Polymorphism in the flanking regions of the PbGP43 gene from the human pathogen Paracoccidioides brasiliensis: search for protein binding sequences and poly(A) cleavage sites

<p>Abstract</p> <p>Background</p> <p><it>Paracoccidioides brasiliensis </it>is a thermo-dimorphic fungus that causes paracoccidiodomycosis (PCM). Glycoprotein gp43 is the fungal main diagnostic antigen, which can also protect against murine PCM and interact with extracellular matrix proteins. It is structurally related to glucanases, however not active, and whose expression varies considerably. We have presently studied polymorphisms in the Pb<it>GP43 </it>flanking regions to help understand such variations.</p> <p>Results</p> <p>we tested the protein-binding capacity of oligonucleotides covering the Pb<it>GP43 </it>proximal 5' flanking region, including overlap and mutated probes. We used electrophoretic mobility shift assays and found DNA binding regions between positions -134 to -103 and -255 to -215. Only mutation at -230, characteristic of <it>P. brasiliensis </it>phylogenetic species PS2, altered binding affinity. Next, we cloned and sequenced the 5' intergenic region up to position -2,047 from <it>P. brasiliensis </it>Pb339 and observed that it is composed of three tandem repetitive regions of about 500 bp preceded upstream by 442 bp. Correspondent PCR fragments of about 2,000 bp were found in eight out of fourteen isolates; in PS2 samples they were 1,500-bp long due to the absence of one repetitive region, as detected in Pb3. We also compared fifty-six Pb<it>GP43 </it>3' UTR sequences from ten isolates and have not observed polymorphisms; however we detected two main poly(A) clusters (1,420 to 1,441 and 1,451 to 1,457) of multiple cleavage sites. In a single isolate we found one to seven sites.</p> <p>Conclusions</p> <p>We observed that the amount of Pb<it>GP43 </it>transcripts accumulated in <it>P. brasiliensis </it>Pb339 grown in defined medium was about 1,000-fold higher than in Pb18 and 120-fold higher than in Pb3. We have described a series of features in the gene flanking regions and differences among isolates, including DNA-binding sequences, which might impact gene regulation. Little is known about regulatory sequences in thermo-dimorphic fungi. The peculiar structure of tandem repetitive fragments in the 5' intergenic region of Pb<it>GP43</it>, their characteristic sequences, besides the presence of multiple poly(A) cleavage sites in the 3' UTR will certainly guide future studies.</p

Aplicação clínica da ressonância magnética em pacientes com traumatismo craniencefálico agudo

PURPOSE: To evaluate the clinical applications of magnetic resonance imaging (MRI) in patients with acute traumatic brain injury (TBI): to identify the type, quantity, severity; and improvement clinical-radiological correlation. METHOD: Assessment of 55 patients who were imaged using CT and MRI, 34 (61.8%) males and 21 (38.2%) females, with acute (0 to 5 days) and closed TBI. RESULTS: Statistical significant differences (McNemar test): ocurred fractures were detected by CT in 29.1% and by MRI in 3.6% of the patients; subdural hematoma by CT in 10.9% and MRI in 36.4 %; diffuse axonal injury (DAI) by CT in 1.8% and MRI in 50.9%; cortical contusions by CT in 9.1% and MRI in 41.8%; subarachnoid hemorrhage by CT in 18.2% and MRI in 41.8%. CONCLUSION: MRI was superior to the CT in the identification of DAI, subarachnoid hemorrhage, cortical contusions, and acute subdural hematoma; however it was inferior in diagnosing fractures. The detection of DAI was associated with the severity of acute TBI.PROPÓSITO: Avaliar a aplicação clínica da ressonância magnética (RM) em pacientes vítimas de traumatismo craniencefálico (TCE) agudo, na identificação do tipo, número, gravidade e correlação clínica-radiológica. MÉTODO: Foram estudados prospectivamente 55 pacientes vítimas de TCE agudo fechado (0-5 dias), por TC e RM, sendo 34 do sexo masculino e 21 do feminino. RESULTADOS: Houve diferença estatisticamente significante (teste McNemar): fraturas de crânio foram detectadas em 29,1% pacientes na TC e 3,6% pela RM; hematoma subdural 10,9% na TC e 36,4% pela RM; lesão axonal difusa (LAD) 1,8% pela TC e 50,9% na RM; contusões corticais 9,1% na TC e 41,8% pela RM, hemorragia subaracnóidea 18,2% na TC e 41,8% pela RM. CONCLUSÃO: A RM foi superior à TC na identificação da LAD, hemorragia subaracnóidea, contusões corticais e hematoma subdural agudo, porém inferior no diagnóstico de fraturas. A detecção de LAD pela RM foi associada com maior gravidade do TCE agudo

Some interesting features of new massive gravity

A proof that new massive gravity - the massive 3D gravity model proposed by Bergshoeff, Hohm and Townsend (BHT) - is the only unitary system at the tree level that can be constructed by augmenting planar gravity through the curvature-squared terms, is presented. Two interesting gravitational properties of the BHT model, namely, time dilation and time delay, which have no counterpart in the usual Einstein 3D gravity, are analyzed as well.Comment: Submitted to Classical and Quantum Gravit

α-particle production in 6He + 120Sn collisions

The collision 6He+120Sn has been investigated at four energies near the Coulomb barrier. A large yield of α particles has been detected, with energies around the energy of the scattered 6He beam. The energy and angular distributions of the α particles have been analyzed and compared with breakup and neutron transfer calculations.Ministerio de Ciencia e Innovación (España) FPA2006-13807-c02-01Junta de Andalucía P07-FQM-02894Ministerio de Educación y Ciencia (España) PCI2006-A7-0654Programa Consolider-Ingenio 2010 (España) CSD2007-0004

Identifying unmet needs in SSc-ILD by semi-qualitative in-depth interviews

Objectives Interstitial lung disease is frequent in SSc (SSc-ILD) and associates with significantly reduced quality of life. Here we aimed to analyse patient pathways, and experiences of patients and healthcare providers (HCPs) in order to identify unmet needs in the management of SSc-ILD patients. Methods Semi-structured qualitative interviews conducted in eight European countries looked at HCP (n = 95) and patient perspectives (n = 47) using two sets of 70 research questions. Pre-diagnostic, diagnostic and post-diagnostic phases of the patient pathway were systematically explored. Results (i) In the pre-diagnostic phase several gaps were identified by HCPs and patients in all participating countries: limited disease knowledge among primary care physicians and specialists, lack of accurate patient information, and delayed and/or inappropriate referral. (ii) The diagnostic phase is in most countries coordinated by rheumatologists, who are also the main point of care. Depending on the local health system, organization of multidisciplinary collaboration varies. HCPs issued lack of national guidelines, while patients stated difficulties obtaining disease-related information. (iii) In the post-diagnostic phase, HCPs and patients indicated lack of curative treatment, specialized nurses, and paramedical and psychological support. Patients and caregivers additionally expressed the need for clear information on SSc-ILD. Conclusion Lack of disease specific knowledge, gaps in national healthcare systems and insufficient information and support for patients and caregivers were identified as unmet needs to ensure timely diagnosis, provide better patient management and to improve quality of life in SSc-ILD patients.Peer reviewe

Arrays on disc for screening and quantification of pollutants

A rapid compact disc based methodology for screening and quantification of organic pollutants in mandarin juices is presented. The assay is established on the coating conjugate indirect competitive principle and developed under disc-array configuration. The detection is based on the acquisition of attenuated reflective signals that were proportional to optical density of the immunoreaction product. The competitive assay is applied to quantify simultaneously, in a selective manner, non-systemic insecticides in mandarin juices. The detection limits were 0.2 and 0.1 ¿g L¿1 and the sensitivity 2.1 and 1.5 ¿g L¿1, for chlorpyrifos and fenthion, respectively. Pollutants were directly quantified after sample dilution in a total time of 40 min. Also, the implementation of positive and negative controls into the array configuration served as an automatic quality control test. The effect of thermal treatment on pesticide dissipation was studied and found that it was insignificant under the studied conditions. Recovery intervals ranged from 96¿105% to 94¿103%, for chlorpyrifos and fenthion, respectively and were similar to those obtained with gas chromatography coupled to mass spectrometry. In the current configuration, 64 samples can be simultaneously analyzed on a disc at a very competitive value, demonstrating its potential for high-throughput multiplexed screening applications for controlled monitoring programs in low-level labs or outside the lab settingThis work was funded by the projects FEDER CTQ2010-15943 (CICYT, Spain), and PROMETEO 2010/008 and ACOMP/2012/158 (Generalitat Valenciana).Navarro, P.; Morais Ezquerro, SB.; Gavaldón, JA.; Pérez, AJ.; Puchades Pla, R.; Maquieira Catala, Á. (2013). Arrays on disc for screening and quantification of pollutants. Analytica Chimica Acta. 784:59-64. https://doi.org/10.1016/j.aca.2013.04.041S596478

Antiparasitic Activity of Natural and Semi-Synthetic Tirucallane Triterpenoids from Schinus terebinthifolius (Anacardiaceae): Structure/Activity Relationships

Leishmaniasis and Chagas are diseases caused by parasitic protozoans that affect the poorest population in the World, causing a high mortality and morbidity. As a result of highly toxic and long-term treatments, the discovery of novel, safe and more efficacious drugs is essential. in this work, the in vitro antiparasitic activity and mammalian cytotoxicity of three natural tirucallane triterpenoids, isolated from leaves of Schinus terebinthifolius (Anacardiaceae), and nine semi-synthetic derivatives were investigated against Leishmania (L.) infantum and Trypanosoma cruzi. Trypomastigotes of T. cruzi were the most susceptible parasites and seven compounds demonstrated a trypanocidal activity with IC50 values in the range between 15 and 58 mu g/mL. Four compounds demonstrated selectivity towards the intracellular amastigotes of Leishmania, with IC50 values in the range between 28 and 97 mu g/mL. the complete characterization of triterpenoids was afforded after thorough analysis of nuclear magnetic resonance (NMR) data as well as electrospray ionization mass spectrometry (ESI-MS). Additionally, structure-activity relationships were performed using Decision Trees.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)Universidade Federal de São Paulo, Inst Ciencias Ambientais Quim & Farmaceut, BR-09972270 Diadema, SP, BrazilAdolfo Lutz Inst, Ctr Parasitol, BR-01246902 São Paulo, BrazilUniv Fed Paraiba, Ctr Ciencias Aplicadas & Educ, BR-58297000 Rio Tinto, BrazilUniv Fed Uberlandia, Inst Quim, BR-38400902 Uberlandia, MG, BrazilUniversidade Federal de São Paulo, Inst Ciencias Ambientais Quim & Farmaceut, BR-09972270 Diadema, SP, BrazilCNPq: 300546/2012-2CNPq: 471458/2012-0FAPESP: 2011/51739-0FAPESP: 2012/18756-1FAPESP: 2013/16320-4Web of Scienc

USE OF COMBINATION OF FLUORESCENT PROBES TO IDENTIFY SPERM SUBPOPULATIONS FOR QUALITY ASSESSMENT OF FRESH AND CRYOPRESERVED CANINE SEMEN. PRELIMINARY RESULTS

The use of fluorescent markers in the evaluation of sperm morphophysiology allows a better accuracy, compared to the subjective nature of some routine tests in semen qualification. In this study was used the combination of fluorescence probes: propionate iodide, Hoechst 33342 and FITC-PSA in fresh and thawed dog semen, to the identification of the following morphological subpopulations: II (intact plasma and acrosomal membranes), IL (intact plasma membrane and lesioned acrosomal membrane), LI (lesioned plasma membrane and intact acrosomal membrane) and LL (both membranes lesioned). When comparing the results obtained with the results of the tests used conventionally in semen evaluation (sperm motility and vigor, hypoosmotic test and morphological alterations), little correlation was observed. The II population declined from fresh semen to thawed, while LL population increased (p <0.05). The IL population was composed of extremely small numbers of cells but increased (p <0.05) from fresh semen to thawed semen. In the thawed semen the major defects had a positive correlation with the LL population (p <0.01). For the thawed semen, the results of the hypoosmotic test (number of cells that reacted to the medium) correlated positively with population II (p <0.025), that is, different from that observed in fresh semen. Although all tests were able to detect decrease in sperm quality post-thawing (p <0.05). The use of this fluorescent probe association allowed qualification and more accurately quantification of plasma membrane and acrosomal insults mediated by cryopreservation. El uso de marcadores fluorescentes en la evaluación de la morfofisiología espermática permite una mayor precisión, comparada con la naturaleza subjetiva de algunas pruebas de rutina en la valoración del semen. En este estudio se usó la combinación de pruebas fluorescentes: yoduro de propidio; Hoechst 33342 y FITC-PSA en semen fresco y descongelado de perro, para la identificación de las siguientes subpoblaciones morfológicas: II (membranas plasmática y acrosomal intactas), IL (membrana plasmática intacta y membrana acrosomal dañada), LI (membrana plasmática dañada y membrana acrosomal intacta) y LL (ambas membranas dañadas). Cuando se comparan los resultados obtenidos con los resultados de las pruebas usadas convencionalmente en la evaluación seminal (motilidad y vigor espermáticos, prueba hipoosmótica y alteraciones morfológicas), se observó poca correlación. La población II disminuyó desde el semen fresco al descongelado, mientras que la población LL se incrementó (p<0.05). la población IL estuvo compuesta por un número extremadamente pequeño de células, pero incremento (p<0.05) desde el semen fresco al descongelado. En el semen descongelado los defectos mayores tuvieron una correlación positiva con la población LL (p<0.01). En el semen descongelado, los resultados de la prueba hipoosmótica (número de células que reaccionan al medio) se correlacionaron positivamente con la población II (p<0.05). El uso de esta asociación de pruebas fluorescentes permitió la valoración y la cuantificación más precisa de los daños a la membrana plasmática y acrosomal mediados por la criopreservación.