Rapid determination of Phytophthora infestans sporangia using a surface plasmon resonance immunosensor

Phytophthora infestans is the cause of late blight disease in potato and is an economically important pathogen worldwide. Early disease detection is important to implement disease control measures. In this study a surface plasmon resonance (SPR) immunosensor for detection of P. infestans sporangia is presented. The specificity of an existing mouse monoclonal antibody (phyt/G1470 mAb) against P. infestans was investigated in plate-trapped antigen ELISA and in subtractive inhibition ELISA. No or only limited cross-reactivity was observed against representatives having air-borne spores from Ascomycetes, Deuteromycetes as well as Basidiomycetes. phyt/G1470 mAb was incorporated in a subtractive inhibition SPR assay, consisting of a pre-incubation of mAb and sporangia, a centrifugation step to remove sporangia-bound phyt/G1470 mAb and quantification of remaining phyt/G1470 mAb by SPR. Good intra- and interday assay variability was observed and the assay had a detection limit of 2.2x10(6) sporangia/ml. Analysis time was 75 min, which is superior to existing P. infestans detection methods.     

Detection of fungal spores using a generic surface plasmon resonance immunoassay

This paper describes a biosensor-based method for detection of fungal spores using surface plasmon resonance (SPR). The approach involves the use of a mouse monoclonal antibody (Pst mAb8) and a SPR sensor for label-free detection of urediniospores from the model organism Puccinia striiformis f.sp. tritici (Pst). In the subtractive inhibition assay, urediniospores and Pst mAb8 were mixed, urediniospore-bound Pst mAb8 removed by centrifugation and the remaining Pst mAb8 quantified using the SPR sensor. Assay conditions were optimised and a detection limit of 3.1 x 10(5)urediniospores/ml was achieved. Spiked Pst samples were further examined in a background of a related spore and it was found that Pst detection was possible in this mixture. This study represent the first use of SPR technology for fungal spore detection as well as the first report of a successful biosensor-based detection strategy for Pst.     

A predictive model for respiratory syncytial virus (RSV) hospitalisation of premature infants born at 33–35 weeks of gestational age,based on data from the Spanish FLIP study

Background

The aim of this study, conducted in Europe, was to develop a validated risk factor based model to predict RSV-related hospitalisation in premature infants born 33–35 weeks'' gestational age (GA).

Methods

The predictive model was developed using risk factors captured in the Spanish FLIP dataset, a case-control study of 183 premature infants born between 33–35 weeks'' GA who were hospitalised with RSV, and 371 age-matched controls. The model was validated internally by 100-fold bootstrapping. Discriminant function analysis was used to analyse combinations of risk factors to predict RSV hospitalisation. Successive models were chosen that had the highest probability for discriminating between hospitalised and non-hospitalised infants. Receiver operating characteristic (ROC) curves were plotted.

Results

An initial 15 variable model was produced with a discriminant function of 72% and an area under the ROC curve of 0.795. A step-wise reduction exercise, alongside recalculations of some variables, produced a final model consisting of 7 variables: birth ± 10 weeks of start of season, birth weight, breast feeding for ≤ 2 months, siblings ≥ 2 years, family members with atopy, family members with wheeze, and gender. The discrimination of this model was 71% and the area under the ROC curve was 0.791. At the 0.75 sensitivity intercept, the false positive fraction was 0.33. The 100-fold bootstrapping resulted in a mean discriminant function of 72% (standard deviation: 2.18) and a median area under the ROC curve of 0.785 (range: 0.768–0.790), indicating a good internal validation. The calculated NNT for intervention to treat all at risk patients with a 75% level of protection was 11.7 (95% confidence interval: 9.5–13.6).

Conclusion

A robust model based on seven risk factors was developed, which is able to predict which premature infants born between 33–35 weeks'' GA are at highest risk of hospitalisation from RSV. The model could be used to optimise prophylaxis with palivizumab across Europe.     

Sponge OAS has a distinct genomic structure within the 2-5A synthetase family

2′,5′-Oligoadenylate synthetases (2-5A synthetases, OAS) are enzymes that play an important role in the interferon-induced antiviral defense mechanisms in mammals. Sponges, the evolutionarily lowest multicellular animals, also possess OAS; however, their function is presently unclear. Low homology between primary structures of 2-5A synthetases from vertebrates and sponges renders their evolutionary relationship obscure. The genomic structure of vertebrate OASs has been thoroughly examined, making it possible to elucidate molecular evolution and expansion of this gene family. Until now, no OAS gene structure was available from sponges to compare it with the corresponding genes from higher organisms. In the present work, we determined the exon/intron structure of the OAS gene from the marine sponge Geodia cydonium and found it to be completely different from the strictly conserved exon/intron pattern of the OAS genes from vertebrates. This finding was corroborated by the analysis of OAS genes from another sponge, Amphimedon queenslandica, whose genome was recently sequenced. Our data suggest that vertebrate and sponge OAS genes have no direct common intron-containing ancestor and two (sub)types of OAS may be discriminated. This study opens new perspectives for understanding the phylogenesis and evolution of 2-5A synthetases as well as functional aspects of this multigene family. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Abbreviations  The nomenclature of particular OASs in the present paper is based on the level of their sequence similarities. OAS1 refers to the OAS consisting of a single OAS domain. Capital letters (OAS1X) are used to differentiate between OAS1 types with sequence homologies of less than 50%; their variants are additionally marked in small letters (OASXx, sequence homology ~70 to ~95%). Labels prime and double prime denote sponge OAS1Xx gene haplotypes (sequence homology close to 100%, a few amino acid substitutions).     

The interferon alpha induced protein ISG12 is localized to the nuclear membrane.

Interferons exert their biological function mainly through the activation of interferon-stimulated genes (ISGs). ISG12 (originally designated p27) belongs to a family of small, interferon alpha inducible genes of unknown function. We have determined the 5' end sequence of ISG12 cDNA from the human cell lines HeLa and AMA by RACE. Comparing this sequence to ISG12 sequences in the expressed sequence tag (EST) database revealed the presence of two alternative splice variants of ISG12 in human cells exhibiting the same open reading frame. We have sequenced the promoter region of the ISG12 gene and found ISRE, IRF1/IRF2, and STAT elements correlating to the interferon alpha inducibility of the gene. Subsequently, we have expressed human ISG12, a 12-kDa hydrophobic protein in the baculovirus expression system and with a C-terminal FLAG-tag in the human cell line 293. Recombinant ISG12 sediments in the nuclear envelope in both cell types. Finally, we have been able to demonstrate the prevalence of the ISG12 gene product in the nuclear envelope of HeLa cells treated with interferon alpha by immunocytochemical analyses. ISG12 is the first interferon induced protein found localizing to the nuclear envelope.     

Mycoplasma hominis septicaemia.

Mycoplasma hominis septicaemia occurred in a patient with a malignant lymphoma of lymphoblastic type in leukaemic phase. M hominis was isolated several times from blood cultures with antibody titres against the micro-organism rising to a high level despite severe immunosuppression. M hominis was detected in the blood after subculture of the blood culture bottles despite their macroscopically normal appearance. The patient''s pyrexia resolved without treatment with antibiotics effective against M hominis.     

Inhibition of mouse fibroblast interferon by gangliosides. Differential effects on biological activity and on induction of (2'--5')oligoadenylate synthetase

Gangliosides are potent inhibitors of the antiviral activity of mouse fibroblasts and other beta-interferons. We have compared the effects of gangliosides on antiviral and antigrowth activities of mouse fibroblast interferon and on the induction of (2'--5')oligoadenylate synthetase, one of the enzymes implicated in the antiviral state induced by interferon. Whereas both biological effects appear to be inhibited by gangliosides in an analogous fashion, inhibition of induction of (2'--5')oligoadenylate synthetase does not correlate with inhibition of vesicular stomatitis virus replication. Ganglioside concentrations that inhibit the interferon-induced (2'--5')oligoadenylate synthetase to levels close to those of uninduced cells, still allow for a 100--1000-fold reduction of viral yield. Significantly higher ganglioside concentrations are required to prevent completely the antiviral effect. This biphasic relationship between (2'--5')oligoadenylate synthetase levels and inhibition of viral yield suggests that no or very small increases in synthetase levels are involved in inhibition of virus by between two and three orders of magnitude.