A synthetic de-greening gene circuit provides a reporting system that is remotely detectable and has a re-set capacity

Plants have evolved elegant mechanisms to continuously sense and respond to their environment, suggesting that these properties can be adapted to make inexpensive and widely used biological monitors, or sentinels, for human threats. For a plant to be a sentinel, a reporting system is needed for large areas and widespread monitoring. The reporter or readout mechanism must be easily detectable, allow remote monitoring and provide a re-set capacity; all current gene reporting technologies fall short of these requirements. Chlorophyll is one of the best-recognized plant pigments with an already well-developed remote imaging technology. However, chlorophyll is very abundant, with levels regulated by both genetic and environmental factors. We designed a synthetic de-greening circuit that produced rapid chlorophyll loss on perception of a specific input. With induction of the de-greening circuit, changes were remotely detected within 2 h. Analyses of multiple de-greening circuits suggested that the de-greening circuit functioned, in part, via light-dependent damage to photosystem cores and the production of reactive oxygen species. Within 24–48 h of induction, an easily recognized white phenotype resulted. Microarray analysis showed that the synthetic de-greening initiated a process largely distinct from normal chlorophyll loss in senescence. Remarkably, synthetically de-greened white plants re-greened after removal of the inducer, providing the first easily re-settable reporter system for plants and the capacity to make re-settable biosensors. Our results showed that the de-greening circuit allowed chlorophyll to be employed as a simple but powerful reporter system useful for widespread areas.     

Influence of artificial accelerated ageing on the colour stability of paints used for ocular prosthesis iris painting

doi: 10.1111/j.1741‐2358.2010.00473.x
Influence of artificial accelerated ageing on the colour stability of paints used for ocular prosthesis iris painting Objectives: To evaluate the colour stability of paints used for ocular prosthesis iris painting submitted for accelerated artificial ageing (AAA). Materials and methods: Forty specimens of acrylic resin for sclera (16 × 2 mm) were made and separated into eight groups (n = 10) according to the type of paint (gouache, GP; oil, OP; acrylic AP; and composite resin for characterisation, CR) and the colours used (blue/brown). After drying (72 h), a new layer of colourless acrylic resin was applied and the initial colour readout was performed (Spectrophotometer PCB 6807). New colour readouts were performed after AAA, and ΔE was calculated. Results: Statistical analysis (two‐way anova –Bonferroni, p < 0.05) demonstrated that the brown colour showed lower ΔE means in comparison with the blue colour, with statistically significant difference for AP only. Blue colour showed no statistically significant difference with regard to the type of paint used. Brown AP showed lower ΔE than the other groups, with significant difference for OP and GP. GP showed greater alteration in ΔE for the brown colour, being statistically similar only to OP. Conclusions: Only the AP group for brown pigment shows clinically acceptable values for colour stability after AAA.     

Involvement of GABAergic and glutamatergic systems in the anticonvulsant activity of 3-alkynyl selenophene in 21?day-old rats

In this study, we investigated the role of GABAergic and glutamatergic systems in the anticonvulsant action of 3-alkynyl selenophene (3-ASP) in a pilocarpine (PC) model of seizures. To this purpose, 21 day-old rats were administered with an anticonvulsant dose of 3-ASP (50 mg/kg, per oral, p.o.), and [(3)H]γ-aminobutyric acid (GABA) and [(3)H]glutamate uptakes were carried out in slices of cerebral cortex and hippocampus. [(3)H]GABA uptake was decreased in cerebral cortex (64%) and hippocampus (58%) slices of 21 day-old rats treated with 3-ASP. In contrast, no alteration was observed in [(3)H]glutamate uptake in cerebral cortex and hippocampus slices of 21 day-old rats that received 3-ASP. Considering the drugs that increase synaptic GABA levels, by inhibiting its uptake or catabolism, are effective anticonvulsants, we further investigated the possible interaction between sub-effective doses of 3-ASP and GABA uptake or GABA transaminase (GABA-T) inhibitors in PC-induced seizures in 21 day-old rats. For this end, sub-effective doses of 3-ASP (10 mg/kg, p.o.) and DL-2,4-diamino-n-butyric acid hydrochloride (DABA, an inhibitor of GABA uptake--2 mg/kg, intraperitoneally; i.p.) or aminooxyacetic acid hemihydrochloride (AOAA; a GABA-T inhibitor--10 mg/kg, i.p.) were co-administrated to 21 day-old rats before PC (400 mg/kg; i.p.) treatment, and the appearance of seizures was recorded. Results demonstrated that treatment with AOAA and 3-ASP or DABA and 3-ASP significantly abolished the number of convulsing animals induced by PC. The present study indicates that 3-ASP reduced [(3)H]GABA uptake, suggesting that its anticonvulsant action is related to an increase in inhibitory tonus.     

Technetium-99m-labeled ceftizoxime loaded long-circulating and pH-sensitive liposomes used to identify osteomyelitis

Osteomyelitis is an infectious disease located in the bone or bone marrow. Long-circulating and pH-sensitive liposomes containing a technetium-99m-labeled antibiotic, ceftizoxime, (SpHL-(99m)Tc-CF) were developed to identify osteomyelitis foci. Biodistribution studies and scintigraphic images of bone infection or non infection-bearing rats that had been treated with these liposomes were performed. A high accumulation in infectious foci and high values in the target-non target ratio could be observed. These results indicate the potential of SpHL-(99m)Tc-CF as a potential agent for the diagnosis of bone infections.     

Exposure to rabies virus in a population of free-ranging capuchin monkeys (Cebus apella nigritus) in a fragmented, environmentally protected area in southeastern Brazil

The aim of this study is to assess the frequency of rabies antibodies in free-ranging capuchin monkeys (Cebus apella nigritus) in a fragmented, environmentally protected, rural area of southeastern Brazil. Thirty-six free-ranging monkeys were tested by the rapid fluorescent focus inhibition test for detection of antibodies against rabies virus. Four individuals (11.11 %) had neutralizing antibody titers ≥ 0.25 IU/mL, demonstrating rabies virus exposure.     

Inflammatory cytokines in vitro production are associated with Ala16Val superoxide dismutase gene polymorphism of peripheral blood mononuclear cells

Obesity is considered a chronic low-grade inflammatory state associated with a chronic oxidative stress caused by superoxide production (O(2)(-)). The superoxide dismutase manganese dependent (SOD2) catalyzes O(2)(-) in H(2)O(2) into mitochondria and is encoded by a single gene that presents a common polymorphism that results in the replacement of alanine (A) with a valine (V) in the 16 codon. This polymorphism has been implicated in a decreased efficiency of SOD2 transport into targeted mitochondria in V allele carriers. Previous studies described an association between VV genotype and metabolic diseases, including obesity and diabetes. However, the causal mechanisms to explain this association need to be more elucidated. We postulated that the polymorphism could influence the inflammatory response. To test our hypothesis, we evaluated the in vitro cytokines production by human peripheral blood mononuclear cells (PBMCs) carrier's different Ala16Val-SOD2 genotypes (IL-1, IL-6, IL-10, TNF-α, IFN-γ). Additionally, we evaluated if the culture medium glucose, enriched insulin, could influence the cytokine production. Higher levels of proinflammatory cytokines were observed in VV-PBMCs when compared to AA-PBMCs. However, the culture medium glucose and enriched insulin did not affect cytokine production. The results suggest that Ala16Val-SOD2 gene polymorphism could trigger the PBMCs proinflammatory cytokines level. However, discerning if a similar mechanism occurs in fat cells is an open question.     

Activation of Haem-Oxidized Soluble Guanylyl Cyclase with BAY 60-2770 in Human Platelets Lead to Overstimulation of the Cyclic GMP Signaling Pathway

Background and Aims

Nitric oxide-independent soluble guanylyl cyclase (sGC) activators reactivate the haem-oxidized enzyme in vascular diseases. This study was undertaken to investigate the anti-platelet mechanisms of the haem-independent sGC activator BAY 60-2770 in human washed platelets. The hypothesis that sGC oxidation potentiates the anti-platelet activities of BAY 60-2770 has been tested.

Methods

Human washed platelet aggregation and adhesion assays, as well as flow cytometry for α_IIbβ₃ integrin activation and Western blot for α1 and β1 sGC subunits were performed. Intracellular calcium levels were monitored in platelets loaded with a fluorogenic calcium-binding dye (FluoForte).

Results

BAY 60-2770 (0.001–10 µM) produced significant inhibition of collagen (2 µg/ml)- and thrombin (0.1 U/ml)-induced platelet aggregation that was markedly potentiated by the sGC inhibitor ODQ (10 µM). In fibrinogen-coated plates, BAY 60-2770 significantly inhibited platelet adhesion, an effect potentiated by ODQ. BAY 60-2770 increased the cGMP levels and reduced the intracellular Ca²⁺ levels, both of which were potentiated by ODQ. The cell-permeable cGMP analogue 8-Br-cGMP (100 µM) inhibited platelet aggregation and Ca²⁺ levels in an ODQ-insensitive manner. The cAMP levels remained unchanged by BAY 60-2770. Collagen- and thrombin-induced α_IIbβ₃ activation was markedly inhibited by BAY 60-2770 that was further inhibited by ODQ. The effects of sodium nitroprusside (3 µM) were all prevented by ODQ. Incubation with ODQ (10 µM) significantly reduced the protein levels of α1 and β1 sGC subunits, which were prevented by BAY 60-2770.

Conclusion

The inhibitory effects of BAY 60-2770 on aggregation, adhesion, intracellular Ca²⁺ levels and α_IIbβ₃ activation are all potentiated in haem-oxidizing conditions. BAY 60-2770 prevents ODQ-induced decrease in sGC protein levels. BAY 60-2770 could be of therapeutic interest in cardiovascular diseases associated with thrombotic complications.     

DNA barcodes identify marine fishes of São Paulo State,Brazil

Anthropogenic impacts are an increasing threat to the diversity of fishes, especially in areas around large urban centres, and many effective conservation actions depend on accurate species identification. Considering the utility of DNA barcoding as a global system for species identification and discovery, this study aims to assemble a DNA barcode reference sequence library for marine fishes from the coastal region of São Paulo State, Brazil. The standard 652 bp ‘barcode’ fragment of the cytochrome c oxidase subunit I (COI) gene was PCR amplified and bidirectionally sequenced from 678 individuals belonging to 135 species. A neighbour‐joining analysis revealed that this approach can unambiguously discriminate 97% of the species surveyed. Most species exhibited low intraspecific genetic distances (0.31%), about 43‐fold less than the distance among species within a genus. Four species showed higher intraspecific divergences ranging from 2.2% to 7.6%, suggesting overlooked diversity. Notably, just one species‐pair exhibited barcode divergences of <1%. This library is a first step to better know the molecular diversity of marine fish species from São Paulo, providing a basis for further studies of this fauna – extending the ability to identify these species from all life stages and even fragmentary remains, setting the stage for a better understanding of interactions among species, calibrating the estimations about species composition and richness in an ecosystem, and providing tools for authenticating bioproducts and monitoring illegal species exploitation.     

Malaria liver stage susceptibility locus identified on mouse chromosome 17 by congenic mapping

Host genetic variants are known to confer resistance to Plasmodium blood stage infection and to control malaria severity both in humans and mice. This work describes the genetic mapping of a locus for resistance to liver stage parasite in the mouse. First, we show that decreased susceptibility to the liver stage of Plasmodium berghei in the BALB/c mouse strain is attributable to intra-hepatic factors and impacts on the initial phase of blood stage infection. We used QTL mapping techniques to identify a locus controlling this susceptibility phenotype (LOD score 4.2) on mouse chromosome 17 (belr1 locus). Furthermore, analysis of congenic mouse strains delimited the belr1 locus boundaries distally to the H2 region. Quantification of parasites in the liver of infected congenic mice strongly suggested that the belr1 locus represents a genetic factor controlling the expansion of P. berghei in the hepatic tissue. The mapping of belr1 locus raises the hypothesis that host gene variation is able to control the progression of Plasmodium liver stage infection and opens the possibility that the human genomic region orthologue to belr1 may contain genes that confer resistance to the human malaria liver stage.