old immunocompetent woman. Intracameral cultures isolated Magnusiomyces capitatus, a dimorphic commensal microbe with limited pathogenicity, in an immune competent patient. Repeated corneal cultures for bacteria and fungus were negative. Initial broad-spectrum topical fortified antibiotics were eventually escalated to include topical natamycin and voriconazole and oral voriconazole. The patient underwent emergent corneal transplantation, but recurrent infection eventually required anterior chamber washout and intracameral injection of amphotericin B. A combination of topical, systemic, intracameral administration of voriconazole, intracameral administration of amphotericin B, anterior chamber washout, and penetrating keratoplasty were required to clear the infection.

Identification of an elongation factor 1Bγ protein with glutathione transferase activity in both yeast and mycelial morphologies from human pathogenic Blastoschizomyces capitatus.

Blastoschizomyces capitatus is an uncommon, opportunistic pathogenic fungus, which causes invasive and disseminated infections. This microorganism is normally present in both environmental and normal human flora. Within a host, B. capitatus is able to grow in both unicellular yeast and multicellular filamentous growth forms. In this study, we obtained in vitro morphological conversion of B. capitatus from yeast-to-mycelial phase to investigate the presence and expression of glutathione transferase (GST) enzymes in both cell forms. A protein with GST activity using the model substrate 1-chloro-2,4-dinitrobenzene was detected in both morphologies and identified by tandem mass spectrometry as a eukaryotic elongation factor 1Bγ (eEF1Bγ) protein, a member of the GST superfamily. No significant difference in GST-specific activity and kinetic constants were observed between mycelial and yeast forms, indicating that eEF1Bγ protein did not show differential expression between the two phases.

[Multi-organ failure in a patient with fungaemia due to Saprochaete capitata]. / Fallo multiorgánico en paciente con fungemia por Saprochaete capitata.

BACKGROUND: The significant increase in systemic fungal infections is mainly due to the increase in immunocompromised patients. The high morbimortality of these infections, along with the high hospitalization costs they generate, makes them a problem of great importance in our hospital practice. Saprochaete capitata is a rare fungus that causes invasive infections, usually in immunocompromised patients, and for which there is still no consensus on the treatment regimen to be used. CASE REPORT: We present a case of disseminated infection by this fungus in a heavily immunosuppressed patient, who died as a result of multiple organ failure despite the life support measures taken and the wide spectrum antibiotics. CONCLUSIONS: It is vital to begin the antibiotic treatment as soon as possible, as well as the monitoring and follow-up cultures to test for fungi in neutropenic patients.