Screening for Abiotic Stress Response in Rice.

Rice (Oryza sativa L.) is the staple food for over half of the world population. However, most rice varieties are severely injured by abiotic stresses, with strong social and economic impacts. Understanding rice responses to stress may guide breeding for more tolerant varieties. However, the lack of consistency in the design of the stress experiments described in the literature limits comparative studies and output assessments. The use of identical setups is the only way to generate comparable data. This chapter comprises three sections, describing the experimental conditions established at the Genomics of Plant Stress (GPlantS) unit of ITQB NOVA to assess the response of rice plants to different abiotic stresses-high salinity, cold, drought, simulated drought, and submergence-and their recovery capacity when intended. All sections include a detailed description of the materials and methodology and useful notes gathered from our team experience. We use seedlings since rice plants at this stage show high sensitivity to abiotic stresses. For the salt, cold, and simulated drought (PEG, polyethylene glycol) stress assays, we grow rice seedlings in a hydroponic system, while for the drought assay, plants are grown in soil and subjected to water withholding. For submergence, we use water-filled Magenta boxes. All setups enable visual score determination and are suitable for sample collection during stress imposition and also recovery. The proposed methodologies are affordable and straightforward to implement in most labs, allowing the discrimination of several rice genotypes at the molecular and phenotypic levels.

Illuminating Olea europaea L. endophyte fungal community.

A wide array of fungal endophytes is known to inhabit plant tissues and were recently recognized as essential for plant health. A better description of the scarcely known endophyte microbiota in olive tree phyllosphere is the first step for elucidating the microbial interactions that lead to olive disease establishment. In this work, the fungal endophytic community of the phyllosphere of different olive tree cultivars (Cobrançosa, Galega vulgar, Madural, Picual, Verdeal Transmontana) is revealed by using a metabarcoding strategy targeting ITS1 barcode. A total of 460 OTUs were obtained, increasing the broad view of fungal endophytes inhabiting the olive tree phyllosphere, in particular yeast endophytes. New endophytes were persistently found in all cultivar tissues. Different olive tree cultivars depicted distinct endophyte communities. Olive cultivars exhibited dissimilar amounts of fungi with distinct ecological functions, which could explain at least in part their differential susceptibility/tolerance to olive diseases.

A global multinational survey of cefotaxime-resistant coliforms in urban wastewater treatment plants.

The World Health Organization Global Action Plan recommends integrated surveillance programs as crucial strategies for monitoring antibiotic resistance. Although several national surveillance programs are in place for clinical and veterinary settings, no such schemes exist for monitoring antibiotic-resistant bacteria in the environment. In this transnational study, we developed, validated, and tested a low-cost surveillance and easy to implement approach to evaluate antibiotic resistance in wastewater treatment plants (WWTPs) by targeting cefotaxime-resistant (CTX-R) coliforms as indicators. The rationale for this approach was: i) coliform quantification methods are internationally accepted as indicators of fecal contamination in recreational waters and are therefore routinely applied in analytical labs; ii) CTX-R coliforms are clinically relevant, associated with extended-spectrum ß-lactamases (ESBLs), and are rare in pristine environments. We analyzed 57 WWTPs in 22 countries across Europe, Asia, Africa, Australia, and North America. CTX-R coliforms were ubiquitous in raw sewage and their relative abundance varied significantly (<0.1% to 38.3%), being positively correlated (p < 0.001) with regional atmospheric temperatures. Although most WWTPs removed large proportions of CTX-R coliforms, loads over 103 colony-forming units per mL were occasionally observed in final effluents. We demonstrate that CTX-R coliform monitoring is a feasible and affordable approach to assess wastewater antibiotic resistance status.

Comparison of Culture- and Quantitative PCR-Based Indicators of Antibiotic Resistance in Wastewater, Recycled Water, and Tap Water.

Standardized methods are needed to support monitoring of antibiotic resistance in environmental samples. Culture-based methods target species of human-health relevance, while the direct quantification of antibiotic resistance genes (ARGs) measures the antibiotic resistance potential in the microbial community. This study compared measurements of tetracycline-, sulphonamide-, and cefotaxime-resistant presumptive total and fecal coliforms and presumptive enterococci versus a suite of ARGs quantified by quantitative polymerase chain reaction (qPCR) across waste-, recycled-, tap-, and freshwater. Cross-laboratory comparison of results involved measurements on samples collected and analysed in the US and Portugal. The same DNA extracts analysed in the US and Portugal produced comparable qPCR results (variation <28%), except for blaOXA-1 gene (0%-57%). Presumptive total and fecal coliforms and cefotaxime-resistant total coliforms strongly correlated with blaCTX-M and intI1 (0.725 ≤ R2 ≤ 0.762; p < 0.0001). Further, presumptive total and fecal coliforms correlated with the Escherichia coli-specific biomarkers, gadAB, and uidA, suggesting that both methods captured fecal-sourced bacteria. The genes encoding resistance to sulphonamides (sul1 and sul2) were the most abundant, followed by genes encoding resistance to tetracyclines (tet(A) and tet(O)) and ß-lactams (blaOXA-1 and,blaCTX-M), which was in agreement with the culture-based enumerations. The findings can help inform future application of methods being considered for international antibiotic resistance surveillance in the environment.

Removal of microorganisms and antibiotic resistance genes from treated urban wastewater: A comparison between aluminium sulphate and tannin coagulants.

The presence of antibiotic resistant-bacteria (ARB) and antibiotic resistance genes (ARG) in treated effluents of urban wastewater treatment plants (WWTP) may represent a threat to the environment and public health. Therefore, cost-effective technologies contributing to minimize loads of these contaminants in the final effluents of WWTP are required. This study aimed at assessing the capacity of coagulation to reduce the ARB&ARG load in secondary treated urban wastewater (STWW), as well as the impact of the process on the structure and diversity of the bacterial community. Coagulation performance using aluminium sulphate, a synthetic substance, and tannins, a biowaste, was compared. Samples were analysed immediately before (STWW) and after the coagulation treatment (Alu, Tan), as well as after 3-days storage in the dark at room temperature (RSTWW, RAlu, RTan), to assess possible reactivation events. Both coagulants decreased the turbidity and colour and reduced the bacterial load (16S rRNA gene copy number, total heterotrophs (HET), and ARB (faecal coliforms resistant to amoxicillin (FC/AMX) or ciprofloxacin (FC/CIP) up to 1-2 log immediately after the treatment. Both coagulants reduced the load of intl1, but in average, aluminium sulphate was able to decrease the content of the analysed ARGs (blaTEM and qnrS) to lower levels than tannin. Reactivation after storage was observed mainly in RTan. In these samples the load of the culturable populations and qnrS gene prevalence increased, sometimes to values higher than those found in the initial wastewater. Reactivation was also characterized by an increment in Gammaproteobacteria relative abundance in the bacterial community, although with distinct patterns for RTan and RAlu. Curvibacter, Undibacterium and Aquaspirillum were among the most abundant genera in RAlu and Aeromonas, Pseudomonas and Stenotrophomonas in RTan. These bacterial community shifts were in agreement with the variations in the culturable bacterial counts of HET for RTan and FC/CIP for RAlu. In summary, the overall performance of aluminium sulphate was better than that of tannins in the treatment of treated urban wastewater.

Neighbor urban wastewater treatment plants display distinct profiles of bacterial community and antibiotic resistance genes.

Urban wastewater treatment plants (UWTPs) are among the major recipients of antibiotic-resistant bacteria (ARB), antibiotic resistance genes (ARGs), and antibiotic residues in urban environments. Although during treatment, bacteria of human and animal origin are removed, some are able to survive, persisting in the final effluent. The occurrence of these bacteria, especially those harboring ARGs, may have a direct impact on the quality of the treated wastewater that is returned to the environment. In this study, we aimed to assess if the final effluent bacterial communities of three UWTPs (PT1, PT2, and PT3) located next to each other were distinct and if such differences were related with the antibiotic resistance profiles.It was observed that the bacterial community (16S rRNA gene Illumina sequencing) and load of selected ARGs of final effluent differed among the three UWTPs, irrespective of sampling time. Members of the families Aeromonadaceae, Campylobacteraceae, Veillonellaceae, [Weeksellaceae], and Porphyromonadaceae were observed to be positively correlated with some ARGs (blaCTX-M, blaOXA-A, blaSHV) and intI1 (p < 0.05), while Intrasporangiaceae were observed to be negatively correlated. While Aeromonadaceae are recognized relevant ARG harbors, the other bacterial families may represent bacteria that co-exist with the ARG hosts, which may belong to minor bacterial groups omitted in the analyses. These findings suggest the importance of bacterial dynamics during treatment to the ARB&ARGs removal, a rationale that may contribute to design new strategies to apply in the UWTPs to prevent the spread of antibiotic resistance.

Assessment of full-scale tertiary wastewater treatment by UV-C based-AOPs: Removal or persistence of antibiotics and antibiotic resistance genes?

This research reports for the first time the full-scale application of different homogeneous Advanced Oxidation Processes (AOPs) (H2O2/UV-C, PMS/UV-C and PMS/Fe(II)/UV-C) for the removal of antibiotics (ABs) and antibiotic resistance genes (ARGs) from wastewater effluent at Estiviel wastewater treatment plant (WWTP) (Toledo, Spain). AOPs based on the photolytic decomposition of H2O2 and peroxymonosulfate tested at low dosages (0.05-0.5 mM) and with very low UV-C contact time (4-18 s) demonstrated to be more efficient than UV-C radiation alone on the removal of the analyzed ABs. PMS (0.5 mM) combined with UV-C (7 s contact time) was the most efficient treatment in terms of AB removal: 7 out of 10 ABs detected in the wastewater were removed more efficiently than using the other oxidants. In terms of ARGs removal efficiency, UV-C alone seemed the most efficient treatment, although H2O2/UV-C, PMS/UV-C and PMS/Fe(II)/UV-C were supposed to generate higher concentrations of free radicals. The results show that treatments with the highest removal of ABs and ARGs did not coincide, which could be attributed to the competition between DNA and oxidants in the absorption of UV photons, reducing the direct photolysis of the DNA. Whereas the photolytic ABs removal is improved by the generation of hydroxyl and sulfate radicals, the opposite behavior occurs in the case of ARGs. These results suggest that a compromise between ABs and ARGs removal must be achieved in order to optimize wastewater treatment processes.

Performance of secondary wastewater treatment methods for the removal of contaminants of emerging concern implicated in crop uptake and antibiotic resistance spread: A review.

Contaminants of emerging concern (CEC) discharged in effluents of wastewater treatment plants (WWTPs), not specifically designed for their removal, pose serious hazards to human health and ecosystems. Their impact is of particular relevance to wastewater disposal and re-use in agricultural settings due to CEC uptake and accumulation in food crops and consequent diffusion into the food-chain. This is the reason why the chemical CEC discussed in this review have been selected considering, besides recalcitrance, frequency of detection and entity of potential hazards, their relevance for crop uptake. Antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) have been included as microbial CEC because of the potential of secondary wastewater treatment to offer conditions favourable to the survival and proliferation of ARB, and dissemination of ARGs. Given the adverse effects of chemical and microbial CEC, their removal is being considered as an additional design criterion, which highlights the necessity of upgrading conventional WWTPs with more effective technologies. In this review, the performance of currently applied biological treatment methods for secondary treatment is analysed. To this end, technological solutions including conventional activated sludge (CAS), membrane bioreactors (MBRs), moving bed biofilm reactors (MBBRs), and nature-based solutions such as constructed wetlands (CWs) are compared for the achievable removal efficiencies of the selected CEC and their potential of acting as reservoirs of ARB&ARGs. With the aim of giving a picture of real systems, this review focuses on data from full-scale and pilot-scale plants treating real urban wastewater. To achieve an integrated assessment, technologies are compared considering also other relevant evaluation parameters such as investment and management costs, complexity of layout and management, present scale of application and need of a post-treatment. Comparison results allow the definition of design and operation strategies for the implementation of CEC removal in WWTPs, when agricultural reuse of effluents is planned.

Propofol affinity to mitochondrial membranes does not alter mitochondrial function.

The molecular mechanisms of hepatotoxicity after propofol anaesthesia have not been fully elucidated, although there is a relation with mitochondrial dysfunction. The action of propofol on mitochondrial hepatic functions in a rat model was evaluated by infusion for 4h with 25 and 62.5mg/kg/h propofol or 3.125ml/kg/h (vehicle). Liver mitochondrial respiratory rates were evaluated as well as mitochondrial transmembrane potential (ΔΨ), calcium fluxes, mitochondrial enzymatic activities (Complex I-V) and oxidative stress biomarkers (superoxide dismutase, catalase, glutathione reductase, glutathione S-transferase, lipid peroxidation and the oxidised/reduced glutathione ratio). Biophysical interactions with membrane models were also performed. The mitochondrial transmembrane potential was decreased and the opening time of the mitochondrial permeability transition pore was slightly reduced for the highest dose. The activity of complex II was stimulated by propofol, which also causes fluctuations on some respiratory parameters, whereas the antioxidant system was affected in a nonspecific manner. Fluorescence quenching studies suggested that propofol is preferably located in deeper regions of the bilayer and has a high affinity to mitochondrial membranes. It is suggested that propofol interacts with liver mitochondrial membranes with mild modification in mitochondrial function.

Evaluation of RT-PCR and hemi-nested RT-PCR in brain samples from dogs with neurologic signs compatible with distemper / Avaliação das técnicas de RT-PCR e heminested RT-PCR em cérebros de cães com sinais neurológicos compatíveis com cinomose

The diagnostic value of RT-PCR and hemi-nested RT-PCR (hnRT-PCR) was compared in brain samples of dogs presenting neurological signs compatible with canine distemper. Samples of central nervous system (CNS) were collected from 68 dogs and tested by direct immunofluorescence test (RFID) and, independent of the results, they were stored at -20°C for at least three years. They were submitted to the RT-PCR and hnRT-PCR techniques aiming to determine the gene responsible for the viral nucleoprotein decoding. Fifty-nine samples were positive for RIFD, 40 for RT-PCR (Kappa = 0.358) and 54 for hnRT-PCR (Kappa = 0.740). All nine RIFD negative samples were also negative for RT-PCR and hnRT-PCR. In spite of the storage duration and proper sample conditions, the estimated accordance between hnRT-PCR and RIFD demonstrated that hnRT-PCR technique can be applied in retrospective studies...

Foi comparado o valor diagnóstico das técnicas de RT-PCR e heminested RT-PCR (hnRT-PCR) em amostras de cérebro de cães com sintomatologia nervosa compatível com cinomose. Fragmentos do sistema nervoso central (SNC) colhidos de 68 animais foram testados pela Imunofluorescência direta (IFD) e, independentemente do resultado, foram armazenados a -20°C por pelo menos três anos. Após esse período, foram submetidos a RT-PCR e a hnRT-PCR com oligonucleotídeos iniciadores direcionados ao gene codificador da nucleoproteína N. As proporções de resultados positivos/examinados foram: 59/68 para a IFD, 40/68 para a RT-PCR (Kappa = 0,358) e 54/68 quando associada à heminested PCR (Kappa = 0,740). Houve nove resultados negativos nas três técnicas empregadas. Os resultados do coeficiente Kappa entre a IFD e hnRT-PCR demonstram que apesar das condições de armazenamento, a hnRT-PCR pode ser utilizada em estudos retrospectivos...

Drug testing data from the 2007 Pan American Games: delta13C values of urinary androsterone, etiocholanolone and androstanediols determined by GC/C/IRMS.

The main purpose of this article is to show the application of the CG/C/IRMS in real time during competition in the steroid confirmation analysis. For this reason, this paper summarizes the results obtained from the doping control analysis during the period of the 2007 Pan American Games held in Rio de Janeiro, Brazil. Approximately 5600 athletes from 42 different countries competed in the games. Testing was performed in accordance to World Anti-Doping Agency (WADA) technical note for prohibited substances. This paper reports data where abnormal urinary steroid profiles, have been found with the screening procedures. One 8 mL urine sample was used for the analysis of five steroid metabolites with two separate analyses by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Urine samples were submitted to GC/C/IRMS for confirmation analysis to determine the (13)C/(12)C ratio of selected steroids. Fifty-seven urine samples were analyzed by GC/C/IRMS and the delta(13)C values ( per thousand) of androsterone, etiocholanolone, 5beta-androstane-3alpha, 17beta-diol (5beta-diol), 5alpha-androstane-3alpha, 17beta-diol (5alpha-diol) and 5beta-pregnane-3alpha, 20alpha-diol (5beta-pdiol), the endogenous reference compound are presented. One urine sample with a testosterone/epitestosterone (T/E) ratio of 4.7 was confirmed to be positive of doping by GC/C/IRMS analysis. The delta values of 5beta-diol and 5alpha-diol were 3.8 and 10.8, respectively, compared to the endogenous reference compound 5beta-pdiol, which exceeded the WADA limit of 3 per thousand. The results obtained by CG/C/IRMS confirmation analyses, in suspicious samples, were conclusive in deciding whether or not a doping steroid violation had occurred.