Phylogenetic revision of Agapophytinae subf.n. (Diptera : Therevidae) based on molecular and morphological evidence

Agapophytinae subf.n. is a highly diverse lineage of Australasian Therevidae, comprising eight described and two new genera: Agapophytus Guerin-Meneville, Acupalpa Krober, Acraspisa Krober, Belonalys Krober, Bonjeania Irwin & Lyneborg, Parapsilocephala Krober, Acatopygia Krober, Laxotela Winterton & Irwin, Pipinnipons gen.n. and Patanothrix gen.n. A genus-level cladistic analysis of the subfamily was undertaken using sixty-eight adult morphological characters and c. 1000 base pairs of the elongation factor-1 alpha (EF-1 alpha) protein coding gene. The morphological data partition produced three most parsimonious cladograms, whereas the molecular data partition gave a single most parsimonious cladogram, which did not match any of the cladograms found in the morphological analysis. The level of congruence between the data partitions was determined using the partition homogeneity test (HTF) and Wilcoxon signed ranks rest. Despite being significantly incongruent in at least one of the incongruence tests, the partitions were combined in a simultaneous analysis. The combined data yielded a single cladogram that was better supported than that of the individual partitions analysed separately. The relative contributions of the data partitions to support for individual nodes on the combined cladogram were investigated using Partitioned Bremer Support. The level of support for many nodes on the combined cladogram was non-additive and often greater than the sum of support for the respective nodes on individual partitions. This synergistic interaction between incongruent data partitions indicates a common phylogenetic signal in both partitions. It also suggests that criteria for partition combination based solely on incongruence may be misleading. The phylogenetic relationships of the genera are discussed using the combined data. A key to genera of Agapophytinae is presented, with genera diagnosed and figured. Two new genera are described: Patanothrix with a new species (Pat. skevingtoni) and Pat. wilsoni (Mann) transferred from Parapsilocephala, and Pipinnipons with a new species (Pip. kroeberi). Pipinnipons fascipennis (Krober) is transferred from Squamopygin Krober and Pip. imitans (Mann) is transferred from Agapophytus. Agapophytus bicolor (Krober) is transferred from Parapsilocephala. Agapophytus varipennis Mann is synonymised with Aga, queenslandi Krober and Aga. flavicornis Mann is synonymised with Aga. pallidicornis (Krober).

Induction of immune tolerance by dendritic cells: implications for preventative and therapeutic immunotherapy of autoimmune disease

Dendritic cells (DC) have a key role in controlling the immune response, by determining the outcome of antigen presentation to T cells. Through costimulatory molecules and other factors, DC are involved in the maintenance of peripheral tolerance through modulation of the immune response. This modulation occurs both constitutively, and in inflammation, in order to prevent autoimmunity and to control established immune responses. Dendritic cell control of immune responses may be mediated through cytokine or cell-contact dependent mechanisms. The molecular and cellular basis of these controls is being understood at an increasingly more complex level. This understanding is reaching a level at which DC-based therapies for the induction of immune regulation in autoimmunity can be tested in vivo. This review outlines the current state of knowledge of DC in immune tolerance, and proposes how DC might control both T cell responses, and themselves, to prevent autoimmunity and maintain peripheral tolerance.

Molecular and functional analyses of Kunjin virus infectious cDNA clones demonstrate the essential roles for NS2A in virus assembly and for a nonconservative residue in NS3 in RNA replication

A number of full-length cDNA clones of Kunjin virus (KUN) were previously prepared; it was shown that two of them, pAKUN and FLSDX, differed in specific infectivities of corresponding in vitro transcribed RNAs by similar to100,000-fold (A. A. Khromykh et al., J. Virol. 72:7270-7279, 1998). In this study, we analyzed a possible genetic determinant(s) of the observed differences in infectivity initially by sequencing the entire cDNAs of both clones and comparing them with the published sequence of the parental KUN strain MRM61C. We found six common amino acid residues in both cDNA clones that were different from those in the published MRM61C sequence but were similar to those in the published sequences of other flaviviruses from the same subgroup. pAKUN clone had four additional codon changes, i.e., Ile59 to Asn and Arg175 to Lys in NS2A and Tyr518 to His and Ser557 to Pro in NS3. Three of these substitutions except the previously shown marker mutation, Arg175 to Lys in NS2A, reverted to the wild-type sequence in the virus eventually recovered from pAKUN RNA-transfected BHK cells, demonstrating the functional importance of these residues in viral replication and/or viral assembly. Exchange of corresponding DNA fragments between pAKUN and FLSDX clones and site-directed mutagenesis revealed that the Tyr518-to-His mutation in NS3 was responsible for an similar to5-fold decrease in specific infectivity of transcribed RNA, while the Ile59-to-Asn mutation in NS2A completely blocked virus production. Correction of the Asn59 in pAKUN NS2A to the wild-type lie residue resulted in complete restoration of RNA infectivity. Replication of KUN replicon RNA with an Ile59-to-Asn substitution in NS2A and with a Ser557-to-Pro substitution in NS3 was not affected, while the Tyr518-to-His substitution in NS3 led to severe inhibition of RNA replication. The impaired function of the mutated NS2A in production of infectious virus was complemented in trans by the helper wild-type NS2A produced from the KUN replicon RNA. However, replicon RNA with mutated NS2A could not be packaged in trans by the KUN structural proteins. The data demonstrated essential roles for the KUN nonstructural protein NS2A in virus assembly and for NS3 in RNA replication and identified specific single-amino-acid residues involved in these functions.

Biological activity and cellular uptake of [Ru(eta(5)-C5H5)(PPh3)(Me(2)bpy)][CF3SO3] complex

Anticancer activity of the new [Ru(eta(5)-C5H5)(PPh3)(Me(2)bpy)][CF3SO3] (Me(2)bpy = 4,4'-dimethyl-2,2'-bipyridine) complex was evaluated in vitro against several human cancer cell lines, namely A2780, A2780CisR, HT29, MCF7, MDAMB231 and PC3. Remarkably, the IC50 values, placed in the nanomolar and sub-micromolar range, largely exceeded the activity of cisplatin. Binding to human serum albumin, either HSA (human serum albumin) or HSA(faf) (fatty acid-free human serum albumin) does not affect the complex activity. Fluorescence studies revealed that the present ruthenium complex strongly quench the intrinsic fluorescence of albumin. Cell death by the [Ru(eta(5)-C5H5)(PPh3)(Me(2)bpy)][CF3SO3] complex was reduced in the presence of endocytosis modulators and at low temperature, suggesting an energy-dependent mechanism consistent with endocytosis. On the whole, the biological activity evaluated herein suggests that the complex could be a promising anticancer agent. (C) 2013 Elsevier Inc. All rights reserved.

A comprehensive high-throughput FTIR spectroscopy-based method for evaluating the transfection event: estimating the transfection efficiency and extracting associated metabolic responses

Reporter genes are routinely used in every laboratory for molecular and cellular biology for studying heterologous gene expression and general cellular biological mechanisms, such as transfection processes. Although well characterized and broadly implemented, reporter genes present serious limitations, either by involving time-consuming procedures or by presenting possible side effects on the expression of the heterologous gene or even in the general cellular metabolism. Fourier transform mid-infrared (FT-MIR) spectroscopy was evaluated to simultaneously analyze in a rapid (minutes) and high-throughput mode (using 96-wells microplates), the transfection efficiency, and the effect of the transfection process on the host cell biochemical composition and metabolism. Semi-adherent HEK and adherent AGS cell lines, transfected with the plasmid pVAX-GFP using Lipofectamine, were used as model systems. Good partial least squares (PLS) models were built to estimate the transfection efficiency, either considering each cell line independently (R 2 ≥ 0.92; RMSECV ≤ 2 %) or simultaneously considering both cell lines (R 2 = 0.90; RMSECV = 2 %). Additionally, the effect of the transfection process on the HEK cell biochemical and metabolic features could be evaluated directly from the FT-IR spectra. Due to the high sensitivity of the technique, it was also possible to discriminate the effect of the transfection process from the transfection reagent on KEK cells, e.g., by the analysis of spectral biomarkers and biochemical and metabolic features. The present results are far beyond what any reporter gene assay or other specific probe can offer for these purposes.

Molecular and seroepidemiologic studies of Enterovirus 71 infection in the State of Pará, Brazil

In many countries, the Enterovirus 71 (EV-71) Picornaviridae family is associated to hand, foot and mouth disease in addition to acute neurological diseases while in Brazil these viruses are more closely associated to the latter group. The aim of this research was to use the first EV-71 isolate of the Northern region of Brazil in molecular and seroepidemiologic studies. Two (2.2%) out of 88 stool samples (44 cases of AFP), collected from January 1998 to December 2000 were positive for EV-71 isolation (73442/PA/99). Nucleotide sequence of the gen that codifies the VP1 protein showed that isolate 73442/PA/99 was similar to the EV-71 strains belonging to genotype B - more closely identified with EV-71 from North America. Neutralization test with 389 sera samples collected from January 1998 to November 2001, from individuals ranging from 0 to 15 years of age living in the city of Belém, State of Pará showed the following results in relation to isolate 73442/PA/99 and prototype BrCr: a total of 207 individuals (53.2%) had neutralization antibodies to both viruses, 167 (42.9%) had no antibodies and 15 showed the presence of neutralizing antibodies to one of the two viruses. Only 20.2% of the children aged 0 to 3 had neutralizing antibodies to EV-71, indicating that these children were more susceptible to the infection. Both the seroprevalence study and VP1 sequencing were important to demonstrate the spread and the molecular pattern of the EV-71 circulating in the Northern Region of Brazil.

Molecular and biological characterization of Trypanosoma cruzi strains isolated from children from Jequitinhonha Valley, State of Minas Gerais, Brazil

Introduction The biological diversity of Trypanosoma cruzi strains plays an important role in the clinical and epidemiological features of Chagas disease. Methods Eight T. cruzi strains isolated from children living in a Chagas disease vector-controlled area of Jequitinhonha Valley, State of Minas Gerais, Brazil, were genetically and biologically characterized. Results The characterizations demonstrated that all of the strains belonged to T. cruzi II, and showed high infectivity and a variable mean maximum peak of parasitemia. Six strains displayed low parasitemia, and two displayed moderate parasitemia. Later peaks of parasitemia and a predominance of intermediate and large trypomastigotes in all T. cruzi strains were observed. The mean pre-patent period was relatively short (4.2±0.25 to 13.7±3.08 days), whereas the patent period ranged from 3.3±1.08 to 34.5±3.52 days. Mortality was observed only in animals infected with strain 806 (62.5%). Histopathological analysis of the heart showed that strains 501 and 806 caused inflammation, but fibrosis was observed only in animals infected with strain 806. Conclusions The results indicate the presence of an association between the biological behavior in mice and the genetic characteristics of the parasites. The study also confirmed general data from Brazil where T. cruzi II lineage is the most prevalent in the domiciliary cycle and generally has low virulence, with some strains capable of inducing inflammatory processes and fibrosis.

Antioxidant factors, nitric oxide levels, and cellular damage in leprosy patients

Introduction The immune response caused by Mycobacterium leprae is a risk factor for the development of oxidative stress (OS) in leprosy patients. This study aimed to assess OS in leprosy patients before the use of a multidrug therapy. Methods We evaluated the nitric oxide (NO) concentration; antioxidant capacity; levels of malondialdehyde, methemoglobin and reduced glutathione; and the activity of catalase and superoxide dismutase (SOD) in leprosy patients. Results We observed lower SOD activity in these leprosy patients; however, the NO levels and antioxidant capacity were increased. Conclusions The infectious process in response to M. leprae could primarily be responsible for the OS observed in these patients.

Description of microsporidia in simulids: molecular and morphological characterization of microsporidia in the larvae of Simulium pertinax Kollar (Diptera: Simuliidae)

IntroductionMicrosporidia constitute the most common black fly pathogens, although the species' diversity, seasonal occurrence and transmission mechanisms remain poorly understood. Infections by this agent are often chronic and non-lethal, but they can cause reduced fecundity and decreased longevity. The objective of this study was to identify microsporidia infecting Simulium (Chirostilbia) pertinax (Kollar, 1832) larvae from Caraguatatuba, State of São Paulo, Brazil, by molecular and morphological characterization.MethodsLarvae were collected at a single point in a stream in a rural area of the city and were kept under artificial aeration until analysis. Polydispyrenia spp. infection was characterized by the presence of at least 32 mononuclear spores measuring 6.9 ± 1.0 × 5.0 ± 0.7µm in persistent sporophorous vesicles. Similarly, Amblyospora spp. were characterized by the presence of eight uninucleate spores measuring 4.5 × 3.5µm in sporophorous vesicles.ResultsThe molecular analysis confirmed the presence of microsporidian DNA in the 8 samples (prevalence of 0.51%). Six samples (Brazilian larvae) were related to Polydispyrenia simulii and Caudospora palustris reference sequences but in separate clusters. One sample was clustered with Amblyospora spp. Edhazardia aedis was the positive control taxon.ConclusionsSamples identified as Polydispyrenia spp. and Amblyospora spp. were grouped with P. simulii and Amblyospora spp., respectively, corroborating previous results. However, the 16S gene tree showed a considerable distance between the black fly-infecting Amblyospora spp. and the mosquito-infecting spp. This distance suggests that these two groups are not congeneric. Additional genomic region evaluation is necessary to obtain a coherent phylogeny for this group.

Indicators of inflammation and cellular damage in chronic asymptomatic or oligosymptomatic alcoholics: correlation with alteration of bilirubin and hepatic and pancreatic enzymes

Biochemical and hematimetric indicators of inflammation and cell damage were correlated with bilirubin and hepatic and pancreatic enzymes in 30 chronic male alcoholics admitted into psychiatric hospital for detoxification and treatment of alcoholism. Aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, alkaline phosphatase, and total bilirubin were altered, respectively, in 90%, 63%, 87%, 23% and 23% of the cases. None of the indicators of inflammation (lactic dehydrogenase, altered in 16% of the cases; alpha-1 globulin, 24%; alpha-2 globulin, 88%; leucocyte counts, 28%) was correlated with alterations of bilirubin or liver enzymes. Lactic dehydrogenase was poorly sensitive for detection of hepatocytic or muscular damage. Alterations of alpha-globulins seemed to have been due more to alcohol metabolism-induced increase of lipoproteins than to inflammation. Among indicators of cell damage, serum iron, increased in 40% of the cases, seemed to be related to liver damage while creatine phosphokinase, increased in 84% of the cases, related to muscle damage. Hyperamylasemia was found in 20% of the cases and significantly correlated with levels of bilirubin, alkaline phosphatase and gamma-glutamyltransferase. It was indicated that injuries of liver, pancreas, salivary glands, and muscle occurred in asymptomatic or oligosymptomatic chronic alcoholics.

Exploring female mice interstrain differences relevant for models of depression

Depression is an extremely heterogeneous disorder. Diverse molecular mechanisms have been suggested to underlie its etiology. To understand the molecular mechanisms responsible for this complex disorder, researchers have been using animal models extensively, namely mice from various genetic backgrounds and harboring distinct genetic modifications. The use of numerous mouse models has contributed to enrich our knowledge on depression. However, accumulating data also revealed that the intrinsic characteristics of each mouse strain might influence the experimental outcomes, which may justify some conflicting evidence reported in the literature. To further understand the impact of the genetic background, we performed a multimodal comparative study encompassing the most relevant parameters commonly addressed in depression, in three of the most widely used mouse strains: Balb/c, C57BL/6, and CD-1. Moreover, female mice were selected for this study taken into account the higher prevalence of depression in women and the fewer animal studies using this gender. Our results show that Balb/c mice have a more pronounced anxious-like behavior than CD-1 and C57BL/6 mice, whereas C57BL/6 animals present the strongest depressive-like trait. Furthermore, C57BL/6 mice display the highest rate of proliferating cells and brain-derived neurotrophic factor (Bdnf) expression levels in the hippocampus, while hippocampal dentate granular neurons of Balb/c mice show smaller dendritic lengths and fewer ramifications. Of notice, the expression levels of inducible nitric oxide synthase (iNos) predict 39.5% of the depressive-like behavior index, which suggests a key role of hippocampal iNOS in depression. Overall, this study reveals important interstrain differences in several behavioral dimensions and molecular and cellular parameters that should be considered when preparing and analyzing experiments addressing depression using mouse models. It further contributes to the literature by revealing the predictive value of hippocampal iNos expression levels in depressive-like behavior, irrespectively of the mouse strain.

The influence of kaolin application on key metabolic pathways associated with grape berry quality: a molecular and biochemical analysis

Dissertação de mestrado em Biologia Molecular, Biotecnologia e Bioempreendedorismo em Plantas

Aspectos bioquímicos y celulares de la reproducción de los vectores de la enfermedad de Chagas: regulación e importancia fisiológica. Biochemical and cellular aspects of the reproduction of Chagas’ disease vectors: regulation and physiologycal impact

En la hipótesis de trabajo del presente proyecto se considera la importancia del metabolismo de lípidos y proteínas en los insectos hematófagos, en particular en los vectores de la enfermedad de Chagas, para afrontar exitosamente la demanda energética de la reproducción. Las hembras de estas especies pueden ingerir una comida de sangre abundante en lípidos y proteínas, los que son modificados en el intestino para su utilización y posterior almacenamiento en estructuras organizadas en el tejido ovárico, sustentando así el rápido crecimiento de los ovocitos. Estos aspectos resultan críticos para el ciclo de vida del insecto y para el mantenimiento de la cadena epidemiológica de la enfermedad. En estas especies, recientemente hemos caracterizado a nivel bioquímico y celular la interacción entre lipoproteínas y tejidos [Fruttero y col., Insect Biochem. Mol. Biol. 39: 322-331 (2009); Fruttero y col. Biocel 33 (3): 260 (2009)] y las fases del ciclo reproductivo [Aguirre y col., J. Insect Physiol. 54: 393-402 (2008)]. No obstante, los factores que participan en su regulación son aún escasamente conocidos. En este contexto, el estudio propone emplear dos especies de triatominos con el objeto de: (1) caracterizar los factores involucrados en la formación y regulación de reservas nutricionales en los ovocitos; (2) analizar los eventos que participan en la regresión del tejido ovárico: atresia folicular y mecanismos de muerte celular. (3) evaluar el impacto de productos naturales (ureasas vegetales y péptidos derivados) en el desarrollo del tejido ovárico. Para la ejecución de los objetivos se llevarán a cabo ensayos in vivo e in vitro con trazadores fluorescentes, fraccionamiento subcelular, estudios de expresión de proteínas (mRNA y proteína), estudios histo-morfológicos, ultraestructurales e inmunocitoquímicos, microscopía láser confocalizada, ensayos de actividad enzimática, ELISA, western-blot, electroforesis bidimensional, espectrometria de masas en tándem, etc. También se evaluarán los mecanismos de muerte celular (apoptosis/autofagia) mediante microscopía electrónica, detección de apoptosis in situ (TUNEL), inmunofluorescencia, etc. Los resultados obtenidos permitirán un mejor conocimiento sobre la fisiología y bioquímica de estos vectores, los que resultan indispensables en el diseño de nuevas estrategias para su control. Debido a la carencia de un tratamiento específico para la enfermedad y a la falta de métodos preventivos (vacuna), el control del vector es una de las vías más importantes para reducir la incidencia de la enfermedad. Actualmente, la situación socio-económica que sufren amplios núcleos de nuestra población propicia condiciones de vida que facilitan la reproducción de los vectores y la transmisión vectorial del parásito. El estudio permitirá además explorar aspectos bioquímicos y celulares básicos, generando conocimientos que podrían ser extensivos a otros insectos de importancia económica en la ganadería y/o agricultura. The aim of this project is to analyze the biochemical and cellular events involved in the lipid and protein metabolism in Chagas' disease vectors, and to evaluate their impact on the physiology of reproduction, particularly in the formation of nutritional resources in developing oocytes. At present, little is known about these critical aspects for the life cycle of the insect and for the epidemiology of the disease. The experimental approaches, which will be carried out using two species of triatomines, were designed: (1) to characterize factors involved in the formation and regulation of nutritional resources in developing oocytes; (2) to analyze the biochemical and cellular events that play a role during the regression of ovarian tissue, including the processes of oocyte resorption and programmed cell death. (3) to evaluate the impact of natural products (ureases from jackbean and related peptides) in the development of ovarian tissue. Methods and techniques involved in the project are: in vivo and in vitro assays with fluorescent tracers, ELISA, chemical assays, enzyme activities, western-blot; protein expression (mRNA), histological techniques, immunohistochemical and ultrastructural studies. Cell death will be analyzed by detection of apoptosis in situ (TUNEL), immunofluorescence (for autophagy), among others. The results obtained from the study will offer the opportunity to explore important aspects in the biology and physiology of Chagas' disease vectors that could be of potential utility in designing alternative strategies for the control of the insect.

Postnatal maternal separation affects hippocampal longterm potentiation in adult stressed rats : molecular and hormonal mechanisms

Magdeburg, Univ., Fak. für Naturwiss., Diss., 2013