Modelling seasonal variations in the age and incidence of Kawasaki disease to explore possible infectious aetiologies

The average age of infection is expected to vary during seasonal epidemics in a way that is predictable from the epidemiological features, such as the duration of infectiousness and the nature of population mixing. However, it is not known whether such changes can be detected and verified using routinely collected data. We examined the correlation between the weekly number and average age of cases using data on pre-vaccination measles and rotavirus. We show that age-incidence patterns can be observed and predicted for these childhood infections. Incorporating additional information about important features of the transmission dynamics improves the correspondence between model predictions and empirical data. We then explored whether knowledge of the age-incidence pattern can shed light on the epidemiological features of diseases of unknown aetiology, such as Kawasaki disease (KD). Our results indicate KD is unlikely to be triggered by a single acute immunizing infection, but is consistent with an infection of longer duration, a non-immunizing infection or co-infection with an acute agent and one with longer duration. Age-incidence patterns can lend insight into important epidemiological features of infections, providing information on transmission-relevant population mixing for known infections and clues about the aetiology of complex paediatric diseases.     

A simplified molecular method for distinguishing among species and ploidy levels in European water frogs (Pelophylax)

Western Palearctic water frogs in the genus Pelophylax are a set of morphologically similar anuran species that form hybridogenetic complexes. Fully reliable identification of species and especially of hybrid ploidy depends on karyological and molecular methods. In central Europe, native water frog populations consist of the Pelophylax esculentus complex, that is, P. lessonae (LL), P. ridibundus (RR) and the hybrid form P. esculentus that can have different karyotypes (RL, LLR and RRL). We developed existing molecular methods further and propose a simple PCR method based on size‐differences in the length of the serum albumin intron‐1 and the RanaCR1, a non‐LTR retrotransposon of the chicken repeat (CR) family. This PCR yields taxon‐specific banding patterns that can easily be screened by standard agarose gel electrophoresis and correctly identify species in all of the 160 samples that had been identified to karyotype with other methods. To distinguish ploidy levels in LR, LLR and RRL specimens, we used the ratio of the peak heights of the larger (ridibundus specific) to the smaller (lessonae specific) bands of fluorescently labelled PCR products resolved on a capillary DNA sequencer and obtained a correct assignment of the karyotype in 93% of cases. Our new method will cut down time and expenses drastically for a reliable identification of water frogs of the P. esculentus complex and potentially for identification of other hybridogenetic complexes and/or taxa, and it even serves as a good indicator of the ploidy status of hybrid individuals.     

A cyclic peptidylic inhibitor of murine urokinase-type plasminogen activator: changing species specificity by substitution of a single residue

The variance of the U-box domain in 64 Arabidopsis thaliana (thale cress) E3s (ubiquitin-protein ligases) was used to examine the interactions between E3s and E2s (ubiquitin-conjugating enzymes). E2s and E3s are components of the ubiquitin protein degradation pathway. Seven U-box proteins were analysed for their ability to ubiquitinate proteins in vitro in co-operation with different E2s. All U-box domains exhibited ubiquitination activity and interacted productively with UBC4/5-type E2s. Three and four of the U-box domains mediated ubiquitin addition in the presence of UBC13 and UBC7 E2s respectively, but no productive interaction was observed with the UBC15 E2 tested. The activity of AtPUB54 [Arabidopsis thaliana (thale cress) plant U-box 54 protein] was dependent on Trp(266) in the E2-binding cleft, and the E2 selectivity was changed by substitution of this position. The function of the distant U-box protein, AtPUB49, representing a large family of eukaryotic proteins containing a U-box linked to a cyclophilin-like peptidyl-prolyl cis-trans isomerase domain, was characterized biochemically. AtPUB49 functioned both as a prolyl isomerase and a chaperone by catalysing cis-trans isomerization of peptidyl-prolyl bonds and dissolving protein aggregates. In conclusion, both typical and atypical Arabidopsis U-box proteins were active E3s. The overlap in the E3/E2 selectivity suggests that in vivo specificity is not determined only by the E3-E2 interactions, but also by other parameters, e.g. co-existence or interactions with additional domains. The biochemical functions of AtPUB49 suggest that the protein can be involved in folding or degradation of protein substrates. Similar functions can also be retained within a protein complex with separate chaperone and U-box proteins.     

Drosophila MFAP1 is required for pre-mRNA processing and G2/M progression

The mammalian spliceosome has mainly been studied using proteomics. The isolation and comparison of different splicing intermediates has revealed the dynamic association of more than 200 splicing factors with the spliceosome, relatively few of which have been studied in detail. Here, we report the characterization of the Drosophila homologue of microfibril-associated protein 1 (dMFAP1), a previously uncharacterized protein found in some human spliceosomal fractions ( Jurica, M. S., and Moore, M. J. (2003) Mol. Cell 12, 5-14 ). We show that dMFAP1 binds directly to the Drosophila homologue of Prp38p (dPrp38), a tri-small nuclear ribonucleoprotein component ( Xie, J., Beickman, K., Otte, E., and Rymond, B. C. (1998) EMBO J. 17, 2938-2946 ), and is required for pre-mRNA processing. dMFAP1, like dPrp38, is essential for viability, and our in vivo data show that cells with reduced levels of dMFAP1 or dPrp38 proliferate more slowly than normal cells and undergo apoptosis. Consistent with this, double-stranded RNA-mediated depletion of dPrp38 or dMFAP1 causes cells to arrest in G(2)/M, and this is paralleled by a reduction in mRNA levels of the mitotic phosphatase string/cdc25. Interestingly double-stranded RNA-mediated depletion of a wide range of core splicing factors elicits a similar phenotype, suggesting that the observed G(2)/M arrest might be a general consequence of interfering with spliceosome function.     

Non-cultured adipose-derived CD45- side population cells are enriched for progenitors that give rise to myofibres in vivo

Side population (SP) cells are highly able to exclude the Hoechst 33342 dye through membrane transporters, a feature associated with cell immaturity and therefore proposed as a marker of stem cells. Herein we demonstrate that the adipose tissue derived stromal vascular fraction (SVF) contains a novel population of non-haematopoietic “side population” (SPCD45⁻) cells. Simultaneous qRT-PCR of 64 genes revealed that the freshly isolated SPCD45⁻ was highly enriched for cells expressing genes related to stem cells, the Notch pathway, and early vascular precursors. Notably, the expression of smooth muscle actin, C-met and Cd34 together with Angpt2, Flk1, VE-cadherin, and Cd31 suggested a phenotypic resemblance to pericytes and aorta-derived mesoangioblasts. Recent evidence suggests that cells residing within the vascular niche may participate in regeneration of skeletal muscle and although skeletal muscle repair mainly relies on the satellitecell, several reports have shown that vessel-associated cells may adopt a myogenic phenotype when exposed to a muscle environment. In accordance with these findings, we also observed invitro myogenic specification of SPCD45⁻ cells when cocultured with myoblasts. Furthermore, immediate intramuscular engraftment of non-cultured SPCD45⁻ cells gave rise to myofibres andcells lining blood vessels, whereas the SVF only provided donor derived mononuclear cells. We therefore conclude that the SPCD45⁻ fraction of adipose-derived SVF is enriched for cells expressing vascular associated markers and that the myogenic differentiation potential of these cells does not depend on prior in vitro expansion.     

Effect of European Clinical Trials Directive on academic drug trials in Denmark: retrospective study of applications to the Danish Medicines Agency 1993-2006

Objective To determine the impact of the European Union’s Clinical Trials Directive on the number of academic drug trials carried out in Denmark.Design Retrospective review of applications for drug trials to the Danish Medicines Agency, 1993-2006.Review methods Applications for drug trials for alternate years were classified as academic or commercial trials. A random subset of academic trials was reviewed for number of participants in and intended monitoring of the trials.Results Academic and commercial drug trials showed an identical steady decline from 1993 to 2006 and no noticeable change after 2004 when good clinical practice became mandatory for academic trials.Conclusion The Clinical Trials Directive introduced in May 2004 to ensure good clinical practice for academic drug trials was not associated with a decline in research activity in Denmark; presumably because good clinical practice units had already been in place in Danish universities since 1999. With such an infrastructure academic researchers can do drug trials under the same regulations as drug companies.     

The Cladistic Race Concept: A Defense

Many contemporary race scholars reject the biological reality of race.Elsewhere I have argued that they have been too quick to do so. Part ofthe reason is that they have overlooked the possibility that races canbe defined cladistically. Since the publication of the cladistic raceconcept, a number of questions and objections have been raised. My aimin this paper is to address these objections.     

Comparative Expression Profiling Reveals an Essential Role for Raldh2 in Epimorphic Regeneration

Zebrafish have the remarkable ability to regenerate body parts including the heart and fins by a process referred to as epimorphic regeneration. Recent studies have illustrated that similar to adult zebrafish, early life stage larvae also possess the ability to regenerate the caudal fin. A comparative microarray analysis was used to determine the degree of conservation in gene expression among the regenerating adult caudal fin, adult heart, and larval fin. Results indicate that these tissues respond to amputation/injury with strikingly similar genomic responses. Comparative analysis revealed raldh2, a rate-limiting enzyme for the synthesis of retinoic acid, as one of the most highly induced genes across the three regeneration platforms. In situ localization and functional studies indicate that raldh2 expression is critical for the formation of wound epithelium and blastema. Patterning during regenerative outgrowth was considered to be the primary function of retinoic acid signaling; however, our results suggest that it is also required for early stages of tissue regeneration. Expression of raldh2 is regulated by Wnt and fibroblast growth factor/ERK signaling.     

P-57 is a neural specific calmodulin-binding protein

P-57 is a novel calmodulin-binding protein which has recently been isolated from bovine cerebral cortex (Andreasen, T. J., Luetje, C. W., Heideman, W., and Storm, D. R. (1983) Biochemistry 22, 4615-4618). In contrast to all other calmodulin-binding proteins characterized thus far, P-57 has equivalent or higher affinity for calmodulin in the absence of free Ca2+ compared to the presence of Ca2+. In this study, the distribution of P-57 in other tissues and within brain was examined using a radioimmune assay and photoaffinity labeling with azido-125I-calmodulin. P-57 was not found in tissues other than brain, retina, and spinal cord. Within brain, P-57 levels varied from 0.1% of the total protein in white matter regions to about 0.5% in cell body-rich fractions. The protein was found in both membrane and soluble fractions. P-57 is the most abundant calmodulin-binding protein in brain and appears to be neural specific. The concentrations of P-57 in brain and its affinity for calmodulin in the absence of Ca2+ are sufficient to complex a significant fraction of the total calmodulin present.     

Disagreement between Human Papillomavirus Assays: An Unexpected Challenge for the Choice of an Assay in Primary Cervical Screening

We aimed to determine the disagreement in primary cervical screening between four human papillomavirus assays: Hybrid Capture 2, cobas, CLART, and APTIMA. Material from 5,064 SurePath samples of women participating in routine cervical screening in Copenhagen, Denmark, was tested with the four assays. Positive agreement between the assays was measured as the conditional probability that the results of all compared assays were positive given that at least one assay returned a positive result. Of all 5,064 samples, 1,679 (33.2%) tested positive on at least one of the assays. Among these, 41% tested positive on all four. Agreement was lower in women aged ≥30 years (30%, vs. 49% at <30 years), in primary screening samples (29%, vs. 38% in follow-up samples), and in women with concurrent normal cytology (22%, vs. 68% with abnormal cytology). Among primary screening samples from women aged 30–65 years (n = 2,881), 23% tested positive on at least one assay, and 42 to 58% of these showed positive agreement on any compared pair of the assays. While 4% of primary screening samples showed abnormal cytology, 6 to 10% were discordant on any pair of assays. A literature review corroborated our findings of considerable disagreement between human papillomavirus assays. This suggested that the extent of disagreement in primary screening is neither population- nor storage media-specific, leaving assay design differences as the most probable cause. The substantially different selection of women testing positive on the various human papillomavirus assays represents an unexpected challenge for the choice of an assay in primary cervical screening, and for follow up of in particular HPV positive/cytology normal women.