Understanding the reaction balances behind the viability PCR protocols based on photoreactive dyes.

Viability-PCR (vPCR) protocols are mainly based on photo-reactive dyes impermeant to intact cell membranes. The absence of cell barriers allows the reagent's interaction with the genetic material after a short incubation period. By light-induced reaction, DNA becomes the unsuitable mould for the polymerases and thus cannot be amplified and detected by PCR. General rules and consensus exist on critical aspects of successful vPCR protocol development. However, the understanding of the vPCR reaction concerning how much reagent is really effective or the proper amount of light has been poorly studied. The convenience of using 600 times more dye than bases pairs exist suggests that although these dyes are DNA intercalating reagents, many organic molecules can adsorb it. Concerning light, no exact references exist about how much energy is needed to activate the azide group of reagents such as propidium monoazide. Therefore, it cannot be calculated in terms of energy how much light needs a vPCR protocol. The general rule is to provide reagents and energy in excess. This work provides different responses (based on experimental results) to both questions, which can contribute to a better understanding of the theoretical basis of vPCR protocols.

Improvement of the Pour Plate Method by Separate Sterilization of Agar and Other Medium Components and Reduction of the Agar Concentration.

Although the pour plate method is widely employed in microbiological quality control, it has certain drawbacks, including having to melt the culture medium before seeding. In this study, the preparation of the culture medium was modified by using a lower concentration of agar (10 g/L), which was separated from the nutrients during sterilization. The new protocol was assessed in media frequently used in microbiological quality control of food, cosmetics, and pharmaceutical products, with tryptic soy agar (TSA), Sabouraud 4% dextrose agar (SDA), and violet red bile glucose agar (VRBG). In comparison with the conventionally produced media, the modifications significantly improved the growth of Saccharomyces cerevisiae in SDA, Staphylococcus aureus, Salmonella enterica subsp. enterica serovar Typhimurium, and Candida albicans in TSA and Escherichia coli ATCC 8739 and ATCC 25922 and S. Typhimurium in VRBG. The modified VRBG was also more selective for Pseudomonas aeruginosa. Regarding physicochemical properties, a significantly lower pH was observed in TSA and VRBG and lower strength values in TSA. Sterilizing agar separately from the other components of the medium and reducing the agar concentration to 10 g/L can improve microorganism growth and enhance the selectivity of differential media in the pour plate method. These modifications could facilitate the automation of this culture technique. IMPORTANCE In the era of rapid microbiological methods, there is a need to improve long-established culture techniques. Drawbacks of the pour plate method include having to melt each medium separately before seeding. For this technique, we demonstrate that separating the agar from the other components of commonly used media during sterilization and reducing the agar concentration to 10 g/L can enhance microbial growth. The new protocol could have advantages in routine laboratory practice because less agar is required and the same molten agar suspension can be used to prepare different media. Moreover, these modifications could facilitate the automation of the pour plate method.

Improvement of Mueller-Kauffman Tetrathionate-Novobiocin (MKTTn) enrichment medium for the detection of Salmonella enterica by the addition of ex situ-generated tetrathionate.

The detection of Salmonella in food is based on the use of a selective enrichment broth such as Muller-Kauffman Tetrathionate-Novobiocin (MKTTn), in which tetrathionate plays a key role by providing Salmonella with a growth advantage. As sodium tetrathionate is unstable, it is generated in situ by the addition of iodine (Lugol's solution) before seeding. This step is cumbersome as the solution is easily spilled, compromising the performance of the medium and hindering the work of technicians. The aim of this study was to optimize MKTTn broth by generating tetrathionate ex situ through an external reaction between iodine and thiosulphate followed by lyophilization. Quality control procedures were performed to compare the modified and original media, testing pure productivity (enrichment with 50-120 CFU of Salmonella Thyphimurium ATCC 14028 and Salmonella Enteritidis ATCC 13076 and plating on Xylose Lysine Deoxycholate agar, XLD), mixed productivity (50-120 CFU of Salmonella strains and Pseudomonas aeruginosa and Escherichia coli at ≥104 CFU and XLD plating) and selectivity (≥104 CFU of P. aeruginosa and Enterococcus faecalis and plating on Tryptone Casein Soy agar, TSA). The modified MKTTn medium (S/L) performed comparably with the original medium in terms of growth of both Salmonella strains (>300 colonies in XLD), alone or with P. aeruginosa and E. coli. Quantitative assays showed no statistically significant differences in the number of colonies grown on XLD after 10-5 dilution (p = 0.7015 with S. Thyphimurium ATCC 14028 and p = 0.2387 with S. Enteritidis ATCC 13076; ANOVA test). MKTTn medium (S/L) was also selective against E. coli (≤100 colonies) and E. faecalis (<10 colonies). These results suggest that adding tetrathionate as a lyophilisate (S/L) is a feasible alternative to the use of Lugol's solution for the preparation of MKTTn enrichment broth and does not affect the properties of the medium.

Global patterns of phosphatase activity in natural soils.

Soil phosphatase levels strongly control the biotic pathways of phosphorus (P), an essential element for life, which is often limiting in terrestrial ecosystems. We investigated the influence of climatic and soil traits on phosphatase activity in terrestrial systems using metadata analysis from published studies. This is the first analysis of global measurements of phosphatase in natural soils. Our results suggest that organic P (Porg), rather than available P, is the most important P fraction in predicting phosphatase activity. Structural equation modeling using soil total nitrogen (TN), mean annual precipitation, mean annual temperature, thermal amplitude and total soil carbon as most available predictor variables explained up to 50% of the spatial variance in phosphatase activity. In this analysis, Porg could not be tested and among the rest of available variables, TN was the most important factor explaining the observed spatial gradients in phosphatase activity. On the other hand, phosphatase activity was also found to be associated with climatic conditions and soil type across different biomes worldwide. The close association among different predictors like Porg, TN and precipitation suggest that P recycling is driven by a broad scale pattern of ecosystem productivity capacity.

Pitfalls and variants in pediatric chest imaging. / Pitfalls y variantes del tórax pediátrico.

Most pitfalls in the interpretation of pediatric chest imaging are closely related with the technique used and the characteristics of pediatric patients. To obtain a quality image that will enable the correct diagnosis, it is very important to use an appropriate technique. It is important to know how technical factors influence the image and to be aware of the possible artifacts that can result from poor patient cooperation. Moreover, radiologists need to be familiar with the normal anatomy in children, with the classic radiologic findings, and with the anatomic and developmental variants to avoid misinterpreting normal findings as pathological.

Biogenic volatile emissions from the soil.

Volatile compounds are usually associated with an appearance/presence in the atmosphere. Recent advances, however, indicated that the soil is a huge reservoir and source of biogenic volatile organic compounds (bVOCs), which are formed from decomposing litter and dead organic material or are synthesized by underground living organism or organs and tissues of plants. This review summarizes the scarce available data on the exchange of VOCs between soil and atmosphere and the features of the soil and particle structure allowing diffusion of volatiles in the soil, which is the prerequisite for biological VOC-based interactions. In fact, soil may function either as a sink or as a source of bVOCs. Soil VOC emissions to the atmosphere are often 1-2 (0-3) orders of magnitude lower than those from aboveground vegetation. Microorganisms and the plant root system are the major sources for bVOCs. The current methodology to detect belowground volatiles is described as well as the metabolic capabilities resulting in the wealth of microbial and root VOC emissions. Furthermore, VOC profiles are discussed as non-destructive fingerprints for the detection of organisms. In the last chapter, belowground volatile-based bi- and multi-trophic interactions between microorganisms, plants and invertebrates in the soil are discussed.

[Persistence of antibodies three years after vaccination with the 23-valent pneumococcal vaccine]. / Persistencia de anticuerpos a los 3 años de la inmunización antineumocócica 23-valente en pacientes con síndrome nefrótico.

OBJECTIVE: To study the persistence of antibodies after primary immunization with the 23-valent pneumococcal vaccine in children with nephrotic syndrome. METHODS: Sera from 14 of 26 children with nephrotic syndrome were obtained 3 years after vaccination. We used an ELISA method to measure IgG antibody levels to pneumococcal serotypes 14, 23F, 3 and 6B. Antibody levels before vaccination, 1 month and 3 years after vaccination were compared. RESULTS: Significant increases in specific antibody concentrations were observed 30 days after vaccination for all serotypes except serotype 3. Differences in response according to serotype were found. The highest serological response was observed for serotype 14 (78.5 % of the patients showed a good-to-moderate response). Three years after vaccination antibody levels were significantly decreased (27.3 % of children for serotype 14, 46.2 % for serotype 23F and 50 % for serotype 6B). CONCLUSIONS: The 23-valent pneumococcal vaccine is immunogenic in children aged more than 2 years with nephrotic syndrome. Response varied according to serotype. Persistence of vaccine-induced antibodies is short-term, with low antibody levels 3 years after immunization. The results of this study suggest that these patients require revaccination 3 years after the first dose.

[Smoking of school children in rural Castilla-León environment. Attitudes of the school population]. / Tabaquismo escolar en el medio rural de Castilla-León. Actitudes de la población escolar.

Tobacco smoking is the most important health problem among school-age children and educational programs aiming at prevention must be taken up by all members of the school community (parents, teachers and students) as well as by health providers. We designed an anti-smoking campaign lasting three years directed towards sixth, seventh and eight-grade elementary school students and secondary school pupils attending classes in the area of Vitigudino (Salamanca). We report the results of student attitude surveys done at the beginning of the program. Sixty-eight percent of boys and 50.7% of girls have smoked at some time. The average age of first contact with tobacco was 12.1 +/- 2.4 years, with boys starting to smoke at a significantly younger age (p < 0.001) than girls. At present, 13.7% of school children smoke. The main reasons given for starting to smoke were that friends smoked (56.9%) and curiosity (55.4%). Tobacco smoking in the family was mentioned by only 29.5% of the students surveyed. Knowledge of medical problems related to smoking was limited. Intention to smoke upon leaving school was reported by 9%, while 46.7% did not know if they would smoke. The fact that only 44.1% of school children believe they will not smoke upon leaving school highlights the need to provide support information that will help students abstain. Programs aimed at school children have been shown to help reduce tobacco smoking in this population. An adolescent who learns to avoid tobacco is unlikely to be a smoker in adulthood.(ABSTRACT TRUNCATED AT 250 WORDS)

Drug screening of meconium in infants of drug-dependent mothers: an alternative to urine testing.

Meconium specimens (first 3 days' stool) obtained from 20 infants of drug-dependent mothers and five control infants were analyzed by radioimmunoassay for the metabolites of three commonly abused drugs, heroin, cocaine, and cannabinoids. Control stools contained no drug. Meconium from the infants of drug-dependent mothers showed the presence of at least one drug metabolite: 80% of the infants of drug-dependent mothers showed cocaine (range 0.14 to 19.91 micrograms/gm stool), 55% showed morphine (range 0.41 to 14.97 micrograms/gm stool), and 60% showed cannabinoid (range 0.05 to 0.67 micrograms/gm stool). The concentrations of metabolites were highest during the first 2 days; some stools tested positive up to the third day. In contrast, only 37% of the infants had positive results on a urine screen (fluorescent polarization immunoassay method). Paired urine and meconium specimens, both analyzed by radioimmunoassay, showed a higher concentration of drug metabolites in the latter; eight urine samples had no detectable drugs despite a corresponding positive stool test result. We conclude that meconium is useful for drug screening in the neonate.