Analysis of Milk Using a Portable Potentiometric Electronic Tongue Based on Five Polymeric Membrane Sensors.

A portable potentiometric electronic tongue (PE-tongue) was developed and applied to evaluate the quality of milk with different fat content (skimmed, semi-skimmed, and whole) and with different nutritional content (classic, calcium-enriched, lactose-free, folic acid-enriched, and enriched in sterols of vegetal origin). The system consisted of a simplified array of five sensors based on PVC membranes, coupled to a data logger. The five sensors were selected from a larger set of 20 sensors by applying the genetic algorithm (GA) to the responses to compounds usually found in milk including salts (KCl, CaCl2, and NaCl), sugars (lactose, glucose, and galactose), and organic acids (citric acid and lactic acid). Principal component analysis (PCA) and support vector machine (SVM) results indicated that the PE-tongue consisting of a five-electrode array could successfully discriminate and classify milk samples according to their nutritional content. The PE-tongue provided similar discrimination capability to that of a more complex system formed by a 20-sensor array. SVM regression models were used to predict the physicochemical parameters classically used in milk quality control (acidity, density, %proteins, %lactose, and %fat). The prediction results were excellent and similar to those obtained with a much more complex array consisting of 20 sensors. Moreover, the SVM method confirmed that spoilage of unsealed milk could be correctly identified with the simplified system and the increase in acidity could be accurately predicted. The results obtained demonstrate the possibility of using the simplified PE-tongue to predict milk quality and provide information on the chemical composition of milk using a simple and portable system.

The Practice of Paediatric Radiation Oncology in Low- and Middle-income Countries: Outcomes of an International Atomic Energy Agency Study.

AIMS: Childhood cancer survival is suboptimal in most low- and middle-income countries (LMICs). Radiotherapy plays a significant role in the standard care of many patients. To assess the current status of paediatric radiotherapy, the International Atomic Energy Agency (IAEA) undertook a global survey and a review of practice in eight leading treatment centres in middle-income countries (MICs) under Coordinated Research Project E3.30.31; 'Paediatric radiation oncology practice in low and middle income countries: a patterns-of-care study by the International Atomic Energy Agency.' MATERIALS AND METHODS: A survey of paediatric radiotherapy practices was distributed to 189 centres worldwide. Eight leading radiotherapy centres in MICs treating a significant number of children were selected and developed a database of individual patients treated in their centres comprising 46 variables related to radiotherapy technique. RESULTS: Data were received from 134 radiotherapy centres in 42 countries. The percentage of children treated with curative intent fell sequentially from high-income countries (HICs; 82%) to low-income countries (53%). Increasing deficiencies were identified in diagnostic imaging, radiation staff numbers, radiotherapy technology and supportive care. More than 92.3% of centres in HICs practice multidisciplinary tumour board decision making, whereas only 65.5% of centres in LMICs use this process. Clinical guidelines were used in most centres. Practice in the eight specialist centres in MICs approximated more closely to that in HICs, but only 52% of patients were treated according to national/international protocols whereas institution-based protocols were used in 41%. CONCLUSIONS: Quality levels in paediatric radiotherapy differ among countries but also between centres within countries. In many LMICs, resources are scarce, coordination with paediatric oncology is poor or non-existent and access to supportive care is limited. Multidisciplinary treatment planning enhances care and development may represent an area where external partners can help. Commitment to the use of protocols is evident, but current international guidelines may lack relevance; the development of resources that reflect the capacity and needs of LMICs is required. In some LMICs, there are already leading centres experienced in paediatric radiotherapy where patient care approximates to that in HICs. These centres have the potential to drive improvements in service, training, mentorship and research in their regions and ultimately to improve the care and outcomes for paediatric cancer patients.

Analysis of musts and wines by means of a bio-electronic tongue based on tyrosinase and glucose oxidase using polypyrrole/gold nanoparticles as the electron mediator.

A bioelectronic tongue (bioET) based on combinations of enzymes (tyrosinase and glucose oxidase) and polypyrrole (Ppy) or polypyrrole/AuNP (Ppy/AuNP) composites was build up and applied to the analysis and discrimination of musts and wines. Voltammetric responses of the array of sensors demonstrated the effectiveness of polymers as electron mediators and the existence of favorable synergistic effects between Ppy and the AuNPs. Using Principal Component Analysis and Parallel Factor Analysis it was possible to discriminate musts according to the °Brix and TPI (Total Polyphenol Index), and wines according to the alcoholic degree and TPI. Partial Least Squares provided good correlations between the bioET output and traditional chemical parameters. Moreover, Support Vector Machines permitted to predict the TPI and the alcoholic degree of wines, from data provided by the bioET in the corresponding grapes. This result opens the possibility to predict wine characteristics from the beginning of the vinification process.

Molecular characterisation of Salmonella strains isolated from outbreaks and sporadic cases of diarrhoea occurred in Paraná State, South of Brazil.

A total of 46 strains of Salmonella isolated from patients with sporadic diarrhoea or involved in foodborne outbreaks were analysed by PCR for genus identification and serotyping. Subtyping was performed using pulsed-field gel electrophoresis (PFGE) and multiple amplification of phage locus typing (MAPLT) for seven variable loci. Bacteria were identified as belonging to serotype Enteritidis (33 strains; 71·7%) or Typhimurium (13 strains; 28·3%). A high similarity coefficient (94·6%) was observed in the Salmonella Enteritidis group for which were found three related PFGE profiles and only one MAPLT; strains representing profile PA/P1/MI were prevalent (27; 81·8%). Two Salmonella Typhimurium isolates were untypeable by PFGE. The remaining 11 strains had eight PFGE and three MAPLT profiles. The discriminatory power of MAPLT was lower than that of PFGE. Salmonella Enteritidis of clonal nature is predominant in Paraná State, with the most prevalent profile PA/P1/M1 associated with sporadic diarrhoea and with seven of nine reported outbreaks. In conclusion, PFGE shows higher discriminatory power among Salmonella strains.

Development and validation of the first SSR markers for Mimosa scabrella Benth.

Mimosa scabrella Benth., popularly known as ''bracatinga'', is a pioneer and endemic species of Brazil, occurring in Mixed Ombrophilous Forest associated with Brazilian Atlantic Rainforest biomes. It is a fast-growing tree of the Fabaceae family that facilitates the dynamics of ecological succession. SSR development, when there is no genome sequence, is time and labor intensive and there are no molecular markers for M. scabrella. We developed and validated the first microsatellite markers for this tetraploid species, evaluating mother trees and progenies. Using Illumina sequencing, we identified 290 SSR loci and 211 primer pairs. After 31 SSR loci PCR/agarose electrophoresis selection, a subset of 11 primer pairs was synthetized with fluorescence in the forward primer for PCR and capillary electrophoresis validation with leaf DNA of 33 adult and 411 progeny individuals. Polymorphic locus percentage was 36, 4 in 11 loci, 3 chloroplast SSRs, and 1 nuclear SSR. Allele number of polymorphic loci ranged from 2 to 11 alleles considering all sampling. All 11 primer pairs were also tested for cross-species amplification for five Fabaceae-Mimosoideae species, ranging from 2 loci transferred to Calliandra tweedii Benth. and all 11 loci transferred to Mimosa taimbensis Burkart. The assessed and validated SSR markers for M. scabrella are suitable and useful for analysis and population genetic studies.

A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry.

The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC) is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB) production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i) cell permeabilization, ii) Nile red staining, and iii) analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99) compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots.

A broad pH range and processive chitinase from a metagenome library.

Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked ß(1-4) present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N'-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin.

A NodD-like protein activates transcription of genes involved with naringenin degradation in a flavonoid-dependent manner in Herbaspirillum seropedicae.

Herbaspirillum seropedicae is an associative, endophytic non-nodulating diazotrophic bacterium that colonises several grasses. An ORF encoding a LysR-type transcriptional regulator, very similar to NodD proteins of rhizobia, was identified in its genome. This nodD-like gene, named fdeR, is divergently transcribed from an operon encoding enzymes involved in flavonoid degradation (fde operon). Apigenin, chrysin, luteolin and naringenin strongly induce transcription of the fde operon, but not that of the fdeR, in an FdeR-dependent manner. The intergenic region between fdeR and fdeA contains several generic LysR consensus sequences (T-N11 -A) and we propose a binding site for FdeR, which is conserved in other bacteria. DNase I foot-printing revealed that the interaction with the FdeR binding site is modified by the four flavonoids that stimulate transcription of the fde operon. Moreover, FdeR binds naringenin and chrysin as shown by isothermal titration calorimetry. Interestingly, FdeR also binds in vitro to the nod-box from the nodABC operon of Rhizobium sp. NGR234 and is able to activate its transcription in vivo. These results show that FdeR exhibits two features of rhizobial NodD proteins: nod-box recognition and flavonoid-dependent transcription activation, but its role in H. seropedicae and related organisms seems to have evolved to control flavonoid metabolism.

RNA-seq transcriptional profiling of Herbaspirillum seropedicae colonizing wheat (Triticum aestivum) roots.

Herbaspirillum seropedicae is a diazotrophic and endophytic bacterium that associates with economically important grasses promoting plant growth and increasing productivity. To identify genes related to bacterial ability to colonize plants, wheat seedlings growing hydroponically in Hoagland's medium were inoculated with H. seropedicae and incubated for 3 days. Total mRNA from the bacteria present in the root surface and in the plant medium were purified, depleted from rRNA and used for RNA-seq profiling. RT-qPCR analyses were conducted to confirm regulation of selected genes. Comparison of RNA profile of root attached and planktonic bacteria revealed extensive metabolic adaptations to the epiphytic life style. These adaptations include expression of specific adhesins and cell wall re-modeling to attach to the root. Additionally, the metabolism was adapted to the microxic environment and nitrogen-fixation genes were expressed. Polyhydroxybutyrate (PHB) synthesis was activated, and PHB granules were stored as observed by microscopy. Genes related to plant growth promotion, such as auxin production were expressed. Many ABC transporter genes were regulated in the bacteria attached to the roots. The results provide new insights into the adaptation of H. seropedicae to the interaction with the plant.

Effect of point mutations on Herbaspirillum seropedicae NifA activity.

NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a ß-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.

Analysis of organic acids and phenols of interest in the wine industry using Langmuir-Blodgett films based on functionalized nanoparticles.

A chemically modified electrode consisting of Langmuir-Blodgett (LB) films of n-dodecanethiol functionalized gold nanoparticles (SDODAuNP-LB), was investigated as a voltammetric sensor of organic and phenolic acids of interest in the wine industry. The nanostructured films demonstrated interfacial properties being able to detect the main organic acids present in grapes and wines (tartaric, malic, lactic and citric). Compared to a bare ITO electrode, the modified electrodes exhibited a shift of the reduction potential in the less positive direction and a marked enhancement in the current response. Moreover, the increased electrocatalytic properties made it possible to distinguish between the different dissociable protons of polyprotic acids. The SDODAuNP-LB sensor was also able to provide enhanced responses toward aqueous solutions of phenolic acids commonly found in wines (caffeic and gallic acids). The presence of nanoparticles increased drastically the sensitivity toward organic acids and phenolic compounds. Limits of detection as low as 10(-6) mol L(-1) were achieved. Efficient catalytic activity was also observed in mixtures of phenolic acid/tartaric in the range of pHs typically found in wines. In such mixtures, the electrode was able to provide simultaneous information about the acid and the phenol concentrations with a complete absence of interferences. The excellent sensing properties shown by these sensors could be attributed to the electrocatalytic properties of the nanoparticles combined with the high surface to volume ratio and homogeneity provided by the LB technique used for the immobilization. Moreover, the LB technique also provided an accurate method to immobilize the gold nanoparticles giving rise to stable and reproducible sensors showing repeatability lower than 2% and reproducibility lower than 4% for all the compounds analyzed.

Rapid quantification of rice root-associated bacteria by flow cytometry.

UNLABELLED: To understand the mechanism of plant-bacterium interaction, it is critical to enumerate epiphytic bacteria colonizing the roots of the host. We developed a new approach, based on flow cytometry, for enumerating these bacteria and used it with rice plants, 7 and 20 days after colonization with Herbaspirillum rubrisubalbicans and Azospirillum brasilense. The results were compared with those obtained with the traditional plate count method. Both methods gave similar numbers of H. rubrisubalbicans associated with rice roots (c. 10(9) CFU g(-1) ). However, flow cytometry gave a number of viable cells of rice-associated A. brasilense that was approx. 10-fold greater than that obtained with the plate count method. These results suggest that the plate count method can underestimate epiphytic populations. Flow cytometry has the additional advantage that it is more precise and much faster than the plate count method. SIGNIFICANCE AND IMPACT OF THE STUDY: Determination of precise number of root-associated bacteria is critical for plant-bacteria interaction studies. We developed a flow cytometry approach for counting bacteria and compared it with the plate count method. Our flow cytometry assay solves two major limitations of the plate count method, namely that requires long incubation times of up to 48 h and only determines culturable cells. This flow cytometry assay provides an efficient, precise and fast tool for enumerating epiphytic cells.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of Escherichia coli categories.

The mass profiles of cell-free extracts of 180 commensal and pathogenic strains of Escherichia coli were determined by MALDI-TOF mass spectrometry (MS). While some peaks were highly conserved in all E. coli, several peaks occurred only in some strains, showing heterogeneity among them. We did not detect strain-specific peaks for any of the E. coli categories tested. However, review of the fully conserved and the variable peaks suggested that MALDI-TOF MS has the potential to distinguish commensal and uropathogenic E. coli strains. Additionally, eight Shigella sonnei isolates were tested and found to be indistinguishable from E. coli by MALDI-TOF MS under the test conditions.

Proteins differentially expressed by Shiga toxin-producing Escherichia coli strain M03 due to the biliar salt sodium deoxycholate.

Shiga toxin-producing Escherichia coli (STEC) can cause conditions ranging from diarrhea to potentially fatal hemolytic uremic syndrome. Enteropathogen adaptation to the intestinal environment is necessary for the development of infection, and response to bile is an essential characteristic. We evaluated the response of STEC strain M03 to the bile salt sodium deoxycholate through proteomic analysis. Cell extracts of strain M03 grown with and without sodium deoxycholate were analyzed by two-dimensional electrophoresis; the differentially expressed proteins were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Three proteins were found to be differentially expressed due to sodium deoxycholate. Glycerol dehydrogenase and phosphate acetyltransferase, which are involved in carbon metabolism and have been associated with virulence in some bacteria, were downregulated. The elongation factor Tu (TufA) was upregulated. This protein participates in the translation process and also has chaperone activities. These findings help us understand strategies for bacterial survival under these conditions.

Comparative proteomic analysis between early developmental stages of the Coffea arabica fruits.

Coffee is one of the most valuable agricultural commodities. There is much agronomic research on coffee, but molecular knowledge of its fruit development and ripening is limited. This study reports a comparative proteomic investigation of immature coffee fruits in two early developmental stages: stage 1, cell division and elongation of the perisperm; and stage 2, early growth of the endosperm progressively replacing the perisperm. Proteins were extracted using a modified SDS-phenol method and two-dimensional electrophoresis gels stained with Coomassie blue revealed about 300 well-resolved polypeptide spots in the pH range of 3 to 10. The differentially expressed polypeptides spots were excised, trypsin-digested, and analyzed by MALDI-TOF mass spectrometry. Peptide MS data were searched against the coffee EST database. Most of the identified protein spots are involved in the glycolytic pathway and energy reserve, and are more highly expressed at stage 2.

End-of-life Zn-MnO2 batteries: electrode materials characterization.

Physical and chemical characterization of several sizes and shapes of alkaline and saline spent Zn-MnO2 batteries was carried out, aiming at contributing for a better definition of the applicable recycling processes. The characterization essays included the mass balance of the components, cathode and anode elemental analysis, the identification of zinc and manganese bearing phases and the morphology analysis of the electrode particles. The electrode materials correspond to 64-79% of the total weigh of the batteries, with the cathodes having clearly the highest contribution (usually more than 50%). The steel components, mainly from the cases, are also important (17-30%). Elemental analysis showed that the electrodes are highly concentrated in zinc (from 48-87% in anodes) and manganese (from 35-50% in cathodes). X-Ray powder diffraction allowed for identifying several phases in the electrodes, namely zinc oxide, in the anodes of all the types of saline and alkaline batteries tested, while zinc hydroxide chloride and ammine zinc chloride only appear in some types of saline batteries. The manganese found in the cathode materials is present as two main phases, MnO x Mn2O3 and ZnO x Mn2O3, the latter corroborating that zinc migration from anode to cathode occurs during the batteries lifespan. A unreacted MnO2 phase was also found presenting a low crystalline level. Leaching trials with diluted HCI solutions of alkaline and saline battery samples showed that all zinc species are reactive attaining easily over than 90% leaching yields, and about 30% of manganese, present as Mn(II/III) forms. The MnO2 phase is less reactive and requires higher temperatures to achieve a more efficient solubilization.

Culture-independent analysis of endophytic bacterial communities associated with Brazilian sugarcane.

Sugarcane is an economically important culture in Brazil. Endophytic bacteria live inside plants, and can provide many benefits to the plant host. We analyzed the bacterial diversity of sugarcane cultivar RB-72454 by cultivation-independent techniques. Total DNA from sugarcane stems from a commercial plantation located in Paraná State was extracted. Partial 16S rRNA genes were amplified and sequenced for library construction. Of 152 sequences obtained, 52% were similar to 16S rRNA from Pseudomonas sp, and 35.5% to Enterobacter sp. The genera Pantoea, Serratia, Citrobacter, and Klebsiella were also represented. The endophytic communities in these sugarcane samples were dominated by the families Enterobacteriaceae and Pseudomonadaceae (class Gammaproteobacteria).

Naringenin degradation by the endophytic diazotroph Herbaspirillum seropedicae SmR1.

Several bacteria are able to degrade flavonoids either to use them as carbon sources or as a detoxification mechanism. Degradation pathways have been proposed for several bacteria, but the genes responsible are not known. We identified in the genome of the endophyte Herbaspirillum seropedicae SmR1 an operon potentially associated with the degradation of aromatic compounds. We show that this operon is involved in naringenin degradation and that its expression is induced by naringenin and chrysin, two closely related flavonoids. Mutation of fdeA, the first gene of the operon, and fdeR, its transcriptional activator, abolished the ability of H. seropedicae to degrade naringenin.

The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae

DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX Hs) can interact with the H. seropedicaeRecA protein (RecA Hs) and that RecA Hs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX Hs inhibited 90% of the RecA Hs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA Hs. RecA Hs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX Hs was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX Hs protein negatively modulates the RecA Hs activities by protein-protein interactions and also by DNA-protein interactions.

Effect of ATP and 2-oxoglutarate on the in vitro interaction between the NifA GAF domain and the GlnB protein of Azospirillum brasilense

Azospirillum brasilense is a diazotroph that associates with important agricultural crops and thus has potential to be a nitrogen biofertilizer. The A. brasilense transcription regulator NifA, which seems to be constitutively expressed, activates the transcription of nitrogen fixation genes. It has been suggested that the nitrogen status-signaling protein GlnB regulates NifA activity by direct interaction with the NifA N-terminal GAF domain, preventing the inhibitory effect of this domain under conditions of nitrogen fixation. In the present study, we show that an N-terminal truncated form of NifA no longer required GlnB for activity and lost regulation by ammonium. On the other hand, in trans co-expression of the N-terminal GAF domain inhibited the N-truncated protein in response to fixed nitrogen levels. We also used pull-down assays to show in vitro interaction between the purified N-terminal GAF domain of NifA and the GlnB protein. The results showed that A. brasilense GlnB interacts directly with the NifA N-terminal domain and this interaction is dependent on the presence of ATP and 2-oxoglutarate.