Multiplex PCR system for fungal pathogen detection

Introduction: The rapid and correct identification of pathogenic species is crucial for several reasons that differ according to the area of interest. In clinical laboratories it is important since knowing the pathogen guides appropriate treatment, dose and duration of therapy. In clinical microbiology laboratories culture and microscopic examination remain the “gold standard”. Biochemical and mass spectometry identification systems, such as the Vitek systems (BioMerieux®) or MALDI-TOF are examples of commercial systems for microbial identification with the advantage of being semi automatic. However, these methods are based on the regrowth and isolation of the microorganism from samples, which in some cases, such as from blood, is time consuming and the success rate of cultivation can be as low as 20%. Recently, several techniques based on the polymerase chain reaction (PCR) have been developed for microbial pathogenic detection and identification particularly Real-Time PCR methods. In this study we developed an alternative PCR based method for the detection of fungal pathogens involved in systemic infections. This is a multiplex PCR method that attributes to each species specific amplicon lengths and a fluoresce dye, according to previously design panels, enabling a fast and reproducible co-amplifying of several loci in a single PCR reaction. Materials and methods: First a panel for species identification, combining fluorescence with molecular weight of specific PCR fragments for Candida and Asprgillus was designed. The PCR fragments obtained are then analysed by capillary electrophoresis and GeneScan fragment analysis. This methodology was optimized using DNA extracted from strains previously identified and, in order to optimize the methodology to clinical samples, we also used serum from healthy donors spiked with different concentrations of fungal DNA. Then the method was tested in DNA extracted from different types of clinical samples, including blood, biopsies and bronchoalveolar lavages of patients with invasive fungal infections (IFI). Results: The optimization of the method, by using DNA from known strains belonging to the target species and strains form other species, showed 100% of specificity. The calculated yield from DNA extracted from serum spiked with fungal DNA was of around 80%. This DNA was then used to determine the sensitivity of the technique and results showed that we were able to obtain amplification products within a range of 1 to 10 pg of the total DNA extracted. Results obtained with DNA extracted from samples of IFI patients, showed that we were able to detect the specific fungal species in 75% of the samples. Several optimizations are being performed. Discussion and conclusions: This new methodology is a promising method since interpretation of results is easy, based on presence/absence of a particular peak of the panel, it is fast, accurate and reproducible and due to the design of the identification panel it is able to identify the pathogenic fungal species involved in mixed infections. Several optimizations are being performed to enhance sensitivity of the method.FEDER through the Programa Operacional Regional Norte, Project U. Norte Inova. JCarvalhoPereira acknowledges FCT for the SFRH/BD/113384/2015 Gran

Phenotypic Diversity of Seminal Root Traits in Bread Wheat Germplasm from Different Origins

Publisher Copyright: © 2022 by the authors.Bread wheat (Triticum aestivum L.) is a major staple crop, and more adapted varieties are needed to ensure productivity under unpredictable stress scenarios resulting from climate changes. In the development of new genotypes, root system traits are essential since roots have a key function in water and nutrient uptake, and root architecture determines the plant’s ability to spatially explore the soil resources. Genetic variation in wheat root system may be assessed at the early stages of development. This study evaluates in vitro and at the seedling stage, the genetic diversity of root growth angle (RGA), seminal root number (SRN), and radicle length (RadL) in 30 bread wheat genotypes from different origins and belonging to distinct evolutive or breeding groups. SRN and RadL were analyzed at 1, 2, 3 and 6 days after sowing (DAS) and RGA was measured through the angle between the first pair of seminal roots. A large variability was found in RGA values that ranged from 63° to 122°. Although differences were found between genotypes within the same groups, the narrower angles tended to occur among landraces, while the higher RGA values were observed in advanced lines and Australian varieties. Differences were also observed as regards the SRN (1.0–3.0, 2.7–4.7, 3.2–5.0 and 4.4–6.3 at 1, 2, 3 and 6 DAS, respectively) and RadL (0.1–1.5, 2.1–5.0, 4.0–7.5 and 5.1–13.7 cm at 1, 2, 3 and 6 DAS, respectively). Genetic variability in root traits at seedling stage allows more rapid selection of genotypes better adapted to environmental and soil constraints, necessary to Portuguese Wheat Breeding Program. It will also contribute to the definition of wheat ideotypes with improved performance under Mediterranean climate conditions.publishersversionpublishe

Grain Composition and Quality in Portuguese Triticum aestivum Germplasm Subjected to Heat Stress after Anthesis

Funding Information: Funding: This work was supported by national funds from Fundação para a Ciência e a Tecnologia (FCT), Portugal, through the Research Unit UIDP/04035/2020 (GeoBioTec). Funding Information: This work was supported by national funds from Funda??o para a Ci?ncia e a Tecnologia (FCT), Portugal, through the Research Unit UIDP/04035/2020 (GeoBioTec).The authors acknowledge GeoBioTec (UIDB/04035/2020) Research Center for support facilities. Publisher Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.Bread wheat (Triticum aestivum) is a major crop worldwide, and it is highly susceptible to heat. In this work, grain production and composition were evaluated in Portuguese T. aestivum germplasm (landraces and commercial varieties), which was subjected to heat after anthesis (grain filling stage). Heat increased the test weight (TW) in Nabão, Grécia and Restauração, indicating an improved flour-yield potential. Mocho de Espiga Branca (MEB) and Transmontano (T94) showed higher thousand-kernel weight (TKW). Gentil Rosso presented increased soluble sugars, which are yeast substrates in the bread-making process. Ardila stood out for its protein increase under heat. Overall SDS was unaffected by higher temperature, but increased in T94, indicating a better dough elasticity for bread-making purposes. Under heat, lipid content was maintained in most genotypes, being endogenous fatty acids (FAs) key players in fresh bread quality. Lipid unsaturation, evaluated through the double bond index (DBI), also remained unaffected in most genotypes, suggesting a lower flour susceptibility to lipoperoxidation. In Grécia, heat promoted a higher abundance of monounsaturated oleic (C18:1) and polyunsaturated linoleic (C18:2) acids, which are essential fatty acids in the human diet. This work highlighted a great variability in most parameters both under control conditions or in response to heat during grain filling. Cluster analysis of traits revealed a lower susceptibility to heat during grain filling in Ardila, Restauração, and Ruivo, in contrast to MEQ, which seems to be more differentially affected at this stage. Characterization and identification of more favorable features under adverse environments may be relevant for agronomic, industrial, or breeding purposes, in view of a better crop adaptation to changing climate and an improved crop sustainability in agricultural systems more prone to heat stress.publishersversionpublishe

Will Casuarina glauca Stress Resilience Be Maintained in the Face of Climate Change?

Actinorhizal plants have been regarded as promising species in the current climate change context due to their high tolerance to a multitude of abiotic stresses. While combined salt-heat stress effects have been studied in crop species, their impact on the model actinorhizal plant, Casuarina glauca, has not yet been fully addressed. The effect of single salt (400 mM NaCl) and heat (control at 26/22 C, supra optimal temperatures at 35/22 C and 45/22 C day/night) conditions on C. glauca branchlets was characterised at the physiological level, and stress-induced metabolite changes were characterised by mass spectrometry-based metabolomics. C. glauca could withstand single salt and heat conditions. However, the harshest stress condition (400 mM NaCl, 45 C) revealed photosynthetic impairments due to mesophyll and membrane permeability limitations as well as major stress-specific differential responses in C and N metabolism. The increased activity of enzymatic ROS scavengers was, however, revealed to be sufficient to control the plant oxidative status. Although C. glauca could tolerate single salt and heat stresses, their negative interaction enhanced the effects of salt stress. Results demonstrated that C. glauca responses to combined salt-heat stress could be explained as a sum of the responses from each single applied stressinfo:eu-repo/semantics/publishedVersio

Shade and Altitude Implications on the Physical and Chemical Attributes of Green Coffee Beans from Gorongosa Mountain, Mozambique

Coffea arabica L. is as a tropical crop that can be grown under monocrop or agroforestry (AFS) systems, usually at altitudes greater than 600 m, with suitable environmental conditions to bean quality. This study aimed to assess the effect of altitude (650, 825, and 935 m) and light conditions (deep shade—DS, and moderate shade—MS provided by native trees, and full Sun—FS) on the physical and chemical attributes of green coffee beans produced in the Gorongosa Mountain. Regardless of altitude, light conditions (mainly MS and FS) scarcely affected most of the studied physical and chemical attributes. Among the few exceptions in physical attributes, bean mass tended to lower values under FS in all three altitudes, whereas bean density increased under FS at 650 m. As regards the chemical compound contents, sporadic changes were found. The rises in trigonelline (MS and FS at 935 m), soluble sugars (FS at 935 m), and the decline in p-coumaric acid (MS and FS at 825 m), may indicate an improved sensory profile, but the rise in FQAs (FS at 825 m) could have a negative impact. These results highlight a relevant uncertainty of the quality changes of the obtained bean. Altitude (from 650 to 935 m) extended the fruit maturation period by four weeks, and altered a larger number of bean attributes. Among physical traits, the average sieve (consistent tendency), bean commercial homogeneity, mass, and density increased at 935 m, whereas the bean became less yellowish and brighter at 825 and 935 m (b*, C* colour attributes), pointing to good bean trade quality, usually as compared with beans from 650 m. Furthermore, at 935 m trigonelline and 5-CQA (MS and FS) increased, whereas FQAs and diCQAs isomers declined (in all light conditions). Altogether, these changes likely contributed to improve the sensory cup quality. Caffeine and p-coumaric acid showed mostly inconsistent variations. Overall, light conditions (FS, MS, or DS) did not greatly and consistently altered bean physical and chemical attributes, whereas altitude (likely associated with lower temperature, greater water availability (rainfall/fog), and extended maturation period) was a major driver for bean changes and improved qualityinfo:eu-repo/semantics/publishedVersio

High-resolution shotgun proteomics reveals that increased air [CO2] amplifies the acclimation response of coffea species to drought regarding antioxidative, energy, sugar, and lipid dynamics

Funding Information: This work received funding support from the European Union's Horizon 2020 research and innovation program (grant agreement No 727934 , project BreedCAFS), and from national funds from Fundação para a Ciência e a Tecnologia, I.P. (FCT) , Portugal, through the project PTDC/ASP-AGR/31257/2017 , and the research units UIDB/00239/2020 ( CEF ), and UIDP/04035/2020 (GeoBioTec) and under the Scientific Employment Stimulus - Individual Call (CEEC Individual) - 2021.01107.CEECIND/CP1689/CT0001 (IM) . Fellowships from the Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil (CNPq) , (to F.M. DaMatta and F.L. Partelli), and Fundação de Amparo à Pesquisa do Estado de Minas Gerais, Brazil (FAPEMIG, projects CRA-RED-00053-16 and APQ 01512-18 , to F.M. DaMatta) are also greatly acknowledged. Publisher Copyright: © 2022 Elsevier GmbHAs drought threatens crop productivity it is crucial to characterize the defense mechanisms against water deficit and unveil their interaction with the expected rise in the air [CO2]. For that, plants of Coffea canephora cv. Conilon Clone 153 (CL153) and C. arabica cv. Icatu grown under 380 (aCO2) or 700 μL L−1 (eCO2) were exposed to moderate (MWD) and severe (SWD) water deficits. Responses were characterized through the activity and/or abundance of a selected set of proteins associated with antioxidative (e.g., Violaxanthin de-epoxidase, Superoxide dismutase, Ascorbate peroxidases, Monodehydroascorbate reductase), energy/sugar (e.g., Ferredoxin-NADP reductase, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, sucrose synthase, mannose-6-phosphate isomerase, Enolase), and lipid (Lineolate 13S-lipoxygenase) processes, as well as with other antioxidative (ascorbate) and protective (HSP70) molecules. MWD caused small changes in both genotypes regardless of [CO2] level while under the single imposition to SWD, only Icatu showed a global reinforcement of most studied proteins supporting its tolerance to drought. eCO2 alone did not promote remarkable changes but strengthened a robust multi-response under SWD, even supporting the reversion of impacts already observed by CL153 at aCO2. In the context of climate changes where water constraints and [CO2] levels are expected to increase, these results highlight why eCO2 might have an important role in improving drought tolerance in Coffea species.publishersversionpublishe

Cytotoxicity assessment of endodontic sealers: metabolic activity, morphology and chromosomal alterations

Introduction: Endodontic treatment aims to eliminate infection of the root canals and fill the dental pulp space, being, the obturation of root canals an important step. The study of the toxicity/biocompatibility of the sealers used to fill the root canals is crucial since they are applied into direct contact with periradicular tissues.There are several types of sealers, categorized according to their main chemical constituents. The aim of this study was to evaluate the cytotoxicity of three root canal sealers, AH Plus, Bio MTA+ and Bio C, on immortalized human gingival fibroblasts. Methods: To study the cytotoxicity of the sealers we performed a Methyltetrazolium (MTT) assay, a study of cell's morphology and a cytogenetic study. Cells were placed in contact with material-conditioned media, for 24 h, at three different concentrations (1, 10 and 100 mg/ml) for the MTT assay. Cell morphology and cytogenetic studies were performed at 100 mg/ml. Cells in normal culture medium were analyzed as control group. Results: MTT assay revealed a cytotoxic effect of Bio MTA+ and Bio C with a growing decrease of metabolic activity with increasing compound concentration, reaching 50% with 100 mg/ml. Regarding the cells morphology, Bio C was the compound that showed a more drastic effect, with a decrease in cell confluence and several morphological changes. AH Plus and Bio MTA+ did not seem to affect the cell confluence, however morphology changes were observed, as compromised cell membranes and loss of cell content. Cytogenetic study was thus far only performed with AH Plus. Since there was a severe decrease of mitotic index after treatment, it was not yet possible to obtain sufficient metaphases, even after several cytogenetic harvesting procedures, but, so far, no relevant structural or numerical changes were observed. Discussion: This preliminary study allowed us to verify that these root canal sealers exhibit some cytotoxicity, depending on the concentration used. Although more studies are still needed, this work could be important to both, help in the selection of the most appropriate compounds for clinical practice and to determine the maximum recommended amounts of each sealer.info:eu-repo/semantics/publishedVersio

Mitigation of the Negative Impact of Warming on the Coffee Crop: The Role of Increased Air [CO2] and Management Strategies

Crop sustainability can be threatened by new environmental challenges regarding predicted climate changes and global warming. Therefore, the study of real biological impacts of future environmental conditions (e.g., increased air [CO2], supra-optimal temperature and water scarcity) on crop plants, as well as the re-evaluation of management procedures and strategies, must be undertaken in order to improve crop adaptation and promote mitigation of negative environmental impacts, thus affording crop resilience. Coffee is a tropical crop that is grown in more than 80 countries, making it one of the world’s most traded agricultural products, while involving millions of people worldwide in the whole chain of value. It has been argued that this crop will be highly affected by climate changes, resulting in decreases in both suitable areas for cultivation and productivity, as well as impaired beverage quality in the near future. Here, we report recent findings regarding coffee species exposure to combined supra-optimal air temperatures and enhanced air [CO2], and impacts of drought stress on the crop. Ultimately, we discuss key strategies to improve coffee performance in the context of new environmental scenarios. The recent findings clearly show that high [CO2] has a positive impact on coffee plants, increasing their tolerance to high temperatures. This has been related to a better plant vigor, to the triggering of protective mechanisms, and to a higher functional status of the photosynthetic machinery. Even so, coffee plant is expected to suffer from water scarcity in a changing world. Therefore, discussion is focused on some important management strategies (e.g., shade systems, crop management and soil covering and terracing), which can be implemented to improve coffee performance and sustain coffee production in a continually changing environment

Lipid profile adjustments may contribute to warming acclimation and to heat impact mitigation by elevated [CO2] in Coffea spp

This work was supported by European Union, Program Horizon 2020, call H2020-SFS-2016-2, action RIA, and Portuguese national funds from Fundacao para a Ciencia e a Tecnologia (project PTDC/ASP-AGR/31257/2017; Funding from CNPq (fellowships to E. Campostrini, F.L. Partelli, and F.M. DaMatta) is also acknowledged.An unexpected heat resilience, and the mitigation of heat impacts by elevated [CO2] were recently reported in Coffea spp. Plants must maintain membrane fluidity and integrity to cope with temperature changes, which requires an adequate lipid dynamics. This work provides the lipid profile (galactolipids, GL; phospholipids, PL; sulfolipids, SL) of chloroplast membranes, and the expression of a set of genes related to lipid metabolism in Coffea arabica L. (cv. Icatu and IPR108) and C. canephora cv. Conilon CL153, under elevated [CO2] (380 or 700 μL L−1), heat (25/20, 31/25, 37/30 and 42/34 °C, day/night) and their interaction. Major membrane lipids alterations, different among genotypes, included: A) responsiveness of total fatty acids (TFAs) synthesis to [CO2] (except IPR108) and heat (except CL153); stronger remodeling (unsaturation degree) in the 700-plants from 37/30 °C to 42/34 °C, coordinated at transcriptional level with the down-regulation of fatty acid desaturase FAD3 gene (C. arabica) and up-regulation of lipoxygenase genes LOX5A (CL153 and Icatu) and LOX5B (Icatu) at the highest temperature; B) quantitative and qualitative modifications in GL (monogalactosyldiacylglycerol, MGDG; digalactosyldiacylglycerol, DGDG), PL (phosphatidylcholine, PC; phosphatidylglycerol, PG), and SL (sulfoquinovosyldiacylglycerol, SQDG) classes, prompted by heat, elevated [CO2], and, especially, the interaction, in CL153 and Icatu. Overall membrane enrichment with MGDG and DGDG as a result of heat and [CO2] interaction in these genotypes, but at the highest temperature only in Icatu the high [CO2] maintained greater contents and unsaturation values of these GLs than in the 380-plants. C) Among PL classes, PG seems to play an active role in heat acclimation of C. arabica genotypes, increasing in 700-plants at 42/34 °C. Globally, Icatu often showed changes closer to those of heat tolerant cv. CL153 than to cv. IPR108. Overall, lipid profile adjustments in chloroplast membranes, from TFAs bulk until FA unsaturation within each class, are expected to contribute to long-term acclimation to climate changes in coffee plant.publishersversionpublishe

The legal and ethical framework governing Body Donation in Europe-1st update on current practice

Previously, we have reported on the legal and ethical aspects and current practice of body donation in several European countries, reflecting cultural and religious variations as well as different legal and constitutional frameworks. We have also established good practice in body donation. Here we shall further extend the legal and ethical frameworks in place and also focus on novelties in the law and different directives. Of particular interest are points that address the commercialization of human bodies and body parts and weaknesses in the legal directives. Therefore, it is important to define what is ethical and what needs to be considered unethical in body donation and the subsequent utilisation of human bodies for teaching and research.peer-reviewe