In vitro selection of DNA aptamers against staphylococcal enterotoxin A.

Staphylococcal enterotoxin A (SEA) is the most frequently reported in staphylococcal food poisoning (SFP) outbreaks. Aptamers are single-stranded nucleic acids that are seen as promising alternatives to antibodies in several areas, including diagnostics. In this work, systematic evolution of ligands by exponential enrichment (SELEX) was used to select DNA aptamers against SEA. The SELEX protocol employed magnetic beads as an immobilization matrix for the target molecule and real-time quantitative PCR (qPCR) for monitoring and optimizing sequence enrichment. After 10 selection cycles, the ssDNA pool with the highest affinity was sequenced by next generation sequencing (NGS). Approximately 3 million aptamer candidates were identified, and the most representative cluster sequences were selected for further characterization. The aptamer with the highest affinity showed an experimental dissociation constant (KD) of 13.36 ± 18.62 nM. Increased temperature negatively affected the affinity of the aptamer for the target. Application of the selected aptamers in a lateral flow assay demonstrated their functionality in detecting samples containing 100 ng SEA, the minimum amount capable of causing food poisoning. Overall, the applicability of DNA aptamers in SEA recognition was demonstrated and characterized under different conditions, paving the way for the development of diagnostic tools.

Contribution of nanotechnology to greater efficiency in animal nutrition and production.

Feed costs present a major burden in animal production for human consumption, representing a key opportunity for cost reduction and profit improvement. Nanotechnology offers potential to increase productivity by creating higher-quality and safer products. The feed sector has benefited from the use of nanosystems to improve the stability and bioavailability of feed ingredients. The development of nanotechnology products for feed must consider the challenges raised by biological barriers as well as regulatory requirements. While some nanotechnology-based products are already commercially available for animal production, the exponential growth and application of these products requires further research ensuring their safety and the establishment of comprehensive legislative frameworks and regulatory guidelines. Thus, this article provides an overview of the current state of the art regarding nanotechnology solutions applied in feed, as well as the risks and opportunities aimed to help researchers and livestock producers.

The potential of tailed amplicons for SARS-CoV-2 detection in Nucleic Acid Lateral Flow Assays.

Nucleic Acid Lateral Flow Assays (NALFAs) are a promising solution for the point-of-care detection of viruses like SARS-CoV-2. However, they show some drawbacks, such as the great dependency on the use of antibodies and the need for post-amplification protocols that enable the preparation of amplicons for effective readings, as well as low sensitivity. Here, we developed amplicons of a specific SARS-CoV-2 gene tailed with single-strand DNA (ssDNA) sequences to hybridize with DNA probes immobilized on the NALFA strips, thus overcoming the aforementioned problems. Results have shown that tailed primers have not compromised the amplification efficiency and allowed the correct detection of the amplicons in the lateral flow strip. This approach has presented a limit of detection (LOD) of 25 RNA copies /reaction mix (1 copy/µL) and the test of cross-reactivity with other related viruses has not shown any cross-reactivity. Twenty clinical samples were evaluated by NALFA and simultaneously compared with the gold standard RT-qPCR protocol, originating equal results. Although the number of clinical specimens tested being relatively small, this indicates a sensitivity and specificity both of 100%. In short, an alternative NALFA was successfully implemented, rendering an accurate route for SARS-CoV-2 diagnosis, compatible with low-resource settings.

SARS-CoV-2 Diagnostics Based on Nucleic Acids Amplification: From Fundamental Concepts to Applications and Beyond.

COVID-19 pandemic ignited the development of countless molecular methods for the diagnosis of SARS-CoV-2 based either on nucleic acid, or protein analysis, with the first establishing as the most used for routine diagnosis. The methods trusted for day to day analysis of nucleic acids rely on amplification, in order to enable specific SARS-CoV-2 RNA detection. This review aims to compile the state-of-the-art in the field of nucleic acid amplification tests (NAATs) used for SARS-CoV-2 detection, either at the clinic level, or at the Point-Of-Care (POC), thus focusing on isothermal and non-isothermal amplification-based diagnostics, while looking carefully at the concerning virology aspects, steps and instruments a test can involve. Following a theme contextualization in introduction, topics about fundamental knowledge on underlying virology aspects, collection and processing of clinical samples pave the way for a detailed assessment of the amplification and detection technologies. In order to address such themes, nucleic acid amplification methods, the different types of molecular reactions used for DNA detection, as well as the instruments requested for executing such routes of analysis are discussed in the subsequent sections. The benchmark of paradigmatic commercial tests further contributes toward discussion, building on technical aspects addressed in the previous sections and other additional information supplied in that part. The last lines are reserved for looking ahead to the future of NAATs and its importance in tackling this pandemic and other identical upcoming challenges.

Prevalence and Diversity of Staphylococcus aureus and Staphylococcal Enterotoxins in Raw Milk From Northern Portugal.

Staphylococcus aureus and staphylococcal enterotoxins are a serious public health concern associated with hospital and community-acquired illnesses. Dairy animals frequently shed S. aureus into the milk supply which can lead to food poisoning in humans. This study aims to investigate the prevalence and genetic diversity of S. aureus and staphylococcal enterotoxins in raw milk from the main dairy region of mainland Portugal. S. aureus was found in 53.0% (95% CI: 40.6-65.4%) of 100 raw cow's milk samples collected from bulk cooling tanks. The highest contamination level was 3.4 log10 CFU.mL-1, and in some samples more than one S. aureus strain was identified. Staphylococcal enterotoxins (SEA-SEE) were detected in one sample. Spa typing revealed 62 distinct S. aureus isolates, being t529 (17.7%, 95% CI: 8.2-27.3%) and t1403 (16.1%, 95% CI: 7.0-25.3%) the predominant types, commonly associated with livestock infection or carriage. The antimicrobial susceptibility test showed that 35.5% of the S. aureus isolates were resistant to at least one antimicrobial agent, with resistance to penicillin being the highest (32.3%, 95% CI: 20.6-43.9%) followed by tetracycline (24.2%, 95% CI: 13.5-34.9%), ciprofloxacin (16.1%, 95% CI: 7.0-25.3%) and chloramphenicol (16.1%, 95% CI: 7.0-25.3%). Moreover, five isolates (8.1%, 95% CI: 1.3-14.8%) were identified as methicillin-resistant S. aureus (MRSA, cefoxitin resistant). Regarding virulence/resistance genes, 46,8% (95% CI: 34.4-59.2%) isolates harbored at least one enterotoxin-encoding gene, and the seg gene was the most frequently detected (41.9%, 95% CI: 29.7-54.2%) followed by the sei (40.3%, 95% CI: 28.1-52.5%), sec (6.5%, 95% CI: 0.3-12.6%), seh (4.8%, 95% CI: 0.0-10.2%), and sea (1.6%, 95% CI: 0.0-4.7%) genes. Five (8.1%, 95% CI: 1.3-14.8%) non-enterotoxigenic isolates carried the mecA gene (corresponding to isolates phenotypically classified as MRSA), and 4.8% (95% CI: 0.0-10.2%) enterotoxigenic strains also had the tsst-1 gene. Our study confirm that raw milk can be a zoonotic source of S. aureus, including enterotoxigenic and MRSA strains. Furthermore, the majority of enterotoxigenic isolates were found to contain genes encoding SEs (SEG, SEH and SEI) not routinely screened. This shows the need for a broader SE screening in food safety control, as well as the relevance of risk mitigation measures to control S. aureus transmission along the food chain in Portugal.

Modelling aptamers with nucleic acid mimics (NAM): From sequence to three-dimensional docking.

Aptamers are single-stranded oligonucleotides, formerly evolved by Systematic Evolution of Ligands by EXponential enrichment (SELEX), that fold into functional three-dimensional structures. Such conformation is crucial for aptamers' ability to bind to a target with high affinity and specificity. Unnatural nucleotides have been used to develop nucleic acid mimic (NAM) aptamers with increased performance, such as biological stability. Prior knowledge of aptamer-target interactions is critical for applying post-SELEX modifications with unnatural nucleotides since it can affect aptamers' structure and performance. Here, we describe an easy-to-apply in silico workflow using free available software / web servers to predict the tertiary conformation of NAM, DNA and RNA aptamers, as well as the docking with the target molecule. Representative 2'-O-methyl (2'OMe), locked nucleic acid (LNA), DNA and RNA aptamers, with experimental data deposited in Protein Data Bank, were selected to validate the workflow. All aptamers' tertiary structure and docking models were successfully predicted with good structural similarity to the experimental data. Thus, this workflow will boost the development of aptamers, particularly NAM aptamers, by assisting in the rational modification of specific nucleotides and avoiding trial-and-error approaches.

Improving aptamer performance with nucleic acid mimics: de novo and post-SELEX approaches.

Aptamers are structural single-stranded oligonucleotides generated in vitro to bind to a specific target molecule. Aptamers' versatility can be enhanced with nucleic acid mimics (NAMs) during or after a selection process, also known as systematic evolution of ligands by exponential enrichment (SELEX). We address advantages and limitations of the technologies used to generate NAM aptamers, especially the applicability of existing engineered polymerases to replicate NAMs and methodologies to improve aptamers after SELEX. We also discuss the limitations of existing methods for sequencing NAM sequences and bioinformatic tools to predict NAM aptamer structures. As a conclusion, we suggest that NAM aptamers might successfully compete with molecular tools based on proteins such as antibodies for future application.

Evaluation of antibacterial activity of caffeic acid encapsulated by ß-cyclodextrins.

CONTEXT: Caffeic acid is described as antibacterial, but this bioactive molecule has some issues regarding solubility and stability to environmental stress. Thus, encapsulation devices are required. OBJECTIVE: The aim of this work was to study the effect of the caffeic acid encapsulation by cyclodextrins on its antibacterial activity. MATERIALS AND METHODS: The interactions between the caffeic acid and three cyclodextrins (ß-cyclodextrin (ßCD), 2-hydroxypropyl-ß-cyclodextrin (HPßCD) and methyl-ß-cyclodextrin were study. RESULTS AND DISCUSSION: The formation of an aqueous soluble inclusion complex was confirmed for ßCD and HPßCD with a 1:1 stoichiometry. The ßCD/caffeic acid complex showed higher stability than HPßCD/caffeic acid. Caffeic acid antibacterial activity was similar at pH 3 and pH 5 against the three bacteria (K. pneumoniae, S. epidermidis and S. aureus). CONCLUSIONS: The antibacterial activity of the inclusion complexes was described here for the first time and it was shown that the caffeic acid activity was remarkably enhanced by the cyclodextrins encapsulation.

Caffeic acid loading wound dressing: physicochemical and biological characterization.

AIM: Caffeic acid has been described as active against bacteria commonly isolated from wound infections. However, its low stability and poor solubility reduce caffeic acid applicability as a pharmaceutical product. These parameters can be enhanced by complexation with cyclodextrin. The main goal of the present work was to incorporate caffeic acid on cyclodextrin-based hydrogels capable of controlled delivery, in order to be used as antibacterial wound dressing. MATERIALS & METHODS: Cyclodextrin-based hydrogels were prepared by direct crosslinking of ß-cyclodextrin or hydroxypropyl-ß-cyclodextrin with 1,4-butanediol diglycidyl ether in the presence of hydroxypropyl methylcellulose. RESULTS: The hydrogels obtained combine good physicochemical properties (viscoelasticity, superabsorbency and high ability to retain and deliver caffeic acid) with the preservation of caffeic acid' antibacterial activity and effect on fibroblasts, with gel-ß-cyclodextrin the most suited. CONCLUSION: The hydrogels obtained could be useful as caffeic acid delivery-system devices for the treatment of wound infections.

Antibacterial potential of northeastern Portugal wild plant extracts and respective phenolic compounds.

The present work aims to assess the antibacterial potential of phenolic extracts, recovered from plants obtained on the North East of Portugal, and of their phenolic compounds (ellagic, caffeic, and gallic acids, quercetin, kaempferol, and rutin), against bacteria commonly found on skin infections. The disk diffusion and the susceptibility assays were used to identify the most active extracts and phenolic compounds. The effect of selected phenolic compounds on animal cells was assessed by determination of cellular metabolic activity. Gallic acid had a higher activity, against gram-positive (S. epidermidis and S. aureus) and gram-negative bacteria (K. pneumoniae) at lower concentrations, than the other compounds. The caffeic acid, also, showed good antibacterial activity against the 3 bacteria used. The gallic acid was effective against the 3 bacteria without causing harm to the animal cells. Gallic and caffeic acid showed a promising applicability as antibacterial agents for the treatment of infected wounds.

Cyclodextrins as encapsulation agents for plant bioactive compounds.

Plants possess a wide range of molecules capable of improve healing: fibre, vitamins, phytosterols, and further sulphur-containing compounds, carotenoids, organic acid anions and polyphenolics. However, they require an adequate level of protection from the environmental conditions to prevent losing their structural integrity and bioactivity. Cyclodextrins are cyclic oligosaccharides arising from the degradation of starch, which can be a viable option as encapsulation technique. Cyclodextrins are inexpensive, friendly to humans, and also capable of improving the biological, chemical and physical properties of bioactive molecules. Therefore, the aim of this review is to highlight the use of cyclodextrins as encapsulating agents for bioactive plant molecules in the pharmaceutical field.

Cyclodextrin-based hydrogels toward improved wound dressings.

Optimal wound dressings should be capable of mechanical wound protection and also facilitate the healing process via maintenance of suitable environmental conditions and the controlled delivery of bioactive molecules. Hydrogels present suitable properties for wound-dressing applications such as good biocompatibility, together with a high water content, the latter of which is important for the maintenance of a moist environment and ready removal from the wound with a minimal level of associated pain. However, their properties as drug delivery systems can be improved by the use of cyclodextrins as cross-linking agents. Cyclodextrins have been extensively used as "carriers" on food, textile, cosmetic and, most especially, in the pharmaceutical industry in view of their powerful complexation abilities and biocompatibilities, together with further desirable characteristics. The conjugation of cyclodextrins with hydrogels may allow the achievement of an optimal wound-dressing material, because the hydrogel component will maintain the moist environment required for the healing process, and the cyclodextrin moiety has the ability to protect and modulate the release of bioactive molecules. Therefore, this review aims to gather information regarding cyclodextrin-based hydrogels for possible wound-dressing applications.

Candida albicans virulence and drug-resistance requires the O-acyltransferase Gup1p.

BACKGROUND: GUP1 gene was primarily identified in Saccharomyces cerevisiae being connected with glycerol uptake defects in association with osmotic stress response. Soon after, Gup1p was implicated in a complex and extensive series of phenotypes involving major cellular processes. These include membrane and wall maintenance, lipid composition, bud-site selection, cytoskeleton orientation, vacuole morphology, secretory/endocytic pathway, GPI anchors remodelling, and lipid-ordered domains assembly, which is compatible with their inclusion in the Membrane Bound O-acyl transferases (MBOAT) family. In mammals, it has been described as a negative regulator of the Sonic hedgehog pathway involved in morphogenesis, differentiation, proliferation, among other processes. RESULTS: We show that Candida albicans Gup1p strongly interferes with the capacity of cells to develop hyphae, to adhere, to invade, and to form a biofilm, all of which are significant virulence factors. Furthermore, the mutant colonies exhibited an aberrant morphology/differentiation pattern. Identically to S. cerevisiae, Cagup1Δ null mutant was more resistant to antifungals like fluconazole, ketoconazole, and clotrimazole, and displayed an abnormal even sterol distribution at the plasma membrane. CONCLUSIONS: This work is the first study in the opportunistic yeast Candida albicans, showing a role for the GUP1 gene in virulence as well as in the mechanisms underlying antifungal resistance. Moreover, its impact is even more significant since these results, taken together with all the knowledge about GUP1 gene (from S. cerevisiae and mammals) give consistence to the possibility that Gup1p may be part of a yeast morphogenic pathway parallel to the mammalian Hedgehog.