State of emergency medicine in Spain

Spain has universal public health care coverage. Emergency care provisions are offered to patients in different modalities and levels according to the characteristics of the medical complaint: at primary care centers (PCC), in an extrahospital setting by emergency medical services (EMS) and at hospital emergency departments (ED). We have more than 3,000 PCCs, which are run by family doctors (general practitioners) and pediatricians. On average, there is 1 PCC for every 15,000 to 20,000 inhabitants, and every family doctor is in charge of 1,500 to 2,000 citizens, although less populated zones tend to have lower ratios. Doctors spend part of their duty time in providing emergency care to their own patients. While not fully devoted to emergency medicine (EM) practice, they do manage minor emergencies. However, Spanish EMSs contribute hugely to guarantee population coverage in all situations. These EMS are run by EM technicians (EMT), nurses and doctors, who usually work exclusively in the emergency arena. EDs dealt with more than 25 million consultations in 2008, which implies, on average, that one out of two Spaniards visited an ED during this time. They are usually equipped with a wide range of diagnostic tools, most including ultrasonography and computerized tomography scans. The academic and training background of doctors working in the ED varies: nearly half lack any structured specialty residence training, but many have done specific master or postgraduate studies within the EM field. The demand for emergency care has grown at an annual rate of over 4% during the last decade. This percentage, which was greater than the 2% population increase during the same period, has outpaced the growth in ED capacity. Therefore, Spanish EDs become overcrowded when the system exerts minimal stress. Despite the high EM caseload and the potential severity of the conditions, training in EM is still unregulated in Spain. However, in April 2009 the Spanish Minister of Health announced the imminent approval of an EM specialty, allowing the first EM resident to officially start in 2011. Spanish emergency physicians look forward to the final approval, which will complete the modernization of emergency health care provision in Spain

Is the expression of the gene coding for cerato-platanin modulated by biotic and abiotic factors?

Ceratocystis platani is the causal agent of canker stain, the most dangerous disease affecting the plane trees. The fungus produces cerato-platanin (CP), a protein of about 12.4 kDa acting as a PAMP in host and non-host plants. On plane leaves CP elicits the transcription of defence-related genes earlier than C. platani does. The amino acid sequence 1-119 of CP is a new protein domain, called "cerato-platanin domain”; thus, CP is the founding member of the cerato-platanin family (pfam PF07249). To date, a number of highly conserved proteins produced by Ascomycetes and Basidiomycetes have been identified with this domain. They have been reported to interact with plants and humans, but very little is known about the regulation of the genes coding for these proteins and about their primary role in the lifestyle of producing fungi. In the present work we demonstrated that CP is released by the fungus during the interaction with the host plant, and the expression of the cp gene is highly modulated. The gene was expressed more rapidly when the fungus was inoculated on the plane leaves than when it was grown in axenic culture. Potential abiotic and biotic stressors have been investigated: temperature, H2O2, umbelliferone (the reference plane phytoalexin), matric water stress, light (presence/absence), growth on sawdust of susceptible and resistant plane or elm trees, and co-culture with Trichoderma atroviride P1 and T. harzianum T22. The gene expression has been evaluated by qRT-PCR using TaqMan probes. The promoter region has been isolated and studied

Cerato-platanin elicts transcription of defence-related genes earlier than Ceratocystis platani on Platanus acerifolia

The ascomycete Ceratocystis platani (Walter) Engelbrecht & Harrington is the causal agent of canker stain, the most dangerous disease affecting the plane tree. Cerato-platanin (CP) is a protein elicitor from C. platani that acts as a pathogen-associated molecular pattern (PAMP), eliciting defence responses in both host and non-host plants. Using plane leaves as an experimental model, we compared the capacity of CP and C. platani to cause alterations in the transcription level of five Platanus acerifolia (Ait.) Willd. genes, three strictly related to a defence response, and two non-defence related. The results suggest that CP elicits the transcription of defence-related genes earlier than does C. platani. The expression of the cp gene in C. platani inoculated on plane leaves and grown in an axenic culture was also studied. For the first time CP has been shown to be released by C. platani when inoculated on P. acerifolia. The cp gene was expressed sooner when C. platani was inoculated on the leaves, than when it was grown in axenic culture, suggesting that the host-tree has a positive effect on the expression of this elicitor protei

Isolation of orthologues of the cp gene and characterization of the protein PoP from Ceratocystis populicola

Cerato-platanin (CP) is a protein of 120 amino acids, produced by the Ascomycete Ceratocystis platani, the agent of plane canker stain. CP is the founder member of the CP family (pfam07249), a group of proteins involved in the interaction between the producing fungi (pathogenic to plants or humans) and the respective hosts. CP is located in the cell walls of the C. platani mycelium, is able to self-aggregate, and is early released in vitro. Leaves of Platanus acerifolia react to CP in a resistance-like manner, activating various defence-related events including the over-expression of many defence-related genes and inhibition of the C. platani growth. The coding cp gene sequence has been cloned in Pichia pastoris, and the 3D solution structure of recombinant CP is now available. Recently our research group has isolated and sequenced the hortologues of the cp gene in various species and clones of the genus Ceratocystis (C. populicola, C. cacaofunesta, C. variospora, C. fimbriata clones from Coffea arabica, Mangifera indica, Fagus sp., Crotolaria juncea, and Ipomea batatas). The protein PoP from C. populicola has been purified and has been characterized structurally and functionally

Progressive pearl necklace collapse mechanism for cerato-ulmin aggregation film

Cerato-ulmin (CU) is a fungal toxin class II hydrophobin, involved in Dutch elm disease. The formation of hydrophobin films at the air–water interface is a key mechanism which plays a role of paramount importance at different stages of the fungal development. We present a study on the precursor stages of growth towards the self-assembly aggregation film of CU. Atomic force microscopy images of CU dropped on mica substrates indicate that the system self-organizes in almost one-dimensional pearl-necklace-like chains, which subsequently collapse and possibly merge to form extended and rather compact planar films. We propose and verify a simple model to describe the self-aggregation mechanism in terms of progressive thickening of the pearl chains due to the successive merging and collapse of the elementary constitutive units

Ophiostoma quercus produces a cerato-ulmin-homologous protein

Cerato-ulmin (CU) is a class II hydrophobin protein of about 7600 Da, produced by the Ascomycota Ophiostoma ulmi (Buisman) Nannf., O. novo-ulmi Brasier and O. himal-ulmi Brasier M. D. Mehrotra. O. ulmi and O. novo-ulmi are responsible for the Dutch elm disease (DED) that in the 20th century destroyed most of the elms native in Europe and North America (Ulmus minor Miller, U. glabra Huds., U. procera Salisb., U. americana L., U. rubra Muhl.), whereas O. himal-ulmi is present in Asia. CU accumulates in the cell walls of DED fungi and is abundantly released in the liquid culture medium with the exception of O. ulmi known to be a scarce or nil CU producer. It has been suggested that CU plays a key role in different phases of DED. However, the relationship between CU production and virulence of DED pathogens is still debated. In another ophiostomatoid species, O. quercus, non pathogenic towards elm trees, an immunologically CU-related protein was present in the mycelial cell wall, but not in the culture medium. Moreover, a DNA sequence that cross-hybridized with an O. novo-ulmi cu gene fragment was detected, thus suggesting the presence of a cu-ortologous gene in O. quercus. In the present work we report the cloning and sequencing of the cu gene from O. quercus. Furthermore, the protein has been partially purified from the mycelium and characterized

Gene induction in Platanus acerifolia after treatments with cerato-platanin and conidia of Ceratocystis fimbriata sp. Platani

Cerato-platanin (CP) is a 120 amino acids-long protein purified from the culture filtrate of Ceratocystis fimbriata (Ell. and Halst.) Davidson f. sp. platani Walter (Cfp), the causative agent of the canker stain of the plane trees (Pazzagli et al., 1999). CP contains 4 cysteines forming two S-S bridges, Cys20-57 and Cys60-115, and has a high percentage (40%) of hydrophobic residues. It is the founder member of the cerato- platanin family, and its N-terminal region is very similar to cerato-ulmin, a class II hydrophobin involved in the pathogenesis of Dutch elm disease (Del Sorbo et al., 2002). CP is located in the Cfp cell walls, and is early-detected in Cfp culture filtrates. In in vitro experimental conditions CP self-assembles, and interacts with the host plane leaves by eliciting phytoalexin synthesis, extended cell plasmolysis and crushing, and abundant starch accumulation in the chloroplasts (Bennici et al., 2005; Boddi et al., 2004; Pazzagli et al., 2005; Scala et al., 2004). cDNA libraries were constructed from RNA extracted from leaves treated with CP (treated) and with water (control) for 48 hours using the Suppressive Subtractive Hybridisation (SSH) method, a powerful technique that enables to compare two population of mRNA and to obtain clones of genes that are expressed in one population of mRNA but not in the other. The positive clones were sequenced and the sequences were analysed using the FASTA, BLAST, ProDom, BLOCK software for their identification in GenBank, EMBL. Many clones from the forward library contain sequences involved: i) in synthesis, processing and translation of proteins, i.e., the DEAD box ATPase/RNA helicase protein, the oligouridylate binding protein, an unknown protein with the RIBOSOMAL S8E domain, the elongation factor 1 alpha, the translation factor erF3, the ribosomal protein PRPL5; ii) in signal transfer in the cell, i.e. the Pto-like serine-threonine kinase; iii) in transcription control, i.e. the histone deacetilase, the DEAD-box helicase; iv) in photosynthesis, i.e. the PSI-D subunit, the ferredoxin, the chlorophyll a/b binding protein. Someone else interesting are: the TUBBY-like protein (TULP); the Fatty Acid Elongase (FAE), in particular, a keto-acyl CoA synthase; the Inosine 5’-phosphate-dehydrogenase; the PEP-carboxyl-kinase; the alanine-amino-transferase; the selenium binding protein; the RAR1; a protein with a DUF597 domain; the thaumatin protein. Moreover, many clones were analysed by Real Time RT-PCR and/or relative PCR in order to study their regulation after treatments with CP or with conidia of Cfp wild strain. Analysis of forward clones showed that many genes are positively regulated. The knowledge of the gene expression after treatments could open the possibility of inducing resistance to Cfp in plane trees

Plant resistance-related responses are elicited by cerato-platanin, a small protein from C. fimbriata f. sp. platani

Cerato-platanin (CP) is a 12.4 kDa protein from the Ascomycete Ceratocystis fimbriata (Ell. and Halst.) Davidson f. sp. platani Walter, the causative agent of the canker stain of the plane trees. CP has the 40% percentage of hydrophobic residues, and contains 4 cysteines forming two S-S bridges at Cys20-57 and Cys60-115. CP is the founder member of the cerato-platanin family, and its N-terminal region presents a high similarity with that of cerato-ulmin, an Ophiostoma class II hydrophobin involved in Dutch elm disease. CP is able to self-assembly to give large, thioflavin T-positive molecular aggregates showing some similarities with those observed in the moderately hydrophobic hydrophobins. CP is located in the fungal cell walls, and is early-secreted in culture filtrates. The cp gene was cloned in Pichia pastoris, and the recombinant protein is properly folded and maintains its biological activity. CP interacts with the plane leaves by eliciting phytoalexin synthesis, extended cell plasmolysis and crushing, and abundant starch accumulation in the chloroplasts. Various plane genes induced by CP treatments have been identified by the suppression subtractive hybridisation method