Real-time polymerase chain reaction as a rapid and efficient alternative to estimation of picornavirus titers by tissue culture infectious dose 50% or plaque forming units

Quantification of viral infectious units is traditionally measured by methods based on forming plaques in semisolid media (PFU) or endpoint dilution of a virus-containing solution (TCID₅₀), methods that are laborious, time-consuming and take on average 3–7 days to carry out. Quantitative real-time PCR is an established method to quantify nucleic acids at high accuracy and reproducibility, routinely used for virus detection and identification. In the present study, a procedure was developed using a two-step real-time PCR and the SYBR Green detection method to study whether there are correlations between TCID₅₀/ml, PFU/ml and Ct values generated by real-time PCR enabling rapid and efficient calculation of titer equivalents when working with viruses in the research laboratory. In addition, an external standard with known concentrations was included using in vitro transcribed viral RNA, thus allowing the calculation of the amount of RNA copies needed for various applications (i.e. per plaque or TCID₅₀).The results show that there is a correlation between the three quantification methods covering a wide range of concentration of viruses. Furthermore, a general regression line between TCID₅₀ and Ct values was obtained for all viruses included in the study, which enabled recording titer equivalents using real-time PCR. Finally, by including an external standard, the amount of RNA genomes generating one TCID₅₀ or PFU for each enterovirus serotype included was determined.     

Immunohistochemical localization to pyloric antral G cells of peptides derived from porcine preprogastrin

Antibodies to the peptides (designated cryptic A and B) that flank the G34 region of pig progastrin were used in immunohistochemical studies of the gastrointestinal tract. In elution and restaining experiments, the same cells were revealed by the cryptic peptide antibodies, and by antibodies specific for C-terminus of G17 and N-terminus of G34. The cells reacting with the cryptic peptide antibodies were localized predominantly to antral mucosa. They were found in pig, ferret, dog and cat but not in man, guinea pig, rat or mouse; presumably in the latter species there are amino acid substitutions in the cryptic peptides that influence immunoreactivity with the present antibodies. The results indicate that progastrin production occurs only in G cells in the gut, and that a single population of cells produces all the predicted regions of progastrin.     

The influence of autotrophy,heterotrophy and temperature on pelagic food web efficiency in a brackish water system

Climate change has been suggested to lead to higher temperature and increased heterotrophy in aquatic systems. The aim of this study was to test how these two factors affect metazooplankton and food web efficiency (FWE was defined as metazooplankton production divided by basal production). We tested the following hypotheses: (1) that lower metazooplankton production and lower FWE would be found in a food web based on heterotrophic production (bacteria) relative to one based on autotrophic production (phytoplankton), since the former induces a larger number of trophic levels; (2) the metazooplankton in the heterotrophic food web would contain less essential fatty acids than those from the autotrophic food web; and (3) that higher temperature would lead to increased FWE. To test these hypotheses, a mesocosm experiment was established at two different temperatures (5 and 10°C) with a dominance of either autotrophic (NP) or heterotrophic basal production (CNP). Metazooplankton production increased with temperature, but was not significantly affected by differences in basal production. However, increased heterotrophy did lead to decreased fatty acid content and lower individual weight in the zooplankton. FWE increased with autotrophy and temperature in the following order: 5CNP < 10CNP < 5NP < 10NP. Our results indicate that in the climate change scenario we considered, the temperature will have a positive effect on FWE, whereas the increase in heterotrophy will have a negative effect on FWE. Furthermore, the quality and individual weight of the metazooplankton will be reduced, with possible negative effects on higher trophic levels.     

Determination of Leb-active oligosaccharides in urine of pregnant and lactating women by radioimmunoassay.

Antiserum obtained by immunizing a goat with lacto-N-difucohexaose I, a Leb blood-group hapten, coupled to poly-L-lysine was used in a radioimmunoassay to detect Leb-active oligosaccharides (chiefly lacto-N-difucohexaose I) in urine of 138 pregnant and lactating women of different ABO and Lewis blood groups. Specificity of the method was determined by comparing inhibitory activities of 18 oligosaccharides. Only women who belonged to the Leb blood group excreted Leb-active oligosaccharides in urine. Leb-active oligosaccharides increase during pregnancy, reaching levels up to approximately 70 mumol lacto-N-difucohexaose I equivalents per pmol creatinine in the third trimester and early post-partum period. Excretion varies considerably but tends to be highest in those individuals who strongly express the Leb antigen on their red blood cells or who belong to blood group O. Leb-active oligosaccharides were detected in plasma of a few individuals but at concentrations 1000-fold lower than in urine.     

Quantified proarrhythmic potential of selected human embryonic stem cell-derived cardiomyocytes

To improve proarrhythmic predictability of preclinical models, we assessed whether human ventricular-like embryonic stem cell-derived cardiomyocytes (hESC-CMs) can be selected following a standardized protocol. Also, we quantified their arrhythmogenic response and compared this to a contemporary used rabbit Purkinje fiber (PF) model. Multiple transmembrane action potentials (AP) were recorded from 164 hESC-CM clusters (9 different batches), and 12 isolated PFs from New Zealand White rabbits. AP duration (APD), early afterdepolarizations (EADs), triangulation (T), and short-term variability of repolarization (STV) were determined on application of the I_Kr blocker E-4031 (0.03/0.1/0.3/1 μM). Isoproterenol (0.1 μM) was used to assess adrenergic response. To validate the phenotype, RNA isolated from atrial- and ventricular-like clusters (n = 8) was analyzed using low-density Taqman arrays. Based on initial experiments, slow beating rate (< 50 bpm) and long APD (> 200 ms) were used to select 31 ventricular-like clusters. E-4031 (1 μM) prolonged APD (31/31) and induced EADs only in clusters with APD90 > 300 ms (11/16). EADs were associated with increased T (1.6 ± 0.2 vs 2.0 ± 0.3?) and STV (2.7 ± 1.5 vs 6.9 ± 1.9?). Rabbit PF reacted in a similar way with regards to EADs (5/12), increased T (1.3 ± 0.1 vs 1.9 ± 0.4?), and STV (1.2 ± 0.9 vs 7.1 ± 5.6?). According to ROC values, hESC-CMs (STV 0.91) could predict EADs at least equivalent to PF (STV 0.69). Isoproterenol shortened APD and completely suppressed EADs. Gene expression analysis revealed that HCN1/2, KCNA5, and GJA5 were higher? in atrial/nodal-like cells, whereas KCNJ2 and SCN1B were higher? in ventricular-like cells (?P < 0.05). Selection of hESC-CM clusters with a ventricular-like phenotype can be standardized. The proarrhythmic results are qualitatively and quantitatively comparable between hESC-CMs and rabbit PF. Our results indicate that additional validation of this new safety pharmacology model is warranted.     

Determination of small amounts of noradrenaline using a fluorescence histochemical method

Summary Using the Hayashi method, the distribution of glucosaminidase was studied throughout sequential molar development. Conspicuous activity was observed in certain cellular components of the stellate reticulum and dental papilla. The outer enamel epithelium, stratum intermedium and ameloblasts displayed less intense reactions.Supported by PHS Grant No. 2800-02 —Tooth Germ Development, National Institute of Dental Research, National Institutes of Health.Lieutenant Commander, Dental Corps, United States Navy.     

Effects of the parkinsonism-inducing neurotoxin MPTP and its metabolite MPP+ on sympathetic adrenergic nerves in mouse iris and atrium

The effect of systemic administration of the parkinsonism-inducing neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and its metabolite MPP+ (1-methyl-4-phenylpyridine) on sympathetic adrenergic nerves in mouse iris and atrium has been investigated employing histo- and neurochemical techniques. The results indicate that MPTP does not have any potent neurotoxic effects on sympathetic adrenergic nerves. The effects of MPTP noted appear mainly to be restricted to a noradrenaline (NA) -depleting action and an acutely transient impairment of the NA uptake mechanism. This latter effect could be counteracted by monoamine oxidase inhibition. MPP+ was found to have more potent neurotoxic actions than MPTP as reflected i.e. by a patchy loss of histochemically demonstrable adrenergic nerves in iris which persisted for at least 7 days. Pretreatment with the NA uptake blocker desipramine antagonised the effects of MPP+, indicating that neurotoxicity is mediated via the NA uptake mechanism. The difference in neurotoxic potency of MPTP between sympathetic adrenergic nerves and central catecholamine neurons might be related to differences in metabolism of MPTP in the CNS and the periphery and/or due to the sympathetic adrenergic nerves being more resistant towards the cytotoxic actions following MPTP administration.