Beckwith–Wiedemann syndrome prenatal diagnosis by methylation analysis in chorionic villi

Beckwith–Wiedemann syndrome (BWS) is an imprinting disorder that can be prenatally suspected or diagnosed based on established clinical guidelines. Molecular confirmation is commonly performed on amniocytes. The possibility to use fresh (CVF) and cultured (CVC) chorionic villi has never been investigated. To verify whether CVF and CVC are reliable sources of DNA to study fetal methylation, we used pyrosequencing to test the methylation level of a number of differentially methylated regions (DMRs) at several imprinted loci (ICR1, ICR2, H19, PWS/AS-ICR, GNASXL, GNAS1A, ZAC/PLAGL1, and MEST) and at non-imprinted MGMT and RASSF1A promoters. We analyzed these regions in 19 healthy pregnancies and highlighted stable methylation levels between CVF and CVC at ICR1, ICR2, GNASXL, PWS/AS-ICR, and MEST. Conversely, the methylation levels at H19 promoter, GNAS1A and ZAC/PLAGL1 were different in CVC compared to fresh CV. We also investigated ICR1 and ICR2 methylation level of CVF/CVC of 2 BWS-suspected fetuses (P1 and P2). P1 showed ICR2 hypomethylation, P2 showed normal methylation at both ICR1 and ICR2. Our findings, although limited to one case of BWS fetus with an imprinting defect, can suggest that ICR1 and ICR2, but not H19, could be reliable targets for prenatal BWS diagnosis by methylation test in CVF and CVC. In addition, PWS/AS-ICR, GNASXL, and MEST, but not GNAS1A and ZAC/PLAGL1, are steadily hemimethylated in CV from healthy pregnancies, independently from culture. Thus, prenatal investigation of genomic imprinting in CV needs to be validated in a locus-specific manner.     

Transient TLR activation restores inflammatory response and ability to control pulmonary bacterial infection in germfree mice

Mammals are colonized by an astronomical number of commensal microorganisms on their environmental exposed surfaces. These symbiotic species build up a complex community that aids their hosts in several physiological activities. We have shown that lack of intestinal microbiota is accompanied by a state of active IL-10-mediated inflammatory hyporesponsiveness. The present study investigated whether the germfree state and its hyporesponsive phenotype alter host resistance to an infectious bacterial insult. Experiments performed in germfree mice infected with Klebsiella pneumoniae showed that these animals are drastically susceptible to bacterial infection in an IL-10-dependent manner. In germfree mice, IL-10 restrains proinflammatory mediator production and neutrophil recruitment and favors pathogen growth and dissemination. Germfree mice were resistant to LPS treatment. However, priming of these animals with several TLR agonists recovered their inflammatory responsiveness to sterile injury. LPS pretreatment also rendered germfree mice resistant to pulmonary K. pneumoniae infection, abrogated IL-10 production, and restored TNF-α and CXCL1 production and neutrophil mobilization into lungs of infected germfree mice. This effective inflammatory response mounted by LPS-treated germfree mice resulted in bacterial clearance and enhanced survival upon infection. Therefore, host colonization by indigenous microbiota alters the way the host reacts to environmental infectious stimuli, probably through activation of TLR-dependent pathways. Symbiotic gut colonization enables proper inflammatory response to harmful insults to the host, and increases resilience of the entire mammal-microbiota consortium to environmental pressures.     

A leucine-rich diet and exercise affect the biomechanical characteristics of the digital flexor tendon in rats after nutritional recovery

An increase in the capacity of athletic performance depends on adequate nutrition, which ensures optimal function of the musculoskeletal system, including tendon stability. However, little is known about the status of tendons and extracellular matrix modifications during malnutrition and nutritional recovery when leucine is used in response to exercise conditioning. The purpose of this study was to evaluate the collagen content and biomechanical aspects of the deep digital flexor tendon (DDFT) in malnourished rats submitted to nutritional recovery (control diet or leucine-rich diet) and aerobic physical activity. After 60 days of undernourishment (6% protein diet), the malnourished rats were subsequently nutritionally recovered with a control diet or leucine-rich diet and trained or not (swimming, without overload) for 5 weeks. The biomechanical analysis and quantification of hydroxyproline were assessed in the DDFT in all experimental groups. The leucine-rich diet increased hydroxyproline content in the tension region, independently of the training. In the compression region, hydroxyproline content was higher in the malnourished and leucine-trained groups. Biomechanical analysis showed a lower load in the malnourished and all-trained groups. The lowest stress was observed with control-trained animals. The nutritional-recovered groups showed higher strain values corresponding to control group, while the lowest values were observed in malnourished and trained groups. The results suggest that a leucine-rich diet stimulates collagen synthesis of the DDFT, especially when in combination with physical exercise, and seems to determine the increase of resistance and the biomechanical characteristics of tendons.     

The synthetic purine reversine selectively induces cell death of cancer cells

The synthetic purine reversine has been shown to possess a dual activity as it promotes the de‐differentiation of adult cells, including fibroblasts, into stem‐cell‐like progenitors, but it also induces cell growth arrest and ultimately cell death of cancer cells, suggesting its possible application as an anti‐cancer agent. Aim of this study was to investigate the mechanism underneath reversine selectivity in inducing cell death of cancer cells by a comparative analysis of its effects on several tumor cells and normal dermal fibroblasts. We found that reversine is lethal for all cancer cells studied as it induces cell endoreplication, a process that malignant cells cannot effectively oppose due to aberrations in cell cycle checkpoints. On the other hand, normal cells, like dermal fibroblasts, can control reversine activity by blocking the cell cycle, entering a reversible quiescent state. However, they can be induced to become sensitive to the molecule when key cell cycle proteins, e.g., p53, are silenced. J. Cell. Biochem. 113: 3207–3217, 2012. © 2012 Wiley Periodicals, Inc.     

Interactions between PII proteins and the nitrogenase regulatory enzymes DraT and DraG in Azospirillum brasilense

In Azospirillum brasilense ADP-ribosylation of dinitrogenase reductase (NifH) occurs in response to addition of ammonium to the extracellular medium and is mediated by dinitrogenase reductase ADP-ribosyltransferase (DraT) and reversed by dinitrogenase reductase glycohydrolase (DraG). The P(II) proteins GlnB and GlnZ have been implicated in regulation of DraT and DraG by an as yet unknown mechanism. Using pull-down experiments with His-tagged versions of DraT and DraG we have now shown that DraT binds to GlnB, but only to the deuridylylated form, and that DraG binds to both the uridylylated and deuridylylated forms of GlnZ. The demonstration of these specific protein complexes, together with our recent report of the ability of deuridylylated GlnZ to be sequestered to the cell membrane by the ammonia channel protein AmtB, offers new insights into the control of NifH ADP-ribosylation.     

Population Genetic Structure of Astyanax scabripinnis (Teleostei, Characidae) from an Urban Stream

Despite the great anthropogenic interference on urban streams, information is still scarce about the genetic variability and structure of native fish populations inhabiting such streams. In the present study, random amplified polymorphic DNA (RAPD) markers were used to analyze genetic variability and structure of populations assigned to the Neotropical fish species Astyanax scabripinnis from an urban stream located in Londrina, Paraná State, southern Brazil. Thirty individuals of this species were collected from three sites throughout the upper Cambé stream. A total of 10 primers amplified 159 loci, of which 128 (80.5%) were polymorphic. Each of the three populations showed very similar proportions of polymorphic loci, which ranged from 63.5 to 64.8%. Unbiased genetic distances varied from 0.0612 to 0.0646. Theta_p-test values indicated moderate to high genetic differentiation among individuals from different localities. The number of migrants varied from 1.34 to 1.46, suggesting a low gene flow between populations. The genetic similarity among all individuals studied ranged from 0.424 to 0.848. The results suggest that populations of A. scabripinnis in Cambé stream are undergoing genetic differentiation.     

ADP-ribosylation of dinitrogenase reductase in Azospirillum brasilense is regulated by AmtB-dependent membrane sequestration of DraG

Nitrogen fixation in some diazotrophic bacteria is regulated by mono-ADP-ribosylation of dinitrogenase reductase (NifH) that occurs in response to addition of ammonium to the extracellular medium. This process is mediated by dinitrogenase reductase ADP-ribosyltransferase (DraT) and reversed by dinitrogenase reductase glycohydrolase (DraG), but the means by which the activities of these enzymes are regulated are unknown. We have investigated the role of the P(II) proteins (GlnB and GlnZ), the ammonia channel protein AmtB and the cellular localization of DraG in the regulation of the NifH-modification process in Azospirillum brasilense. GlnB, GlnZ and DraG were all membrane-associated after an ammonium shock, and both this membrane sequestration and ADP-ribosylation of NifH were defective in an amtB mutant. We now propose a model in which membrane association of DraG after an ammonium shock creates a physical separation from its cytoplasmic substrate NifH thereby inhibiting ADP-ribosyl-removal. Our observations identify a novel role for an ammonia channel (Amt) protein in the regulation of bacterial nitrogen metabolism by mediating membrane sequestration of a protein other than a P(II) family member. They also suggest a model for control of ADP-ribosylation that is likely to be applicable to all diazotrophs that exhibit such post-translational regulation of nitrogenase.     

CFD simulation of an internal spin-filter: evidence of lateral migration and exchange flow through the mesh

In the present work Computational Fluid Dynamics (CFD) was used to study the flow field and particle dynamics in an internal spin-filter (SF) bioreactor system. Evidence of a radial exchange flow through the filter mesh was detected, with a magnitude up to 130-fold higher than the perfusion flow, thus significantly contributing to radial drag. The exchange flow magnitude was significantly influenced by the filter rotation rate, but not by the perfusion flow, within the ranges evaluated. Previous reports had only given indirect evidences of this exchange flow phenomenon in spin-filters, but the current simulations were able to quantify and explain it. Flow pattern inside the spin-filter bioreactor resembled a typical Taylor-Couette flow, with vortices being formed in the annular gap and eventually penetrating the internal volume of the filter, thus being the probable reason for the significant exchange flow observed. The simulations also showed that cells become depleted in the vicinity of the mesh due to lateral particle migration. Cell concentration near the filter was approximately 50% of the bulk concentration, explaining why cell separation achieved in SFs is not solely due to size exclusion. The results presented indicate the power of CFD techniques to study and better understand spin-filter systems, aiming at the establishment of effective design, operation and scale-up criteria.     

Assessing the environmental profile of orange production in Brazil

Background, aim, and scope

Brazil is the world’s largest orange producing country, with a total planted area of more than 820,000 hectares. The bulk of the oranges produced in Brazil (70%) is processed into frozen concentrated orange juice (FCOJ) by large processing companies. Exports represent around 97% of the total FCOJ produced, making Brazil the largest world producer and exporter of FCOJ. The Brazilian citrus sector accounts for half of the world’s supply of orange juice and for 80% of the juice traded on the international market. The goal of this study is to characterize the Brazilian orange producers in terms of farm size, cultivated varieties, watering system and tillage practices. In addition, the study presents some aspects of the LCI of oranges grown specifically for the production of FCOJ in the two major orange-growing areas in Brazil (the Northern and Southern regions of the State of São Paulo) during reference crop year 2002/03 in order to generate detailed production inventory data and identify the potential environmental impacts of tillage, both of which are key to enable the formulation of sustainability parameters for the production of FCOJ in Brazil.

Methods

This study was performed in compliance with the guidelines and requirements of the ISO 14040 standard series. All information and data considered and evaluated in this study (the use of water, energy, fertilizers, pesticides and soil correctors) were gathered through in-depth questionnaires either filled in directly by the farm manager or completed by the farm manager and sent in by mail. The data cover a total of 367,200 metric tons of oranges produced by 4 million plants in commercial production from a total evaluated area that accounts for 19.5% of the overall production of oranges in the State of São Paulo. The two major orange-producing areas in Brazil located respectively in the Northern and Southern regions of the State of São Paulo were evaluated. Only the inputs and outputs associated with tillage practices and technology were considered in this paper. The environmental aspects of the production of fertilizers and pesticides were not included within the boundaries. The functional unit selected for this study was 1,000 kg of oranges for FCOJ. Farm-specific data have been combined with agricultural production data to construct an orange cultivation model.

Results

The orange varieties for the production of FCOJ evaluated in this study were Pêra, Valência and Natal. The farms investigated had a cultivated area varying from 22 to 7,000 ha, with a plant density ranging from 170 to 500 plants/ha and an average yield of 30,500 kg/ha for mature trees. Depending on the region, the production of 1,000 kg of oranges requires approx. 120 to 4,400 MJ of energy; 0.3 to 36 kg of diesel; 1,100 to 54,500 kg of water for irrigation; 0.3 to 65 kg of fertilizers (NPK); 0.1 to 13.5 kg of pesticides; 8 to 650 kg of soil correctors. The distribution of some inputs across the farms located in the two main Brazilian orange-producing regions is presented, in addition to the total weighted average.

Discussion

In addition to the variations among the regions evaluated, it was generally observed that there were very considerable differences in order of magnitude between the data collected from farms located in the same region. Although the inputs are directly related to the specific characteristics of each farm and the climatic and topographic conditions of its location, this study has identified some farms that are in a good position to reduce the amounts of some inputs and enhance their environmental and economical performance.

Conclusions

Taking into account the aspects evaluated in this study, only 21% of the orange growers showed good performance, i.e., consumption of pesticides, fertilizers and soil correction compounds equal to or lower than the weighted averages, except for “land use”, which was above the average for the majority of these farms. This study provided important results that allow for a better understanding of the agricultural practices involved and the potential environmental impacts of this product.

Recommendation and perspective

The authors would like to suggest that the five best-performing farms average values be set as the maximum allowable level and point of departure for the discussion on the good environmental performance of Brazilian FCOJ. Besides the total inputs, the role of good agricultural practices to achieve good yields and, at the same time, improve the sustainability of orange growing should also be taken into consideration. Future updates of this study will show the evolution of natural resource management as a result of improved land use, new agricultural practices, reduced use of fertilizers and agrochemicals.     

Low and high-dose intradermal infection with Leishmania major and Leishmania amazonensis in C57BL/6 mice

A model of skin infection with Leishmania amazonensis with low doses of parasites is compared to infection with high doses of L. amazonensis and low and high doses of Leishmania major. C57BL/6 mice were infected with 103 or 10(6) parasites in the ear and the outcome of infection was assessed. The appearance of lesions in mice infected with 103 parasites was delayed compared to mice infected with 10(6) Leishmania and parasites were detectable at the infection site before lesions became apparent. Mice infected with L. amazonensis displayed persistent lesions, whereas infection with L. major spontaneously healed in all groups, although lymphocytes persisted at the site of infection after healing. Macrophages persisted only in L. amazonensis-infected mice. High-dose L. amazonensis-infected mice produced lower levels of IFN-γ and TNF than mice infected with L. major. No correlation between the persistence of parasites and IL-10 levels and the production of nitric oxide or urea by macrophages was found. We conclude that infection with low doses of L. amazonensis in the dermis changes the course of infection by delaying the appearance of lesions. However, low-dose infection does not change the outcomes of susceptibility and cytokine production described for subcutaneous infection with high numbers of parasites.