Alterations of the resin canal system of <Emphasis Type="Italic">Pinus pinaster</Emphasis> seedlings after fertilization of a healthy and of a <Emphasis Type="Italic">Hylobius abietis</Emphasis> attacked stand

Changes in resource availability and biotic and abiotic stress may alter the defensive mechanisms of pine trees. The effect of fertilisation on the resin canal structure of Pinus pinaster seedlings established in two trials in NW Spain, one attacked by Hylobius abietis and the other non-attacked, was studied. The leaders of 50 plants were destructively sampled and the resin canal density, the canal area and its relative conductive area in the phloem and xylem were assessed. Experimentally increased nutrient availability significantly decreased resin canal density in the phloem of the seedlings in the two analysed trials, where unfertilised seedlings presented up to 30% more resin canal density than the fertilised seedlings (mean value ± SEM = 0.32 ± 0.02 resin canals mm⁻² in the fertilised plants versus 0.45 ± 0.04 resin canals mm⁻² in the control plants). Fertilisation had no effect on the resin canal system in the xylem, but significantly increased tracheid size. Significant differences of resin canals among sites were observed mainly in the xylem; the resin canal density was 1.7-fold greater in the attacked site than in the non-attacked site. The similar structure of phloem resin canals in both sites supports that phloem resin canals are constitutive mechanisms of defence in P. pinaster, whereas xylem resin canals would be constitutive mechanisms but also inducible mechanisms of resistance following the attack of pine weevils or bark beetles. XM and LS equally contributed to this paper.     

Incidence of dermatophytosis in a public hospital of São Bernardo do Campo,São Paulo State,Brazil

The incidence of cases of dermatophytosis in dermatology outpatient departments has increased in recent years. Infection control is essential to prevent transmission, and accurate diagnosis of this type of infection is important to avoid confusion with other dermatological processes caused by non-fungal agents. The objective of this study was to determine the incidence of dermatophytosis and its etiological agents in a public hospital of São Bernardo do Campo, Brazil. From February 2005 to May 2006, 273 samples were collected from 191 patients in a public dermatology outpatient department. The samples were collected according to conventional methods used in clinical mycology and direct examinations and cultures were performed. Of the samples suspected of fungal infection, 19% produced positive cultures, and the largest number of samples was from female patients between 31 and 40 years old. The most commonly infected area was the toenails, and the most common infective agents according to microscopic analysis and biochemical tests were the Candida species, which were responsible for 61.6% of the infections. These data agree with findings reported in the literature indicating a lower prevalence of filamentous fungi, such as Epidermophyton, Trichophyton, and Microsporum, in dermatomycosis.     

Reproductive status of endemic felid species in Latin American zoos and implications for ex situ conservation

Reproductive evaluations were conducted on 185 male cats representing eight endemic Latin American species that were maintained in 44 zoos and private facilities in 12 Latin American countries. Reproductive assessments (testicular measures, ejaculate quality, and blood testosterone/cortisol concentration) were used to establish normative values for large‐ and small‐sized cats in Latin American collections. Data also were analyzed using multiple regression to study the impact of proven breeder status, diet, and various animal housing combinations. Most felids (>95%) in the survey were of wild‐born origin, and <20% had produced offspring in captivity. Larger felids had bigger testes and produced more semen, but tended to produce low‐sperm‐density ejaculates. The ejaculates of small felids were more sperm‐concentrated, but contained fewer total spermatozoa. Sperm motility was unrelated to species size, and certain species (puma, margay, tigrina, and jaguarundi) consistently produced few (<40%) normal sperm forms. Across species, >50% of males had low sperm counts (<1 million total sperm per ejaculate). Among large cats (jaguars and pumas), proven breeders had larger (P<0.05) testes, greater semen volume, and more normal sperm than nonbreeders. Males on adequate diets had higher (P<0.05) circulating cortisol. Among small‐sized felids, proven breeders had higher (P<0.05) testosterone, and males housed alone or paired with a conspecific female had more (P<0.05) total sperm per ejaculate and greater (P<0.05) seminal and testicular volumes. Fifty‐nine ejaculates (potentially representing ～100 artificial insemination (AI) or 26,000 in vitro fertilization (IVF) procedures) were cryopreserved for a felid genome resource bank. In conclusion, breeding success and reproductive traits for many endemic felids in Latin American zoos appear to be suboptimal, and likely would benefit from improvements in diet and exhibitry. Technology transfer and continued training of zoo staff and scientists in Latin American countries are essential if these zoos are to achieve their tremendous conservation potential for felids and other threatened endemic species. Zoo Biol 22:421–441, 2003. © 2003 Wiley‐Liss, Inc.     

A density functional theory investigation of the interaction of the tetraaqua calcium cation with bidentate carbonyl ligands

Calcium complexes with bidentate carbonyl ligands are important in biological systems, medicine and industry, where the concentration of Ca²⁺ is controlled using chelating ligands. The exchange of two water molecules of [Ca(H₂O)₆]²⁺ for one bidentate monosubstituted and homo disubstituted dicarbonyl ligand was investigated using the B3LYP/6-311++G(d,p) method. The ligand substituents NH₂, OCH₃, OH, CH₃, H, F, Cl, CN and NO₂ are functional groups with distinct electron-donating and -withdrawing effects that bond directly to the sp² C atom of the carbonyl group. The geometry, charge and energy characteristics of the complexes were analyzed to help understand the effects of substituents, spacer length and chelation. Coordination strength was quantified in terms of the enthalpy and free energy of the exchange reaction. The most negative enthalpies were calculated for the coordination of bidentate ligands containing three to five methylene group spacers between carbonyls. The chelate effect contribution was analyzed based on the thermochemistry. The electronic character of the substituent modulates the strength of binding to the metal cation, as ligands containing electron-donor substituents coordinate stronger than those with electron-acceptor substituents. This is reflected in the geometric (bond length and chelating angle), electronic (atomic charges) and energetic (components of the total interacting energy) characteristics of the complexes. Energy decomposition analysis (EDA)—an approach for partitioning of the energy into its chemical origins—shows that the electrostatic component of the coordination is predominant, and yields relevant contribution of the covalent term, especially for the electron-withdrawing substituted ligands. The chelate effect of the bidentate ligands was noticeable when compared with substitution by two monodentate ligands.

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Graphical abstract The affinity of 18 bidentate carbonyl ligands toward the [Ca(H₂O)₄]²⁺ cation is evaluated in terms of energetic, geometric and electronic parameters of the isolated ligands and the substituted aqua complexes. The electronic effects—inductive and mesomeric—intrinsic to the molecular structure of each ligand are found to modulate the strength of the metal-ligand interaction. The effects of polysubstitution, chelation and the length of the alkyl spacers between the anchor points of the ligand are also analyzed.

     

Morphological and Genetic Diversity of Opisthosporidia: New Aphelid Paraphelidium tribonemae gen. et sp. nov.

Aphelids are a poorly known group of parasitoids of algae that have raised considerable interest due to their pivotal phylogenetic position. Together with Cryptomycota and the highly derived Microsporidia, they have been recently re‐classified as the Opisthosporidia, which constitute the sister group to the fungi within the Holomycota. Despite their huge diversity, as revealed by molecular environmental studies, and their phylogenetic interest, only three genera have been described (Aphelidium, Amoeboaphelidium, and Pseudaphelidium), from which 18S rRNA gene sequences exist only for Amoeboaphelidium and Aphelidium species. Here, we describe the life cycle and ultrastructure of a new representative of Aphelida, Paraphelidium tribonemae gen. et sp. nov., and provide the first 18S rRNA gene sequence obtained for this genus. Molecular phylogenetic analysis indicates that Paraphelidium is distantly related to both Aphelidium and Amoebaphelidium, highlighting the wide genetic diversity of aphelids. Paraphelidium tribonemae has amoeboflagellate zoospores containing a lipid‐microbody complex, dictyosomes, and mitochondria with rhomboid cristae, which are also present in trophonts and plasmodia. The amoeboid trophont uses pseudopodia to feed from the host cytoplasm. Although genetically distinct, the genus Paraphelidium is morphologically indistinguishable from other aphelid genera and has zoospores able to produce lamellipodia with subfilopodia like those of Amoeboaphelidium.     

ChromaPipe: a pipeline for analysis, quality control and management for a DNA sequencing facility

Optimizing and monitoring the data flow in high-throughput sequencing facilities is important for data input and output, for tracking the status of results for the users of the facility, and to guarantee a good, high-quality service. In a multi-user system environment with different throughputs, each user wants to access his/her data easily, track his/her sequencing history, analyze sequences and their quality, and apply some basic post-sequencing analysis, without the necessity of installing further software. Recently, Fiocruz established such a core facility as a "technological platform". Infrastructure includes a 48-capillary 3730 DNA Sequence Analyzer (Applied Biosystems) and supporting equipment. The service includes running samples for large-scale users, performing DNA sequencing reactions and runs for medium and small users, and participation in partial or full genome projects. We implemented a workflow that fulfills these requirements for small and high throughput users. Our implementation also includes the monitoring of data for continuous quality improvement (reports by plate, month and user) by the sequencing staff. For the user, different analyses of the chromatograms, such as visualization of good quality regions, as well as processing, such as comparisons or assemblies, are available. So far, 180 users have made use of the service, generating 155,000 sequences, 35% of which were produced for the BCG Moreau-RJ genome project. The pipeline (named ChromaPipe for Chromatogram Pipeline) is available for download by the scientific community at the url http://bioinfo.pdtis.fiocruz.br/ChromaPipe/. The support for assembly is also configured as a web service: http://bioinfo.pdtis.fiocruz.br/Assembly/.     

Ferric species of the giant extracellular hemoglobin of Glossoscolex paulistus as function of pH: an EPR study on the irreversibility of the heme transitions

The present article is focused on the transitions of ferric heme species of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) induced by successive alterations in pH, involving alkaline and acid mediums. Electron paramagnetic resonance (EPR) is the spectroscopy used to evaluate the transitions that occur in the first coordination sphere of ferric ion as a consequence of ligand changes in a wide range of pH, since this tool is very sensitive to slight changes that occur in the heme pocket of paramagnetic species. This approach is adequate to obtain information regarding the reversibility/irreversibility that involves the heme transitions induced by pH, since the degree of reversibility is associated to the intensity of the changes that occur in the spatial configuration of the polypeptide chains, which is clearly associated to the first coordination sphere. The results demonstrate a significant degree of irreversibility of heme transitions, since the final species, which do not present any change after 6 h of its respective formations, are quite different of the initial species. The results denote that the more stable species are the bis-histidine (hemichrome) and pentacoordinate species, due to the properties of their ligands and to the mechanical influence of the respective subunits. EPR spectra allow to distinguish the types of hemichrome species, depending on the reciprocal orientation between the histidine axial ligands, in agreement with Walker's Classification [Walker, F.A., 1999. Magnetic spectroscopic (EPR, ESEEM, M?ssbauer, MCD and NMR) studies of low-spin ferriheme centers and their corresponding heme proteins. Coord. Chem. Rev. 185-186, 471-534]. However, these transitions are not completed, i.e., the appearance of a determined species does not mean the total consumption of its precursor species, implying the coexistence of several types of species, depending on pH. Furthermore, it is possible to conclude that a "pure" EPR spectrum of aquomet ferric species is an important indicator of a high level of conservation referent to the "native" configuration of whole hemoglobin, which is only encountered at pH 7.0. The results allow to infer important physico-chemical properties as well as to evaluate aspects of the structure-activity relationship of this hemoprotein, furnishing information with respect to the denaturation mechanism induced by drastic changes in pH. These data are very useful since HbGp has been proposed as prototype of substitute of blood, thus requiring wide knowledge about its structural and chemical properties.     

The involvement of CD4+CD25+ T cells in the acute phase of Trypanosoma cruzi infection

The infection with Trypanosoma cruzi leads to a vigorous and apparently uncontrolled inflammatory response in the heart. Although the parasites trigger specific immune response, the infection is not completely cleared out, a phenomenon that in other parasitic infections has been attributed to CD4+CD25+ T cells (Tregs). Then, we examined the role of natural Tregs and its signaling through CD25 and GITR in the resistance against infection with T. cruzi. Mice were treated with mAb against CD25 and GITR and the parasitemia, mortality and heart pathology analyzed. First, we demonstrated that CD4+CD25+GITR+Foxp3+ T cells migrate to the heart of infected mice. The treatment with anti-CD25 or anti-GITR resulted in increased mortality of these infected animals. Moreover, the treatment with anti-GITR enhanced the myocarditis, with increased migration of CD4+, CD8+, and CCR5+ leukocytes, TNF-alpha production, and tissue parasitism, although it did not change the systemic nitric oxide synthesis. These data showed a limited role for CD25 signaling in controlling the inflammatory response during this protozoan infection. Also, the data suggested that signaling through GITR is determinant to control of the heart inflammation, parasite replication, and host resistance against the infection.     

The outer membrane protein TolC from Sinorhizobium meliloti affects protein secretion, polysaccharide biosynthesis, antimicrobial resistance, and symbiosis

Sinorhizobium meliloti is capable of establishing a symbiotic nitrogen fixation relationship with Medicago sativa. During this process, it must cope with diverse environments and has evolved different types of transport systems that help its propagation in the plant roots. TolC protein family members are the outer-membrane components of several transport systems involved in the export of diverse molecules, playing an important role in bacterial survival. In this work, we have characterized the protein TolC from S. meliloti 2011. An insertional mutation in the tolC gene strongly affected the resistance phenotype to antimicrobial agents and induced higher susceptibility to osmotic and oxidative stresses. Immunodetection experiments and comparison of the extracellular proteins present in the supernatant of the wild-type versus tolC mutant strains showed that the calcium-binding protein ExpE1, the endoglycanase ExsH, and the product of open reading frame SMc04171, a putative hemolysin-type calcium-binding protein, are secreted by a TolC-dependent secretion system. In the absence of TolC, neither succinoglycan nor galactoglucan were detected in the culture supernatant. Moreover, S. meliloti tolC mutant induced a reduced number of nonfixing nitrogen nodules in M. sativa roots. Taken together, our results confirm the importance of TolC in protein secretion, exopolysaccharide biosynthesis, antimicrobials resistance, and symbiosis.