Intracellular poly-P assessment by DAPI staining and image analysis

In wastewater treatment, enhanced biological phosphorus removal (EBPR) is considered a well-established process to remove phosphate (P). EBPR is based on the activity of polyphosphate-accumulating organisms (PAOs) able to take up and store large amounts of P as intracellular (poly-P) granules. However, monitoring poly-P in mixed cultures is usually performed by a laborious and time consuming off-line chemical analysis. Thus, there is a clear need to develop new techniques to rapidly monitor these processes, such as image analysis coupled to sample staining and microscopy inspection. A lab-scale sequencing batch reactor (SBR) was fed with synthetic wastewater containing acetate and propionate as main carbon sources and an orthophosphate solution was added. A COD/P ratio of 10 mg COD mg P-PO4-1 was used to provide selective advantages to PAOs. The SBR was operated with a cycle time of 6 h: 120 min anaerobic including 5 min feed, 180 min aerobic and 60 min wasting/settling. Biomass samples were collected at the end of the aerobic stage. Bulk P concentration was determined by segmented flow analysis and total P concentration was similarly measured following acid digestion at 100oC. Intracellular poly-P concentration was determined by subtracting the bulk P from the total P. Intracellular poly-P granules were observed in epifluorescence microscopy using DAPI staining with a 25 ìg mL-1 DAPI solution. A long pass filter was used with an excitation bandpass of 365-370 nm and emission cut off at 421 nm. A specially developed program in Matlab was used for image analysis. A total of 41 samples were collected. Two thirds were fed as training data to the partial least squares (PLS) model and the remaining used for validation. Both absolute (in mg poly-P / L) and relative (in mg poly-P / g MLSS) intracellular poly-P concentrations were studied. This procedure was found to predict, at some extent, the relative intracellular poly-P concentration (real poly-P = 0.971 x predicted poly-P, R2 of 0.744). Regarding the absolute intracellular poly-P concentration, a total of 3 samples needed to be discarded in order to obtain a similar result (real poly-P = 1.005 x predicted poly-P, R2 of 0.731)

Novel method to quantify intracellular accumulation of polyphosphate in EBPR systems

A new method for intracellular storage polyphosphate (poly-P)identification and quantification in enhanced biological phosphorus removal (EBPR) systems is proposed based on image analysis. In EBPR systems, 4',6-diamidino-2-phenylindole (DAPI) is usually combined with fluororescent in situ hybridization (FISH) to evaluate the microbial community. The proposed technique is based on an image analysis procedure specifically developed for determining poly-P inclusions within biomass suspension using solely DAPI by epifluorescence microscopy. Due to contradictory literature DAPI concentrations used for poly-P detection, the present work assessed the optimal DAPI concentration for samples acquired at the end of the EBPR aerobic stage when the accumulation is performed. Digital images were then acquired and processed by means of image processing and analysis. Regarding image analysis results and considering the current operational conditions, a promising correlation could be found between average poly-P intensity values and the analytical determination, although presenting a correlation coefficient somewhat far from the ideal. The proposed methodology can be seen as a promising alternative procedure to quantify intracellular poly-P accumulation in a faster and less labor intensive way

Biopolymer monitoring using quantitative image analysis techniques

Polyhydroxyalkanoates (PHAs) are intracellular biopolymers with many applications, particularly as substitutes of polypropylene and polyethylene, due to their thermoplastic properties and biocompatible nature. Furthermore, glycogen is a polysaccharide of glucose with high importance in the metabolism of microbial communities and polyphosphate is a microbial storage compound that should be recovered in order to offset the worldwide depletion of phosphorus sources. The determination of these biopolymers by chemical analysis is a laborious task, often involving digestion processes prior to gas and high-performance liquid chromatography, which are time consuming and difficult to apply in industry. Currently, it is important to develop new, rapid and simple techniques to monitor these polymers. Image analysis is a non-invasive and rapid technique that has the potential to be used to quantify these intracellular polymers quickly, in real-time. Mesquita et al. (2013) showed that it is possible to predict the concentration of glycogen and PHAs by quantitative image analysis, using aniline blue and nile blue staining, respectively. Polyphosphate can also be predicted by this technique through DAPI staining, which is currently under development. These biopolymers are produced by several different microorganisms, and combining their quantification with fluorescence in situ hybridization (FISH) techniques for microbial identification can enable the determination of organisms that store high quantities of each biopolymer. In this work, an advanced quantitative technique is developed to perform real time monitoring of these three biopolymers in a bioreactor performing biological phosphorus removal. Image analysis of the biopolymers was combined with FISH to determine the storage level of each compound within the different microbial populations. This technique will further enable the assessment of biopolymer levels within microbial communities, which can be applied in the biopolymer production industry

Spread of vaccinia virus to cattle herds, Argentina, 2011

To the Editor: Since 1999, several zoonotic outbreaks of vaccinia virus (VACV) infection have been reported in cattle and humans in rural areas of Brazil. The infections have caused exanthematous lesions on cows and persons who milk them, and thus are detrimental to the milk industry and public health services (1,2). In Brazil during the last decade, VACV outbreaks have been detected from the north to the extreme south of the country (1–4). Because Brazil shares extensive boundaries with other South American countries, humans and cattle on dairy and beef-producing farms in those countries may be at risk of exposure to VACV. To determine if VACV has spread from Brazil to Argentina, we investigated the presence of VACV in serum samples from cattle in Argentina.Facultad de Ciencias VeterinariasComisión de Investigaciones Científicas de la provincia de Buenos Aire

Spread of vaccinia virus to cattle herds, Argentina, 2011

To the Editor: Since 1999, several zoonotic outbreaks of vaccinia virus (VACV) infection have been reported in cattle and humans in rural areas of Brazil. The infections have caused exanthematous lesions on cows and persons who milk them, and thus are detrimental to the milk industry and public health services (1,2). In Brazil during the last decade, VACV outbreaks have been detected from the north to the extreme south of the country (1–4). Because Brazil shares extensive boundaries with other South American countries, humans and cattle on dairy and beef-producing farms in those countries may be at risk of exposure to VACV. To determine if VACV has spread from Brazil to Argentina, we investigated the presence of VACV in serum samples from cattle in Argentina.Facultad de Ciencias VeterinariasComisión de Investigaciones Científicas de la provincia de Buenos Aire

The Use of the International Classification of Functioning, Disability and Health (ICF) Framework on Educational Planning: Promoting an Environmental Approach

Portugal was the first country decreeing the formal use of the International Classification of Functioning, Disability and Health (ICF) framework as basis for guiding assessment and planning processes within the special education field. Compared to traditional approaches, the ICF places a greater emphasis on understanding environmental contexts and their influence on human functioning. The aim of this study was to analyze the impact of its use on improving the environmental approach on assessment and intervention processes. A content and semantic analysis was carried out on 280 IEPs designed before and after the ICF use for 140 students in need of special supports. Results revealed that the assessment process of a same student included a wider spectrum of environmental factors when designed using ICF framework. This step forward on assessment processes still needs to trigger the person-environment fit perspective on intervention planning. The analysis of environmental factors’ influence on students functioning (Body Functions and Activities and Participation) is seen as the guiding principle for the ICF use improvement supporting the IEPs design

Spread of vaccinia virus to cattle herds, Argentina, 2011

To the Editor: Since 1999, several zoonotic outbreaks of vaccinia virus (VACV) infection have been reported in cattle and humans in rural areas of Brazil. The infections have caused exanthematous lesions on cows and persons who milk them, and thus are detrimental to the milk industry and public health services (1,2). In Brazil during the last decade, VACV outbreaks have been detected from the north to the extreme south of the country (1–4). Because Brazil shares extensive boundaries with other South American countries, humans and cattle on dairy and beef-producing farms in those countries may be at risk of exposure to VACV. To determine if VACV has spread from Brazil to Argentina, we investigated the presence of VACV in serum samples from cattle in Argentina.Facultad de Ciencias VeterinariasComisión de Investigaciones Científicas de la provincia de Buenos Aire

Valuing health states: is the MACBETH approach useful for valuing EQ-5D-3L health states?

Background Quality Adjusted Life Years (QALYs) are a key outcome measure widely used within health technology assessment and health service research studies. QALYs combine quantity and quality of life, with quality of life calculations relying on the value of distinct health states. Such health states’ values capture the preferences of a population and have been typically built through numerical elicitation methods. Evidence points to these value scores being influenced by methods in use and individuals reporting cognitive difficulties in eliciting their preferences. Evidence from other areas has further suggested that individuals may prefer using distinct elicitation techniques and that this preference can be influenced by their numeracy. In this study we explore the use of the MACBETH (Measuring Attractiveness by a Categorical Based Evaluation Technique) non-numerical preference elicitation approach for health states’ evaluation. Methods A new protocol for preference elicitation based on MACBETH (only requiring qualitative judgments) was developed and tested within a web survey format. A sample of the Portuguese general population (n=243) valued 25 EQ-5D-3L health states with the MACBETH protocol and with a variant of the time trade-off (TTO) protocol, for comparison purposes and for understanding respondents’ preference for distinct protocols and differences in inconsistent evaluations. Respondents answered to a short numeracy test, and basic socio-economic information collected. Results Results show that the mean values derived from MACBETH and the TTO variant are strongly correlated; however, there are substantial differences for several health states’ values. Large and similar numbers of logical inconsistencies were found in respondents’ answers with both methods. Participants with higher levels of numeracy according to the test preferred expressing value judgments with MACBETH, while participants with lower levels were mostly indifferent to both methods. Higher correlations between MACBETH and TTO variant evaluations were observed for individuals with higher numeracy. Conclusion Results suggest that it is worth researching the use of non-numerical preference elicitation methods. Numeracy tests more appropriate for preference elicitation when no explicit considerations of uncertainty are made need to be explored and used. Further behavioural research is needed to fully understand the potential for using these methods in distinct settings (e.g. in different evaluation contexts and in face-to-face and non-face-to-face environments), as well as to explore the effect of literacy on assessments and on respondents’ preferences.UID/MULTI/4066/2016info:eu-repo/semantics/publishedVersio

Cardiac Actions of a Small Molecule Inhibitor Targeting GATA4-NKX2-5 Interaction

Transcription factors are fundamental regulators of gene transcription, and many diseases, such as heart diseases, are associated with deregulation of transcriptional networks. In the adult heart, zinc-finger transcription factor GATA4 is a critical regulator of cardiac repair and remodelling. Previous studies also suggest that NKX2-5 plays function role as a cofactor of GATA4. We have recently reported the identification of small molecules that either inhibit or enhance the GATA4-NKX2-5 transcriptional synergy. Here, we examined the cardiac actions of a potent inhibitor (3i-1000) of GATA4-NKX2-5 interaction in experimental models of myocardial ischemic injury and pressure overload. In mice after myocardial infarction, 3i-1000 significantly improved left ventricular ejection fraction and fractional shortening, and attenuated myocardial structural changes. The compound also improved cardiac function in an experimental model of angiotensin II-mediated hypertension in rats. Furthermore, the up-regulation of cardiac gene expression induced by myocardial infarction and ischemia reduced with treatment of 3i-1000 or when micro-and nanoparticles loaded with 3i-1000 were injected intramyocardially or intravenously, respectively. The compound inhibited stretch- and phenylephrine-induced hypertrophic response in neonatal rat cardiomyocytes. These results indicate significant potential for small molecules targeting GATA4-NKX2-5 interaction to promote myocardial repair after myocardial infarction and other cardiac injuries

Transcriptome assembly and microarray construction for Enchytraeus crypticus, a model oligochaete to assess stress response mechanisms derived from soil conditions

The soil worm Enchytraeus crypticus (Oligochaeta) is an ecotoxicology model species that, until now, was without genome or transcriptome sequence information. The present research aims at studying the transcriptome of Enchytraeus crypticus, sampled from multiple test conditions, and the construction of a high-density microarray for functional genomic studies. Over 1.5 million cDNA sequence reads were obtained representing 645 million nucleotides. After assembly, 27,296 contigs and 87,686 singletons were obtained, from which 44% and 25% are annotated as protein-coding genes, respectively, sharing homology with other animal proteomes. Concerning assembly quality, 84% of the contig sequences contain an open reading frame with a start codon while E. crypticus homologs were identified for 92% of the core eukaryotic genes. Moreover, 65% and 77% of the singletons and contigs without known homologs, respectively, were shown to be transcribed in an independent microarray experiment. An Agilent 180 K microarray platform was designed and validated by hybridizing cDNA from 4 day zinc- exposed E. crypticus to the concentration corresponding to 50% reduction in reproduction after three weeks (EC50). Overall, 70% of all probes signaled expression above background levels (mean signal + 1x standard deviation). More specifically, the probes derived from contigs showed a wider range of average intensities when compared to probes derived from singletons. In total, 522 significantly differentially regulated transcripts were identified upon zinc exposure. Several significantly regulated genes exerted predicted functions (e.g. zinc efflux, zinc transport) associated with zinc stress. Unexpectedly, the microarray data suggest that zinc exposure alters retro transposon activity in the E. crypticus genome. An initial investigation of the E. crypticus transcriptome including an associated microarray platform for future studies proves to be a valuable resource to investigate functional genomics mechanisms of toxicity in soil environments and to annotate a potentially large number of lineage specific genes that are responsive to environmental stress conditions