Impact Melt Facies in the Moon's Crisium Basin: Identifying, Characterizing, and Future Radiogenic Dating.

Both Earth and the Moon share a common history regarding the epoch of large basin formation, though only the lunar geologic record preserves any appreciable record of this Late Heavy Bombardment. The emergence of Earth's first life is approximately contemporaneous with the Late Heavy Bombardment; understanding the latter informs the environmental conditions of the former, which are likely necessary to constrain the mechanisms of abiogenesis. While the relative formation time of most of the Moon's large basins is known, the absolute timing is not. The timing of Crisium Basin's formation is one of many important events that must be constrained and would require identifying and dating impact melt formed in the Crisium event. To inform a future lunar sample dating mission, we thus characterized possible outcrops of impact melt. We determined that several mare lava-embayed kipukas could contain impact melt, though the rim and central peaks of the partially lava-flooded Yerkes Crater likely contain the most pure and intact Crisium impact melt. It is here where future robotic and/or human missions could confidently add a key missing piece to the puzzle of the combined issues of early Earth-Moon bombardment and the emergence of life.

The perverse impact of external reference pricing (ERP): a comparison of orphan drugs affordability in 12 European countries. A call for policy change.

Objective: The study compared the relative cost differences of similar orphan drugs among high and low GDP countries in Europe: Bulgaria, France, Germany, Greece, Hungary, Italy, Norway, Poland, Romania, Spain, Sweden, UK. Methods: Annual treatment costs per patient were calculated. Relative costs were computed by dividing the costs by each economic parameter: nominal GDP per capita, GDP in PPP per capita, % GDP contributed by the government, government budget per inhabitant, % GDP spent on healthcare, % GDP spent on pharmaceuticals, and average annual salary. An international comparison of the relative costs was done using UK as the reference country and results were analysed descriptively. Results: 120 orphan drugs were included. The median annual costs of orphan drugs in all countries varied minimally (cost ratios: 0.87 to 1.08). When the costs were adjusted using GDP per capita, the EU-5 and Nordic countries maintained minimal difference in median cost. However, the lower GDP countries showed three to six times higher relative costs. The same pattern was evident when costs were adjusted using the other economic parameters. Conclusion: When the country's ability to pay is taken into consideration, lower GDP countries pay relatively higher costs for similarly available orphan drugs in Europe.

Evaluation of anthelmintic activity in captive wild ruminants by fecal egg reduction tests and a larval development assay.

The effectiveness of anthelmintics was evaluated in four herds of captive ruminants, wapiti (Cervus elaphus), Armenian red sheep (Ovis orientalis), giraffe (Giraffa camelopardalis), and pronghorn (Antilocapra americana), by the use of fecal egg reduction tests (FERTs) and a commercial larval development assay (LDA) designed to evaluate susceptibility or resistance of nematodes to anthelmintics. Haemonchus sp. was the predominant nematode in the red sheep, giraffe, and pronghorn herds, whereas Ostertagia sp. and Trichostrongylus sp. were predominant in the wapiti. The LDA data indicated susceptibility by the worms to benzimidazoles except in the red sheep flock, which showed a high level of resistance. High levels of resistance to levamisole were seen in the worm populations from the wapiti and red sheep, moderate resistance in the pronghorn herd, and susceptibility in the giraffe herd. Worms were susceptible in all four herds to a combination of benzimidazole/levamisole. There was suspected avermectin resistance by Trichostrongylus sp. in the wapiti herd and by Haemonchus sp. in the giraffe. The FERTs agreed with the LDA in showing the Haemonchus in the giraffe was susceptible to fenbendazole and had suspected resistance to ivermectin, whereas Haemonchus in the red sheep and pronghorn were susceptible to ivermectin. There was correlation between the tests evaluating anthelmintics. The LDA is useful as a screening test in the selection of an anthelmintic for use in grazing ruminants, but the effectiveness of a drug in a host species may depend as much on the dose used, and the method of administration, as it does on the parasite's sensitivity to the anthelmintic.

Parasite diversity and anthelmintic resistance in two herds of horses.

Diversity of parasite populations was compared between two herds of horses, one a regularly treated herd the other a feral herd which has bad no anthelmintic treatment for at least 25 years. Eggs obtained from fecal samples of both herds were tested for anthelmintic resistance by use of an in-vitro larval hatch/development assay (LDA), DrenchRite. A fecal egg reduction test was also performed with the domesticated herd using fenbendazole, pyrantel pamoate and ivermectin. Cyathostomes were the predominant group of worms present in both herds. Trichostrongylus axei was seen in both herds, but Strongylus equinus, Strongylus vulgaris, Gyalocephalus capitatus, Poteriostomum spp. and Strongyloides westeri were only found in the feral horses. Larvae of Strongylus edentatus were found in a single domesticated horse. Fecal egg reduction tests with the domesticated herd showed a 32% egg count reduction for fenbendazole, a 93% reduction with pyrantel, and a 99% reduction with ivermectin. From the LDA, anthelmintic resistance was evaluated by determining the resistance ratio of the domesticated herd compared with the feral herd. For benzimidazoles in the domesticated herd, 45% of the cyathostome population was 9.4 times more tolerant than the feral herd's parasite population. The parasite population in the domesticated herd was 1.5 times more tolerant to Levamisole, and 1.7 times more tolerant to the benzimidazole/levamisole combination than the parasite population within the feral herd. 9% of the parasite population in the domesticated herd was 90 times more tolerant to avermectins than the feral herd's parasite population, even though a subpopulation of worms in the feral herd were tolerant to low concentrations of avermectins despite never being previously exposed to this class of anthelmintic.

Teratogen-mediated inhibition of target tissue response to Shh signaling.

Veratrum alkaloids and distal inhibitors of cholesterol biosynthesis have been studied for more than 30 years as potent teratogens capable of inducing cyclopia and other birth defects. Here, it is shown that these compounds specifically block the Sonic hedgehog (Shh) signaling pathway. These teratogens did not prevent the sterol modification of Shh during autoprocessing but rather inhibited the response of target tissues to Shh, possibly acting through the sterol sensing domain within the Patched protein regulator of Shh response.

Crystal structure of a Hedgehog autoprocessing domain: homology between Hedgehog and self-splicing proteins.

The approximately 25 kDa carboxy-terminal domain of Drosophila Hedgehog protein (Hh-C) possesses an autoprocessing activity that results in an intramolecular cleavage of full-length Hedgehog protein and covalent attachment of a cholesterol moiety to the newly generated amino-terminal fragment. We have identified a 17 kDa fragment of Hh-C (Hh-C17) active in the initiation of autoprocessing and report here its crystal structure. The Hh-C17 structure comprises two homologous subdomains that appear to have arisen from tandem duplication of a primordial gene. Residues in the Hh-C17 active site have been identified, and their role in Hedgehog autoprocessing probed by site-directed mutagenesis. Aspects of sequence, structure, and reaction mechanism are conserved between Hh-C17 and the self-splicing regions of inteins, permitting reconstruction of a plausible evolutionary history of Hh-C and the inteins.

Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function.

Targeted gene disruption in the mouse shows that the Sonic hedgehog (Shh) gene plays a critical role in patterning of vertebrate embryonic tissues, including the brain and spinal cord, the axial skeleton and the limbs. Early defects are observed in the establishment or maintenance of midline structures, such as the notochord and the floorplate, and later defects include absence of distal limb structures, cyclopia, absence of ventral cell types within the neural tube, and absence of the spinal column and most of the ribs. Defects in all tissues extend beyond the normal sites of Shh transcription, confirming the proposed role of Shh proteins as an extracellular signal required for the tissue-organizing properties of several vertebrate patterning centres.

Cholesterol modification of hedgehog signaling proteins in animal development.

Hedgehog (Hh) proteins comprise a family of secreted signaling molecules essential for patterning a variety of structures in animal embryogenesis. During biosynthesis, Hh undergoes an autocleavage reaction, mediated by its carboxyl-terminal domain, that produces a lipid-modified amino-terminal fragment responsible for all known Hh signaling activity. Here it is reported that cholesterol is the lipophilic moiety covalently attached to the amino-terminal signaling domain during autoprocessing and that the carboxyl-terminal domain acts as an intramolecular cholesterol transferase. This use of cholesterol to modify embryonic signaling proteins may account for some of the effects of perturbed cholesterol biosynthesis on animal development.

Hedgehog patterning activity: role of a lipophilic modification mediated by the carboxy-terminal autoprocessing domain.

Autocatalytic processing mediated by the carboxyterminal domain of the hedgehog (hh) protein precursor (Hh) generates an amino-terminal product that accounts for all known signaling activity. The role of autoprocessing biogenesis of the hh signal has been unclear, since a truncated unprocessed protein lacking all carboxy-terminal domain sequences retains signaling activity. Here, we present evidence that the autoprocessing reaction proceeds via an internal thioester intermediate and results in a covalent modification that increases the hydrophobic character of the signaling domain and influences its spatial and subcellular distribution. We demonstrate that truncated unprocessed amino-terminal protein causes embryonic mispatterning, even when expression is localized to cells that normally express Hh, thus suggesting a role for autoprocessing in spatial regulation of hh signaling. This type of processing also appears to operate in the biogenesis of other novel secreted proteins.

Control of Drosophila tracheal branching by the novel homeodomain gene unplugged, a regulatory target for genes of the bithorax complex.

We have identified a novel Drosophila homeodomain gene, unplugged (unp), whose function is required for formation of the tracheal branches that penetrate the CNS. In unp mutant embryos the segmentally repeated ganglionic branches stall and fail to penetrate the CNS and the segment-specific cerebral branch and associated cerebral anastomosis fail to form. Expression of unp in the founder cells for the cerebral branch within the first tracheal metamere is repressed in posterior segments by Ubx and other bithorax complex genes. This pattern of expression and homeotic gene regulation is reproduced by an unusual 2.6 kb cis-regulatory sequence located downstream of the unp transcription unit. Since the unp protein is localized to the nucleus of tracheal precursor cells as they migrate and extend, unp protein appears to play a regulatory role in neural branching of the tracheae, and the segment-specific aspects of these neural branching patterns appear to be generated by homeotic regulation of unp expression.

The product of hedgehog autoproteolytic cleavage active in local and long-range signalling.

The secreted protein products of the hedgehog (hh) gene family are associated with local and long-range signalling activities that are responsible for developmental patterning in multiple systems, including Drosophila embryonic and larval tissues and vertebrate neural tube, limbs and somites. In a process that is critical for full biological activity, the hedgehog protein (Hh) undergoes autoproteolysis to generate two biochemically distinct products, an 18K amino-terminal fragment, N, and a 25K carboxy-terminal fragment, C (ref. 16); mutations that block autoproteolysis impair Hh function. We have identified the site of autoproteolytic cleavage and find that it is broadly conserved throughout the hedgehog family. Knowing the site of cleavage, we were able to test the function of the N and C cleavage products in Drosophila assays. We show here that the N product is the active species in both local and long-range signalling. Consistent with this, all twelve mapped hedgehog mutations either affected the structure of the N product directly or otherwise blocked the release of N from the Hh precursor as a result of deletion or alteration of sequences in the C domain.

The novel homeodomain gene buttonless specifies differentiation and axonal guidance functions of Drosophila dorsal median cells.

We have identified a novel homeodomain gene, buttonless (btn), that is specifically expressed in 20 cells of a single type during Drosophila embryonic development. These cells, the dorsal median (DM) cells, are arranged as a single pair within each segment along the dorsal midline of the CNS. Distinctive features of the DM cells include a large cell body and a long thick process extending laterally to the muscle attachment site. In the absence of btn gene function the initial commitment to the DM cell fate is made but differentiation fails to occur and the DM cells are lost. The btn mutation thus specifically eliminates the DM cells, and this genetic ablation in turn reveals a requirement for DM cells as cellular cues for axonal guidance during transverse nerve outgrowth and bifurcation of the median nerve.

The degree of variation in DNA sequence recognition among four Drosophila homeotic proteins.

The homeodomain has been implicated as a major determinant of biological specificity for the homeotic selector (HOM) genes. We compare here the DNA sequence preferences of homeodomains encoded by four of the eight Drosophila HOM proteins. One of the four, Abdominal-B, binds preferentially to a sequence with an unusual 5'-T-T-A-T-3' core, whereas the other three prefer 5'-T-A-A-T-3'. Of these latter three, the Ultrabithorax and Antennapedia homeodomains display indistinguishable preferences outside the core while Deformed differs. Thus, with three distinct binding classes defined by four HOM proteins, differences in individual site recognition may account for some but not all of HOM protein functional specificity. We further show that amino acid residues within the N-terminal arm are responsible for the sequence specificity differences between the Ultrabithorax and Abdominal-B homeodomains. Similarities and differences at the corresponding positions within the N-terminal arms are conserved in the vertebrate Abdominal-B-like HOM proteins, which play critical roles in limb specifications as well as in regional specification along the anterior-posterior axis. This and other patterns of residue conservation suggest that differential DNA sequence recognition may play a role in HOM protein function in a wide range of organisms.

Cooperative binding of an Ultrabithorax homeodomain protein to nearby and distant DNA sites.

Cooperativity in binding of regulatory proteins to multiple DNA sites can heighten the sensitivity and specificity of the transcriptional response. We report here the cooperative DNA-binding properties of a developmentally active regulatory protein encoded by the Drosophila homeotic gene Ultrabithorax (Ubx). We show that naturally occurring binding sites for the Ubx-encoded protein contain clusters of multiple individual binding site sequences. Such sites can form complexes containing a dozen or more Ubx-encoded protein molecules, with simultaneous cooperative interactions between adjacent and distant DNA sites. The distant mode of interaction involves a DNA looping mechanism; both modes appear to enhance transcriptional activation in a simple yeast assay system. We found that cooperative binding is dependent on sequences outside the homeodomain, and we have identified regions predicted to form coiled coils carboxy terminal to the homeodomains of the Ubx-encoded protein and several other homeotic proteins. On the basis of our findings, we propose a multisite integrative model of homeotic protein action in which functional regulatory elements can be built from a few high-affinity sites, from many lower-affinity sites, or from sites of some intermediate number and affinity. An important corollary of this model is that even small differences in binding of homeotic proteins to individual sites could be summed to yield large overall differences in binding to multiple sites. This model is consistent with reports that homeodomain protein targets contain multiple individual binding site sequences distributed throughout sizable DNA regions. Also consistent is a recent report that sequences carboxy terminal to the Ubx homeodomain can contribute to segmental specificity.

Stimulation of rat spleen cells by staphylococcal enterotoxins.

There is much interest in staphylococcal enterotoxins as T cell mitogens in humans, mice and rabbits. Rat spleen cells were shown to proliferate in response to staphylococcal enterotoxins A and B and toxic shock syndrome toxin-1 at concentrations (5 to 500 ng ml-1) which also stimulate mouse spleen cells. The proliferative response to all these enterotoxins was inhibited by cyclosporin A, indicating the response to be predominantly that of T cells. These results indicate that the rat provides another convenient model for the analysis of T cell responses to enterotoxins.

Infestation of northern spotted owls by hippoboscid (Diptera) flies in northwestern California.

Hippoboscid flies were found on 62 (17%) of 382 northern spotted owls (Strix occidentalis caurina) captured between April and September, 1986 through 1990. Two species of hippoboscids were identified: Icosta americana and Ornithomya anchineuria. Male and female adult spotted owls had similar prevalences and relative densities of hippoboscid flies. Juvenile owls had lower prevalence and relative densities than adults. There were no significant differences in mean intensity of hippoboscids on adult male, adult female and juvenile spotted owls. Relative densities of flies infesting adult owls were significantly greater during years of increased fall temperatures, decreased winter precipitation, and decreased summer temperatures.