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The ISME Journal (2015), 1–12 © 2015 International Society for Microbial Ecology All rights reserved 1751-7362/15 www.nature.com/ismej
Molecular mechanisms underlying the close association between soil Burkholderia and fungi Nejc Stopnisek1,2, Daniela Zühlke3, Aurélien Carlier1, Albert Barberán4, Noah Fierer4,5, Dörte Becher3, Katharina Riedel3, Leo Eberl1 and Laure Weisskopf1,2 1
Institute of Plant Biology, University of Zurich, Zürich, Switzerland; 2Institute for Sustainability Sciences, Agroscope, Zürich, Switzerland; 3Institute of Microbiology, University of Greifswald, Greifswald, Germany; 4 Cooperative Institute for Research in Environmental Sciences, University of Colorado, Boulder, CO, USA and 5 Department of Ecology and Evolutionary Biology, University of Colorado, Boulder, CO, USA
Bacterial species belonging to the genus Burkholderia have been repeatedly reported to be associated with fungi but the extent and specificity of these associations in soils remain undetermined. To assess whether associations between Burkholderia and fungi are widespread in soils, we performed a co-occurrence analysis in an intercontinental soil sample collection. This revealed that Burkholderia significantly co-occurred with a wide range of fungi. To analyse the molecular basis of the interaction, we selected two model fungi frequently co-occurring with Burkholderia, Alternaria alternata and Fusarium solani, and analysed the proteome changes caused by cultivation with either fungus in the widespread soil inhabitant B. glathei, whose genome we sequenced. Co-cultivation with both fungi led to very similar changes in the B. glathei proteome. Our results indicate that B. glathei significantly benefits from the interaction, which is exemplified by a lower abundance of several starvation factors that were highly expressed in pure culture. However, co-cultivation also gave rise to stress factors, as indicated by the increased expression of multidrug efflux pumps and proteins involved in oxidative stress response. Our data suggest that the ability of Burkholderia to establish a close association with fungi mainly lies in the capacities to utilize fungalsecreted metabolites and to overcome fungal defense mechanisms. This work indicates that beneficial interactions with fungi might contribute to the survival strategy of Burkholderia species in environments with sub-optimal conditions, including acidic soils. The ISME Journal advance online publication, 19 May 2015; doi:10.1038/ismej.2015.73
Introduction Members of the genus Burkholderia belong to the class β-Proteobacteria and are widely distributed in the environment. Burkholderia are particularly abundant in soil where they can be associated with a wide range of plants (Elliott et al., 2009; Carlier and Eberl, 2012), invertebrates (Kikuchi et al., 2005) and fungi (Warmink et al., 2009; Uroz et al., 2012; Scherlach et al., 2013). Since Burkholderia are mostly found in acidic soils (Stopnisek et al., 2014), interactions with fungi might be of particular relevance as fungi also favour acidic environments. From in vitro studies, we know that Burkholderia can form either antagonistic or mutualistic interactions with fungi. Antagonistic behaviour of Burkholderia species is well described and is largely due to
Correspondence: L Weisskopf, Institute for Sustainability Sciences, Agroscope, Reckenholzstrasse 191, CH-8046 Zürich, Switzerland. E-mail:
[email protected]
Received 10 October 2014; revised 24 March 2015; accepted 26 March 2015
the production of multiple antifungal compounds (Lewenza and Sokol, 2001; Partida-Martinez and Hertweck, 2007; Schmidt et al., 2009) that can be inhibitory to a wide range of phytopathogenic fungi (Quan et al., 2006; Kilani-Feki et al., 2011; Groenhagen et al., 2013). It has been reported that many environmental Burkholderia strains have beneficial effects on fungi, suggesting symbiotic and/or mutualistic interactions. Benefits from such interactions were mainly studied in a model system comprised of B. terrae and Lyophyllum sp., where it was shown that the bacteria (i) colonize the hyphae and use them for transportation and dispersal (Warmink et al., 2011), (ii) survive better in acidic soils (Nazir et al., 2010a) and (iii) use several fungal exudates as nutrients (Warmink et al., 2009; Nazir et al., 2010b, 2013). Along these lines, Drigo et al. (2013) reported that Burkholderia strains were among the main consumers of carbon released from arbuscular mycorrhizal fungi in a rhizosphere community. In addition, it has been suggested that the presence of fungi is essential for colonization of sterile soils by Burkholderia (Nazir et al., 2010a; Warmink et al., 2011). Despite these reports indicating that Burkholderia benefits from partnering with fungi, the following
Molecular basis of Burkholderia-fungi interactions N Stopnisek et al 2
questions remain unanswered: (i) how widespread are such associations in soils, (ii) how specific are they, (iii) what is their molecular basis and (iv) what benefits do the bacteria receive from the interaction? To answer these questions, we designed a comprehensive study in which co-occurrence network analysis, cultivation-based methods and proteomics were used. Co-occurrence network analysis was carried out on soils from a continentalscale study to determine the extent, the specificity and the distribution of such interactions in the soil. Model Burkholderia strains previously identified as major soil inhabitants (Stopnisek et al., 2014) were tested further for their ability to translocate and disperse with fungi. Finally, the widespread soil inhabitant B. glathei was grown alone or in the presence of either Fusarium solani or Alternaria alternata and the bacterial proteome was analysed under each situation to identify the molecular and physiological basis of the interaction.
Materials and methods Co-occurrence analysis
We analysed the microbial communities of 266 soil samples from the Nutrient Network (NutNet) globally distributed experiment (Borer et al., 2014). Details of this data set and the methods used to characterize the bacterial and fungal communities are available in Prober et al. (2015). In total, 37 393 ITS fungal operational taxonomic units (OTUs) and 223 693 16S rRNA bacterial OTUs (with OTUs defined at 497% sequence similarity level for ITS1 and 16S rRNA reads for fungi and bacteria, respectively) were included to the analysis. Co-occurrence patterns between Burkholderia and fungal OTUs were tested using Spearman's rank correlations between OTUs that occurred in at least 5% of the samples and had a rho of 40.3 and P-valueo0.01 adjusted using the false discovery rate method (Barberan et al., 2012). Monitoring the interactions between fungi and Burkholderia sp.
Fungi used in this experiment belonged to the species Alternaria alternata, Fusarium solani,
Rhizoctonia solani and Lyophyllum sp. Karsten, while Burkholderia species applied were B. glathei, B. hospita, B. fungorum and B. caledonica (Table 1). To visualize the interaction with fungi under the fluorescent microscope, all Burkholderia strains were tagged with either the green fluorescent protein (GFP) or the dsRED protein using the electroporation protocol described by Choi et al. (2006). In short, overnight cultures of Burkholderia strains were washed twice in 0.3 M sucrose and subsequently electroporated with the plasmid pBBR1MCS-2-gfp mut3 Kmr or pin62: DsRed Cmr. Electroporated cells were transferred on Pseudomonas isolation agar and Luria-Bertani (LB) plates with the corresponding antibiotics (50 mg l − 1). To follow the interactions, fungi were inoculated on water agar plates (15 g l − 1) or LB agar at least 3 days before spotting the bacteria on the mycelium. For that overnight liquid Burkholderia cultures were prepared, washed three times in 0.9% NaCl, OD600 was adjusted to 0.1 and 4 drops of 2 μl were spotted on the fungal mycelium pre-grown for 3 days. Plates were incubated for up to 10 days in the dark and the growth of Burkholderia as well as interactions with fungi were followed daily under the fluorescence microscope (Leica M165 FC, Mannheim, Germany) and binocular (Leica DM6000 B, Mannheim, Germany). To monitor dispersal and attachment of Burkholderia strains on hyphae, a sterile iron ring was placed before pouring in the middle of the plate, which served as a physical barrier for bacterial cells. Both sides were filled up to 5 mm below the edge of the iron circle. Selected fungi and Burkholderia strains were inoculated in the inner side of the ring, so that only bacterial cells which could attach and/or disperse would be detected on the outer side of the ring. All experiments were performed in triplicates.
Proteome analysis
For proteomic analyses, B. glathei LMG14190 and two fungi, A. alternata and F. solani, were used. Co-cultures with A. alternata (AB) and F. solani (FB) as well as the B. glathei alone (B) were grown in triplicates as described above with some modifications: as growth medium 1/3 Difco Malt Extract
Table 1 Bacterial and fungal strains used in this study Strains
Burkholderia fungorum LMG16225 Burkholderia glathei LMG14190
Interaction studies Interaction studies, genome sequencing, proteomics Interaction studies Interaction studies
Coenye et al. (2001) Vandamme et al. (1997)
Burkholderia hospita LMG20598 Burkholderia phytofirmans LMG22487 Alternaria alternata Fusarium solani Rhizoctonia solani Lyophyllum sp. Karsten DSM 2979
The ISME Journal
Interaction studies, proteomics Interaction studies, proteomics Interaction studies Interaction studies
Goris et al. (2002) Sessitsch et al. (2005) Phytopathology group Institute of Plant Sciences Federal Institute of Technology, Zurich, Switzerland Leibniz Institute DSMZ
Molecular basis of Burkholderia-fungi interactions N Stopnisek et al 3
Agar (MEA) (BD, Franklin Lakes, NJ, USA) with 7 g agar l − 1 medium was used and plates were supplemented with a cellophane membrane (Bio-Rad, Hercules, CA, USA; 165-0963-MSDS). After 10 days at room temperature, the biomass was collected and samples were treated as described by Carlier and Eberl (2012) with some modifications: to separate bacterial and fungal cells, mixed samples were homogenized in phosphatebuffered saline solution with a pestle, debris were pelleted at 1000 g for 3 min and the top layer was transferred to 50% Percoll (Sigma-Aldrich, St Louis, MO, USA). After ultracentrifugation at 15 000 g for 3 h, fractions were collected and analysed by phasecontrast light microscopy (Leica DM6000 B) and bacterial fractions were pooled and washed twice in phosphate-buffered saline solution. To extract proteins, a bead-beating protocol was used. Fractions were transferred into 2 ml tubes (Sarstedt, Nürnbrecht, Germany) with 0.25 ml of 0.1 mm glass beads (BioSpec Products, Bartlesville, OK, USA) and placed into FastPrep-24 lyser tubes (MP Biomedicals, Santa Ana, CA, USA). Eight rounds of 30 s lysis of 6.5 m s − 1 followed by 5 min incubation on ice were used to extract proteins in concentrations from 2 to 8 mg ml − 1. In all, 25 μg of cytosolic protein extract (RotiNanoquant, Roth, Karlsruhe, Germany) was resolved on 1D SDS-PAGE, lanes were cut in equidistant pieces and in-gel digested with trypsin as recently described (Grube et al., 2014). Peptide mixtures were separated by RP chromatography using an EASYnLC 1000 (Thermo Fisher Scientific, Waltham, MA, USA) with self-packed columns. Peptides were loaded and desalted on the separating column following resolution with a binary non-linear 76 min-gradient from 5% to 75% ACN in 0.1% acetic acid at a constant flow rate of 300 nl min − 1. The liquid chromatography system was coupled online to an Orbitrap Elite equipped with a nanoelectrospray ion source (Thermo Fisher Scientific) performing mass spectrometry (MS) and MS/MS experiments of the eluted peptides. Survey scans were recorded in m/z range from 300 to 1700 with a resolution of 60 000 and with lock mass option enabled. MS/MS experiments were performed for the 20 most intensive precursor ions as determined in the survey scan excluding unassigned charge states or singly charged ions from the MS/MS experiments in the Linear ion trap. Proteins were identified by searching all MS/MS spectra against a database containing protein sequences of Burkholderia glathei LMG14190, Fusarium solani and Alternaria alternata (version 13-Dec-17) with added laboratory contaminants using Sorcerer-SEQUEST (Sequest v. 27 rev. 11, Thermo Scientific) including Scaffold_4.0.5 (Proteome Software, Portland, OR, USA). SEQUEST was searched with a parent ion tolerance of 10 ppm and a fragment ion mass tolerance of 1.00 Da. Up to two missed tryptic cleavages were allowed. Methionine oxidation (+15.99492 Da) was set as variable modification. For protein identification, a stringent SEQUEST filter for peptides was used (Xcorr versus
charge state: 2,2, 3,3 and 3,8 for doubly, triply and quadruply charged peptides and deltaCn value greater than 0.10) and at least two peptides per protein were required. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. The protein false discovery rate, based on a decoy database, was 0.4%. Relative quantification was based on spectral counting of exclusive spectra using normalized spectral abundance factors (Zhang et al., 2010). Statistical analysis using MeV v4.8.1 (Saeed et al., 2003) was done for proteins that were present in at least two out of three biological replicates. Hierarchical clustering and Student's t-test of z-transformed normalized exclusive spectra were performed with the following settings: unequal group variances were assumed (Welch approximation), P-values based on permutation (1000) with P = 0.05, significance determined by standard bonferroni correction. Only proteins showing at least twofold changes in addition to statistical significance were considered for further analysis. So-called 'off/on' proteins needed to be detected in all replicates of one treatment and absent from all replicates of another treatment. Functional prediction and assignment of proteins to cluster of orthologous groups and TIGRFAMs (Haft et al., 2013), respectively, was accomplished by the in-house developed analysis pipeline 'Prophane 2.0' (http://www.prophane.de; Schneider et al., 2011). Voronoi treemaps were generated using Paver (Decodon, Greifswald, Germany, http://www.decodon.com/). Burkholderia glathei LMG14190 genome sequencing
High molecular mass DNA was extracted following a standard protocol (Wilson 2001). Paired-end insert libraries were constructed at the FGCZ (Functional Genomics Center Zurich) using the Nextera XT kit (Illumina, San Diego, CA, USA). Sequencing of the 2 × 250 bp inserts was performed on the Illumina MiSeq platform at the FGCZ. The sequencing reads were assembled using the SPADES v2.5 software (Nurk et al., 2013). The resulting 138 contigs were examined and edited for misassemblies in GAP5 (Bonfield and Whitwham, 2010). Ribosomal RNA and tRNA genes were predicted using RNAmmer and tRNAscan (Schattner et al., 2005; Lagesen et al., 2007). The predicted RNA genes were masked using the maskfeat utility of the EMBOSS software package (Rice et al., 2000) before protein-coding gene finding using the Prodigal software (Hyatt et al., 2010). The annotated contigs, containing CDS and RNA gene predictions, were submitted to the RAST online service for functional annotation (Aziz et al., 2008). The annotations were curated using the Artemis software suite (Carver et al., 2008). The annotated sequences were deposited at the European Nucleotide Archive (accession numbers CCNS01000001–CCNS01000139). The ISME Journal
Molecular basis of Burkholderia-fungi interactions N Stopnisek et al 4
Results and Discussion Burkholderia co-occur with fungi in soils
The frequency of fungi co-occurring with Burkholderia was assessed with co-occurrence analysis from a trans-continental soil sample collection. The analysis supported our hypothesis that members of the genus Burkholderia significantly co-occur with multiple fungal taxa, suggesting broad and frequent associations between fungi and Burkholderia in soils (Figure 1, Supplementary Table S1). Apart from Burkholderia, other bacterial genera also showed high co-occurrence with fungi. Whether these patterns reflect an intimate interaction with the fungi or result from their shared preference for low pH or other environmental parameters remains to be investigated. For Acidobacteria and Verrucomicrobia, two phyla containing OTUs that showed high co-occurrence with fungi, low pH dependency has been demonstrated but to our knowledge there is no evidence for beneficial interactions between members of those phyla and fungi. In contrast, while the genus Burkholderia also shows strong preference for acidic soils (Stopnisek et al., 2014), mounting evidence suggests that many Burkholderia species establish direct associations with fungi and may often live directly on or inside fungal cells (Warmink et al., 2011; Nazir et al., 2012; Uroz et al., 2012). Thus, we speculate that the strong co-occurrence of Burkholderia and fungi indicates that the two organisms interact with each other in the soil, rather than just reflecting shared niche preferences.
an iron ring (Figure 2). The fungus and the bacteria were inoculated together inside the ring and only those bacteria able to attach to the hyphae were able to cross the ring to reach the outer compartment. This experiment was carried out both with water agar, on which bacteria alone could not grow, and with LB as growth media in the outer compartment. Interestingly, even when the rich LB medium was used, Burkholderia strains were mainly found in close vicinity to fungal hyphae, suggesting that the benefits provided by the fungi were not restricted to transportation, but included other goods that caused bacteria to stay on the hyphae rather than spread onto the rich medium. Our experiments showed that (i) for all four tested Burkholderia strains, growth on the hyphae was favoured over growth on the medium (Figure 2) and (ii) all tested Burkholderia strains were able to cross the iron ring and be translocated with the hyphae of the fungi tested, with the exception of Lyophyllum sp. Karsten. Under the conditions used, the mycelium of Lyophyllum showed hydrophobicity, thus inhibiting the formation of a water film around the hyphae, which might be necessary for bacterial attachment and dispersal (Pion et al., 2013). However, Warmink et al. (2011) showed that B. terrae BS001 could migrate together with Lyophyllum sp. Karsten in soil microcosms,
Burkholderia sp. attach, translocate and disperse on fungal hyphae
To test the specificity of the interactions and the ability of Burkholderia to attach, translocate or disperse on hyphae of different fungal species, we designed an in vitro experiment. Four Burkholderia strains that were previously identified as widespread soil inhabitants (Stopnisek et al., 2014), and four fungi that were among those taxa which most frequently co-occurred with Burkholderia were grown in dual cultures in Petri dishes containing
Figure 1 Co-occurrence of fungal (grey boxes) and Burkholderia (black boxes) OTUs from 266 soil samples. Box size represents the number of significant (rho40.3, P-valueo0.01 after false discovery rate (FDR)) associations for each OTU. The ISME Journal
Figure 2 Monitoring Burkholderia–fungi interactions. Plates with sterile iron rings and either water agar, LB or a combination of both were used to monitor the ability to translocate, to disperse and to form biofilms on the hyphae. For microscope observations, Burkholderia cells were tagged with gfp or DsRed (on the pictures B. glathei LMG14190 tagged with DsRed), co-cultivated with fungi (here F. solani) and monitored on a daily basis. Microscopy pictures represent different stages of dispersal on F. solani hyphae over time. (a) Network of hyphae is densely colonised by bacteria (red) in the inner part of the ring. (b) Crossing of the iron ring by fungal hyphae enables Burkholderia cells to cross too when attached to the hyphae. (c) Colonization of the hyphae by transported Burkholderia occurs also on the outer part of the iron ring. The same behaviour was visible when water agar was replaced by LB.
Molecular basis of Burkholderia-fungi interactions N Stopnisek et al 5
suggesting that the physiology of this fungus may change when growing in soil. Our results are consistent with the results reported by Nazir et al. (2012), who found that 19 Burkholderia strains were able to co-migrate with Lyophyllum sp. Karsten, but their abilities differed depending on the soils used. No visible deleterious effects of the bacteria on mycelial growth or morphology were observed. Global analyses to elucidate the molecular basis of Burkholderia–fungi interactions
We used a proteomic approach to characterize the molecular nature of the interaction between Burkholderia strains and fungi. Among the fungal OTUs that co-occurred with Burkholderia, those belonging to the genera Alternaria and Fusarium were among the most frequently detected ones in our analysis (Supplementary Table S1), and thus we selected A. alternata and F. solani as model species. As a representative of the genus Burkholderia, B. glathei LMG14190 (T) (Vandamme et al., 1997) was chosen for these interaction studies, since members of this species were not only the most frequently detected in a previous soil survey (Stopnisek et al., 2014), but have also been demonstrated to interact with fungi (Koele et al., 2009). Genetic features of B. glathei LGM14190 possibly involved in interactions with fungi
To obtain insights into the reasons for the ubiquitous distribution of B. glathei in soils, as well as for better interpretation of proteomics data, we sequenced the genome of B. glathei LMG14190, which was not available at the time of our analyses. The draft genome is 8'049'485 bp in size, which is consistent with already sequenced strains of the genus Burkholderia (Xu et al., 2013; Carlier et al., 2014, Liu et al., 2014). Annotation of the draft genome predicted 7216 protein coding genes, 50 tRNAs and 3 rRNAs (PRJEB6934). The genome of B. glathei LMG14190 encodes a large number of genes involved in transport and utilization of various carbon sources. In addition to the utilization of simple sugars, B. glathei LMG14190 has the genetic potential to degrade polymeric carbohydrates such as chitin, cellulose, glycogen, as well as alcohols, including ethylene glycol, ethanol and butanediol. B. glathei LMG14190 contains genes coding for enzymes involved in the degradation of glycerol, which is predicted to be one of the C sources fungi secrete (Nazir et al., 2013). Additionally, fungi produce a number of aromatic compounds that could represent another source of nutrients for Burkholderia (Gutiérrez et al., 1994). Degradation of aromatic compounds is a commonly observed feature of various Burkholderia strains (Pérez-Pantoja et al., 2012; Andreolli et al., 2013; Schamfuss et al., 2013; Xu et al., 2013) and the corresponding genes were also identified in the
genome of B. glathei LMG14190. Beside pathways for the utilization of toluene, phenolic, benzoate and catechol compounds, the genome also encodes the complete salicylate degradation pathway. Given the antibacterial and antifungal properties of many of these aromatic compounds, the capacity to degrade such substances could also serve as a protection mechanism for the bacteria and/or their fungal partners. The type three secretion system has been described as essential to the interaction between B. terrae BS001 and Lyophyllum sp. Karsten (Warmink and van Elsas, 2008). However, homologous genes could not be detected in the genome of B. glathei LMG14190. We also tested for the presence of the hrcR gene, a highly conserved component of the type three secretion system (Warmink and van Elsas, 2008), in the four Burkholderia strains used in our dual culture assays. Surprisingly, the hrcR gene could only be amplified from one of the strains tested, B. hospita, but was not detected in B. glathei, B. fungorum or B. phytofirmans (data not shown). This suggests that type three secretion system is not essential for the interaction with the fungi tested here. Nonetheless, the genome of B. glathei LMG14190 harbours genes that have been described to have a role in biological interactions, such as those involved in the synthesis of the exopolysaccharide cepacian (BGLT00414-BGLT00424), which is produced by most Burkholderia species and is important for interactions with other organisms as well as for resistance to different stresses (Ferreira et al., 2011). Global changes in the proteome of B. glathei when co-cultivated with fungi
A total of 2406 unique B. glathei LMG14190 proteins and only 3 fungal proteins were detected in the present study, indicating an efficient enrichment of bacterial cells from the co-cultures (Supplementary Table S2). After applying stringent quality filters and considering only proteins identified in at least 2 out of 3 biological replicates, 1583 proteins remained, the expression of which could be semi-quantitatively assessed by spectral counting (Supplementary Table S3). Among those, 923 proteins (almost 60%) were identified in both, single and co-cultures, while 282, 47 and 29 proteins were found to be exclusively expressed during single culturing or co-cultivation with A. alternata and F. solani, respectively (Figure 3). Overall, 742 were differentially expressed (expression change of more than twofold) during co-cultivation compared to the single cultures with the majority of these proteins expressed in lower amounts in the presence of either of the fungi. Most of the proteins identified in this study were assigned to functions associated with the production and conversion of energy, as well as with transport and metabolism of carbohydrates and amino acids (Supplementary Table S2). This corresponds to the high proportion The ISME Journal
Molecular basis of Burkholderia-fungi interactions N Stopnisek et al 6
Figure 3 Venn diagram depicting quantified proteins of B. glathei LMG14190 grown in single culture (B) and in co-cultivation with A. alternata (BA) and F. solani (BF), respectively.
of predicted genes with the same function in the genome of B. glathei LMG14190 (approximately 20% of the whole genome). In contrast, functional classes such as transcription, defence and signalling were under-represented in the B. glathei proteome when considering their contribution to the total number of predicted genes (Supplementary Table S2). To shed light on the molecular mechanisms involved in bacterial–fungal interactions, we focussed mainly on the B. glathei proteins whose abundance changed significantly during fungal co-cultivation. As shown in Figures 4 and 5 co-cultivation affected protein expression across all functional categories. Notably, a particular high percentage of differentially expressed proteins were involved in cell motility and translation (Figures 4 and 5). The putative relevance of the observed protein expression changes during co-cultivation to the biological interaction between B. glathei and fungi is highlighted below.
Attenuation of starvation and stress responses in B. glathei when co-cultivated with fungi
B. glathei grown in the absence of fungi was limited to the nutrients available in the growth medium (1/3 MEA) and this resulted in the expression of many transcriptional regulators related to starvation and stress response. The expression rates of most of these regulators and stress factors were significantly reduced in the co-cultivations, suggesting multiple beneficial effects of fungi on B. glathei (Supplementary Table S3). From a total of 255 predicted proteins in signal transduction mechanisms, 36 and 32 were present in co-cultivations with A. alternata (AB) and F. solani (FB), respectively, with 29 of them shared between the co-cultivations (Supplementary Table S3), suggesting similar metabolic response and regulation of B. glathei when growing with both fungi. Most of these proteins were factors related to stress or starvation linked to important nutrients such as nitrogen, phosphate and carbon, which were all relieved upon co-cultivation. For instance, the attenuation of nitrogen (N) starvation The ISME Journal
in both co-cultivations was supported by the decrease of the nitrogen regulation protein NRI (BGLT03875) and of the glutamine synthetase (BGLT01582). The nitrogen regulation protein NRI is the major activator of nitrogen-controlled genes such as the glutamine synthetase (Reitzer and Magasanik, 1985; Reitzer et al., 1989). It has been shown that nitrogen starvation in E. coli leads also to an increase of proteins involved in amino acid and polyamine degradation (Zimmer et al., 2000). Decreased expression of various amino-acid transport systems as well as of amino acid and polyamine degradation pathways in B. glathei in co-cultivations additionally supports the idea that the fungi alleviate the N starvation response (Figures 4 and 5). Even though most of the pathways and transport systems showed lower abundances compared with the single culture, subunits of histidine ABC transporters (BGLT00146, BGLT01156) were significantly increased in co-cultivations. Histidine could act as N source provided by fungi to B. glathei, since many enzymes of the histidine degradation pathway, such as histidine ammonia-lyase (BGLT04159), urocanate hydratase (BGLT03664) and N-formylglutamate deformylase (BGLT04163), were detected (Supplementary Table S3). Beside N starvation, phosphate (P) starvation seems to have been alleviated in co-cultivation, as indicated by a significant decrease or absence of proteins such as phosphate starvation-inducible protein (BGLT03521) and glycerol-3-phosphate uptake system (BGLT04691, BGLT05290, BGLT00157) in the co-cultivation with fungi, compared with the control. Expression of proteins associated with the UgpBAEC glycerol-3phosphate uptake system, a member of the ABC superfamily, was among the most strongly decreased in the co-cultivation experiments. In E. coli, glycerol-3phosphate can be used as C and/or P source and is also an essential intermediate in phospholipid biosynthesis (Schweizer et al., 1982; Boos, 1998). Glycerol-3phosphate is an insufficient carbon source, but a sufficient P source for E. coli (Boos, 1998). Thus, the Ugp system that is used for scavenging phosphatecontaining compounds in E. coli may also serve this purpose in Burkholderia. The limited amount of carbon contained in the growth medium could explain the detection of proteins indicative of C starvation in the pure culture control. In both co-cultivations, however, most of these proteins were absent or strongly decreased, such as the Stringent starvation protein A, SspA (BGLT04645) for instance. In E. coli, SspA expression is induced by starvation for glucose, nitrogen, phosphate, amino acids as well as by a decreased growth rate (Williams et al., 1994). In addition to SspA, the carbon starvation protein A, CstA (BGLT04027), a protein that is more specifically expressed during C starvation was also detected and significantly decreased in expression when F. solani was present, while no change was detected in co-cultivation with A. alternata. The decreased amount of SspA and other C starvation proteins
Molecular basis of Burkholderia-fungi interactions N Stopnisek et al 7
Figure 4 Impact of co-cultivation on proteome composition of B. glathei LMG14190. The percentage of proteins in relation to the theoretical proteome with at least twofold change in co-cultivation with either fungus compared with the control (B. glathei alone) is depicted. BA, co-cultivation of B. glathei with A. alternata; BF, co-cultivation of B. glathei with F. solani. Black bars indicate proteins with changed amount in both co-cultivations, white bars indicate proteins with changed amounts in the BF co-cultivation, grey bars indicate proteins with changed amounts in the BA co-cultivation. Functional annotation is based on cluster of orthologous groups (COG).
during co-cultivation suggests a preference of B. glathei for fungal exudates over the C source present in the growth medium (MEA), which is mainly maltose. Indeed, the maltose ABC transporter (BGLT01992), which is responsible for the uptake of maltose, was decreased in co-cultivations. Additionally, a xylose ABC transporter (BGLT06434) was also strongly decreased in co-cultivation. D-xylose is the most abundant sugar in nature after glucose (Ladisch et al., 1983), and it can be utilized by E. coli as a sole carbon source and metabolized through the pentose phosphate pathway (Song and Park, 1998). That xylose was used when B. glathei grew alone on MEA is suggested by the expression of xylose isomerase (BGLT06433), which catalyses the first reaction in the catabolism of D-xylose (Schellenberg et al., 1984) and which was not present in the co-cultivations with either fungus (Supplementary Table S3). While our results suggest that B. glathei may preferentially utilize fungal exudates in the cocultivation experiments, we could not unambiguously identify the primary C source of B. glathei in the exudates. A likely candidate appears to be ribose, as the amounts of proteins involved in ribose uptake
(BGLT04604, BGLT04606 and BGLT05567) were strongly increased in co-cultivation. Burkholderia proteins involved in detoxification processes in the presence of both fungi
In contrast to the positive effects of fungi on alleviation of nutrient starvation, proteins indicating stress were also detected when B. glathei was grown with the fungi. An increase in various catalases and peroxidases (BGLT01132, BGLT04737) in co-cultivations suggests that bacteria experienced oxidative stress when grown with fungi (Supplementary Table S3). Increased expression of 5-methyltetrahydropteroyltriglutamate homocysteine methyltransferase, MetE (BGLT01007) in co-cultivation with A. alternaria also suggests oxidative stress, since this protein is very highly expressed in E. coli cells exposed to oxidative stress (Hondorp and Matthews, 2004). Antibacterial compounds released by the fungi could cause oxidative stress (Ammar et al., 1979; Hellwig et al., 2002; Deshmukh et al., 2014; Soltani and Hosseyni Moghaddam, 2014). An increase of the RND (resistance nodulation division) family of efflux pumps The ISME Journal
Molecular basis of Burkholderia-fungi interactions N Stopnisek et al 8
Figure 5 Voronoi treemaps visualizing changes in the proteome composition of B. glathei LMG14190 during fungal co-cultivation. Expression ratios of B. glathei LMG14190 proteins in single cultures (B) compared to co-cultivation with A. alternata (AB) or F. solani (FB). Functional classification of proteins was carried out by Prophane 2.0 and is based on cluster of orthologous groups (COG). Red colour indicates higher expression of the respective protein in single cultivation and blue colour indicates higher expression in co-cultivations. Each cell represents a single protein, functional classes are separated by thicker black lines.
(BGLT02584, BGLT02585, BGLT02586, BGLT04622, BGLT05365) in co-cultivations further supports the idea of fungal release of antibacterial molecules. The detected proteins show high homology to members of the NodT family of outer membrane transport proteins from the genus Rhizobium (Rivilla et al., 1995) and the CmeABC multidrug efflux system of Campylobacter jejuni (Lin et al., 2002), which are involved in the export of a wide range of drugs. In addition to drug resistance, RND pumps have an important role in biological interactions such as nodulation, colonization and host persistence (Piddock, 2006). In B. cenocepacia, RND efflux pumps also influence phenotypic traits involved in pathogenesis, such as motility and chemotaxis (Bazzini et al., 2011). In addition to these detoxification systems, other proteins with a putative function in detoxification were found to be increased in cocultivations: the proteins YagS (BGLT01120), YagT (BGLT01121) and YagR (BGLT00865), which are part of the yagTSRQ operon, were significantly increased in both co-cultivations (Supplementary Table S3). This operon was shown to encode a periplasmatic aldehyde oxidoreductase that oxidizes a broad spectrum of aldehydes to their respective acids, thereby contributing to the detoxification of the cells (Neumann et al., 2009). RND efflux pumps were not the only proteins that were increased and that are associated with cell envelope biogenesis and outer membrane structures: many outer membrane porines and exporters The ISME Journal
(BGLT02029, BGLT00225, BGLT02300, BGLT00513 and BGLT04032) were also highly increased in co-cultivations. Although the functions of most of these proteins remain unknown, a recent transcriptomic study of B. cenocepacia J2315 stress responses revealed that most of the homologous proteins were increased under nutrient starvation and low oxygen conditions, or when exposed to oxidative stress (Sass et al., 2013). Thus, the expression of membraneassociated proteins observed in co-cultivation might be due to stresses induced by the fungi, although changes in membrane composition could also originate from osmotic stress. The primary response to osmotic stress in most bacteria is the uptake of potassium (Paul 2013). Interestingly, the two homologous potassium uptake systems (BGLT02399BGLT02401 and BGLT06537-BGLT06539) encoded in B. glathei genome were expressed in both cocultivations (Supplementary Table S3). B. glathei proteins involved in motility are not expressed in fungal co-culture
Using fungal hyphae for dispersal has been shown in previous studies to be advantageous in the soil, where heterogeneity of soil particles and the lack of water films represent major obstacles to motility. In our in vitro experiments, dispersal of Burkholderia strains on fungal hyphae was observed but many proteins involved in motility, which were expressed in the control, were undetected in the presence of the
Molecular basis of Burkholderia-fungi interactions N Stopnisek et al 9
Figure 6 Schematic overview of the most important changes in B. glathei LMG14190 proteome during co-cultivation with A. alternata and F. solani. The overview includes proteins involved in major pathways that have been detected in our analysis and proteins whose amounts were either decreased (red) or increased (green) in both co-cultivations. Dashed lines represent the effects of hypothetical stress factors (lightning symbols) or substrates that could be produced by fungi. All flagellar proteins were absent in the co-cultivation setups, thus the whole flagella apparatus is marked in red.
two fungi (Figures 4 and 5, Supplementary Table S3). This might indicate that bacterial cells encountering a fungal hypha cease to invest resources into their own motility and rather rely on the fungus for transport.
Conclusion Evidence has emerged over the past years that Burkholderia are often associated with fungi in soils (Warmink et al., 2009; Lepleux et al., 2012; Nazir et al., 2012). The co-occurrence analysis presented here fully supports this notion and extends our understanding of the ability of Burkholderia to form associations with a broad range of fungal taxa. The widespread soil inhabitants Burkholderia glathei, B. terrae, B. fungorum and B. phytofirmans could all interact and disperse with A. alternata, F. solani and R. solani, suggesting that these broadly occurring interactions might also occur in the environment. Previous reports indicated that the interactions with fungi might be beneficial for the bacteria, yet knowledge of the nature of these benefits is still scarce (Nazir et al., 2010a, 2013; Warmink et al., 2011). Our proteome analysis, which gave a snapshot view of a dynamic interaction, revealed that in a
nutrient-limited medium, B. glathei was able to use multiple substrates provided by the fungi, which attenuated the starvation response observed when grown alone (Figure 6). However, B. glathei encountered new stresses when co-cultivated with fungi, as revealed by the higher expression of proteins associated with various defence and tolerance mechanisms. These functions seem important for the successful colonization and persistence of Burkholderia sp. on fungal hyphae. The benefits that Burkholderia appear to gain from the interaction with fungi are likely to outweigh the costs involved in the co-existence and thus these associations might represent a successful strategy to permit the survival of Burkholderia in the soil environment.
Conflict of Interest The authors declare no conflict of interest.
Acknowledgements We are grateful to Sandra Maass and Andreas Otto for helping with protein identification by mass spectrometry. Financial support of the Swiss National Science Foundation (grant 31003A-130089 to LW) is gratefully The ISME Journal
Molecular basis of Burkholderia-fungi interactions N Stopnisek et al 10
acknowledged. AB is supported by a James S. McDonnell (JSMF) Postdoctoral Fellowship and NF was supported by a grant from the US National Science Foundation (DEB-0953331).
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Supplementary Information accompanies this paper on The ISME Journal website (http://www.nature.com/ismej)
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Mo Farah Biography
Nationality: British
Famous: Philanthropists Athletes
Age: 37 Years, 37 Year Old Males
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Also Known As: Mohammed Farah
Born in: Mogadishu
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father: Muktar Farah
mother: Amran Farah
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Mohamed Muktar Jama "Mo" Farah is a British distance runner who is the 2012 and 2016 Olympic gold medalist in both the 5000 m and 10,000 m. He is just the second athlete in modern Olympic Games history to successfully defend the 5000 m and 10,000 m titles; the first one was Lasse Virén. Though he mostly competes in the 5000 m and 10,000 m, he has also enjoyed considerable success in the 1500 m and the marathon. He also holds the British indoor record in the 3000 m and the current indoor world record for the two miles. Born in Mogadishu, Somalia, he moved with his family to the UK as a child in order to escape the politically chaotic situation in his home country. His initial years in the UK were difficult as he had trouble fitting in. A well-built and athletic lad, he took to sports at a young age, his first love being football. He was also very talented in athletics, an option he eventually chose. He won the English schools cross country championship – his first major national title at the age of 15 and there was no looking back. His national and international running career flourished over the years and he became one of the most decorated athletes in British athletics history.
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- Mo Farah initially trained under coach Alan Storey in 2001 and won the 5000 m at the European Athletics Junior Championship title that year.
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- As a senior, Mo Farah clocked a time of 13 minutes 9.40 seconds for 5000 m to become Britain's second-fastest runner after Dave Moorcroft, in 2006. The same year he won the silver medal in the European Championship 5000 m in Gothenburg and the 2006 European Cross Country Championships in San Giorgio su Legnano, Italy.
- Over the next few years he also trained in Kenya and Ethiopia following which he won European titles in 2010 in 5000 m and 10,000 m. He moved to Portland, Oregon, in 2011 to train under American coach Alberto Salazar.
- The year 2011 was a highly successful one for Mo Farah. He won the gold in the 3000 m at the European Indoor Championships. He then proceeded to win the silver medal in the 10,000 m and then the gold in the 5000 m at the 2011 World Championships in Athletics in Daegu, South Korea, becoming the first British man to win a World Championships medal over either distance.
- His career was poised to touch greater heights in 2012. At the London 2012 Olympics, he won Great Britain's first Olympic gold medal in the 10,000 m, in a time of 27:30.42. He made a long-distance double at the 2012 Olympics, when he won the 5000 m in a time of 13:41.66.
- In 2013, Mo Farah broke the European 1500 m record with a time of 3:28.81 at the Herculis meeting in Monaco. With this feat, he effectively broke Steve Cram's 28-year-old British record and Fermín Cacho's 16-year-old European record. The same year, he also won the 10,000 m and 5000 m event at the World Championships in Moscow.
- He was supposed to compete at the 2014 Commonwealth Games in Glasgow but an illness forced him to withdraw.
- In 2015, he broke the indoor two-mile world record at the Birmingham Indoor Grand Prix by running an 8:03.4 to break Kenenisa Bekele's record. He proceeded to win the long-distance gold medal double at the 2015 World Championships in Athletics, at the age of 32.
- His amazing form continued at the 2016 Rio Olympics where he won gold medals at the 10,000 m and 5000 m, becoming the second athlete in modern Olympic Games history, after Lasse Virén, to successfully defend the 5000 m and 10,000 m titles.
- He was named the European Athlete of the Year in both 2011 and 2012.
- Mo Farah was appointed Commander of the Order of the British Empire (CBE) in the 2013 New Year Honours for services to athletics.
- Mo Farah married his longtime girlfriend Tania Nell in 2010. The couple has three children. The family also includes his wife' daughter from a previous relationship.
- He launched the Mo Farah Foundation after a trip to Somalia in 2011. The following year he participated in ITV's 'The Cube' and won £250,000 which he contributed to the foundation.
- Farah took part in the 2012 Olympic hunger summit at 10 Downing Street hosted by Prime Minister David Cameron. The purpose of the summit was to raise awareness and activity in tackling the problem of world hunger.
- This Olympic gold medalist is noted for his unique victory celebration dance known as the "Mobot".
- This British athlete is the first person to win the full jackpot on ITV's game show 'The Cube', presented by Philip Schofield.
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In Vienne, the Saint Maurice cathedral built between the 12th twelfth and 16th sixteenth century. Both Roman and Gothic in style. The portico and stained glass window above the main entrance. The three front porticos were date from the 14th fourteenth century Vienne, Isère Isere, France, Europe
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Copyright: Per Karlsson, BKWine Photography
040513-108-CRW_0851.tif, access, arch, architectural, architecture, archway, basilica, BKWine, building, cathedral, Catholic, Catholicism, Christian, Christianity, church, city, door, doorway, du, entrance, Europe, European, France, French, heritage, isere, landmark, legacy, Maurice, monument, patrimony, post, religion, religious, rhone, sacred, Saint, Saint Maurice cathedral, spiritual, st, town, vertical, vienne


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=> M.A. Folklore
=> M.Phil. Folklore
=> Ph.D. Folklore
Department of Fine Arts and Aesthetics
Madurai Kamaraj University,
Palkalai Nagar,
Madurai-625 021
Tamil Nadu
Contact NO : +91-0452-2458471
Fax No : +91-0452-2459181
Email : [email protected]
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Spring 2019 Community Grants
Community Grants Program Application Deadline is February 15, 2019.
If you are new to applying to our Community Grants Program or would like a refresher on what we are looking for in applications, please contact Penny, our Grants Coordinator at 613.546.9696 or email@example.com
Both new and seasoned applicants are encouraged to apply. Please review the updated application forms and guidelines now on the website.
The Spring 2019 Community Grants deadline for applications is February 15, 2019.
Further details and the forms are available on our website here: Apply for a Grant
275 Ontario Street Suite #100
Kingston, ON K7K 2X5
Phone: 613.546.9696
Fax: 613.531.9238
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The Tac Force Arctic Commander assisted open knife boasts a fast opening tanto blade that locks up tight. The combo edge blade features a grey and white skull finish. The blade has a finger choil so you can grip up and make precision cuts when you need to. The comfortable polymer handle scales have the same grey and white skull motif. Open the blade with the spine flipper or thumb studs. It locks in place with a liner lock. Lanyard hole for alternate carry. The butt of the handle comes to a point. Tip up pocket clip.
- Knife Category: Spring Asissted
- Blade Style: Tanto Point, Combo Edge
- Action: Side Open Spring Assist
- Blade Length: 3"
- Blade Material: 440 Stainless Steel
- Open Length: 8 3/4"
- Closed Length: 5"
- Weight: 5.9 Ounces


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Sacred Banyan Wall Art
Sanjhi Work,
This eye-catching picture of an enchanting banyan tree set off against a jade green backdrop is ideal for your kitchen, balcony, or living room. Looking closer, you can see that a stencil was used in creating it; this is a characteristic of the Sanjhi school of art that originated in Uttar Pradesh. Spike your decor by bringing this one home.
Enter pincode to check delivery details
Product Index
- Heritage
- Contemporary
This craft, which involves the cutting of an intricate stencil has motifs ranging from depicting scenes from the life of Lord Krishna to creating a rangoli or floor decoration to modern cotemporary patterns.
Process of Making
Anatomy of the Product
Process of Making
The art depicts Indian mythological stories in numerous forms, with predominant focus on Krishna?s Leela. Motifs created are taken inspiration from the folklores of Krishna and stories from Krishnaleela.The desired designs are cut using a cutting tool directly on the paper.Nowadays, the stencils are put up as wall arts. Hence, having cut the paper, it is framed in glass to increase the commercial use as well increase the longevity....
Anatomy of the Product
Process of Making
Anatomy of the Product
Dimensions(cm): Length: 32.5 Width: 1.5 Height: 41 Type of Material:Paper


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Wu Guangqiang
THE municipal government of Yueqing City in Zhejiang Province recently issued a notice, which triggered heated disputes.
The notice listed specific provisions on funeral arrangements, including the duration of funeral services no longer than three days "in principle," and no more than four wreaths being placed.
I called up one of my university classmates living in Yueqing to learn about the background information on the matter.
"The local funeral and interment customs are indeed annoyingly outdated," said my classmate. Rich families hold extravagant funerals, which usually last seven to 10 days. Every day sees feasts consisting of dozens or even hundreds of tables and Buddhist or Taoist ceremonies. Dined and wined, mourners and other funeral attendees sit around the table playing mahjong, or gambling, to be more accurate.
Tons of fake paper banknotes and various "goods" are burned as offerings to the dead. Truckloads of firecrackers are set off. The funeral procession will cover a distance of dozens of miles and the dead will be interred in their coffin twice. To avoid being called unfilial, many not-so-wealthy, or even poor families, are forced to follow suit.
Needless to say, the bad practice must be eradicated. The question is how.
Like other folk customs, funerary customs date back to thousands of years ago, and are still deeply ingrained in vast rural areas. "The Book of Rites" or "Liji," a collection of texts describing the social forms, administration and ceremonial rites of the Zhou Dynasty, prescribes concrete rituals and formalities that all ranks of society must observe.
Concerning filial duty, it offers three criteria: supporting parents when they are alive, mourning them after they die and holding funeral services to mourn. Over time, observing these time-honored rituals has become a must-do tradition for Chinese.
It takes a long time to get rid of old customs and establish new ones. After the founding of the PRC, governments at all levels have been striving to educate the people to take on new funerary and interment practices. As a result, cremation has taken the place of burial in the majority of places in the country, and funeral ceremonies have become much simpler and more unadorned in cities and most rural areas.
It's not hard for open-minded people, me included, to accept more enlightened arrangements after death. I bought a grave for my parents, but I will not allow my remains to take up precious land after my death. Scattering my ashes into the sea or burying them beneath a tree is my wish.
Yet it is ridiculous for a government to limit the number of wreaths or days for funerals. It's alright to ban Party members, officials and civil servants from being involved in lavish funerals, but to require grass-roots masses to do so is unreasonable.
Impractical measures are as bad as no measures at all. What if five or six wreaths are found at a funeral? The confiscation of the extra ones could result in conflicts while doing nothing about the breach will reduce the regulation into a piece of waste paper, thus undermining the government's credibility.
The three-day limit for funeral services is even more stupid. To me, one day is enough, but it is very likely that some villagers have relatives and friends who come from other provinces or even overseas to attend the funeral, so three days are really too short.
Moreover, by doing so the Yueqing government may have exceeded its authority. China's Administrative Licensing Law stipulates that only a provincial government or higher executive bodies have the right to issue administrative regulations.
Personally, I'm opposed to the liberal stance of minimizing the government's role. On the other hand, I'm also against the attempt to create an omnipotent government. The government should know very well the boundary between its authority and law as well as the freedom of citizens.
(The author is an English tutor and freelance writer.)
深圳报业集团版权所有, 未经授权禁止复制; Copyright 2010, All Rights Reserved.
Shenzhen Daily E-mail:email@example.com


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A few months before his hospitalization with an aggressive tumor, Mr. B was the picture of health, enjoying semi-retirement after an accomplished career. He would have accepted any treatment if it could have provided meaningful time with his family. I would have done anything to help him.
Unfortunately, right-to-try legislation, which President Donald Trump highlighted at the beginning of his recent drug pricing speech, is a step in the wrong direction to help individuals like Mr. B.
Federal right-to-try legislation would provide a pathway for terminally ill patients to obtain experimental drugs, bypassing the Food and Drug Administration (FDA). This week, the House plans to vote on a right-to-try bill that passed in the Senate by unanimous consent last August.
A right-to-try bill that's an attack on the FDA hiding behind a supposed compassionate law is about to get a congressional vote.
Right-to-try legislation may seem appealing and intuitive at first glance. After all, some may ask, what is there to lose from giving terminally ill patients a last chance at a longer life? However, this legislation is unlikely to meaningfully improve their access to treatment options, and may diminish the safety of their treatment and degrade their quality of life in their final days.
What right-to-try advocates do not emphasize is that our current regulatory system already gives terminally ill patients safer and more effective avenues for trying experimental treatments. Indeed, when patients have exhausted approved medications and are unable to enroll in clinical trials, they can obtain investigational drugs through the FDA's expanded access program. Right-to-try supporters claim that the FDA is a barrier in this process. Yet according to testimony by FDA Commissioner Scott Gottlieb, his agency authorizes 99 percent of the over 1,000 expanded access applications that it receives annually. An application takes 45 minutes to complete and typically receives authorization within a few days (or immediately by phone, in emergencies).
Instead of being a barrier, the FDA plays an instrumental role in giving terminally ill patients a chance at better outcomes. Whereas the FDA may have confidential information about ongoing, blinded trials of investigational drugs, a patient's physician would not have access to this information. In about 10 percent of cases, the FDA modifies an expanded access application for safety reasons, for instance, changing the dose of a medication.
Betsy Davis threw herself a party before becoming one of the first people to use a California law allowing her to take her own life in 2016.
Rather than prioritizing what's best for the terminally ill, some leaders of the right-to-try movement may be more invested in chipping away at the FDA's authority. The movement's champions, including Americans for Prosperity and the Goldwater Institute, have worked to reduce federal regulatory authority on other fronts. Indeed, the Goldwater Institute is concurrently campaigning to diminish FDA authority by advocating for legislation to rollback restrictions on how companies communicate about off-label uses of drugs.
As a witness to the day-in, day-out suffering that terminal illness can cause, I believe that policies relating to the terminally ill must honor their needs first and foremost, rather than serving a larger legislative agenda. Moreover, the FDA's regulatory authority today has been shaped by past tragedies caused by once-promising drugs with unintended consequences. Given my commitment to the Hippocratic oath of "do no harm," I believe that a strong FDA is vital.
Regrettably, advocacy for right-to-try may already be causing harm through statements that imply investigational drugs are always beneficial. Darcy Olsen, former president of the Goldwater Institute, entitled her book on the topic "The Right to Try: How Federal Government Prevents Americans from Getting the Life-Saving Treatments They Need." By the very definition of the word "investigational," we don't yet know whether a given drug is effective, or whether its effectiveness is sufficient to justify potential harms. In one study, only 43 percent of the drugs given to patients under expanded access received FDA approval within the following five years.
Notably, investigational drugs may cause suffering or hasten death. Some individuals feel that these risks are worthwhile, but for many, they are inconsistent with how they would like to spend what may be their last days with family. The decision is personal. The right-to-try movement's false promise of benefit and insufficient attention to the risks of investigational drugs may mislead individuals who are trying to make important decisions about their remaining time.
Before passing away, Mr. B returned home. I hope his family's last days together were peaceful. As much as I would have wanted to offer him an investigational drug that had a chance of prolonging his life, I would not have wanted to worsen his suffering, at least not without a well-informed consideration of the risks and benefits.
Helen Knight (email@example.com) will graduate this week with a Doctor of Medicine degree from the Johns Hopkins University School of Medicine.


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A former church which is a Listed Building and had stood derelict for 30 years is nearing the end of a dramatic transformation to provide a brand new service as a distinctive community centre, with a whole host of facilities.
St Patrick's old church in Claudy, Londonderry is undergoing a wide-ranging programme of refurbishment and conversion which has won commendation for the performance of the contract team.
"The scheme is going well and we are pleased with the work which is due to be completed this October or November," said Mr Philip O'Kane of scheme funders Rapid NI.
Main contractors JPM Contracts moved on site in June May 2014, working with Lead consultants and Architects Farren Architects and quantity surveyors AD Surveyors.
Works within the building included a comprehensive initial strip out and the creation of a complete new interior. This comprises a community space, kitchen facilities, meeting rooms and toilets. New partitioning overlaid with feature tongue and groove boarding has been installed to create the new layout, with the same boarding also being installed behind the altar area. The building has also been re-plastered, re-wired and comprehensively re-decorated. New lighting, and new surface-tiled sandwich concrete flooring has been installed, together with a new heating system.
The church is cruciform in plan and tiered seating on mezzanine level balconies on the left and right wings of the building has been removed and a new floor installed in these areas, with the staircase and balustrading leading up to them being retained. The mezzanine areas will now provide storage space.
The building's Listed status has provided a number of challenges said Project Architect Mr Eoighin Farren of Farren Architects, who told Premier Construction: "Many elements had to be retained, restored or replaced 'like for like`. All the works were carried out in close consultation with the Northern Ireland Environment Agency – The Historic Environment Division of which is responsible for the conservation of the built heritage (monuments and buildings) in Northern Ireland."
He said that additional works on the project include the removal of the building's external sand/cement and plaster render which acted as a water membrane and did not allow water penetration from external elements but also trapped any water retained within the walls. This was replaced with lime render which allows the walls to breathe.
Lime render is porous and allows the building to 'breath' or release any moisture retained within the walls. The original stained glass windows have been removed, refurbished and replaced and the mouldings on the bell tower, which were in a poor state critical condition, have been replaced with exact reproductions. The building has also been completely re-roofed. A large percentage of the roof timbers and roof slates have been restored and repaired.
"JPM Contractors have done a superb job and are very meticulous in their work," added Mr Farren.
In 1817 James Ross gave the site of the former St Patrick's church to Father McFeely PP of Claudy, on which the latter built the first Saint Patrick's Church at a cost of £500 with the building estimated to hold 600 people.
The church was consecrated in 1820 and was roofless for several years until 1827, when Father Donnelly had it roofed.
The building was renovated in the 1930's and blessed by Dr O'Kane, Bishop of Derry – between 1931 and 1934 – possibly in 1932.
Rapid NI (the Rural Area Partnership in Derry), consists of representatives from community organisations, statutory authorities, the private sector and special interest groups and its primary objective is: To halt the social and economic decline of the rural communities within the Derry City Council Area and to enable these communities, in partnership with all interested parties, to devise locally-led strategies to facilitate sustainable social, economic and cultural development.
The role of JPM Contracts Ltd in most projects is in the capacity of Principal Contractor on major commercial building contracts. JPM Contracts Ltd has amassed a wealth of expertise in areas such as refurbishments, new builds, listed buildings and civil engineering.


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Items Tagged: AI
IT pros get a handle on machine learning and big data
Despite its benefits, machine learning can also go very wrong. Beginners need to understand their input data, project scope and purpose, and the machine learning algorithms at work.
- Premiered: 07/15/15
- Author: Mike Matchett
- Published: TechTarget: Search Data Center
Big data and IoT benefit from machine learning, AI apocalypse not Imminent
Enterprise gets more from its data and better predictive analytics with capable machine learning, but this AI still isn't good enough at finding meaningful patterns in data.
- Premiered: 05/19/16
- Author: Mike Matchett
- Published: TechTarget: Search IT Operations
Four big data and AI trends to keep an eye on
AI is making a comeback - and it's going to affect your data center soon.
- Premiered: 11/17/16
- Author: Mike Matchett
- Published: TechTarget: Search IT Operations
SQL Server machine learning goes full throttle on operational data
Artificial intelligence is a hot topic in IT, and Microsoft has made strides to synchronize SQL Server with machine learning tools for use in analyzing operational data pipelines.
- Premiered: 03/06/17
- Author: Mike Matchett
- Published: TechTarget: Search SQL Server
IBM Cloud Object Storage Provides the Scale and Integration Needed for Modern Genomics Infra.
For hospitals and medical research institutes, the ability to interpret genomics data and identify relevant therapies is key to provide better patient care through personalized medicine. Many such organizations are racing forward, analyzing patients' genomic profiles to match more clinically actionable treatments using artificial intelligence (AI).
These rapid advancements in genomic research and personalized medicine are very exciting, but they are creating enormous data challenges for healthcare and life sciences organizations. High-throughput DNA sequencing machines can now process a human genome in a matter of hours at a cost approaching one thousand dollars. This is a huge drop from a cost of ten million dollars ten years ago and means the decline in genome sequencing cost has outpaced Moore's Law (see chart). The result is an explosion in genomic data – driving the need for solutions that can affordably and securely store, access, share, analyze and archive enormous amounts of data in a timely manner.
Challenges include moving large volumes of genomic data from cost-effective archival storage to low latency storage for analysis to reduce the time needed to analyze genetic data. Currently, it takes days to do a comprehensive DNA sequence analysis.
Sharing and interpreting vast amounts of unstructured data to find relationships between a patient's genetic characteristics and potential therapies adds another layer of complexity. Determining connections requires evaluating data across numerous unstructured data sources, such as genomic sequencing data, medical articles, drug information and clinical trial data from multiple sources.
Unfortunately, the traditional file storage within most medical organizations doesn't meet the needs of modern genomics. These systems can't accommodate massive amounts of unstructured data and they don't support both data archival and high-performance compute. They also don't facilitate broad collaboration. Today, organizations require a new approach to genomics storage, one that enables:
- Scalable and convenient cloud storage to accommodate rapid unstructured data growth
- Seamless integration between affordable unstructured data storage, low latency storage, high performance compute, big data analytics and a cognitive healthcare platform to quickly analyze and find relationships among complex life science data types
- A multi-tenant hybrid cloud to share and collaborate on sensitive patient data and findings
- Privacy and protection to support regulatory compliance
Smarter storage starts with analytics
Storage smartens up to keep pace with data-intensive business applications embedding operational analytics capabilities.
- Premiered: 04/03/17
- Author: Mike Matchett
- Published: TechTarget: Search Storage
Data center storage architecture gets smarter with AI
Trends, such as event-triggered computing, as exemplified by Lambda Architectures, converge on data center storage to hasten data center intelligence evolution.
- Premiered: 05/04/17
- Author: Mike Matchett
- Published: TechTarget: Search Storage
Object storage use cases coming to a data service near you
Object storage, unlike traditional file and block storage, is well-suited to manage vast amounts of unstructured data.
- Premiered: 07/06/17
- Author: Steve Ricketts
- Published: TechTarget: Search Storage
Accelerate predictive analytics software with flash technology
For organizations that must analyze large amounts of data quickly and efficiently, nothing beats solid-state storage. Predictive analytics applications perform better with flash.
- Premiered: 09/19/17
- Author: Taneja Group
- Published: TechTarget: Search Storage
IBM Cognitive Storage Support Promises to Reduce Customers' Technical Issues and Support Costs
Real-time insights from installed storage systems--combined with IBM storage support capabilities--should lead to more reliable and performant storage as well as happier customers
- Premiered: 11/13/17
- Author: Jeff Byrne
SoftNAS Improves Cloud Storage Price/Performance Barriers and Fulfills Its Cloud Fabric Vision
With up to 67% cost savings, SoftNAS Cloud 4 connects any kind of customer data, anywhere in the world to the cloud
- Premiered: 07/24/18
- Author: Jeff Kato
- Published: BusinessWire
HPE 3PAR Performance Insights: Bringing InfoSight Analytics to the Edge
In an era in which every tech company claims to have an AI offering, HPE InfoSight stands out as the genuine article. HPE InfoSight is a best-in-class AI solution that uses cloud-based machine learning to provide global insights into the status and health of infrastructure, removing much of the management burden and helping customers to solve some of their most challenging IT problems. In particular, HPE InfoSight delivers cross-stack insights into a storage array's health, configuration, capacity and performance based on near-real time analytics and the knowledge gained from a vast treasure trove of field data collected over many years.
Among a seemingly endless set of over-hyped AI solutions, HPE InfoSight is delivering remarkable results and significantly enriching the customer experience. The solution has reduced support incidents across more than 50,000 connected HPE 3PAR storage systems by 85%, while lowering operating expenses by nearly 80%. With its unmatched track record, HPE InfoSight has become the leader in AI-driven operations for the Hybrid Cloud and an essential asset for HPE 3PAR customers.
Now InfoSight technology is being incorporated into a new solution at the edge, which is enabling HPE 3PAR customers to better understand, anticipate and improve array performance. As we'll see, HPE Performance Insights for 3PAR Storage takes an innovative approach to helping IT managers track storage performance and deliver it when and where it's needed most, based on the power and intelligence of InfoSight's AI and machine learning technologies.


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Looking for an easy, reliable, and convenient way to replace your tires? Zohr will get your tires delivered and installed. We cover all of Mesquite!
You're busy? We get it. That's why we bring the tire shop to you. Zohr is the easiest, most convenient way for tire replacement.
We are pleased to service the entire Dallas-Ft. Worth metro area. The map below shows where in Mesquite, TX we're able to deliver and install tires. Please click here to see all the cities in which we offer tire service.
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J. Ebner Graphische Betriebe GmbH & Co. KG was a German printing company, founded 1817 in Ulm as J. Ebner'sche Buchhandlung und Buchdruckerei.
Sometimes credited as Ebner Graphische Betriebe or J. Ebner, Ulm but mostly just as Ebner, Ulm.
In 2001 the company was acquired by the CPI Group.
in 2002, CPI merged Ebner and Franz Spiegel Buch GmbH, a printing company founded in 1930, which is also based in Ulm and was also acquired in 2001, to CPI - Ebner & Spiegel.


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herbal alternatives to soliris?
View Single Post
Thu Aug 22, 2013, 10:23 PM
Join Date: Jul 2013
Location: Santa Fe, NM
Posts: 25
Just wanted to give an update. The local hospital has taken Karin on as a charity case so we could do Soliris (Eculizumab - we are calling it E-mab :-)) and that may happen. It does not seem to address the marrow failure though when I read the papers. We are trying Papaya Leaf extract for the thrombocytopenia - just been on it 10 days but last hematologist visit the count was 17000 platelets - same as the previous week's count and the hematologist asked whether we had started the Papaya Leaf extract. Well whether it holds up or not we were able to skip a platelet transfusion. Her LDH is 630, her neutrophils are 0.75 x 10^9 / Liter, marrow cellularity is normal, her PNH clone size is 39%, so seems to be moderate AA/PNH. I have seen a couple of case studies where the PNH clone was reduced considerably after treatment for other things with Rituximab (R-mab), one for AA/PNH and the other for non-Hodgkin's lymphoma/PNH so am looking into that as a treatment.
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28 Minutes for 3 Million Dollars (1967) Maurizio Pradeaux
Labels : 60's , Action , Italy , Maurizio Pradeaux , 1 com
28 Minutes for 3 Million Dollars (1967)
aka 28 minuti per 3 milioni di dollari
Genre: Action | Crime
Country: Italy | Director: Maurizio Pradeaux
Language: English or Italian or German
(3 separate audio tracks) | Subtitles: None
(3 separate audio tracks) | Subtitles: None
Aspect ratio: Cinemascope 2.35:1 | Length: 85mn
Dvdrip H264 Mkv - 1024x448 - 25fps - 1.47gb
The famous "Grand Mogul" diamond is stolen by a group of experts on the payroll of a millionaire diamond collector. But his butler, too, proves an equally efficient thief. Richard Harrison, Franca Polesello, Claudio Biava and Ferruccio Viotti star in this 1967 crime caper from director Maurizio Pradeaux, who would later go on to direct the Giallo "Death Carries A Cane".
28 Minutes for 3 Million Dollars (1967)
If you enjoy this blog, you can support it by buying a NITROFLARE premium account from the links above. For just a few bucks, you will get full speed and unlimited downloads from a great filehost. If you find a dead link, just leave a com on the post REUPS AND REQUESTS and I'll try to fix it as soon as possible. Cheers.


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Now Only God Knows Why I Meet Hijras At My Shoots, a photo by firoze shakir photographerno1 on Flickr.
I shot this hijra at Banganga on the top near a Temple , a beggar hijra , humble polite and I shot a few frames , but you will see one as the others I shall remove from public view...
The one place hija pictures are stolen is Flickr.com and you can do nothing about it Yahoo Copyright has no time for human emotions of Flickr members like me whose pictures are on other Flickr members site with my name too .. stolen but shot by Firoze Shakir.
So my Hijra documentary is not for general public and I dont sell my pictures .. and the Flickr Space Ball toll is all bunkum.. you can breach it anytime you want .. and I am a Pro member .. makes no difference ..
But all said and done there is no alternative to photo management .. better than Flickr.
I cross blog from here to my Blogspot site and Twitter and Facebook like famous Pinterest .. I pimp my links manually on Facebook.