The use of dyes in modern biomedicine.

The discovery of the aniline dyes in the 19th century and contemporary investigation of their use as biological stains by scientists such as Koch and Ehrlich led to the idea of selectivity and formed the basis of modern chemotherapy; several of these dyes remain in pharmacopoeias. While the development of therapeutics has tended to avoid colored compounds due to unwanted coloration, the modern application of photosensitizing dyes, both in the fields of cancer therapy and anti-infection, depends on this phenomenon. In addition, the fluorescence of some anticancer photosensitizers allows their use as tumor localizing agents, which is particularly useful in precancerous conditions. It is also fitting that dyes employed in Ehrlich's original studies, such as the phenothiazinium dye, methylene blue, are now in clinical use for disinfecting donated blood products.     

Isolation of a further anonymous informative DNA sequence from chromosome seven closely linked to cystic fibrosis.

A library prepared from flow-sorted chromosomes was used to isolate single-copy sequences from chromosome seven. One such sequence 7C22 has been shown to be polymorphic for an EcoRI restriction site and to be informative for the study of CF in approximately 35% of matings. The segregation of the 7C22 alleles was followed through nineteen informative families with more than one child affected by cystic fibrosis. We report that the locus for 7C22 is linked to the locus for cystic fibrosis at a recombination fraction of 0.045. This marker will prove useful in improving the accuracy and informativeness of prenatal diagnosis and in constructing a fine genetic map around the cystic fibrosis gene.     

DAPK catalytic activity in the hippocampus increases during the recovery phase in an animal model of brain hypoxic-ischemic injury

Death-associated protein kinase (DAPK) is a pro-apoptotic, calmodulin (CaM)-regulated protein kinase whose mRNA levels increase following cerebral ischemia. However, the relationship between DAPK catalytic activity and cerebral ischemia is not known. This knowledge is critical as DAPK function is dependent on the catalytic activity of its kinase domain. Consequently, we examined DAPK catalytic activity in a rat model of neonatal cerebral hypoxia-ischemia (HI). An increase in DAPK specific activity was found in homogenates of the hippocampus from the injured right hemisphere, compared to the uninjured left hemisphere, 7 days after injury. The results raised the possibility that an upregulation of DAPK activity might be associated with the recovery phase of HI, during which neuronal repair and differentiation are initiated. Therefore, we examined the change of DAPK in an experimentally tractable cell culture model of neuronal differentiation. We found that DAPK catalytic activity and protein levels increase after nerve growth factor (NGF)-induced differentiation of rat PC12 cells. These results suggest that DAPK may have a previously unappreciated role in neuronal development or recovery from injury, and that potential future therapies targeting DAPK should consider a restricted time window.     

Identification of methyl farnesoate in the cypris larva of the barnacle, Balanus amphitrite, and its role as a juvenile hormone

Previous investigations have shown that insect juvenile hormone (JH) and its analogues induce precocious metamorphosis of barnacle cypris larvae. In the present study, methyl farnesoate (MF; structurally identical to JH III, except for the absence of an epoxide group) has been shown to have a concentration-dependent effect on the development of cyprids of the barnacle Balanus amphitrite. Analysis of cypris extracts by gas chromatography-mass spectrometry with selected ion monitoring (GC-MS-SIM) confirmed the presence of endogenous MF. These data provide evidence that MF functions as a juvenilizing hormone in barnacle cyprids, an effect that hitherto has not been noted.     

Recombinations between IRP and cystic fibrosis.

A candidate gene for cystic fibrosis was recently isolated by selective cloning of HpaII-tiny-fragment islands; it maps considerably closer to CF than does MET or D7S8 (pJ3.11), and DNA polymorphisms from this region are in marked disequilibrium with CF. cDNA cloning has shown that this protein has a growth factor-like structure and shows homology to the murine and human proto-oncogene int-1; it is designated IRP (int-1-related protein). DNA sequences from the IRP locus that recognize RFLPs are proving to be highly informative for prenatal diagnosis. We report five crossovers that have been identified which occur either within the IRP locus or between IRP and CF; these recombinants demonstrate that CF maps between the DNA markers D7S8 and KM.19.     

BMP-regulated exosomes from Drosophila male reproductive glands reprogram female behavior

Male reproductive glands secrete signals into seminal fluid to facilitate reproductive success. In Drosophila melanogaster, these signals are generated by a variety of seminal peptides, many produced by the accessory glands (AGs). One epithelial cell type in the adult male AGs, the secondary cell (SC), grows selectively in response to bone morphogenetic protein (BMP) signaling. This signaling is involved in blocking the rapid remating of mated females, which contributes to the reproductive advantage of the first male to mate. In this paper, we show that SCs secrete exosomes, membrane-bound vesicles generated inside late endosomal multivesicular bodies (MVBs). After mating, exosomes fuse with sperm (as also seen in vitro for human prostate-derived exosomes and sperm) and interact with female reproductive tract epithelia. Exosome release was required to inhibit female remating behavior, suggesting that exosomes are downstream effectors of BMP signaling. Indeed, when BMP signaling was reduced in SCs, vesicles were still formed in MVBs but not secreted as exosomes. These results demonstrate a new function for the MVB–exosome pathway in the reproductive tract that appears to be conserved across evolution.     

Accelerated mitochondrial evolution and "Darwin's corollary": asymmetric viability of reciprocal F1 hybrids in Centrarchid fishes

Reciprocal crosses between species can yield hybrids with different viabilities. The high frequency of this asymmetric hybrid viability ("Darwin's corollary") places it alongside Haldane's rule and the "large-X effect" as a general feature of postmating reproductive isolation. Recent theory suggests that reciprocal cross asymmetries can arise from stochastic substitutions in uniparentally inherited loci such as mitochondrial genomes, although large systematic differences in mitochondrial substitution rates can also contribute to asymmetries. Although the magnitude of asymmetry will be relatively insensitive to unequal rates of mitochondrial evolution in diverging species, we show here that rate asymmetries can have a large effect on the direction of viability asymmetries. In reciprocal crosses between species, the maternal parent with faster mitochondrial evolution will tend to produce less viable F(1) hybrids owing to an increased probability of mito-nuclear incompatibilities. We test this prediction using data on reciprocal hybrid viability and molecular evolution rates from a clade of freshwater fishes, Centrarchidae. As predicted, species with accelerated mitochondrial evolution tend to be the worse maternal parent for F(1) hybrids, providing the first comparative evidence for a systematic basis to Darwin's corollary. This result is consistent with the hypothesis that mito-nuclear incompatibilities can play an important role in reproductive isolation. Such asymmetrical reproductive isolation may help explain the asymmetrical mitochondrial introgression observed between many hybridizing species. However, as with any comparative study, we cannot rule out the possibility that our results arise from a mutual correlation with a third variable such as body size.     

Measurement of the Infection and Dissemination of Bluetongue Virus in Culicoides Biting Midges Using a Semi-Quantitative RT-PCR Assay and Isolation of Infectious Virus

Background

Culicoides biting midges (Diptera: Ceratopogonidae) are the biological vectors of globally significant arboviruses of livestock including bluetongue virus (BTV), African horse sickness virus (AHSV) and the recently emerging Schmallenberg virus (SBV). From 2006–2009 outbreaks of BTV in northern Europe inflicted major disruption and economic losses to farmers and several attempts were made to implicate Palaearctic Culicoides species as vectors. Results from these studies were difficult to interpret as they used semi-quantitative RT-PCR (sqPCR) assays as the major diagnostic tool, a technique that had not been validated for use in this role. In this study we validate the use of these assays by carrying out time-series detection of BTV RNA in two colony species of Culicoides and compare the results with the more traditional isolation of infectious BTV on cell culture.

Methodology/Principal Findings

A BTV serotype 1 strain mixed with horse blood was fed to several hundred individuals of Culicoides sonorensis (Wirth & Jones) and C. nubeculosus (Mg.) using a membrane-based assay and replete individuals were then incubated at 25°C. At daily intervals 25 Culicoides of each species were removed from incubation, homogenised and BTV quantified in each individual using sqPCR (C_q values) and virus isolation on a KC-C. sonorensis embryonic cell line, followed by antigen enzyme-linked immunosorbent assay (ELISA). In addition, comparisons were also drawn between the results obtained with whole C. sonorensis and with individually dissected individuals to determine the level of BTV dissemination.

Conclusions/Significance

C_q values generated from time-series infection experiments in both C. sonorensis and C. nubeculosus confirmed previous studies that relied upon the isolation and detection of infectious BTV. Implications on the testing of field-collected Culicoides as potential virus vectors by PCR assays and the use of such assays as front-line tools for use in diagnostic laboratories in this role are discussed.     

Erythropoiesis in cultures of dispersed cell preparations from early chick blastodiscs

Optimal conditions were determined for differentiation of erythroid cells in liquid cultures of dispersed cell preparations from chick blastodiscs at the primitive streak and head-fold stages of development. Most of the unattached erythroid cells free in the medium were mature erythrocytes.