Evaluation of the Redesigned Enterotube—a System for the Identification of Enterobacteriaceae

The redesigned Enterotube has been evaluated with 414 unknown Enterobacteriaceae cultures from the stock culture collection of the Center for Disease Control. When the Enterotube was used as recommended by the manufacturer, an average of 96.4% of these cultures were correctly identified. Only two groups (Salmonella and Edwardsiella) were identified with less than 90% accuracy (89.2 and 87.5%, respectively). The Enterotube now provides a convenient, rapid, and accurate test system for the identification of typically reacting enteric bacteria.     

Cell-based imaging of sodium iodide symporter activity with the yellow fluorescent protein variant YFP-H148Q/I152L

The sodium iodide symporter (NIS) mediates iodide (I^–) transport in the thyroid gland and other tissues and is of increasing importance as a therapeutic target and nuclear imaging reporter. NIS activity in vitro is currently measured with radiotracers and electrophysiological techniques. We report on the development of a novel live cell imaging assay of NIS activity using the I^–-sensitive and genetically encodable yellow fluorescent protein (YFP) variant YFP-H148Q/I152L. In FRTL-5 thyrocytes stably expressing YFP-H148Q/I152L, I^– induced a rapid and reversible decrease in cellular fluorescence characterized by 1) high affinity for extracellular I^– (35 µM), 2) inhibition by the NIS inhibitor perchlorate, 3) extracellular Na⁺ dependence, and 4) TSH dependence, suggesting that fluorescence changes are due to I^– influx via NIS. Individual cells within a population of FRTL-5 cells exhibited a 3.5-fold variation in the rate of NIS-mediated I^– influx, illustrating the utility of YFP-H148Q/I152L to detect cell-to-cell difference in NIS activity. I^– also caused a perchlorate-sensitive decrease in YFP-H148Q/I152L fluorescence in COS-7 cells expressing NIS but not in cells lacking NIS. These results demonstrate that YFP-H148Q/I152L is a sensitive biosensor of NIS-mediated I^– uptake in thyroid cells and in nonthyroidal cells following gene transfer and suggest that fluorescence detection of cellular I^– may be a useful tool by which to study the pathophysiology and pharmacology of NIS. thyroid; fluorescence microscopy; FRTL-5 cells     

Evaluation of the Improved Auxotab 1 System for Identifying Enterobacteriaceae

The improved Auxotab Enteric 1 System is more accurate in identifying Enterobacteriaceae than is the original product, and results correlate better with those obtained with conventional tests.     

Aerobic training reduces oxidative stress in skeletal muscle of rats exposed to air pollution and supplemented with chromium picolinate

Objective: The purpose of this study was to investigate the effects of chromium picolinate (CrPic) supplementation associated with aerobic exercise using measures of oxidative stress in rats exposed to air pollution.

Methods: Sixty-one male Wistar rats were divided into eight groups: residual oil fly ash (ROFA) exposure and sedentary (ROFA-SED); ROFA exposure, sedentary and supplemented (ROFA-SED-CrPic); ROFA exposure and trained (ROFA-AT); ROFA exposure, supplemented and trained (ROFA-AT-CrPic); sedentary (Sal-SED); sedentary and supplemented (Sal-SED-CrPic); trained (Sal-AT); and supplemented and trained (Sal-AT-CrPic). Rats exposed to ROFA (air pollution) received 50?µg of ROFA daily via intranasal instillation. Supplemented rats received CrPic (1?mg/kg/day) daily by oral gavage. Exercise training was performed on a rat treadmill (5×/week). Oxidative parameters were evaluated at the end of protocols.

Results: Trained groups demonstrated lower gain of body mass (P?P?P?P?=?.0014), although CAT activity did not differ among groups (P?=?.4487).

Conclusion: Air pollution exposure did not lead to alterations in oxidative markers in lungs and heart, and exercise training was responsible for decreasing oxidative stress of the gastrocnemius.     

Nano-biolistics: a method of biolistic transfection of cells and tissues using a gene gun with novel nanometer-sized projectiles

Background

Biolistic transfection is proving an increasingly popular method of incorporating DNA or RNA into cells that are difficult to transfect using traditional methods. The technique routinely uses 'microparticles', which are ~1 μm diameter projectiles, fired into tissues using pressurised gas. These microparticles are efficient at delivering DNA into cells, but cannot efficiently transfect small cells and may cause significant tissue damage, thus limiting their potential usefulness. Here we describe the use of 40 nm diameter projectiles - nanoparticles - in biolistic transfections to determine if they are a suitable alternative to microparticles.

Results

Examination of transfection efficiencies in HEK293 cells, using a range of conditions including different DNA concentrations and different preparation procedures, reveals similar behaviour of microparticles and nanoparticles. The use of nanoparticles, however, resulted in ~30% fewer damaged HEK293 cells following transfection. Biolistic transfection of mouse ear tissue revealed similar depth penetration for the two types of particles, and also showed that < 10% of nuclei were damaged in nanoparticle-transfected samples, compared to > 20% in microparticle-transfected samples. Visualising details of small cellular structures was also considerably enhanced when using nanoparticles.

Conclusions

We conclude that nanoparticles are as efficient for biolistic transfection as microparticles, and are more appropriate for use in small cells, when examining cellular structures and/or where tissue damage is a problem.     

Low Concentrations of Selenium and Zinc in Nails are Associated with Childhood Asthma

The purpose of this study was to investigate possible associations between Zn, Se, Cu, Mn, and Co concentrations in nails and asthma in a young population from a Southern Brazil city. Additionally, correlations between these chemical elements among asthmatic and non-asthmatic children were evaluated. Before nail collection (n?=?165), children were asked to complete the International Study of Asthma and Allergies in Childhood questionnaire. The concentrations of trace elements were determined by inductively coupled plasma mass spectrometry. The chi-square test was used to evaluate the association between element concentrations in nails and the respiratory outcome. To evaluate correlations between the elements, we used the Spearman correlation test. For all tests, the significance level was set at 95% (P?≤?0.05). Children included in the highest quartile of nail Se and Zn concentration presented a fivefold decrease in the prevalence ratio of asthma while children in the lowest Se range presented an almost 2.5-fold increase in the asthma prevalence ratio. There were weak to strong correlations between Cu vs. Zn, Cu vs. Co, Cu vs. Se, Zn vs. Se, Zn vs. Mn, and Mn vs. Co in both asthmatic and non-asthmatic children. Interestingly, non-asthmatics also presented correlations between Co vs. Se and Zn. Taken together, our results clearly demonstrated an association between concentrations of selenium and zinc and childhood asthma and the usefulness of nail as a noninvasive matrix to detect minerals imbalance in asthma patients.     

Assessment of Trace Elements in Scalp Hair of a Young Urban Population in Brazil

The aim of this study was to establish background values for toxic and essential elements in hair, interelement correlations and the differences in levels between genders in a healthy young population from Southern Brazil. Hair samples (n = 167) were collected from healthy students aged 12–18 years. Trace element concentrations in hair were determined by inductively coupled plasma mass spectrometry. The study provided relatively low values for toxic elements and balanced concentrations for the essential elements in the adolescents' hair with reliable reference data. Interestingly, this study also demonstrated statistical correlations considered newfound between the elements in hair. Hair mercury levels were influenced by gender; with males presenting higher values. The overall findings of the present study, with respect to the estimated chemical elements, are of prime importance in the evaluation of reference values for determining environmental effects on children living in urban areas.     

Functional characterization and evolution of PTH/PTHrP receptors: insights from the chicken

ABSTRACT: BACKGROUND: The parathyroid hormone (PTH)-family consists of a group of structurally related factors that regulate calcium and bone homeostasis and are also involved in development of organs such as the heart, mammary gland and immune system. They interact with specific members of family 2 B1 G-protein coupled receptors (GPCRs), which have been characterised in teleosts and mammals. Two PTH/PTHrP receptors, PTH1R and PTH2R exist in mammals and in teleost fish a further receptor PTH3R has also been identified. Recently in chicken, PTHfamily members involved in calcium transport were characterized and specific PTHRs are suggested to exist although they have not yet been isolated or functionally characterized. The aim of this study is to further explore the evolution and function of the vertebrate PTH/PTHrP system through the isolation, phylogenetic analysis and functional characterization of the chicken receptors. RESULTS: Two PTHRs were isolated in chicken and sequence comparison and phylogenetic analysis indicate that the chicken receptors correspond to PTH1R and PTH3R, which emerged prior to the teleost/tetrapod divergence since they are present in cartilaginous fish. The vertebrate PTH2R receptor and its ligand TIP39 have been lost from bird genomes. Chicken PTH1R and PTH3R have a divergent and widespread tissue expression and are also evident in very early embryonic stages of development. Receptor stimulation studies using HEK293 cells stably expressing the chicken PTH1R and PTH3R and monitoring cAMP production revealed they are activated by chicken 1-34 N-terminal PTH-family peptides in a dose dependent manner. PTH-L and PTHrP were the most effective peptides in activating PTH1R (EC50 = 7.7 nM and EC50 = 22.7 nM, respectively). In contrast, PTH-L (100 nM) produced a small cAMP accumulation on activation of PTH3R but PTHrP and PTH (EC50 = 2.5 nM and EC50 = 22.1 nM, respectively) readily activated the receptor. PTHrP also stimulated intracellular Ca2+ accumulation on activation of PTH1R but not PTH3R. CONCLUSION: Two PTHR homologues of the vertebrate PTH1R and PTH3R were isolated and functionally characterized in chicken. Their distinct pattern of expression during embryo development and in adult tissues, together with their ligand preference, suggests that they have acquired specific functions, which have contributed to their maintenance in the genome. PTH2R and its activating ligand, TIP39, are absent from bird genomes. Nonetheless identification of putative PTH2R and TIP39 in the genome of an ancient agnathan, lamprey, suggests the PTH/PTHrP ligand and receptor family was already present in an early basal paraphyletic group of vertebrates and during the vertebrate radiation diverged via gene/genome duplication and deletion events. Knowledge of the role PTH/PTHrP system in early vertebrates will help to establish evolution of function.