Florfenicol Binding to Molecularly Imprinted Polymer Nanoparticles in Model and Real Samples.

A simple and straightforward technique for coating microplate wells with molecularly imprinted polymer nanoparticles (nanoMIPs) to develop assays similar to the enzyme-linked immunosorbent (ELISA) assay to determine and quantify florfenicol (FF) in real food samples such as liquid milk and salmon muscle is presented here. The nanoMIPs were synthesized by a solid-phase approach with an immobilized FF (template) and characterized using dynamic light scattering, a SPR-2 biosensor system and transmission electron microscopy. Immobilization of nanoMIPs was conducted by preparing a homogenous solution of FF-nanoMIPs in water mixed with polyvinyl alcohol (PVA) 0.2% (w/v) in each well of a microplate. The detection of florfenicol was achieved in competitive binding experiments with a horseradish peroxidase-florfenicol (FF-HRP) conjugate. The assay made it possible to measure FF in buffer and in real samples (liquid milk and salmon muscle) within the range of 60-80 and 90-100 ng/mL, respectively. The immobilized nanoMIPs were stored for six weeks at room temperature and at 5 °C. The results indicate good signal recovery for all FF concentrations in spiked milk samples, without any detrimental effects to their binding properties. The high affinity of nanoMIPs and the lack of a requirement for cold chain logistics make them an attractive alternative to traditional antibodies used in ELISA.

Isolation and identification of compounds from the resinous exudate of Escallonia illinita Presl. and their anti-oomycete activity.

The resinous exudates from Escallonia illinita by products was characterized by FT-IR, NMR and HRMS. Six compounds were isolated and identified as follows: 1,5-diphenylpent-1-en-3-one (1), 4-(5-hydroxy-3,7-dimethoxy-4-oxo-4H-chromen-2-yl)phenyl acetate (2), pinocembrin (3), kaempferol 3-O-methylether (4), (3S,5S)-(E)-1,7-diphenylhept-1-ene-3,5-diol (5) and the new diarylheptanoid (3S,5S)-(E)-5-hydroxy-1,7-diphenylhept-1-en-3-yl acetate (6). The anti-oomycete potential of the resinous exudate, as well as the main compounds, was tested in vitro against Saprolegnia parasitica and Saprolegnia australis. The resinous exudate showed a strong anti-oomycete activity. In addition, the compounds 6, 1 and 3 demonstrated significant inhibition of Saprolegnia strains development. These findings strongly suggest that E. illinita is a potential biomass that could be used as a natural anti-oomycete product.

Effect of microalgae on intestinal inflammation triggered by soybean meal and bacterial infection in zebrafish.

Soybean meal has been used in many commercial diets for farm fish; despite this component inducing intestinal inflammation. On the other hand, microalgae have increasingly been used as dietary supplements in fish feed. Nevertheless, the vast quantity of microalgae species means that many remain under- or unstudied, thus limiting wide scale commercial application. In this work, we evaluated the effects to zebrafish (Danio rerio) of including Tetraselmis sp (Ts); Phaeodactylum tricornutum (Pt); Chlorella sp (Ch); Nannochloropsis oculata (No); or Nannochloropsis gaditana (Ng) as additives in a soybean meal-based diet on intestinal inflammation and survival after Edwardsiella tarda infection. In larvae fed a soybean meal diet supplemented with Ts, Pt, Ch, or Ng, the quantity of neutrophils present in the intestine drastically decreased as compared to larvae fed only the soybean meal diet. Likewise, Ts or Ch supplements in soybean meal or fishmeal increased zebrafish survival by more than 20% after being challenged. In the case of Ts, the observed effect correlated with an increased number of neutrophils present at the infection site. These results suggest that the inclusion of Ts or Ch in fish diets could allow the use of SBM and at the same time improve performance against pathogen.

Reconstruction of the microalga Nannochloropsis salina genome-scale metabolic model with applications to lipid production.

BACKGROUND: Nannochloropsis salina (= Eustigmatophyceae) is a marine microalga which has become a biotechnological target because of its high capacity to produce polyunsaturated fatty acids and triacylglycerols. It has been used as a source of biofuel, pigments and food supplements, like Omega 3. Only some Nannochloropsis species have been sequenced, but none of them benefit from a genome-scale metabolic model (GSMM), able to predict its metabolic capabilities. RESULTS: We present iNS934, the first GSMM for N. salina, including 2345 reactions, 934 genes and an exhaustive description of lipid and nitrogen metabolism. iNS934 has a 90% of accuracy when making simple growth/no-growth predictions and has a 15% error rate in predicting growth rates in different experimental conditions. Moreover, iNS934 allowed us to propose 82 different knockout strategies for strain optimization of triacylglycerols. CONCLUSIONS: iNS934 provides a powerful tool for metabolic improvement, allowing predictions and simulations of N. salina metabolism under different media and genetic conditions. It also provides a systemic view of N. salina metabolism, potentially guiding research and providing context to -omics data.

Global transcriptional responses of Acidithiobacillus ferrooxidans Wenelen under different sulfide minerals.

In order to provide new information about the adaptation of Acidithiobacillus ferrooxidans during the bioleaching process, the current analysis presents the first report of the global transcriptional response of the native copper mine strain Wenelen (DSM 16786) oxidized under different sulfide minerals. Microarrays were used to measure the response of At. ferrooxidans Wenelen to shifts from iron supplemented liquid cultures (reference state) to the addition of solid substrates enriched in pyrite or chalcopyrite. Genes encoding for energy metabolism showed a similar transcriptional profile for the two sulfide minerals. Interestingly, four operons related to sulfur metabolism were over-expressed during growth on a reduced sulfur source. Genes associated with metal tolerance (RND and ATPases type P) were up-regulated in the presence of pyrite or chalcopyrite. These results suggest that At. ferrooxidans Wenelen presents an efficient transcriptional system developed to respond to environmental conditions, namely the ability to withstand high copper concentrations.

Comparative genomic analysis of carbon and nitrogen assimilation mechanisms in three indigenous bioleaching bacteria: predictions and validations.

BACKGROUND: Carbon and nitrogen fixation are essential pathways for autotrophic bacteria living in extreme environments. These bacteria can use carbon dioxide directly from the air as their sole carbon source and can use different sources of nitrogen such as ammonia, nitrate, nitrite, or even nitrogen from the air. To have a better understanding of how these processes occur and to determine how we can make them more efficient, a comparative genomic analysis of three bioleaching bacteria isolated from mine sites in Chile was performed. This study demonstrated that there are important differences in the carbon dioxide and nitrogen fixation mechanisms among bioleaching bacteria that coexist in mining environments. RESULTS: In this study, we probed that both Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans incorporate CO2 via the Calvin-Benson-Bassham cycle; however, the former bacterium has two copies of the Rubisco type I gene whereas the latter has only one copy. In contrast, we demonstrated that Leptospirillum ferriphilum utilizes the reductive tricarboxylic acid cycle for carbon fixation. Although all the species analyzed in our study can incorporate ammonia by an ammonia transporter, we demonstrated that Acidithiobacillus thiooxidans could also assimilate nitrate and nitrite but only Acidithiobacillus ferrooxidans could fix nitrogen directly from the air. CONCLUSION: The current study utilized genomic and molecular evidence to verify carbon and nitrogen fixation mechanisms for three bioleaching bacteria and provided an analysis of the potential regulatory pathways and functional networks that control carbon and nitrogen fixation in these microorganisms.

Structure-function relationship between the tobamovirus TMV-Cg coat protein and the HR-like response.

The tobamovirus TMV-Cg induces an HR-like response in Nicotiana tabacum cv. Xanthi nn sensitive plants lacking the N or N' resistance genes. This response has been characterized by the appearance of necrotic lesions in the inoculated leaf and viral systemic spread, although the defence pathways are activated in the plant. A previous study demonstrated that the coat protein (CP) of TMV-Cg (CPCg) was the elicitor of this HR-like response. We examined the influence of four specific amino acid substitutions on the structure of CPCg, as well as on the development of the host response. To gain insights into the structural implications of these substitutions, a set of molecular dynamic experiments was performed using comparative models of wild-type and mutant CPCg as well as the CP of the U1 strain of TMV (CPU1), which is not recognized by the plants. A P21L mutation produces severe changes in the three-dimensional structure of CPCg and is more unstable when this subunit is laterally associated in silico. This result may explain the observed incapacity of this mutant to assemble virions. Two other CPCg mutations (R46G and S54K) overcome recognition by the plant and do not induce an HR-like response. A double CPCg mutant P21L-S54K recovered its capacity to form virions and to induce an HR-like response. Our results suggest that the structural integrity of the CP proteins is important for triggering the HR-like response.

Tobamovirus coat protein CPCg induces an HR-like response in sensitive tobacco plants.

When inoculated into sensitive tobacco Xanthi-nn plants, the crucifer and garlic-infecting Tobacco mosaic virus (TMV-Cg) induces local necrotic lesions that resemble those seen in the hypersensitive response (HR) of resistant tobacco plants. However, unlike these, tobacco Xanthi-nn plants do not become resistant to infection and the virus spreads systemically causing a severe disease characterized by necrotic lesions throughout the plant. To identify the viral protein that elicits this necrotic response, we used a set of hybrid viruses constructed by combination of TMV-Cg and the tobacco mosaic virus strain U1 (TMV-U1). In this study we present evidence that the coat protein of TMV-Cg (CPCg) is the elicitor of the necrotic response in tobacco Xanthi-nn plants. Local and systemic necrotic lesions induced by TMV-Cg and by the hybrid U1-CPCg -that carries CPCg in a TMV-U1 context- are characterized by cell death and by the presence of autoflorescent phenolic compounds and H2O2, just like the HR lesions. In addition, defense-related genes and detoxifying genes are induced in tobacco Xanthi-nn plants after TMV-Cg and U1-CPCg inoculation. We postulate that in our system, CPCg is recognized by sensitive tobacco plants that mount an incomplete defense response. We call this an HR-like since it is not enough to induce plant resistance.

Replicase mediated resistance against potato leafroll virus in potato Desirée plants.

Potato leafroll virus (PLRV) is a major menace for the potato production all over the world. PLRV is transmitted by aphids, and until now, the only strategy available to control this pest has been to use large amounts of insecticides. Transgenic approaches involving the expression of viral replicases are being developed to provide protection for plants against viral diseases. The purpose of this study was to compare the protection afforded by the differential expression of PLRV replicate transgene in potato plants cv. Desirée, Plants were genetically modified to express the complete sense PLRV replicase gene. Two constructions were used, one containing the constitutive 35SCaMV promoter and the other the phloem-specific RolA promoter from Agrobacterium rhizogenes. Transgenic plants were infected with PLRV in vitro, using infested aphids. In plants in which 35SCaMV controlled the expression of the PLRV replicase gene, signs of infection were initially detected, although most plants later developed a recovery phenotype showing undetectable virus levels 40 days after infection. In turn, those plants with the RolA promoter displayed an initial resistance that was later overcome. Different molecular mechanisms are likely to participate in the response to PLRV infection of these two types of transgenic plants.

Replicase mediated resistance against Potato Leafroll Virus in potato Desiree plants

Potato leafroll virus (PLRV) is a major menace for the potato production all over the world. PLRV is transmitted by aphids, and until now, the only strategy available to control this pest has been to use large amounts of insecticides. Transgenic approaches involving the expression of viral replicases are being developed to provide protection for plants against viral diseases. The purpose of this study was to compare the protection afforded by the differential expression of PLRV replicate transgene in potato plants cv. Desirée, Plants were genetically modified to express the complete sense PLRV replicase gene. Two constructions were used, one containing the constitutive 35SCaMV promoter and the other the phloem-specific RolA promoter from Agrobacterium rhizogenes. Transgenic plants were infected with PLRV in vitro, using infested aphids. In plants in which 35SCaMV controlled the expression of the PLRV replicase gene, signs of infection were initially detected, although most plants later developed a recovery phenotype showing undetectable virus levels 40 days after infection.