Publications by authors named "Luca Roscini"

35 Publications

Single Strain High-Depth NGS Reveals High rDNA (ITS-LSU) Variability in the Four Prevalent Pathogenic Species of the Genus .

Microorganisms 2021 Feb 2;9(2). Epub 2021 Feb 2.

Department of Pharmaceutical Sciences, University of Perugia, 06121 Perugia, Italy.

Ribosomal RNA in fungi is encoded by a series of genes and spacers included in a large operon present in 100 tandem repeats, normally in a single locus. The multigene nature of this locus was somehow masked by Sanger sequencing, which produces a single sequence reporting the prevalent nucleotide of each site. The introduction of next generation sequencing led to deeper knowledge of the individual sequences (reads) and therefore of the variants between the same DNA sequences located in different tandem repeats. In this framework, NGS sequencing of the rDNA region was used to elucidate the extent of intra- and inter-genomic variation at both the strain and species level. Specifically, the use of an innovative NGS technique allowed the high-throughput high-depth sequencing of the ITS1-LSU D1/D2 amplicons of 252 strains belonging to four opportunistic yeast species of the genus . Results showed the presence of a large extent of variability among strains and species. These variants were differently distributed throughout the analyzed regions with a higher concentration within the Internally Transcribed Spacer (ITS) region, suggesting that concerted evolution was not able to totally homogenize these sequences. Both the internal variability and the SNPs between strain can be used for a deep typing of the strains and to study their ecology.
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http://dx.doi.org/10.3390/microorganisms9020302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7912828PMC
February 2021

Do Metabolomics and Taxonomic Barcode Markers Tell the Same Story about the Evolution of Complex in Fermentative Environments?

Microorganisms 2020 Aug 15;8(8). Epub 2020 Aug 15.

Department of Pharmaceutical Sciences, University of Perugia, 06121 Perugia, Italy.

Yeast taxonomy was introduced based on the idea that physiological properties would help discriminate species, thus assuming a strong link between physiology and taxonomy. However, the instability of physiological characteristics within species configured them as not ideal markers for species delimitation, shading the importance of physiology and paving the way to the DNA-based taxonomy. The hypothesis of reconnecting taxonomy with specific traits from phylogenies has been successfully explored for Bacteria and Archaea, suggesting that a similar route can be traveled for yeasts. In this framework, thirteen single copy loci were used to investigate the predictability of complex Fourier Transform InfaRed spectroscopy (FTIR) and High-performance Liquid Chromatography-Mass Spectrometry (LC-MS) profiles of the four historical species of the group, both on resting cells and under short-term ethanol stress. Our data show a significant connection between the taxonomy and physiology of these strains. Eight markers out of the thirteen tested displayed high correlation values with LC-MS profiles of cells in resting condition, confirming the low efficacy of FTIR in the identification of strains of closely related species. Conversely, most genetic markers displayed increasing trends of correlation with FTIR profiles as the ethanol concentration increased, according to their role in the cellular response to different type of stress.
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http://dx.doi.org/10.3390/microorganisms8081242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463906PMC
August 2020

Delta-Integration of Single Gene Shapes the Whole Metabolomic Short-Term Response to Ethanol of Recombinant Strains.

Metabolites 2020 Apr 3;10(4). Epub 2020 Apr 3.

Department of Pharmaceutical Sciences, University of Perugia, 06121 Perugia, Italy.

In yeast engineering, metabolic burden is often linked to the reprogramming of resources from regular cellular activities to guarantee recombinant protein(s) production. Therefore, growth parameters can be significantly influenced. Two recombinant strains, previously developed by the multiple δ-integration of a glucoamylase in the industrial 27P, did not display any detectable metabolic burden. In this study, a Fourier Transform InfraRed Spectroscopy (FTIR)-based assay was employed to investigate the effect of δ-integration on yeast strains' tolerance to the increasing ethanol levels typical of the starch-to-ethanol industry. FTIR fingerprint, indeed, offers a holistic view of the metabolome and is a well-established method to assess the stress response of microorganisms. Cell viability and metabolomic fingerprints have been considered as parameters to detecting any physiological and/or metabolomic perturbations. Quite surprisingly, the three strains did not show any difference in cell viability but metabolomic profiles were significantly altered and different when the strains were incubated both with and without ethanol. A LC/MS untargeted workflow was applied to assess the metabolites and pathways mostly involved in these strain-specific ethanol responses, further confirming the FTIR fingerprinting of the parental and recombinant strains. These results indicated that the multiple δ-integration prompted huge metabolomic changes in response to short-term ethanol exposure, calling for deeper metabolomic and genomic insights to understand how and, to what extent, genetic engineering could affect the yeast metabolome.
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http://dx.doi.org/10.3390/metabo10040140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7241245PMC
April 2020

Metabolomic Alterations Do Not Induce Metabolic Burden in the Industrial Yeast M2n[pBKD2-]-C1 Engineered by Multiple δ-Integration of a Fungal β-Glucosidase Gene.

Front Bioeng Biotechnol 2019 28;7:376. Epub 2019 Nov 28.

Department of Pharmaceutical Sciences-Microbiology, University of Perugia, Perugia, Italy.

In the lignocellulosic yeast development, metabolic burden relates to redirection of resources from regular cellular activities toward the needs created by recombinant protein production. As a result, growth parameters may be greatly affected. Noteworthy, M2n[pBKD2-]-C1, previously developed by multiple δ-integration of the β-glucosidase , did not show any detectable metabolic burden. This work aims to test the hypothesis that the metabolic burden and the metabolomic perturbation induced by the δ-integration of a yeast strain, could differ significantly. The engineered strain was evaluated in terms of metabolic performances and metabolomic alterations in different conditions typical of the bioethanol industry. Results indicate that the multiple δ-integration did not affect the ability of the engineered strain to grow on different carbon sources and to tolerate increasing concentrations of ethanol and inhibitory compounds. Conversely, metabolomic profiles were significantly altered both under growing and stressing conditions, indicating a large extent of metabolic reshuffling involved in the maintenance of the metabolic homeostasis. Considering that four copies of gene have been integrated without affecting any parental genes or promoter sequences, deeper studies are needed to unveil the mechanisms implied in these metabolomic changes, thus supporting the optimization of protein production in engineered strains.
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http://dx.doi.org/10.3389/fbioe.2019.00376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6893308PMC
November 2019

Candida palmioleophila isolation in Italy from two cases of systemic infection, after a CHROMagar and Vitek system mis-identification as C. albicans.

New Microbiol 2020 Jan 9;43(1):47-50. Epub 2019 Dec 9.

University of Perugia - Department of Pharmaceutical Sciences.

A correct, fast, reliable identification method is pivotal in nosocomial environments to guide treatment strategies, whereas misidentification might lead to treatment failure. For routine identifications the Vitek system and CHROMagar are widely used but not always reliable, especially now with an increasing number of new emerging fungal pathogens that need careful identification. Here we describe two cases of candidemia, due to Candida palmioleophila previously misidentified as Candida albicans by using the Vitek2 system and CHROMagar. The first case is a 54-year-old man with an infected ulcer in the lower right limb, treated with a targeted therapy using a central venous catheter (CVC). After two months he developed a CVC-related candidemia MDR identified as C. albicans. The second case is a 2-month-old male baby that was admitted to the neonatal unit with acute respiratory failure due to a severe community-acquired bilateral pneumonia; blood cultures were all positive for C. albicans MDR. The isolated strains where re-identified with Maldi-Tof and DNA sequencing as C. palmioleophila. From the identification point of view, CHROMagar can be clearly misleading, especially because CHROMagar types currently available are not designed to discriminate new emerging species, suggesting that systems other than MALDI-TOF and marker sequencing may be inadequate even for routine identification and could contribute to producing misleading identifications and therapeutically wrong practices, leading to failures and patient death.
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January 2020

Nanostructured zinc oxide on silica surface: Preparation, physicochemical characterization and antimicrobial activity.

Mater Sci Eng C Mater Biol Appl 2019 Nov 17;104:109977. Epub 2019 Jul 17.

Department of Pharmaceutical Sciences, University of Perugia, via del Liceo 1, 06123 Perugia, Italy.

Zinc oxide nanoparticles were synthesized using two silica supports largely used in pharmaceutical field as excipients, Cab-O-Sil-H5 and Syloid 244 FP characterized by high surface area and different porosity. In order to evaluate the effects of different silica on nanoparticle chemical physical properties, composites (ZnO-SiO) containing different amounts of ZnO nanoparticles were obtained and characterized by X-ray Powder Diffraction (XRPD), Transmission Electron Microscopy (TEM), Attenuated Transmission Reflectance (ATR), UV-vis spectroscopy and finally Photoluminescence (PL). Composites showed the presence of quite uniformly distributed zinc nanostructures on the silica surface with size in the range of 30-50 nm with an estimated specific surface area ranged from ca. 20 to 70 m/g. The formation of a Zn-O-Si interface in ZnO-SiO was observed as well. Photoluminescence studies revealed that ZnO-SiO samples based on Cab-O-Sil present a higher contribution of oxygen vacancies per unit volume. Finally, the resulting composites were tested for antibacterial and antifungal activities. Whereas silica supports did not show any antibacterial and antifungal activities, most of the prepared composites, both with Cab-O-Sil-5H and Syloid 244 FP supports, resulted active against both bacteria and fungi. In particular the contingency analysis showed that the amount of zinc oxide in the composites was partly related to MIC results in bacteria (p = 0.059), whereas it showed an interesting p = 0.022 in yeast in the case of low amount of ZnO (10%). Thus, the described ZnO-SiO composites can be proposed for the preparation of both pharmaceutical formulations and medical disposals with antibacterial and antifungal activities.
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http://dx.doi.org/10.1016/j.msec.2019.109977DOI Listing
November 2019

Spectroscopic Characterization of Bovine, Avian and Johnin Purified Protein Derivative (PPD) with High-Throughput Fourier Transform InfraRed-Based Method.

Pathogens 2019 Aug 29;8(3). Epub 2019 Aug 29.

Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche "Togo Rosati", Via Gaetano Salvemini 1, 06126 Perugia, Italy.

Tuberculins purified protein derivatives (PPDs) are obtained by precipitation from heat treated mycobacteria. PPDs are used in diagnosis of mycobacterial infections in humans and animals. Bovine PPD (PPDB) is obtained from ( complex), while Avian PPD (PPDA) and Johnin PPD (PPDJ) are extracted, respectively, from and ( complex). PPDB and PPDA are used for bovine tuberculosis diagnosis, while PPDJ is experimentally used in the immunodiagnosis of paratuberculosis. Although PPDs date back to the 19th Century, limited knowledge about their composition is currently available. The goal of our study was to evaluate Fourier Transform InfraRed (FTIR) spectroscopy as a tool to differentiate PPDB, PPDA, and three PPDJs. The results highlighted that the three PPDs have specific profiles, correlated with phylogenetic characteristics of mycobacteria used for their production. This analysis is eligible as a specific tool for different PPDs batches characterization and for the assessment of their composition. The entire PPD production may be efficiently controlled, since the N content of each preparation is related to IR spectra, with a reference spectrum for each PPD and a standardized analysis protocol.
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http://dx.doi.org/10.3390/pathogens8030136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789744PMC
August 2019

Biofilm Specific Activity: A Measure to Quantify Microbial Biofilm.

Microorganisms 2019 Mar 7;7(3). Epub 2019 Mar 7.

Department of Pharmaceutical Sciences⁻Microbiology, University of Perugia, 06123 Perugia, Italy.

Microbes growing onto solid surfaces form complex 3-D biofilm structures characterized by the production of extracellular polymeric compounds and an increased resistance to drugs. The quantification of biofilm relays currently on a number of different approaches and techniques, often leading to different evaluations of the ability to form biofilms of the studied microbial strains. Measures of biofilm biomass were carried out with crystal violet (CV) and a direct reading at 405 nm, whereas the activity was assessed with the XTT ((2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2-tetrazolium-5-carboxanilide) method. The strains of four pathogenic species of the genus (, , and ) and of were employed to determine the effective relatedness among techniques and the specific activity of the biofilm, as a ratio between the XTT and the CV outcomes. Since the ability to form biomass and to be metabolically active are not highly related, their simultaneous use allowed for a categorization of the strains. This classification is putatively amenable of further study by comparing the biofilm type and the medical behavior of the strains.
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http://dx.doi.org/10.3390/microorganisms7030073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6463164PMC
March 2019

A yeast metabolome-based model for an ecotoxicological approach in the management of lignocellulosic ethanol stillage.

R Soc Open Sci 2019 Jan 16;6(1):180718. Epub 2019 Jan 16.

Department of Agronomy Food Natural resources Animals and Environment (DAFNAE), University of Padova, Legnaro, Italy.

Lignocellulosic bioethanol production results in huge amounts of stillage, a potentially polluting by-product. Stillage, rich in heavy metals and, mainly, inhibitors, requires specific toxicity studies to be adequately managed. To this purpose, we applied an FTIR ecotoxicological bioassay to evaluate the toxicity of lignocellulosic stillage. Two weak acids and furans, most frequently found in lignocellulosic stillage, have been tested in different mixtures against three strains. The metabolomic reaction of the test microbes and the mortality induced at various levels of inhibitor concentration showed that the strains are representative of three different types of response. Furthermore, the relationship between concentrations and FTIR synthetic stress indexes has been studied, with the aim of defining a model able to predict the concentrations of inhibitors in stillage, resulting in an optimized predictive model for all the strains. This approach represents a promising tool to support the ecotoxicological management of lignocellulosic stillage.
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http://dx.doi.org/10.1098/rsos.180718DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366221PMC
January 2019

High-Throughput Rapid and Inexpensive Assay for Quantitative Determination of Low Cell-Density Yeast Cultures.

Microorganisms 2019 Jan 24;7(2). Epub 2019 Jan 24.

Department of Pharmaceutical Sciences, University of Perugia, 06123 Perugia, Italy.

A procedure for microbial cell density determination with a high-throughput densitometric assay was developed to allow a precise quantification of both free and sessile cells, such as those of a biofilm, with a large range from low to high cell densities. Densitometry was chosen because it allows fast, rapid and cost-effective measures; it is non-disruptive; and has an easy learning curve. The method setup, and the further validation, was carried out with strains of and Equations were developed at the level of the single strains, of the three species and finally a general one applicable to all three species. In the cross validation, with strains absent from the training set, the method was shown to be robust and flexible. The best results were obtained with species specific equations, although the global equation performed almost as well in terms of correlation between real and estimated density values. In all cases, a correlation around 0.98 between effective and predicted density was obtained with figures ranging from 10² to 10⁸ cells mL. The entire analytical part of the procedure can be accomplished with a MS Excel macro provided free of charge.
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http://dx.doi.org/10.3390/microorganisms7020032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6406537PMC
January 2019

Early Ongoing Speciation of Within the Grape Ecosystem Revealed by the Internal Variability Among the rDNA Operon Repeats.

Front Microbiol 2018 3;9:1687. Epub 2018 Aug 3.

Institute of Sciences of Food Production (ISPA), National Research Council (CNR), Lecce, Italy.

A yeast strain was isolated during a study on vineyard-associated yeast strains from Apulia in Southern Italy. ITS and LSU D1/D2 rDNA sequences showed this strain not to belong to any known species and was described as the type strain of , a close relative of . Several secondary peaks appeared in the sequences, suggesting internal heterogeneity among the copies of the rDNA. This hypothesis was tested by sequencing single clones of the marker region. The analyses showed different levels of variability throughout the operon with differences between the rRNA encoding genes and the internally transcribed regions. and share high frequency variants, i.e., variants frequently found in many clones, whereas there is a large variability of the low frequency polymorphisms, suggesting that the mechanism of homogenization is more active with the former than with the latter type of variation. These findings indicate that low frequency variants are detected in Sanger sequencing as secondary peaks whereas in Next Generation Sequencing (NGS) of metagenomics DNA would lead to an overestimate of the alpha diversity. For the first time in our knowledge, this investigation shed light on the variation of the copy number of the rDNA cistron during the yeast speciation process. These polymorphisms can be used to investigate on the processes occurring in these taxonomic markers during the separation of fungal species, it being a genetic process highly frequent in the complex microbial ecosystem existing in grape, must and wine.
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http://dx.doi.org/10.3389/fmicb.2018.01687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6085423PMC
August 2018

NGS barcode sequencing in taxonomy and diagnostics, an application in "" pathogenic yeasts with a metagenomic perspective.

IMA Fungus 2018 Jun 22;9(1):91-105. Epub 2018 May 22.

Microbiology Section, Department of Pharmaceutical Sciences, University of Perugia, 06121, Italy.

Species identification of yeasts and other Fungi is currently carried out with Sanger sequences of selected molecular markers, mainly from the ribosomal DNA operon, characterized by hundreds of tandem repeats of the 18S, ITS1, 5.8S, ITS2 and LSU . The ITS region has been recently proposed as a primary barcode marker making this region the most used one in taxonomy, phylogeny and diagnostics. The introduction of NGS is providing tools of high efficacy and relatively low cost to amplify two or more markers simultaneously with great sequencing depth. However, the presence of intra-genomic variability between the repeats requires specific analytical procedures and pipelines. In this study, 286 strains belonging to 11 pathogenic yeasts species were analysed with NGS of the region spanning from ITS1 to the D1/D2 domain of the LSU encoding ribosomal DNA. Results showed that relatively high heterogeneity can hamper the use of these sequences for the identification of single strains and even more of complex microbial mixtures. These observations point out that the metagenomics studies could be affected by species inflection at levels higher than currently expected.
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http://dx.doi.org/10.5598/imafungus.2018.09.01.07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6048569PMC
June 2018

Yeast Biofilm as a Bridge Between Medical and Environmental Microbiology Across Different Detection Techniques.

Infect Dis Ther 2018 Mar 16;7(Suppl 1):27-34. Epub 2018 Mar 16.

Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy.

Medical and environmental microbiology have two distinct, although very short, histories stemming, the first from the pioneering works of Sommelweiss, Pasteur, Lister and Koch, the second mainly from the studies of Bejerink and Winogradsky. These two branches of microbiology evolved and specialized separately producing distinct communities and evolving rather different approaches and techniques. The evidence accumulated in recent decades indicate that indeed most of the medically relevant microorganisms have a short circulation within the nosocomial environment and a larger one involving the external, i.e. non-nosocomial, and the hospital environments. This evidence suggests that the differences between approaches should yield to a convergent approach aimed at solving the increasing problem represented by infectious diseases for the increasingly less resistant human communities. Microbial biofilm is one of the major systems used by these microbes to resist the harsh conditions of the natural and anthropic environment, and the even worse ones related to medical settings. This paper presents a brief outline of the converging interest of both environmental and medical microbiology toward a better understanding of microbial biofilm and of the various innovative techniques that can be employed to characterize, in a timely and quantitative manner, these complex structures. Among these, micro-Raman along with micro-Brillouin offer high hopes of describing biofilms both at the subcellular and supercellular level, with the possibility of characterizing the various landscapes of the different biofilms. The possibility of adding a taxonomic identification of the cells comprising the biofilm is a complex aspect presenting several technical issues that will require further studies in the years to come.
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http://dx.doi.org/10.1007/s40121-018-0191-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5856731PMC
March 2018

Merging FT-IR and NGS for simultaneous phenotypic and genotypic identification of pathogenic Candida species.

PLoS One 2017 4;12(12):e0188104. Epub 2017 Dec 4.

Department of Pharmaceutical Sciences-Microbiology, University of Perugia, Perugia (Italy).

The rapid and accurate identification of pathogen yeast species is crucial for clinical diagnosis due to the high level of mortality and morbidity induced, even after antifungal therapy. For this purpose, new rapid, high-throughput and reliable identification methods are required. In this work we described a combined approach based on two high-throughput techniques in order to improve the identification of pathogenic yeast strains. Next Generation Sequencing (NGS) of ITS and D1/D2 LSU marker regions together with FTIR spectroscopy were applied to identify 256 strains belonging to Candida genus isolated in nosocomial environments. Multivariate data analysis (MVA) was carried out on NGS and FT-IR data-sets, separately. Strains of Candida albicans, C. parapsilosis, C. glabrata and C. tropicalis, were identified with high-throughput NGS sequencing of ITS and LSU markers and then with FTIR. Inter- and intra-species variability was investigated by consensus principal component analysis (CPCA) which combines high-dimensional data of the two complementary analytical approaches in concatenated PCA blocks normalized to the same weight. The total percentage of correct identification reached around 97.4% for C. albicans and 74% for C. parapsilosis while the other two species showed lower identification rates. Results suggested that the identification success increases with the increasing number of strains actually used in the PLS analysis. The absence of reliable FT-IR libraries in the current scenario is the major limitation in FTIR-based identification of strains, although this metabolomics fingerprint represents a valid and affordable aid to rapid and high-throughput to clinical diagnosis. According to our data, FT-IR libraries should include some tens of certified strains per species, possibly over 50, deriving from diverse sources and collected over an extensive time period. This implies a multidisciplinary effort of specialists working in strain isolation and maintenance, molecular taxonomy, FT-IR technique and chemo-metrics, data management and data basing.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0188104PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5714347PMC
December 2017

Toll Like Receptor 4 Affects the Cerebral Biochemical Changes Induced by MPTP Treatment.

Neurochem Res 2017 Feb 21;42(2):493-500. Epub 2017 Jan 21.

Department of Pharmaceutical Sciences, Section of Biochemical and Health Sciences, University of Perugia, Via del Giochetto, 06123, Perugia, Italy.

The etiology and pathogenesis of Parkinson's disease (PD) are still unclear. However, multiple lines of evidence suggest a critical role of the toll like receptor 4 (TLR4) in inflammatory response and neuronal death. Neuroinflammation may be associated with the misfolding and aggregation of proteins accompanied by a change in their secondary structure. Recent findings also suggest that biochemical perturbations in cerebral lipid content could contribute to the pathogenesis of central nervous system (CNS) disorders, including PD. Thus, it is of great importance to determine the biochemical changes that occur in PD. In this respect, Fourier Transform Infrared (FTIR) spectroscopy represents a useful tool to detect molecular alterations in biological systems in response to stress stimuli. By relying upon FTIR approach, this study was designed to elucidate the potential role of TLR4 in biochemical changes induced by methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin in a mouse model of PD. The analysis of the FTIR spectra was performed in different brain regions of both wild type (WT) and toll like receptor 4-deficient (TLR4) mice. It revealed that each brain region exhibited a characteristic molecular fingerprint at baseline, with no significant differences between genotypes. Conversely, WT and TLR4 mice showed differential biochemical response to MPTP toxicity, principally related to lipid and protein composition. These differences appeared to be characteristic for each brain area. Furthermore, the present study showed that WT mice resulted more vulnerable than TLR4 animals to striatal dopamine (DA) depletion following MPTP treatment. These results support the hypothesis of a possible involvement of TLR4 in biochemical changes occurring in neurodegeneration.
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http://dx.doi.org/10.1007/s11064-016-2095-6DOI Listing
February 2017

Exploring ecological modelling to investigate factors governing the colonization success in nosocomial environment of Candida albicans and other pathogenic yeasts.

Sci Rep 2016 06 1;6:26860. Epub 2016 Jun 1.

Infectious Diseases Clinic, Santa Maria Misericordia University Hospital, Piazzale Santa Maria della Misericordia, 15, 33100 Udine, Italy.

Two hundred seventy seven strains from eleven opportunistic species of the genus Candida, isolated from two Italian hospitals, were identified and analyzed for their ability to form biofilm in laboratory conditions. The majority of Candida albicans strains formed biofilm while among the NCAC species there were different level of biofilm forming ability, in accordance with the current literature. The relation between the variables considered, i.e. the departments and the hospitals or the species and their ability to form biofilm, was tested with the assessment of the probability associated to each combination. Species and biofilm forming ability appeared to be distributed almost randomly, although some combinations suggest a potential preference of some species or of biofilm forming strains for specific wards. On the contrary, the relation between biofilm formation and species isolation frequency was highly significant (R(2) around 0.98). Interestingly, the regression analyses carried out on the data of the two hospitals separately were rather different and the analysis on the data merged together gave a much lower correlation. These findings suggest that, harsh environments shape the composition of microbial species significantly and that each environment should be considered per se to avoid less significant statistical treatments.
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http://dx.doi.org/10.1038/srep26860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4887984PMC
June 2016

Ionic Conductivity as a Tool To Study Biocidal Activity of Sulfobetaine Micelles against Saccharomyces cerevisiae Model Cells.

Langmuir 2016 Feb 25;32(4):1101-10. Epub 2016 Jan 25.

Department of Pharmaceutical Sciences-Microbiology, University of Perugia , Borgo XX Giugno 74, I-06121 Perugia, Italy.

Zwitterionic sulfobetaine surfactants are used in pharmaceutical or biomedical applications for the solubilization and delivery of hydrophobic molecules in aqueous medium or in biological environments. In a screening on the biocidal activity of synthetic surfactants on microbial cells, remarkable results have emerged with sulfobetaine amphiphiles. The interaction between eight zwitterionic sulfobetaine amphiphiles and Saccharomyces cerevisiae model cells was therefore analyzed. S. cerevisiae yeast cells were chosen, as they are a widely used unicellular eukaryotic model organism in cell biology. Conductivity measurements were used to investigate the interaction between surfactant solution and cells. Viable counts measurements were performed, and the mortality data correlated with the conductivity profiles very well, in terms of the inflection points (IPs) observed in the curves and in terms of supramolecular properties of the aggregates. A Fourier transform infrared (FTIR)-based bioassay was then performed to determine the metabolomic stress-response of the cells subjected to the action of zwitterionic surfactants. The surfactants showed nodal concentration (IPs) with all the techniques in their activities, corresponding to the critical micellar concentrations of the amphiphiles. This is due to the pseudocationic behavior of sulfobetaine micelles, because of their charge distribution and charge densities. This behavior permits the interaction of the micellar aggregates with the cells, and the structure of the surfactant monomers has impact on the mortality and the metabolomic response data observed. On the other hand, the concentrations that are necessary to provoke a biocidal activity do not promote these amphiphiles as potential antimicrobial agents. In fact, they are much higher than the ones of cationic surfactants.
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http://dx.doi.org/10.1021/acs.langmuir.5b04077DOI Listing
February 2016

First Case of Trichoderma longibrachiatum CIED (Cardiac Implantable Electronic Device)-Associated Endocarditis in a Non-immunocompromised Host: Biofilm Removal and Diagnostic Problems in the Light of the Current Literature.

Mycopathologia 2016 Apr 20;181(3-4):297-303. Epub 2015 Nov 20.

Cardiovascular Medicine Unit 2, Azienda Ospedaliera Universitaria Pisana, Via Paradisa 2, Cisanello, 56100, Pisa, Italy.

Background: Trichoderma species are saprophytic filamentous fungi producing localized and invasive infections that are cause of morbidity and mortality, especially in immunocompromised patients, causing up to 53% mortality. Non-immunocompromised patients, undergoing continuous ambulatory peritoneal dialysis, are other targets of this fungus. Current molecular diagnostic tools, based on the barcode marker ITS, fail to discriminate these fungi at the species level, further increasing the difficulty associated with these infections and their generally poor prognosis.

Case Report: We report on the first case of endocarditis infection caused by Trichoderma longibrachiatum in a 30-year-old man. This patient underwent the implantation of an implantable cardioverter defibrillator in 2006, replaced in 2012. Two years later, the patient developed fever, treated successfully with amoxicillin followed by ciprofloxacin, but an echocardiogram showed large vegetation onto the ventricular lead. After CIED extraction, the patient had high-grade fever. The culturing of the catheter tip was positive only in samples deriving from sonication according to the 2014 ESCMID guidelines, whereas the simple washing failed to remove the biofilm cells from the plastic surface. Subsequent molecular (ITS sequencing) and microbiological (macromorphology) analyses showed that the vegetation was due to T. longibrachiatum.

Conclusions: This report showed that T. longibrachiatum is an effective threat and that sonication is necessary for the culturing of vegetations from plastic surfaces. Limitations of the current barcode marker ITS, and the long procedures required by a multistep approach, call for the development of rapid monophasic tests.
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http://dx.doi.org/10.1007/s11046-015-9961-7DOI Listing
April 2016

Phenotypic and molecular diversity of Meyerozyma guilliermondii strains isolated from food and other environmental niches, hints for an incipient speciation.

Food Microbiol 2015 Jun 20;48:206-15. Epub 2015 Jan 20.

Department of Pharmaceutical Sciences - Microbiology, University of Perugia, Borgo 20 Giugno 74, 06121 Perugia, Italy; CEMIN, Centre of Excellence on Nanostructured Innovative Materials, Department of Chemistry, Biology and Biotechnology, University of Perugia, via Elce di Sotto 8, I-06123 Perugia, Italy. Electronic address:

Meyerozyma guilliermondii is a yeast species widely isolated from several natural environments and from fruit; in medical microbiology it is known as the teleomorph of the opportunistic pathogen Candida guilliermondii, which causes about 2% of the human blood infections. This yeast is also promising in a variety of biotechnological applications as vitamins production and post-harvest control. The question if isolates from different sources are physiologically and genetically similar, or if the various environments induced significant differences, is crucial for the understanding of this species structure and to select strains appropriate for each application. This question was addressed using LSU and ITS sequencing for taxonomic assignment, i-SSR (GACA4) for the molecular characterization and FTIR for the metabolomic fingerprint. All data showed that fruit and environmental isolates cluster separately with a general good agreement between metabolomics and molecular analysis. An additional RAPD analysis was able to discriminate strains according to the isolation position within the pineapple fruit. Although all strains are members of the M. guilliermondii species according to the current standards, the distribution of large variability detected suggests that some specialization occurred in the niches inhabited by this yeast and that food related strains can be differentiated from the medical isolates.
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http://dx.doi.org/10.1016/j.fm.2014.12.014DOI Listing
June 2015

FTIR metabolomic fingerprint reveals different modes of action exerted by structural variants of N-alkyltropinium bromide surfactants on Escherichia coli and Listeria innocua cells.

PLoS One 2015 14;10(1):e0115275. Epub 2015 Jan 14.

Department of Pharmaceutical Sciences-Microbiology, University of Perugia, Borgo XX Giugno 74, I-06121 Perugia, Italy; CEMIN, Centre of Excellence on Nanostructured Innovative Materials, Department of Chemistry, Biology and Biotechnology, University of Perugia, via Elce di Sotto 8, I-06123 Perugia, Italy.

Surfactants are extremely important agents to clean and sanitize various environments. Their biocidal activity is a key factor determined by the interactions between amphiphile structure and the target microbial cells. The object of this study was to analyze the interactions between four structural variants of N-alkyltropinium bromide surfactants with the Gram negative Escherichia coli and the Gram positive Listeria innocua bacteria. Microbiological and conductometric methods with a previously described FTIR bioassay were used to assess the metabolomic damage exerted by these compounds. All surfactants tested showed more biocidal activity in L. innocua than in E. coli. N-tetradecyltropinium bromide was the most effective compound against both species, while all the other variants had a reduced efficacy as biocides, mainly against E. coli cells. In general, the most prominent metabolomic response was observed for the constituents of the cell envelope in the fatty acids (W1) and amides (W2) regions and at the wavenumbers referred to peptidoglycan (W2 and W3 regions). This response was particularly strong and negative in L. innocua, when cells were challenged by N-tetradecyltropinium bromide, and by the variant with a smaller head and a 12C tail (N-dodecylquinuclidinium bromide). Tail length was critical for microbial inhibition especially when acting against E. coli, maybe due the complex nature of Gram negative cell envelope. Statistical analysis allowed us to correlate the induced mortality with the metabolomic cell response, highlighting two different modes of action. In general, gaining insights in the interactions between fine structural properties of surfactants and the microbial diversity can allow tailoring these compounds for the various operative conditions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115275PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4294686PMC
December 2015

Candida milleri species reveals intraspecific genetic and metabolic polymorphisms.

Food Microbiol 2014 Sep 27;42:72-81. Epub 2014 Feb 27.

Università degli Studi di Milano, Department of Food, Environmental and Nutritional Science, via Celoria 2, 20133 Milan, Italy. Electronic address:

Candida milleri, together with Candida humilis, is the most representative yeast species found in type I sourdough ecosystems. In this work, comparison of the ITS region and the D1/D2 domain of 26S rDNA gene partial sequences, karyotyping, mtDNA-RFLP analysis, Intron Splice Site dispersion (ISS-PCR) and (GTG)5 microsatellite analyses, assimilation test of different carbohydrates, and metabolome assessment by FT-IR analysis, were investigated in seventeen strains isolated from four different companies as well as in type strains CBS6897(T) and CBS5658(T). Most isolates were ascribed to C. milleri, even if a strong relatedness was confirmed with C. humilis as well, particularly for three strains. Genetic characterization showed a high degree of intraspecific polymorphism since 12 different genotypes were discriminated. The number of chromosomes varied from 9 to 13 and their size ranged from less than 0.3 to over 2 Mbp. Phenotypic traits let to recognize 9 different profiles of carbon sources assimilation. FT-IR spectra from yeast cells cultivated in different media and collected at different growth phases revealed a diversity of behaviour among strains in accordance with the results of PCR-based fingerprinting. A clear evidence of the polymorphic status of C. milleri species is provided thus representing an important feature for the development of technological applications in bakery industries.
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http://dx.doi.org/10.1016/j.fm.2014.02.011DOI Listing
September 2014

A novel, rapid and automated conductometric method to evaluate surfactant-cells interactions by means of critical micellar concentration analysis.

Chem Biol Interact 2014 Jul 6;218:20-7. Epub 2014 May 6.

CEMIN - Centro di Eccellenza Materiali Innovativi e Nanostrutturati, Dipartimento di Chimica, Biologia e Biotecnologie, Via Elce di Sotto n.8, Italy; Dipartimento di Biologia Applicata, Via Borgo XX Giugno, 74 Università degli Studi di Perugia, I 06100 Perugia, Italy.

Conductometry is widely used to determine critical micellar concentration and micellar aggregates surface properties of amphiphiles. Current conductivity experiments of surfactant solutions are typically carried out by manual pipetting, yielding some tens reading points within a couple of hours. In order to study the properties of surfactant-cells interactions, each amphiphile must be tested in different conditions against several types of cells. This calls for complex experimental designs making the application of current methods seriously time consuming, especially because long experiments risk to determine alterations of cells, independently of the surfactant action. In this paper we present a novel, accurate and rapid automated procedure to obtain conductometric curves with several hundreds reading points within tens of minutes. The method was validated with surfactant solutions alone and in combination with Saccharomyces cerevisiae cells. An easy-to use R script, calculates conductometric parameters and their statistical significance with a graphic interface to visualize data and results. The validations showed that indeed the procedure works in the same manner with surfactant alone or in combination with cells, yielding around 1000 reading points within 20 min and with high accuracy, as determined by the regression analysis.
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http://dx.doi.org/10.1016/j.cbi.2014.04.012DOI Listing
July 2014

Neuroinflammation and endoplasmic reticulum stress are coregulated by cyclo(His-Pro) to prevent LPS neurotoxicity.

Int J Biochem Cell Biol 2014 Jun 31;51:159-69. Epub 2014 Mar 31.

Dipartimento di Medicina Sperimentale Università di Perugia, polo Unico S. Andrea delle Fratte, 06123 Perugia, Italy.

Many neurological and neurodegenerative diseases are associated with oxidative stress and glial inflammation, all related to endoplasmic reticulum stress. Cyclo(His-Pro) is an endogenous cyclic dipeptide that exerts cytoprotection by interfering with the Nrf2-NF-κB systems, the former presiding the antioxidant and the latter the pro-inflammatory cellular response. Here we investigated whether the cyclic dipeptide inhibits glial inflammation thus reducing the detrimental effect of inflammatory neurotoxins on neurons. We found that systemic administration of cyclo(His-Pro) exerts in vivo anti-inflammatory effects in the central nervous system by down-regulating hepatic and cerebral TNFα expression thereby counteracting LPS-induced gliosis. Mechanistic studies indicated that the cyclic dipeptide-mediated effects are achieved through the activation of Nrf2-driven antioxidant response and the inhibition of the pro-inflammatory NF-κB pathway. Moreover, by up-regulating Bip, cyclo(His-Pro) increases the ER stress sensitivity and triggers the unfolded protein response to alleviate the ER stress. These results unveil a novel potential therapeutic use of cyclo(His-Pro) against neuroinflammatory-related diseases and we might now consider its potential anti-inflammatory role in other neuropathological conditions.
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http://dx.doi.org/10.1016/j.biocel.2014.03.023DOI Listing
June 2014

FTIR analysis of the metabolomic stress response induced by N-alkyltropinium bromide surfactants in the yeasts Saccharomyces cerevisiae and Candida albicans.

Colloids Surf B Biointerfaces 2014 Apr 7;116:761-71. Epub 2014 Feb 7.

Department of Pharmaceutical Sciences - Microbiology, University of Perugia, Borgo XX Giugno 74, I-06121 Perugia, Italy. Electronic address:

The activity of surfactants against fungal cells has been studied less than against bacteria, although the medical and industrial importance of the former is of paramount importance. In this paper the surfactant biocidal effect was measured in the yeasts Saccharomyces cerevisiae and Candida albicans with a previously described FTIR bioassay which estimates the stress level as function of the FTIR spectra variation of the cells upon exposition to the chemicals. N-tetradecyltropinium bromide was chosen as stressing agent on the basis of previous preliminary study demonstrating its ability to kill prokaryotic and especially eukaryotic cells at concentration around or over the critical micellar concentration (c.m.c.). Here we show that this surfactant is able to inactivate S. cerevisiae cells at 0.4mM and C. albicans cells at 0.6mM after 1h exposition. FTIR analysis revealed that the surfactant induced metabolomics reactions of S. cerevisiae cells in the regions of amides (W2) and fatty acids (W1). In the same way C. albicans cells showed the maximum stress response in amides (W2) and mixed (W3) regions. Variations of the hydrophobic tail of this surfactant produced a reduced level of cell stress with both the 12C and 16C variants; although these two compounds were more effective in inducing cell mortality in S. cerevisiae but not in C. albicans. In conclusion, this paper has shown that, for this surfactant, the n-alkyl chain must vary between 12C and 16C and that the hydrophilic head size is not as critical as the tail length.
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http://dx.doi.org/10.1016/j.colsurfb.2014.01.054DOI Listing
April 2014

Biocidal and inhibitory activity screening of de novo synthesized surfactants against two eukaryotic and two prokaryotic microbial species.

Colloids Surf B Biointerfaces 2013 Nov 26;111:407-17. Epub 2013 Jun 26.

Department of Applied Biology - Microbiology, University of Perugia, Borgo XX Giugno 74, I-06121 Perugia, Italy. Electronic address:

Thirty-six quaternary ammonium salts, of which 28 structurally different non-commercially available surfactants, were tested to screen their biocidal and inhibitory antimicrobial activity. Their activity was compared to commercially available amphiphiles as well as to non-amphiphilic quaternary ammonium salts. As target of these compounds four microbial species were employed of which two (Saccharomyces cerevisiae and Candida albicans) were important yeast in the food and clinical environment and the other two (Escherichia coli and Listeria innocua) represented the Gram negative and positive bacteria, respectively. The surfactants showed the ability to kill the microbial cells in water solution and to variably hamper their growth onto agar medium. The non-amphiphilic compounds (which represent analogues of some surfactants used in this study, since they have the same head group but no hydrophobic portion) had little effect in solution and no effect against the microbial growth on plate. Amphoteric and non-amphoteric zwitterionic surfactants showed reduced biocidal activity. The most active antimicrobial agent was N-tetradecyltropinium bromide (23S) surfactant. The presence of cells did not significantly affect the ability to form micelles, as demonstrated by comparative conductometric measurements.
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http://dx.doi.org/10.1016/j.colsurfb.2013.06.033DOI Listing
November 2013

Assessment of safety and efficiency of nitrogen organic fertilizers from animal-based protein hydrolysates--a laboratory multidisciplinary approach.

J Sci Food Agric 2014 Jan 12;94(2):235-45. Epub 2013 Jul 12.

Department of Applied Biology-Microbiology, University of Perugia, Borgo XX Giugno 74, I-06121, Perugia, Italy.

Background: Protein hydrolysates or hydrolysed proteins (HPs) are high-N organic fertilizers allowing the recovery of by-products (leather meal and fluid hydrolysed proteins) otherwise disposed of as polluting wastes, thus enhancing matter and energy conservation in agricultural systems while decreasing potential pollution. Chemical and biological characteristics of HPs of animal origin were analysed in this work to assess their safety, environmental sustainability and agricultural efficacy as fertilizers. Different HPs obtained by thermal, chemical and enzymatic hydrolytic processes were characterized by Fourier transform infrared spectroscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis, and their safety and efficacy were assessed through bioassays, ecotoxicological tests and soil biochemistry analyses.

Results: HPs can be discriminated according to their origin and hydrolysis system by proteomic and metabolomic methods. Three experimental systems, soil microbiota, yeast and plants, were employed to detect possible negative effects exerted by HPs. The results showed that these compounds do not significantly interfere with metabolomic activity or the reproductive system.

Conclusion: The absence of toxic and genotoxic effects of the hydrolysates prepared by the three hydrolytic processes suggests that they do not negatively affect eukaryotic cells and soil ecosystems and that they can be used in conventional and organic farming as an important nitrogen source derived from otherwise highly polluting by-products.
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http://dx.doi.org/10.1002/jsfa.6239DOI Listing
January 2014

Furanodien-6-one from Commiphora erythraea inhibits the NF-κB signalling and attenuates LPS-induced neuroinflammation.

Mol Immunol 2013 Jul 26;54(3-4):347-54. Epub 2013 Jan 26.

Dipartimento di Medicina Sperimentale Scienze Biochimiche, Università di Perugia, 06123 Perugia, Italy.

We investigated the in vitro anti-inflammatory activity of 1(10),4-furanodien-6-one, one the most active compounds of the hexane extract of Commiphora erythraea (Ehrenb.) Engl., by exposing microglial BV-2 cells to lipopolysaccharide. We showed that furanodien-6-one pre-treatment restored cell viability and ROS to control levels while halving NO generation. Production of pro-inflammatory IL-6, IL-23, IL-17, TGF-β, and INF-γ, significantly induced by LPS, was also markedly reduced by furanodien-6-one treatment. We further showed that furanodien-6-one protects primary neuronal cultures against the inflammatory/toxic insults of LPS-treated BV-2 conditioned media, indicating that furanodien-6-one exerts anti-inflammatory/cytoprotective effects in neuronal cells. We then investigated whether furanodien-6-one exerts anti-inflammatory properties in an in vivo model of microglial activation. In adult mice ip-injected with LPS we found that furanodien-6-one had strong cerebral anti-inflammatory properties by inhibiting liver and brain TNFα as well as IL-1β expression. Results were not unexpected since FTIR-metabolomic analyses showed that furanodien-6-one-treated mice had a reduced dissimilarity to control animals and that the response to LPS treatment was markedly modified by furanodien-6-one. In conclusion our data provide strong evidence of the anti-inflammatory properties of furanodien-6-one that could be exploited to counteract degenerative pathologies based on neuroinflammation.
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http://dx.doi.org/10.1016/j.molimm.2013.01.003DOI Listing
July 2013

Yamadazyma terventina sp. nov., a yeast species of the Yamadazyma clade from Italian olive oils.

Int J Syst Evol Microbiol 2013 Jan 5;63(Pt 1):372-376. Epub 2012 Oct 5.

Department of Applied Biology - Microbiology, University of Perugia, Perugia 06123, Italy.

During an investigation of olive oil microbiota, three yeast strains were found to be divergent from currently classified yeast species according to the sequences of the D1/D2 domain of the gene encoding the rRNA large subunit (LSU) and the internal transcribed spacer region including the gene for 5.8S rRNA. Phylogenetic analysis revealed that these strains, designated CBS 12509, CBS 12510(T) and CBS 12511, represent a novel anascosporogenous species described herein as Yamadazyma terventina sp. nov; the type strain is DAPES 1924(T) (= CBS 12510(T) = NCAIM Y.02028(T)). This novel species was placed in the Yamadazyma clade, with Yamadazyma scolyti, Candida conglobata and Candida aaseri as closest relatives. Y. terventina differs from the above-mentioned species in the ability to strongly assimilate dl-lactate and weakly assimilate ethanol.
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http://dx.doi.org/10.1099/ijs.0.045898-0DOI Listing
January 2013

Effect of pH on potassium metabisulphite biocidic activity against yeast and human cell cultures.

Food Chem 2012 Oct 16;134(3):1327-36. Epub 2012 Mar 16.

Dipartimento Biologia Applicata - Microbiologia, Università degli Studi di Perugia, Via Borgo 20 Giugno, 74, I-06121 Perugia, Italy.

Potassium metabisulphite (PMB) is a common antimicrobial additive in the food industry. In aqueous solutions, PMB leads to complex equilibria according to its concentration, pH and temperature, and different chemical species can be present. In winemaking, PMB is used at low pH, suggesting that the biocidic activity is exerted by sulphur dioxide while, in other applications, it is employed at higher pH values with little if any dissociation. This observation leads to the question of which chemical form is biologically active. For this reason, Saccharomyces cerevisiae cells were subjected to PMB solutions at different pH values and analysed with a Fourier transform infrared spectroscopy (FTIR)-based bioassay, to assess the entity and the type of stress. Cell viability was determined and compared to the metabolomics (FTIR) stress indices, which revealed that the metabolomics fingerprint was an effective description of the cell health state. GC-MS metabolite profiles were obtained to describe (in detail) the changes caused by PMB in the fatty acids region. Human dermal fibroblasts (HDF) were also subjected to PMB stress at pH 7.0 and analysed with the FTIR protocol, in order to compare the response spectra of yeast and human cell cultures.
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http://dx.doi.org/10.1016/j.foodchem.2012.03.025DOI Listing
October 2012

Candida coquimbonensis sp. nov., a link between Australian and Nearctic/Neotropical Phaffomyces.

Int J Syst Evol Microbiol 2012 Dec 22;62(Pt 12):3067-3071. Epub 2012 Jun 22.

Department of Biology, Tennessee State University, 3500 John Merritt Blvd., Nashville, TN 37209, USA.

A novel species of ascomycetous yeast, Candida coquimbonensis sp. nov., from the necrotic tissue of cacti in Chile and Australia is described. C. coquimbonensis sp. nov. is closely related and phenotypically similar to Phaffomyces opuntiae. There is no overlap in the geographical distribution between C. coquimbonensis and any species in the Phaffomyces clade. However, this is the first member of the clade to be collected in both native (Chile) and non-native (Australia) cactus habitats. The type strain of C. coquimbonensis sp. nov. is TSU 00-206.4B(T) ( = CBS 12348(T) = USCFST 12-103(T)).
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http://dx.doi.org/10.1099/ijs.0.045294-0DOI Listing
December 2012
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