Alzheimer's β-Amyloid Peptide 1–42 Induces a Phagocytic Response in Murine Microglia

Abstract: β-Amyloid (Aβ) peptides are a key component of the senile plaques that characterize Alzheimer's disease. Cytokine-producing microglia have been shown to be intimately associated with amyloid deposits and have also been implicated as scavengers responsible for clearing Aβ deposits. However, little is known about the initial activation of these microglia or the effect of Aβ on phagocytosis. Murine BV-2 microglia were used to assess the effect of synthetic Aβ 1–42 on phagocytosis by quantifying uptake of fluorescent microspheres, acetylated low-density lipoproteins, and zymosan particles by flow cytometry. Aβ 1–42 stimulated microglial phagocytosis in a time- and dose-dependent manner. Aβ fibrils produced the greatest potentiation, and once activated, phagocytosis remained elevated after removal of Aβ from the cultures. Aβ-stimulated phagocytosis could be blocked if proteoglycans were first complexed to Aβ fibrils. These data suggest that Aβ fibrils act as an immune signal to stimulate microglial phagocytosis and that extracellular matrix molecules may modify Aβ function.     

Genomic Structure of the Human Gene for Spinocerebellar Ataxia Type 2 (SCA2) on Chromosome 12q24.1

Spinocerebellar ataxia type 2 (SCA2) is a member of a group of neurodegenerative diseases that are caused by instability of a DNA CAG repeat. We report the genomic structure of theSCA2gene. Its 25 exons, encompassing approximately 130 kb of genomic DNA, were mapped onto the physical map of the region. Exonic sizes varied from 37 to 890 bp, and intronic sizes ranged from 323 bp to more than 15 kb. The CAG repeat was contained in the 5′ coding region of the gene in exon 1. Determination of the splice junction sequences indicated the presence of only one deviation from the GT-AG rule at the donor splice site of intron 9, which contained a GC instead of a GT dinucleotide. Exon 10, immediately downstream from this rare splice donor site, was alternatively spliced. Alternative splicing does not affect the reading frame and is predicted to encode an isoform containing 70 amino acids less.     

Regulation of Alternative Polyadenylation by U1 snRNPs and SRp20

Although considerable information is currently available about the factors involved in constitutive vertebrate polyadenylation, the factors and mechanisms involved in facilitating communication between polyadenylation and splicing are largely unknown. Even less is known about the regulation of polyadenylation in genes in which 3′-terminal exons are alternatively recognized. Here we demonstrate that an SR protein, SRp20, affects recognition of an alternative 3′-terminal exon via an effect on the efficiency of binding of a polyadenylation factor to an alternative polyadenylation site. The gene under study codes for the peptides calcitonin and calcitonin gene-related peptide. Its pre-mRNA is alternatively processed by the tissue-specific inclusion or exclusion of an embedded 3′-terminal exon, exon 4, via factors binding to an intronic enhancer element that contains both 3′ and 5′ splice site consensus sequence elements. In cell types that preferentially exclude exon 4, addition of wild-type SRp20 enhances exon 4 inclusion via recognition of the intronic enhancer. In contrast, in cell types that preferentially include exon 4, addition of a mutant form of SRp20 containing the RNA-binding domain but missing the SR domain inhibits exon 4 inclusion. Inhibition is likely at the level of polyadenylation, because the mutant SRp20 inhibits binding of CstF to the exon 4 poly(A) site. This is the first demonstration that an SR protein can influence alternative polyadenylation and suggests that this family of proteins may play a role in recognition of 3′-terminal exons and perhaps in the communication between polyadenylation and splicing.     

Detection of resistance genes and susceptibility patterns in Bacteroides and Parabacteroides strains

Susceptibility to five antimicrobials was determined for Bacteroides spp. (n = 52) and Parabacteroides distasonis (n = 8). All isolates were susceptible to metronidazole. The resistance rates to ampicillin, cefoxitin, tetracycline and clindamycin were 98%, 9.6%, 65.3% and 19.2% of the Bacteroides strains, respectively. The genes cepA, cfiA, cfxA, tetQ, ermF and nim were found in 69.2%, 17.3% 9.6%, 50%, 7.7% and 3.8% for these strains respectively. All P. distasonis strains were resistant to ampicilin. Cefoxitin, tetracycline and clindamycin resistance rates were 75%, 87.5% and 50%, respectively. The ermF and nim genes were absent and 37.5%, 12.5%, 12.5% and 87.5% of this strains possessed cepA, cfiA, cfxA and tetQ genes, respectively. Ten cfiA gene positive strains of Bacteroides and Parabacteroides were submitted to E-test with imipenem and amoxicillin–clavulanate. The resistance rate to imipenem was 4.1% and 8.3% to amoxicillin–clavulanate. This feature is for the first time described in Brazil.     

An Entangled Model for Sustainability Indicators

Nowadays the challenge for humanity is to find pathways towards sustainable development. Decision makers require a set of sustainability indicators to know if the sustainability strategies are following those pathways. There are more than one hundred sustainability indicators but they differ on their relative importance according to the size of the locality and change on time. The resources needed to follow these sustainability indicators are scarce and in some instances finite, especially in smaller regions. Therefore strategies to select set of these indicators are useful for decision makers responsible for monitoring sustainability. In this paper we propose a model for the identification and selection of a set of sustainability indicators that adequately represents human systems. In developing this model, we applied evolutionary dynamics in a space where sustainability indicators are fundamental entities interconnected by an interaction matrix. we used a fixed interaction that simulates the current context for the city of Cuernavaca, México as an example. We were able to identify and define relevant sets indicators for the system by using the Pareto principle. In this case we identified a set of sixteen sustainability indicators with more than 80% of the total strength. This set presents resilience to perturbations. For the Tangled Nature framework we provided a manner of treating different contexts (i.e., cities, counties, states, regions, countries, continents or the whole planet), dealing with small dimensions. This model provides decision makers with a valuable tool to select sustainability indicators set for towns, cities, regions, countries, continents or the entire planet according to a coevolutionary framework. The social legitimacy can arise from the fact that each individual indicator must be selected from those that are most important for the subject community.     

Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

Background

Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR).

Methods/Principal Findings

The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm.

Conclusions/Significance

Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.     

Vasopressin Infusion with Small-Volume Fluid Resuscitation during Hemorrhagic Shock Promotes Hemodynamic Stability and Survival in Swine

Introduction

Current management of hemorrhagic shock (HS) in the battlefield and civilian settings favors small-volume fluid resuscitation before controlling the source of bleeding. We investigated in a swine model of HS the effects of vasopressin infusion along with small-volume fluid resuscitation; with erythropoietin (EPO) and HS severity as additional factors.

Methods

HS was induced in 24 male domestic pigs (36 to 41 kg) by blood withdrawal (BW) through a right atrial cannula modeling spontaneous bleeding by a mono-exponential decay function. The initial 12 pigs received no fluids; the last 12 pigs received normal saline (NS) half the BW volume. Pigs were randomized 2:1 to receive intraosseously vasopressin (0.04 U/kg·min^-1) or vehicle control from minute 7 to minute 210. Pigs assigned to vasopressin were further randomized 1:1 to receive EPO (1,200 U/kg) or vehicle control and 1:1 to have 65% or 75% BW of their blood volume. Shed blood was reinfused at 210 minutes and the pigs recovered from anesthesia.

Results

Survival at 72 hours was influenced by vasopressin and NS but not by EPO or % BW. Vasopressin with NS promoted the highest survival (8/8) followed by vasopressin without NS (3/8), NS without vasopressin (1/4), and neither treatment (0/4) with overall statistical significance (log-rank test, p = 0.009) and each subset different from vasopressin with NS by Holm-Sidak test. Vasopressin increased systemic vascular resistance whereas NS increased cardiac output.

Conclusion

Vasopressin infusion with small-volume fluid resuscitation during severe HS was highly effective enabling critical hemodynamic stabilization and improved 72 hour survival.     

Resistance to Bemisia tabaci biotype B of Solanum pimpinellifolium is associated with higher densities of type IV glandular trichomes and acylsugar accumulation

Advances in tomato breeding for pest resistance have been achieved via gene introgression from wild Solanum (section Lycopersicon) species (Solanaceae). Ninety‐nine F₃ families derived from an interspecific cross using as parental lines Solanum lycopersicum L. ‘LAM‐148' (susceptible standard) and Solanum pimpinellifolium L. ‘TO‐937‐15’ (multiple pest resistance accession with type IV glandular trichomes and acylsugar accumulation) were evaluated for their resistance against the whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) biotype B in free‐choice and no‐choice tests for oviposition and adult colonization. The parental lines and eight F₃ families with contrasting levels of resistance against the whitefly were selected and investigated in additional assays, which included the estimation of trichome densities and foliar acylsugar levels. The F₃ families BTR‐302 and BTR‐331 exhibited low amounts of eggs of whitefly and transgressive segregation for type IV glandular trichome density with values greater than that of TO‐937‐15 plants. However, the tested families did not surpass the total foliar acylsugar content found in TO‐937‐15. BTR‐331 exhibited low colonization in the free‐choice test and it was the least preferred F₃ family in the no‐choice test. The higher resistance levels of BTR‐331 were associated with a positive combination of higher type IV trichome density and higher acylsugar levels. Some F₃ families displayed reduced fruit set due to the presence of flowers with style exertion of the antheridial‐cone. Fruit weight at harvest stage of the selected families (from 4.9 to 14.5 g) was lower than that of LAM‐148 (139.5 g) but higher than that of TO‐937‐15 plants (1.3 g). Therefore, although difficult to reach due to the simultaneous segregation of many polygenic traits, the combination of high B. tabaci resistance levels with superior horticultural traits is feasible. These results confirm TO‐937‐15 as a source of biotype B resistance. From the breeding standpoint, the genetic similarity between S. lycopersicum and S. pimpinellifolium would allow a more efficient resistance introgression by facilitating recombination and minimizing the potentially undesirable linkage drag associated with this trait.     

Relevance of disease- and organ-specific endothelial cells for in vitro research

The endothelium is a dynamic, heterogeneous, disseminated organ that possesses vital secretory, synthetic, metabolic and immunological functions. Endothelial dysfunction has been implicated as a key factor in the development of organ-specific vascular diseases. This minireview gives a brief overview on EC (endothelial cell) biomarkers in arterial and venous endothelium and critically discusses the different sources of ECs that are most frequently applied in in vitro assays and research. The relevance of organ- and disease-specific endothelial cell cultures for studying cellular responses as a basis for improving therapeutic interventions is highlighted with particular emphasis on endothelial dysfunction in transplant-associated coronary artery disease, in atherosclerotic lesions and in response to diabetes mellitus.     

Biodegradation of epoxyconazole and piraclostrobin fungicides by Klebsiella sp. from soil

Three bacterial strains have been isolated from soil in which soybean had been continuously cropped and treated with Opera^®, a fungicide containing epoxyconazole and pyraclostrobin. The three strains (1,805, 2,801 and 3,803), obtained from soil at 80–100 cm depth, were selected on medium containing 0.03% Opera^®. Morphological examination revealed that the strains were Gram-negative, and two of them (1,805 and 2,801) exhibited polymorphism. The growth profiles demonstrated that 1,805 and 3,803 were more efficient growing in the presence of Opera^® than 2,801. Maximum growth was reached between 24 and 48 h, however, 2,801 was not able to survive after this period. The total protein content produced by 1,805, 2,801 and 3,803 in liquid selective medium containing Opera^® were 111.0 ± 0.02, 80.0 ± 0.05 and 130.5 ± 0.07 μg/ml, respectively. According to its biochemical and molecular features, strain 1,805 was identified as Klebsiella sp. On the basis of the characteristics presented (facultative anaerobic nature, polymorphic character and capacity of growing in the presence of Opera^®) strain 1,805 seems to be able to degrade the epoxyconazole and pyraclostrobin.