Nonrecurrent MECP2 duplications mediated by genomic architecture-driven DNA breaks and break-induced replication repair

Recurrent submicroscopic genomic copy number changes are the result of nonallelic homologous recombination (NAHR). Nonrecurrent aberrations, however, can result from different nonexclusive recombination-repair mechanisms. We previously described small microduplications at Xq28 containing MECP2 in four male patients with a severe neurological phenotype. Here, we report on the fine-mapping and breakpoint analysis of 16 unique microduplications. The size of the overlapping copy number changes varies between 0.3 and 2.3 Mb, and FISH analysis on three patients demonstrated a tandem orientation. Although eight of the 32 breakpoint regions coincide with low-copy repeats, none of the duplications are the result of NAHR. Bioinformatics analysis of the breakpoint regions demonstrated a 2.5-fold higher frequency of Alu interspersed repeats as compared with control regions, as well as a very high GC content (53%). Unexpectedly, we obtained the junction in only one patient by long-range PCR, which revealed nonhomologous end joining as the mechanism. Breakpoint analysis in two other patients by inverse PCR and subsequent array comparative genomic hybridization analysis demonstrated the presence of a second duplicated region more telomeric at Xq28, of which one copy was inserted in between the duplicated MECP2 regions. These data suggest a two-step mechanism in which part of Xq28 is first inserted near the MECP2 locus, followed by breakage-induced replication with strand invasion of the normal sister chromatid. Our results indicate that the mechanism by which copy number changes occur in regions with a complex genomic architecture can yield complex rearrangements.

Evaluation of Quantitative RT-PCR Using Nonamplified and Amplified RNA

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a standard assay in molecular medicine for gene expression analysis. Samples from incisional/needle biopsies, laser-microdissected tumor cells and other biologic sources, normally available in clinical cancer studies, generate very small amounts of RNA that are restrictive for expression analysis. As a consequence, an RNA amplification procedure is required to assess the gene expression levels of such sample types. The reproducibility and accuracy of relative gene expression data produced by sensitive methodology as qRT-PCR when cDNA converted from amplified (A) RNA is used as template has not yet been properly addressed. In this study, to properly evaluate this issue, we performed 1 round of linear RNA amplification in 2 breast cell lines (C5.2 and HB4a) and assessed the relative expression of 34 genes using cDNA converted from both nonamplified (NA) and A RNA. Relative gene expression was obtained from beta actin or glyceraldehyde 3-phosphate dehydrogenase normalized data using different dilutions of cDNA, wherein the variability and fold-change differences in the expression of the 2 methods were compared. Our data showed that 1 round of linear RNA amplification, even with suboptimal-quality RNA, is appropriate to generate reproducible and high-fidelity qRT-PCR relative expression data that have similar confidence levels as those from NA samples. The use of cDNA that is converted from both A and NA RNA in a single qRT-PCR experiment clearly creates bias in relative gene expression data.

Occurrence and characterization of injuries caused by Mycotretus apicalis Lacordaire, 1842 (Coleoptera: Erotylidae) on cultivation of Pleurotus sajor-caju

When carrying out experiments on the production of the edible mushroom Pleurotus sajor-caju in the Laboratory of Edible Mushrooms, Universidade Federal de Lavras, Lavras, Brazil, in the second half of 2007, the presence of beetles later identified as belonging to the species Mycotretus apicalis was verified. This is the first recorded instance of this insect in cultures of P. sajor-caju in Brazil. The larvae and adults of this insect feed on the fruiting bodies of commercial harvests, resulting in reduction in mushroom quality. To provide evaluation of the injuries caused by these insects, substrates colonized by P. sajor-caju were infested with 4, 8, 16, 32 and 64 insects per block of substrate being the qualitative and quantitative losses then noted. Despite the lack of an observed decrease in biological efficiency, the injuries caused by these insects affected the commercial quality of the mushrooms, which may result in economic losses. The results showed that infestations of 32 insects per 0.8 kg of substrate led to a depreciation in the prices of mushrooms meant to be sold.

Long-Term Culture of Mouse Embryonic Stem Cell-Derived Adherent Neurospheres and Functional Neurons

Innumerous protocols, using the mouse embryonic stem (ES) cells as model for in vitro study of neurons functional properties and features, have been developed. Most of these protocols are short lasting, which, therefore, does not allow a careful analysis of the neurons maturation, aging, and death processes. We describe here a novel and efficient long-lasting protocol for in vitro ES cells differentiation into neuronal cells. It consists of obtaining embryoid bodies, followed by induction of neuronal differentiation with retinoic acid of nonadherent embryoid bodies (three-dimensional model), which further allows their adherence and formation of adherent neurospheres (AN, bi-dimensional model). The AN can be maintained for at least 12 weeks in culture under repetitive mechanical splitting, providing a constant microenvironment (in vitro niche) for the neuronal progenitor cells avoiding mechanical dissociation of AN. The expression of neuron-specific proteins, such as nestin, sox1, beta III-tubulin, microtubule-associated protein 2, neurofilament medium protein, Tau, neuronal nuclei marker, gamma-aminobutyric acid, and 5-hydroxytryptamine, were confirmed in these cells maintained during 3 months under several splitting. Additionally, expression pattern of microtubule-associated proteins, such as lissencephaly (Lis1) and nuclear distribution element-like (Ndel1), which were shown to be essential for differentiation and migration of neurons during embryogenesis, was also studied. As expected, both proteins were expressed in undifferentiated ES cells, AN, and nonrosette neurons, although presenting different spatial distribution in AN. In contrast to previous studies, using cultured neuronal cells derived from embryonic and adult tissues, only Ndel1 expression was observed in the centrosome region of early neuroblasts from AN. Mature neurons, obtained from ES cells in this work, display ionic channels and oscillations of membrane electrical potential typical of electrically excitable cells, which is a characteristic feature of the functional central nervous system (CNS) neurons. Taken together, our study demonstrated that AN are a long-term culture of neuronal cells that can be used to analyze the process of neuronal differentiation dynamics. Thus, the protocol described here provides a new experimental model for studying neurological diseases associated with neuronal differentiation during early development, as well as it represents a novel source of functional cells that can be used as tools for testing the effects of toxins and/or drugs on neuronal cells.

Comparison of signal-to-cutoff values in first, second, and third generation enzyme immunoassays for the diagnosis of HTLV-1/2 infection in ""at-risk"" individuals from Sao Paulo, Brazil

Data obtained during routine diagnosis of human T-cell lymphotropic virus type 1 (HTLV-1) and 2 (HTLV-2) in ""at-risk"" individuals from Sao Paulo, Brazil using signal-to-cutoff (S/C) values obtained by first, second, and third generation enzyme immunoassay (EIA) kits, were compared. The highest S/C values were obtained with third generation EIA kits, but no correlation was detected between these values and specific antibody reactivity to HTLV-1, HTLV-2, or untyped HTLV (p = 0.302). In addition, use of these third generation kits resulted in HTLV-1/2 false-positive samples. In contrast, first and second generation EIA kits showed high specificity, and the second generation EIA kits showed the highest efficiency, despite lower S/C values. Using first and second generation EIA kits, significant differences in specific antibody detection of HTLV-1, relative to HTLV-2 (p = 0.019 for first generation and p < 0.001 for second generation EIA kits) and relative to untyped HTLV (p = 0.025 for first generation EIA kits), were observed. These results were explained by the composition and format of the assays. In addition, using receiver operating characteristics (ROC) analysis, a slight adjustment in cutoff values for third generation EIA kits improved their specificities and should be used when HTLV ""at-risk"" populations from this geographic area are to be evaluated. (C) 2009 Elsevier B.V. All rights reserved.

Mesenchymal Stem Cells Attenuate Renal Fibrosis Through Immune Modulation and Remodeling Properties in a Rat Remnant Kidney Model

Mesenchymal stem cells (MSCs) have regenerative properties in acute kidney injury, but their role in chronic kidney diseases is still unknown. More specifically, it is not known whether MSCs halt fibrosis. The purpose of this work was to investigate the role of MSCs in fibrogenesis using a model of chronic renal failure. MSCs were obtained from the tibias and femurs of male Wistar-EPM rats. Female Wistar rats were subjected to the remnant model, and 2 vertical bar x vertical bar 10(5) MSCs were intravenously administrated to each rat every other week for 8 weeks or only once and followed for 12 weeks. SRY gene expression was observed in female rats treated with male MSCs, and immune localization of CD73(+)CD90(+) cells at 8 weeks was also assessed. Serum and urine analyses showed an amelioration of functional parameters in MSC-treated animals at 8 weeks, but not at 12 weeks. Masson`s trichrome and Sirius red staining demonstrated reduced levels of fibrosis in MSC-treated animals. These results were corroborated by reduced vimentin, type I collagen, transforming growth factor beta, fibroblast specific protein 1 (FSP-1), monocyte chemoattractant protein 1, and Smad3 mRNA expression and alpha smooth muscle actin and FSP-1 protein expression. Renal interleukin (IL)-6 and tumor necrosis factor alpha mRNA expression levels were significantly decreased after MSC treatment, whereas IL-4 and IL-10 expression levels were increased. All serum cytokine expression levels were decreased in MSC-treated animals. Taken together, these results suggested that MSC therapy can indeed modulate the inflammatory response that follows the initial phase of a chronic renal injury. The immunosuppressive and remodeling properties of MSCs may be involved in the decreased fibrosis in the kidney. STEM CELLS 2009;27:3063-3073

Expression and purification of human respiratory syncytial virus recombinant fusion protein

The Human Respiratory Syncytial Virus (HRSV) fusion protein (F) was expressed in Escherichia call BL21A using the pET28a vector at 37 degrees C. The protein was purified from the soluble fraction using affinity resin. The structural quality of the recombinant fusion protein and the estimation of its secondary structure were obtained by circular dichroism. Structural models of the fusion protein presented 46% of the helices in agreement with the spectra by circular dichroism analysis. There are only few studies that succeeded in expressing the HRSV fusion protein in bacteria. This is a report on human fusion protein expression in E. call and structure analysis, representing a step forward in the development of fusion protein F inhibitors and the production of antibodies. (c) 2008 Elsevier Inc. All rights reserved.

Analysis of the genome of Spodoptera frugiperda nucleopolyhedrovirus (SfMNPV-19) and of the high genomic heterogeneity in group II nucleopolyhedroviruses

The genome of the most virulent among 22 Brazilian geographical isolates of Spodoptera frugiperda nucleopolyhedrovirus, isolate 19 (SfMNPV-1 9), was completely sequenced and shown to comprise 132 565 bp and 141 open reading frames (ORFs). A total of 11 ORFs with no homology to genes in the GenBank database were found. Of those, four had typical baculovirus; promoter motifs and polyadenylation sites. Computer-simulated restriction enzyme cleavage patterns of SfMNPV-1 9 were compared with published physical maps of other SfMNPV isolates. Differences were observed in terms of the restriction profiles and genome size. Comparison of SfMNPV-1 9 with the sequence of the SfMNPV isolate 3AP2 indicated that they differed due to a 1427 bp deletion, as well as by a series of smaller deletions and point mutations. The majority of genes of SfMNPV-1 9 were conserved in the closely related Spodoptera exigua NPV (SeMNPV) and Agrotis segetum NPV (AgseMNPV-A), but a few regions experienced major changes and rearrangements. Synthenic maps for the genomes of group 11 NPVs revealed that gene collinearity was observed only within certain clusters. Analysis of the dynamics of gene gain and loss along the phylogenetic tree of the NPVs showed that group 11 had only five defining genes and supported the hypothesis that these viruses form ten highly divergent ancient lineages. Crucially, more than 60% of the gene gain events followed a power-law relation to genetic distance among baculoviruses, indicative of temporal organization in the gene accretion process.

Occurrence of herpes simplex virus 1 and three periodontal bacteria in patients with chronic periodontitis and necrotic pulp

Viral and bacterial associations appear to be implicated in the development of periodontal infections. Little information is available describing the periodontopathic agents in root canals with necrotic pulp. In this study, the occurrence and the combinations among herpes simplex virus type 1 (HSV-1) and Dialister pneumosintes, Tannerella forsythia.. and Treponema denticola in patients with chronic periodontitis and necrotic pulp were evaluated. Clinical samples from healthy subjects and patients with periodontal or pulp infections were analyzed using a nested polymerase chain reaction PCR to detect HSV and PCR to detect the 3 periodontal bacteria. The presence of Tannerella forsythia and Treponema denticola was observed in healthy, periodontitis, and necrotic pulp patients. HSV was observed in periodontitis and necrotic pulp patients, and no healthy subject harbored D. pneumosintes or HSV. The occurrence of Tannerella forsythia was not statistically significant in patients with necrotic pulp (P = 0.704). Periodontal bacteria were observed varying from 10.3% to 20.7% in periodontitis and necrotic pulp patients. The presence of Treponema denticola - HSV association was predominant in patients showing necrotic pulp (24.1%); however, HSV alone was observed in one patient with periodontitis and in another patient with necrotic pulp. The presence of double association among bacteria or bacteria - HSV could indicate a role in both periodontitis and necrotic pulp, and Tannerella forsythia - Treponenta denticola - HSV and Tannerella forsythia - D. pneumosintes - Treponema denticola - HSV associations might be important in periodontitis.

Accelerated testing of mold growth on traditional and recycled book paper

The growth of molds on paper containing cellulose is a frequent occurrence when the level of relative air humidity is high or when books become wet due to water leaks in libraries. The aim of this study is to differentiate the bioreceptivity of different types of book paper for different fungi. Laboratory tests were performed with strains of Aspergillus niger, Cladosporium sp., Chaetomium globosum and Trichoderma harzianum isolated from books. Four paper types were evaluated: couche Men (offset), recycled and a reference paper containing only cellulose. The tests were carried out in chambers with relative air humidity of 95% and 100%. Mold growth was greatest in the tests at 100% relative humidity. Results of stereoscopic microscopy observation showed that Cladosporium sp. grew in 74% of these samples, A. niger in 75%, T. harzianum in 72% and C. globosum in 60%. In the chambers with 95% air humidity Cladosporium sp. grew in only 9% of the samples, A. niger in 1%, T harzianum in 3% and C globosum did not grow in any sample. The most bioreceptive paper was couche and the least receptive was recycled paper. The composition of the recycled paper, however, varies depending on the types of waste materials used to make it. (C) 2011 Elsevier Ltd. All rights reserved.

A Metal-Repressed Promoter From Gram-Positive Bacillus subtilis Is Highly Active and Metal-Induced in Gram-Negative Cupriavidus metallidurans

A synthetic version of the metal-regulated gene A (mrgA) promoter from Bacillus subtilis, which in this Gram-positive bacterium is negatively regulated by manganese, iron, cobalt, or copper turned out to promote high level of basal gene expression that is further enhanced by Co(II), Cd(II), Mn(II), Zn(II), Cu(II), or Ni(II), when cloned in the Gram-negative bacterium Cupriavidus metallidurans. Promoter activity was monitored by expression of the reporter gene coding for the enhanced green fluorescent protein (EGFP), and cellular intensity fluorescence was quantified by flow cytometry. Expression levels in C. metallidurans driven by the heterologous promoter, here called pan, ranged from 20- to 53-fold the expression level driven by the Escherichia coli lac promoter (which is constitutively expressed in C. metallidurans), whether in the absence or presence of metal ions, respectively. The pan promoter did also function in E. coli in a constitutive pattern, regardless of the presence of Mn(II) or Fe(II). In conclusion, the pan promoter proved to be a powerful tool to express heterologous proteins in Gram-negative bacteria, especially in C. metallidurans grown upon high levels of toxic metals, with potential applications in bioremediation. Biotechnol. Bioeng. 2010; 107: 469-477. (C) 2010 Wiley Periodicals, Inc.

Refolded dengue virus type 2 NS1 protein expressed in Escherichia coli preserves structural and immunological properties of the native protein

The dengue virus NS1 protein has been shown to be a protective antigen under different experimental conditions but the recombinant protein produced in bacterial expression systems is usually not soluble and loses structural and immunological features of the native viral protein In the present study, experimental conditions leading to purification and refolding of the recombinant dengue virus type 2 (DENV-2) NS1 protein expressed in Escherichia coil are described The refolded recombinant protein was recovered as heat-stable soluble dimers with preserved structural features, as demonstrated by spectroscopic methods In addition, antibodies against epitopes of the NS1 protein expressed in eukaryotic cells recognized the refolded protein expressed in E coli but not the denatured form or the same protein submitted to a different refolding condition Collectively, the results demonstrate that the recombinant NS1 protein preserved important conformation and antigenic determinants of the native virus protein and represents a valuable reagent either for the development of vaccines or for diagnostic methods. (C) 2010 Elsevier B V All rights reserved

Evaluation of Fungal Burden and Aflatoxin Presence in Packed Medicinal Plants Treated by Gamma Radiation

This study was developed to evaluate the fungal burden, toxigenic molds, and mycotoxin contamination and to verify the effects of gamma radiation in four kinds of medicinal plants stored before and after 30 days of irradiation treatment. Eighty samples of medicinal plants (Peumus boldus, Camellia sinensis, Maytenus ilicifolia. and Cassia angustifolia) purchased from drugstores, wholesale, and open-air markets in Sao Paulo city, Brazil, were analyzed. The samples were treated using a (60)Co gamma ray source (Gammacell) with doses of 5 and 10 kGy. Nonirradiated samples were used as controls of fungal isolates. For enumeration of fungi on medicinal plants, serial dilutions of the samples were plated in duplicate onto dichloran 18% glycerol agar. The control samples revealed a high burden of molds, including toxigenic fungi. The process of gamma radiation was effective in reducing the number of CFU per gram in all irradiated samples of medicinal plants after 30 days of storage, using a dose of 10 kGy and maintaining samples in a protective package. No aflatoxins were detected. Gamma radiation treatment can be used as an effective method for preventing fungal deterioration of medicinal plants subject to long-term storage.

Identification of enteropathogenic and enterohaemorrhagic Escherichia coli strains by immunoserological detection of intimin

Aims: To evaluate the sensitivity and specificity of polyclonal and monoclonal antibodies (Mabs) against intimin in the detection of enteropathogenic and enterohaemorrhagic Escherichia coli isolates using immunoblotting. Methods and Results: Polyclonal and Mabs against the intimin-conserved region were raised, and their reactivities were compared in enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) isolates using immunoblotting analysis. In comparison with rat antiserum, rabbit anti-intimin IgG-enriched fraction had a stronger recognition pattern to a wide spectrum of intimin types in different EPEC and EHEC serotypes. On the other hand, murine monoclonal IgG2b specific to intimin, with dissociation constant of 1 center dot 3 x 10-8 mol l-1, failed in the detection of some of these isolates. Conclusion: All employed antibodies showed 100% specificity, not reacting with any of the eae-negative isolates. The sensitivity range was according to the employed antisera, and 97% for rabbit anti-intimin IgG-enriched fraction, followed by 92% and 78% sensitivity with rat antisera and Mab. Significance and Impact of the Study: The rabbit anti-intimin IgG-enriched fraction in immunoblotting analysis is a useful tool for EPEC and EHEC diagnoses.

Relation of genetic phylogeny and geographical distance of tick-borne encephalitis virus in central Europe

Tick-borne encephalitis virus (TBEV) is the most important arboviral agent causing disease of the central nervous system in central Europe. In this study, 61 TBEV E gene sequences derived from 48 isolates from the Czech Republic, and four isolates and nine TBEV strains detected in ticks from Germany, covering more than half a century from 1954 to 2009, were sequenced and subjected to phylogenetic and Bayesian phylodynamic analysis to determine the phylogeography of TBEV in central Europe. The general Eurasian continental east-to-west pattern of the spread of TBEV was confirmed at the regional level but is interlaced with spreading that arises because of local geography and anthropogenic influence. This spread is reflected by the disease pattern in the Czech Republic that has been observed since 1991. The overall evolutionary rate was estimated to be approximately 8x10(-4) substitutions per nucleotide per year. The analysis of the TBEV E genes of 11 strains isolated at one natural focus in Zd`ar Kaplice proved for the first time that TBEV is indeed subject to local evolution.