The genome sequence of the gram-positive sugarcane pathogen Leifsonia xyli subsp. xyli

The genome sequence of Leifsonia xyli subsp. xyli, which causes ratoon stunting disease and affects sugarcane worldwide, was determined. The single circular chromosome of Leifsonia xyli subsp. xyli CTCB07 was 2.6 Mb in length with a GC content of 68% and 2,044 predicted open reading frames. The analysis also revealed 307 predicted pseudogenes, which is more than any bacterial plant pathogen sequenced to date. Many of these pseudogenes, if functional, would likely be involved in the degradation of plant heteropolysaccharides, uptake of free sugars, and synthesis of amino acids. Although L. xyli subsp. xyli has only been identified colonizing the xylem vessels of sugarcane, the numbers of predicted regulatory genes and sugar transporters are similar to those in free-living organisms. Some of the predicted pathogenicity genes appear to have been acquired by lateral transfer and include genes for cellulase, pectinase, wilt-inducing protein, lysozyme, and desaturase. The presence of the latter may contribute to stunting, since it is likely involved in the synthesis of abscisic acid, a hormone that arrests growth. Our findings are consistent with the nutritionally fastidious behavior exhibited by L. xyli subsp. xyli and suggest an ongoing adaptation to the restricted ecological niche it inhabits.     

Role of Cytoskeleton Proteins in the Morphological Changes During Apoptotic Cell Death of Cerebellar Granule Neurons

Cytoskeleton proteins are substrates for proteases and further apoptotic death. We evaluated the participation of cytoskeleton in morphological changes during cell death induced by two apoptotic conditions, potassium deprivation (K5) and staurosporine, in cerebellar granule neurons (CGC). We found that K5 induced somatic damage, but neurites were relatively preserved, which corresponded to the reorganization of actin and α-tubulin in neurites. Staurosporine (STS) induced an early alteration of neurites with reorganization of cytoskeleton proteins in somas. Caspase inhibitor ZVAD totally inhibited STS-induced α-tubulin reorganization and partially blocked STS-induced actin reorganization. α-tubulin and actin reorganization induced by K5 was affected by ZVAD. Calpain inhibitor (IC1) did not affect α-tubulin or actin reorganization induced by STS, K5 or ionomycin. Neither ZVAD, nor IC1 changed α-tubulin or actin levels upon K5 treatment. STS increased α-tubulin and actin levels, but neither ZVAD nor IC1 changed α-tubulin levels upon STS treatment. In contrast, ZVAD reduced the STS-induced increase of actin. These results suggest that CGC cytoskeleton proteins undergo a differential expression and reorganization depending on the apoptotic condition.     

Dispersion patterns of Diachasmimorpha longicaudata (Hymenoptera: Braconidae) in citrus orchards in southeast Brazil

We studied the dispersion patterns of the exotic endoparasitoid, Diachasmimorpha longicaudata (Ahsmed) (Hymenoptera: Braconidae), in 1999 (summer) and in 2000 (winter) in a citrus orchard in southeast Brazil. Different population densities of D. longicaudata were released in the centre of the orchard, and their dispersion was determined by using yellow, sticky, rectangular traps, placed in various distances and heights around the release point. Our results suggest that during summer, climatic conditions did not affect dispersion. However, in winter, dispersion rates were positively affected by temperature, and negatively by rainfall. Both estimated dispersal distance and surface were higher in summer than in winter for all release densities. Dispersion peaked at 2000 parasitoids ha^?1 in summer and 8000 parasitoids ha^?1 in winter. The importance of our results for the biological control of fruit flies by augmented or innoculative releases of D. longicaudata in southeast Brazil is discussed.     

Phytochrome in etiolated annual rye

Summary During a seven-fold increase in length the content of the coleoptile in photoreversible phytochrome increased four-fold and that of the primary leaf nine-fold. The phytochrome content, during growth, expressed on a fresh- or dry-weight basis did not vary greatly for either organ. Phytochrome per mg dry weight (OD⁷³⁰/mg=0.5) was nearly the same in the leaf as in the coleoptile. Coleoptiles studied had a constant DNA content of 4.1 g per organ. DNA content of the leaf increased with age. Phytochrome per DNA was much higher in the coleoptile than in the primary leaf and increased with growth in each of these organs. Thus, there was not a constant amount of phytochrome per cell in either tissue with increasing age and there was not the same amount of phytochrome per cell in the coleoptile as in the primary leaf at any age.This work was supported in part by U.S. Atomic Energy Commission Contract No. AT (30-I)2373.     

Comparative Study and Mutational Analysis of Distinctive Structural Elements of Hyperthermophilic Enzymes

Comparison of the three-dimensional structure of hyperthermophilic and mesophilic β-glycosidases shows differences in secondary structure composition. The enzymes from hyperthermophilic archaea have a significantly larger number of β-strands arranged in supernumerary β-sheets compared to mesophilic enzymes from bacteria and other organisms. Amino acid replacements designed to alter the structure of the supernumerary β-strands were introduced by site directed mutagenesis into the sequence encoding the β-glycosidase from Sulfolobus solfataricus. Most of the replacements caused almost complete loss of activity but some yielded enzyme variants whose activities were affected specifically at higher temperatures. Far-UV CD spectra recorded as a function of temperature for both wild type β-glycosidase and mutant V349G, one of the mutants with reduced activity at higher temperatures, were similar, showing that the protein structure of the mutant was stable at the highest temperatures assayed. The properties of mutant V349G show a difference between thermostability (stability of the protein structure at high temperatures) and thermophilicity (optimal activity at high temperatures).     

Proteomic Profiling of Triple-negative Breast Carcinomas in Combination With a Three-tier Orthogonal Technology Approach Identifies Mage-A4 as Potential Therapeutic Target in Estrogen Receptor Negative Breast Cancer

Breast cancer is a very heterogeneous disease, encompassing several intrinsic subtypes with various morphological and molecular features, natural history and response to therapy. Currently, molecular targeted therapies are available for estrogen receptor (ER)⁻ and human epidermal growth factor receptor 2 (Her2)-positive breast tumors. However, a significant proportion of primary breast cancers are negative for ER, progesterone receptor (PgR), and Her2, comprising the triple negative breast cancer (TNBC) group. Women with TNBC have a poor prognosis because of the aggressive nature of these tumors and current lack of suitable targeted therapies. As a consequence, the identification of novel relevant protein targets for this group of patients is of great importance. Using a systematic two dimensional (2D) gel-based proteomic profiling strategy, applied to the analysis of fresh TNBC tissue biopsies, in combination with a three-tier orthogonal technology (two dimensional PAGE/silver staining coupled with MS, two dimensional Western blotting, and immunohistochemistry) approach, we aimed to identify targetable protein markers that were present in a significant fraction of samples and that could define therapy-amenable sub-groups of TNBCs. We present here our results, including a large cumulative database of proteins based on the analysis of 78 TNBCs, and the identification and validation of one specific protein, Mage-A4, which was expressed in a significant fraction of TNBC and Her2-positive/ER negative lesions. The high level expression of Mage-A4 in the tumors studied allowed the detection of the protein in the tumor interstitial fluids as well as in sera. The existence of immunotherapeutics approaches specifically targeting this protein, or Mage-A protein family members, and the fact that we were able to detect its presence in serum suggest novel management options for TNBC and human epidermal growth factor receptor 2 positive/estrogen receptor negative patients bearing Mage-A4 positive tumors.Breast cancer, although a very heterogeneous disease, can be divided into three therapeutically relevant fundamental disease entities, simply based on estrogen receptor (ER) and human epidermal growth factor receptor 2 (Her2)¹ status (i.e. ER⁺ and/or Her2⁺, and ER⁻Her2⁻), as the major currently available breast cancer therapeutic options are based on the ability to target these proteins. Hormone receptor positive and hormone receptor negative breast cancers are disease entities with distinct morphological, genetic and biological behavior (1). Hormone receptor negative tumors, which constitute ∼30% of primary breast cancers, tend to be high-grade, more frequently BRCA1 and TP53 mutated, and, more importantly, are not amenable to endocrine therapy. Her2 is amplified in ∼18–20% of breast cancers, and is more frequently observed in hormone receptor negative tumors. Her2 amplification is associated with worse prognosis (higher rate of recurrence and mortality) in patients with newly diagnosed breast cancer who do not receive any adjuvant systemic therapy. Her2 status is also predictive for several systemic therapies, particularly for agents that target Her2. The development of a humanized monoclonal antibody against Her2 (trastuzumab) has resulted in reduction of the risk of recurrence and mortality in patients with Her2 amplification (2, 3). Although trastuzumab is considered one of the most effective targeted therapies currently available in oncology, a significant number of patients with Her2-overexpressing breast cancer do not benefit from it (4, 5).Breast tumors that do not express ER, PgR, or Her2 (ER⁻ PgR⁻ Her2⁻), as determined by immunohistochemistry (IHC), are generally referred to as triple negative breast cancers (TNBCs), and they are not candidates for targeted therapies (endocrine therapy or trastuzumab). Although TNBCs account for a relatively small proportion of breast cancer cases (10–15%), they are responsible for a disproportionate number of breast cancer deaths. TNBC tumors form a recognizable prognostic group of breast cancer with aggressive behavior that currently lacks the benefit of available systemic therapy (6–8). Given the need to develop molecular criteria to reproducibly categorize molecular breast tumor subtypes at the protein level and the lack of targeted therapies available to treat patients bearing TNBCs, we have implemented a systematic proteomics approach to identify, characterize, and evaluate proteins present in triple-negative tumors that could constitute an appropriate therapeutic target for the clinical management of this group of patients. To this end, based on the analysis of 78 individual TNBC samples, we have established a large, cumulative, 2D-PAGE database of proteins expressed by TNBCs, including some that could be of potential therapeutic value. Comparison of this TNBC protein database with protein databases of other breast cancer subtypes previously established by our laboratory allowed us to single out a number of proteins preferentially expressed in TNBCs for which targeted therapeutics exist. In this report we further focused on the characterization of one such target, the cancer/testis antigen, melanoma-associated antigen 4 - Mage-A4.Cancer/testis antigens (CTAs) are expressed in a large variety of tumor types, whereas their expression in normal tissues is restricted to male germ cells, which are immune-privileged because of their lack of or low expression of human leukocyte antigen (HLA) molecules (9). Several studies have shown the existence of natural cellular and humoral responses against some CTAs, indicating that they are appropriate targets for vaccine-based cancer immunotherapy (10–12). So far, the use of CTAs in immunotherapeutic approaches to cancer treatment has been tested in more than 60 early phase clinical trials, with varying success, and a few candidate products have reached late-stage clinical trials. One such candidate vaccine, Astuprotimut-R (GSK-249553), a Mage-A3 antigen-specific cancer immunotherapeutic agent, is currently under clinical evaluation by GlaxoSmithKline in the largest-ever treatment trial in lung cancer, called MAGRIT (Mage-A3 as Adjuvant nonsmall cell lunG canceR ImmunoTherapy) (13).At present, CTAs comprise about 150 members, more than half of which are encoded by large, recently expanded families on chromosome X (14; see also CTDatabase at www.cta.lncc.br; last accessed 01.09.2012). These genes are organized into clusters and have undergone rapid evolution, possibly because of positive selection. The biological functions of CTAs are not fully understood, but emerging evidence suggest that they direct the proliferation, differentiation, and survival of human germ line cells and may have similar effect in cancer cells. Mage-A4 protein belongs to the Mage-A family of CT antigens. The Mage-A family is composed by 12 proteins (14, 15) and many members of the Mage-A family of CTAs have been associated with cancer, including breast cancer (14, 16, 17). However, past studies reported mostly on MAGE genes rather than protein expression, or on the expression of Mage protein families and not on any given specific protein.In this paper we describe the identification of Mage-A4 in breast tumor biopsies using 2D PAGE coupled with MS proteomics, and follow the protein localization from the tumor cells, to the tumor microenvironment, and to the serum of a patient. Using a three-tier orthogonal technology approach that combined 2D PAGE silver staining coupled with MS, with 2D Western blotting, and IHC, we showed that high level Mage-A4 expression in breast tumors occurs almost exclusively in the receptor negative disease (TNBC and Her2⁺ER⁻PgR⁻). The existence of immunotherapeutic approaches targeting MAGE protein family members (Mage-A4 specific or with broader specificity) and the fact that we were able to detect its presence in serum suggest novel management options for patients bearing Mage-A4 positive TNBCs and Her2⁺ER⁻PgR⁻ tumors.     

Macrolide use in the previous years is associated with failure to eradicate Helicobacter pylori with clarithromycin‐containing regimens

Background

There is some evidence that prior use of macrolide antibiotics is a useful predictor of the likelihood of standard triple therapy failure in Helicobacter pylori eradication. In this study, we have evaluated whether previous intake of macrolides correlates with failure to eradicate H. pylori using two different first‐line clarithromycin‐containing regimens.

Materials and Methods

Retrospective study of 212 patients with H. pylori infection treated with one of two first‐line clarithromycin‐containing regimens: 108 patients treated with triple therapy for 10 days and 104 patients treated with concomitant therapy for 10 days. The intake of macrolides (clarithromycin, azithromycin, and other macrolides) prior to the eradication therapy was obtained from the electronic medical record, which contains information regarding all the medication prescribed to the patients since the year 2004.

Results

One hundred of 212 patients (47.2%) had received at least one treatment with macrolides during the years prior to the eradication therapy. H. pylori eradication rates were significantly lower in patients with previous use compared to patients without previous use of macrolides, both with triple therapy (60.8% vs 92.9%; P < .0001) and with concomitant therapy (85.7% vs 98.2%; P = .024).

Conclusions

Previous use of macrolides correlates with a low H. pylori eradication rate with triple and concomitant clarithromycin‐containing regimens. In addition, our study shows that in patients without previous use of macrolides, triple therapy achieves per‐protocol eradication rates over 90%.     

No evidence for transmission of antibiotic-resistant Escherichia coli strains from humans to wild western lowland gorillas in Lopé National Park, Gabon

The intensification of human activities within the habitats of wild animals is increasing the risk of interspecies disease transmission. This risk is particularly important for great apes, given their close phylogenetic relationship with humans. Areas of high human density or intense research and ecotourism activities expose apes to a high risk of disease spillover from humans. Is this risk lower in areas of low human density? We determined the prevalence of Escherichia coli antibiotic-resistant isolates in a population of the critically endangered western lowland gorilla (Gorilla gorilla gorilla) and other wild mammals in Lopé National Park (LNP), Gabon, and we tested whether the observed pattern could be explained by bacterial transmission from humans and domestic animals into wildlife populations. Our results show a high prevalence of antibiotic-resistant bacterial isolates in humans and low levels in gorillas and other wildlife. The significant differences in the genetic background of the resistant bacteria isolated from humans and gorillas suggest that transmission is low or does not occur between these two species. These findings indicate that the presence of antibiotic-resistant strains in wildlife do not imply direct bacteria transmission from humans. Thus, in areas of low human density, human-wildlife E. coli transmission seems to be low. The presence of antibiotic-resistant isolates in gorillas may be better explained by other mechanisms for resistance acquisition, such as horizontal gene exchange among bacteria or naturally acquired resistance.