Phylogeny and taxonomy of the genera of Erysiphaceae, part 5: Erysiphe (the "Microsphaera lineage" part 1).

In this contribution, we offer the fifth installment of a series focusing on the phylogeny and taxonomy of powdery mildews. This paper is the second segment evaluating the genus Erysiphe. The first treatment of Erysiphe focused on phylogenetically basal species in the "Uncinula lineage." This research presents a phylogenetic-taxonomic assessment of species that form the group previously referred to as the "Microsphaera lineage." Given the size of the group, we split the treatment of this lineage of Erysiphe species into two parts based on their phylogenetic placement. Phylogenetic trees based on ITS+28S data are supplemented by sequences of additional markers (CAM, GADPH, GS, RPB2, and TUB). Included in the analysis of the Microsphaera lineage is the "Erysiphe aquilegiae complex" (group, clade, cluster), which encompasses sequences obtained from an assemblage of Erysiphe species with insufficient resolution in rDNA analyses. Attempts have been made to resolve this group at the species level by applying a multilocus approach. A detailed discussion of the "Erysiphe aquilegiae complex" is provided. Sequences are provided for the first time for several species, particularly North American species, such as Erysiphe aggregata, E. erineophila, E. parnassiae, and E. semitosta. Ex-type sequences for Microsphaera benzoin and M. magnusii have been retrieved. Alphitomorpha penicillata, Microsphaera vanbruntiana, and M. symphoricarpi are epitypified with ex-epitype sequences. The new species Erysiphe alnicola, E. deutziana, E. cornigena, E. lentaginis, and E. sambucina are described, the new combinations E. lauracearum, E. passiflorae, and E. sambucicola are introduced, and the new name E. santali is proposed.

Phylogeny and taxonomy of the genera of Erysiphaceae, part 3: Cystotheca.

This contribution is part of a series devoted to the phylogeny and taxonomy of powdery mildews, with an emphasis on North American taxa. An overview of Cystotheca species is given, including references to ex-type sequences or, if unavailable, proposals for representative reference sequences for phylogenetic-taxonomic purposes. The new species C. mexicana is described, based on Mexican collections on Quercus glaucoides × Quercus microphylla and Quercus liebmannii × Q. microphylla. Cystotheca lanestris is reported for the first time worldwide on Quercus laceyi (Collected in Mexico) and on Q. toumeyi (collected in Arizona, USA). Cystotheca lanestris on Q. agrifolia and on Q. cerris is reported for the first time in Mexico. Epitypes with ex-epitype sequences are designated for Cystotheca wrightii, Lanomyces tjibodensis (= C. tjibodensis), Sphaerotheca kusanoi, and S. lanestris (C. lanestris).

The Unexpected Identity of Tympanis vagabunda.

Tympanis species (Leotiales) are plant pathogens distributed mostly in northern temperate ecosystems. The diversity and identity of some species remains unclear. Tympanis vagabunda, found in Sicilia (Italy) on dry twigs of Rosa, Rubus, and Pistacia, is one example of an obscure and poorly known species. During the study of its type specimen in S, which contained one twig with a wood anatomy fitting neither of the three mentioned hosts, the microanatomic structures indicated that it belongs to the genus Rutstroemia (Helotiales). To investigate its identity, the types of R. fruticeti, R. juniperi, R. urceolus, and R. longiasca were studied for comparison. The species for which molecular data were available were included in a dataset that contained identified species of Rutstroemia, along with other select species from the families Rutstroemiaceae and Sclerotiniaceae. R. fruticeti, a saprobe frequently reported from Rubus fruticosus in Europe, is found to be a later synonym of T. vagabunda, and the combination Rutstroemia vagabunda is proposed. R. juniperi is an infrequently reported European species on twigs of Juniperus and is morphologically hard to distinguish from R. vagabunda; available molecular data support its recognition as a distinct species. R. longiasca differs from R. vagabunda in its black apothecia, smaller asci, and narrower ascospores. R. urceolus differs from R. vagabunda in having black apothecia and smaller inamyloid asci, and excipulum at the flanks and margin is composed of dark-walled hyphae.

Mielinosis extrapontina: reporte de un caso de síndrome de desmielinización osmótica

Introducción: El síndrome de desmielinización osmótica es una entidad clínica la cual supone un reto diagnóstico por su curso en muchos casos silentes y por su cuadro clínico que se presenta típicamente de manera tardía. Se describen dos tipos de desmielinización osmótica que pueden presentarse de manera aislada o simultánea: la mielinosis central pontina y la mielinosis extrapontina. Caso clínico: femenina de 21 años sin antecedentes personales con episodio convulsivo tónico clónico por primera vez en su vida. Se le encuentra a su ingreso un nivel de sodio sérico disminuido, razón por la cual se inicia la corrección intravenosa del mismo. Cursó con cefaleas persistentes y vómitos a las 48 horas posterior a la corrección del sodio, y se realizó una resonancia magnética cerebral que evidenció datos de mielinosis extrapontina. Conclusión: La corrección rápida de los niveles de sodio se asocia a la posibilidad de desarrollar mielinosis pontina. La precaución en la corrección de la hiponatremia es la piedra angular para prevenir el desarrollo de esta condición. (provisto por Infomedic International)

Introduction: Osmotic demyelination syndrome is a clinical entity which poses a diagnostic challenge because of its often-silent course and its clinical picture which typically presents late. We describe two types of osmotic demyelination that can occur in isolation or simultaneously: central pontine myelinosis and extrapontine myelinosis. Case report: 21-year-old female with no personal history with tonic-clonic seizure episode for the first time in her life. On admission she was found to have a decreased serum sodium level, for which reason intravenous sodium correction was started. He presented with persistent headaches and vomiting 48 hours after sodium correction, and a brain MRI was performed, which showed evidence of extrapontine myelinosis. Conclusion: Rapid correction of sodium levels is associated with the possibility of developing pontine myelinosis. Caution in correcting hyponatremia is the cornerstone in preventing the development of this condition. (provided by Infomedic International)

A re-assessment of Taxomyces andreanae, the alleged taxol-producing fungus, using comparative genomics.

The monotypic "bulbilliferous hyphomycete" genus Taxomyces was erected in 1993 for a fungal endophyte isolated from the Yew tree Taxus brevifolia and named Taxomyces andreanae. This fungus was reported to produce the plant-derived anti-cancer drug taxol. The original description of the fungus was not conclusive as to its taxonomic position because no sporulation or other salient morphological features were reported. Consequently, the taxonomic affinities of this fungus have remained obscure. However, a full genome sequence of this strain was generated by a German research group in 2013, in an unsuccessful attempt to detect the biosynthesis genes encoding for taxol. This prompted us to search for phylogenetic marker genes and compare those with the data that recently have become available from state-of-the-art polyphasic taxonomic studies. Surprisingly, the strain turned out to belong to the phlebioid clade of wood-destroying Basidiomycota as inferred from a comparison of its partial ITS, the 28S rDNA (LSU), the RNA polymerase II largest subunit (rpb1), the RNA polymerase II second largest subunit (rpb2), and the translation elongation factor 1-α (tef1) sequences. A multi gene genealogy based on these loci revealed that the closest relative is Ceriporiopsis (syn. Mycoacia) gilvescens. Even though such wood-destroying Basidiomycota are regularly encountered among the endophytic isolates after surface-disinfection of plant organs, the vast majority of the reported endophytic fungi belong to the Ascomycota. Nevertheless, the data available now allow for synonymizing Taxomyces with Ceriporiopsis, and the necessary new combination is made.

Species of the common discomycete genus Bisporella reassigned to at least four genera.

Bisporella as typically conceived is a genus of noticeable, bright yellow inoperculate discomycetes. This interpretation of the genus, however, is at odds with Bisporella pallescens, the current name of the type species of the genus; furthermore, the genus has been interpreted as including the unusual species Bisporella resinicola. By comparing morphological and molecular traits of species traditionally included in Bisporella, we show that the genus is polyphyletic, with many "typical" members of the genus belonging instead in Calycina in Pezizellaceae. Bisporella pallescens is conclusively linked with its asexual morph, Bispora antennata, and the genus Bisporella is abandoned as a later synonym of the monotypic genus Bispora (previously applied only to asexual fungi) and placed as sister to Hymenoscyphus in Helotiaceae. Bisporella resinicola is shown to represent an independent monotypic genus, Eustilbum, which so far is placed incertae sedis in Helotiales. Finally, "Bisporella" subpallida, like Bispora, belongs to Helotiaceae but is instead related to "Phaeohelotium" epiphyllum.

Apothecial Ancestry, Evolution, and Re-Evolution in Thelebolales (Leotiomycetes, Fungi).

Closed cleistothecia-like ascomata have repeatedly evolved in non-related perithecioid and apothecioid lineages of lichenized and non-lichenized Ascomycota. The evolution of a closed, darkly pigmented ascoma that protects asci and ascospores is conceived as either an adaptation to harsh environmental conditions or a specialized dispersal strategy. Species with closed ascomata have mostly lost sterile hymenial elements (paraphyses) and the capacity to actively discharge ascospores. The class Leotiomycetes, one of the most speciose classes of Ascomycota, is mainly apothecioid, paraphysate, and possesses active ascospore discharge. Lineages with closed ascomata, and their morphological variants, have evolved independently in several families, such as Erysiphaceae, Myxotrichaceae, Rutstroemiaceae, etc. Thelebolales is a distinctive order in the Leotiomycetes class. It has two widespread families (Thelebolaceae, Pseudeurotiaceae) with mostly closed ascomata, evanescent asci, and thus passively dispersed ascospores. Within the order, closed ascomata dominate and a great diversity of peridia have evolved as adaptations to different dispersal strategies. The type genus, Thelebolus, is an exceptional case of ascomatal evolution within the order. Its species are the most diverse in functional traits, encompassing species with closed ascomata and evanescent asci, and species with open ascomata, active ascospore discharge, and paraphyses. Open ascomata were previously suggested as the ancestral state in the genus, these ascomata depend on mammals and birds as dispersal agents. In this scheme, species with closed ascomata, a lack of paraphyses, and passive ascospore discharge exhibit derived traits that evolved in adaptation to cold ecosystems. Here, we used morphological and phylogenetic methods, as well as the reconstruction of ancestral traits for ascomatal type, asci dehiscence, the presence or absence of paraphyses, and ascospore features to explore evolution within Thelebolales. We demonstrate the apothecial ancestry in Thelebolales and propose a new hypothesis about the evolution of the open ascomata in Thelebolus, involving a process of re-evolution where the active dispersal of ascospores appears independently twice within the order. We propose a new family, Holwayaceae, within Thelebolales, that retains the phenotypic features exhibited by species of Thelebolus, i.e., pigmented capitate paraphyses and active asci discharge with an opening limitation ring.

Sareomycetes: more diverse than meets the eye.

Since its resurrection, the resinicolous discomycete genus Sarea has been accepted as containing two species, one with black apothecia and pycnidia, and one with orange. We investigate this hypothesis using three ribosomal (nuITS, nuLSU, mtSSU) regions from and morphological examination of 70 specimens collected primarily in Europe and North America. The results of our analyses support separation of the traditional Sarea difformis s.lat. and Sarea resinae s.lat. into two distinct genera, Sarea and Zythia. Sarea as circumscribed is shown to conservatively comprise three phylospecies, with one corresponding to Sarea difformis s.str. and two, morphologically indistinguishable, corresponding to the newly combined Sarea coeloplata. Zythia is provisionally maintained as monotypic, containing only a genetically and morphologically variable Z. resinae. The new genus Atrozythia is erected for the new species A. klamathica. Arthrographis lignicola is placed in this genus on molecular grounds, expanding the concept of Sareomycetes by inclusion of a previously unknown type of asexual morph. Dating analyses using additional marker regions indicate the emergence of the Sareomycetes was roughly concurrent with the diversification of the genus Pinus, suggesting that this group of fungi emerged to exploit the newly-available resinous ecological niche supplied by Pinus or another, extinct group of conifers. Our phylogeographic studies also permitted us to study the introductions of these fungi to areas where they are not native, including Antarctica, Cape Verde, and New Zealand and are consistent with historical hypotheses of introduction.

Notes on Trochila (Ascomycota, Leotiomycetes), with new species and combinations.

Studies of Trochila (Leotiomycetes, Helotiales, Cenangiaceae) are scarce. Here, we describe two new species based on molecular phylogenetic data and morphology. Trochilabostonensis was collected at the Boston Harbor Islands National Recreation Area, Massachusetts. It was found on the stem of Asclepiassyriaca, representing the first report of any Trochila species from a plant host in the family Apocynaceae. Trochilaurediniophila is associated with the uredinia of the rust fungus Ceroteliumfici. It was discovered during a survey for rust hyperparasites conducted at the Arthur Fungarium, in a single sample from 1912 collected in Trinidad. Macro- and micromorphological descriptions, illustrations, and molecular phylogenetic analyses are presented. The two new species are placed in Trochila with high support in both our six-locus (SSU, ITS, LSU, rpb1, rpb2, tef1) and two-locus (ITS, LSU) phylogenetic reconstructions. In addition, two species are combined in Trochila: Trochilacolensoi (formerly placed in Pseudopeziza) and T.xishuangbanna (originally described as the only species in Calycellinopsis). This study reveals new host plant families, a new ecological strategy, and a new country record for the genus Trochila. Finally, our work emphasizes the importance of specimens deposited in biological collections such as fungaria.

Importancia de protocolo FASTTRACK en pacientes sometidos a derivación biliodigestiva, revisión bibliográfica / FASTTRACK protocol importance in patients undergoing biliodigestive diversión, bibliographic review

Los protocolos ERAS por sus siglas en inglés (Enhanced Recovery after Surgery) se centran en reducir las complicaciones postoperatorias y la respuesta al estrés, optimizando la recuperación postoperatoria, dentro de las intervenciones el inicio temprano de la alimentación ha demostrado ser eficaz para reducir la morbilidad, los costos generales y la satisfacción del paciente. El presente estudio, revisión bibliográfica, plantea la importancia de tomar este tipo de medidas en pacientes con derivación biliodigestiva en el Hospital Médico Quirúrgico del Instituto Salvadoreños del Seguro Social. Cuantificar este aspecto en el Hospital Médico Quirúrgico del ISSS permitirá profundizar en estudios clínicos o analíticos para posteriormente implementar y protocolizar la intervención

Placement of Triblidiaceae in Rhytismatales and comments on unique ascospore morphologies in Leotiomycetes (Fungi, Ascomycota).

Triblidiaceae is a family of uncommonly encountered, non-lichenized discomycetes. A recent classification circumscribed the family to include Triblidium (4 spp. and 1 subsp.), Huangshania (2 spp.) and Pseudographis (2 spp. and 1 var.). The apothecia of these fungi are persistent and drought-tolerant; they possess stromatic, highly melanized covering layers that open and close with fluctuations of humidity. Triblidialean fungi occur primarily on the bark of Quercus, Pinaceae and Ericaceae, presumably as saprobes. Though the type species of Huangshania is from China, these fungi are mostly known from collections originating from Western Hemisphere temperate and boreal forests. The higher-rank classification of triblidialean fungi has been in flux due in part to an overemphasis on ascospore morphology. Muriform ascospores are observed in species of Triblidium and in Pseudographiselatina. An intense, dark blue/purple ascospore wall reaction in iodine-based reagents is observed in species of Pseudographis. These morphologies have led, in part, to these genera being shuffled among unrelated taxa in Hysteriaceae (Dothideomycetes, Hysteriales) and Graphidaceae (Lecanoromycetes, Ostropales). Triblidiaceae has been placed within the monofamilial order Triblidiales (affinity Lecanoromycetes). Here, we demonstrate with a three-gene phylogenetic approach that triblidialean fungi are related to taxa in Rhytismatales (Leotiomycetes). We synonymize Triblidiales under Rhytismatales and emend Triblidiaceae to include Triblidium and Huangshania, with Pseudographis placed within Rhytismataceae. A history of Triblidiaceae is provided along with a description of the emended family. We discuss how the inclusion of triblidialean fungi in Rhytismatales brings some rarely observed or even unique ascospore morphologies to the order and to Leotiomycetes.

A multigene phylogeny toward a new phylogenetic classification of Leotiomycetes.

Fungi in the class Leotiomycetes are ecologically diverse, including mycorrhizas, endophytes of roots and leaves, plant pathogens, aquatic and aero-aquatic hyphomycetes, mammalian pathogens, and saprobes. These fungi are commonly detected in cultures from diseased tissue and from environmental DNA extracts. The identification of specimens from such character-poor samples increasingly relies on DNA sequencing. However, the current classification of Leotiomycetes is still largely based on morphologically defined taxa, especially at higher taxonomic levels. Consequently, the formal Leotiomycetes classification is frequently poorly congruent with the relationships suggested by DNA sequencing studies. Previous class-wide phylogenies of Leotiomycetes have been based on ribosomal DNA markers, with most of the published multi-gene studies being focussed on particular genera or families. In this paper we collate data available from specimens representing both sexual and asexual morphs from across the genetic breadth of the class, with a focus on generic type species, to present a phylogeny based on up to 15 concatenated genes across 279 specimens. Included in the dataset are genes that were extracted from 72 of the genomes available for the class, including 10 new genomes released with this study. To test the statistical support for the deepest branches in the phylogeny, an additional phylogeny based on 3156 genes from 51 selected genomes is also presented. To fill some of the taxonomic gaps in the 15-gene phylogeny, we further present an ITS gene tree, particularly targeting ex-type specimens of generic type species. A small number of novel taxa are proposed: Marthamycetales ord. nov., and Drepanopezizaceae and Mniaeciaceae fams. nov. The formal taxonomic changes are limited in part because of the ad hoc nature of taxon and specimen selection, based purely on the availability of data. The phylogeny constitutes a framework for enabling future taxonomically targeted studies using deliberate specimen selection. Such studies will ideally include designation of epitypes for the type species of those genera for which DNA is not able to be extracted from the original type specimen, and consideration of morphological characters whenever genetically defined clades are recognized as formal taxa within a classification.

Overview of Phacidiales, including Aotearoamyces gen. nov. on Nothofagus.

The new genus Aotearoamyces is proposed to accommodate a single species that was repeatedly collected on fallen wood in Nothofagaceae forests of New Zealand and was previously misidentified as a Claussenomyces species. This monotypic genus belongs to Tympanidaceae, a recently erected family in Phacidiales. Aotearoamyces is differentiated from other Tympanidaceae by phragmospores that do not form conidia either in or outside the asci, an exciple of textura intricata with hyphae widely spaced and strongly gelatinized (plectenchyma), and apically flexuous, partly helicoid paraphyses. The asexual morph was studied in pure culture. Phylogenetic analyses of combined SSU, ITS and LSU sequences strongly support a sister relationship between the sexually typified Aotearoamyces and the asexually typified "Collophorina" paarla characterized morphologically by forming endoconidia, a feature not found in the genetically distinct type species of Collophorina. Based on our molecular results, we place the genus Epithamnolia in the Mniaecia lineage within Phacidiales.

Efectividad a 3 y 6 meses de una intervención integral para caries dental en escolares de Paraíso de Osorio, La Paz; La Laguna, Chalatenango; Juayúa, Sonsonate; Mizata y San Pablo Tacachico, La Libertad / Effectiveness at 3 and 6 months of a comprehensive intervention for dental caries in schoolchildren from Paraíso de Osorio, La Paz; La Laguna, Chalatenango; Juayúa, Sonsonate; Mizata and San Pablo Tacachico, La Libertad

OBJETIVO: Determinar la efectividad a 3 y 6 meses de una intervención integral para la prevención y tratamiento de caries en dientes permanentes, en escolares de 9 a 11 años atendidos por cinco Unidades Comunitarias de Salud Familiar en el año 2017. METODOLOGÍA: Esta investigación fue de tipo intervención cuasi experimental, dirigida a la prevención y limitación del daño por caries en dientes permanentes de 150 escolares de centros educativos públicos cubiertos por 5 UCSF. Fueron atendidas en total 1741 superficies, en las que se aplicaron 1398 sellantes, 253 obturaciones y 90 remineralizaciones con flúor. Posterior a 3 y 6 meses, se midió con los índices correspondientes la efectividad a través de la reducción de placa bacteriana, la ausencia de caries y la supervivencia de los tratamientos. Se utilizaron las pruebas de Anova, F de Fisher y Kaplan Meier para el análisis de los datos. RESULTADOS: En la evaluación inicial se encontró el 46% de la población con higiene oral óptima y un promedio de 3 dientes cariados (2.91) por paciente. A los 6 meses de realizada la intervención, el 83.21% presentó higiene oral óptima, se determinó 1 diente cariado por sujeto (1.07), el 41.20% de supervivencia de sellantes de fosas y fisuras (SFF) y el 82.60% de supervivencia de obturaciones. CONCLUSIÓN: La intervención a 3 y 6 meses mostró ser efectiva para prevenir y limitar el daño por caries dental en dientes permanentes en escolares entre 9 y 11 años de edad

OBJECTIVE: To determine effectiveness after 3 and 6 months of being applied an integral intervention for prevention and treatment of dental caries in permanent teeth, in school children from 9 to 11 years; being attended in five Family Health Community Centers (UCSF Unidades Comunitarias de Salud Familiar for its acronym in Spanish) at 2017. METHODOLOGY: This research was a quasi-experimental intervention, directed to the prevention and limitation of damage by dental caries in permanent teeth of 150 school children, from public schools, being attended in five Family Health Community Centers (UCSF). In total, 1741 surfaces were intervened in which 1398 pits and fissures sealant, 253 fillings and 90 remineralizations with fluor, were applied. After 3 and 6 months, the evaluation of the effectiveness through reduction of dentobacterial plaque, the absence of dental cavities and the survival of treatments, was assessed with the corresponding indices. The statistical tests used for the analysis of data were Anova, F de Fisher and Kaplan Meier. RESULTS: In the initial evaluation it was found 46% of population with optimal oral hygiene and an average of 3 decayed teeth (2.91) per patient. Six months after the intervention, 83.21% presented optimal oral hygiene, 1 (1.07) decayed tooth was determined per subject, 41.20% permanence of pits and fissures sealants and 82.60% permanence of dental fillings. CONCLUSION: Intervention showed effectiveness in prevention and limitation of damage by dental cavities in permanent teeth after 3 and 6 months of being executed in school children between 9 and 11 years old.

An RNAi screen of the RRM-domain proteins of Trypanosoma brucei.

In eukaryotes, proteins containing RNA Recognition Motifs (RRMs) are involved in many different RNA processing reactions, RNA transport, and mRNA decay. Kinetoplastids rely extensively on post-transcriptional mechanisms to control gene expression, so RRM domain proteins are expected to play a prominent role. We here describe the results of an RNA interference screen targeting 37 of the 72 RRM-domain proteins of Trypanosoma brucei. RNAi targeting 8 of the genes caused clear growth inhibition in bloodstream trypanosomes, and milder effects were seen for 9 more genes. The small, single-RRM protein TbRBP3 specifically associated with 10 mRNAs in trypanosome lysates, but RBP3 depletion did not affect the transcriptome.

Detecting tagged Hedgehog with intracellular and extracellular immunocytochemistry for functional analysis.

In this chapter, we explain different strategies to analyze the extracellular Hedgehog (Hh) morphogen distribution and Hh intracellular trafficking by immunohistochemistry techniques. For this purpose, it has been very useful to have a transgenic fly line that expresses a Hh-green fluorescent protein (GFP) fusion protein. These flies can be used to study the way Hh spreads through the anterior compartment where it signals, and analyze in detail how Hh is internalized by its receptor Patched. In addition, this Hh-GFP fusion made without lipid modifications (cholesterol or palmitic acid) can be used to investigate the function of these lipids on Hh in terms of spreading, internalization, and signaling abilities.

Hedgehog lipid modifications are required for Hedgehog stabilization in the extracellular matrix.

The Hedgehog (Hh) family of morphogenetic proteins has important instructional roles in metazoan development. Despite Hh being modified by Ct-cholesterol and Nt-palmitate adducts, Hh migrates far from its site of synthesis and programs cellular outcomes, depending on its local concentrations. We show that in the receiving cells of the Drosophila wing imaginal disc, lipid-unmodified Hh spreads across many more cell diameters than the wild type and this spreading leads to the activation of low but not high threshold responses. Unlipidated Hh forms become internalized through the apical plasma membrane, while wild-type Hh enters through the basolateral cell surface - in all cases via a dynamin-dependent mechanism. Full activation of the Hh pathway and the spread of Hh throughout the extracellular matrix depend on the ability of lipid-modified Hh to interact with heparan sulfate proteoglycans (HSPG). However, neither Hh-lipid modifications nor HSPG function are required to activate the targets that respond to low levels of Hh. All these data show that the interaction of lipid-modified Hh with HSPG is important both for precise Hh spreading through the epithelium surface and for correct Hh reception.

The translational efficiencies of the two Leishmania infantum HSP70 mRNAs, differing in their 3'-untranslated regions, are affected by shifts in the temperature of growth through different mechanisms.

Exposure of Leishmania promastigotes to the temperature of their mammalian hosts induces a typical heat-shock response. In Leishmania infantum, HSP70 is encoded by two types of genes that differ in their 3'-untranslated regions (3'-UTRs). Previously, we have shown that specific transcripts for each gene are present in promastigotes growing at normal temperature (26 degrees C), but only transcripts with 3'-UTR-type I (3'-UTRI) accumulate in a temperature-dependent manner. Here, we have investigated the translational efficiencies of both types of HSP70 transcripts at the different temperatures that the parasite encounters in the insect (26 degrees C, normal temperature) or in the mammalian host (heat-shock temperatures). Interestingly, 3'-UTRI-bearing transcripts (HSP70-I) were found associated with ribosomes in promastigotes at normal and heat-shock temperatures, whereas the HSP70-II transcripts appear to be preferentially translated at heat-shock temperatures but not at 26 degrees C. We have analyzed the function of these UTRs in the translational control by use of plasmid constructs in which the CAT reporter gene was flanked by UTRs of the HSP70 genes. Unexpectedly, it was found that CAT transcripts with 3'-UTRII bind to ribosomes at 26 degrees C, and, indeed, the CAT protein is synthesized. A valid conclusion of these experiments was that both types of 3'-UTRs are essential for translation of HSP70 mRNAs at heat shock temperatures, although the 3'-UTRII is more efficient during severe heat shock (39 degrees C). In addition, these results suggest that sequence region other than the 3'-UTR of HSP70-II gene is involved in the translational silent state of HSP70-II transcripts at 26 degrees C. Finally, a null mutant has been created by targeted disruption of both HSP70-II alleles. Remarkably, the deltaHSP70 mutant synthesizes HSP70 at a lower rate than the wild-type parasites. Overall, our data suggest that the biological function of the HSP70-II gene is to top up HSP70 levels under conditions of stress.

Genomic DNA macroarrays as a tool for analysis of gene expression in Leishmania.

Gene-array technologies have been applied in a wide number of organisms to study gene expression profiling under several physiological and experimental conditions. Gene-array implementations combined with the information arising from emerging genome sequencing projects are expected to be in the near future a major tool to characterize genes involved in different processes. So far, gene expression profile studies in trypanosomatids have been performed in microarrays that use a glass support to immobilize fragments of genomic DNA followed by fluorescent detection. Here, we wanted to test the potential of genomic DNA macroarrays of Leishmania infantum using nylon membranes and radioactive detection. Nylon macroarrays present a number of advantages since the processing of the membranes is based on standard Southern blotting protocols familiar to molecular biologists, and the data acquisition equipment is available to most research institutions. Nylon macroarrays were employed to search for genes showing increased mRNA abundance during an axenic differentiation of L. infantum promastigotes to amastigotes. Several clones were rescued and, after validation by Northern blot assays, these L. infantum sequences were used to screen the Leishmania major gene database. The L. major contigs with high homology to the L. infantum sequences allowed a consistent identification of the regulated genes.