PCR and real time PCR for the detection of Cryptosporidium parvum oocyst DNA

Three DNA extraction kits were used, all without preliminary procedures, then DNA extraction was preceded with freeze/thaw cycles in three versions. A lack of desired effect resulted in the application of liquid nitrogen/water bath cycles before the use of the extractions in further experiments. The effectiveness of DNA extraction was measured by PCR signal and C(T) values of real time PCR. A comparison of the efficiency of various Cryptosporidium parvum undiluted oocyst treatments prior to DNA extraction with the use of three kits has shown that the best results were obtained after extraction of DNA with the QIAamp DNA Tissue Mini Kit (T kit), preceded by triple liquid nitrogen/water bath in 100 degrees C for 2 minutes and with overnight proteinase K digestion. After extraction with the T kit, the detection limit was 50 oocysts per 200 microl when effectiveness was evaluated with PCR and 10 oocysts in the case of real time PCR.     

Density-dependent and inter-specific interactions affecting European eel settlement in freshwater habitats

Identifying the factors influencing the settlement of European eel (Anguilla anguilla) juveniles in continental habitats is crucial to designing effective management and conservation measures for this endangered species. A long-term data series (1993–2008) of European eel and European catfish (Silurus glanis) abundance in a freshwater canal of the Camargue water system (southern France), along with parallel data on water salinity and glass eel abundance in the adjacent Vaccarès lagoon, was analysed to identify the possible causes of decline in eel abundance observed in the canal during the last two decades. A model including glass eel recruitment and catfish abundance as covariates explained 78% of the observed variation in eel settlement success. Results suggest that (1) salinity does not play a significant role in determining the fraction of eels moving from the brackish lagoon to the canal; (2) density dependence affects settlement success, possibly through a reduction of juvenile survival in the adjacent lagoon; and (3) catfish abundance is negatively correlated with eel settlement. We discuss this latter point in terms of possible predation of catfish upon eels and/or inter-specific competition between the two species.     

An anti-inflammatory role for carbon monoxide and heme oxygenase-1 in chronic Th2-mediated murine colitis

Cigarette smoking is a significant environmental factor in the human inflammatory bowel diseases, remarkably, conferring protection in ulcerative colitis. We previously demonstrated that a prominent component of cigarette smoke, CO, suppresses Th17-mediated experimental colitis in IL-10(-/-) mice through a heme oxygenase (HO)-1-dependent pathway. In this study, homeostatic and therapeutic effects of CO and HO-1 were determined in chronic colonic inflammation in TCR-α-deficient ((-/-)) mice, in which colitis is mediated by Th2 cytokines, similar to the cytokine milieu described in human ulcerative colitis. TCRα(-/-) mice exposed to CO or treated with the pharmacologic HO-1 inducer cobalt protoporphyrin demonstrated amelioration of active colitis. CO and cobalt protoporphyrin suppressed colonic IL-1β, TNF, and IL-4 production, whereas IL-10 protein secretion was increased. CO induced IL-10 expression in macrophages and in vivo through an HO-1-dependent pathway. Bacterial products regulate HO-1 expression in macrophages through MyD88- and IL-10-dependent pathways. CO exposure and pharmacologic HO-1 induction in vivo resulted in increased expression of HO-1 and IL-10 in CD11b(+) lamina propria mononuclear cells. Moreover, induction of the IL-10 family member IL-22 was demonstrated in CD11b(-) lamina propria mononuclear cells. In conclusion, CO and HO-1 induction ameliorated active colitis in TCRα(-/-) mice, and therapeutic effects correlated with induction of IL-10. This study provides further evidence that HO-1 mediates an important homeostatic pathway with pleiotropic anti-inflammatory effects in different experimental models of colitis and that targeting HO-1, therefore, is a potential therapeutic strategy in human inflammatory bowel diseases.     

Cutting edge: Tissue-retentive lung memory CD4 T cells mediate optimal protection to respiratory virus infection

We identify in this article a new class of lung tissue-resident memory CD4 T cells that exhibit tissue tropism and retention independent of Ag or inflammation. Tissue-resident memory CD4 T cells in the lung did not circulate or emigrate from the lung in parabiosis experiments, were protected from in vivo Ab labeling, and expressed elevated levels of CD69 and CD11a compared with those of circulating memory populations. Importantly, influenza-specific lung-resident memory CD4 T cells served as in situ protectors to respiratory viral challenge, mediating enhanced viral clearance and survival to lethal influenza infection. By contrast, memory CD4 T cells isolated from spleen recirculated among multiple tissues without retention and failed to mediate protection to influenza infection, despite their ability to expand and migrate to the lung. Our results reveal tissue compartmentalization as a major determining factor for immune-mediated protection in a key mucosal site, important for targeting local protective responses in vaccines and immunotherapies.     

Avian coronavirus in wild aquatic birds

We detected a high prevalence (12.5%) of novel avian coronaviruses in aquatic wild birds. Phylogenetic analyses of these coronaviruses suggest that there is a diversity of gammacoronaviruses and deltacoronaviruses circulating in birds. Gammacoronaviruses were found predominantly in Anseriformes birds, whereas deltacoronaviruses could be detected in Ciconiiformes, Pelecaniformes, and Anseriformes birds in this study. We observed that there are frequent interspecies transmissions of gammacoronaviruses between duck species. In contrast, deltacoronaviruses may have more stringent host specificities. Our analysis of these avian viral and host mitochondrial DNA sequences also suggests that some, but not all, coronaviruses may have coevolved with birds from the same order.     

Model parameter uncertainties in a dual-species biofilm competition model affect ecological output parameters much stronger than morphological ones

Bacterial biofilms are complex microbial depositions on immersed interfaces that form wherever the environmental conditions sustain microbial growth. Despite their name, biofilms can develop in highly irregular structures. Recently several mathematical concepts have been introduced to model these spatially structured microbial populations. Regardless of the type of model, they all have, even for microbially relatively simple systems, many parameters which generally are known at most approximately. We investigate the effect of uncertainties in model parameters on four morphological and four ecological output parameters using a nonlinear diffusion model for a biofilm in which two species compete for a shared nutrient. To this end we conduct an extensive computer simulation experiment for two different levels of data uncertainty, three different hydrodynamic conditions, and two different scenarios of bulk substrate availability. Our results indicate that input model parameter uncertainties have a much larger effect on ecological than on morphological output parameters.     

Nostophycin biosynthesis is directed by a hybrid polyketide synthase-nonribosomal peptide synthetase in the toxic cyanobacterium Nostoc sp. strain 152

Cyanobacteria are a rich source of natural products with interesting pharmaceutical properties. Here, we report the identification, sequencing, annotation, and biochemical analysis of the nostophycin (npn) biosynthetic gene cluster. The npn gene cluster spans 45.1 kb and consists of three open reading frames encoding a polyketide synthase, a mixed polyketide nonribosomal peptide synthetase, and a nonribosomal peptide synthetase. The genetic architecture and catalytic domain organization of the proteins are colinear in arrangement, with the putative order of the biosynthetic assembly of the cyclic heptapeptide. NpnB contains an embedded monooxygenase domain linking nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) catalytic domains and predicted here to hydroxylate the nostophycin during assembly. Expression of the adenylation domains and subsequent substrate specificity assays support the involvement of this cluster in nostophycin biosynthesis. Biochemical analyses suggest that the loading substrate of NpnA is likely to be a phenylpropanoic acid necessitating deletion of a carbon atom to explain the biosynthesis of nostophycin. Biosyntheses of nostophycin and microcystin resemble each other, but the phylogenetic analyses suggest that they are distantly related to one another.     

Estimating the lowest detectable dose of ionizing radiation by FISH whole-chromosome painting

Chromosome translocations are the hallmark of exposure to ionizing radiation, but they also occur spontaneously, and their frequencies increase dramatically with age. This complicates dosimetry unless a pre-exposure sample is available for each putatively exposed individual. Here we use published values for translocations in unexposed subjects from a wide range of ages, together with data from an in vitro (137)Cs dose-response curve, to estimate the minimum dose of whole-body radiation that is detectable by translocation analyses in individuals of a given age. For subjects aged 20 to 69 years, we show that the minimum detectable acute dose increases linearly with age at a rate of 0.179, 0.218 and 0.256 cGy per year for significance levels of P = 0.05, P = 0.01 and P = 0.001, respectively. For chronic exposures, the corresponding minimum detectable doses are 1.591, 2.270 and 3.055 cGy per year. For newborns, the 95th and 99th percentiles of translocation frequencies are 0.20 and 0.31 per 100 cell equivalents, respectively, indicating that values greater than these are consistent with exposures at P = 0.05 and P = 0.01, respectively. These results improve our understanding of the requirements and limitations for performing biological dosimetry when only the age of the exposed individual is known.     

High sensitivity to short wavelengths in a lizard and implications for understanding the evolution of visual systems in lizards

Progress in developing animal communication theory is frequently constrained by a poor understanding of sensory systems. For example, while lizards have been the focus of numerous studies in visual signalling, we only have data on the spectral sensitivities of a few species clustered in two major clades (Iguania and Gekkota). Using electroretinography and microspectrophotometry, we studied the visual system of the cordylid lizard Platysaurus broadleyi because it represents an unstudied clade (Scinciformata) with respect to visual systems and because UV signals feature prominently in its social behaviour. The retina possessed four classes of single and one class of double cones. Sensitivity in the ultraviolet region (UV) was approximately three times higher than previously reported for other lizards. We found more colourless oil droplets (associated with UV-sensitive (UVS) and short wavelength-sensitive (SWS) photoreceptors), suggesting that the increased sensitivity was owing to the presence of more UVS photoreceptors. Using the Vorobyev-Osorio colour discrimination model, we demonstrated that an increase in the number of UVS photoreceptors significantly enhances a lizard's ability to discriminate conspecific male throat colours. Visual systems in diurnal lizards appear to be broadly conserved, but data from additional clades are needed to confirm this.