jejuni real-time PCR assays), each Lapatinib dilution point was tested in duplicate and the mean standard curves were used for quantity estimation. The CV of the Ct values were calculated for the ten different inter-assay experiments. They illustrate the variability of the Ct values obtained between experiments including the specific DNA extraction procedure and the amplification step. Use of the standard curves The standard curves were thus used (i) to evaluate the sensitivity of the real-time PCR assays, (ii) to assess the intra- and inter-assay variabilities, and (iii) to allow a reliable

quantification of C. jejuni and C. coli in pure cultures or in the field samples. Statistical analysis PCR amplification efficiency (E) was estimated using the slope of the standard curve and the formula E = 10(-1/slope)-1. A reaction with 100% efficiency will generate a slope of -3.32. Data analysis Gefitinib price was performed using the SDS software (Applied Biosystems).

The 119 field samples from the experimental infection were evaluated in parallel with the real-time PCR assays and the bacterial culture described in this study. All data analyses were performed with Microsoft excel and SAS Systems version 8 (SAS, Cary, N.C.). Specificity and sensitivity were assessed using the bacterial culture as a gold standard. The sensitivity was calculated as a/(a+c), where a is the number of samples found positive by both real-time PCR and bacterial culture (direct inoculation or after selective enrichment) and c is the number of samples positive by bacterial culture but negative by real-time Urease PCR. The specificity was calculated as d/(b+d), where d is the number of samples negative by both methods and b is the number

of samples positive by real-time PCR but negative by bacterial culture. Kappa-statistic was used to measure the agreement between the microaerobic cultivation and each species-specific real-time PCR assay [64]. Acknowledgements The authors thank Sebastien Tessier for technical assistance during his practice training period and the staff of the BioEpAR and MAE units at the Veterinary School of Nantes, notably Jean-Yves Audiart, Françoise Armand, Emmanuelle Blandin, and Françoise Leray. We thank especially Francis Mégraud and Philippe Lehours of the French National Reference Center for Campylobacter and Helicobacter (Bordeaux, France) for providing us reference strains from their collection and field strains from clinical cases. This work was supported by grants from INRA, Anses, and the Region Pays de La Loire. References 1. Moore JE, Corcoran D, Dooley JS, Fanning S, Lucey B, Matsuda M, McDowell DA, Megraud F, Millar BC, O’Mahony R, O’Riordan L, O’Rourke M, Rao JR, Rooney PJ, Sails A, Whyte P: Campylobacter. Vet Res 2005,36(3):351–382.PubMedCrossRef 2. EFSA: The Community Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents, Antimicrobial Resistance and Foodborne Outbreaks in the European Union in 2006. The EFSA Journal 2007, 130. 3.

05); these observations correlated

with a significant reduction in lesion intensity (p < 0.001) on mushrooms treated with 2.9 × 106 and 1.4 × 107 PFU B. bacteriovorus Doxorubicin cost HD100 (mean = 0.010 1/PV in both cases) compared with mushrooms inoculated with P. tolaasii 2192T alone (mean = 0.014 1/PV). Despite this significant reduction in lesion intensity, the total number of CFU recovered from B. bacteriovorus HD100 treated mushrooms onto King’s Medium B was high, suggesting that the bacteria recovered from seemingly similar, beige-coloured colonies on the King’s Medium B plates were not solely pathogenic P. tolaasii 2192T, but might include other species indigenous to the mushroom pileus surface that are not well preyed upon by B. bacteriovorus HD100, as observed in SEM images of mushroom tissue to which King’s medium B broth was added alone. Figure 4 Bacterial CFU numbers recovered from P. tolaasii -inoculated mushrooms in the presence and absence of Bdellovibrio . Lesion intensities and number of bacterial colony forming units (CFU) recovered from mushroom pilei subject to three different treatments detailed to the right. Each P. tolaasii

2192T inoculation contained 1.7 × 106 CFU. Images of mushrooms with typical: high, mean, and low intensity lesions in each group are shown below the graph. Horizontal black bars indicate the mean values for GPCR Compound Library lesion intensity/CFU count in each treatment group. Student’s t-test of significance between B .bacteriovorus-treated and non-treated mushrooms inoculated with P. tolaasii 2192T: *p <0.05, ***p <0.001. Enterobacterspecies are present on the surface of some commercially produced supermarket mushrooms The number of CFU recovered from the mushrooms that were treated with B. bacteriovorus HD100 after inoculation

with P. tolaasii was relatively high compared to mushrooms inoculated with P. tolaasii alone. To confirm the identity of the bacteria seen in Figures 3d and e and recovered from supermarket mushroom tissue pre-treated with B. bacteriovorus HD100 before P. tolaasii 2192T at both 2.9 × 106 and 1.4 × 107 PFU ml−1, 20 colonies taken from the King’s medium B agar plates used to enumerate bacterial CFU, recovered from the treated mushroom tissue of two mushrooms in each group, were grown on Coliform Chromogenic agar (oxoid). This agar contains two chromogenic substrates that turn Nutlin-3 in vitro purple when cleaved by the enzymes glucorinidase and galactosidase, which are both present in coliforms such as E. coli, and absent from Pseudomonads (including P. tolaasii); all 20 colonies were pigmented purple indicating them as coliform, closely related to E. coli, and therefore as indigenous species to the mushroom pileus, and distinctly different to P. tolaasii 2192T , which produced straw coloured colonies on the agar. Three of these coliform isolates were identified by 16 s rDNA sequencing as members of the Enterobacter genus using the BLAST online tool (http://​blast.​ncbi.​nlm.​nih.​gov/​Blast.

Phillips A, Sudbery I, Ramsdale M: Apoptosis induced by environmental stresses and amphotericin B in Candida albicans. Proc Natl Acad Sci USA 2003, 100:14327–14332.CrossRefPubMed 30. Bahmed K, Bonaly R, Benallaoua S, Coulon J: Effect of sub-inhibitory concentrations of amphotericin B on the yeast surface and phagocytic killing activity. Process Biochem 2005, 40:759–765.CrossRef 31. Vivas JJ, Urbina JA, de Souza W: Ultrastructural alterations in Trypanosoma (Schizotrypanum) cruzi induced by Δ 24(25) -sterol

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bud. J Cell Biol 1997, 139:985–994.CrossRefPubMed 38. Palmié-Peixoto IV, Rocha M, Urbina JA, de Souza W, Einicker-Lamas M, Motta MC: Effects of sterol biosynthesis inhibitors on endosymbiont-bearing trypanosomatids. FEMS Microbiol Letters 2006, 255:33–42.CrossRef 39. Urbina JA, Vivas J, Lazardi K, Molina J, Payares G, Piras MM, Piras R: Antiproliferative effects of delta 24(25) sterol methyl transferase inhibitors on Trypanosoma (Schizotrypanum) cruzi : in vitro and in vivo studies. Chemotherapy 1996,42(4):294–307.CrossRefPubMed 40. Lorente SO, Rodrigues JC, Jimenez C, Joyce-Menekse M, Rodrigues C, Croft SL, Yardley V, de Luca-Fradley K, Ruiz-Perez LM, Urbina J, de Souza W, Gonzalez Pacanowska D, Gilbert IH: Novel azasterols as potential agents for treatment of leishmaniasis and trypanosomiasis. Antimicrob Agents Chemother 2004, 48:2937–2950.CrossRefPubMed 41.

Phylogenetic tree showing the position of 16S rDNA OTU’s recovered from stool sample of S3 individual was constructed using neighbor-joining method based on partial 16S rDNA sequences. The bootstrap values (expressed as percentages of 1000 replications) Napabucasin clinical trial are shown at branch points. The scale bar represents genetic distance (2 substitutions per 100 nucleotides). GenBank accession numbers are in parentheses. (PDF 2 MB) Additional file 5: Figure S4. Phylogenetic tree showing the position of 16S rDNA OTU’s recovered from stool sample of T1 individual was constructed using neighbor-joining method

based on partial 16S rDNA sequences. The bootstrap values (expressed as percentages of 1000 replications) are shown at branch points. The scale bar represents genetic distance (2 substitutions per 100 nucleotides). GenBank accession numbers are in parentheses. (PDF 935 KB) Additional file 6: Figure S5. Phylogenetic tree showing the position of 16S rDNA OTU’s recovered from stool sample of T2 individual was constructed using neighbor-joining method based on partial 16S rDNA sequences. The bootstrap values (expressed as percentages of 1000 replications) are shown at branch points. The scale bar represents genetic distance (5 substitutions per 100 nucleotides). GenBank accession numbers are in parentheses.

(PDF 2 MB) Additional file 7: Figure S6. Phylogenetic tree showing the position of 16S rDNA OTU’s recovered from stool sample of T3 individual was constructed using neighbor-joining method based Rucaparib purchase on partial 16S rDNA sequences. The bootstrap values (expressed as percentages of 1000 replications) are shown at branch points. The scale bar represents genetic distance (5 substitutions per 100 nucleotides). GenBank accession numbers are in parentheses. (PDF 1 MB) References 1. Vrieze A, Holleman F, Zoetendal EG, de Vos WM, Hoekstra JBL, Nieuwdorp M: The environment within: how gut microbiota (-)-p-Bromotetramisole Oxalate may influence metabolism and body composition. Diabetologia 2010, 53:606–613.PubMedCrossRef 2. Backhed F, Ding H, Wang T, Hooper LV, Koh GY, et al.: The gut microbiota as an environmental factor that regulates fat storage. Proc Natl Acad Sci USA 2004, 101:15718–15723.PubMedCrossRef

3. Hooper LV, Midtvedt T, Gordon JI: How host-microbial interactions shape the nutrient environment of the mammalian intestine. Annu Rev Nutr 2002, 22:283–307.PubMedCrossRef 4. Ley RE, Hamady M, Lozupone C, Turnbaugh P, Ramey RR, Bircher JS, Schlegel ML, Tucker TA, Schrenzel MD, Knight R, Gordon JI: Evolution of mammals and their gut microbes. Science 2008,320(5883):1647–1651.PubMedCrossRef 5. Neish AS, Denning TL: Advances in understanding the interaction between the gut microbiota and adaptive mucosal immune responses. F1000 Biology Reports 2010, 2:27.PubMed 6. Hopkins MJ, Sharp R, Macfarlane GT: Age and disease related changes in intestinal bacterial populations assessed by cell culture, 16S rRNA abundance, and community cellular fatty acid profiles. Gut 2001, 48:198–205.PubMedCrossRef 7.

J Clin Microbiol 2011,49(9):3114–3121.PubMedCrossRef 53. Borch K, Jonsson KA, Petersson F, Redeen S, Mardh S, Franzen LE: Prevalence of gastroduodenitis and Helicobacter pylori infection in a general population sample: relations to symptomatology and life-style. Dig Dis Sci 2000,45(7):1322–1329.PubMedCrossRef 54. Monstein HJ, Olsson C, Nilsson I, Grahn N, Benoni C, Ahrne S: find more Multiple displacement

amplification of DNA from human colon and rectum biopsies: bacterial profiling and identification of Helicobacter pylori-DNA by means of 16S rDNA-based TTGE and pyrosequencing analysis. J Microbiol Methods 2005,63(3):239–247.PubMedCrossRef 55. CLC bio; http://​wwwclcbiocom 56. NCBI-Entrez Nucleotide; http://​wwwncbinlmnihgov​/​nucleotide Competing selleck products interests The authors declare that they have no competing interests. Authors’ contributions AK, AR, KB and HJM participated in the conception, design

and data interpretation and drafting of the manuscript. AK, AR, MND performed the practical molecular biology procedures. KB collected and selected the biopsy specimens. All authors have been involved in drafting of the manuscript and approved the final version.”
“Background Over the past decade, Clostridium difficile has emerged as an important gut pathogen, causing hospital- and community-acquired diarrhea. The number of patients and the severity of disease have increased dramatically, due to the emergence of two hypervirulent PCR ribotype, 027 [1] and 078 [2, 3]. Traditionally, PCR ribotype 027 has been linked to nosocomial outbreaks. In contrast, PCR ribotype 078 has been detected frequently in farming animals, especially pigs [2, 4], and is found more during community acquired infection. The increase in C. difficile infections (CDI) of humans

has boosted interest in C. difficile biology, diagnostics and pathogenesis. In the past few years, multiple genome sequences of several Forskolin nmr PCR ribotypes have been determined [5–8]. The analyses of the genomes, aided by comparative genomics of DNA-DNA microarrays [9, 10] has shown that the genomes of C. difficile are highly variable with inserts of mobile DNA from phage, plasmid or transposon origin. These mobile DNA elements are actively moving within C. difficile genomes and are frequently passed on to neighboring bacteria, harboring mosaic genomes [7, 11]. It is unclear what role the mobile elements play in the virulence of C. difficile. Some virulence linked genes, for example the holin-like tcdE, have a phage origin [12]. In fact, it has been suggested that the whole pathogenicity locus (PaLoc), encoding the major C. difficile virulence factors TcdA and TcdB, is of phage origin [13, 14]. Recently, phages have been shown to upregulate toxin production in C. difficile, thereby increasing the virulence [15]. C.

aureus . Modified after Marilley et al. [19] Additionally, pyruvate or citrate are starting materials for the formation of short-chain flavor compounds such as acetoin, 2,3-butanedione, 1-butanol, 2-propanol, acetic acid, acetaldehyde and ethanol through glycolytic, lactate converting and non-glycolytic carbohydrates fermentations or fermentations of nitrogenous compounds [44]. The catabolism

of pyruvate (presented on Figure 3) seems to play an important role in case of S. aureus since the products of this metabolic pathway were found in the headspace of this bacterium in our study and also by other researchers, inter alia ethanol, acetaldehyde, acetic acid [11] and acetoin [6, 40]. Figure 3 Simplified scheme of pyruvate DNA Damage inhibitor metabolism via R788 research buy glycolytic fermentations and lactate converting

fermentations, modified after Michal et al.[44]. Exclusively, pathways which lead to the production of VOCs significantly released by S. aureus in this study (underlined with solid line) are presented, including acetoin (3-hydroxy-2-butanone), acetaldehyde, ethanol, 1-butanol, acetone, 2-propanol. In case of P. aeruginosa the metabolism of amino acids rather than glycolysis of carbohydrates yields pyruvate as starting material (significantly released or taken up products are underlined with dotted line). Detailed investigation of the subspecies of the genus Staphylococcus shows that

acetoin is produced by the subspecies aureus and not by the subspecies anaerobius. On the other hand, Pseudomonads are described as organisms with strictly respiratory metabolism mostly with oxygen and in some species nitrate as terminal electron acceptor [45], hence the release of alcohols and acids from these microorganisms is not expected. Indeed, carboxylic acids were not observed to be released by P. aeruginosa in our in vitro study, but a very week production of 2-butanol and substantially stronger Cell press of ethanol and 3-methyl-1-butanol were found. These may be related to altered activity of aldehyde- and alcoholdehydrogenase as reported by Nosova et al. [46] while the metabolism of amino acids [44] rather than glycolysis of carbohydrates via Entner-Doudoroff pathway [1] yields pyruvate as starting material under conditions applied in our study. Nevertheless, it seems that the most dominant metabolic process in P. aeruginosa cultures is the catabolism of organic compounds such as aldehydes as carbon and energy sources. The versatile nutritional requirements of Pseudomonas are commonly known and some of its subspecies utilize over 100 different compounds of diverse chemical classes what makes them particularly important organisms of bioremediation in environment (degradation of oil spills, pesticides and other xenobiotics) [1, 47].

Unfortunately, few novel drugs have been developed specifically for MDR/PDR Gram-negative bacteria in recent years [8–10]. The development of new antimicrobial agents cannot keep up with the evolution of bacterial resistance. Thus, more efforts should be placed on discovering and developing new antimicrobial agents. As a source of new antibiotics, food-associated microorganisms have recently received increased attention. The well-known active compounds produced by these strains are peptide antibiotics, such as lantibiotics and lipopeptides [11–13]. Many of them are potentially useful in medical and food applications due to their low intestinal toxicity. To obtain antimicrobial

agents that are novel safe and

potent, a lot of food bacteria were isolated and screened for their antimicrobial activity. In this work, strain B7, a new bacterial isolate from a sample of dairy waste, was Navitoclax research buy found to produce antibiotics against both Gram-positive Selisistat and Gram-negative human pathogens. Based on the 16S rRNA gene sequence analysis as well as physiological and biochemical characterization, strain B7 was identified as Paenibacillus ehimensis. After isolation and purification of the fermentation products, the chemical structure and biological characteristics of the active compounds produced by P. ehimensis B7 were determined. Methods Strains and culture conditions Samples of dairy waste were collected from a local dairy industry in Wuxi. The

dairy waste samples were suspended in 0.1% sterile peptone water and antibiotic producing strains were isolated using a competitive inhibition method as previously described [14]. Nutrition broth was used for routine culture. The active compounds were produced in synthetic Katznelson and Lochhead (KL) medium, which had the following composition (in g/L): glucose, 5; (NH4)2SO4, 1.5; MgSO4 .7H2O, 0.2; NaCl, 0.1; CaC12, 0.1; FeSO4 .7H2O, 0.01; ZnSO4, 0.01; MnSO4 .H2O, 0.0075; and KH2PO4 2.7. The medium was autoclaved and brought to a pH of 7.2. Staphylococcus epidermis CMCC 26069 was purchased from the National Center for Medical Culture Collections. S. aureus ATCC 43300, S. aureus ATCC 25923, E. coli ATCC 35218, and P. aeruginosa ATCC 27853 were purchased from the American Type Culture Collection Epothilone B (EPO906, Patupilone) (ATCC). Clinical isolates (P. aeruginosa 5215 and E. coli 5539) were isolated from patients at the Fourth People’s Hospital of Wuxi, Wuxi, China. The tested strains that were used to determine the sensitivity to the active compounds were routinely grown at 37°C on a nutrient agar or in a nutrient broth. For long-term storage, all of the strains were stored in 20% (v/v) glycerol at −80°C. This study was approved by the Ethics Committee of the Fourth People’s Hospital of Wuxi. Strain identification The morphology of strain B7 was examined by light microscopy after Gram-staining and spore staining.

The growing concept that microbial multicellular aggregates form functional and higher organized structures, as a kind of proto-tissue, supports the notion that PCD may be a much more spread and conserved mechanism of cellular altruistic behaviour. The characteristic apoptotic markers, as DNA fragmentation, phosphatidylserine externalization, chromatin condensation, release

of cytochrome C, and/or caspases activation are MLN8237 cost also valid to assess apoptotic yeast cells [1, 8]. Furthermore, an increasing list of homologues of apoptotic regulators in metazoans has been identified in yeast, such as Yca1p, the proposed yeast caspase [9]; Aifp, the apoptosis inducing factor [10]; EndoG, an endonuclease which regulates not only life but also death in yeast [11]; Nma111p, a yeast HtrA-like protein [12]; Bir1p, an inhibitor-of-apoptosis

protein [13] and Ybh3p, a yeast protein that interacts with Bcl-xL and harbours a functional BH3 domain [14]. Additionally, the expression in S. cerevisiae of the mammalian Bcl-2 family and PKC isoforms [15], led to the same phenotypes observed in mammalian cells, see more providing evidence that apoptosis is an evolutionarily conserved mechanism. Several agents can induce yeast PCD, like hydrogen peroxide, UV radiation, the absence of nutrients, hyper-osmotic stress, acetic acid [8] and aging [6]. Aging in yeast can be studied assessing either replicative or chronological lifespan. Replicative lifespan is defined as the number Rucaparib chemical structure of daughter cells a single yeast mother cell produces before senescence; chronological lifespan is defined by the length of time cells can survive in a non-dividing, quiescence-like state [16]. Chronological aged yeast cells also exhibit typical apoptotic markers. During

chronological aging, the old yeasts die and release certain substances (nutrients) into the medium in order to promote survival of other aged cells, yet fitter ones [6]. On the other hand, it has been demonstrated that apoptotic S. cerevisiae cells display changes in the expression of some genes associated with the sphingolipids metabolism [17], which is consistent with changes in the proportions of the various sphingolipid types in dying cells [18]. Carmona-Guitierrez and co-authors [19] observed the apoptosis induction by external addition of C2-ceramide, whereas Barbosa and co- authors reported changes in sphingolipids during chronological aging, namely a decrease of dihydrosphingosine levels and an increase of dihydro-C(26) -ceramide and phyto-C(26) -ceramide levels [20]. Also, a role in apoptosis and aging of Ydc1p ceramidase was described [18], and a yeast homologue of mammalian neutral sphingomyelinase 2 was associated with apoptosis [21]. Moreover, some intermediates in sphingolipids biosynthesis act as signalling molecules and growth regulators [22, 23].

J Colloid Interf Sci 2002, 248:376–382. 10.1006/jcis.2002.8238CrossRef 17. Kolská Z, Řezníčková A, Švorčík V: Surface characterization of polymer foils. e-polymers 2012, 83:1–6. 18. Yin J, Yang Y, Hu ZQ, Deng BL: Attachment of silver nanoparticles (AgNPs) onto thin-film composite (TFC) membranes through covalent bonding to reduce membrane

biofouling. J Membrane Sci 2013, 441:73–82.CrossRef 19. Kim JS, Kuk E, Yu KN, Kim JH, Park SJ, Lee HJ, Kim SH, Park YK, Park YH, Hwang CY, Kim YK, Lee YS, Jeong DH, Cho MH: Antimicrobial effects of silver nanoparticles. Nanomed-Nanotechnol 2007, 3:95–101. 10.1016/j.nano.2006.12.001CrossRef 20. Mayoral A, Barron H, Estrada-Salas R, Vazquez-Duran A, Jose-Yacamán M: Nanoparticle stability from the nano to the meso interval. Nanoscale 2010, 2:335–342. 10.1039/b9nr00287a20644815CrossRef KU-57788 in vivo 21. Chu PK, Chen JY, Wang LP, Huang N: Plasma-surface modification of biomaterials. Mater Sci Eng R 2002, 36:143–206. 10.1016/S0927-796X(02)00004-9CrossRef 22. Webb HK, Crawford RJ, Sawabe T, Ivanova EP: The systems studied may

have potential application e.g. in medicine as prevention of creation of bacterial biofilm. Microbs Environ 2009, 24:39–42. 10.1264/jsme2.ME08538CrossRef Competing interests The authors declare that they have no competing interests. Authors’ contributions AR carried out the AFM analysis, evaluated the surface morphology and roughness, and wrote and designed the study. ZN analyzed the chemical Erlotinib and optical properties of AgNPs and silver-grafted PET. ZK performed zeta potential measurement. VS participated in the study coordination and paper correction. All authors read and approved the final manuscript.”
“Background The molecular imaging (MI) of tumors has recently gained widespread use [1–4] due to its ability to facilitate quantitative and

repetitive imaging of targeted molecules and biological processes in living organisms [2, 5, 6]. Contrast agents are generally required for Abiraterone datasheet high-quality MI diagnosis. Advances in nanotechnology enable the development of various nanoparticles (NPs) as contrast agents for effective MI in the diagnosis or analysis of diseases. Superparamagnetic iron oxide nanoparticles (SPIONs) are a promising form of imaging probe that can accumulated in cells and generate a strong magnetic resonance (MR) imaging contrast in T2- or T2*-weighted images [7]. To date, SPIONs have been used to investigate several pathophysiological processes in tumor cells [8, 9], transplanted cells [1, 7, 10], or precursor cells in vivo[11–13]. SPION probes are generally comprised of superparamagnetic iron oxide cores of magnetite or maghemite NPs encased in various coatings. Cellular uptake of SPIONs may be achieved by phagocytosis, macropinocytosis, or receptor-mediated endocytosis [2, 14, 15].

Structural elements are in capital letters with the name of the corresponding feature underneath them. Underlined and in italics:

possible transmembrane helix. In bold and italics: alpha helices. Underlined: Beta-sheets. In white letters and highlighted in black: meander loop and Cys pocket. The asterisks (*) indicate the three totally conserved amino acids among cytochromes P450, and the exclamation points (!) show the amino acid variation found in the deduced CYP61 from different X. dendrorhous strains. The CYP61 gene mutation To study the function of the CYP61 gene in X. dendrorhous, mutant cyp61 – strains were generated. The wild-type strains UCD 67–385 and CBS 6938 were transformed with plasmid pBS-cyp61/Hyg, and strain AVHN2 was transformed Adriamycin nmr with plasmid pBS-cyp61/Zeo. All transformations were performed with linearized plasmids as indicated in Figure  4. Through a double homologous recombination event, the donor DNA fragment containing the CYP61 gene Ivacaftor nmr interrupted by one of the two resistance markers replaced the CYP61 gene in the yeast chromosome. In this way, we obtained the transformant strains 385-cyp61 hph , CBS-cyp61 hph and Av2-cyp61 zeo (Table  2). The genotype modifications in the transformant strains were validated

by PCR reactions using specific primers for the CYP61 gene, zeocin or hygromycin B resistance cassettes (Table  1) and genomic DNA from the parental and transformant strains. The amplicons confirmed the CYP61 gene interruption (Figure  5). However, as strain UCD 67–385 is diploid [30] and we were able to detect a CYP61 wild-type allele, the resulting strain 385-cyp61 hph is heterozygous (385-CYP61/cyp61 hph ). For this reason, strain 385-CYP61/cyp61 hph was transformed with the linearized plasmid pBS-cyp61/Zeo obtaining the cyp61 – homozygote mutant strain 385-cyp61 hph Carteolol HCl /cyp61 zeo (Figure  5). The ploidy levels of strains CBS 6938 and AVHN2 are unknown; based on random mutagenesis experiments

and by transformation of carotenogenic genes performed at our laboratory [21, 31], we estimate that these strains are aneuploid. In these cases, the PCR-based genotype analysis determined that a unique CYP61 gene copy was mutated in strains CBS-cyp61 hph and Av2-cyp61 zeo (Figure  5), indicating that these strains are hemizygous, so a second transformation event was not necessary in these mutants. Interestingly, a clear difference in the color phenotype could be distinguished among all the cyp61 – mutants and their corresponding parental strains, indicating alterations in carotenoid biosynthesis (see below). Figure 4 Plasmids constructed in this work. In each plasmid illustration, relevant features for this work, such as endonuclease recognition sites and primer binding sites (thin arrows), are shown. Some elements of the original plasmid (pBluescript SK-) were kept and shown in gray. Plasmid pBS-gCyp61 harbors the genomic version of the CYP61 gene from X.