Development of a loop-mediated isothermal amplification assay for the rapid detection of Styphnolobium japonicum (L.) Schott as an adulterant of Ginkgo biloba (L.)

Species adulteration is a concern in herbal products, especially when plant substitutes of lower economic value replace valuable botanicals. Styphnolobium japonicum is well known as a potential adulterant of Ginkgo biloba, which is one of the most demanded medicinal plants due to its wide use in pharmaceuticals, food supplements, and traditional medicine. Despite bearing some resemblance to ginkgo's flavonol composition, S. japonicum lacks many of G. biloba's desired therapeutic properties. To prevent adulteration practices, it is crucial to implement rigorous quality control measures, including fast and simple diagnostic tools that can be used on-field. Purpose: This study aims to develop for the first time a species-specific loop-mediated isothermal amplification (LAMP) method for the fast identification of S. japonicum in ginkgo-containing products. Methods: A set of four specific primers (SjF3, SjB3, SjFIP, and SjBIP) and loop primers (SjLF and SjLB) were designed for a LAMP based assay using the 5.8S partial sequence and the internal transcribed spacer 2 of nuclear ribosomal DNA of S. japonicum. Results: The successful amplification of the LAMP assay was inspected through visual detection, with the highest intensity recorded at the optimal conditions set at 68 °C for 40 min. The primers showed high specificity and were able to accurately discriminate S. japonicum from G. biloba and 49 other species of medicinal plants. Furthermore, the proposed LAMP assay proved to be fast, selective, and highly sensitive, as demonstrated by the absolute and relative limits of detection, which were reached at 0.5 pg for S. japonicum DNA and 0.01 % S. japonicum in G. biloba, respectively. Conclusions: This novel approach allows easy identification and discrimination of S. japonicum as a potential adulterant of G. biloba, thus being a useful tool for quality control. Compared to chromatographic or PCR-based methods, the assay proved to be fast, sensitive and did not require expensive equipment, thus offering the possibly usage in field analysis.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through the project “POIROT: novel methods and approaches for detecting the illegal addition of Pharmaceutical drugs and bOtanIcal adulteRatiOn in planT food supplements” (PTDC/SAU-PUB/3803/2021) and through national funds FCT/MCTES (PIDDAC) to CIMO (UIDB/00690/2020 and UIDP/ 00690/2020) and SusTEC (LA/P/0007/2020). M. Honrado is grateful to FCT for PhD grant 2021.08119.BD financed by POPH-QREN (subsidised by FSE and MCTES).info:eu-repo/semantics/publishedVersio

Engineering a biosynthetic pathway for high-value glycosaminoglycans production

info:eu-repo/semantics/publishedVersio

Effect of sub-lethal chemical disinfection on the biofilm forming ability, resistance to antibiotics and expression of virulence genes of Salmonella Enteritidis biofilm-surviving cells

Although disinfection procedures are widely implemented in food environments, bacteria can survive and present increased virulence/resistance. Since little is known about these phenomena regarding biofilms, this study aimed to investigate the effect of chemical disinfection on biofilm-derived cells of Salmonella Enteritidis. Using a reference strain (NCTC 13349) and a food isolate (350), biofilm susceptibility to benzalkonium chloride (BAC), sodium hypochlorite (SH) and hydrogen peroxide (HP) was evaluated and biofilms were exposed to sub-lethal concentrations of each disinfectant. Biofilm-derived cells were characterized for their biofilm forming ability, antibiotic resistance and expression of virulence-associated genes. Except for a few instances, disinfectant exposure did not alter antibiotic susceptibility. However, SH and HP exposure enhanced the biofilm forming ability of Salmonella Enteritidis NCTC 13349. After BAC and HP exposure, biofilm-derived cells presented a down-regulation of rpoS. Exposure to BAC also revealed an up-regulation of invA, avrA and csgD on Salmonella Enteritidis NCTC 13349. The results obtained suggest that biofilm-derived cells that survive disinfection may represent an increased health risk.This study was supported by the Portuguese Foundation forScience and Technology (FCT) under the scope of the stra-tegic funding of unit UIDB/04469/2020 and BioTecNorteoperation (NORTE-01-0145-FEDER-000004) funded by theEuropean Regional Development Fund under the scope ofNorte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Structural MRI texture analysis for detecting Alzheimer's disease

Purpose:: Alzheimer’s disease (AD) has the highest worldwide prevalence of all neurodegenerative disorders, no cure, and low ratios of diagnosis accuracy at its early stage where treatments have some effect and can give some years of life quality to patients. This work aims to develop an automatic method to detect AD in 3 different stages, namely, control (CN), mild-cognitive impairment (MCI), and AD itself, using structural magnetic resonance imaging (sMRI). Methods:: A set of co-occurrence matrix and texture statistical measures (contrast, correlation, energy, homogeneity, entropy, variance, and standard deviation) were extracted from a two-level discrete wavelet transform decomposition of sMRI images. The discriminant capacity of the measures was analyzed and the most discriminant ones were selected to be used as features for feeding classical machine learning (cML) algorithms and a convolution neural network (CNN). Results:: The cML algorithms achieved the following classification accuracies: 93.3% for AD vs CN, 87.7% for AD vs MCI, 88.2% for CN vs MCI, and 75.3% for All vs All. The CNN achieved the following classification accuracies: 82.2% for AD vs CN, 75.4% for AD vs MCI, 83.8% for CN vs MCI, and 64% for All vs All. Conclusion:: In the evaluated cases, cML provided higher discrimination results than CNN. For the All vs All comparison, the proposedmethod surpasses by 4% the discrimination accuracy of the state-of-the-art methods that use structural MRI.info:eu-repo/semantics/publishedVersio

Pathway expression optimization using the Ribosome Binding Site (RBS) Calculator tool

Hydroxycinnamic acids and curcumin are plant metabolites with great therapeutic potential, including anti-inflammatory and anticancer activities. In this study, p-coumaric acid, caffeic acid and curcumin were produced in Escherichia coli using an artificial biosynthetic pathway [1]. Their production was induced by heat using the dnaK and ibpA heat shock promoters [2]. The ribosome binding sites (RBSs) used were tested and further optimized for each gene to assure an efficient translation. To optimize the RBSs we used the bioinformatic design tool RBS Calculator (v1.1) developed by Salis Lab (Penn State University) [3]. This tool predicts the translation initiation rate (TIR) of mRNAs and designs synthetic RBS with specific TIRs. This allows to improve the translation efficiency and to reach a desired response and therefore obtain the expected production using novel genes or biosynthetic pathways. Tyrosine ammonia lyase from Rhodotorula glutinis was used to produce p-coumaric acid from tyrosine. p-Coumaric acid was converted to caffeic acid using 4-coumarate 3-hydroxylase from Saccharothrix espanaensis or cytochrome P450 CYP199A2 from Rhodopseudomonas palustris. Curcumin was produced from ferulic acid using 4-coumarate-CoA ligase from Arabidopsis thaliana, diketide-CoA synthase and curcumin synthase from Curcuma longa. The optimization of the RBSs lead to an increase in the production of p-coumaric acid, caffeic acid and curcumin up to 97.8, 11.7 and 14.4 times, respectively. The highest p-coumaric acid, caffeic acid and curcumin production obtained were 2.5 mM, 370 µM and 17 µM, respectively. These results demonstrate that it is of utmost importance to consider the strength of the RBS when designing a biosynthetic pathway and user-friendly bioinformatic tools such as RBS Calculator can be very useful for that purpose. References: [1] J. L. Rodrigues, M. R. Couto, R. G. Araújo, K. L. J. Prather, L. D. Kluskens, L. R. Rodrigues. Hydroxycinnamic acids and curcumin production in engineered Escherichia coli using heat shock promoters, Biochemical Engineering Journal, 125, 41-49, 2017. [2] J. L. Rodrigues, M. Sousa, K. L. J. Prather, L. D. Kluskens, L. R. Rodrigues. Selection of Escherichia coli heat shock promoters toward their application as stress probes, Journal of Biotechnology, 188, 61-71, 2014. [3] A. E. Borujeni, A. S. Channarasappa, H. M. Salis. Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites, Nucleic Acids Research, 42, 26462659, 2014.info:eu-repo/semantics/publishedVersio

Hyperthermia produced by magnetic nanoparticles as an alternative method to control a major foodborne pathogen

NanoSpain 2017Salmonella enterica is responsible for the majority of the reported foodborne outbreaks, and that is why it is considered one of the most important foodborne pathogens nowadays [1]. Like many others bacteria, S. enterica can survive disinfection and resist a wide variety of biocidal agents [2]. Nowadays, the synthesis of superparamagnetic nanoparticles (MNPs) and its application in magnetic hyperthermia (MH) is of great interest, with MH being recently reported as a viable alternative to traditional disinfection methods against bacteria [3]. However, fundamental studies comprising the MH effect on different populations of planktonic cells and biofilm cells are scarce. Therefore, this work aimed at evaluating the effect of MH on different populations of planktonic cells and biofilms of S. enterica. The work was performed using a S. enterica collection strain (NCTC 13349), which different planktonic cell populations (lag, exponential, and stacionary phase) were adjusted to a final concentration of 1 × 108 cells/ml, while biofilms were formed in silicone coupons. Samples containing both magnetite nanoparticles and S. enterica cells or biofilms have been subjected to an alternating magnetic field of chosen amplitude 100 Oe with frequency of 873 kHz until different temperatures were reached. In order to evaluate the bactericidal effect of MH, survival of planktonic and biofilm cells was determined by colony forming unit (CFU) enumeration. Based on the most relevant results, cell membrane integrity and the effects of MH on cells surface and biofilm structure were analysed through microscopy techniques. Results showed that the high structural-magnetic quality magnetite nanoparticles used were effective against all planktonic cell populations and biofilms under an oscillating magnetic field. In fact, MNPs-based hyperthermia was able to promote a significant cell viability reduction on all planktonic cell populations both bacterial lyfe styles. Nonetheless, planktonic cells were more tolerant to MH than biofilms, possibly due to diffusion limitations along these bacterial communities. Microscopy images of planktonic cells and biofilms showed that MH can affect cell membrane integrity as well as the biofilms structure. In conclusion, this work presents evidences of the bactericidal effect of MH produced by MNPs against S. enterica, both regarding planktonic populations and biofilms. This ability of MH to control a major foodborne pathogen constitutes a novel contribution to the finding of new useful applications of hyperthermia.info:eu-repo/semantics/publishedVersio

Lactobacillus crispatus represses vaginolysin expression by BV associated Gardnerella vaginalis and reduces cell cytotoxicity

Using a chemically-defined medium simulating genital tract secretions, we have shown that pre-adhering Lactobacillus crispatus to Hela epithelial cells reduced cytotoxicity caused by Gardnerella vaginalis. This effect was associated to the expression of vaginolysin and was specific to L. crispatus interference, as other vaginal facultative anaerobes had no protective effect.This work was supported by Portuguese National Funds (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684). JC, and MER acknowledge the financial support of individual Grants SFRH/BD/93963/2013, and SFRH/BPD/95401/2013 respectively. NC is an Investigador FCT.info:eu-repo/semantics/publishedVersio

Saccharomyces cerevisiae as a host for chondroitin production

Chondroitin is a glycosaminoglycan that has gained widespread use in nutraceuticals and pharmaceuticals, mainly for treating osteoarthritis. Traditionally, it has been extracted from animal cartilage but recently, biotechnological processes have emerged as a commercial alternative to avoid the risk of viral or prion contamination and offer a vegan-friendly source. Typically, these methods involve producing the chondroitin backbone using pathogenic bacteria and then modifying it enzymatically through the action of sulfotransferases. Despite the challenges of expressing active sulfotransferases in bacteria, the use of eukaryotic microorganisms is still limited to a few works using Pichia pastoris. To create a safer and efficient biotechnological platform, we constructed a biosynthetic pathway for chondroitin production in S. cerevisiae as a proof-of-concept. Up to 125 mg/L and 200 mg/L of intracellular and extracellular chondroitin were produced, respectively. Furthermore, as genome-scale models are valuable tools for identifying novel targets for metabolic engineering, a stoichiometric model of chondroitin-producing S. cerevisiae was developed and used in optimization algorithms. Our research yielded several novel targets, such as uridine diphosphate (UDP)-Nacetylglucosamine pyrophosphorylase (QRI1), glucosamine-6-phosphate acetyltransferase (GNA1), or N-acetylglucosamine-phosphate mutase (PCM1) overexpression, that might enhance chondroitin production and guide future experimental research to develop more efficient host organisms for the biotechnological production process.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit with DOI 10.54499/UIDB/04469/2020. The authors acknowledge FCT for funding MRC doctoral grant SFRH/BD/132998/2017 and further extension COVID/BD/152454/2022.info:eu-repo/semantics/publishedVersio

Caracterização do património geomorfológico do Parque Natural do Douro Internacional (NE de Portugal) com vista à sua valorização

O Parque Natural do Douro Internacional (PNDI) é uma área protegida pertencente à Rede Nacional de Áreas Protegidas, sob a alçada do Instituto da Conservação da Natureza e da Biodiversidade. O Parque localiza-se no nordeste transmontano, numa área de 851 km2, que acompanha longitudinalmente os rios Douro e Águeda, através de um troço fronteiriço e ao longo de 130 km. Os vales do tipo canhão fluvial com as sua vertentes abruptas, as arribas, destacam-se entre outras geoformas no PNDI. O presente trabalho visa caracterizar e quanti!car a relevância do Património Geomorfológico, inserido no riquíssimo Património Geológico existente na área, bem como a apresentação de propostas de valorização. Foi feita a caracterização dos geomorfossítios inventariados no âmbito dum projecto anterior, tendo-se concluído que os aspectos de maior importância da paisagem do PNDI são o Planalto Mirandês, os relevos residuais, as geoformas graníticas e os vales profundos do rio Douro e afluentes. De seguida, procedeu-se à quantificação da relevância, utilizando uma adaptação dos métodos propostos por Cendrero (2000) e Brilha (2005) para o Património Geológico, obtendo-se uma seriação dos geomorfossítios quanti!cados, o que permite concluir quais os locais com maior potencial para valorização e divulgação. Para a valorização destes geomorfossítios propõem-se várias estratégias como a implementação de painéis interpretativos temáticos, inseridos num percurso rodoviário com o tema “Rota das Arribas”, passando pelos miradouros mais emblemáticos dos rios Douro e Águeda. O Património Geomorfológico do PNDI é um dos ex-libris do Parque, pelo que deverá ser valorizado, constituindo uma importante valência para o impulso do geoturismo na região.The International Douro Natural Park (IDNP) is a protected area that belongs to the Protected Areas National Network, managed by the Nature and Biodiversity Conservation Institute. It is located in northeastern Portugal, with an area of 851 km2. The Park follows the Douro and Águeda rivers, through the border with Spain, along 130 km. The fluvial canyons and cliffs associated with these rivers are important landscape elements in the IDNP. The present work intends to characterize and quantify the relevance of Geomorphological Heritage, inserted in the rich Geological Heritage of this area, as also to present valorization strategies. A characterization of potential geomorphosites identified in a previous project was developed, which highlights the most important elements of the IDNP landscape, namely the Miranda Plain, residual reliefs, granitic landforms and deep valleys of the Douro and the Águeda rivers. A quantitative assessment was also applied, based on a modified version of the models proposed by Cendrero (2000) and Brilha (2005) for the Geological Heritage. A final ranking of the geomorphosites was proposed which establishes the valuable sites that must be included in conservation strategies or selected for geotourism and educational programs. Several strategies were proposed to value these geomorphosites, such as thematic interpretative panels and a car route, the “Arribas Route”, joining the most important viewpoints of the Douro and the Águeda rivers. The Geomorphological Heritage of the IDNP is an ex-libris of this Natural Park. It must be recognized, valued and considered as a major contribution to the geotourism in the region

Synthetic biology approaches to engineer polyphenols microbial cell factories

Polyphenols are secondary metabolites isolated from plants that can be divided into flavonoids, stilbenoids, curcuminoids, coumarins, polyphenolic amides and lignans. These exhibit diverse biological and potential therapeutic activities including antioxidant, anti-inflammatory and anticancer, among others. Despite all this potential, extracting polyphenols from plants is not straightforward given the low yields of the process. The extracted amounts are not sufficient to respond to the increasing demand for polyphenols, the process is expensive and unfriendly for the environment. Hence, developing microbial cell factories to effectively produce polyphenols arises as an attractive way to address the mentioned limitations and produce high amounts of these compounds. Advances in the metabolic engineering and synthetic biology fields have been key in the design of efficient and robust microbial cell factories, mainly due to the development of proper molecular biology tools, as well as to the unravelling of new enzymes in plants or other organisms to better engineer such heterologous pathways. Several hosts have been explored as potential polyphenols microbial cell factories. However, there is still a long way before this production at an industrial scale can become a reality. The perspectives and current challenges resulting from these developments will be discussed.info:eu-repo/semantics/publishedVersio