Non-competitive cyclic peptides for targeting enzyme-substrate complexes.

Affinity reagents are of central importance for selectively identifying proteins and investigating their interactions. We report on the development and use of cyclic peptides, identified by mRNA display-based RaPID methodology, that are selective for, and tight binders of, the human hypoxia inducible factor prolyl hydroxylases (PHDs) - enzymes crucial in hypoxia sensing. Biophysical analyses reveal the cyclic peptides to bind in a distinct site, away from the enzyme active site pocket, enabling conservation of substrate binding and catalysis. A biotinylated cyclic peptide captures not only the PHDs, but also their primary substrate hypoxia inducible factor HIF1-α. Our work highlights the potential for tight, non-active site binding cyclic peptides to act as promising affinity reagents for studying protein-protein interactions.

Assessment of the Effectiveness of Combat Eyewear Protection Against Blast Overpressure.

It is unclear whether combat eyewear used by U. S. Service members is protective against blast overpressures (BOPs) caused by explosive devices. Here, we investigated the mechanisms by which BOP bypasses eyewear and increases eye surface pressure. We performed experiments and developed three-dimensional (3D) finite element (FE) models of a head form (HF) equipped with an advanced combat helmet (ACH) and with no eyewear, spectacles, or goggles in a shock tube at three BOPs and five head orientations relative to the blast wave. Overall, we observed good agreement between experimental and computational results, with average discrepancies in impulse and peak-pressure values of less than 15% over 90 comparisons. In the absence of eyewear and depending on the head orientation, we identified three mechanisms that contributed to pressure loading on the eyes. Eyewear was most effective at 0 deg orientation, with pressure attenuation ranging from 50 (spectacles) to 80% (goggles) of the peak pressures observed in the no-eyewear configuration. Spectacles and goggles were considerably less effective when we rotated the HF in the counter-clockwise direction around the superior-inferior axis of the head. Surprisingly, at certain orientations, spectacles yielded higher maximum pressures (80%) and goggles yielded larger impulses (150%) than those observed without eyewear. The findings from this study will aid in the design of eyewear that provides better protection against BOP.

A comparison of electronic and manual fracture risk assessment tools in screening elderly male US veterans at risk for osteoporosis.

This study compares four screening tools in their ability to predict osteoporosis. We found that there was no significant difference between the tools. These results provide support for the use of automated screening tools which work in conjunction with the electronic medical record and help improve screening rates for osteoporosis. INTRODUCTION: The purpose of this study is to compare the performance of four fracture risk assessment tools (FRATs) in identifying osteoporosis by bone mineral density (BMD) T-score: Veterans Affairs Fracture Absolute Risk Assessment Tool (VA-FARA), World Health Organization's Fracture Risk Assessment Tool (FRAX), electronic FRAX (e-FRAX), and Osteoporosis Self-Assessment Screening Tool (OST). METHODS: We performed a cross-sectional analysis of all patients enrolled in the VA Salt Lake City bone health team (BHT) who had completed a DXA scan between February 1, 2012, and February 1, 2013. DXA scan results were obtained by chart abstraction. For calculation of FRAX, osteoporosis risk factors were obtained from a screening questionnaire completed prior to DXA. For VA-FARA and e-FRAX, risk factors were derived from the electronic medical record (EMR). Clinical risk scores were calculated and compared against the gold standard of DXA-based osteoporosis. Sensitivity, specificity, and predictive values were calculated. Receiver operator characteristic (ROC) curves were plotted, and areas under the curve (AUC) were compared. RESULTS: A cohort of 463 patients met eligibility criteria (mean age 80.4 years). One hundred twelve patients (24%) had osteoporosis as defined by DXA T-score ≤-2.5. Sensitivity, specificity, and predictive values were calculated. ROC statistics were compared and did not reach statistical significance difference between FRATs in identifying DXA-based osteoporosis. CONCLUSIONS: Our study suggests that all FRATs tested perform similarly in identifying osteoporosis among elderly, primarily Caucasian, male veterans. If these electronic screening methods perform similarly for fracture outcomes, they could replace manual FRAX and thus improve efficiency in identifying individuals who should be sent for DXA scan.

Curious bivalves: Systematic utility and unusual properties of anomalodesmatan mitochondrial genomes.

Mitogenomic trees for Bivalvia have proved problematic in the past, but several highly divergent lineages were missing from these analyses and increased representation of these groups may yet improve resolution. Here, we add seven new sequences from the Anomalodesmata and one unidentified semelid species (Bryopa lata, Euciroa cf. queenslandica, Laternula elliptica, Laternula truncata, Lyonsia norwegica, Myadora brevis, Tropidomya abbreviata, "Abra" sp.). We show that relationships in a mitogenomic tree for the Class are improved by the addition of seven anomalodesmatans from this highly divergent clade, but are still not completely consistent with relationships recovered in studies of nuclear genes. We suggest that some anomalous relationships (for instance the non-monophyly of Bivalvia) may be partially explained by compositional heterogeneity in the mitogenome and suggest that the addition of more taxa may help resolve both this effect and possible instances of long branch attraction. We also identify several curious features about anomalodesmatan mitogenomes. For example, many protein-coding gene boundaries are poorly defined in marine bivalves, but particularly so in anomalodesmatans, primarily due to non-conserved boundary sequences. The use of transcriptomic and genomic data together enabled better definition of gene boundaries, the identification of possible pseudogenes and suggests that most genes are translated monocistronically, which contrasts with many other studies. We also identified a possible case of gene duplication of ND5 in Myadora brevis (Myochamidae). Mitogenome size in the Anomalodesmata ranges from very small compact molecules, with the smallest for Laternula elliptica (Laternulidae) only 14,622bp, to Bryopa lata (Clavagellidae) which is at least 31,969bp long and may be >40,000bp. Finally, sampled species show a high degree of sequence divergence and variable gene order, although intraspecific variation in Laternula elliptica is very low.

Identification of Shell Colour Pigments in Marine Snails Clanculus pharaonius and C. margaritarius (Trochoidea; Gastropoda).

Colour and pattern are key traits with important roles in camouflage, warning and attraction. Ideally, in order to begin to understand the evolution and ecology of colour in nature, it is important to identify and, where possible, fully characterise pigments using biochemical methods. The phylum Mollusca includes some of the most beautiful exemplars of biological pigmentation, with the vivid colours of sea shells particularly prized by collectors and scientists alike. Biochemical studies of molluscan shell colour were fairly common in the last century, but few of these studies have been confirmed using modern methods and very few shell pigments have been fully characterised. Here, we use modern chemical and multi-modal spectroscopic techniques to identify two porphyrin pigments and eumelanin in the shell of marine snails Clanculus pharaonius and C margaritarius. The same porphyrins were also identified in coloured foot tissue of both species. We use high performance liquid chromatography (HPLC) to show definitively that these porphyrins are uroporphyrin I and uroporphyrin III. Evidence from confocal microscopy analyses shows that the distribution of porphyrin pigments corresponds to the striking pink-red of C. pharaonius shells, as well as pink-red dots and lines on the early whorls of C. margaritarius and yellow-brown colour of later whorls. Additional HPLC results suggest that eumelanin is likely responsible for black spots. We refer to the two differently coloured porphyrin pigments as trochopuniceus (pink-red) and trochoxouthos (yellow-brown) in order to distinguish between them. Trochopuniceus and trochoxouthos were not found in the shell of a third species of the same superfamily, Calliostoma zizyphinum, despite its superficially similar colouration, suggesting that this species has different shell pigments. These findings have important implications for the study of colour and pattern in molluscs specifically, but in other taxa more generally, since this study shows that homology of visible colour cannot be assumed without identification of pigments.

The complete mitochondrial genome of a turbinid vetigastropod from MiSeq Illumina sequencing of genomic DNA and steps towards a resolved gastropod phylogeny.

A need to increase sampling of mitochondrial genomes for Vetigastropoda has been identified as an important step towards resolving relationships within the Gastropoda. We used shotgun sequencing of genomic DNA, using an Illumina MiSeq, to obtain the first mitochondrial genome for the vetigastropod family Turbinidae, doubling the number of genomes for the species-rich superfamily Trochoidea. This method avoids the necessity of finding suitable primers for long PCRs or primer-walking amplicons, resulting in a timely and cost-effective method for obtaining whole mitochondrial genomes from ethanol-preserved tissue samples. Bayesian analysis of amino acid variation for all available gastropod genomes including the new turbinid mtgenome produced a well resolved tree with high nodal support for most nodes. Major clades within Gastropoda were recovered with strong support, with the exception of Littorinimorpha, which was polyphyletic. We confirm here that mitogenomics is a useful tool for molluscan phylogenetics, especially when using powerful new models of amino acid evolution, but recognise that increased taxon sampling is still required to resolve existing differences between nuclear and mitochondrial gene trees.

Cenozoic climate change and diversification on the continental shelf and slope: evolution of gastropod diversity in the family Solariellidae (Trochoidea).

Recent expeditions have revealed high levels of biodiversity in the tropical deep-sea, yet little is known about the age or origin of this biodiversity, and large-scale molecular studies are still few in number. In this study, we had access to the largest number of solariellid gastropods ever collected for molecular studies, including many rare and unusual taxa. We used a Bayesian chronogram of these deep-sea gastropods (1) to test the hypothesis that deep-water communities arose onshore, (2) to determine whether Antarctica acted as a source of diversity for deep-water communities elsewhere and (3) to determine how factors like global climate change have affected evolution on the continental slope. We show that although fossil data suggest that solariellid gastropods likely arose in a shallow, tropical environment, interpretation of the molecular data is equivocal with respect to the origin of the group. On the other hand, the molecular data clearly show that Antarctic species sampled represent a recent invasion, rather than a relictual ancestral lineage. We also show that an abrupt period of global warming during the Palaeocene Eocene Thermal Maximum (PETM) leaves no molecular record of change in diversification rate in solariellids and that the group radiated before the PETM. Conversely, there is a substantial, although not significant increase in the rate of diversification of a major clade approximately 33.7 Mya, coinciding with a period of global cooling at the Eocene-Oligocene transition. Increased nutrients made available by contemporaneous changes to erosion, ocean circulation, tectonic events and upwelling may explain increased diversification, suggesting that food availability may have been a factor limiting exploitation of deep-sea habitats. Tectonic events that shaped diversification in reef-associated taxa and deep-water squat lobsters in central Indo-West Pacific were also probably important in the evolution of solariellids during the Oligo-Miocene.

Hand injuries from tools in domestic and leisure settings: relative incidence and patterns of initial management.

A search of the UK Department of Trade and Industry's Home and Leisure Accident database found 16,003 emergency hospital attendances in 2000-2002 following accidents with tools. The hand was the site of injury in 9535 cases (60%). The tool most commonly involved was a Stanley knife, causing as many hand injuries (21%) as all power tools combined. The power tools most frequently causing hand injury were circular saws (28% of power tool injuries), hedge trimmers (21%) and electric drills (17%). Compared to injuries from manual tools, power tool hand injuries were more than twice as likely to be referred to specialists and three times more likely to be admitted to hospital. Specialist referral/admission most commonly occurred following hand injury from mowers (51% admitted/referred), routers (50%) and circular saws (48%). The rate for manual blade injuries was 14%. Missed diagnoses following manual blade injuries may stem from comparatively low rates of specialist assessment.

A molecular phylogenetic framework for the Muricidae, a diverse family of carnivorous gastropods.

With over 1600 extant described species, the Muricidae are one of the most species-rich and morphologically diverse families of molluscs. As predators of molluscs, polychaetes, anthozoans barnacles and other invertebrates, they form an important component of many benthic communities. Traditionally, the classification of muricids at specific and generic levels has been based primarily on shells, while subfamilies have been defined largely by radular morphology, although the composition and relationships of suprageneric groups have never been studied exhaustively. Here we present the phylogenetic relationships of 77 muricid species belonging to nine of the ten currently recognized subfamilies, based on Bayesian inference and Maximum Likelihood analyses of partial sequences of three mitochondrial (12S, 16S and COI) and one nuclear (28S) genes. The resulting topologies are discussed with respect to traditional subfamilial arrangements, and previous anatomical and molecular findings. We confirm monophyly of each of the subfamilies Ergalataxinae, Rapaninae, Coralliophilinae, Haustrinae, Ocenebrinae and Typhinae as previously defined, but earlier concepts of Muricinae, Trophoninae and Muricopsinae are shown to be polyphyletic. Based on our phylogenetic hypothesis, a new arrangement of these subfamilies is proposed.

Molecular systematics of the marine gastropod families Trochidae and Calliostomatidae (Mollusca: Superfamily Trochoidea).

This study is the most extensive molecular study of the gastropod families Trochidae and Calliostomatidae published to date, in terms of both numbers of taxa and of gene sequences. As a result of Bayesian phylogenetic analyses of molecular sequence data from one nuclear gene and three mitochondrial genes, we propose dramatic changes to Trochidae family systematics, present the first molecular phylogeny for Calliostomatidae and include the first published sequence data for the enigmatic subfamily Thysanodontinae. Our phylogeny demonstrates that within the family Trochidae there is strong support for three subfamilies new to traditional classifications: Alcyninae subfam. nov., Fossarininae and Chrysostomatinae subfam. nov. As proposed, Alcyninae consists only of the nominotypical genus Alcyna, which is sister to all other trochids. The subfamily Fossarininae, as defined here, includes Fossarina, Broderipia, Synaptocochlea and "Roya"eximia and probably also Clydonochilus and Minopa. The subfamily Chrysostomatinae comprises the genera Chrysostoma and Chlorodiloma. Additional molecular support is also obtained for recently redefined Trochinae, Monodontinae, and Cantharidinae and for the traditionally recognised subfamilies Umboniinae and Stomatellinae. The subfamily Lirulariinae is not supported by the molecular data, but rather is incorporated into Umboniinae. We also demonstrate that the current concept of the subfamily Margaritinae (previously a trochid subfamily, but recently and provisionally assigned to Turbinidae) is not monophyletic. We provide preliminary evidence that whereas Margarella rosea (previously a member of Margaritinae) belongs in the trochid subfamily Cantharidinae, its presumptive congener M. antarctica is not a trochid, but instead clusters with the thysanodontine genus Carinastele. Based on the phylogenetic placement of C. kristelleae, we agree with previous proposals based on morphological data that Thysanodontinae are more closely related to Calliostomatidae than Trochidae. Both Calliostoma and Carinastele are carnivorous and if a sister relationship can be confirmed between Carinastele and Margarella antarctica it might mean that carnivory evolved twice in Trochoidea. The direction of dietary changes was not investigated in this study, but mapping diet onto the phylogeny suggests that true herbivory is predominantly a derived character. The new classification system also means that five trochid subfamilies are predominantly associated with hard substrata, one with soft substrata (Umboniinae) and two with algae and seagrass (Alcyninae and Cantharidinae). There has been a shift back to hard substrata in one umboniine clade. Two of three clades within Calliostomatidae were predominantly associated with hard substrata, but one Japanese clade is associated with sand. The finding of three new, unidentified species from very deep water means that Trochidae, like Calliostomatidae, now includes species found at bathyal depths. More deep-water species may be found as increased sampling leads to the discovery of new species.

Global diversification of mangrove fauna: a molecular phylogeny of Littoraria (Gastropoda: Littorinidae).

The genus Littoraria is one of very few molluscan groups that are closely associated with mangroves. We document its global evolutionary radiation and compare biogeographic patterns with those of mangrove plants, based on phylogenetic and fossil evidence. Using sequences from three genes (nuclear 28S rRNA, mitochondrial 12S rRNA and COI) we reconstruct a phylogeny of 37 of the 39 living morphospecies. Six monophyletic subgenera are defined (Bulimilittorina, Lamellilitorina, Littoraria, Palustorina, Protolittoraria, Littorinopsis) and we synonymize L. coccinea and L. glabrata. A deep division between Palustorina from the Indo-West Pacific and Littoraria from the Atlantic and Eastern Pacific is estimated by a Bayesian relaxed-clock method to be of Middle Eocene to Palaeocene age (43.2-62.7 Ma), which far predates the Early Miocene (18 Ma) closure of the Tethyan Seaway; this, as in mangrove plants, may reflect vicariance by climatic cooling, rather than tectonic processes. The age of Littoraria angulifera in the Atlantic is, however, consistent with Early Miocene vicariance of a Tethyan ancestor. We infer that speciation events are mainly of Miocene or older age, and that diversification has not been driven by depletion of mangrove habitats during recent glacial intervals. Parsimonious reconstruction of ancestral habitats suggests that the genus has inhabited mangrove or wood substrates since its origin, while the rock-dwelling habit of the four members of Protolittoraria is derived. Three species span the Eastern Pacific Barrier, and one is amphi-Atlantic, consistent with a long larval phase of up to 10 weeks. Allopatric speciation is inferred, but usually with subsequent range overlap. Ovoviviparity (interpreted as an adaptation to life in mangroves) has arisen twice.

Mudwhelks and mangroves: the evolutionary history of an ecological association (Gastropoda: Potamididae).

Most of the 29 living species of Potamididae show a close association with mangroves. The trees provide the snails with shelter, protection from predators, a solid substrate and sometimes food. Using sequences from three genes (nuclear 18S rRNA and 28S rRNA, mitochondrial COI) we derive a molecular phylogeny and recognize six living genera (Terebralia, Telescopium, Tympanotonos, Cerithidea, Cerithideopsis, Cerithideopsilla). The oldest modern genera (Terebralia, Cerithideopsis) appeared in the Tethyan realm in the Middle Eocene, shortly after the origin of mangrove trees. Whereas most potamidid genera are now restricted to either the Indo-West Pacific (IWP) or to the eastern Pacific plus Atlantic (EPA), sister clades of Cerithideopsis survive in both realms. Based on a reinterpretation of the fossil record (particularly of the monotypic Tympanotonos and extinct Potamides), and parsimonious reconstruction of ancestral habitats, we suggest that the living potamidids are an adaptive radiation that has always been closely associated with mangroves. The specialized tree-climbing groups Cerithidea and Cerithideopsis were independently derived from mud-dwelling ancestors. Cerithideopsilla cingulata (a species complex in the IWP) and 'Potamides' conicus (in the Mediterranean and Indian Ocean) form a single clade within the genus Cerithideopsilla. This refutes the hypothesis that 'P.'conicus is the sole relict of the Tethyan Potamides that has occurred in the Mediterranean region since the Palaeocene. Instead, the phylogeny and fossil record suggest that an ancestor of Cerithideopsilla conica with planktotrophic larvae dispersed from the IWP to the Mediterranean in the Middle Miocene, that its direct development evolved in the Mediterranean during the Pliocene, and that it reinvaded the Indian Ocean during the Plio-Pleistocene.

Molecular phylogeny suggests polyphyly of both the turban shells (family Turbinidae) and the superfamily Trochoidea (Mollusca: Vetigastropoda).

Bayesian and parsimony phylogenetic analyses of sequence from two nuclear and two mitochondrial genes suggest that neither the molluscan superfamily Trochoidea, nor the family Turbinidae are monophyletic. The family Turbinidae s.l. divides into two main groups. The first group includes taxa previously referred to the five subfamilies Angariinae, Colloniinae, Phasianellinae, Tricoliinae, Gabrieloninae, and the liotiine genus Cinysca; these subfamilies are here recognized as Angariidae, Colloniidae, and Phasianellidae (with subfamilies Phasianellinae, Tricoliinae, and Gabrieloninae). The second group, which corresponds to Turbinidae sensu stricto, includes Prisogasterinae, Turbininae, and the liotiine genus Liotina, all of which are more closely related to trochids than they are to the first group. Several morphological studies have suggested previously that the family Phasianellidae is distinct from Turbinidae. However, this is the first study to suggest that Phasianellidae forms a group with some taxa previously thought of as turbinids, but excluding the nominotypical genus Turbo and its allies. The family Turbinidae has traditionally been described as the only family in the Vetigastropoda group that has a calcified operculum. The non-monophyly of Turbinidae suggests that calcareous opercula may have arisen independently more than once within the Vetigastropoda.

Speciation and diversity on tropical rocky shores: a global phylogeny of snails of the genus Echinolittorina.

A phylogenetic approach to the origin and maintenance of species diversity ideally requires the sampling of all species within a clade, confirmation that they are evolutionarily distinct entities, and knowledge of their geographical distributions. In the marine tropics such studies have mostly been of fish and reef-associated organisms, usually with high dispersal. In contrast, snails of the genus Echinolittorina (Littorinidae) are restricted to rocky shores, have a four-week pelagic development (and recorded dispersal up to 1400 km), and show different evolutionary patterns. We present a complete molecular phylogeny of Echinolittorina, derived from Bayesian analysis of sequences from nuclear 28S rRNA and mitochondrial 12S rRNA and COI genes (nodal support indicated by posterior probabilities, maximum likelihood, and neighbor-joining bootstrap). This consists of 59 evolutionarily significant units (ESUs), including all 50 known taxonomic species. The 26 ESUs found in the Indo-West Pacific region form a single clade, whereas the eastern Pacific and Atlantic species are basal. The earliest fossil occurred in the Tethys during the middle Eocene and we suggest that the Indo-West Pacific clade has been isolated since closure of the Tethyan seaway in the early Miocene. The geographical distributions of all species (based on more than 3700 locality records) appear to be circumscribed by barriers of low temperature, unsuitable sedimentary habitat, stretches of open water exceeding about 1400 km, and differences in oceanographic conditions on the continuum between oceanic and continental. The geographical ranges of sister species show little or no overlap, indicating that the speciation mode is predominantly allopatric. Furthermore, range expansion following speciation appears to have been limited, because a high degree of allopatry is maintained through three to five branching points of the phylogeny. This may be explained by infrequent long-distance colonization, habitat specialization on the oceanic/continental gradient, and perhaps by interspecific competition. In the eastern Pacific plus Atlantic we identify five cases of divergence on either side of the Isthmus of Panama, but our estimates of their ages pre-date the emergence of the Isthmus. There are three examples of sister relationships between species in the western Atlantic and eastern Atlantic, all resulting from dispersal to the east. Within the Indo-West Pacific, we find no geographical pattern of speciation events; narrowly endemic species of recent origin are present in both peripheral and central parts of the region. Evidence from estimated divergence times of sister species, and from a plot of the number of lineages over time, suggest that there has been no acceleration of diversification during the glacio-eustatic cycles of the Plio-Pleistocene. In comparison with reefal organisms, species of Echinolittorina on rocky shores may be less susceptible to extinction or isolation during sea-level fluctuations. The species richness of Echinolittorina in the classical biogeographic provinces conforms to the common pattern of highest diversity (11 species) in the central "East Indies Triangle" of the Indo-West Pacific, with a subsidiary focus in the eastern Pacific and western Atlantic, and lowest diversity in the eastern Atlantic. The diversity focus in the East Indies Triangle is produced by a mosaic of restricted allopatric species and overlap of a few widespread ones, and is the result of habitat specialization rather than historical vicariance. This study emphasizes the plurality of biogeographic histories and speciation patterns in the marine tropics.

Identification of the primary caspase 3 cleavage site in alpha II-spectrin during apoptosis.

Alpha II-spectrin is one of the major proteins responsible for maintaining the cytoskeletal integrity of the cell. The caspase 3-mediated cleavage of alpha II-spectrin during apoptotic cell death may play an important role in altering membrane stability and the formation of apoptotic bodies. In this study, we identified the primary caspase 3 cleavage site in alpha II-spectrin. We found that the transcriptional inhibitor, actinomycin D, induced caspase 3 activation and that caspase 3 activation is coincident with the cleavage of alpha II-spectrin protein at a primary cleavage site. Deletion analysis and site directed mutagenesis identified the primary cleavage site in alpha II spectrin at amino acid 1185 (DETD). The primary caspase 3 cleavage site in alpha II spectrin is conserved in immature and mature B cells. Our results indicate that alpha II-spectrin is initially cleaved at a caspase 3 consensus site and this primary event likely alters the structural conformation of the protein exposing subsequent cleavage sites and altering cytoskeletal integrity. Identification of the primary cleavage site for caspase 3 may help to elucidate the role of alpha II-spectrin in membrane stability and apoptosis as well as provide new insights into alpha II-spectrin autoantibody formation associated with the autoimmune disease, Sjögren's syndrome.

A molecular phylogeny of the Littorininae (Gastropoda: Littorinidae): unequal evolutionary rates, morphological parallelism, and biogeography of the Southern Ocean.

A molecular phylogeny is presented for the subfamily Littorininae (including representatives of all subgeneric taxa and all members of a group of southern-temperate species formerly classified as 'Nodilittorina'), based on sequence data from two nuclear (18S rRNA, 28S rRNA) and two mitochondrial (12S rRNA, CO1) genes. The phylogeny shows considerable disagreement with earlier hypotheses derived from morphological data. In particular, 'Nodilittorina' is polyphyletic and is here divided into four genera (Echinolittorina, Austrolittorina, Afrolittorina new genus, and the monotypic Nodilittorina s.s.). The phylogenetic relationships of 'Littorina' striata have been controversial and it is here transferred to the genus Tectarius, a surprising relationship for which there is little morphological support. The relationships of the enigmatic Mainwaringia remain poorly resolved, but it is not a basal member of the subfamily. The two living species of Mainwaringia are remarkable for a greatly elevated rate of evolution in all four genes examined; it is suggested that this may be connected with their protandrous hermaphroditism, which is unique in the family. The molecular phylogeny provides a new framework for the adaptive radiation of the Littorininae, showing more frequent shifts between habitats and climatic regimes than previously suspected, and striking parallelism of morphological characters. The fossil record of littorinids is poor, but ages of clades are estimated using a calibration based on a Lower Eocene age of the genus Littoraria. Using these estimates, the antitropical distribution of Littorina and Afrolittorina is an ancient pattern of possibly Cretaceous age. The five members of Austrolittorina show a Gondwanan distribution in Australia, New Zealand, and South America. Based on the morphological uniformity within this clade, relatively recent (Plio-Pleistocene) trans-Pacific dispersal events seemed a likely explanation, as proposed for numerous other congeneric marine taxa. However, molecular estimation of ages of divergence suggest an initial vicariance between Australian and South American lineages at 40-73Ma, contemporary with the later stages of fragmentation of the Gondwanan supercontinent, followed by more recent (but still mid-Cenozoic) dispersal events across the Tasman Sea and the Pacific Ocean. Afrolittorina is another Cretaceous clade, now restricted to southern Africa and southern Australia, but divergence between these lineages (29-55Ma) post-dates Gondwanan fragmentation. Within both Austrolittorina and Afrolittorina all sister-species divergences are estimated to fall in the range 10-47Ma, so that there is no evidence for speciation events in the Plio-Pleistocene.

The marine indo-west pacific break: contrasting the resolving power of mitochondrial and nuclear genes.

Simultaneous studies of both nuclear and mitochondrial markers were undertaken in two widespread Indo-West Pacific (IWP) marine invertebrates to compare and contrast the ability of these markers to resolve genetic structure. In particular, we were interested in the resolution of a genetic break between the Indian and Pacific Oceans due to historical isolation. Sequence variation from the nuclear gene encoding myosin heavy chain (MyHC) and the mitochondrial gene cytochrome oxidase I (COI) were examined for the snapping shrimp Alpheus lottini from wide-ranging populations throughout the Indian and Pacific Oceans. A previously identified genetic break between oceans based on COI sequences appears to have been an artifact caused by the inadvertent inclusion of pseudogene sequences; our new COI data provide evidence only of a break between IWP and East Pacific populations. Distribution of a single nucleotide polymorphism in MyHC, on the other hand, shows evidence of a cline between Indian and Pacific Oceans. New allozyme and mtDNA sequence data were also obtained for the starfish Linckia laevigata. Allozyme data show a clear genetic break between Indian Ocean populations and Pacific (including western Australian) populations, whereas the distribution of mtDNA haplotypes shows a region of overlap in the central IWP. Comparisons of our data for both Alpheus and Linckia with data from other population genetic studies in the IWP suggest that nuclear markers (allozymes, sequence data and morphological characters) may in some instances reveal historical patterns of genetic population structure whereas mtDNA variation better reflects present day patterns of gene flow.

Evidence for three major clades within the snapping shrimp genus Alpheus inferred from nuclear and mitochondrial gene sequence data.

The snapping shrimp genus Alpheus is among the most diverse of caridean shrimps, and analyses of taxa separated by the Isthmus of Panama have been used to estimate rates of molecular evolution. Although seven morphological groups have been informally suggested, no formal phylogenetic analysis of the genus has been previously attempted. Here we infer the phylogenetic relationships within Alpheus using sequence data from two nuclear genes, glucose-6-phosphate isomerase and elongation factor-1alpha, and from the mitochondrial gene cytochrome oxidase I. Three major clades corresponding to previously noted morphological features were identified. Discrepancies between earlier informal morphological groupings and molecular analyses largely consisted of species whose morphologies were not entirely typical of the group to which they had been assigned. The traditional placements of shrimp with highly sessile lifestyles and consequently simplified morphologies were also not supported by molecular analyses. Phylogenies for Alpheus suggest that specialized ecological requirements (e.g., symbiotic associations and estuarine habitats) and modified claw morphologies have evolved independently several times. These new analyses also support the sister species status of transisthmian pairs analyzed previously, although very similar pairs were not always resolved with the more slowly evolving nuclear loci. In addition, six new cryptic species were identified in the course of these studies plus a seventh whose status remains to be determined.

Mitochondrial pseudogenes are pervasive and often insidious in the snapping shrimp genus Alpheus.

Here we show that multiple DNA sequences, similar to the mitochondrial cytochrome oxidase I (COI) gene, occur within single individuals in at least 10 species of the snapping shrimp genus Alpheus. Cloning of amplified products revealed the presence of copies that differed in length and (more frequently) in base substitutions. Although multiple copies were amplified in individual shrimp from total genomic DNA (gDNA), only one sequence was amplified from cDNA. These results are best explained by the presence of nonfunctional duplications of a portion of the mtDNA, probably located in the nuclear genome, since transfer into the nuclear gene would render the COI gene nonfunctional due to differences in the nuclear and mitochondrial genetic codes. Analysis of codon variation suggests that there have been 21 independent transfer events in the 10 species examined. Within a single animal, differences between the sequences of these pseudogenes ranged from 0.2% to 20.6%, and those between the real mtDNA and pseudogene sequences ranged from 0.2% to 18.8% (uncorrected). The large number of integration events and the large range of divergences between pseudogenes and mtDNA sequences suggest that genetic material has been repeatedly transferred from the mtDNA to the nuclear genome of snapping shrimp. Unrecognized pseudogenes in phylogenetic or population studies may result in spurious results, although previous estimates of rates of molecular evolution based on Alpheus sister taxa separated by the Isthmus of Panama appear to remain valid. Especially worrisome for researchers are those pseudogenes that are not obviously recognizable as such. An effective solution may be to amplify transcribed copies of protein-coding mitochondrial genes from cDNA rather than using genomic DNA.

Differential effects of cyclosporin and tacrolimus on the expression of fibrosis-associated genes in isolated glomeruli from renal transplants.

BACKGROUND: Chronic allograft nephropathy is characterized by an excessive accumulation of extracellular matrix proteins leading to glomerular and interstitial fibrosis. The aim of this study was to determine the effects of two different immunosuppressive agents (cyclosporin and tacrolimus) on the expression of the genes controlling extracellular matrix deposition in renal transplant glomeruli. METHODS: Fifty-one renal transplant recipients were randomized to receive immunosuppression with either microemulsion cyclosporin or tacrolimus. Isolated glomeruli were plucked from protocol transplant biopsies performed 1 week, 3 months and 6 months after transplantation. Expression of the genes for collagen IValpha2, collagen III, matrix metalloproteinase 2, tissue inhibitor of metalloproteinases (TIMP) 1 and TIMP-2, tenascin and transforming growth factor (TGF) beta1 was studied by quantitative reverse transcriptase-polymerase chain reaction. RESULTS: The expression of messenger RNA (mRNA) for collagen III and TIMP-1 was significantly higher in patients receiving cyclosporin therapy than in those having tacrolimus (P < 0.01); this finding was accounted for by differences in the biopsy material at 1 week. A significant difference in collagen III, TIMP-1 and TIMP-2 mRNA expression was also detected between patients depending on the source of renal donor (cadaveric or living). There were no significant differences in the level of glomerular TGF-beta1. CONCLUSION: The data provide new in vivo evidence that tacrolimus may exert a less fibrogenic influence on transplant glomeruli than cyclosporin.