Novel “nano-phage” interfaces for wireless biosensors

The prevention of food-borne illness has become a very important factor in public health. Meantime, the conventional microbiological detection techniques are time consuming, require proficiency and appropriate laboratory conditions. Recently, there has been an extensive work undertaken towards the development of diagnostic biosensor devices for on-site detection of biological threats that explore a diversity of transduction mechanisms and bio-recognition elements. In particular, the environmentally robust filamentous phages have been successfully used as an alternative to fragile antibodies in wireless biosensor system for real-time pathogen detection. However, when phages are used as interface, they can aggregate forming bundles of fibers that cannot cover completely the sensor’s interface leading to the decrease in sensor’s performance. In this work we developed novel wireless magnetoelastic biosensors with interface formed by biorecognition nanoparticles called “nano-phage”. “Nano-phage” comprises nanoparticles with diameter ~11 nm composed of self-assembled fusion major coat protein of landscape phages selected against the target analyte. For proof-of-concept, we investigated interfaces formed by three model phages selected from landscape libraries: streptavidin binders 7b1 and SAE10 and clone E2 highly specific and selective for S. typhimurium. Beside food borne pathogens, this new approach can be used to develop biosensors with increased performance for early detection of cancer diseases and other pathologies

The Use of Dietary Additives in Fish Stress Mitigation: Comparative Endocrine and Physiological Responses

In the last years, studies on stress attenuation in fish have progressively grown. This is mainly due to the interest of institutions, producers, aquarists and consumers in improving the welfare of farmed fish. In addition to the development of new technologies to improve environmental conditions of cultured fish, the inclusion of beneficial additives in the daily meal in order to mitigate the stress response to typical stressors (netting, overcrowding, handling, etc.) has been an important research topic. Fish are a highly diverse paraphyletic group (over 27,000 species) though teleost infraclass include around 96% of fish species. Since those species are distributed world-wide, a high number of different habitats and vital requirements exist, including a wide range of environmental conditions determining specifically the stress response. Although the generalized endocrine response to stress (based on the release of catecholamines and corticosteroids) is detectable and therefore provides essential information, a high diversity of physiological effects have been described depending on species. Moreover, recent omics techniques have provided a powerful tool for detecting specific differences regarding the stress response. For instance, for transcriptomic approaches, the gene expression of neuropeptides and other proteins acting as hormonal precursors during stress has been assessed in some fish species. The use of different additives in fish diets to mitigate stress responses has been deeply studied. Besides the species factor, the additive type also plays a pivotal role in the differentiation of the stress response. In the literature, several types of feed supplements in different species have been assayed, deriving in a series of physiological responses which have not focused exclusively on the stress system. Immunological, nutritional and metabolic changes have been reported in these experiments, always associated to endocrine processes. The biochemical nature and physiological functionality of those feed additives strongly affect the stress response and, in fact, these can act as neurotransmitters or hormone precursors, energy substrates, cofactors and other essential elements, implyingmulti-systematic and multi-organic responses. In this review, the different physiological responses among fish species fed stress-attenuating diets based on biomolecules and minerals have been assessed, focusing on the endocrine regulation and its physiological effects

European Sea Bass (Dicentrarchus labrax) immune status and disease resistance are impaired by arginine dietary supplementation

Infectious diseases and fish feeds management are probably the major expenses in the aquaculture business. Hence, it is a priority to define sustainable strategies which simultaneously avoid therapeutic procedures and reinforce fish immunity. Currently, one preferred approach is the use of immunostimulants which can be supplemented to the fish diets. Arginine is a versatile amino acid with important mechanisms closely related to the immune response. Aiming at finding out how arginine affects the innate immune status or improve disease resistance of European seabass (Dicentrarchus labrax) against vibriosis, fish were fed two arginine-supplemented diets (1% and 2% arginine supplementation). A third diet meeting arginine requirement level for seabass served as control diet. Following 15 or 29 days of feeding, fish were sampled for blood, spleen and gut to assess cell-mediated immune parameters and immune-related gene expression. At the same time, fish from each dietary group were challenged against Vibrio anguillarum and survival was monitored. Cell-mediated immune parameters such as the extracellular superoxide and nitric oxide decreased in fish fed arginine-supplemented diets. Interleukins and immune-cell marker transcripts were down-regulated by the highest supplementation level. Disease resistance data were in accordance with a generally depressed immune status, with increased susceptibility to vibriosis in fish fed arginine supplemented diets. Altogether, these results suggest a general inhibitory effect of arginine on the immune defences and disease resistance of European seabass. Still, further research will certainly clarify arginine immunomodulation pathways thereby allowing the validation of its potential as a prophylactic strategy.European Union's Seventh Framework Programme AQUAEXCEL (Aquaculture Infrastructures for Excellence in European Fish Research) [262336]; AQUAIMPROV [NORTE-07-0124-FEDER-000038]; North Portugal Regional Operational Programme (ON. 2 - O Novo Norte) , under the National Strategic Reference Framework, through the European Regional Development Fund; North Portugal Regional Operational Programme (ON. 2 - O Novo Norte), under the National Strategic Reference Framework through the COMPETE - Operational Competitiveness Programme; Fundacao para a Ciencia e Tecnologia; Fundacao para a Ciencia e Tecnologia [SFRH/BD/89457/2012, SFRH/BPD/77210/2011]; Generalitat Valenciana through the project REVIDPAQUA [ISIC/2012/003]; [PEst-C/MAR/LA0015/2013]; [UID/Multi/04423/2013]info:eu-repo/semantics/publishedVersio

Susceptibility testing of Candida albicans and Candida glabrata to Glycyrrhiza glabra L.

Medicinal plants and their botanical formulations have gained a pivotal attention among scientific researchers mainly due to its action as health promoters. Licorice (Glycyrrhiza glabra L.) constitutes a great example, with an increasingly evidenced antimicrobial potential. Opportunistic yeast infections constitute an alarming public health problem, highly exacerbated by the inefficacy of antifungal drugs and the increase of drug-resistant microorganisms, being Candida species one of the most common invaders. The present work aims to assess Candida glabrata and Candida albicans susceptibility to G. glabra methanol: water extract by using flow cytometry and transmission electron microscopy techniques. After 5 minutes, licorice extract (1.5 mg/mL) altered Candida membrane potential. Within an hour, it induced primary damages on Candida species cells, causing cell cytoplasm disorganization with high evidence of cell membrane invaginations, making cells turgid. Thus, based on the current findings, licorice extract seems to be a promising anti-Candida agent, without presenting any toxic potential at the effective concentrations used.The authors are grateful to Foundation for Science and Technology (FCT, Portugal) for N. Martins grant (SFRH/BD/87658/2012) and financial support to the research centre CIMO (strategic project PEst-OE/ AGR/UI0690/2014). This work was also supported by the Programa Operacional, Fatores de competitividade – COMPETE and by national funds through FCT – Fundação para a Ciência e a Tecnologia on the scope of the projects FCT PTDC/SAU-MIC/119069/2010, RECI/EBBEBI/0179/2012 and PEst-OE/EQB/LA0023/2013. The authors thank the Project “BioHealth – Biotechnology and Bioengineering approaches to improve health quality”, Ref. NORTE-07-0124-FEDER-000027, cofunded by the Programa Operacional Regional do Norte (ON.2–O Novo Norte), QREN, FEDER.info:eu-repo/semantics/publishedVersio

A genotypic analysis of five P. aeruginosa strains after biofilm infection by phages targeting different cell surface receptors

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2017.01229/full#supplementary-materialAntibiotic resistance constitutes one of the most serious threats to the global public health and urgently requires new and effective solutions. Bacteriophages are bacterial viruses increasingly recognized as being good alternatives to traditional antibiotic therapies. In this study, the efficacy of phages, targeting different cell receptors, against Pseudomonas aeruginosa PAO1 biofilm and planktonic cell cultures was evaluated over the course of 48h. Although significant reductions in the number of viable cells were achieved for both cases, the high level of adaptability of the bacteria in response to the selective pressure caused by phage treatment resulted in the emergence of phage-resistant variants. To further investigate the genetic makeup of phage-resistant variants isolated from biofilm infection experiments, some of these bacteria were selected for phenotypic and genotypic characterization. Whole genome sequencing was performed on five phage-resistant variants and all of them carried mutations affecting the galU gene as well as one of pil genes. The sequencing analysis further revealed that three of the P. aeruginosa PAO1 variants carry large deletions (> 200 kbp) in their genomes. Complementation of the galU mutants with wild-type galU in trans restored LPS expression on the bacterial cell surface of these bacterial strains and rendered the complemented strains to be sensitive to phages. This provides unequivocal evidence that inactivation of galU function was associated with resistance to the phages that uses LPS as primary receptors. Overall, this work demonstrates that P. aeruginosa biofilms can survive phage attack and develop phage-resistant variants exhibiting defective LPS production and loss of type IV pili that are well adapted to the biofilm mode of growth.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the project PTDC/BBB-BSS/6471/2014; the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER006684) and BioTecNorte operation (NORTE-01-0145-FEDER000004)funded by European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte. SS is an FCT investigator (IF/01413/2013). Work done in the lab of JL is supported by Canadian Institutes of Health Research (Grant MOP-14687). YH was a recipient of a postdoctoral fellowship award from Cystic Fibrosis, and JL holds a Canada Research Chair in Cystic Fibrosis and Microbial Glycobiology funded by the Canadian Foundation of Innovation. Work done in the lab of CK is supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada(#371639).info:eu-repo/semantics/publishedVersio

Processed beetroot (Beta vulgaris L.) as a natural antioxidant in mayonnaise: Effects on physical stability, texture and sensory attributes

AbstractThe oxidative and physical stability of the reformulated mayonnaise with processed beetroot was investigated and compared with a control (mayonnaise without beetroot) and a commercially available product. Processing of beetroot had an impact on the structural integrity of the antioxidants present. Microwaving (960W for 7min) was advantageous for preserving the betalain and polyphenol content of beetroot compared to roasting (180°C for 90min) and boiling (100°C for 30min). The oxidative stability of mayonnaise samples was determined by Rancimat and the thiobarbituric (TBA) assay. The addition of microwaved beetroot significantly enhanced the oxidative stability of mayonnaise at the end of a storage period of 4 weeks (4°C). Although no significant differences (p>0.05) were detected between the mayonnaise samples containing beetroot and the commercial control, the latter was less susceptible to oxidation during storage. The turbiscan stability index (TSI) revealed that the commercial mayonnaise was less prone to destabilization phenomena. All the textural parameters increased with the incorporation of beetroot. The sensory evaluation revealed that, with the exception of graininess and uniformity, most of the sensory attributes are preserved if not improved with the addition of beetroot

Dietary Tryptophan Induces Opposite Health-Related Responses in the Senegalese Sole (Solea senegalensis) Reared at Low or High Stocking Densities With Implications in Disease Resistance

High rearing densities are typical conditions of both inland and onshore intensive aquaculture units. Despite obvious drawbacks, this strategy is nonetheless used to increase production profits. Such conditions inflict stress on fish, reducing their ability to cope with disease, bringing producers to adopt therapeutic strategies. In an attempt to overcome deleterious effects of chronic stress, Senegalese sole, Solea senegalensis, held at low (LD) or high density (HD) were fed tryptophan-supplemented diets with final tryptophan content at two (TRP2) or four times (TRP4) the requirement level, as well as a control and non-supplemented diet (CTRL) for 38 days. Fish were sampled at the end of the feeding trial for evaluation of their immune status, and mortalities were recorded following intra-peritoneal infection with Photobacterium damselae subsp. piscicida. Blood was collected for analysis of the hematological profile and innate immune parameters in plasma. Pituitary and hypothalamus were sampled for the assessment of neuro-endocrine-related gene expression. During the feeding trial, fish fed TRP4 and held at LD conditions presented higher mortalities, whereas fish kept at HD seemed to benefit from this dietary treatment, as disease resistance increased over that of CTRL-fed fish. In accordance, cortisol level tended to be higher in fish fed both supplemented diets at LD compared to fish fed CTRL, but was lower in fish fed TRP4 than in those fed TRP2 under HD condition. Together with lower mRNA levels of proopiomelanocortin observed with both supplementation levels, these results suggest that higher levels of tryptophan might counteract stress-induced cortisol production, thereby rendering fish better prepared to cope with disease. Data regarding sole immune status showed no clear effects of tryptophan on leucocyte numbers, but TRP4-fed fish displayed inhibited alternative complement activity (ACH50) when held at LD, as opposed to their HD counterparts whose ACH50 was higher than that of CTRLfed fish. In conclusion, while dietary tryptophan supplementation might have harmful effects in control fish, it might prove to be a promising strategy to overcome chronic stress-induced disease susceptibility in farmed Senegalese sole