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44. Bjarnsholt T, Kirketerp-Moller K, Jensen PO, Madsen KG, Phipps R, Krogfelt K, Hoiby N, Givskov M: Why chronic wounds will not heal: a novel hypothesis. Wound Repair Regen 2008,16(1):2–10.CrossRefPubMed 45. Walker TS, Tomlin KL, Worthen GS, Poch KR, Lieber JG, Saavedra MT, Fessler MB, Malcolm KC, Vasil ML, Nick JA: Enhanced Pseudomonas aeruginosa biofilm development

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by heat shock of the gene encoding the alternative sigma factor AlgU (sigma E) which controls mucoidy in cystic fibrosis isolates of Pseudomonas aeruginosa. J Bacteriol 1995,177(19):5670–5679.PubMed 51. Ojha A, Anand M, Bhatt A, Kremer L, Jacobs J, William R, Hatfull GF: GroEL1: A dedicated chaperone involved in mycolic acid biosynthesis during biofilm formation in Mycobacteria. Cell 2005,123(5):861–873.CrossRefPubMed 52. Frees D, Chastanet A, Qazi S, Sorensen K, Hill P, Msadek T, Ingmer H: Clp ATPases are required for stress tolerance, intracellular replication and biofilm formation in Staphylococcus aureus. Mol Microbiol 2004,54(5):1445–1462.CrossRefPubMed 53. Pruss BM, Besemann C, Denton A, Wolfe AJ: A complex transcription network controls the early stages of biofilm development by Escherichia coli. J Bacteriol 2006,188(11):3731–3739.CrossRefPubMed 54. Ren D, Bedzyk LA, Thomas SM, Ye RW, Wood TK: Gene expression in Escherichia coli biofilms. Appl Microbiol Biotechnol 2004,64(4):515–524.CrossRefPubMed 55.

JR performed most of the experiments involving silencing of GSTT1 and helped with midgut dissections and oocyst counting. GN and GJ-G performed the P. yoelii infections in An. gambiae and An. stephensi. MP and GJ-G silenced TEP1, LRIM1, and LRIM2 in P. yoelii-infected An. gambiae. A M-C prepared the P. falciparum gametocyte cultures. C B-M contributed with experimental design, data analysis, image processing, assembly of final figures, and writing the manuscript.”
“Background Nowadays low-cost

energy bio-industrial processes in biotechnology are Givinostat highly desired. This has led to increased interest in the production of cold adapted enzymes. One class of such enzymes includes cold-adapted β-D-galactosidases (EC 3.2.1.23) that can find many applications in industrial biotechnology. These enzymes are capable of hydrolyzing 1,4-β-D-galactoside linkages and can sometimes catalyse the synthesis of oligosaccharides. The production of lactose-free milk and synthetic oligosaccharides like lactulose are only examples of this cutting edge enzyme class application. Currently, commercially available β-galactosidase preparations (e.g. Lactozym – Novo Nordisk, Maxilact

– DSM Food Specialties) applied for lactose hydrolysis contain Kluyveromyces lactis β-galactosidase naturally intracellularly biosynthesized by K. lactis strains. This enzyme is optimally active at approximately 50°C and displays PFT�� low activity at 20°C while an ideal enzyme Suplatast tosilate for treating milk should work well at 4–8°C. Besides, the latter enzyme should be optimally active at pH 6.7–6.8 and cannot be inhibited

by sodium, calcium or glucose. Such β-galactosidases are still highly desired. Only several enzymes optimally hydrolyzing lactose at low selleck temperatures have been characterized till now [1–14], however, none of them have been produced on the commercial scale. The β-galactosidases were obtained from different microbial sources, including those from Arthrobacter sp. [1, 2, 7, 8, 12], Arthrobacter psychrolactophilus [9, 13]Carnobacterium piscicola [3], Planococcus sp. [4, 14], Pseudoalteromonas haloplanktis [5], and Pseudoalteromonas sp. [10, 11]. Additionally, in order to make progress in cheaper production of β-D-galactosidases of industrial interest, high efficiency yeast expression systems must be taken into consideration. On the other hand extracellular production must occur to allow easy and fast isolation of target protein. There are several studies in literature related to the extracellular production of the Aspergillus niger β-galactosidase by recombinant Saccharomyces cerevisiae strains [15–19], although this enzyme is mainly interesting for lactose hydrolysis in acid whey, because of their acidic pH optimum as well as their activity at elevated temperatures. The S. cerevisiae expression system was also used for the production of K.

1953) Brenner et al. 1973 and Brenneria paradisiaca to the genus Dickeya gen. nov. as Dickeya chrysanthemi comb. nov. and Dickeya paradisiaca comb. nov. and delineation of four novel species, Dickeya dadantii sp. nov., Dickeya dianthicola sp. nov., Dickeya dieffenbachiae sp. nov. and Dickeya zeae sp. nov. Int J Syst Evol Microbiol 2005,55(4):1415–1427.PubMedCrossRef 26. Darrasse A, Priou S, Kotoujansky A, Bertheau Y: PCR and restriction fragment length polymorphism of a pel gene as a tool

to identify Erwinia carotovora GS-4997 in relation to potato diseases. Appl Environ Microbiol 1994,60(5):1437–1443.PubMed 27. Duarte V, De Boer SH, Ward LJ, De Oliveira AMR: Characterization of atypical Erwinia carotovora strains causing blackleg of potato in Brazil. J Appl Microbiol 2004,96(3):535–545.PubMedCrossRef 28. Yap MN, Barak JD, Charkowski AO: Genomic diversity of Erwinia carotovora subsp carotovora and its correlation with virulence. Appl Environ Microbiol 2004,70(5):3013–3023.PubMedCrossRef

29. Glasner JD, Marquez-Villavicencio M, Kim HS, Jahn CE, Ma B, Biehl BS, Rissman AI, Mole B, Yi X, Yang CH, et al.: Niche-specificity and the variable fraction of the GSK2399872A in vivo pectobacterium pan-genome. Mol Plant Microbe Interact 2008,21(12):1549–1560.PubMedCrossRef 30. Terta M, Kettani-Halabi Pexidartinib M, Ibenyassine K, Tran D, Meimoun P, M’Hand RA, El-Maarouf-Bouteau H, Val F, Ennaji MM, Bouteau F: Arabidopsis thaliana cells: a model to evaluate the virulence of pectobacterium carotovorum. Mol Plant Microbe Fludarabine solubility dmso Interact

2010,23(2):139–143.PubMedCrossRef 31. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, et al.: Clustal W and Clustal X version 2.0. Bioinformatics 2007,23(21):2947–2948.PubMedCrossRef 32. Tamura K, Nei M, Kumar S: Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci USA 2004,101(30):11030–11035.PubMedCrossRef 33. Saitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987,4(4):406–425.PubMed 34. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S: MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods . Mol Biol Evol 2011,28(10):2731–2739.PubMedCrossRef Competing interests The authors declare that they have no competing interests. Authors’ contributions MK-H designed the study, performed the experiments, data analyses and wrote the paper, MT and MA participated in the sample preparation and preliminary examination, EE participated in the design of the study, FB drafted the manuscript, MME coordinated the study, designed and participated in manuscript preparation. All authors read and approved the manuscript.”
“Background When grown in spatially structured environments several Pseudomonas species are known to produce variants with altered phenotypic properties.

Figure 5 Surface roughness Selleckchem Ipatasertib by AFM. (a) 2D and (b) 3D AFM images of the smooth surface, and (c) 2D and (d) 3D AFM images

of the self-assembled nanotip W BE surface. Figure 6 Cumulative probability of HRS/LRS. Cumulative probability of 4 × 4, 20 × 20, and 50 × 50 μm2 cross-point resistive switching Quizartinib manufacturer memory devices. Figure 7 Data retention and endurance. (a) Good data retention and (b) excellent ac endurance with every cycle reading of >105 are obtained. All switching devices have such a long endurance. Conclusions Improvement in the resistive switching and self-compliance behaviors of a forming-free resistive memory stack of Ir/TaO x /W in a cross-point structure has been obtained. The cross-sectional TEM image confirms the amorphous TaO x /WO x film. The AFM image shows the presence GW786034 ic50 of nanotips on the W bottom electrode surface. The device has shown excellent switching uniformity during 100 consecutive dc sweeps with set/reset voltages of ±2.5 V and a resistance ratio of >100. The self-compliance behavior which comes from the bulk resistance of the stack shows the built-in capability of the device

to minimize current overshoot during switching. The improvement in the switching is attributed to the formation of a defective switching layer and bottom electrode surface morphology with nanoscale tips which can enhance the electric field resulting in Tenofovir the uniform formation/rupture

of the oxygen vacancy conducting filament. The device has exhibited an ac cycle endurance of >105 cycles and a data retention of >104 s. It is expected that this self-compliance, low-voltage-operated cross-point resistive memory device could be useful for the development of future nanoscale nonvolatile memory devices. Acknowledgements This work was supported by the National Science Council (NSC), Taiwan, under contract number NSC-102-2221-E-182-057-MY2. References 1. Waser R, Aono M: Nanoionics-based resistive switching memories. Nat Mater 2007, 6:833.CrossRef 2. Lee MJ, Lee CB, Lee D, Lee SR, Chang M, Hur JH, Kim YB, Kim CJ, Seo DH, Seo S, Chung UI, Yoo IK, Kim K: A fast, high-endurance and scalable non-volatile memory device made from asymmetric Ta 2 O 5− x /TaO 2− x bilayer structures. Nat Mater 2011, 10:625.CrossRef 3. Liu Q, Sun J, Lv H, Long S, Yin K, Wan N, Li Y, Sun L, Liu M: Real-time observation on dynamic growth/dissolution of conductive filaments in oxide-electrolyte-based ReRAM. Adv Mater 1844, 2012:24. 4. Park J, Lee W, Choe M, Jung S, Son M, Kim S, Park S, Shin J, Lee D, Siddik M, Woo J, Choi G, Cha E, Lee T, Hwang H: Quantized conductive filament formed by limited Cu source in sub-5 nm era. In Proceedings of the 2011 IEEE International Electron Devices Meeting (IEDM): Dec 5–7 2011; Washington, DC. Piscataway: IEEE; 2011:63. 5.

N Engl J Med 1995,333(1):32–41.PubMedCrossRef Competing selleck products interests The authors declared that they ICG-001 cell line have no competing interests. Authors’ contributions Z-SZ, Z-YY and Y-YW design the study, LL, Y-XW, and H-QT carried out the Realtime quantitative RT-PCR and immunohistochemistry, Y-SS drafted the manuscript. All authors read and approved the final manuscript.”
“Background Hepatocellular carcinoma (HCC) is currently the fifth most common malignancy worldwide [1], and its overall incidence is steadily rising. In spite of the therapeutic

options for HCC such as hepatic resection [2], radiofrequency ablation [3], transcatheter arterial chemoembolization [4], and sorafenib [5], the prognosis of patients with advanced HCC remains poor [6, 7]. Therefore, research to clarify the mechanisms of hepatocarcinogenesis is urgently required [8]. Gene expression microarray analysis has revealed many cancer-related genes in HCC [9]. This method enables the expression status of all genes to be investigated simultaneously [10]. Furthermore, single nucleotide polymorphism (SNP) arrays

have made it possible to detect copy number changes R788 datasheet as well as copy-neutral loss of heterozygosity (LOH) [11]. Recently we developed a double combination array analysis consisting of gene expression array and SNP array analysis, and reported a number of tumor suppressor genes in HCC [12–17]. In these studies, we hypothesized that DNA methylation of the promoter region of these genes downregulated gene expression, causing HCC progression. In addition to this double combination array analysis, we obtained further data from the same specimens using methylation array analysis to make this association of DNA methylation more conclusive. We named it triple combination array analysis; this method seems

to be an efficient procedure for the detection of tumor suppressor genes of HCC [18]. Doublecortin domain-containing 2 (DCDC2) is a candidate tumor suppressor gene detected by this triple combination array analysis. This gene second encodes a member of the doublecortin family [19], and contains two doublecortin domains. The doublecortin domain has been demonstrated to bind tubulin and enhance microtubule polymerization [19, 20], and mutations in this gene have been associated with dyslexia [21–24]. However, there are only a few reports of the relationship between DCDC2 and cancer [25]. In addition, no previous study has researched the role of DCDC2 in HCC. Although it had been considered that DCDC2 gene had an impotrtant role in neuroendocrine systems, the expression of the gene was reported in GeneCards relatively strongest in liver in whole human organs including brain. Therefore, we selected this gene for this study, because we predicted the gene might have some role in liver.

1. This tool can help local governments promote and tailor sustainable activities to meet the needs and wants of residents. By applying the PAIRS metric and conducting an assessment survey, municipalities can gain a sense of what kinds of sustainability initiatives are viable and effective and which are likely to earn broad support or meet resistance. This knowledge can enable staff to effectively communicate environmentally focused projects to residents.   2. PAIRS can help cities identify highly effective and readily implemented practices which can leverage local resources or sustainability capital between two cities and even lead them full

circle back to more traditional sister city exchanges of informal cultural LY411575 chemical structure Epacadostat cell line capital.   3. Jointly pursuing sustainable development, as this method suggests, helps actors to effectively share the burden of developing and implementing new sustainable strategies. By utilizing PAIRS to locate a partner city to leverage existing resources and reap benefits not currently enjoyed, both cities can address their needs in a way that might be more cost-effective than pursuing them in isolation.   4. The PAIRS metric does not show bias toward any single sector and thus could encourage reciprocity in different sectors. One partner

might seek a collaboration to boost its sustainable water supply and offer a reciprocal exchange of compostable waste with a partner city. Thus, the balance of sustainable improvement is equal for both participating cities.   5. The PAIRS metric can be utilized over time to measure improvement and identify new areas in which to address sustainability as circumstances and needs evolve.   PAIRS represents an important innovation in sustainability science and an achievement in transdisciplinary research—the type of research needed to remediate Dipeptidyl peptidase global problems (Stokols 2006). Unlike the more common interdisciplinary approach, which looks to short-term problem-solving and tends to have minimal impact on theory and the ever changing state of society,

this research draws on easily identifiable theoretical frameworks to provide a comprehensive analysis that goes beyond any singular discipline’s approach (Rosenfield 1992). Using the sister city model to foster cooperation among cities, a team of MDV3100 mw researchers from different disciplines produced a data-driven mathematical tool that cities can use to evaluate the prospects for improving sustainability practices by leveraging existing resources and establishing synergistic partnerships along key sustainability dimensions with neighboring cities. This project will serve not only to inspire more scholarly work on exploring new ways to increase sustainability in urban and rural settings, but also to implement changes in the manner in which sustainability objectives are pursued at the municipal level.

Also, lack of financial support may have contributed to delay in procuring abortion. Women’s reasons for seeking abortion were discussed in several studies [9, 24, 29–31]. These included inappropriate timing of the pregnancy, fear of expulsion from school, financial difficulties, and uncertainties

about the Pritelivir partner. In this study, fear of expulsion from school was the most common reason for terminating pregnancy. As reported by many authors [15, 17, 30, 31], majority of patients in the present study presented late in poor general condition. This was found to be the most important factor influencing the outcome of surgical procedure as also emphasized by a number of authors [9, 15, 30]. In resource-poor countries, difficulties in diagnosis, lack of awareness of the disease and delayed referral to tertiary hospital often result in delayed presentation to a hospital Surgical intervention is considered to be the gold standard treatment for patients with bowel perforation following induced abortion [9]. In this study, all patients underwent surgical treatment which is in keeping with other studies [9, 11, 16–20, 26, 32, 33]. One of the many factors affecting the surgical outcome in patients with bowel perforation is time interval from perforation to laparotomy [9, 15]. Early

Selleckchem Doramapimod surgery can minimize the complications while delayed surgery leads to severe peritonitis and septic shock. In the present study, the majority of patients were operated more than 24 h after the onset of illness. Similar observation TH-302 mw was reported by other studies done in developing countries [4, 9, 30]. Delayed definitive surgery in the present study 4��8C may be attributed to late presentation due to lack of accessibility to health care facilities, lack of awareness of the disease as a result some patients with bowel perforation following induced abortion may decide to take medications in the pre-hospital period with hope that the symptoms will abate. It is also possible that some clinicians managing the patients initially

may not have considered perforation as a possible diagnosis leading to delayed referral to tertiary care hospital. In keeping with other studies [9, 16–20], the ileum and the sigmoid colon were the most common parts of the bowel affected. The relative fixity of these portions of the bowel has been suggested as a possible reason for this. Early surgical interference is the optimal treatment option for perforation. However, the type of surgery to be applied is controversial [9]. The surgical management of small intestinal injuries is fairly straightforward with minimal sequalae. Our practice in managing these patients is a simple closure in solitary perforations and segmental intestinal resection and primary anastomosis in multiple perforations or gangrenous bowel.

Considering the possible harmful effects of having medical students working in an emergency department alone, all activities developed must be under supervision, what help their practical training process that will never be achieved only by books.[5, 9] Nevertheless, replacing curricular activities by extra-curricular ones shall be always discouraged. Not only but also, many Universities unfortunately do not offer a good enough plan of activities for their medical students, making regular lectures not a priority in their schedules (an issue that shall be addressed in a different paper). Despite the better

quality of medical care that can be offered by dedicated doctors compared to medical students, in Brazilian busy public emergency rooms most of the time it is impossible to dedicate the appropriate Akt inhibitor time on consultations to each patient (what may be the reality in most of the countries worldwide). Then, a team of committed medical students can be extremely helpful on patient care. Even being

non-licensed not-fully-trained, if properly supervised, they can play an important role in this environment. Tutors must be always aware of eventual medical errors that if not promptly approached will be under their legal responsibility as well as a threat to patients’ safety. Since it is a surgical clerkship, it is expected LY3039478 price that the vast majority of students aim to follow a surgical career beforehand (70.6%). However, data concerning the influence of the extra-curricular activity in their decision should be analyzed carefully. Most of the students that do not have interest Amobarbital on a surgical career and find the practical activities a bad influence for them may abandon it before its completion (500 hours), or even before 200 hours, and would not participate in the present study. Conclusions Our data suggests that 200 hours seems to be a suitable threshold in medical students’ learning surgical manual dexterity in an Emergency Department clerkship, even in the

absence of objective parameters to further evaluate this theory. Last but not least, maturity and quality of medical care significantly improved proportional to the number of hours served in the ED clerkship. Therefore the practice of leaving before 200 hours should be actively discouraged. Further comparative studies with objective criteria to evaluate students and residents’ manual dexterity and their own perception of their abilities should be performed in order to assess our initial findings. Acknowledgements We thank Iwan Augusto Collaço, MD, PhD, TCBC, for his substantial academic contributions in the field of Trauma, and for his efforts by which this study was made possible. We also thank Kenneth Stahl, MD, FACS, for his contribution with the discussion of this study. This article has been published as part of World Journal of Emergency Surgery Volume 7 Supplement 1, 2012: Proceedings of the World Trauma Congress 2012.

LB9 (GenBank: JQ864377.1) matching 99% identity. This explains the relatively high number of total bacterial colonies recovered from mushroom tissue treated with Bdellovibrio, despite the reduction in the dark lesions characteristic of P. tolaasii infection: Bdellovibrio predation rapidly reduces P. tolaasii population numbers on the mushroom surface, but does not necessarily reduce those of other non-disease

causing, likely mushroom-indigenous species, such as the Enterobacter isolated in this study. The King’s Medium B in which P. tolaasii 2192T and B. bacteriovorus HD100 were added to the surface of the Fludarabine cost mushroom during test inoculations, and the cell-lysate debris left behind after P. tolaasii death due to predation, may then allow these indigenous Enterobacter to occupy the niche caused by Bdellovibrio predation of P. tolaasii. Discussion We showed

that B. bacteriovorus HD100 is a predator of P. tolaasii 2192T in vitro and in vivo (in funga), suppressing population growth GDC-0994 of the strain over a 24-hour period where 4 × 106 or 1.6 × 107 PFU B. bacteriovorus HD100 were added to pathogen on post-harvest mushrooms (Figures 1 and 4). P. tolaasii is a difficult pathogen to control in mushroom grow-houses due to its ability to persist in nutrient-poor soils and the ease with which it spreads through mushroom compost, through flagellar swimming, and via the hands of pickers during the manual harvesting process [8]. Furthermore, commensal bacterial species in the mushroom casing soil play a key role in mushroom growth initiation, and therefore any treatment to prevent or treat P. tolaasii infection must not result in a completely sterile growth environment, which may result from broad antibiotic or antiseptic treatment. Thus it is beneficial to explore post-harvest anti- P. tolaasii treatments, such as this study with B. bacteriovorus. Our SEM images confirmed that B. bacteriovorus HD100 survived on the post-harvest supermarket mushroom surfaces after 48 hours, and was therefore unaffected by any

pre-treatment of those mushrooms for commercial purposes to promote growth and extend shelf-life in the film-covered plastic trays they were sold in (Figure 3c). B. bacteriovorus is therefore a viable treatment for bacterial selleck chemicals llc diseases of mushrooms, such as brown blotch disease. Previous studies of mushroom infections have found that a ‘threshold’ number of P. tolaasii cells are required for the initiation of infection, which includes production of tolaasin, the chemical mediator of the brown blotch symptom development [8]. We found that when B. bacteriovorus HD100 was applied to the surface of post-harvest, commercially grown mushrooms before or after inoculation with P. tolaasii, both the intensity of the brown blotch symptoms of disease and the number of P. tolaasii 2192T present the mushroom surface were significantly reduced (Figures 2 and 4), supporting the threshold hypothesis.

To quantitate the productivity of actinorhodin, equal amounts of spores of M145 and 4F containing pCWH74 were inoculated into R2YE liquid medium

lacking KH2PO4 and CaCl2, and 1 ml culture was harvested in a time-course. As shown in Figure 4, actinorhodin was produced in 4F at both 30 and 37°C, earlier than in M145 at 30°C. At 100 h, productivity of actinorhodin in 4F at 30°C was ~2.8 times higher than in M145 at 30°C. Strains M145 and 4F grew better in TSB than in R2YE liquid media (data no shown), but no actinorhodin was detected when cultured in TSB medium at 30 and 37°C. Growth curves of the two VX-680 molecular weight strains in R2 lacking KH2PO4 and CaCl2 at 30°C showed that their biomass values were similar from 20 to 120 hours (data not shown). Thus, better growth of M145 and 4F in TSB medium (Figure 3) did not correlate with delayed and less production of actinorhodin in R2YE medium (Figure 4). Like in 4F, M145 produced more actinorhodin in R2YE medium at 30°C than at 37°C, suggesting that expression of the actinorhodin biosynthetic genes might be temperature-dependent. Temperature-dependent antibiotic gene clusters have been reported in Streptomyces, for example, much higher productivity Smad phosphorylation of validamycin A produced by Streptomyces hygroscopicus was found at 37°C than at 30°C [40]. We infer that by replacement of thermophilic-specific promoters, many single genes and especially antibiotic

genes clusters of mesophilic Streptomyces should be heterologously expressed in the fast-growing and thermophilic Streptomyces. Heterologous expression of the anthramycin biosynthetic gene cluster of the

thermophilic S. refuineus subsp. thermotolerans in strain 4F Expression of the anthramycin biosynthetic genes of S. refuineus subsp. thermotolerans could be detected at high temperature (i.e. 47°C), but not at 30 or 37°C [22]. An integrating cosmid, 024COA-3, containing the whole anthramycin biosynthetic gene cluster was introduced by conjugation from E. coli into strain 4F. PCR amplification experiments confirmed the presence of the anthramycin genes in the clone of 4F. Aldehyde dehydrogenase After culturing in AP1 medium at 30, 37 and 47°C for 24 h, mycelium was extracted, dried and re-dissolved in MeOH. Thin-layer chromatography, followed by a bio-assay by overlaying with LB agar containing as indicator strain a Bacillus sp., revealed a zone of growth inhibition on 4F at 47°C, but no inhibition zone was found at 30 and 37°C (data not shown). A spot on a TLC plate was further purified for HPLC-MS analysis. As shown in Figure 5, an anthramycin-specific peak (ES+ = 316 Dalton, see ref [41]) was detected. Thus the anthramycin biosynthetic gene cluster of the thermophilic S. refuineus subsp. thermotolerans was heterologously expressed in strain 4F. We introduced the same cosmid 024COA-3 containing the anthramycin gene cluster into strain 2C, but no transformants were obtained.